Patent Application Publication Oo) Pub. No.: US 2012/0301400 Al Williams Et Al

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US 20120301400A1 (19) United States (12) Patent Application Publication oo) Pub. No.: US 2012/0301400 Al Williams et al. (43) Pub. Date: Nov. 29,2012

(54) MEDITOPES AND MEDITOPE-BINDING (30) Foreign Application Priority Data ANTIBODIES AND USES THEREOF Oct. 10, 2011 (US) ...... PCT/US2011/055656 (76) Inventors: John C. Williams, Monrovia, CA (US); David A. Horne, Duarte, CA Publication Classification (US); Yuelong Ma, Duarte, CA (51) Int. Cl. (US); Heng Wei Chang, Foster C07K16/18 (2006.01) City, CA (US); Joshua Michael C07K 7/08 (2006.01) Donaldson, Lumberton, NJ (US); C07K1/14 (2006.01) Cindy Zer, Duarte, CA (US); A61K 39/395 (2006.01) Krzysztof Bzymek, Pasadena, CA A61K 49/00 (2006.01) (US); Kendra Nicole Avery, C12N 5/071 (2010.01) Pasadena, CA (US); Jun Xie, C07K 7/06 (2006.01) Duarte, CA (US) C07K2/00 (2006.01) (21) Appl. No.: 13/443,804 (52) U.S. Cl...... 424/9.1; 530/387.9; 530/330; 530/329; 530/328; 530/327; 530/326; 530/321; 424/139.1; (22) Filed: Apr. 10, 2012 424/178.1; 435/331

Related U.S. Application Data (57) ABSTRACT (63) Continuation-in-part of application No. 13/270,207, Antibodies and meditopes that bind to the antibodies are filed on Oct. 10, 2011. provided, as well as complexes, compositions and combina (60) Provisional application No. 61/391,558, filed on Oct. tions containing the meditopes and antibodies, and methods 8, 2010, provisional application No. 61/597,708, filed of producing, using, testing, and screening the same, includ on Feb. 10, 2012. ing therapeutic and diagnostic methods and uses. Patent Application Publication Nov. 29, 2012 Sheet I of 79 US 2012/0301400 Al

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MEDITOPES AND MEDITOPE-BINDING aspects, the antibodies bind to a meditope that is a cyclic ANTIBODIES AND USES THEREOF peptide derived from a peptide having an amino acid sequence of SEQ ID NO: I. PRIORITY CLAIM [0007] In some aspects, the antibodies bind to a meditope [0001] This application claims the benefit of U.S. Provi that is a cyclic peptide derived from a peptide having an sional Patent Application Ser. No. 61/597,708, filed Feb. 10, amino acid sequence of SEQ ID NO: 2. In some aspects, the 2012; U.S. application Ser. No. 13/270,207, filed Oct. 10, antibodies bind to a meditope that is a peptide having an 2011, which claims the benefit of U.S. Provisional Applica amino acid sequence selected from the group consisting of tion Ser. No. 61/391,558 filed Oct. 8, 2010; and to Interna the sequences set forth in SEQ ID NO: I, 2,16-18,23, 29,31, tional Application Number PCT/US2011/055656 filed Oct. 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, and 55, or a cyclic 10, 2011, the contents of which are incorporated herein by peptide derived therefrom, or the sequences set forth in SEQ reference in their entirety. ID NOs: I, 2, and 15-55, or a cyclic peptide derived there from, or to any of the meditopes described herein. In some BACKGROUND cases, the meditope is a cyclic peptide derived from a peptide [0002] Monoclonal antibodies (mAbs) are used in a num of the amino acid sequence set forth in SEQ ID NO: I or 2. ber of therapeutic, diagnostic, and research applications. [0008] In some aspects, the antibody or fragment binds to Therapeutic and diagnostic areas include cancer, antiviral the meditope with a particular affinity, such as an affinity that treatment, autoimmune and inflammatory disease, allergy, is equal to or substantially equal to one of the exemplary cardiovascular disease, osteoporosis, and rheumatology. antibodies described herein, such as cetuximab, meditope- [0003] Protein engineering and other efforts have generated enabled trastuzumab, meditope-enabled MSA, or other mAbs with improved efficacy and targeting (e.g., bispecific exemplified antibody. In some examples, the antibodies bind mAbs), improved localization, tissue penetration, and blood to the meditope or meditopes with a dissociation constant of clearance (e.g., single chain Fab variable fragments (scFvs), less than at or about 10 μΜ, less than at or about 5 μΜ, or less diabodies, minibodies, and other fragments), and altered than at or about 2 μΜ, less than at or about I μΜ, less than at immunostimulatory, safety, toxicity, and/or pharmacokinetic/ or about 500, 400, 300, 200, 100 nM, or less, such as at or pharmacodynamics properties, such as those containing about 200 picomolar or less, for example, with such a disso modified Fe regions (e.g., through mutation or glycosyla- ciation constant, as measured by a particular technique, such tion). mAbs have been reengineered to permit site-specific as surface plasmon resonance (SPR), Isothermal Titration conjugation of small molecules for improved delivery (e.g., calorimetry (ITC), fluorescence, fluorescence polarization, ThioMABs) or to irreversibly bind to their antigen (e.g., NMR, IR, calorimetry titrations; Kinetic exclusion; Circular infinite affinity mAbs). mAbs have also been developed to dichroism, differential scanning calorimetry, or other known improve the circulation and presentation of bioactive peptides method, e.g., by SPR. and other biologies (e.g., CovX-bodies). Conjugationto vari [0009] In some aspects, the antibodies include a heavy ous agents has allowed targeted immunotherapy and diagnos chain variable (VH) region and/or a light chain variable (VL) tic methods. Hetero-multimeric scFvs and scFvs or mAbs region. In some aspects, the VL region has an amino acid fused to avidin have been developed for pre-targeted therapy sequence comprising a threonine, serine, or aspartate at posi and to improve the detection limits for tumor imaging. tion 40, a residue other than glycine at position 41, and/or an [0004] Although mAbs can be effective and have advan aspartate or asparagine at position 85, according to Kabat tages over small molecule approaches, existing antibodies numbering, and/or comprises an isoleucine or leucine at posi and methods have various limitations. These can include tion 10 and isoleucine at position 83, according to Kabat adverse side effects resulting from off-taiget interactions, numbering, and/or comprises a valine or isoleucine at posi and/or collateral damage due to, among other things, long tion 9 and a residue other than glutamine at position 100, circulation times of antibody-drug conjugates). There is a according to Kabat numbering. In some examples, the amino need for improved antibodies and associated compounds, acid sequence of the VL region has a threonine at position 40, including those providing improved efficacy, synergy, speci an asparagine at position 41, and an aspartate at position 85, ficity, and safety, and methods and uses of the same. Provided according to Kabat numbering. are antibodies, compounds and compositions including pep [0010] In some aspects, the VH region has an amino acid tides and other molecules, and related methods that address sequence comprising a serine or proline at position 40 and an such needs. isoleucine, tyrosine, methionine, phenylalanine, or tryp tophan at position 89, according to Kabat numbering. In some SUMMARY examples, the amino acid sequence of the VH region has a [0005] Among the provided embodiments are antibodies, serine at position 40 and an isoleucine at position 89, accord compounds and compositions, including peptides and other ing to Kabat numbering. molecules for use with the antibodies, as well as methods and [0011] In some examples, the amino acid sequence of the uses thereof and for producing the same, as well as com VL region has a valine or isoleucine at position 9, an isoleu pounds, compositions, complexes, mixtures, and systems, cine or leucine at position 10, an arginine at position 39, a e.g., kits, containing the same. In some aspects, the provided threonine at position 40, an asparagine at position 41, a gly embodiments afford improved efficacy, syneigy, specificity, cine at position 42, a serine at position 43, an isoleucine at and/or safety, compared with available antibodies and asso position 83, an aspartate at position 85, and an alanine at ciated compounds, compositions, methods and uses. position 100, according to Kabat numbering; and/or the [0006] Provided are antibodies, including meditope-en- amino acid sequence of the VH region has a serine at position abled antibodies (including fragments thereof), which bind to 40 and an isoleucine at position 89, according to Kabat num (i.e., are capable of binding to) one or more meditope. In some bering. US 2012/0301400 Al Nov. 29, 2012 2

[0012] In some aspects of the provided antibodies, the VL [0019] In some examples, the VL region comprises the region does not contain a proline at position 40, a glycine at amino acid sequence of SEQ ID NO: 73. In some examples, position 41, and/or a threonine at position 85, according to the VH region comprises the amino acid sequence of SEQ ID Kabat numbering, and/or the VH region does not contain an NO: 74. asparagine or alanine at position 40 and/or a valine at position [0020] In some aspects, the antibody or fragment has a VL 89, according to Kabat numbering. region with an amino acid sequence comprising a light chain [0013] In some aspects, the VL region does not contain an framework region (FR) I (FR-L1), an FR-L2, an FR-L3, serine at position 10, a proline at position 40, a glycine at and/or an FR-L4 of the light chain sequence set forth in SEQ position 41, an phenylalanine at position 83, and/or a threo ID NO: 68 (or an FR-Ll, FR-L2, FR-L3, and/or FR-L4 that is nine at position 85, according to Kabat numbering, and/or the at least at or about 75, 80, 85, 90, 91,92,93,94,95,96,97,98, VH region does not contain an asparagine or alanine at posi or 99% identical to the FR-Ll, FR-L2, FR-L3, and/or FR-L4 tion 40 and/or a valine at position 89, according to Kabat of the light chain of SEQ ID NO: 68). In some examples, the numbering. VL region comprises at least one complementarity determin [0014] In some aspecst, the antibodies (including frag ing region (CDR) that is distinct from the CDRs of the light ments) further include one or more constant regions, typically chain sequence set forth in SEQ ID NO: 69; and/or the VH human constant region(s), such as a CL and/or CHI, e.g., region comprises at least one CDR that is distinct from the human CL and/or CHl. CDRs of the heavy chain sequence set forth in SEQ ID NO: [0015] In some aspects, the provided antibody or fragment 70. has a VL region with an amino acid sequence comprising a [0021] In some examples, the VL region comprises the light chain framework region (FR) I (FR-L1), an FR-L2, an amino acid sequence of SEQ ID NO: 75. FR-L3, and/or an FR-L4 of the light chain sequence set forth [0022] In some aspects, the antibody does not specifically in SEQ ID NO: 71 or SEQ ID NO: 61 (or an FR-Ll, FR-L2, bind to the epitope of an EGFR that is specifically bound by FR-L3, and/or FR-L4 that is at least at or about 75, 80, 85, 90, cetuximab, does not contain the CDRs of cetuximab, and/or 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the FR-Ll, does not compete for antigen binding with cetuximab. In FR-L2, FR-L3, and/or FR-L4 of the light chain of SEQ ID other aspects, the antibody is cetuximab. NO: 71 or 61), and in some aspects at least one complemen [0023] In some aspects, the antibodies or fragments com tarity determining region (CDR) that is distinct from the pete for antigen binding with, specifically bind to the same CDRs of the light chain sequence set forth in SEQ ID NO: 71; antigen or epitope as, and/or contain one, more, or all CDRs and/or a VH region with an amino acid sequence having a (or CDRs comprising at least at or about 75,80,85,90, 91,92, heavy chain FRl (FR-H1), an FR-H2, an FR-H3, and/or an 93, 94, 95, 96, 97, 98, or 99% identity to the CDRs), e.g., FR-H4, of the heavy chain sequence set forth in SEQ ID NO: including a heavy chain CDR I, 2, and/or 3 and/or a light 72 or SEQ ID NO: 63 (or an FR-Hl, FR-H2, FR-H3, and/or chain CDRl, 2, and/or 3, of one or more known antibodies, FR-H4 that is at least at or about 75, 80, 85, 90, 91, 92, 93, 94, including any commercially available antibody, such as 95,96, 97, 98, or 99% identical to the FR-H1,FR-H2, FR-H3, abagovomab, abciximab, adalimumab, adecatumumab, ale- and/or FR-H4 of the heavy chain of SEQ ID NO: 72 or 63), mtuzumab, altumomab, altumomab pentetate, anatumomab, and in some aspects at least one CDR that is distinct from the anatumomab mafenatox, arcitumomab, atlizumab, basilix- CDRs of the heavy chain sequence set forth in SEQ ID NO: imab, bectumomab, ectumomab, belimumab, benralizumab, 72. bevacizumab, brentuximab, canakinumab, capromab, capro- [0016] In some aspects, the provided antibody or fragment mab pendetide, catumaxomab, certolizumab, clivatuzumab has a CDR of the light chain sequence set forth in SEQ ID NO: tetraxetan, daclizumab, denosumab, eculizumab, edrecolo- 61, a CDR of the heavy chain sequence set forth in SEQ ID mab, efalizumab, etaracizumab, ertumaxomab, fanolesomab, NO: 63. Fbta05, fontolizumab, gemtuzumab, girentuximab, goli- [0017] In some aspects, the VL comprises the amino acid mumab, ibritumomab, igovomab, infliximab, ipilimumab, sequence of SEQ ID NO: 76 and the VH comprises the amino labetuzumab, mepolizumab, muromonab, muromonab-CD3, acid sequence of SEQ ID NO: 77. natalizumab, necitumumab, nimotuzumab, ofatumumab, [0018] In some aspects, the antibody has a VL region with omalizumab, oregovomab, palivizumab, panitumumab, an amino acid sequence comprising a light chain framework ranibizumab, rituximab, satumomab, sulesomab, ibritumo region (FR) I (FR-L1), anFR-L2, anFR-L3, and/oranFR-L4 mab, ibritumomab tiuxetan, tocilizumab, tositumomab, tras- of the light chain sequence set forth in SEQ ID NO: 9 (or an tuzumab, Trbs07, ustekinumab, visilizumab, votumumab, FR-Ll, FR-L2, FR-L3, and/or FR-L4 that is at least at or zalutumumab, and/or brodalumab; and/or anrukinzumab, about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% bapineuzumab, dalotuzumab, demcizumab, ganitumab, ino- identical to the FR-Ll, FR-L2, FR-L3, and/or FR-L4 of SEQ tuzumab, mavrilimumab, moxetumomab pasudotox, rilotu- ID NO: 9); and/or a VH region with an amino acid sequence mumab, sifalimumab, tanezumab, tralokinumab, tremeli- having a heavy chain FRl (FR-H1), an FR-H2, an FR-H3, mumab, urelumab, the antibody produced by the hybridoma and/ or an FR-H4 of the heavy chain sequence set forth in SEQ 10B5 (see Edelson & Unanue, Curr Opin Immunol, 2000 ID NO: 6 (or an FR-Hl, FR-H2, FR-H3, and/or FR-H4 that is August; 12(4):425-31), B6H12.2 (abeam) or other anti-CD47 at least at or about 75,80,85,90,91,92,93,94,95,96,97,98, antibody (see Chao et ah, Cell, 142, 699-713, Sep. 3, 2010); or 99% identical to the FR-Hl, FR-H2, FR-H3, and/or FR-H4 and/or an antibody or fragment thereof having a sequence set of SEQ ID NO: 9). In some examples, the VL region com forth in any of SEQ ID NOs: 78-124, and/or 125-170. prises at least one complementarity determining region [0024] In some aspects, the antibody or fragment specifi (CDR) that is distinct from the CDRs of the VL sequence set cally binds to an antigen selected from the group consisting forth in SEQ ID NO: 9; and/or the VH region comprises at of: CA-125, glycoprotein (GP) Ilb/IIIa receptor, TNF-alpha, least one CDR that is distinct from the CDRs of the VH CD52, TAG-72, Carcinoembryonic antigen (CEA), interleu- sequence set forth in SEQ ID NO: 6. kin-6 receptor (IL-6R), IL-2, interleukin-2 receptor a-chain US 2012/0301400 Al Nov. 29, 2012 3

(CD25), CD22, B-cell activating factor, interleukin-5 recep residues 39, 89, 105, and/or 108 of the heavy chain of the tor (CD125), VEGF, VEGF-A, CD30, IL-lbeta, prostate spe meditope-enabled antibody, according to Rabat numbering. cific membrane antigen (PSMA), CD3, EpCAM, EGF recep [0030] In some aspecst, the meditope binds to a meditope- tor (EGFR), MUCl, human interleukin-2 receptor, Tac, enabled antibody having: a light chain comprising an amino RANK ligand, a complement protein, e.g., C5, EpCAM, acid sequence set forth in SEQ ID NO: 71 and a heavy chain CDl la, e.g., human CDl la, an integrin, e.g., alpha-v beta-3 comprising an amino acid sequence set forth in SEQ ID NO: integrin, vitronectin receptor alpha v beta 3 integrin, F1ER2, 72; to a meditope-enabled antibody having a light chain hav neu, CD3, CDl 5, CD20 (small and/or large loops), Interferon ing an amino acid sequence set forth in SEQ ID NO: 9 and a gamma, CD33, CA-IX, TNF alpha, CTLA-4, carcinoembry- heavy chain comprising an amino acid sequence set forth in onic antigen, IL-5, CD3 epsilon, CAM, Alpha-4-integrin, SEQ ID NO: 6; and/or to a meditope-enabled antibody having a light chain having an amino acid sequence set forth in SEQ IgE, e.g., IgE Fe region, an RSV antigen, e.g., F protein of ID NO: 68 and/or a heavy chain having an amino acid respiratory syncytial virus (RSV), TAG-72, NCA-90 (granu sequence set forth in SEQ ID NO: 70. locyte cell antigen), IL-6, GD2, GD3, IL-12, IL-23, IL-17, [0031] In some aspects, the peptide is between 5 and 16 CTAA16.88, IL13, interleukin-1 beta, beta-amyloid, IGF-I amino acids in length. receptor (IGF-1R), delta-like ligand 4 (DLL4), alpha subunit [0032] In some aspects, the peptide has the formula: of granulocyte macrophage colony stimulating factor recep tor, hepatocyte growth factor, IFN-alpha, nerve growth factor, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-Xl 2 (Formula I) IL-13, CD326, Programmed cell death I ligand I (PD-L1, wherein: a.k.a. CD274, B7-H1), CD47, and CD137. [0033] Xl=Cys, Gly, β-alanine, 2,3-diaminopropionic [0025] In some aspects, the antibody or fragment has a light acid, β-azidoalanine, or null; chain having P8, V9 or 19, HO or L10, Q38, R39, T40, N41 [0034] X2=Gln or null; G42, S43, P44, R45, D82, 183, A84, D85, Y86, Y87, G99, [0035] X3=Phe, Tyr, β-β'-diphenyl-Ala, His, Asp, A100, G101, T102, K103, L104, E105, R142, 5162, V163, 2-bromo-L-phenylalanine, 3-bromo-L-phenylalanine, or T164, E165, Q166, D167, 5168, and/or Y173, according to 4-bromo-L-phenylalanine, Asn, Gin, a modified Phe, a Kabat numbering, and/or has a heavy chain having Q6, P9, hydratable carbonyl-containing residue; or a boronic acid- R38, Q39, S40, P41, G42, K43, G44, L45, S84, D86, T87, containing residue; A88,189, Y90, Y91, W103, G104, Q105, G106, T107, L108, [0036] X4=AsporAsn; Vlll, T110, Y147, E150, P151, V152, T173, F174, P175, A176, V177, Y185, S186, and/or L187, according to Rabat [0037] X5=Leu; β-β'-diphenyl-Ala; Phe; a non-natural numbering. analog of phenylalanine, tryptophan, or tyrosine; a hydratable carbonyl-containing residue; or a boronic acid-containing [0026] Also provided are complexes containing an anti residue; body or antibodies (e.g., meditope-enabled antibodies) bound [0038] X6=Ser or Cys; to one or more meditope. The antibody or antibody can be any of the meditope-enabled antibodies described herein, such as [0039] X7=Thr or Ser or Cys; any of the aforementioned antibodies (including fragments [0040] X8=Arg, a modified Arg, or a hydratable carbonyl or thereof). The one or more meditope can include any one or boronic acid-containing residue; more of the meditopes described herein, such as those [0041] X9=Arg, Ala; described in this section, including monovalent and multiva [0042] XlO=Leu, Gin, Glu, β-β'-diphenyl-Ala; Phe; a non lent meditopes, and labeled meditopes, as well as meditope natural analog of phenylalanine, tryptophan, or tyrosine; a fusion proteins. hydratable carbonyl-containing residue; or a boronic acid- [0027] Also provided are meditopes, e.g., isolated medi containing residue; topes. Among the provided meditopes are those described [0043] Xll=Lys; and above. Among the provided meditopes are those comprising [0044] X12=Cys, Gly, 7-aminoheptanoic acid, β-alanine, a peptide that binds to a meditope binding site of a meditope- diaminopropionic acid, propargylglycine, isoaspartic acid, or enabled antibody, wherein the peptide is not a peptide of SEQ null, ID NO: I or 2 or cyclic peptide derived therefrom. In other [0045] wherein: aspects, the meditope is a peptide of SEQ ID NO: I or 2, or a [0046] the modified Arg has a structure of the formula cyclic peptide derived therefrom. shown in FIG. 34, [0028] In some asepcts, the meditope (e.g., the peptide) [0047] the modified Phe is a Phe with one or more halogen binds to the meditope binding site with a dissociation con incorporated into the phenyl ring, and stant of less than at or about 10 μΜ, lessthanatorabout 5 μΜ, [0048] formula I is not SEQ ID NO: I or SEQ ID NO: 2 or or less than at or about 2 μΜ, less than at or about I μΜ, less a cyclic peptide derived therefrom. than at or about 500, 400, 300, 200, 100 nM, or less, such as [0049] In some aspects, the peptide is a cyclic peptide, such at or about 200 picomolar or less, for example, with such a as one in which the cyclization is by disulfide bridge, a thio- dissociation constant, as measured by a particular technique, ether bridge, a lactam linkage, cycloaddition. In certain such as surface plasmon resonance (SPR), Isothermal Titra aspects, where the peptide is one of Formula I, the cyclization tion calorimetry (ITC), fluorescence, fluorescence polariza is via a linkage between Xl and X12, Xl and Xl I, X3 and tion, NMR, IR, calorimetry titrations; Kinetic exclusion; Cir Xl I, X4 and Xl I, orX2 andX12. In some aspects, where the cular dichroism, differential scanning calorimetry, or other peptide is one of Formula I, the non-natural amino acid is known method, e.g., by SPR. β-β'-diphenyl-Ala, abranched alkyl, or an extended aromatic. [0029] In some aspects, the meditope binding site includes In some aspects, where the peptide is one of Formula I, each residues 40, 41, 83, and/or 85 of the light chain of the medi of the one or more halogen is an ortho-, meta-, or para-bromo tope-enabled antibody, according to Rabat numbering, and/or phenyl substituent. US 2012/0301400 Al Nov. 29, 2012 4

[0050] Among the provided meditopes are peptides having C(O)C1âlkyl, -CH=CH-CO2C1âlkyl, an amino acid selected from the group consisting of the —CO2H, or—CO2C1âlkyl group; sequences set forth in SEQ IDNOs: 1,2, and 15-55, e.g., 1,2, [0067] Rc is H, C1âlkyl, C3_8cycloalkyl, branched 16-18,23,29,31,32,36,39,42,43,45,46,51,52,54, and55, alkyl, or aryl; e.g, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, [0068] Rrf is H or a C1âlkyl, C2_8alkenyl, C2_8alky- and 55, or a cyclic peptide derived therefrom. nyl, C3_8cycloalkyl, branched alkyl, or aryl group, [0051] In some embodiments, the meditope comprises a each optionally substituted with one or more substitu compound of Formula (X): ents selected from the group consisting of —N3, —NH2, —OH, —SH, halogen, oxo, acetal, ketal, —B(OH)2, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1. 4alkyl, -CH=CH-CO2C1âlkyl, -CO2H, and —CO2C1âlkyl group; and [0069] Re is H; -NHRrf; or a C1âlkyl, C3_8cy- cloalkyl, C2_12alkenyl, C2_8alkynyl, or aryl group, each optionally substituted with one or more substitu ents selected from the group consisting of —N3, —NH2, —OH, —SH, oxo, C2_4acetal, C2_4ketal, —B(OH)2, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1. 4alkyl, -CH=CH-CO2C1âlkyl, and -CO2C1. 4alkyl group; or [0070] (b) a Cj.âlkyl substituted with an oxo, acetal, ketal, —B(OH)2, boronic ester, —SH, —OH, phospho nate ester, ortho ester, —CH=CH—CHO, —CH=CH—C(O)C1âlkyl, -CH=CH-CO2C1. 4alkyl, or —CO2C1âlkyl group; [0071] R9 is C1âlkyl or -C1âlkylene-Rx; [0072] wherein Rx is -CO2H, -CONH2, -CH2NHC (O)NH2, or —CH2NHC(=NH)NH2; [0073] R10 is: [0074] (I) a C1âlkyl optionally substituted with one or wherein: more substituents selected from the group consisting of the center marked with is in the “R” or “S” configuration; oxo, acetal, ketal, —B(OH)2, boronic ester, phospho [0052] R3 and R3 are each, independently, Fl or phenyl, nate ester, ortho ester, —CH=CH—CHO, optionally substituted with one, two, or three substituents —CH=CH—C(O)C1âlkyl, -CH=CH-CO2C1. independently selected from C1âlkyl, —OH, fluoro, 4alkyl, -CO2C1âlkyl, -CO2H, and -CONH2 chloro, bromo, and iodo; group; or [0053] R5 is: [0075] (2) a C1âlkyl group substituted with one or two [0054] (A) C1âlkyl, optionally substituted with one or phenyl groups, or one naphthyl, imidazole, or indole more substituents selected from the group consisting of group, wherein each phenyl is optionally substituted oxo, acetal, ketal, —B(OH)2, boronic ester, phospho- with one, two, or three substituents independently nate ester, ortho ester, —CO2C1âlkyl, —CH=CH— selected from —OH, fluoro, chloro, bromo, and iodo; CHO, —CH=CH-C(O)C1âlkyl, -CH=CH- [0076] n is Oor I; CO2C1âlkyl, —CO2H, and —CONH2 group; or [0077] p is Oor I; [0055] (B) a C1âlkyl group substituted with: [0078] X is C1âlkylene or C2_8alkenylene, each carbon [0056] a) one or two phenyl groups, wherein each thereof optionally substituted with —CO2H, —NH2, or phenyl is optionally substituted with one, two, or —NHC(O)Rx; three substituents independently selected from—OH, [0079] wherein one carbon of said alkylene is optionally fluoro, chloro, bromo, and iodo; or replaced with —C(O)NH—, a 5-membered heteroaryl [0057] b) a naphthyl, imidazole, or indole group; ring, or —S—S—; and [0058] R6 is -C1âlkyl-OH or -C1âlkyl-SH; [0080] Ry is —C1âlkyl or -CH(Rz)CO2H; [0059] R7 is -C1âlkyl-OH or -C1âlkyl-SH; [0081] wherein Rz is —H or —C1âlkyl optionally [0060] m is 0, I, 2, 3, 4, or 5; substituted with —OH, —SH, or —NH2; [0061] R8 is: or a pharmaceutically acceptable salt thereof. [0062] (a) —OH, -NRaR6, -N(Rc)C(O)Re, or [0082] In one embodiment, a meditope contains a cysteine —N(Rc)C(=NRrf)Re; that covalently binds to a cysteine in a meditope binding site. [0063] wherein: Such a meditope may be conjugated to any substance, mol [0064] Ra is H; ecule or compound, which may be a therapeutic molecule, [0065] R6 is H OrC1âlkyl optionally substituted with such as a small molecule diagnostic molecule, such as a one or more substituents selected from the group con marker. In some aspects, the “Cys meditope” directs the con sisting of oxo, acetal, and ketal, jugate to the Ig and binds via a covalent linkage. [0066] —B(OH)2, —SH, boronic ester, phosphonate [0083] Also among the provided meditopes are labeled ester, ortho ester, -CH=CH-CHO, -CH=CH- meditopes, such as those comprising a meditope (such as any US 2012/0301400 Al Nov. 29, 2012 5

of those described above) and a therapeutic or diagnostic one example, the method includes administering to the sub agent. In some aspects, the therapeutic or diagnostic agent is ject one or more meditope-enabled antibody or fragment as selected from the group consisting of: a metal chelator bound described above, and a meditope, e.g., any of those described to a metal ion, a small molecule, a chemotherapeutic agent, a above, including multivalent meditopes and meditopes therapeutic antibody or functional fragment, a toxin, a radio coupled to a therapeutic or diagnostic agent. In one aspect, the isotope, an enzyme, a nuclease, a hormone, an immunomodu- meditope-enabled antibody or fragment and one or more lator, an oligonucleotide, an oiganic or inorganic nanopar meditope are administered sequentially. In another aspect, ticle, an RNAi molecule, an siRNA, a chelator, a boron they are administered simultaneously. Generally, the one or compound, a photoactive agent, a dye, fluorescent or lumi more meditope comprises a peptide that binds to a meditope nescent substance, an enzyme, an enhancing agent, a radio binding site of the meditope-enabled antibody or fragment. In active substance, and a chelator. one aspect, the meditope-enabled antibody or fragment is [0084] Also among the provided meditopes are multivalent bound to the one or more meditope, such that administration meditopes, such as those comprising two or more meditopes of the meditope-enabled antibody and the one or more medi and one or more linker, e.g., where each of the two or more meditopes is a peptide that binds to a meditope binding site of tope comprises administering a complex of the meditope- a meditope-enabled antibody. Such multivalent meditopes enabled antibody and the meditope. In another aspect, the may comprise any of the meditopes described herein, e.g., meditope-enabled antibody is administered prior to adminis above. In one aspect, the two or more meditopes comprise at tration of the one or more meditope. least three meditopes, or at least four meditopes. In one [0091] In some aspects, the one or more meditope is aspect, the one or more linker includes a peptide, a small coupled to a therapeutic agent, such as a therapeutic agent chemical scaffold, a biotin-streptavidin, an organic or inor selected from the group consisting of: drugs, chemotherapeu ganic nanoparticle, a polynucleotide sequence, peptide tic agents, therapeutic antibodies, toxins, radioisotopes, nucleic acid, an organic polymer, or an immunoglobulin Fe enzymes, chelators, boron compounds, photoactive agents, domain. dyes, metals, metal alloys, and nanoparticles. [0085] Also among the provided embodiments are medi [0092] Also provided are diagnostic methods, such as those tope-enabled antibody-meditope complexes, including those carried out by administering to a subject such pharmaceutical containing any of the meditope-enabled antibodies and any of compositions, or any of the meditope-enabled antibodies, the meditopes described herein, e.g., described above. In meditopes, complexes, and/or other compound described some cases, the peptide binds to the meditope binding site herein, e.g., above, and detecting binding of the administered with a dissociation constant of less than 10 μΜ, as measured composition, antibody, meditope, complex, and/or com by surface plasmon resonance (SPR), or another affinity as pound to a substance in the subject, e.g., to an antigen. In described above. some aspects, the diagnostic method includes administering [0086] Also provided are methods using the meditopes and/ to a subject one or more meditope-enabled antibody or frag or antibodies, such as any of the above meditopes and/or ment thereof, e.g., any of those described above. In some antibodies. For example, provided are methods for purifying aspects, it further includes detecting binding of the antibody a meditope-enabled antibody or fragment thereof, or a cell or fragment to an antigen in the subject. In some aspects, the expressing the meditope-enabled antibody or fragment meditope-enabled antibody or fragment and one or more thereof. In one aspect, such methods include a step of con meditope are administered sequentially; in other aspecst, they tacting a composition containing the meditope-enabled anti are administered simultaneously. In one example, the medi body, fragment, or cell with a meditope, under conditions tope-enabled antibody or fragment is bound to the one or whereby the meditope-enabled antibody or fragment binds to more meditope, such that administration of the meditope- the peptide. In some aspect, they further include then isolating enabled antibody and the one or more meditope comprises the antibody or fragment or cell. Such methods in some administering a complex of the meditope-enabled antibody aspects therby purify the antibody, fragment, or cell. and the meditope. In another example, the meditope-enabled [0087] In some aspects, the meditope is coupled to a solid antibody is administered prior to administration of the one or support. In some aspects, the isolation or purification is more meditope. In some aspects, the meditope or meditopes effected by a change in pH. is a multivalent meditope. In some aspects, the meditope is [0088] Also provided are compositions, e.g., pharmaceuti coupled to a diagnostic agent, such as an imaging agent, such cal compositions, comprising the meditopes (including mul as an imaging agent selected from the group consisting of: tivalent and labeled meditopes), meditope-enabled antibod fluorescent substances, luminescent substances, dyes, indica ies, and complexes, and/or other compounds described tors, and radioactive substances. In some cases, the imaging herein, e.g., above. In one example, the composition includes agent is DOTA. the complex, meditope, and/or meditope-enabled antibody, [0093] Also provided are methods for generating medi and a pharmaceutically acceptable carrier. tope-enabled antibodies or fragments thereof, e.g., based on [0089] Also provided are methods of treatment, such as template antibodies. In some aspects, such methods include those carried out by administering to a subject such pharma effecting one or more amino acid substitutions in a template ceutical compositions, or any of the meditope-enabled anti antibody or fragment thereof. In one example, the substitu bodies, meditopes, complexes, and/or other compound tions include a substitution at position 40, 41, or 85 of the VL described herein, e.g., above. In one example, the methods region, and/or position 40 or position 89 of the VH region, include administering to the subject an antibody or fragment according to Rabat numbering. as described above. [0094] In some aspects, the methods generate a meditope- [0090] In one example, the method of treatment includes enabled antibody or fragment thereof which binds to a medi administering to a subject one or more meditope-enabled tope that is a peptide having an amino acid sequence selected antibody or fragment, e.g., any of those described above. In from the group consisting of the sequences set forth in SEQ US 2012/0301400 Al Nov. 29, 2012 6

ID NO: I, 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, combining a meditope and a meditope-enabled antibody or 52, 54, and 55, or a cyclic peptide derived therefrom. fragment thereof, whereby the meditope non-covalently [0095] In some aspects, the template antibody or fragment binds to the antibody or fragment thereof; and (b) adding a is selected from the group consisting of abagovomab, abcix- candidate meditope variant or analog; and (c) measuring dis imab, adalimumab, adecatumumab, alemtuzumab, altumo- placement of the meditope by the candidate meditope variant mab, altumomab pentetate, anatumomab, anatumomab or analog, wherein displacement of the meditope by the can mafenatox, arcitumomab, atlizumab, basiliximab, bectumo- didate variant or analog identifies it as the meditope variant or mab, ectumomab, belimumab, benralizumab, bevacizumab, analog. brentuximab, canakinumab, capromab, capromab pendetide, [0101] Also provided are methods for modifying medi catumaxomab, certolizumab, clivatuzumab tetraxetan, dacli- topes and meditope-enabled antibodies, including a method zumab, denosumab, eculizumab, edrecolomab, efalizumab, for modifying a meditope, comprising effecting one or more etaracizumab, ertumaxomab, fanolesomab, Fbta05, fontoli- amino acid substitutions, insertions, or deletions, to a peptide zumab, gemtuzumab, girentuximab, golimumab, ibritumo- having the sequence set forth in any of SEQ ID NOs: I, 2, mab, igovomab, infliximab, ipilimumab, labetuzumab, 16-18,23,29,31,32,36,39,42,43,45,46,51,52, 54, and55, mepolizumab, muromonab, muromonab-CD3, natalizumab, or cyclic peptide derived therefrom, thereby altering the pH- necitumumab, nimotuzumab, ofatumumab, omalizumab, dependence of the binding affinity of the peptide for a medi oregovomab, palivizumab, panitumumab, ranibizumab, rit- tope-enabled antibody or fragment thereof, such as any of uximab, satumomab, sulesomab, ibritumomab, ibritumomab those described above. In some aspects, the method decreases tiuxetan, tocilizumab, tositumomab, trastuzumab, Trbs07, the binding affinity of the peptide for the antibody or fragment ustekinumab, visilizumab, votumumab, zalutumumab, and at a lysosomal pH level. In other aspects, the method brodalumab, or is the antibody produced by the hybridoma increases the binding affinity in a hypoxic environment. 10B5, oris B6H12.2. [0096] In some aspects, the meditope-enabled antibody [0102] Also provided are methods for modifying a medi binds to one or more meditopes (e.g., one of SEQ ID NO: I, tope-enabled antibody, such as those comprising: effecting 2, or cyclic peptide derived therefrom), e.g., those described one or more modificaitons at position 8, 9, 10, 38, 39, 40, 41 above, with a particular affinity, such as with a dissociation 42, 43, 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, constant of less than 10, 5, or 2 (or other number specified 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, and/or 173 above) μΜ, as measured by SPR, or other method described of the light chain, or 6, 9,38, 39,40,41,42,43,44,45, 84, 86, above. 87,88,89, 90,91,103,104,105,106,107,108,109,110,147, 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and/or 187 [0097] In some aspects, each of the one or more amino acid of the heavy chain, of the meditope-enabled antibody, accord substitutions is at a position selected from the group consist ing to Rabat numbering. In some aspects, the modification ing of: position 8, 9, 10, 38, 39, 40, 41 42, 43, 44, 45, 82, 83, methods are provided in tandem, to produce modified medi 84,85,86,87,99,100,101,102,103,104,105,142,162,163, topes and modified meditope-enabled antibodies that bind to 164, 165, 166, 167, 168, and 173 of the light chain, and/or 6, one another, such as by making modifications in the antibody 9,38,39,40,41,42,43,44,45, 84,86, 87, 88, 89, 90,91,103, based on modifications in a modified meditope, or vice versa. 104, 105, 106, 107, 108, 109, HO, 147, 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and 187 of the heavy chain, [0103] Also provided are meditope analogs, including according to Rabat numbering. In some aspects, the medi those that bind to any of the meditope-enabled antibodies tope-enabled antibody contains a light chain having P8, V9 or described above, and/or with binding properties comparable 19, HO or L10, Q38, R39, T40, N41 G42, S43, P44, R45, D82, to any of the meditopes described above. 183, A84, D85, Y86, Y87, G99, A100, G101, T102, K103, L104, E105, R142, S162, V163, T164, E165, Q166, D167, BRIEF DESCRIPTION OF THE DRAWINGS S168, andY173, according to Rabat numbering and a heavy chain having Q6, P9, R38, Q39, S40, P41, G42, R43, G44, [0104] FIG. I shows meditope peptides binding to frame L45, S84, D86, T87, A88,189, Y90,Y91, W103, G104, Q105, work loops of cetuximab. FIG. 1A: The complex of cetux- G106, T107, L108, Vlll, T110, Y147, E150, P151, V152, imab Fab (light chain is denoted by Vi and Ci; heavy chain is T173, F174, P175, A176, V177, Y185, S186, and L187, denoted by Vh and Ch) and cyclic CQFDLSTRRLRC (de according to Rabat numbering. picted within the shaded area and labeled with the word [0098] Also provided are polynucleotides comprising a “meditope”) (SEQ ID NO: I) indicates that the meditope nucleotide sequence encoding the meditope-enabled antibod binds to an interface of the Fab framework, which is distinct ies and meditopes, as well as vectors including the same, and from the CDR loops of cetuximab. FIG. IB (top) shows the libraries comprising the vectors. stick representation of the cQFD meditope and (bottom) the [0099] Also provided are screening methods, such as those stick representation of the cQYN meditope. The N- and C-ter- including the steps of expressing antibodies or fragments minal cysteines are solvent exposed and display high thermal thereof from the libraries, and selecting an antibody or frag factors. ment thereof from among the expressed antibodies or frag [0105] FIG. 2 shows certain embodiments of the cetuximab ments. In some cases, the selection is based on a binding Fab binding interface, cetuximab is a human-murine chimera affinity, pH tolerance, pH dependence, toxicity, PR, or PD of and, therefore, has a mixture of murine Ig variable domains the selected antibody or fragment thereof, and/or is effected and human constant Ig domains. FIG. 2A shows residues of by contacting the antibodies or fragments thereof with a chi4.18 and the humanized trastuzumab, which correspond meditope and detecting binding to one or more of the anti to those residues of cetuximab that make contact with the bodies or fragments thereto. meditope. FIG. 2B shows a stereoview of Arg9 of the cQFD [0100] Also provided are methods for selecting a meditope meditope that occupies a distinct pocket encoded by the analog or meditope variant, including those comprising: (a) murine sequence of cetuximab (foreground). There is a salt US 2012/0301400 Al Nov. 29, 2012 7

bridge from Asp85 of the cetuximab light chain to the guard - recycling and/or elicits an immune response. The addition of dinium group of the meditope Arg9 and the backbone amide an antibody such as matuzumab which recognizes a different of Leu 10. domain of EGFR in combination with cetuximab can, in some [0106] FIG. 3 shows surface plasmon resonance (SPR) cases be more effective due to daisy chaining of the surface traces of cQFD and cQYN with an immobilized Fab. The receptor (right panel I). A multivalent meditope (in this par cetuximab Fab used for crystallographic studies was coupled ticular example, trivalent) tethers/cross-links the therapeutic to a CM5 chip at low densities. Traces at increasing concen mAh and can enhance its therapeutic potential (right). In trations of the cQFD (FIG. 3A) and cQYN (FIG. 3B) medi- addition, the multivalent meditope (shown here as a trivalent topes are shown. The series of traces are fit to a simple I: I version) can also carry an imaging agent, which can allow for Langmuir binding model. The residuals of the fit are shown both enhanced therapeutics as well as imaging. below each. [0112] FIG. 9 shows an scFv-meditope linker. scFvs are [0107] FIG. 4 illustrates that the cQFD and cQYN medi- created by fusing the light chain variable domain to the heavy topes do not bind to the CDRs of cetuximab, as was previ chain variable domain (or vice versa) through a 12-20 amino ously hypothesized. FIG. 4A shows an overlay of two crystal acid linker (typically {GGGS}3_5). In this example, a portion structures, one of a cetuximab Fab and its antigen, EGFR of the flexible linker sequence can be replaced with the medi domain III, the other showing the cQFD meditope and cetux tope sequence. imab Fab, in which the cQFD meditope binds to the central [0113] FIG. 10 shows results from an SDS-PAGE showing cavity of the cetuximab Fab. The antigen, EGFR domain III, that the cQFD meditope coupled to beads could bind to cetux binds at the complementarity determining regions at a signifi imab. Biotinylated cQFD peptide was added to avidin- cant distance from the meditope binding site. FIG. 4B shows coupled beads, thoroughly washed, and equilibrated in PBS. SDS-PAGE results on the left-hand side and the correspond Cetuximab was then added to the beads (lane I), washed ing size exclusion chromatography results on the right-hand (lanes 2-4), and then eluted (lane 5). The top band is the IgG side. Size exclusion experiments of the individual compo heavy chain and bottom band is the IgG light chain. nents, Fab, EGFR domain III, and SMT-cQFD meditope, as [0114] FIG. 11 is a depiction of an exemplary embodiment well as an admixture of all three, indicate the formation of a of a steric mask. In this exemplary embodiment, the meditope hetero-trimeric complex that coeluted. The non-reducing is fused to the N-terminus of the Fab light or heavy chain SDS-PAGE gel shows the fraction that eluted first, indicating through a flexible linker containing a tumor associated pro the presence of all three components within the new peak (the tease site, to sterically occlude the antigen binding site. left-most peak for “complex,” shaded light gray) observed [0115] FIG. 12 shows SPR-determined binding kinetics for from the admixture. FIG. 4C shows the results of a FACS monovalent (left panel) and tetravalent (right panel) medi- analysis, indicating that the cQFD meditope bound to EGFR topes to cetuximab. The panel above the surface plasmon positive MD-MBA-468 cells only in the presence of cetux resonance traces depicts a cartoon of the monovalent medi- imab (arrows). The meditope alone or the meditope in the topes being passed over the bivalent IgG. As shown in the presence of M425, a murine EGFR antibody, did not bind. right panel, avidin was saturated with biotinylated meditope FIG. 4D shows results of surface plasmon resonance experi and passed over the same immobilized cetuximab IgG. The ments using a sensor chip coupled with a cetuximab scFv or off-rate of the multivalent meditope was reduced by at least Fab. The experiments indicate that saturation of the scFv 36 fold. (Note the time scales between the two experiments could not be achieved at concentrations as high as 100 μΜ of are different.) the cQFD meditope. The same experiments using the cetux [0116] FIG. 13 illustrates the synthesis of dimeric and tri- imab Fab coupled sensor chip indicate full saturation. The meric meditopes according to some embodiments, for medi- dissociation constant from this experiment is 660 nM. Con topes containing lactam linkages. trol SPR experiments (bottom panel) show that the cetuximab [0117] FIG. 14 illustrates the characterization of a fluores scFv readily binds to the soluble EGFR domain III fragment, cein isothiocyanate (FITC)-Iabeled meditope dimer (“14” in indicating that the CDR loops were functional. FIG. 13), with an FlPLC trace of final bivalent meditope and [0108] FIG. 5 is a graph of fluorescence intensity compared its mass spectrum. to cell count and shows fluorescently-labeled cetuximab [0118] FIG. 15 shows the nucleic acid sequence (SEQ ID binding to MDA-MB-468 cells in the presence of cQFD. NO:3) and the corresponding amino acid sequence (SEQ ID Cetuximab binds to MDA-MB-468 cells without meditope, NO:4) for a meditope-Fc fusion protein according to some and in the presence of 6 μΜ and 60 μΜ meditope. As embodiments. expected, the Figure shows that there was no binding of the [0119] FIG. 16 shows an exemplary bivalent meditope. The isotype IgG control to EGFR on MDA-MB468 cells. meditope is directly fused to the N-terminus of a peptide [0109] FIG. 6 shows how meditope and EGFR bind to linker that is directly fused to the Fe Region of an IgG. In this distinct sites. The top images show superposition of the example, as the Fe is naturally homodimerized during expres cQFD-Fab complex and the EGFR-Fab complex (I YY8) in sion, the end product from the meditope-Fc construct is biva stereo. The bottom images show that residues 39-46 of the lent. heavy chain are flexible and accommodate the meditope. [0120] FIG. 17 shows the results of a series of exemplary [0110] FIG. 7 shows Fab frameworkbinders. Superposition FACS analyses monitoring binding of monovalent and biva of Fabs bound to meditope, Protein A, and Protein L indicate lent meditopes to cetuximab pretreated EGFR-expressing that each binds to a unique site on the cetuximab Fab. cells. MDA-MB-468 cells that over-express EGFR were pre [0111] FIG. 8 illustrates a mechanism of action in one treated with 10 nM of cetuximab for 30 minutes, rinsed, and embodiment for enhancing tumor therapy. In the exemplified then treated with either the meditope-Fc construct or the embodiment, bivalent antibodies bound to antigen (e.g., ErbB monomeric meditope at four concentrations. The bottom receptor family) overexpressed on tumor cells (left panel) and trace is a negative control (no antibody). The next four traces blocks receptor signaling, alters endocytosis and receptor show that a monomeric meditope binds to cells pre-treated US 2012/0301400 Al Nov. 29, 2012 8

with cetuximab in a concentration dependent manner. The top [0127] FIG. 24 shows that an antibody containing HER2- four traces also show that the bivalent, meditope Fe binds to binding CDR loops grafted on cetuximab-like framework cells pre-treated with cetuximab in a concentration dependent binds to HER2 and a meditope-Fc. In FIG. 24A, FACS analy manner, but with higher affinity (i.e., more shifted to the sis shows that HER2-CDR grafted meditope-enabled mAh right). This is predicted and consistent with a multivalent binds to SKBR3 cells, which overexpresses HER2 (top three interaction. traces at different concentrations). FIG. 24B shows that while [0121] FIG. 18 is a depiction of an alternative meditope-Fc the meditope-Fc bound to the HER2-CDR grafted, meditope- linker, a coiled coil, which may be used in accordance with enabled mAh (top three traces—peak shifted right in concen certain embodiments. tration dependent manner), it did not bind to wild-type tras [0122] FIG. 19: Fragment screening by NMR. Representa tuzumab (second from bottom) or to the negative control tive NMR spectra of a fragment pool before (top trace) and (bottom trace). after (bottom trace) cetuximab addition. Using this method, [0128] FIG. 25A shows nucleic acid (SEQ ID NO: 11) and lead compounds (e.g., those shown herein) have been identi amino acid (SEQ ID NO: 12) sequences forthe heavy chain of fied and the binding site is being determined using diffraction a trastuzumab with secretion sequence and engineered studies. restriction sites (underlined). FIG. 25B shows nucleic acid [0123] FIG. 20 illustrates exemplary steps to alter and/or (SEQ ID NO: 13) and amino acid (SEQ ID NO: 14) sequences enhance the binding affinity (or alter another property) of a for a light chain of trastuzumab with secretion sequence and mAh for a meditope or compound binding at the meditope site engineered restriction sites (underlined). Shaded areas indi using a directed random library. Specifically, a gene library cate exemplary residues that in some aspects could be altered where codons for mAh residues that line the meditope bind to enhance or alter the specificity of the meditope, or could ing site are replaced with NNK (where N is any nucleotide participate in binding interactions with a modified epitope. and K is a thymidine or guanosine) can be selected using [0129] FIG. 26 illustrates examples of sequence and struc FACS sorting (where the meditope and the antigen are labeled tural alignment of the IgG and IgE Fab domains, with shaded with distinct fluorophores). In another embodiment, the areas indicating certain residues corresponding to resiues codons are substituted with NNN, where N is any nucleotide. near a meditope binding site. The GPI linker can ensure the selected mAh remains associ [0130] FIG. 27 is a graph showing surface plasmon reso ated with the cell transfected with vectors encoding the gene nance (SPR) studies. The binding affinity of different medi- sequences. Sequencing the genes encoding the light and topes to cetuximab [cQFD (meditope I, SEQ ID NO: I), heavy chain from the selected cells will identify high affinity cQYN (meditope 2, SEQ ID NO: 2), and cQYD (meditope mAbs. The method can be repeated to select for higher affin 16, SEQ ID NO: 16)] was determined from pH=4.0 to pH=8. ity meditope or meditope analogs. 0. [0124] FIG. 21 shows surface representation of sequence [0131] FIG. 28 illustrates the results of an MTT assay com differences of cetuximab and trastuzumab. The dark grey paring the efficacy of a bivalent meditope-Fc to that of a regions in the top panel indicate amino acid differences monomeric meditope to inhibit MDA-MB-468 cell growth. between cetuximab and the fully humanized trastuzumab FIG. 28A shows results of a study in which a meditope-Fc, but framework. Certain residues inside the box have been not a monomeric meditope, inhibited cell growth when com mutated onto the trastuzumab framework. The CDR loops of bined with cetuximab. FIG. 28B shows that the meditope-Fc trastuzumab have been identified (dark regions; bottom enhances the cell-killing capacity of cetuximab, similar to the panel) and grafted onto the cetuximab framework. combination of M425 and cetuximab. [0125] FIG. 22 Illustrates that a meditope-enabled trastu [0132] FIG. 29 illustrates binding of a meditope to a cetux zumab binds to a meditope-Fc fusion protein. The meditope imab meditope binding site (“Cetuximab Binding” as shown binding site was created on trastuzumab, a humanized mAh in Figure), but not to a fully humanized framework (“Trastu that binds to the tumor antigen, HER2. FACS analysis in the zumab No binding” as shown in Figure) or another murine- top panel shows that meditope-enabled trastuzumab binds to chimera framework (“Rituximab No binding” as shown in SKBR3 cells that overexpress HER2 (top two traces). In the Figure). The cQFD meditope (meditope I, SEQ ID NO: I) bottom graph, the meditope-Fc binds to meditope-enabled was conjugated to a CM5 chip for surface plasmon resonance trastuzumab (top two traces—peak shifted right), but not to studies and cetuximab (meditope binding Fab), trastuzumab wild-type trastuzumab (second from bottom) or to the nega (fully humanized framework), and rituximab (murine-human tive control (bottom trace). chimeric framework) were tested at concentrations of 0.01, [0126] FIG. 23A shows the nucleic acid (SEQ ID NO:5) 0.05, 0.1, 0.5 and I μΜ. Only cetuximab bound to the medi- sequence of a meditope-enabled trastuzumab heavy chain tope-conjugated chip. sequence. Signal peptide sequence is shaded in grey. FIG. [0133] FIG. 30 shows biophysical data obtained for the 23B shows the amino acid (SEQ ID NO: 6) sequence of a meditopes. FIG. 30A shows a SPR sensogram of an unmodi meditope-enabled trastuzumab heavy chain sequence as fied meditope cQFD (SEQ ID NO: I) and a QYN meditope compared to the wild-type (SEQ ID NO:7). Differences are (SEQ ID NO:2) using a cetuximab Fab chip. FIG. 30B shows highlighted in grey. After the amino acids shown in FIG. 23B, a representative binding isotherm of the meditope (SEQ ID there are no differences remaining in the human sequence of NO: I) and cetuximab Fab (top) and integration (bottom). the heavy chain shown. FIG. 23C shows the nucleic acid FIG. 30C shows superposition of both meditopes. Oval I (SEQ ID NO: 8) sequence of a meditope-enabled trastuzumab indicates the Phe/Tyr position. The arrow indicates a shift of light chain sequence. Signal peptide sequence is shaded in the backbone that leads to a favorable hydrogen bond network grey. FIG. 23D shows the amino acid (SEQ ID NO:9) and may account for a favorable change in the enthalpy. The sequence of a meditope-enabled trastuzumab light chain hydrophobic groups, F/Y3 L5, and LlO are nearly identically sequence as compared to the wild-type (SEQ ID NO:10). positioned, but the hydroxyl group ofY5 in the cQYN medi Differences are highlighted in grey. tope prevents R8 from interacting with the Q105 backbone, as US 2012/0301400 Al Nov. 29, 2012 9

observed in the cQFD meditope (“steric clash”). This rear [0143] FIG. 40 is a table showing the X-ray diffraction data rangement also results in a concomitant change in the back for the structures shown in FIG. 39 and additional meditopes bone residues of the β-turn (‘backbone rotation”). FIG. 30D in Tables 3 and 4. shows individual thermodynamic parameters determined by [0144] FIG. 41 shows representative surface plasmon reso ITC of different meditope variants. The Phe3Tyr variant nance studies of meditope variants. Based on the structural (meditope 2, SEQ ID NO: 2) shows a significant change in Δ1Τ information of the Phe3H is mutation in the cQFD meditope, though lower affinity. Based on the structure, Gln2 in the a variant meditope was synthesized with β,β'-diphenylala- meditope of SEQ ID NO: I was replaced with the D-stere- nine at position 3 (top trace). A significant improvement in the omer. ITC analysis of this meditope, (SEQ ID NO: 15), binding affinity for cetuximab of -4 fold, was observed by revealed a significant increase in entropy and loss in enthalpy. surface plasmon resonance. Aminohexanoic acid was used to [0134] FIG. 31 shows the chemical structure of a meditope replace the disulfide bridge of the original meditope (bottom according to some embodiments. The circles indicate posi trace). While the binding affinity was decreased, these data tions that can be modified, e.g., to improve meditope affinity indicate that modifications can be made to meditopes to for the Fab. The boxes indicate cyclization strategies. ‘Click’ address potential issues with pharmacokinetics, pharmaco chemistry and olefin metathesis, top and bottom far right dynamics and toxicity in animal and human studies. It is boxes, respectively, are additional routes to cyclize the medi noted that this combination can be combined with unnatural tope. amino acids at other positions within a meditope. [0135] FIG. 32 illustrates the synthesis of lactam (2, SEQ [0145] FIG. 42 illustrates structural information that can be ID NO: 32) and fluorescein-labeled lactam (3). useful, e.g., to improve meditope affinity. The top panel [0136] FIG. 33 shows the sites of optimization according to shows a crystal structure of a meditope bound cetuximab, some embodiments. Ovals indicate cavities in the Fab region showing Phe3 bound within the Fab cavity of cetuximab. of the mAh that may be exploited to optimize the affinity of a Substitution of this position with histidine and subsequent meditope. structural analysis indicates the side chain (imidazole) takes [0137] FIG. 34 shows a modified Arg8 residue that may be on a new conformation (middle panel). Based on this obser used to optimize meditope binding parameters, and may be vation, β,β'-diphenylalanine which could place one phenyl used in accordance with the embodiments described herein side chain at the original position and one phenyl side chain at (R=alkyl, substituted alkyl, aromatic or NHR'", where the imidazole ring was substituted at position 3. The crystal R'"=alkyl, substituted alkyl, or aromatic). structure indicates that this substitution mimics both side [0138] FIG. 35 shows the hydrophilic interface in the vicin chain conformations. Surface plasmon resonance studies ity of .Phe3 of a meditope. Halogens incorporated in the shows that this substitute binds with higher affinity (FIG. 41, phenyl ring may form strong noncovalent bonds to the top panel). hydroxyl group of cetuximab Tyr87 or Tyr91, as well as to [0146] FIG. 43 shows results of a study demonstrating that backbone carbonyls. a meditope-enabled trastuzumab provided herein has a simi [0139] FIG. 36 illustrates covalent interactions between a lar binding affinity for antigen as wild-type trastuzumab, as meditope and the backbone of cetuximab. A hydratable side described in Example 4. chain may be incorporated at Arg8 (left) or LeulO (right) to [0147] FIG. 44 shows results of a study described in form a covalent bond to the hydroxyl groups of Ser or Tyr of Example 4, demonstrating that HER2-expressing cells pre the Fab. bound with meditope-enabled trastuzumab (TM), but not [0140] FIG. 37 illustrates a fluorescence polarization assay those pre-bound with wild-type trastuzumab (WT T), bound of dose-dependent inhibition of the interaction between to a cQFD (SEQ ID NO: I) meditope-Protein L fusion cetuximab and a fluorescein-labeled peptide by unlabeled (MPL). Control cells, without pre-bound antibody (MPL peptide. The assay identified small molecule compounds that (WT) only) also did not bind to MPL. could compete with the meditope for mAh binding and thus [0148] FIG. 45 shows results of a study described in can be developed to function in a similar manner as a medi Example 4, demonstrating that HER2-expressing cells tope. As a control, it was demonstrated that a non-labeled (SKBR3) pre-bound with meditope-enabled trastuzumab meditope could displace the fluorescently-labeled meditope. (TM), but not those pre-bound with wild-type trastuzumab Equilibration of the displacement at three times points indi (WT T), bound to a cQFD (SEQ ID NO: I) meditope-Protein cates that the assay is robust and amendable to high through L fusion in which all but one lysine of Protein L were mutated put screening. to aiginine and asparagine (MPL-5K). Control cells, not pre [0141] FIG. 38 shows five lead compounds identified from bound with any antibody (MPL-5K only) also did not bind to the fluorescence polarization screen. MPL-5K. [0142] FIG. 39 shows stick structures for eighteen modified [0149] FIG. 46 shows results of a study described in meditopes, corresponding to SEQ ID NO:22 (A), SEQ ID Example 4, demonstrating that meditope binds to meditope- NO: 16 (B), SEQ ID NO: 17 (C), SEQ ID NO: 18 (D), SEQ ID enabled trastuzumab to a similar degree whether the antibody NO: 15 (E), SEQ ID NO:23 (F), SEQ ID NO:24 (G), SEQ ID is pre-bound to cells expressing antigen prior to incubation NO:25 (H), SEQ ID NO:32 (I), SEQ ID NO:33 (J), SEQ ID with the meditope (left panel), or meditope-Protein L- and NO:34 (K), SEQ ID NO:35 (L), SEQ ID NO:36 (M), SEQ ID antibody are pre-mixed and then incubated with cells express NO:37 (N), SEQ ID NO:38 (O), SEQ ID NO:39 (P), SEQ ID ing antigen (right panel). TM=meditope-enabled trastu NO:40 (Q) and SEQ ID NO:41 (R), SEQ ID NO:42 (S), SEQ zumab. The percentage of MPL-positive cells relative to no ID NO:43 (T), SEQ ID NO:44 (U), SEQ ID NO:46 (V) SEQ treatment control is shown in the legend. IDNO:49 (W), SEQIDNO:51 (X), SEQIDNO:52 (Y), SEQ [0150] FIG. 47A shows light chain nucleic acid (SEQ ID ID NO:53 (Z), SEQ ID NO:54 (AA), and SEQ ID NO:55 NO: 61) and light chain amino acid (SEQ ID NO: 61) (BB). The sequence of these structures is shown in Tables 3 sequences of a CDR-grafted meditope-enabled trastuzumab and 4. (with trastuzumab-like CDRs grafted onto a cetuximab-like US 2012/0301400 Al Nov. 29, 2012 10

framework), with the signal sequence and other residues to bind to meditopes (for reference the heavy chain M5A shaded. FIG. 47B shows heavy chain nucleic acid (SEQ ID sequence is set forth in SEQ ID NO: 70). NO: 62) and heavy chain amino acid (SEQ ID NO: 63) of this [0160] FIG. 57 shows results of FACS analysis, demon antibody. strating binding of the meditope-enabled M5A antibody [0151] FIG. 48 shows the structure of meditope 54 (see (M5A 8M) to the M5A antigen (CEA) on LS174T cells. Table 4) bound to a cetuximab Fab fragment (backside view), [0161] FIG. 58 shows surface plasmon resonance (SPR) with structures of the 5-position β,β'-diphenylalanine medi data for a 5-diphenyl meditope bound to an M5A 8M medi tope (SEQ ID NO: 18, meditope 18) and cQFD (meditope I, tope enabled antibody. SEQ ID NO: I) superimposed. [0162] FIG. 59 illustrates illustrates the characterization of [0152] FIG. 49 shows the results of a study in which an meditope 54, with an FlPLC trace and its mass spectrum. Alexa488-labeled meditope-Fc fusion protein (600 nM, 180 [0163] FIG. 60 illustrates the mass spectrum of DOTA- nM, or 60 nM) was incubated with MDA-MB-468 cells, NFlS-MPL conjugates formed from a starting material ratio pre-labeled with cetuximab or M425 (a mouse anti-EGFR of 120:1 (DOTA-NHS:MPL). antibody) for 30 minutes, and antibody binding and meditope [0164] FIG. 61A shows MDA-MB-468 cells that were pre binding analyzed by FACS analysis. incubated with Alexa 555-cetuximab and then incubated with [0153] FIG. 50 (top panel) shows stick representations of Alexa 488-meditope-Fc. DAPI was used as counter stain. the structures of meditope 18 (SEQ ID NO: 18, shown in Images were taken with an Olympus AX70 Automatic Table 3, with a β,β'-diphenylalanine at position 5) bound to Upright Microscope. White arrows indicate positive medi- cetuximab (dark grey sticks), the same meditope (18) bound tope-Fc staining. FIG. 61B shows quantification of meditope- to meditope-enabled trastuzumab (white sticks), and wild- Fc-positive cells. Bar graph showed shows percentage of type trastuzumab (outline), superimposed. The bottom panel meditope-Fc-positive cells of total cells from cetuximab pre shows a ribbon cartoon comparing wild-type and meditope- treated or untreated samples counted from two fields. enabled trastuzumab. [0154] FIG. 51, in the upper-left panel, shows a superposi DETAILED DESCRIPTION tion of the structures of trastuzumab and trastuzumab memab A. Definitions (in this figure, the trastuzumab memab is labeled as “Medi tope-enabled Memab”) with certain residues involved in [0165] An “antibody,” as used herein, refers to an immuno meditope-binding in the meditope-enabled antibody illus globulin molecule that specifically binds to, or is immuno- trated by sticks. The top right panel shows a superposition of logically reactive with an antigen or epitope, and includes the structures of meditope-enabled trastuzumab (memab) and both polyclonal and monoclonal antibodies, as well as func cetuximab, with the same residues labeled. The bottom panel tional antibody fragments, including but not limited to frag shows a “cartoon/ribbon diagram” of all three of these struc ment antigen binding (Fab) fragments, F(ab')2 fragments, tures superimposed. Fab' fragments, Fv fragments, recombinant IgG (rlgG) frag [0155] FIG. 52 shows the structure of meditope (position ments, single chain variable fragments (scFv) and single β,β' diphenyl), Protein L (left), ProteinA (right) and Fab (grey domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. cartoon). Lysines in Protein L and the meditope that were The term “antibody” includes genetically engineered or oth mutated in MPLs described herein are shown in black. erwise modified forms ofimmunoglobulins, suchas intrabod ies, peptibodies, chimeric antibodies, fully human antibodies, [0156] FIG. 53 shows surface plasmon resonance (SPR) humanized antibodies, meditope-enabled antibodies and het data for a meditope-Protein L fusion (MPL): Top panel shows eroconjugate antibodies (e.g., bispecific antibodies, diabod- the traces of MPL being added to the meditope-enabled tras ies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv). tuzumab at concentrations up to 10 nM. The fit data indicates Unless otherwise stated, the term “antibody” should be a binding affinity of 165 pM. The bottom panel shows the understood to encompass functional antibody fragments traces of Protein L (only) added at the same concentrations to thereof. the meditope-enabled trastuzumab, showing that there is not [0166] The terms “complementarity determining region,” binding at this concentration. and “CDR,” are known in the art to refer to non-contiguous [0157] FIG. 54 shows that a GPI-Iinked meditope-enabled sequences of amino acids within antibody variable regions, trastuzumab binds to a meditope-Protein L (MPL) fusion which confer antigen specificity and binding affinity. In gen protein. Ixl CF 6 cells/sample were used. Cells were removed eral, there are three CDRs in each heavy chain variable region from plates by gentle pipetting and were washed once with (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light 0.1% BSA (w/v) in PBS. AF647-labeled-MPL was diluted to chain variable region (CDR-L1, CDR-L2, CDR-L3). 10 nM in wash buffer and incubated with cells for 30 min at “Framework regions” and “FR” are known in the art to refer RT. Cells were washed twice and then analyzed by FACS. to the non-CDR portions of the variable regions of the heavy [0158] FIG. 55A shows a sequence comparison between and light chains. In general, there are four FRs in each heavy the CFll domain of a human IgG2, IgG4, IgG3, and IgGl chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), (SEQ ID NOs: 64-67). FIG. 55B shows the differences and four FRs in each light chain variable region (FR-L1, between these sequences mapped onto the co-crystal struc FR-L2, FR-L3, and FR-L4). ture of cetuximab and meditope I (SEQ ID NO: I, cQFD). [0167] The precise amino acid sequence boundaries of a [0159] FIG. 56 shows the amino acid sequence of the light given CDR or FR can be readily determined using any of a chain of a meditope-enabled (“medi”) anti-CEA antibody number of well-known schemes, including those described (meditope-enabled M5A) (SEQ ID NO: 68) compared to the by Rabat et al. (1991), “Sequences of Proteins of Immuno wild-type M5A light chain sequence (SEQ ID NO: 69). logical Interest,” 5th Ed. Public Flealth Service, National Shaded residues represent eight point mutations that were Institutes of Flealth, Bethesda, Md. (“Rabat” numbering introduced in the light chain of the M5 A antibody, allowing it scheme), Al-Lazikani et al, (1997) JMB 273, 927-948 US 2012/0301400 Al Nov. 29, 2012 11

(“Chothia” numbering scheme), MacCallum et al., J. Mol. defined by the Rabat, Chothia, or Contact method. In other Biol. 262:732-745 (1996), “Antibody-antigen interactions: cases, the particular amino acid sequence of a CDR or FR is Contact analysis and binding site topography,” J. Mol. Biol. given. 262,732-745(Contact” numbering scheme), Lefranc M P et [0171] The term “meditope-enabled” antibody and ah, “IMGT unique numbering for immunoglobulin and T cell “meMAb” refer to an antibody or functional fragment thereof receptor variable domains and Ig superfamily V-Iike that is able to bind to a meditope, via a meditope binding site. domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 Examples of meditope-enabled antibodies include, but are (“IMGT” numbering scheme), and Honegger A and Pliick- not limited to, cetuximab and others described herein. A thun A, “Yet another numbering scheme for immunoglobulin “meditope binding site” is a region of the meditope-enabled variable domains: an automatic modeling and analysis tool,” antibody containing the amino acid residues that interact with a bound meditope, which residues include framework region J Mol Biol, 2001 June 8; 309(3):657-70, (AHo numbering (FR) residues of the heavy and light chains. With reference to scheme). a Fab fragment or a Fab portion of an antibody, the meditope [0168] The boundaries of a given CDR or FR may vary binding site is located within the central cavity of the Fab depending on the scheme used for identification. For fragment or portion. example, the Rabat scheme is based structural alignments, [0172] The “central cavity,” with respect to the three-di while the Chothia scheme is based on structural information. mensional structure of a Fab, refers to the internal cavity of Numbering for both the Rabat and Chothia schemes is based the F ab, lined by portions of the heavy and light chain variable upon the most common antibody region sequence lengths, and constant regions. The central cavity thus is lined by with insertions accommodated by insertion letters, for residues of the VH, VL, CHI, and CL regions and does not example, “30a,” and deletions appearing in some antibodies. include the antigen binding site. The two schemes place certain insertions and deletions (“in- [0173] In some embodiments, the meditope binding site dels”) at different positions, resulting in differential number includes residues 40, 41, 83, and 85 of the light chain of a ing. The Contact scheme is based on analysis of complex meditope-enabled antibody, according to Rabat numbering, crystal structures and is similar in many respects to the and/or residues 39, 89, 105, and 108 of the heavy chain of the Chothia numbering scheme. meditope-enabled antibody, according to Rabat numbering. [0174] In some embodiments, the meditope binding site is [0169] Table I, below, lists the positions of CDR-Ll, CDR- locatedwithinacavityformedbyresidues 8, 9,10,38,39,40, L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified 41 42,43,44,45,82,83,84,85,86,87,99,100,101,102,103, by the Rabat, Chothia, and Contact schemes, respectively. 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, and 173 of For CDR-H I, residue numbering is given listed using both the the light chain and 6, 9, 38, 39, 40, 41, 42, 43, 44, 45, 84, 86, Rabat and Chothia numbering schemes. It is noted that 87,88,89, 90,91,103,104,105,106,107,108,111,110,147, because the Rabat numbering scheme places insertions at 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and 187 of H35A and H35B, the end of the Chothia CDR-Hl loop when the heavy chain of the antibody, according to Rabat number numbered using the Rabat numbering convention varies ing. between H32 and H34, depending on the length of the loop. [0175] With respect to a Fab portion of a meditope-enabled antibody, the meditope binding site includes residues within TABLE I the central cavity. The meditope-binding site typically further includes constant region residues. CDR Kabat Chothia Contact [0176] The terms “meditope”, as used herein, refers to a CDR-Ll L24-L34 L24-L34 L30-L36 peptide or peptides that binds to a meditope-binding site of a CDR-L2 L50-L56 L50-L56 L46-L55 meditope-enabled antibody, which antibody has a threonine CDR-L3 L89-L97 L89-L97 L89-L96 CDR-Hl H31-H35B H26-H32 . . . 34 H30-H35B at position 40, an asparagine at position 41, and an aspartage (Kabat Numbering1) at position 85 of its light chain, according to Rabat number CDR-Hl H31-H35 H26-H32 H30-H35 ing, or contains a meditope binding site containing residues (Chothia Numbering2) that correspond to those within the meditope-binding site of CDR-H2 H50-H65 H52-H56 H47-H58 CDR-H3 H95-H102 H95-H102 H93-H101 cetuximab, meditope-enabled trastuzumab, or meditope-en abled M5A, disclosed herein. Exemplary meditopes include, 1Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD but are not limited to, the cQFD and cQYN peptides and 2Al-Lazilcani et al., (1997) JMB 273, 927-948 variants thereof (“meditope variants” or “variant medi topes”), as well as multivalent and labeled meditopes. Other [0170] Thus, unless otherwise specified, a “CDR” or molecules may also bind to meditope binding sites of medi “complementary determining region,” or individual specified tope-enabled antibodies, with functional characteristics simi CDRs (e.g., “CDR-H1, CDR-H2), of a given antibody or lar to those of a meditope. Such molecules, meditope analogs, region thereof, such as a variable region thereof, should be may include, but are not limited to, small molecules, aptam- understood to encompass a (or the specific) complementary ers, nucleic acid molecules, peptibodies and any other sub determining region as defined by any of the known schemes stance able to bind to the same meditope binding site as a Likewise, unless otherwise specified, a “FR” or “framework meditope. region,” or individual specified FRs (e.g., “FR-H1, FR-H2), [0177] A “therapeutic agent,” as used herein, is an atom, of a given antibody or region thereof, such as a variable region molecule, or compound that is useful in treatment of a disease thereof, should be understood to encompass a (or the specific) or condition. framework region as defined by any of the known schemes. In [0178] A “therapeutically effective amount,” “therapeuti some instances, the scheme for identification of a particular cally effective concentration” or “therapeutically effective CDR, FR, or FRs or CDRs is specified, such as the CDR as dose” is the amount of a compound that produces a desired US 2012/0301400 Al Nov. 29, 2012 12

therapeutic effect in a subject, such as preventing or treating modalities. Further, the administration of the two or more a taiget condition, alleviating symptoms associated with the agents, drugs, treatment regimens, treatment modalities or a condition, producing a desired physiological effect, or allow combination thereof may be by the same or different routes of ing imaging or diagnosis of a condition that leads to treatment administration. of the disease or condition. The precise therapeutically effec [0182] A “therapeutic antibody” may refer to any antibody tive amount is the amount of the composition that will yield or functional fragment thereof that is used to treat cancer, the most effective results in terms of efficacy of treatment in autoimmune diseases, transplant rejection, cardiovascular a given subject. This amount will vary depending upon a disease or other diseases or conditions such as those variety of factors, including, but not limited to, the character described herein. Examples of therapeutic antibodies that istics of the therapeutic compound (including activity, phar may be used according to the embodiments described herein macokinetics, pharmacodynamics, and bioavailability), the include, but are not limited to murine antibodies, murinized or physiological condition of the subject (including age, sex, humanized chimera antibodies or human antibodies includ disease type and stage, general physical condition, respon ing, but not limited to, Erbitux (cetuximab), ReoPro (abcix- siveness to a given dosage, and type of medication), the nature imab), Simulect (basiliximab), Remicade (infliximab); of the pharmaceutically acceptable carrier or carriers in the Orthoclone OKT3 (muromonab-CD3); Rituxan (rituximab), formulation, and the route of administration. One skilled in Bexxar (tositumomab) Humira (adalimumab), Campath (ale- the clinical and pharmacological arts will be able to deter mtuzumab), Simulect (basiliximab), Avastin (bevacizumab), mine a therapeutically effective amount through routine Cimzia (certolizumab pegol), Zenapax (daclizumab), Soliris experimentation, namely by monitoring a subject’s response (eculizumab), Raptiva (efalizumab), Mylotarg (gemtu- to administration of a compound and adjusting the dosage zumab), Zevalin (ibritumomab tiuxetan), Tysabri (natali- accordingly. For additional guidance, see Remington: The zumab), Xolair (omalizumab), Synagis (palivizumab), Science and Practice of Pharmacy 2 Iit Edition, Univ. of Sci Vectibix (panitumumab), Lucentis (ranibizumab), and Her- ences in Philadelphia (USIP), Lippincott Williams & ceptin (trastuzumab). Wilkins, Philadelphia, Pa., 2005. [0183] “Treating” or “treatment” of a condition may refer [0179] A “pharmaceutically acceptable carrier” refers to a to preventing the condition, slowing the onset or rate of devel pharmaceutically acceptable material, composition, or opment of the condition, reducing the risk of developing the vehicle that is involved in carrying or transporting a com condition, preventing or delaying the development of symp pound of interest from one tissue, organ, or portion of the toms associated with the condition, reducing or ending symp body to another tissue, organ, or portion of the body. For toms associated with the condition, generating a complete or example, the carrier may be a liquid or solid filler, diluent, partial regression of the condition, or some combination excipient, solvent, or encapsulating material, or some com thereof. bination thereof. Each component of the carrier must be [0184] As used herein, the terms “including,” “containing,” “pharmaceutically acceptable” in that it must be compatible and “comprising” are used in their open, non-limiting sense. with the other ingredients of the formulation. It also must be [0185] As used herein, the singular forms “a,” “an,” and suitable for contact with any tissue, organ, or portion of the “the” include plural referents unless the context clearly dic body that it may encounter, meaning that it must not carry a tates otherwise. risk of toxicity, irritation, allergic response, immunogenicity, [0186] To provide a more concise description, some of the or any other complication that outweighs its therapeutic ben quantitative expressions given herein are not qualified with efits. the term “about”. It is understood that, whether the term [0180] A “route of administration” may refer to any admin “about” is used explicitly or not, every quantity given herein istration pathway known in the art, including but not limited is meant to refer to the actual given value, and it is also meant to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, to refer to the approximation to such given value that would transdermal, or vaginal. “Transdermal” administration may reasonably be inferred based on the ordinary skill in the art, be accomplished using a topical cream or ointment or by including equivalents and approximations due to the experi means of a transdermal patch. “Parenteral” refers to a route of mental and/or measurement conditions for such given value. administration that is generally associated with injection, [0187] As used herein, “alkyl” refers to a saturated, including infraorbital, infusion, intraarterial, intracapsular, straight- or branched-chain hydrocarbon group having from I intracardiac, intradermal, intramuscular, intraperitoneal, to 12 carbon atoms. Representative alkyl groups include, but intrapulmonary, intraspinal, intrastemal, intrathecal, intrau are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-me- terine, intravenous, subarachnoid, subcapsular, subcutane thyl-1-propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-me- ous, transmucosal, or transtracheal. thyl-1 -butyl, 2-methyl-3-butyl, 2,2-dimethyl-l -propyl, [0181] “In combination” or “in combination with,” when 2-methyl-l-pentyl, 3-methyl-1-pentyl, 4-methyl-l -pentyl, used herein in the context of multiple agents, therapeutics, or 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, treatments, means in the course of treating the same disease or 2,2-dimethyl-1-butyl, 3,3-dimethyl-l-butyl, 2-ethyl-1-butyl, condition in a subject administering two or more agents, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, drugs, treatment regimens, treatment modalities or a combi n-hexyl, and the like, and longer alkyl groups, such as heptyl, nation thereof (e.g., an antibody in combination with a medi- octyl, and the like. The term 11Cx^alkyl,” where x and y are tope or a multivalent tethering agent), in any order. This integers, refers to an alkyl with x-y carbon atoms. includes simultaneous administration (or “coadministra [0188] As used herein, an “alkenyl” refers to a straight- or tion”), administration of a first agent prior to or after admin branched-chain hydrocarbon group having one or more istration of a second agent, as well as in a temporally spaced double bonds therein and having from 2 to 12 carbon atoms. order of up to several days apart. Such combination treatment Illustrative alkenyl groups include, but are not limited to, may also include more than a single administration of any one ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, or more of the agents, drugs, treatment regimens or treatment pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, US 2012/0301400 Al Nov. 29, 2012 13

4-(2-methyl-3-butene)-pentenyl, and the like. The term “Cx_ âlkenyl,” where x and y are integers, refers to an alkenyl with -continued x-y carbon atoms. [0189] The term “alkylenyl” or “alkylene” refers to a diva lent alkyl group. The term “alkenylene” or “alkenylene” refers to a divalent alkenyl group. [0190] As used herein, “alkynyl” refers to a straight- or branched-chain hydrocarbon group having one or more triple bonds therein and having from 2 to 10 carbon atoms. Exem plary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-l-butynyl, 4-propyl-2-pentynyl, 4-butyl-2-hexy- and nyl, and the like. [0191] “Aryl” means a mono-, bi-, or tricyclic aromatic group, wherein all rings of the group are aromatic. For bi- or tricyclic systems, the individual aromatic rings are fused to one another. Exemplary aryl groups include, but are not lim ited to, phenyl, naphthalene, and anthracene. [0192] The term “boronic ester” refers to a substituent [0199] As used herein, the term “5-membered heteroaryl” —B(OR)2, wherein each R group is independently a refers to a monocyclic, aromatic heterocycle having five ring Câlkyl, orthe two Rgroups taken together form a C2_6alky- atoms that are selected from carbon, oxygen, nitrogen, and lene. sulfur. Examples of 5-membered heteroaryl groups include, [0193] The term “acetal” refers to a —CEi(OR)2 group, but are not limited to, imidazolyl, pyrazolyl, triazolyl, tetra- wherein each R group is independently a Câlkyl, or the two zolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, R groups taken together form a C2_6alkylene. Exemplary isothiazolyl, pyrrolyl, and thiadiazolyl. Particular examples acetal groups include dimethylacetal or diethylacetal, or a of 5-membered heteraryls include those that may be formed cyclic acetal. The term “ketal” refers to a —C(OR)2— group, by 1,3-cycloaddition reactions such as a Eluisgen reaction wherein each R group is IndependentlyaC1âlkyl, or the two between an azide and a propaigyl group. R groups taken together form a C2_6alkylene. Exemplary [0200] As used herein, the term “substituted” means that ketals include dimethylketal or diethylketal, or a cyclic ketal. the specified group or moiety bears one or more suitable [0194] The term “halo” represents chloro, fluoro, bromo, or substituents. As used herein, the term “unsubstituted” means iodo. In some embodiments, halo is chloro, fluoro, or bromo. that the specified group bears no substituents. As used herein, The term “halogen” as used herein refers to fluorine, chlorine, the term “optionally substituted” means that the specified bromine, or iodine. group is unsubstituted or substituted by the specified number of substituents. Where the term “substituted” is used to [0195] The term “ortho ester” refers to a —C(OR)3 group, describe a structural system, the substitution is meant to occur wherein each R group is independently a C1âlkyl, or two of at any valency-allowed position on the system. the R groups taken together form a C2_6alkylene [0201] As used herein, the expression “one or more sub [0196] The term “oxo” means an =O group and may be stituents” denotes one to maximum possible number of sub attached to a carbon atom or a sulfur atom. stitution^) that can occur at any valency-allowed position on [0197] The term “phosphonate ester” refers to a —P(O) the system. In a certain embodiment, “one or more substitu (OR)2 group, wherein each R group is independently a ents” means I, 2, 3, 4, or 5 substituents. In another embodi Câlkyl, orthe two Rgroups taken together form a C2_6alky- ment, one or more substituent means 1, 2, or 3 substituents. Iene [0202] Any atom that is represented herein with an unsat [0198] As used herein, the term “cycloalkyl” refers to a isfied valence is assumed to have the sufficient number of saturated or partially saturated, monocyclic, fused polycyclic, hydrogen atoms to satisfy the atom’s valence. bridged polycyclic, or spiro polycyclic carbocycle having [0203] When any variable, such as alkyl, R3, or R5, appears from 3 to 15 ring carbon atoms. A non limiting category of in more than one place in any formula or description provided cycloalkyl groups are saturated or partially saturated, mono- herein, the definition of that variable on each occurrence is cyclic carbocycles having from 3 to 6 carbon atoms. Illustra independent of its definition at every other occurrence. tive examples of cycloalkyl groups include, but are not lim [0204] Numerical ranges, as used herein, are intended to ited to, the following moieties: include sequential whole numbers. For example, a range expressed as “from 0 to 4” or “0-4” includes 0, I, 2, 3 and 4. [0205] When a multifunctional moiety is shown, the point of attachment to the core is indicated by a line or hyphen. For example, —OFl refers to a moiety in which an oxygen atom is the point of attachment of the hydroxyl group to the remain der of the molecule. [0206] Any formula given herein is intended to represent compounds having structures depicted by the structural for mula as well as certain variations or forms. For example, compounds of any formula given herein may have asymmet O ric or chiral centers and therefore exist in different stereoiso- US 2012/0301400 Al Nov. 29, 2012 14

meric forms. All stereoisomers, including optical isomers, [0212] Compound salts include acidic salts formed with enantiomers, and diastereomers, of the compounds of the inorganic and/or organic acids, as well as basic salts formed general formula, and mixtures thereof, are considered to fall with inoiganic and/or organic bases. In addition, where a within the scope of the formula. Furthermore, certain struc given compound contains both a basic moiety, such as, but not tures may exist as geometric isomers (i.e., cis and trans iso limited to, a pyridine or imidazole, and an acidic moiety, such mers), as tautomers, or as atropisomers. All such isomeric as, but not limited to, a carboxylic acid, one of skill in the art forms, and mixtures thereof, are contemplated herein as part will recognize that the compound may exist as a zwitterion of the present invention. Thus, any formula given herein is (“inner salt”); such salts are included within the term “salt” as intended to represent a racemate, one or more enantiomeric used herein. Salts of the compounds of the invention may be forms, one or more diastereomeric forms, one or more tauto prepared, for example, by reacting a compound with an meric or atropisomeric forms, and mixtures thereof. amount of a suitable acid or base, such as an equivalent [0207] Diastereomeric mixtures may be separated into their amount, in a medium such as one in which the salt precipitates individual diastereomers on the basis of their physical chemi or in an aqueous medium followed by lyophilization. cal differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or frac [0213] Exemplary salts include, but are not limited, to sul tional crystallization. Enantiomers may be separated by con fate, citrate, acetate, oxalate, chloride, bromide, iodide, verting the enantiomeric mixture into a diastereomeric mix nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, ture by reaction with an appropriate optically active lactate, salicylate, acid citrate, tartrate, oleate, tannate, pan compound (e.g., chiral auxiliary such as a chiral alcohol or tothenate, bitartrate, ascorbate, succinate, maleate, gentisi- Mosher’s acid chloride, or formation of a mixture of diaste nate, fumarate, gluconate, glucuronate, saccharate, formate, reomeric salts), separating the diastereomers and converting benzoate, glutamate, methanesulfonate (“mesylate”), ethane- (e.g., hydrolyzing or de-salting) the individual diastereomers sulfonate, benzenesulfonate, p-toluenesulfonate, and pamo to the corresponding pure enantiomers. Enantiomers may ate (i.e., l,l'-methylene-bis(2-hydroxy-3-naphthoate)) salts. also be separated by use of chiral EiPLC column. The chiral A pharmaceutically acceptable salt may involve the inclusion centers of compounds of the present invention may be desig of another molecule such as an acetate ion, a succinate ion or nated as “R” or “S” as defined by the IUPAC 1974 Recom other counterion. The counterion may be any oiganic or inor mendations, or by “D” or “L” designations consistent with the ganic moiety that stabilizes the charge on the parent com peptide literature. pound. Furthermore, a pharmaceutically acceptable salt may [0208] As used herein, a box around a portion of a structure, have more than one charged atom in its structure. Instances drawn with a subscript, indicates that the structural fragment where multiple charged atoms are part of the pharmaceuti that appears within the box is repeated according to the sub cally acceptable salt can have multiple counterions. Flence, a script. For example, the substructure: pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion. [0214] Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, ■ Fragment - butyrates, citrates, camphorates, camphorsulfonates, fuma- rates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, suc [0209] where x is 0,1, or 2, indicates the fragment is absent cinates, sulfates, tartarates, thiocyanates, toluenesulfonates from the structure, or is -Fragment-, or is (also known as tosylates,) and the like. -Fragment-Fragment-. For example, within formula (X), the [0215] Exemplary basic salts include ammonium salts, following substructure: alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magne sium salts, salts with organic bases (for example, oiganic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. V g. I Basic nitrogen-containing groups may be quarternized with X agents such as lower alkyl halides (e.g. methyl, ethyl, and P ^ butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain halides [0210] where p i s 0 or I, means that the —X—NFi— group (e.g. decyl, lauryl, and stearyl chlorides, bromides and within the box is absent from the structure (p is 0), or is iodides), aralkyl halides (e.g. benzyl and phenethyl bro present once (p is I). mides), and others. [0211] The compounds of the invention can form pharma [0216] Additionally, acids and bases which are generally ceutically acceptable salts, which are also within the scope of considered suitable for the formation of pharmaceutically this invention. A “pharmaceutically acceptable salt” refers to useful salts from pharmaceutical compounds are discussed, a salt of a free acid or base of a compound described herein for example, by P. Stahl et al, Camille G. (eds.) Flandbook of that is non-toxic, is physiologically tolerable, is compatible Pharmaceutical Salts. Properties, Selection and Use. (2002) with the pharmaceutical composition in which it is formu Zurich: Wiley-VCFl; S. Berge et al, Journal of Pharmaceuti lated, and is otherwise suitable for formulation and/or admin cal Sciences (1977) 66(1) 1-19; P Gould, International J. of istration to a subject. Reference to a compound herein is Pharmaceutics (1986) 33 201-217; Anderson et al, The Prac understood to include reference to a pharmaceutically accept tice of Medicinal Chemistry (1996), Academic Press, New able salt of said compound unless otherwise indicated. York; and in The Orange Book (Food & DrugAdministration, US 2012/0301400 Al Nov. 29, 2012 15

MD, available from FDA). These disclosures are incorpo C-QFDLSTRRLK-C (cQFD; SEQ ID NO: I) and rated herein by reference thereto. C-QYNLSSRALK-C (cQYN; SEQ ID NO: 2), non-co- [0217] Additionally, any compound described herein is valently bind to a murine-human chimeric antibody, cetux- intended to refer also to any unsolvated form, or a hydrate, imab, by interacting with residues outside the complementa solvate, or polymorph of such a compound, and mixtures rity determining regions (CDRs), including residues in the thereof, even if such forms are not listed explicitly. “Solvate” framework and constant region. Also as described herein, the means a physical association of a compound of the invention ability to bind to these peptides was based on certain aspects with one or more solvent molecules. This physical associa specific to cetuximab and in the studies herein binding was tion involves varying degrees of ionic and covalent bonding, not observed, for example, to fully human antibodies, murine including hydrogen bonding. In certain instances the solvate antibodies, or other chimeric antibodies). As demonstrated will be capable of isolation, for example when one or more herein, these meditopes are capable of binding the chimeric solvent molecules are incorporated in the crystal lattice of a antibody simultaneously with its antigen. See FIG. 4. As crystalline solid. “Solvate” encompasses both solution-phase demonstrated herein, the binding site for these meditopes on and isolatable solvates. Suitable solvates include those cetuximab also are distinct from the binding sites of other formed with pharmaceutically acceptable solvents such as framework-binding antigens such as the superantigens Sta water, ethanol, and the like. In some embodiments, the sol phylococcal ProteinA (SpA) and Pepto streptococcus magnus vent is water and the solvates are hydrates. Protein L (PpL) (FIG. 7). In certain aspects, the provided [0218] Any formula given herein is also intended to repre embodiments build upon this discovery, for example, by sent unlabeled forms as well as isotopically labeled forms of modifying other mAbs to allow their binding to these and the compounds. Isotopically labeled compounds have struc other meditopes (i.e., “meditope-enabling”) and generating tures depicted by the formulas given herein except that one or variant meditopes and/or altered meditope-enabled antibod more atoms are replaced by an atom having a selected atomic ies, for example, those having altered properties, including mass or mass number. Examples of isotopes that can be improved binding affinity, toxicity, PK/PD, and/or pH depen incorporated into compounds of the invention include iso dence. topes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2Fl, 3Fl, 11C, 13C, 14C, C. Meditope-EnabledAntibodies and Complexes 15N, 18O, 17O, 31P, 32P, 35S, 18F, 36Cl, and 125I, respectively. [0221] Provided are meditope-enabled antibodies that are Such isotopically labelled compounds are useful in metabolic capable of binding to one or more meditopes via meditope- studies (for example with 14C), reaction kinetic studies (with, binding sites. In some cases, the meditope-enabled antibody for example 2Fl or 3Fl), detection or imaging techniques [such binds to a cyclic peptide of SEQ ID NO: I or 2 (meditope I or as positron emission tomography (PET) or single-photon 2) and/or to one or more variants thereof, such as meditopes I, emission computed tomography (SPECT)] including drug or 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, or substrate tissue distribution assays, or in radioactive treat 55 (meditopes based on peptides having the sequences set ment of patients. In particular, an 18F or 11C labeled com forth in SEQ ID NOs: I, 2, 16-18, 23, 29, 31, 32, 36, 39, 42, pound may be particularly suitable for PET or SPECT stud 43, 45, 46, 51, 52, 54, or 55), or in some cases, any of ies. Further, substitution with heavier isotopes such as meditopes I, 2, or 15-55. Among the provided meditope- deuterium (i.e., 2Fl) may afford certain therapeutic advan enabled antibodies are those that bind to a meditope or medi tages resulting from greater metabolic stability, for example topes with an affinity similar to that of cetuximab. For increased in vivo half-life or reduced dosage requirements. example, in certain aspects, the antibodies bind to the medi- Isotopically labeled compounds of this invention and pro tope(s) with a dissociation constant of less than at or about 10 drugs thereof can generally be prepared by carrying out the μΜ, less than at or about 5 μΜ, or less than at or about 2 μΜ, procedures disclosed in the schemes or in the examples and less than at or about I μΜ, less than at or about 500,400, 300, preparations described below by substituting a readily avail 200.100 nM, or less, such as atorabout200picomolarorless. able isotopically labeled reagent for a non-isotopically In some cases, the dissociation constant, such as any of those labeled reagent. listed herein, is that measured using a particular technique, B. Antibodies and Antibody-Binding Substances such as surface plasmon resonance (SPR), isothermal titra tion calorimetry (ITC), fluorescence, fluorescence polariza [0219] Provided herein are antibodies, including mono tion, NMR, IR, calorimetry titrations; kinetic exclusion; cir clonal antibodies (mAbs), and fragments, e.g., functional cular dichroism, differential scanning calorimetry, or other fragments, thereof, and compounds and compositions, known method. For example, in some cases, the analog or including peptides, such as meditopes, and meditope analogs, meditope exhibits a binding constant of less than at or about that bind to the antibodies. The substances, e.g., meditopes, 10 μΜ, less than at or about 5 μΜ, or less than at or about 2 generally bind to regions/binding sites of the antibodies and μΜ, less than at or about I μΜ, less than at or about 500, 400, fragments other than (e.g., separate from) the complementa 300.200.100 nm, or less, as measured by SPR or as measured rity determining regions (CDRs), typically to residues within by ITC or as measured by any of these methods. the framework regions (FRs) and/or constant regions of the [0222] In some examples, the meditope-binding site is a antibodies and fragments. Also provided are complexes, com structural feature of the monoclonal antibody, cetuximab, a pounds, and compositions containing the antibodies and sub human-murine chimeric antibody used for the treatment of stances, e.g., meditopes, as well as methods and uses of the EGFR-expressing metastatic colorectal cancer and head and same, including therapeutic, diagnostic, research, and other neck cancers. Thus, in some cases, the meditope-binding site uses of the antibodies and substances, and methods for pro contains residues corresponding to those within the meditope ducing the same. binding site of cetuximab. In a study reported herein, x-ray [0220] In certain aspects, the provided embodiments are crystallographic analysis has revealed that the peptides of based on the discovery described herein that certain peptides, SEQ ID NO: I binds to a meditope-binding site within the US 2012/0301400 Al Nov. 29, 2012 16

central cavity of the cetuximab Fab fragment, defined by heavy and light chain variable region FRs. In some embodi various residues of the heavy and light chains (see FIGS. I ments, such residues are replaced with the corresponding and 4A), with a binding constant of -700 nM (see FIGS. residue present in cetuximab, or comparable amino acid. 30A-30B). Thus, in certain embodiments, residues within the FRs of a [0223] Several interactions between cetuximab’s meditope human or humanized antibody are replaced with correspond binding site and the cQFD (SEQ ID NO: I, meditope I) and ing murine residues; in certain embodiments, they are cQYN (SEQ ID NO: 2, meditope 2) meditopes are based on replaced by other residues, such as those having similar func particular structural features of cetuximab, particularly tional groups or moieties for interacting with the meditopes. within the framework and constant regions of the central Typically, the residues replaced by corresponding murine (or cavity of the Fab fragment. The regions of cetuximab that other) residues are found within the central Fab cavity, and constitute the meditope binding site are unique and appear to thus are not exposed to the immune system. As such, in some be the result of the chimeric nature of this antibody. Specifi embodiments, introducing these amino acid substitutions in a cally, the Fab variable regions (Fvs) are murine and the Fab human or humanized antibody do not increase or do not constant regions (CF! I and CL) are human. The engineering substantially increase the antigenicity of the modified tem of this Fab produced a combination of residues not found in a plate antibody, in the context of delivery to a human subject. sequence alignment of murine and human chimeric antibod In addition, antigenicity prediction algorithms may be further ies. For example, data herein show that the meditopes did not used to indicate that the human sequence with the point muta bind to fully human IgG framework (e.g., trastuzumab), to tions should not be antigenic. other chimeric antibodies such as rituximab (FIG. 29), or to [0226] In some embodiments, the one or more residues that mouse antibodies, confirming that the cetuximab meditope- are replaced, are selected from light chain framework resi binding site is highly specific. In addition to these results, dues 10, 39-43, 83, 85, 100 and 104, according to Rabat superposition of the molecular structure of trastuzumab numbering (see Rabat E. A., et al. (1991) Sequences of Pro (1N8Z; Cho et al., Nature, “Structure of the extracellular teins of Immunological Interest, Fifth Edition. NIH Publica region of HER2 alone and in complex with the Fierceptin tion No. 91-3242, incorporated herein by reference in its Fab,” 2003 Feb. 13; 421(6924):756-60) and rituxumab entirety), and/or heavy chain framework residue numbers 40, (20SL; Du et al, JBiol Chem1 2007 May 18; 282(20): 15073- 89 and 105, according to Rabat numbering (see FIG. 2). In 80. Epub 2007 Mar. 29) Fabs on to the meditope bound general, unless otherwise specified, amino acid positions in cetuximab Fab structure further highlighted the uniqueness of the heavy or light chain of an antibody refer to Rabat num the framework. Superposition of multiple human and murine bering. Also encompassed within the present disclosure are Fabs onto the cetuximab-cyclic peptide (meditope I) struc residues in other antibodies corresponding to the residues of ture indicated that the key interactions between this peptide cetuximab described herein, such as those within the cetux and murine-human chimeric Fab are absent in both human- imab meditope-binding site. In some embodiments, residues only and murine-only IgG structures compared in this study. in the template antibody corresponding to light chain residues Point mutations of key residues within the cQFD cyclic pep 9,10,39,40,41,42,43,83, 85,100, and/or 104, and/or heavy tide (meditope I) reduced its binding affinity for the cetux chain residues 40, 89, and/or 105, are replaced, for example, imab Fab, further confirming the high specificity and struc with amino acids present at those positions within cetuximab. tural model. Thus, the interaction appears to be specific to the [0227] In one embodiment, the one or more residues central cavity of this specific murine-human chimera Fab and replaced are light chain framework residues including, but not the selected meditope. limited to, 40, 41, 83 and 85, according to Rabat numbering. [0224] In certain embodiments, the unique interactions In one embodiment, light chain residue 40 is replaced with between the meditopes and the cetuximab binding site are threonine; light chain residue 41 is replaced with asparagine, exploited to generate additional meditope-enabled antibod light chain residue 83 is replaced with isoleucine or valine, ies. Meditope-enabled antibodies are useful, for example, to and/or light chain residue 85 is replaced with aspartate. In a improve antibody and cell purification methods, improving particular example, light chain framework Pro40 is replaced the therapeutic efficacy of mAbs, enhancing targeted delivery with Thr (P40T) or Ser (P40S), light chain framework Gly41 of imaging or therapeutic agents, including in pre-taigeted is replaced with Asn (G41N), light chain framework residue delivery and imaging, and improving mAb-based imaging Phe83 is replaced with He (F83I) or Val (F83V) and light methods, and in some aspects are broadly applicable to any chain framework residue Thr85 is replaced with Asp (T85D) monoclonal antibody. or Asn (T85N). [0225] In some embodiments, the meditope-enabled anti [0228] Thus, among the provided meditope-enabled anti bodies are generated by modifying an antibody other than bodies are antibodies having one or more modifications, typi cetuximab (sometimes referred to as the template antibody), cally amino acid substitutions, at residues that correspond to such as an antibody having one or more CDRs distinct from positions within the meditope binding site of cetuximab or those of cetuximab, to confer the ability to bind to one or more other meditope-enabled antibody, such as those described of the provided meditopes, such as a meditope of SEQ ID NO: herein, including meditope-enabled trastuzumab and medi I or 2, or variant thereof. The template antibody can be a tope-enabled M5A. Among the antibodies are those having a human or humanized antibody or a mouse antibody. In one VL region with a threonine, serine, or aspartate at position 40, aspect, the modifications include substituting residues within a residue other than glycine at position 41, and an aspartate or the central cavity of the Fab fragment, typically within the asparagine at position 85, according to Rabat numbering, for framework regions (FRs) of the heavy and light chain variable example, an antibody with a VL region having a threonine at regions and/or the constant regions to render the template position 40, an asparagine at position 41, and an aspartate at antibody meditope-enabled. For example, where the template position 85. In some embodiments, the antibody has a VH antibody is a human or humanized antibody, the modifica region with a serine at position 40 and an isoleucine at posi tions generally include substitutions at residues within the tion 89 and/or a VH region with a serine or proline at position US 2012/0301400 Al Nov. 29, 2012 17

40 and an isoleucine, tyrosine, methionine, phenylalanine, or neering methods typically employed to alter antigen binding tryptophan at position 89, according to Kabat numbering. In and other properties include various in vitro randomization, some embodiments, the VL region has an isoleucine or leu affinity maturation, and selection methods, including error- cine at position 10, and/or an isoleucine at position 83. In prone PCR, spiked PCR, site-directed mutagenesis, phage some embodiments, the VL region has a valine or isoleucine display and other selection methods. Also provided are con at position 9 and/or a residue other than glutamine at position structs, libraries, and expression systems, including GPI-

100. linked expression systems, for carrying out such methods. [0229] In some examples, the VL region has a valine or [0234] Thus, in certain embodiments, the provided medi isoleucine at position 9, an isoleucine or leucine at position tope-enabled antibody has a light chain and/or heavy chain 10, an arginine at position 39, a threonine at position 40, an variable region with the framework region or regions (FRs) of asparagine at position 41, a glycine at position 42, a serine at a meditope-enabled antibody, such as cetuximab, a meditope- position 43, an isoleucine at position 83, an aspartate at posi enabled trastuzumab, or a meditope-enabled M5A (or FR(s) tion 85, and an alanine at position 100; and the VHregionhas with at least at or about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, a serine at position 40 and an isoleucine at position 89, 97, 98, or 99% identify to the FR(s) of such an antibody). In according to Kabat numbering. some aspects, such an antibody has one or more CDRs that [0230] In some examples, the VL region does not contain a are distinct from the CDRs of that meditope-enabled anti proline at position 40, a glycine at position 41, or a threonine body. at position 85, according to Kabat numbering, and/or the VH [0235] For example, in some embodiments, the VL region region does not contain an asparagine or alanine at position 40 has an amino acid sequence comprising a light chain frame or a valine at position 89, according to Kabat numbering. In work region (FR) I (FR-L1), an FR-L2, an FR-L3, and/or an some examples, the VL region does not contain an serine at FR-L4 of the light chain sequence set forth in SEQ ID NO: 71 position 10, a proline at position 40, a glycine at position 41, (or an FR-Ll, FR-L2, FR-L3, and/or FR-L4 that is at least at an phenylalanine at position 83, or a threonine at position 85, or about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% according to Kabat numbering, and/or the VH region does not identical to the FR-Ll, FR-L2, FR-L3, and/or FR-L4 of SEQ contain an asparagine or alanine at position 40 or a valine at ID NO: 71), and in some aspects at least one CDR that is position 89, according to Kabat numbering. distinct from the CDRs of the light chain sequence set forth in [0231] In some aspects, the antibody has a light chain hav SEQ ID NO: 71; and/or a VH region with an amino acid ing P8, V9 or 19, HO or L10, Q38, R39, T40, N41 G42, S43, sequence having a heavy chain FRl (FR-H1), an FR-H2, an P44, R45, D82,183, A84, D85, Y86, Y87, G99, A100, GlOl, FR-H3, and/or an FR-H4, of the heavy chain sequence set T102, K103, L104, E105, R142, S162, V163, T164, E165, forth in SEQ ID NO: 72 (or an FR-Hl, FR-H2, FR-H3, and/or Q166, D167, S168, andY173, according to Kabat numbering, FR-H4 that is at least at or about 75, 80, 85, 90, 91, 92, 93, 94, and/or a heavy chain having Q6, P9, R38, Q39, S40, P41, 95, 96, 97, 98, or 99% identical to the FR-Hl, FR-H2, FR-H3, G42, K43, G44, L45, S84, D86, T87, A88, 189, Y90, Y91, and/or FR-H4 of SEQ ID NO: 72), and in some aspects at least W103, Gl04, Q105, G106, T107, L108, V109, T110, Vlll, one CDR that is distinct from the CDRs of the heavy chain Yl47, E150, Pl51, V152, T173, F174, P175, A176, V177, sequence set forth in SEQ ID NO: 72. Y185, S186, and L187, according to Kabat numbering. [0236] In some embodiments, the VL region has an amino [0232] In other embodiments, the meditope-enabled anti acid sequence comprising a light chain framework region bodies are generated via CDR grafting, typically by modify (FR) I (FR-L1), an FR-L2, an FR-L3, and/or an FR-L4 ofthe ing one or more complementarity determining region (CDR) light chain sequence set forth in SEQ ID NO: 9 (or an FR-Ll, (e.g., one or more of CDRs 1-3) of the heavy and/or light FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75,80, chain of a meditope-enabled antibody, such as any of the 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the meditope-enabled antibodies described herein, to replace FR-Ll, FR-L2, FR-L3, and/or FR-L4 of SEQ ID NO: 9), and them with other CDRs, such as CDRs of existing or new in some aspects at least one CDR that is distinct from the antibodies. CDR grafting is standard practice for producing CDRs of the light chain sequence set forth in SEQ ID NO: 9; humanized monoclonal antibodies, e.g., by grafting CDRs of and/or a VH region with an amino acid sequence having a an antibody generated in a non-human species, such as heavy chain FRl (FR-H1), an FR-H2, an FR-H3, and/or an mouse, onto a human antibody framework. See U.S. Pat. Nos. FR-H4, of the heavy chain sequence set forth in SEQ ID NO: 5,558,864 and 8,133,982; Kettleborough et ah, “Humaniza 6 (or an FR-Hl, FR-H2, FR-H3, and/or FR-H4 that is at least tion of a mouse monoclonal antibody by CDR-grafting: the at or about 75,80, 85,90,91,92, 93,94,95,96, 97,98, or99% importance of framework residues on loop conformation,” identical to theFR-Hl, FR-H2, FR-H3, and/or FR-H4 of SEQ Protein Eng., 4:773-783 (1991). Thus, in certain embodi ID NO: 6), and in some aspects at least one CDR that is ments, the antigen specificity of a meditope-enabled antibody distinct from the CDRs of the heavy chain sequence set forth is altered by grafting the CDRs of preexisting or newly- in SEQ ID NO: 6. generated antibodies of interest. Also among the provided [0237] In some embodiments, the VL region has an amino meditope-enabled antibodies are such CDR-grafted medi acid sequence comprising a light chain framework region tope-enabled antibodies. (FR) I (FR-L1), an FR-L2, an FR-L3, and/or an FR-L4 ofthe [0233] In some embodiments, the meditope-enabled anti light chain sequence set forth in SEQ ID NO: 68 (oranFR-Ll, bodies are generated, using one of the antibodies disclosed FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75,80, herein (e.g., cetuximab, meditope-enabled trastuzumab, or 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the meditope-enabled M5A (anti-CEA) antibody) as a template FR-Ll, FR-L2, FR-L3, and/or FR-L4 of SEQ ID NO: 68), and sequence, and carrying out one or more known antibody in some aspects at least one CDR that is distinct from the engineering methods to alter it, for example, to alter its anti- CDRs of the light chain sequence set forth in SEQ ID NO: 68; gen-binding characteristics, producing a meditope-enabled and/or a VH region with an amino acid sequence having a antibody with distinct characteristics. Known antibody engi heavy chain FRl (FR-H1), an FR-H2, an FR-H3, and/or an US 2012/0301400 Al Nov. 29, 2012 18

FR-H4, of the heavy chain sequence set forth in SEQ ID NO: or antigen-binding regions thereof, and/or antibodies that 70 (oranFR-Hl, FR-H2, FR-H3, and/or FR-H4 that is at least compete for binding with such antibodies. at or about 75,80,85, 90,91,92,93, 94,95,96, 97, 98, or99% [0242] Table 2 lists CAS® Registry Numbers (see www. identical to the FR-HI, FR-H2, FR-H3, and/or FR-H4 of SEQ cas.org/expertise/cascontent/registry/regsys.html) for certain ID NO: 70), and in some aspects at least one CDR that is antibodies. distinct from the CDRs of the heavy chain sequence set forth in SEQ ID NO: 70. TABLE 2 [0238] In some embodiments, the VL region has an amino acid sequence comprising a light chain framework region Antibody CAS Registry number (FR) I (FR-L1), anFR-L2, anFR-L3, and/or an FR-L4 ofthe abagovomab 792921-10-9 light chain sequence set forth in SEQ ID NO: 61 (oranFR-Ll, abciximab 143653-53-6 FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75, 80, adalimumab 331731-18-1 adecatumumab 503605-66-1 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the alemtuzumab 216503-57-0 FR-Ll, FR-L2, FR-L3, and/or FR-L4 of SEQ ID NO: 61), and indium (IllIn) 156586-92-4 in some aspects at least one CDR that is distinct from the altumomab pentetate arcitumomab 154361-48-5 CDRs of the light chain sequence set forth in SEQ ID NO: 61; arcitumumab 154361-48-5 and/or a VH region with an amino acid sequence having a atlizumab 375823-41-9 heavy chain FRl (FR-H1), an FR-H2, an FR-H3, and/or an basiliximab 152923-56-3 bectumomab 158318-63-9 FR-H4, of the heavy chain sequence set forth in SEQ ID NO: belimumab 356547-88-1 63 (or an FR-Hl, FR-H2, FR-H3, and/or FR-H4 that is at least benralizumab 1044511-01-4 at or about 75,80,85, 90,91,92,93, 94,95,96, 97, 98, or99% bevacizumab 216974-75-3 identical to the FR-HI, FR-H2, FR-H3, and/or FR-H4 of SEQ brentuximab 914088-09-8 canakinumab 914613-48-2 ID NO: 63), and in some aspects at least one CDR that is capromab pendetide 145464-28-4 distinct from the CDRs of the heavy chain sequence set forth capromab 151763-64-3 in SEQ ID NO: 63. catumaxomab 509077-98-9 certolizumab 428863-50-7 [0239] In some embodiments, the meditope-enabled anti certolizumab 428863-50-7 body has one or more CDRs distinct from the CDRs set forth cetuximab 205923-56-4 in SEQ ID NO: 6,7,9,10,12,14,61,63,68, 69,70,71, and/or clivatuzumab tetraxetan 943976-23-6 72. daclizumab 152923-56-3 denosumab 615258-40-7 [0240] In some embodiments, the meditope is an antibody eculizumab 219685-50-4 other than cetuximab, does not specifically bind to an EGFR, edrecolomab 156586-89-9 binds to an antigen other than EGFR, and/or does not specifi efalizumab 214745-43-4 cally bind to the epitope on EGFR that is specifically bound etaracizumab 892553-42-3 etrumaxomab 509077-99-0 by cetuximab. fanolesomab 225239-31-6 [0241] In some examples, the meditope-enabled antibody FBTA05 Lymphomun/FBTA05 is generated based on a template antibody that is selected fontolizumab 326859-36-3 gemtuzumab 220578-59-6 from among abagovomab, abciximab, adalimumab, adecatu- girentuximab 916138-87-9 mumab, alemtuzumab, altumomab, altumomab pentetate, golimumab 476181-74-5 anatumomab, anatumomab mafenatox, arcitumomab, atli- ibritumomab 174722-31-7 zumab, basiliximab, bectumomab, ectumomab, belimumab, igovomab 171656-50-1 benralizumab, bevacizumab, brentuximab, canakinumab, Infliximab 170277-31-3 capromab, capromab pendetide, catumaxomab, certoli- ipilimumab 477202-00-9 labetuzumab 219649-07-7 zumab, clivatuzumab tetraxetan, daclizumab, denosumab, mepolizumab 196078-29-2 eculizumab, edrecolomab, efalizumab, etaracizumab, ertu- muromonab-CD3 140608-64-6 maxomab, fanolesomab, Fbta05, fontolizumab, gemtu- natalizumab 189261-10-7 zumab, girentuximab, golimumab, ibritumomab, igovomab, nimotuzumab 828933-61-3 infliximab, ipilimumab, labetuzumab, mepolizumab, ofatumumab 679818-59-8 muromonab, muromonab-CD3, natalizumab, necitumumab omalizumab 242138-07-4 oregovomab 213327-37-8 nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab 188039-54-5 palivizumab, panitumumab, ranibizumab, rituximab, satu- panitumumab 339177-26-3 momab, sulesomab, ibritumomab, ibritumomab tiuxetan, ranibizumab 347396-82-1 tocilizumab, tositumomab, trastuzumab, Trbs07, usteki- rituximab 174722-31-7 numab, visilizumab, votumumab, zalutumumab, satumomab 138955-26-7 sulesomab 167747-19-5 brodalumab, anrukinzumab, bapineuzumab, dalotuzumab, tiuxetan (ibritumomab) 174722-31-7 demcizumab, ganitumab, inotuzumab, mavrilimumab, mox- tocilizumab 375823-41-9 etumomab pasudotox, rilotumumab, sifalimumab, tan- tositumomab 192391-48-3 ezumab, tralokinumab, tremelimumab, the antibody pro trastuzumab 180288-69-1 duced by the hybridoma 10B5, B6H12.2, and urelumab, ustekinumab 815610-63-0 fragments thereof, antibodies having the CDRs and/or anti- votumumab 148189-70-2 zalutumumab 667901-13-5 gen-binding regions thereof, and/or antibodies that compete brodalumab 1174395-19-7 for binding with such antibodies; and/or antibodies having a anrukinzumab 910649-32-0 sequence set forth in any of SEQ ID NOs: 78-124, and/or bapineuzumab 648895-38-9 125-170, fragments thereof antibodies having the CDRs and/ US 2012/0301400 Al Nov. 29, 2012 19

tor, hepatocyte growth factor, IFN-alpha, nerve growth factor, TABLE 2-continued IL-13, PD-Ll, CD326, CD47, and CD137. In some examples, the meditope-enabled antigen binds to another Antibody CAS Registry number antigen identified as a target in a disease or condition of dalotuzumab 1005389-60-5 interest, such as cancer or other disease. demcizumab (OMP- 1292853-12-3 [0245] The meditope-enabled antibodies generally further 21M18) include a constant region, typically a heavy and light chain ganitumab 905703-97-1 constant region, which generally are human or partially inotuzumab 635715-01-4 human constant regions. In some aspects, the heavy chain mavrilimumab 1085337-57-0 constant region includes a CHl, or a portion thereof. In some moxetumomab 1020748-57-5 moxetumomab 1020748-57-5 aspects, the light chain constant region includes a CL, or a pasudotox portion thereof. In some embodiments, the portion of the rilotumumab 872514-65-3 constant region is sufficient to render the antibody capable of sifalimumab 1143503-67-6 binding to the meditope, e.g., with the requisite binding affin tanezumab 880266-57-9 tralokinumab 1044515-88-9 ity. In some aspects, the constant regions are the constant tremelimumab 745013-59-6 regions of cetuximab or trastuzumab. Thus, in some aspects, urelumab 934823-49-1 the heavy chain constant regions are one or more human IgGl necitumumab 906805-06-9 constant regions; in some aspects, the light chain constant regions are kappa constant chain regions. In other examples, [0243] In other examples, the template antibody is selected the constant regions can include those of other isotypes, from among: abagovomab, abciximab, adalimumab, adeca- including human (or other organism, e.g., murine or chicken) tumumab, alemtuzumab, altumomab, altumomab pentetate, IgGl, IgG2, IgG3, IgG4, IgEs, IgAl, IgA2, IgD, or IgM, and anatumomab, anatumomab mafenatox, arcitumomab, atli- can include kappa or lambda constant regions. Thus, among zumab, basiliximab, bectumomab, ectumomab, belimumab, the provided meditope-enabled antibodies are those gener bemalizumab, bevacizumab, brentuximab, canakinumab, ated by mutation of residues in other IgGs, such as human, capromab, capromab pendetide, catumaxomab, certoli- murine, or chicken, or other immunoglobulins. In other zumab, clivatuzumab tetraxetan, daclizumab, denosumab, words, the meditope-enabling methods provided herein may eculizumab, edrecolomab, efalizumab, etaracizumab, ertu- be used for any antibody, including IgA, IgE, IgD, and IgM maxomab, fanolesomab, Fbta05, fontolizumab, gemtu- and from any organism that produces antibodies including but zumab, girentuximab, golimumab, ibritumomab, igovomab, not limited to chicken, murine, rat, bovine, rabbit, primates, infliximab, ipilimumab, labetuzumab, mepolizumab, and goat. muromonab, muromonab-CD3, natalizumab, necitumumab, [0246] For example, the sequences of the first constant nimotuzumab, ofatumumab, omalizumab, oregovomab, region (CHl) of a human IgGl, IgG2, IgG3, and IgG4, are palivizumab, panitumumab, ranibizumab, rituximab, satu- compared in FIG. 55A. FIG. 55B shows the sequence differ momab, sulesomab, ibritumomab, ibritumomab tiuxetan, ences in the IgG2-4 CHI, compared to the IgGl CHI, tocilizumab, tositumomab, trastuzumab, Trbs07, usteki- mapped onto the co-crystal structure of cetuximab and the numab, visilizumab, votumumab, zalutumumab, the anti cQFD meditope (meditope I, SEQ ID NO: I). As shown in body produced by the hybridoma 10B5, and brodalumab, or the Figure, the residues that are different among these iso tiuzetan. In some such examples, the one or more CDRs are types are not within the meditope-binding region of cetux CDRs present in these template antibodies, and/or the anti imab, confirming that the meditope-enabling technology is bodies specifically bind to the same antigen or epitope as such applicable to isotypes other than the IgGl of cetuximab. As antibodies, and/or compete for binding with such antibodies another example, the sequence and structural alignment of an to their antigens. IgGl and an IgE Fab domain indicates residues on the IgE [0244] Thus, in some cases, the meditope-enabled antibod near the meditope binding site (FIG. 26). ies (including fragments thereof) specifically binds to an anti [0247] The provided methods for meditope-site grafting of gen selected from the group consisting of: CA-125, glyco the Fab cavity within monoclonal antibodies can be used to protein (GP) Ilb/IIIa receptor, TNF-alpha, CD52, TAG-72, create a unique handle for meditope binding and used with the Carcinoembryonic antigen (CEA), interleukin-6 receptor technology previously disclosed and for newly-generated (IL-6R), IL-2, interleukin-2 receptor a-chain (CD25), CD22, antibodies. In certain embodiments, the meditope binding site B-cell activating factor, interleukin-5 receptor (CD125), can be created on pre-existing and all future monoclonal VEGF, VEGF-A, CD30, IL-lbeta, prostate specific mem antibodies. brane antigen (PSMA), CD3, EpCAM, EGF receptor [0248] As described below in section F, also provided are (EGFR), MUCl, human interleukin-2 receptor, Tac, RANK methods for modifying the meditope-enabled antibodies, for ligand, a complement protein, e.g., C5, EpCAM, CDl la, e.g., example, to alter various properties of the meditope-enabled human CDl la, an integrin, e.g., alpha-v beta-3 integrin, vit antibodies, including aspects of the interaction with the medi- ronectin receptor alpha v beta 3 integrin, HER2, neu, CD3, topes, including affinity, avidity, pH-dependence, as well as CDl5, CD20 (small and/or large loops), Interferon gamma, other aspects, including pharmacokinetics (PK) and pharma CD33, CA-IX, TNF alpha, CTLA-4, carcinoembryonic anti codynamics (PD) of the antibodies. Thus, also among the gen, IL-5, CD3 epsilon, CAM, Alpha-4-integrin, IgE, e.g., provided meditope-enabled antibodies are those modified IgE Fe region, an RSV antigen, e.g., fusion protein of respi antibodies generated according to those methods, e.g., by ratory syncytial virus (RSV), TAG-72, NCA-90 (granulocyte generating a pharmacophore binding model, including anti cell antigen), IL-6, GD2, GD3, IL-12, IL-23, IL-17, bodies having any one or more of the modifications described CTAA16.88, IL13, interleukin-1 beta, beta-amyloid, IGF-I in section F, below. receptor (IGF-1R), delta-like ligand 4 (DLL4), alpha subunit [0249] Also among the antibodies used as template anti of granulocyte macrophage colony stimulating factor recep bodies to generate the meditope-enabled antibodies are modi US 2012/0301400 Al Nov. 29, 2012 20

fied antibodies and portions thereof, such as a CovX-Body™. cQFD and cQYN and others described herein. Suitable modi Thus, among the provided meditope-enabled antibodies are fications include, but are not limited to, any peptide modifi CovX-bodies, modified to allow their binding to one or more cation known in the art, such as, but not limited to, modifica of the provided meditopes, e.g., with an affinity as described tions to the manner and/or position of peptide cyclization, herein. modifications to one or more amino acid components of the [0250] Also provided are complexes containing one or cyclic peptide, or adding or deleting one or more amino acid more meditope bound to a meditope-enabled antibody, such from the cyclic peptide. In a particular example, cQFD may as any of the antibodies described herein. be altered with one or more of the following modifications: a [0251] Also provided are nucleic acids, such as cDNA and modification of Arg8, a modification of Phe3, a modification RNA molecules, encoding the meditope-enabled antibodies, of Leu5, a modification of Leul 0, change to the mode of and vectors and libraries containing the same, as well as peptide cyclization, and/or an incorporation of hydratable methods for their use, including selection and expression carbonyl functionality at one or more positions, and one or methods and methods for generating transgenic animals using such constructs. more amino acid deletions or additions. In the case of cQYN, suitable modifications may include one or more of the fol D. Meditopes lowing: a modification of Arg8, a modification of Leu5, a modification of Leul 0, change to the mode of peptide [0252] Also provided are meditopes, including variant, cyclization, and/or an incorporation of hydratable carbonyl modified, and multivalent meditopes, that bind to meditope- functionality at one or more positions, and one or more dele enabled antibodies, and complexes and compositions con tions or additions. Certain amino acid positions within the taining the same, and methods and uses thereof. In certain meditope may be deleted or replaced with a different natural embodiments, the meditopes include meditopes I and 2 (cQFD (SEQ ID NO: I) and cQYN (SEQ ID NO: 2)), which amino acid or an unnatural amino acid, or the meditope may were originally identified as candidate peptides for binding to be chemically conjugated with a fragment. It is shown herein the CDR region of cetuximab, as described by Riemer, et al. that a meditope in which Arg9 of SEQ ID NO: I has been (2004); JImmunol 173,394-401; Riemer, et al. (2005); JNatl mutated to citrulline binds to cetuximab. In addition, the Cancer Inst 97, 1663-1670. As demonstrated herein, the amino and carboxy termini can be extended with further cQFD and cQYN meditopes bind to sites within cetuximab amino acids beyond (Te., in addition to) the cyclic portion of distinct from the cetuximab CDRs, and thus were not likely the meditope variant in order to make additional contact to the candidates for specific cetuximab-like antibody immunogens Fab. For example, Protein L has been added to the C-terminus for use as a vaccine. of the cQFD meditope and preliminary data shows that this [0253] Nucleic acids encoding the meditopes, including binds with much higher affinity. Such modifications are dis meditope variants and multivalent meditopes, as well as vec cussed further in Examples 6 and 7. tors, cells, libraries, and other systems containing the same, [0257] In some embodiments, the meditopes include those also are provided. listed in Tables 3 and 4, as well as such meditopes with [0254] I. Variant Meditopes additional amino acids, such as those up to 16 amino acids in [0255] Among the provided meditopes are meditope vari length. For example, in some aspects, the meditope is one of ants (also called variant meditopes), having one or more meditopes I, 2, or 15-55, further including a serine before the modifications, e.g., structural modifications, as compared to first residues, i.e., at position zero. The meditopes listed in meditope I or 2 (meditope of SEQ ID NO: I or 2), and Table 3 employ a disulfide linkage to connect the C and N methods for producing the same. In some embodiments, termini (except that meditope 31 contained an additional tail, cQFD and cQYN meditopes are used as starting points in the meaning that the disulfide linkage is not between the two design of meditope variants. In some aspects, the meditope terminal residues); for the peptides in Table 4, a lactam variants are designed to have altered properties, such as bridge, a linkage other than disulfide (such as [3+2] cycload increased or altered affinity, altered pH dependence, or dif dition), or no linkage is used (e.g., an acyclic or linear vari ferent affinities under different physiological conditions for ant). Additional meditopes that may be used according to the one or more of the provided meditope-enabled antibodies, embodiments described herein include any meditope as including cetuximab and other antibodies described herein, defined herein peptide that binds to an antibody framework e.g., as compared to the unmodified meditopes, cQFD and binding interface (i.e., between the Fab light and heavy cQYN. Meditope variants are designed and produced using chains) of cetuximab or any other therapeutic antibody. For various chemical and biophysical methods. example, in addition to the cyclic peptides cQFD and cQYN, [0256] Meditope variants include, but are not limited to, some embodiments include one or more variants of cQFD and variants incorporating modifications to meditopes, e.g., cQYN.

TABLE 3

SEQ ID Meditope NO Number Sequence Modification (red) Linkage method

1 I C-QFDLSTRRLK-C original Disulfide I-Cys: 12-Cys

2 2 C-QYNLSSRALK-C original Disulfide I-Cys: 12-Cys

15 15 C-qFDLSTRRLK-C q = D-glutamine Disulfide 1-Cys: 12-Cys US 2012/0301400 Al Nov. 29, 2012 21

TABLE 3-continued

SEQ ID Meditope NO Number Sequence Modification (red) Linkage method

16 16 C-QYDLSTRRLK-C Y = tyrosine Disulfide I-Cys: 12-Cys

17 17 C-QXDLSTRRLK-C X = β-β'-di-phenyl-Ala Disulfide I-Cys: 12-Cys 18 18 C-QFDXSTRRLK-C X = β-β'-di-phenyl-Ala Disulfide I-Cys: 12-Cys

19 19 C-QFDESTRXLK-C F = phenylalanine, Disulfide I-Cys: 12-Cys X = citrulline

20 20 C-QFDESTRRLK-C F = phenylalanine Disulfide I-Cys: 12-Cys

21 21 C-QFDESTRRLK-C E = glutamic acid Disulfide I-Cys: 12-Cys

22 22 C-QFDXSTRRLK-C Y = tyrosine Disulfide I-Cys: 12-Cys

23 23 C-QFDLSTRRQK-C Q = glutamine Disulfide I-Cys: 12-Cys

24 24 C-QFDLSTRQLK-C Q = glutamine Disulfide I-Cys: 12-Cys

25 25 C-OYNLSTARLK-C Y = tyrosine; Disulfide I-Cys: 12-Cys N = asparagine; A = alanine

26 26 C-QADLSTRRLK-C A = alanine Disulfide I-Cys: 12-Cys

27 27 C-QFDASTRRLK-C A = alanine Disulfide I-Cys: 12-Cys

28 28 C-QFDLSTARLK-C A = alanine Disulfide I-Cys: 12-Cys

29 29 C-QFDLSTRRAK-C A = alanine Disulfide I-Cys: 12-Cys

30 30 C-QFDLSTRREK-C E = glutamic acid Disulfide I-Cys: 12-Cys

31 31 AcC-QFDLSTRRLR- AcC-N-acetylcysteine Disulfide I-AcCys: 12-Cys QGGGSE R = arginine

TABLE 4

SEQ ID NO Sequence Modification (red) Linkage method

32 32 Qi-QFDLSTRRLK-Qi G = glycine Lactam I-Gly:: 12-Gly

33 33 G-QEDLSTRRLK-G H = histidine Lactam I-Gly:: 12-Gly

34 34 G-QNDLSTRRLK-G N = asparagine Lactam I-Gly:: 12-Gly

35 35 G-QQDLSTRRLK-G Q = glutamine Lactam I-Gly:: 12-Gly

36 36 G-QXDLSTRRLK-G X = 2-bromo-L- Lactam I-Gly:: 12-Gly phenylalanine

37 37 G-QXDLSTRRLK-G X = 3-bromo-L- Lactam I-Gly:: 12-Gly phenylalanine

38 38 G-QXDLSTRRLK-G X = 4-bromo-L- Lactam I-Gly:: 12-Gly phenylalanine

39 39 G-QFDLSTRXLK-G X = citrulline Lactam I-Gly:: 12-Gly

40 40 G-OFDLSTXXLK- G X = citrulline Lactam I-Gly:: 12-Gly

41 41 G-QFDLSTNRLK-G X = citrulline Lactam I-Gly:: 12-Gly

42 42 X- FDLSTRRLK-X X = 7-aminoheptanoic acid Lactam I-Gln:: Il-X

43 43 X-QFDLSTRRLK-X X = β-alanine Lactam I-X: 12-X

44 44 X-QFDLSTRRLK-X1X = diaminopropionic acid; Lactam I-X: 12-X X' = iso-aspartic acid US 2012/0301400 Al Nov. 29, 2012 22

TABLE 4-continued

SEQ ΙΓ NO Sequence Modification (red) Linkage method

45 45 X-QFDLSTRRLK-X X = β-alanine; Lactam 1-X: 12-X' X' = iso-aspartic acid

46 46 X-OFDLSTRRLK-X1X = diaminopropionic acid; Lactam 1-X: 12-X' X' = β-alanine

47 47 F-DLSTRRL-K Lactam I-Phe: 9-Lys

48 48 C-QFDLSTRRLK-C Disulfide I-Cys: 12-Cys; Lactam 4-Asp to Il-Lys

49 49 Q-XDLSTRRLK-X Y = tyrosine, Lactam 1-Gln: Il-X X = 7-aminoheptanoic acid

50 50 X-OFDLSTRRLK-X'X = β-azidoalanine, [3 + 2] cycloaddition Azide- X' = propargylglycine 1-X: alkyne-12-X'

51 51 Q-XDLSTRRLK-X' X = β-β'-di-phenyl-Ala, Lactam 1-Gln: Il-X' X' = 7-aminoheptanoic acid

52 52 qFDLSTRRLK-X q = D-glutamine, Lactam 1-Gln: Il-X X = 7-aminoheptanoic acid

53 53 Q-XDXSTRRLK-X' X = β-β'-di-phenyl-Ala, Lactam 1-Gln: Il-X' X' = 7-aminoheptanoic acid

54 54 Q-FDLSTXRLK-X' X = n-butyl-arginine, Lactam 1-Gln: Il-X' X' = 7-aminoheptanoic acid

55 55 £QFDLSTRRLK£ No linkage

[0258] The meditope variants typically have an amino acid 4-bromo-L-phenylalanine, Asn, Gin, a modified Phe, a sequence length of between 5 and 16 amino acids, e.g., 5, 6, hydratable carbonyl-containing residue; or a boronic acid- 7,8, 9,10,11,12,13,14,15, or 16 amino acids in length, such containing residue; as between 8 and 13 amino acids in length, e.g., between 9 and [0265] X4=Asp or Asn; 12 amino acids in length. The meditope can additionally be conjugated to or associated with (e.g., as part of a fusion [0266] X5=Leu; β-β'-diphenyl-Ala; Phe; a non-natural protein or peptide) with another molecule, such as another analog of phenylalanine, tryptophan, or tyrosine; a hydratable peptide, including a linker or agent. Thus, in this case, the carbonyl-containing residue; or a boronic acid-containing compound containing the meditope may contain additional residue; amino acid residues beyond the lengths described in this [0267] X6=Ser; paragraph, where the meditope portion contains between 5 [0268] X7=Thr or Ser; and 16 amino acids and the complex or compound contains [0269] X8=Arg, a modified Arg, or a hydratable carbonyl or additional amino acids. Examples are described herein, e.g., boronic acid-containing residue; SEQ ID NO: 31 above. [0270] X9=Arg, Ala; [0259] In some embodiments, the variant meditopes are [0271] XlO=Leu, Gin, Glu, β-β'-diphenyl-Ala; Phe; a non cyclic peptides. In other embodiments, they are linear or natural analog of phenylalanine, tryptophan, or tyrosine; a acyclic peptides. hydratable carbonyl-containing residue; or a boronic acid- [0260] The meditopes can include peptides, or cyclic pep containing residue; tides derived from such peptides, for example, where the peptides have the formula: [0272] Xll=Lys; and [0273] X12=Cys, Gly, 7-aminoheptanoic acid, β-alanine, X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 (Formula I), diaminopropionic acid, propargylglycine, isoaspartic acid, or [0261] for example, where: null. [0262] Xl=Cys, Gly, β-alanine, diaminopropionic acid, [0274] In some aspects, the modified Arg has a structure of β-azidoalanine, or null; the formula shown in FIG. 34. In some aspects, the modified [0263] X2=Gln or null; Phe is a Phe with one or more halogen incorporated into the [0264] X3=Phe, Tyr, β-β'-diphenyl-Ala, Elis, Asp, phenyl ring. In some aspects, formula I is not SEQ ID NO: I 2-bromo-L-phenylalanine, 3-bromo-L-phenylalanine, or or SEQ ID NO: 2. US 2012/0301400 Al Nov. 29, 2012 23

[0275] In some embodiments, the meditopes are peptides each optionally substituted with one or more substitu having the structure of Formula (X): ents selected from the group consisting of —N3, —NH2, —OH, —SH, halogen, oxo, acetal, ketal, —B(OH)2, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1. 4alkyl, -CH=CH-CO2C1âlkyl, -CO2H, and —CO2C1âlkyl group; and [0294] Re is H; -NHRrf; or a C1âlkyl, C3_8cy- cloalkyl, C2_12alkenyl, C2_8alkynyl, or aryl group, each optionally substituted with one or more substitu ents selected from the group consisting of —N3, —NH2, —OH, —SH, oxo, C2_4acetal, C2_4ketal, —B(OH)2, boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C1. 4alkyl, -CH=CH-CO2C1âlkyl, and -CO2C1. R>° NH 4alkyl group; or [0295] (b) a C1âlkyl substituted with an oxo, acetal, ketal, —B(OH)2, boronic ester, —SH, —OH, phospho nate ester, ortho ester, —CH=CH—CHO, —CH=CH—C(O)C1âlkyl, -CH=CH-CO2C1. 4alkyl, or —CO2C1âlkyl group; [0296] R9 is C1âlkyl or —C1âlkylene-Rx; [0297] wherein IV is -CO2H, -CONH2, -CH2NHC (O)NH2, or —CH2NHC(=NH)NH2; [0298] R10 is: [0299] (I) a C1âlkyl optionally substituted with one or wherein: more substituents selected from the group consisting of [0276] the center marked with is in the “R” or “S” oxo, acetal, ketal, —B(OH)2, boronic ester, phospho configuration; nate ester, ortho ester, —CH=CH—CHO, [0277] R3 and R3 are each, independently, H or phenyl, —CH=CH—C(O)C1âlkyl, -CH=CH-CO2C1. optionally substituted with one, two, or three substituents 4alkyl, CO2C1âlkyl, —CO2H, and —CONH2 group; independently selected from C1âlkyl, —OH, fluoro, or chloro, bromo, and iodo; [0300] (2) a C1âlkyl group substituted with one or two [0278] R5 is: phenyl groups, or one naphthyl, imidazole, or indole [0279] (A) C1âlkyl, optionally substituted with one or group, wherein each phenyl is optionally substituted more substituents selected from the group consisting of with one, two, or three substituents independently oxo, acetal, ketal, —B(OH)2, boronic ester, phospho- selected from —OH, fluoro, chloro, bromo, and iodo; nate ester, ortho ester, —CO2C1âlkyl, —CH=CH— [0301] n is Oor I; CHO, —CH=CH-C(O)C1âlkyl, -CH=CH- [0302] p is Oor I; CO2C1âlkyl, —CO2H, and —CONH2 group; or [0303] X is C1âlkylene or C2_8alkenylene, each carbon [0280] (B) a C1âlkyl group substituted with: thereof optionally substituted with —CO2H, —NH2, or [0281] a) one or two phenyl groups, wherein each —NHC(O)Ry; phenyl is optionally substituted with one, two, or [0304] wherein one carbon of said alkylene is optionally three substituents independently selected from—OH, replaced with —C(O)NH—, a 5-membered heteroaryl fluoro, chloro, bromo, and iodo; or ring, or —S—S—; and [0282] b) a naphthyl, imidazole, or indole group; [0305] Ry is —C1âlkyl or -CH(Rz)CO2H; [0283] R6 is —C alkyl-OH or -C1âlkyl-SH; [0306] wherein Rz is —H or —C1âlkyl optionally [0284] R7 is —C alkyl-OH or -C1âlkyl-SH; substituted with —OH, —SH, or —NH2; [0285] m is 0, 1,2, 3, 4, or 5; or a pharmaceutically acceptable salt thereof. [0286] R8 is: [0307] In some cases, such meditopes are not SEQ ID NO: [0287] (a) —OH, -NRaR6, -N(Rc)C(O)Re, or I or 2, or are not cyclic peptides derived from such sequences, —N(Rc)C(=NRrf)Re; and/or are not meditope I or 2. [0288] wherein: [0308] In some embodiments of the meditope of Formula [0289] Ra is H; (X), m is 0, I, or 2. In other embodiments, R3 is H or phenyl [0290] R6 is H OrC1âlkyl optionally substituted with and R3 is phenyl, 2-bromophenyl, 3-bromophenyl, or 4-bro- one or more substituents selected from the group con mophenyl. In further embodiments, R5 is methyl, ethyl, pro sisting of oxo, acetal, and ketal, pyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl, each [0291] —B(OH)2, —SH, boronic ester, phosphonate optionally substituted with an oxo, —B(OH)2, —CO2H, or ester, ortho ester, -CH=CH-CHO, -CH=CH- —CONH2 group, or with one or two phenyl groups each C(O)C1âlkyl, -CH=CH-CO2C1âlkyl, optionally substituted with a bromo or chloro substituent. In —CO2H, or —CO2C1âlkyl group; further embodiments, R8 is —OH, —NH2, —N(Rc)C(O)Re, [0292] Rcis H, C3_8cycloalkyl, branched alkyl, or aryl; or—N(Rc)C(=NRrf)Re. In still further embodiments, Rc is H [0293] Rrf is H or a C1âlkyl, C2_8alkenyl, C2_8alky- or methyl, Rrf is H or C1âlkyl, and Re is C1âlkyl, or —NH nyl, C3_8cycloalkyl, branched alkyl, or aryl group, (C1âlkyl). In other embodiments, R9 is methyl or ethyl, US 2012/0301400 Al Nov. 29, 2012 24

optionally substituted with —CO2H, —CONH2, Phe3Tyr cQYN (SEQ ID NO: 2) has an alteration in the -CH2NHC(O)NH2, or —CH2NHC(=NH)NH2. In still extended conformation of the Arg8 side chain as compared to other embodiments, R10 is methyl, ethyl, propyl, isopropyl, cQFD (SEQ ID NO: I) (see FIGS. 30C and 35). Data herein butyl, isobutyl, sec-butyl, or tert-butyl, each optionally sub suggest the formation of a favorable hydrogen bond network, stituted with an oxo, —B(OH)2, —CO2H, or —CONH2 with water bound to the Fab. Enthalpy-driven optimization group. In still other embodiments, —X—NH— is -Cys-Cys-, has proven successful in many small-molecule approaches in -Gly-Gly-, -C(O)(CH2)6-NH-, -β-ΑΗ-β-ΑΗ-, -C(O) drug design and there are opportunities in the provided medi CH(NH2)CH2CH=CHCH2CH(C02H)-NH-, -C(O) topes for engineering increases in entropy. In some embodi CH(NH2)CH2NHC(0)CH2CH(C02H)—NH-, -β-Ala-C ments, approaches resulting in enthalpic and/or entropic (O)CH2CH(CO2H)-NH-, or -C(O)CH(NH2)CH2- gains in meditope designs are used to generate the variant triazinyl-CH2—CH(CO2H)-NH-. meditopes, e.g., to optimize binding. [0309] Modifications [0315] For example, when bound to a meditope-enabled [0310] Based on the structural and thermodynamic data, antibody, the hydrophobic phenyl ring of Phe3 is surrounded multiple positions within the meditopes I and 2 described by a fairly polar array of side chain residues of the meditope- herein have been identified as target sites for modification, enabled antibody Fab (FIG. 35). In some embodiments, one e.g., with different natural or non-natural amino acids, to or more halogens is introduced on the phenyl ring of this enhance the overall binding affinity and/or to alter another residue, to allow for a halogen bonding interaction with the property as described herein. Suchmodifications include, but polar side chain residues. A halogen bond is a relatively are not limited to, modification of cQFD or cQYN to generate strong non-covalent bond, similar to a hydrogen bond but a head-to-tail cyclic lactam peptide, modification of Arg8, involving the interaction of a halogen such as bromine or modification of position 3 (e.g., Phe3 of cQFD or variant chlorine (or other halogen), with an oxygen atom. In some thereof), modification of Leu5, modification of LeulO, and/or aspects, the residue at this position is modified to incorporate incorporation of hydratable carbonyl functionality (see FIG. a halogen substituent. In some aspects, Phe3 is replaced with 31). As demonstrated herein, mutation of each of Phe3, Leu5, 2-bromo-, 3-bromo-, or 4-bromophenylalanine, in order to and Arg8 to alanine in the original meditope of cQFD reduced place a bromine atom in a position suitable for halogen bond the affinity of the resulting compounds for the meditope- ing with a meditope-enabled antibody, e.g., at positions Tyr87 enabled antibody binding interface by IO-140-fold. In some (light chain), Gln38, and/or Tyr91 (heavy chain) of a medi aspects, the variant meditopes include those having modifi tope-enabled antibody, respectively. Such phenylalanine cations at one or more of position 1,3,5,8,10, and 12 of the derivatives are commercially available and in some aspects meditope of SEQ ID NO: I or 2, or other meditope listed in are incorporated into the cyclic peptide meditope variant by Table 3 or 4. solid phase peptide synthesis (SPPS). Exemplary variant [0311] Position 8 meditopes include those containing 2-bromo-L-phenylala- [0312] In some embodiments, the meditope variants con nine, 3-bromo-L-phenylalanine, or 4-bromo-L-phenylala- tain a modification in the position corresponding to position 8 nine in the position corresponding to Phe3 of meditope I. (Arg8) of meditope I or 2. In the unmodified meditope [0316] In another example, the meditope incorporates an (cQFD; SEQ ID NO: I), Arg8 is extended, making a hydro additional phenyl group at this position, for example, by gen bond with the heavy chain carbonyl of Q105 of the replacing Phe3 with β,β'-diphenylalanine. meditope-enabled antibody heavy chain. The immediate area [0317] Positions 5 and 10 (e.g., Leu5, LeulO of Meditopes about this residue is hydrophobic yet solvent exposed (FIG. I or 2) 33A). In some aspects, the meditopes contain a modified [0318] In some embodiments, the meditope variants con residue at this position (e.g., modified Arg8). In some tain a modification in the position corresponding to position 5 examples, the modified residue maintains the iminium func or 10 (Leu5 or LeulO) of meditopes I or 2. As shown herein, tionality of the Arg8 residue useful for meditope-enabled the side chains of Leu5 and LeulO of meditope I make hydro- antibody H-bonding, and introduces a substituted or unsub phobic contacts to the meditope-enabled Fab, cetuximab (see stituted hydrophobic arm to fill the cavity. Such modifications FIG. 36, right panel; LeulO). In certain embodiments, one or result in significant gains in binding, due to entropic more properties of the meditopes, e.g., affinity, are altered by increases, as supported by ligand docking calculations. Such incorporating a different natural amino acid, or a non-natural modifications may be incorporated by using an N-alkyl amino acid at one or both of these positions, e.g., thereby guanidinium group, or an alkyl-amidine functionality. In changing the amount of surface area that can be extended. In either case, the substituted group of the terminal N-atom can one embodiment, natural amino acids (Phe/Tyr/Trp) and non be alkyl or aryl, wherein each position of the alkyl or aryl natural analogs (e.g., β,β'-diphenyl-L-alanine, or amino acids group may be optionally substituted with additional function incorporating side chains that include branched alkyl, alities within the group including the terminal position. In one extended aromatics such as napthyl, or other hydrophobic example, a modified Arginine (modified Arg8), having a groups) are systematically introduced via SPPS at one or both structure as shown in FIG. 34, is substituted for Arg8 of the positions. meditope, e.g., of SEQ ID NO: I or 2 with the butyl group on [0319] “Hydratable” Functionality the NH2 (shown in FIG. 34 as NHR). In some aspects, the [0320] In certain embodiments, one or more Fab hydroxyl variant meditope contains an n-butyl-arginine or butylami- bearing side chains surrounding the meditope-binding site of dine modification at position 8. a meditope-enabled antibody is/are exploited through selec [0313] Position 3 tive trapping, by formation of a covalent bond with a medi [0314] In some embodiments, the meditope variants con tope that incorporates a hydratable functionality. Thus, in tain a modification in the position corresponding to position some embodiments, the meditope contains one or more resi 3, such as Phe3 of meditope I. As shown herein with struc dues with hydratable substituents, e.g., in order to create a tural data, the hydroxyl group of the meditope variant highly selective but irreversible interaction with a meditope- US 2012/0301400 Al Nov. 29, 2012 25

enabled antibody or other substance. For example, Arg8 of acid group in one residue with an amine group in another the meditope I extends in proximity to Ser43 of the light residue; (vi) an ester bond made by reaction of an acid group chain (3.5 A) and Tyr91 (3.8 A) of the meditope-enabled in one residue with an alcohol in another residue, such as a antibody heavy chain, according to Kabat numbering (FIG. serine; (vii) a double bond, made by reaction of two residues 36, left panel). Incorporation of a hydratable functionality at each containing a terminal olefin, e.g., by olefin metathesis the end of Arg8 or LeulO of the meditope would allow for (e.g., reaction of two allylglycine residues or two N-allyl selective formation of a serine or tyrosine hemi-acetal. Such substituted amino acids), or (viii) by reaction of any other pair a covalent adduct would essentially afford irreversible bind of suitable residues known in the art. For a review, see, for ing. In addition, residues containing boronic acid may also be example, Davies, J. S., “The Cyclization of Peptides and integrated into the meditope as a hydratable group. Boronic Depsipeptides,” J. Peptide Sci. 2003, 9, 471-501. In one acid plays an important role in the structural activity of embodiment, the meditope may direct a reactive group to an bortezamib (Velcade®), which is used to treat multiple unnatural amino acid incorporated into the Fab, such as myeloma. Further representative examples of hydratable resi p-acetylphenylalanine. dues are also shown in FIG. 34, where R=—CFi2CFiO or [0325] Various methods for cyclization of a meditope may —CFi2B(OFi)2. In some examples, such variants are prepared be used, e.g., to address in vivo stability and to enable by incorporating residues containing such groups using chemoselective control for subsequent conjugation chemis SPPS. try. In some embodiments, the cyclization strategy is a lactam [0321] Such hydratable strategies can be applied to engi cyclization strategy, including head-to-tail (head-tail) lactam neering new amino acid residues within the antibody Fab- cyclization (between the terminal residues of the acyclic pep meditope binding site and introducing the requisite comple tide) and/or lactam linkage between other residues. Lactam mentary “hydratable” functionalities within different formation may also be effected by incorporating residues positions of the meditope. A similar strategy can be applied such as glycine, β-Ala, 7-aminoheptanoic acid, and the like, by introducing cysteine residues into the Fab region and use into the acyclic meditope cyclization precursors to produce this functionality for nucleophilic attack on a “hydratable” different lactam ring sizes and modes of connectivity. Addi functionality such as an electrophilic carbonyl group or tional cyclization strategies such as “click” chemistry and derivative thereof contained within the meditope or for mak olefin metathesis also can be used (see FIG. 31, right boxes). ing selective disulfide bonds (—S—S—) between the Fab Such methods of peptide and peptidomimetic cyclization are region and the meditope containing the requisite thiol or thiol well known in the art. equivalent functionalities. Other embodiments of this idea [0326] In some embodiments, the meditopes containing would include introducing Michael acceptors contained in lactam linkages are more stable, in vivo, e.g., have a linkage the meditope such as α,β-unsaturated carbonyl functional that is more stable in vivo compared to meditopes with other ities. These functionalities are well-known to selectively react linkages. with thiols to form stable covalent carbon-sulfur bonds. [0327] In some embodiments, the terminal residues of an [0322] Alternative Cyclization Strategies and Replacement acyclic peptide are reacted to form a cyclic meditope, e.g., of Disulfide Bridge cyclic meditope variant. In other embodiments, other posi [0323] In certain embodiments, the variant meditopes tions are amenable to cyclization, including between residues include a disulfide bridge, as in cQFD and cQYN. Disulfide 3 and 11 and 4 and 11. Thus, in some aspects, the meditopes bridges may be formed by the reaction of the side chains of contain a linkage formed between residues other than the two cysteine residues. In certain embodiments, the disulfide N-terminal and C-terminal residues, such as between resi bridge in a meditope, e.g., meditope I or 2, is replaced with an dues 3 and 11 and/or 4 and 11, e.g., of a 12-amino acid alternative linkage or is removed. Thus, among the variant peptide. meditopes are those having alternative linkages or lacking the [0328] In some embodiments, the meditopes, e.g., variant disulfide bridge of the original meditopes. meditopes, contain a reactive amine functionality (e.g., [0324] In some aspects, the linkage is made between one or Lysl I), which can be used for subsequent conjugation of the more unnatural amino acids within the amino acid chain. meditope variant, e.g., to a scaffold or linker or to an agent, Examples of linkages that may be made with unnatural amino such as a diagnostic, e.g., imaging, agent or therapeutic agent acids include linkages comprising (i) stable hydrazone or as described herein. For example, FIG. 13 shows a procedure oxime-based linkages made by reaction of a residue compris for conjugation of a meditope variant with fluorescein for ing an aldehyde or ketone with a residue comprising an amine FACS analysis; this strategy can be applied to other imaging group, where the amine nitrogen is substituted with —NF12 or and other agents, including DOTA for in vivo PET imaging. alkyloxy group (e.g., reaction of a p-acetylphenylalanine, m-acetylphenylalanine, or p-(3-oxobutanoyl)-L-phenylala- [0329] In some embodiments, thiol functionalities can be nine residue with a p-(2-amino-3-hydroxyethyl)-phenylala- introduced in any suitable position on the meditope and can nine residue), (ii) thiol reactive by incorporating phenylse- be selectively modified using a number of external reagents lenidylalanine, (iii) a UV crosslinker containing containing imagining agents, other proteins and peptides, benzophenone by incorporating p-benzoyl-L-phenylalanine, metal chelators, siRNAs, nanoparticles, and cytotoxic drugs. (iv) amine reactive by incorporating p-isopropylthiocarbo- [0330] Characterization of Meditopes nyl-phenylalanine or p-ethylthiocarbonyl-phenylalanine, (v) [0331] In some embodiments, the meditopes, such as vari heterocyclic linkages, such as a triazine, thiazole, thiazoli- ant meditopes, are characterized, for example, by ITC, SPR dine, or oxazole linkage, made, for example, by reaction of a and/or diffraction and/or other methods, such as those residue containing an azide group with a residue containing described herein in the Examples. In one example, the syn an alkyne group via Fluisgen cycloaddition (e.g., reaction of a thesis of meditopes and characterization thereof is carried out p-propargyloxyphenylalanine residue with a p-azidopheny- in an iterative fashion, e.g., such that the meditope variant lalanine residue); (v) an amide bond made by reaction of an with the most desirable property, e.g., highest affinity for one US 2012/0301400 Al Nov. 29, 2012 26

or more meditope-enabled antibodies or other desired prop ments, the meMAb-meditope interaction is tailored with erty, such as pH dependence, is subsequently modified to respect to pH. Examples of such meditopes are described improve the desired property. herein. In some aspects, the binding affinities of the meditope [0332] In one example, for characterization of binding of variants are measured as a function of buffer pH. For meditopes to meditope-enabled antibodies, the meditope is example, variant meditopes include meditopes with a binding purified to >95% homogeneity and structurally characterized affinity for one or more meditope-enabled antibody or frag by mass spectrometry. Peptides are dialyzed in water, their ment that is decreased at a lysosomal pH level or is increased concentrations measured by UV-Vis and calibrated with in a hypoxic environment. For example, provided are variant elemental analysis, and diluted (>100—X) into the appro meditopes that exhibit higher binding affinity for a meditope- priate buffer. Binding to a meditope-enabled antibody is rig enabled antibody at a neutral pH (e.g., pH 7-8, e.g., pH orously characterized by ITC, SPR, X-ray diffraction, or a 7.3-7.5) and exhibit a relatively lower binding affinity for the combination thereof. ITC measurements may be performed antibody at a more acidic pH, such as a pH between at or about on a TA Instruments nanoITC, with only I -2 mg of peptide 4 and 6.5, e.g., at an endosomal pH (e.g., between at or about per measurement. In one example, for SPR measurements, 5 and at or about 6.5) or at a lysosomal pH (e.g., between at or low density and high density chips are conjugated with a about 4.5 and at or about 5). See FIG. 27, showing several meditope-enabled antibody, e.g., a Fab or a full IgG. In some examples. In another example, the meditopes have increased cases, the chips are first characterized using an antigen, such binding affinity for the antibody in a hypoxic environment, as a soluble fragment of the entire extracellular domain of such as a tumor environment, for example, as compared to the EGFR (residues 1-621) in the case of cetuximab. In a study affinity observed under other physiological conditions, such reported herein, the cQFD meditope bound with similar as in the blood. In some embodiments, modification of medi enthalpy and entropy to the cetuximab Fab fragment as com tope variants or meditope “analogs” for the specific release at pared to the fully intact cetuximab IgG, e.g., as measured by low pHs (e.g., in lysosomes for drug delivery) is provided; SPR and ITC. Accordingly, binding measurements may be and modification of meditopes so that they bind with higher carried out using the full IgG or Fab fragment of cetuximab or affinity in a hypoxic environment (e.g., tumor stroma pH is other meditope-enabled antibody. In one example, fordiffrac- often lower than normal tissues). Also provided are methods tion, the co-crystallization conditions of the cetuximab Fab for generating such meditope variants. fragment and the meditope of SEQ ID NO: I are well-estab [0336] According to some embodiments, the meditope lished and diffraction quality crystals are typically obtained binding site may be exploited or optimized to enhance bind in I to 3 days, typically I day. Full data sets are collected in 8 ing of, purification of, and/or imaging or other method using to 12 hours with an in-house source (Rigaku 007-HF and an the meditope-enabled antibodies and functional fragments R-Axis IV++) and in 10 min at the Stanford Synchrotron thereof. In a separate embodiment, a meditope may contain Radiation Lightsource, which allows for rapid characteriza one or more cysteine residue that binds to one or more engi tion of the interactions of the meditope variants with medi neered cysteine in the Fab at the meditope binding site (e.g., tope-enabled antibodies. ThioMAbs). The meditope is thereby conjugated to any diag [0333] In some aspects, ITC, SPR and X-ray diffraction nostic and/or therapeutic substance, molecule or compound. data, e.g., collectively, provide atomic detail to guide subse For example, the substance may be a small molecule diag quent chemical modifications and ultimately improve the nostic molecule, such as a marker. The “Cys meditope” affinity of the meditopes and/or make other alterations to the directs the conjugate to the antibody and binds via a covalent meditopes. A calculation based on AG==-RT In Ka shows linkage. Alternatively, the meditope may be conjugated to the that the difference between micromolar and nanomolar affin Fab to one or more unnatural amino acids that are incorpo ity of a meditope for cetuximab results from a change in free rated into the meditope binding site. energy at 300 K of -4 kCal/mol, which is on the order of a [0337] II. Multivalent Meditopes strong hydrogen bond. Thus, the loss of an ordered water [0338] Also among the provided meditopes are multivalent molecule from a protein binding pocket or the reorientation of meditopes. In certain aspects, conjugation of a meditope, to a an amino acid residue-chain may be sufficient to alter binding linker or scaffold (e.g., to form a multivalent tethering entity), by orders of magnitude. significantly improves overall affinity for and targeting to a [0334] In some examples, other approaches are used to alter meditope-enabled mAb bound to an antigen, e.g., a tumor- properties of the meditopes, e.g., to improve the affinity of the associated antigen. meditope-Fab interaction. In one example, structural data, [0339] Full-length monoclonal antibodies (mAbs) and such as those obtained in the studies described above, may be F(Ab)'2 fragments include two Fab domains (in the case of used to replace residues in the Fab, by mutagenesis, for full-length mAbs, coupled to a dimeric Fe). Such bivalent example, to add additional hydrogen bonds, substitute amino antibodies (e.g., IgGs) preferentially bind to cells expressing acids for unnatural amino acids or alter the hydrophobic antigen at high densities. Combinations of monoclonal anti interface, for example, in ways that might better complement bodies (mAbs) that recognize unique epitopes on the same meditope binding. In some examples, fluorescence polariza antigen can produce synergistic effects, including enhance tion assays are used to identify meditope variants that can ment of various antibody effector functions (e.g., ADCC, displace a given meditope, such as SEQ ID NO: I. In other complement-dependent lysis, signaling inhibition), enhance examples, the same technique is used to identify small mol ment of cell death, and in the case of cancer-targeting anti ecules that can displace the meditope and then use these small bodies, enhancement of tumor growth inhibition. See, for molecules as templates to further improve the binding affinity example, Dechant M et ah, “Complement-dependent tumor of the meditopes. cell lysis triggered by combinations of epidermal growth [0335] In some examples, the meditope variants are factor receptor antibodies,” Cancer Res, 2008 Jul. I; 68(13): designed based on pH dependence, e.g., to have differing 4998-5003; Scheuer W et ah, “Strongly enhanced antitumor affinities at different pH conditions. Thus, in certain embodi activity of trastuzumab and pertuzumab combination treat US 2012/0301400 Al Nov. 29, 2012 27

ment on HER2-positive human xenograft tumor models,” peptide linker, e.g., a flexible peptide linker. In one example, Cancer Res, 2009 Dec. 15; 69(24):9330-6; Cardarelli P M et the length of the linker is chosen to roughly match the dis al., “Binding to CD20 by anti-Bl antibody or F(ab')(2) is tance between the Fabs of an IgG, such as of 17 amino acids sufficient for induction of apoptosis in B-cell lines,” Cancer in length. In some aspects, the linker contains glycines, ImmunolImmunother, 2002 March; 51(1):15-24. Epub 2001 serines, and/or a combination thereof. Exemplary “meditope- Dec. 18. While the precise mechanism of this enhanced cell Fc” fusions are shown in FIG. 15 (SEQ ID NO: 3 and 4, death remains debated, studies indicate that both mAbs respectively). See also FIG. 16. should be multivalent (e.g., full antibodies or F(ab)'2) to [0343] In some embodiments, the composition of the linker achieve enhanced effects, suggesting that the second bivalent and/or the distance between the Fe and the meditope is sys mAh (which binds to a unique epitope on the same antigen) tematically altered, e.g., to optimize affinity and specificity. can cluster cell surface antigens and act more efficiently, e.g., In one embodiment, each natural or unnatural residue can be more efficiently kill tumor cells. See FIGS. 8 and 16. substituted at any position within the linker for optimization. [0340] In some embodiments herein, such clustering is In some aspects, the linker is between at or about 2 and 100 recapitulated using multivalent meditopes. In some aspects, residues in length, e.g., at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, the multivalent meditope used instead of a second antibody, in 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28, some aspects to achieve synergy in combination with a medi- 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, tope-enabled antibody. In some aspects, the multivalent 125, 150, 175, or 200 residues (amino acids) in length, or meditopes provide advantages compared to use of a second more. In one example, such linkers are generated using cur antibody recognizing a separate epitope. For example, pro rent and future DNA synthesizers, and inserted between the duction of the multivalent meditopes can be more efficient, meditope and the Fe regions. The linker may also be ‘rigidi- and cost-effective when compared to a second antibody. For fied’ to limit the radius of gyration and to enhance the affinity example, although a number of preclinical/clinical trials are and specificity of the Fc-meditope. For example, a coiled coil investigating the co-administration of two monoclonal anti domain may be placed between the meditope and the Fe (FIG. bodies, the costs of producing and marketing such a thera 18). In other examples, inert protein domains (e.g., immuno peutic is likely to be prohibitive. In some aspects, the multi globulin folds) are substituted for the linker. Multiple immu valent meditopes also are comparatively more easily targeted noglobulin folds can be placed between the meditope and the to disease sites, such as tumors. Given the nature of the Fe domain. In certain embodiments, the composition of the meditope binding site (within the meditope binding site, sepa linker is of human origin to mitigate potential antigenicity. rate from the antibody CDRs), and the broadly applicable [0344] In some examples, the provided meditopes are teth methods provided herein for meditope-enabling any antibody ered to a scaffold to create a multivalent meditope, e.g., for of choice, the multivalent meditopes also have the advantage enhanced selectivity and binding affinity. In some embodi of being readily applicable to a large range of therapeutic ments, multivalent meditope scaffolds are used to scaffold or antibodies, without the need to identify a second antibody or “daisy-chain” meditope-enabled mAbs bound to tumor asso epitope with therapeutically acceptable characteristics. Thus, ciated antigen to enhance ligand antagonism, alter receptor in some aspects, use of a multivalent meditope avoids the endocytosis, and/or improve an immune response through need to identify a second mAh with acceptable characteris ADCC/CDC (see FIG. 8). Thus, in some embodiments, the tics, and the associated and significant cost of its development meditopes are used to tether two or more antibodies or func thereof. tional fragments thereof. Such meditopes may be part of a [0341] Specificity and affinity are often achieved through multivalent tethering agent, for example, to enhance cancer or multivalency. For a bivalent ligand with a linker, this can be tumor therapy and imaging. A multivalent tethering agent expressed as AGrotoZ=AGl+AG2-AGftnfer. In other words, may include two or more meditopes coupled through linker Krotoz=Ki *K2/Kftnfer. In the case where the linker makes no (s), such as through a long linker and biotin to streptavidin, to contribution to the free energy (Kftnfer-I), the apparent affin create a multivalent meditope tethering entity. ity of the bivalent ligand for the bivalent taiget is the product [0345] In one embodiment, the multivalent meditope teth of the monomeric binding constants. Thus, significant gains ering entity is a tetravalent meditope tethering agent. In one in affinity can be achieved through multivalency in general aspect, the tetrameric tethering entity is shown by surface (e.g., for a meditope with Kri=I μΜ the affinity of a ‘theo plasmon resonance to have enhanced binding to an antibody retical’bivalent meditope is I pM). Whilesuchlargegainsare as compared to the monovalent peptide, which is consistent rarely seen in general (primarily due to the geometry of biva- with a multivalent interaction. In one example, use of such lent/trivalent/multivalent receptors), synergy is observed. multivalent meditopes (e.g., tetravalent meditope) produces The geometry of a cell surface-expressed antigen can place enhanced binding affinity of the meditope-enabled antibody strict constraints on the linker of a multivalent meditope, but to the antigen (e.g., in the case of cetuximab, binding affinity can also ensure specificity, which can be an important goal in for EGFR positive cells), compared to use of the antibody some contexts, such as for targeted delivery, e.g., by mini alone or monovalent meditope alone. Such binding affinities mizing the risk of off-target effects. can be determined using well-known methods, e.g., by FACS [0342] In one example, the multivalent meditope contains analysis. See FIG. 12. an Fe region of an antibody or portion thereof, such as sub [0346] In some embodiments, to address the receptor con stantially all of the Fe region. The use of the Fe region to straints on the linker, unmodified or modified, e.g., variant, ‘dimerize’ ligands is established and described, for example, e.g., optimized, meditopes, such as those obtained as by Jazayeri J A & Carroll G J., “Fc-based cytokines: prospects described in Example 6, are coupled to a multivalent scaffold, for engineering superior therapeutics,” BioDrugs, 22(1):11- such as by optimizing the linker. In some aspects, the multi 26 (2008) In some examples, to generate a bivalent meditope, valent meditope is able to “latch-on” to adjacent antibodies, the meditope is fused to an Fe region (e.g., to the N-terminus e.g., IgGs, to form a “daisy-chain”-like array (see FIG. 8), of the Fe region of an IgG) through a linker, typically a which can be used, for example, in antibodies targeting tumor US 2012/0301400 Al Nov. 29, 2012 28

antigens, given the high antigen density of tumor cells. While example, to ensure that conjugation to the multivalent scaf an intramolecular association of a bivalent meditope and anti fold does not affect the meditope-Ig interaction. In some body is possible, the C2 symmetry of the antibody, e.g., IgG, cases, such measurements can be limited in their ability to can place severe geometrical constraints on the linker for such determine mutlivalency and synergistic effects, given that an interaction. Thus, in some aspects, the meditope is based these approaches generally do not involve antigen present on on a trivalent or higher valency scaffold, ensuring that more a cell surface (such as on the surface of a tumor cell). Thus, in than one antibody would be “daisy chained”. By including a some aspects, FACS analysis and/or cell viability assays are third meditope arm, the lifetime of the initial encounter of a used to quantify the effect of the multivalent meditope trivalent meditope to antigen-bound antibody can increase. directly on cells expressing antigen recognized by the medi This, in turn, can increase the probability that an additional tope-enabled antibody (e.g., cells that overexpress EGFR in arm will bind to a neighboring antigen-bound antibody, thus the context of cetuximab). Exemplary protocols are described stabilizing the overall complex. In other embodiments, a mul in Example 7. In general, a cell line expressing (e.g., over tifunctional meditope may be constructed to simultaneously expressing) the antigen recognized by the meditope-enabled bind a memab and other targets, such as other B and T-cell antibody is incubated with the meditope-enabled antibody surface proteins, such as T cell receptor and costimulatory under conditions whereby the antibody binds to the antigen molecules. In some embodiments, multiple meditope-en- expressed on the cells. In some cases, varying concentrations abled antibodies (for example, including one or more modi of the antibody are used. Either simultaneously or subse fied meditope-enabled antibodies) having specificities for quently, the cells are incubated with the multivalent medi different meditopes are used together with such different tope, in some cases in varying concentrations. Appropriate meditopes in such multivalent embodiments. incubation times and washes are carried out. A second anti [0347] Various linkers and scaffolds are known in the art body and monovalent meditopes may be used as positive and and may be used in connection with these embodiments. An negative controls, respectively. The antibodies and meditopes exemplary scaffold synthesis scheme is shown in FIG. 13 and may be labeled with agents detectable by flow cytometry or discussed in the Examples herein. In some aspects, the use of microscopy, which are well known. In some examples, a shift templates 4 and 5 shown in FIG. 13 allows for the formation (in the case of FACS) or increased signal in the presence of of both bi- and trivalent meditopes, respectively. In some multivalent meditope, compared to monovalent meditope, embodiments, different length polyethylene glycol (PEG) indicates a synergistic or additive effect. In another example, and/or other linkers are used, for example, to improve or alter to further confirm the additive effects of the multivalent medi binding or other properties. In some aspects, the PEG length tope, the non-labeled, monovalent meditope is used in a com is between at or about IOA and at or about 1000 A. The petition assay, to determine whether it can compete with the synthetic approach is also amenable to DOTA incorporation labeled multivalent meditope for the antigen-bound medi for radionuclide imaging. For example, a 30 A PEG bifunc tope-enabled antibody. tional arm has been incorporated in the synthesis of a FITC- [0351] In other examples, cell viability assays are used to labeled divalent meditope, namely compound 13 (FIG. 13). determine the ability of a multivalent meditope to enhance The distance between the CDR regions within an IgG is -130 cell-killing effects of a meditope-enabled antibody. For A. The length of the PEG linker may be systematically varied example, a cell expressing the antigen of interest may be to ensure this approach is optimal. End-to-end distances of incubated with varying concentrations of the meditope-en commercially available PEGs extend to 90 A (Pierce), which abled antibody and/or multivalent meditope. Monovalent would exceed the IgG distance. meditopes and second antibodies again are useful as controls. [0348] In other embodiments, other scaffolds and/or linkers In some examples, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- are used, e.g., to generate high affinity multivalent meditopes diphenyltetrazolium bromide), is used to quantify the number and/or to create synergy. For example, DNA may be used as or percentage of viable cells. Other approaches for measuring a scaffold for the meditopes to create a more rigid scaffold. cell viability, proliferation, and/or death are known in the art For example, different scaffolds of biological and chemical and may be used. origin may also be used to achieve multivalency. This [0352] In another example, for multivalent meditopes that includes, but is not limited to, constructing a bivalent or demonstrate activity in such assays, Western blot analysis or trivalent scaffold, using streptavidin or (see European Appli other biochemical or signaling approach is performed to cation, Publication No.: EP 2065402 Al), strepavidin as a investigate inhibition of signaling associated with a particular tetravalent scaffold, unique scaffolds (http://www.sciencedi- antigen, such as a cell surface receptor (e.g., in the case of rect.com/science/article/pii/S0022283611000283), Origami cetuximab, to follow the phosphorylation status of EGFR, DNA (see Gu et ah, Nature Nanotechnology 4, 245-248 AKT, MAP which are part of the EGFR signaling pathway). (2009)) and the like. A chemical scaffold may also be created Data from such studies may be compared with data from cells using molecules including, but not limited to, DNA (single treated only with meditope-enabled antibody (i.e., without strand, duplex, Holliday junctions, aptamers and the like), meditope), with monovalent meditopes, and/or with tyrosine RNA (single strand, hairpin, stem loop, aptamers and the kinase or other known inhibitors (AG1478, etc.). In some like), PNA (peptide nucleic acids), DNA/PNA duplexes and examples, an increase in cell death as a function of multiva triplexes for rigidity, organic and or inorganic nanoparticles lent meditope concentration is observed, demonstrating syn (directly coupled or coupled through organic polymers such ergistic cell killing effects of the multivalent meditopes. as PEG), organic polymers that can form duplexes with them [0353] III. Fusion Proteins selves and/or with DNA or PNA. [0354] Also provided are fusion proteins containing one or [0349] Characterization of Multivalent Meditopes more meditopes. As demonstrated herein by diffraction on the [0350] Binding properties of the multivalent meditopes to meditope-enabled Fab fragment bound to a cQFD meditope meditope-enabled antibodies can be characterized by any of a (meditope I), the N- and C-termini of that antibody-bound number of known methods, including SPR and !TC, for meditope were shown to be juxtaposed to the location of US 2012/0301400 Al Nov. 29, 2012 29

bound Protein L, a bacterial protein that binds to human sequence of exemplary meditope-Protein L fusion proteins antibodies, including IgMs, IgDs, IgGs, IgEs, and IgAs. In are set forth in SEQ ID NO: 57 (His6-Smt3-meditope-Pro- some embodiments, provided are meditope-Protein L fusion teinL) and SEQ ID NO: 58 (containing two cysteins, set forth proteins (MPLs). In some aspects, the Protein L-meditope in bold text, which in some aspects are used to cyclize the fusions exhibit binding to meditope-enabled antibodies with MPL, e.g., by peroxide or overnight with air: greater affinity as compared to meditopes alone and/or Pro tein alone, e.g., via energy additivity. The MPLs also can be advantageous compared to Protein L alone and other multi (SEQ ID NO: 58) valent conjugates of Protein L, Protein A, and/or similar SCOFPT iSTFFTiKCGGGGSEVTTKVNTiTFAPGKTOTAEFKGTFEEATAE

antibody-binding proteins, in that they provide specificity for AYRYAALLAKVNGEYTADLEDGGNHMNIKFAG . meditope-enabled antibodies and will not target endogenous immunoglobulin molecules when administered therapeuti [0359] In some embodiments, the meditope-Protein L cally. (MPL) construct exhibits improved binding affinity for a [0355] Originally isolated from bacterium Peptostrepto- meditope-enabled antibody as compared to meditope alone. coccus magnus, Protein Lisa protein that binds to the kappa For example, the improvement can be an at least or an at or light chain of human and other antibodies. Exemplary medi about 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, tope-Protein L (MPL) fusion proteins include those in which 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, a provided meditope is coupled to a Protein L, which can be 80, 70, 60, 50, 40, 30, 20, or 10-fold improvement in affinity, wild-type Protein L or a variant thereof, such as one of several e.g., in dissociation constant, for the meditope-enabled anti variants known in the art, or a variant modified to improve one body as compared with the corresponding meditope alone or more property, such as to reduce antigenicity. Methods for and/or with the Protein L alone. In one example, the binding modifying similar proteins, such as to reduce antigenicity or constants for interactions between Protein L and the meditope immunogenicity, or to improve “immune tolerance,” are alone with the meditope-enabled antibody are approximately known in the art and described, for example, by Ahlgren et al., 0.5 μΜ and I μΜ, respectively, whereas the binding constant JNudMed, 2010; 51:1131-1138; Feldwisch J et ah, JMol for the MPL to the antibody is approximately 165 pM. Biol, 2010 Apr. 30; 398(2):232-47. Epub 2010 Mar. 10. For [0360] IV. Meditope-Agent Conjugates example, amino acid substitutions can be made, e.g., in an [0361] In certain embodiments, the meditopes, including iterative process, randomly or based on structural modeling, one or more meditope, meditope variants, multivalent medi following by analysis of the desired property, e.g., antigenic topes, meditope-Protein L fusions (MPLs), multivalent teth ity. In another example, mutations are introduced to improve ering agents or multivalent meditope variant tethering agents specificity. In other embodiments, the meditopes are fused are conjugated to one or more therapeutic or diagnostic agent, with other antibody-binding substances, such as other anti e.g., imaging agents, therapeutically effective agents or com body-binding proteins, for example, Protein A or Protein G. pounds in therapeutically effective amounts or both. Provided [0356] The MPLs generally include a linker, linking the are such complexes containing the meditope and one or more meditope portion to the Protein L portion. The linker typically agent. includes at least 2, more typically at least 3, e.g., 3, 4, 5, 6, 7, [0362] In some aspects, the binding of the meditopes or 8, 9, or 10 amino acid residues (e.g., four glycines). In some variants thereof to one or more meditope-enabled antibody aspects, such residues link the C-terminus of the meditope conjugated to a therapeutic or diagnostic (e.g., imaging) agent and the N-terminus of Protein L. In one example, the medi or compound is used to treat, prevent, diagnose or monitor a tope-Protein L fusion protein has the sequence set forth in disease or condition. In one aspect, such conjugation of a SEQ ID NO: 58(SCQFDLSTRRLKCGGGGSEV meditope, such as a multivalent meditope, to an agent, when TI KVNLIFADGKIQ TA EFKGTFE EATA EA used in connection with meditope-enabled monoclonal anti YRYAALLAKVNGEYTADLEDGGNH MN I bodies, provides a highly versatile platform technology that K F A G). will significantly improve mAh based therapeutics and imag [0357] In some embodiments, the MPLs are used in con ing methods to treat and detect disease (see FIG. 8). nection with meditope-enabled antibodies, in the methods [0363] The diagnostic and therapeutic agents include any and uses described herein, including those involving the con such agent, which are well-known in the relevant art. Among jugation of the meditopes to agents, such as therapeutic or the imaging agents are fluorescent and luminescent sub diagnostic agents, such as cytotoxins, radionuclides, DOTA, stances, including, but not limited to, a variety of organic or proteins or other biological molecules, e.g., to taiget disease inorganic small molecules commonly referred to as “dyes,” and/or image disease sites. In some aspects, to facilitate such “labels,” or “indicators.” Examples include fluorescein, conjugation, one or more lysines in the Protein L fusion are rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. mutated, for example, to arginine or asparagine, to generate a Enzymes that may be used as imaging agents in accordance Protein L fusion protein that can be specifically conjugated to with the embodiments of the disclosure include, but are not other molecules, for example, by leaving the N-terminal limited to, horseradish peroxidase, alkaline phosphatase, acid amine and the epsilon amine available for conjugation, the phoshatase, glucose oxidase, β-galactosidase, β-glucoron- latterbeing solvent exposed and more reactive. An exemplary idase or β-lactamase. Such enzymes may be used in combi fusion protein has the sequence set forth in SEQ ID NO: 59 (S nation with a chromogen, a fluorogenic compound or a Iumi- CQFDLSTRRLRCGGGGSEVTI RV NLIFA nogenic compound to generate a detectable signal. DGNIQTAEFRGTF EEATAEAYRYAALLA [0364] Radioactive substances that may be used as imaging RVNGEYTADLEDGGNHMNIKFAG), in which agents in accordance with the embodiments of the disclosure all lysines but one have been removed. include, but are not limited to, 18F, 32P, 33P, 45Ti, 47Sc, 52Fe, [0358] The coding sequence of an exemplary meditope- 59Fe, 62Cu, 64Cu, 67Ga, 67Ga, 68Ga, 77As, 86Y, 90Y 89Sr, 89Zr, Protein L fusion protein is set forth in SEQ ID NO: 56. The 94Tc, 94Tc, 99mTc, "Mo, 105Pd, 105Rh, IllAg, 111In, 123I, US 2012/0301400 Al Nov. 29, 2012 30

124I5 I2Sj5 I3Ij5 I42Pf5 I43pf5 I49pnf5 I53gm5 I54-I58Qd5 ISlxb5 nine, actinomycin-D, adriamycin, aldesleukin, alemtu- 166Dy, 166Ho, 169Er, 175Lu, 177Lu, 186Re, 188Re, 189Re, 194Ir, zumab, alitretinoin, all-transretinoic acid, alpha interferon, 198Au, 199Au, 211At, 211Pb, 212Bi, 212Pb, 213Bi, 223Ra and altretamine, amethopterin, amifostine, anagrelide, anastro- 225Ac. Paramagnetic ions that may be used as additional zole, arabinosylcytosine, arsenic trioxide, amsacrine, ami- imaging agents in accordance with the embodiments of the nocamptothecin, aminoglutethimide, asparaginase, azacyti- disclosure include, but are not limited to, ions of transition dine, bacillus calmette-guerin (BCG), bendamustine, and lanthanide metals (e.g. metals having atomic numbers of bevacizumab, etanercept, bexarotene, bicalutamide, bort- 21-29, 42, 43, 44, or 57-71). Thesemetals include ions of Cr, ezomib, bleomycin, busulfan, calcium leucovorin, citro- V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, vorum factor, capecitabine, canertinib, carboplatin, carmus- Dy, Ho, Er, Tm, Yb and Lu. tine, cetuximab, chlorambucil, cisplatin, cladribine, [0365] When the imaging agent is a radioactive metal or cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, paramagnetic ion, the agent may be reacted with another dasatinib, daunomycin, decitabine, denileukin diftitox, dex- long-tailed reagent having a long tail with one or more chelat amethasone, dexasone, dexrazoxane, dactinomycin, dauno- ing groups attached to the long tail for binding to these ions. rubicin, decarbazine, docetaxel, doxorubicin, doxifluridine, The long tail may be a polymer such as a polylysine, polysac eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, charide, or other derivatized or derivatizable chain having exemestane, estramustine, etoposide, filgrastim, fluoxymes- pendant groups to which the metals or ions may be added for terone, fulvestrant, flavopiridol, floxuridine, fludarabine, binding. Examples of chelating groups that may be used fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab according to the disclosure include, but are not limited to, ozogamicin, goserelin, granulocyte-colony stimulating fac ethylenediaminetetraacetic acid (EDTA), diethylenetri- tor, granulocyte macrophage-colony stimulating factor, hex- aminepentaacetic acid (DTPA), DOTA, ΝΟΤΑ, ΝΕΤΑ, amethylmelamine, hydrocortisone hydroxyurea, ibritumo- TETA, porphyrins, polyamines, crown ethers, bis-thiosemi- mab, interferon alpha, interleukin-2, interleukin-11, carbazones, polyoximes, and like groups. The chelate is nor isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifos- mally linked to the PSMA antibody or functional antibody famide, irinotecan, lapatinib, lenalidomide, letrozole, leuco fragment by a group, which enables the formation of a bond vorin, leuprolide, liposomal Ara-C, lomustine, mechlore- to the molecule with minimal loss of immunoreactivity and thamine, megestrol, melphalan, mercaptopurine, mesna, minimal aggregation and/or internal cross-linking. The same methotrexate, methylprednisolone, mitomycin C, mitotane, chelates, when complexed with non-radioactive metals, such mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, as manganese, iron and gadolinium are useful for MRI, when oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitu- used along with the antibodies and carriers described herein. mumab, PEG Interferon, pegaspargase, pegfilgrastim, PEG- Macrocyclic chelates such as ΝΟΤΑ, DOTA, and TETA are of L-asparaginase, pentostatin, plicamycin, prednisolone, pred use with a variety of metals and radiometals including, but not nisone, procarbazine, raloxifene, rituximab, romiplostim, limited to, radionuclides of gallium, yttrium and copper, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, respectively. Other ring-type chelates such as macrocyclic sunitinib, semustine, streptozocin, tamoxifen, tegafur, polyethers, which are of interest for stably binding nuclides, tegafur-uracil, temsirolimus, temozolamide, teniposide, tha such as 223Ra for RAIT may be used. In certain embodiments, lidomide, thioguanine, thiotepa, topotecan, toremifene, tosi- chelating moieties may be used to attach a PET imaging tumomab, trastuzumab, tretinoin, trimitrexate, alrubicin, vin agent, such as an Al—18F complex, to a targeting molecule cristine, vinblastine, vindestine, vinorelbine, vorinostat, or for use in PET analysis. zoledronic acid. [0366] Exemplary therapeutic agents include, but are not [0368] Therapeutic antibodies and functional fragments limited to, drugs, chemotherapeutic agents, therapeutic anti thereof, that may be used as therapeutic agents in accordance bodies and antibody fragments, toxins, radioisotopes, with the embodiments of the disclosure include, but are not enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic limited to, alemtuzumab, bevacizumab, cetuximab, edreco- agent at the site of the tumor), nucleases, hormones, immu- lomab, gemtuzumab, ibritumomab tiuxetan, panitumumab, nomodulators, antisense oligonucleotides, RNAi molecules rituximab, tositumomab, and trastuzumab and other antibod (e.g., siRNA or shRNA), chelators, boron compounds, pho ies associated with specific diseases listed herein. toactive agents and dyes. The therapeutic agent may also [0369] Toxins that may be used as therapeutic agents in include a metal, metal alloy, intermetallic or core-shell nano accordance with the embodiments of the disclosure include, particle bound to a chelator that acts as a radiosensitizer to but are not limited to, ricin, abrin, ribonuclease (RNase), render the targeted cells more sensitive to radiation therapy as DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral compared to healthy cells. Further, the therapeutic agent may protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, include paramagnetic nanoparticles for MRI contrast agents and Pseudomonas endotoxin. (e.g., magnetite or Fe3O4) and may be used with other types [0370] Radioisotopes that may be used as therapeutic of therapies (e.g., photodynamic and hyperthermal therapies agents in accordance with the embodiments of the disclosure and imaging (e.g., fluorescent imaging (Au and CdSe)). include, but are not limited to, 32P, 89Sr, 90Y. 99mTc, "Mo, [0367] Chemotherapeutic agents are often cytotoxic or 131I, 153Sm, 177Lu, 186RE, 213Bi, 223Ra and 225Ac. cytostatic in nature and may include alkylating agents, anti metabolites, anti-tumor antibiotics, topoisomerase inhibitors, E. Methods of Use and Compositions mitotic inhibitors hormone therapy, targeted therapeutics and immunotherapeutics. In some embodiments the chemothera [0371] Also provided are methods and uses of the medi- peutic agents that may be used as therapeutic agents in accor topes, meditope-enabled antibodies, and complexes contain dance with the embodiments of the disclosure include, but are ing the same, including therapeutic and diagnostic uses, as not limited to, 13-cis-retinoic acid, 2-chlorodeoxyadenosine, well as other uses, including antibody purification. Also pro 5-azacitidine, 5-fluorouracil, 6-mercaptopurine, 6-thiogua- vided are pharmaceutical compositions containing the medi- US 2012/0301400 Al Nov. 29, 2012 31

topes (including variant and multivalent meditopes and medi- expressed by the mRNA component include luciferase, tope fusion proteins) for use in such diagnostic and aequorin and derivatives thereof. Numerous fluorescent and therapeutic methods. luminescent dyes and proteins are known in the art (see, e.g., [0372] I. Therapeutic and Diagnostic Uses and Pharmaceu U.S. Patent Application Publication 2004/0067503; Valeur, tical Compositions B., “Molecular Fluorescence: Principles and Applications,” [0373] In one embodiment, the specificity and binding of JohnWiley and Sons, 2002; Handbook of Fluorescent Probes the meditopes, meditope variants, multivalent meditopes, and Research Products, Molecular Probes, 9.sup.th edition, MPLs, multivalent meditope tethering agents and multivalent 2002; and The Handbook—A Guide to Fluorescent Probes meditope variant tethering agents, etc., are used to deliver and Labeling Technologies, Invitrogen, 10th edition, avail therapeutic agents, diagnostic agents (e.g., imaging agents), able at the Invitrogen web site; both of which are hereby or a combination thereof for treatment, diagnosis (e.g., imag incorporated by reference as if fully set forth herein.) ing) a disease or condition, typically when administered in [0378] In other aspects, the meditope administered in com combination with (either simultaneously or separately) one or more meditope-enabled monoclonal antibodies. bination with a meditope enabled antibody, the antibody- meditope complex, the multivalent tethering agent adminis [0374] In one example, meditopes, e.g., multivalent medi topes, are used for pre-targeted therapy or pre-taigeted imag tered in combination with a meditope enabled antibody, or a ing, as described further below, by administering a meditope- combination thereof may be further conjugated to or other enabled monoclonal antibody before administering the wise associated with a non-protein imaging agent or a deliv meditopes, meditope variants, multivalent meditope tethering ery vehicle such as a nanoparticle, radioactive substances agents or multivalent meditope variant tethering agents. Fur (e.g., radioisotopes, radionuclides, radiolabels or radiotrac ther, the use of multivalent meditopes can enhance selectivity ers), dyes, contrast agents, fluorescent compounds or mol and improve tumor detection as has been demonstrated for ecules, bioluminescent compounds or molecules, enzymes engineered scFvs or chemically conjugated mAbs, but avoids and enhancing agents (e.g., paramagnetic ions). In addition, it potential immunogenicity inherent in these non-human con should be noted that some nanoparticles, for example quan structs. tum dots and metal nanoparticles (described below) may also [0375] Thus, the platform technology described herein has be suitable for use as an imaging agent or a therapeutic agent a broad impact on the mAh delivery field and provides useful (e.g., using hyperthermal and photodynamic therapies as well methods for the treatment and diagnosis of various diseases as imaging agents through fluorescence and or MRI contrast). and conditions, including cancers, such as those against [0379] The meditope-mAb technology allows for a system which use of an antibody is indicated. For example, medi that may be used to generate an antibody-meditope complex tope-enabled antibodies directed against EGFR-positive can that may be further conjugated to one or more meditope- cers, including colorectal and squamous cell carcinoma head enabled antibody, a therapeutic agent, an imaging agent or a and neck cancers, would benefit from use of where cetuximab combination thereof. Thus, a set of meditopes or high affinity and a meditope. Additionally, modifying other therapeutic antibodies to generate meditope-enabled versions of such meditope variants, each conjugated to a unique cytotoxic or antibodies allows forthe platform technology to be utilized in imaging agent, would allow for the co-administration of a methods for the treatment and diagnosis of several other desired meditope conjugate and meditope-enabled mAh for cancers, diseases and other conditions. treatment. The meditope conjugates are an improvement over [0376] Use of the meditopes in such methods is advanta the current antibody-drug conjugates, which have drawbacks geous, for example, by way of specificity, e.g., as compared to such as a reduced specificity due to chemical modification or other means for binding or conjugation to antibodies. For release of payload. A method for enhancing the binding affin example, PpL (Protein L) and SpA (Protein A) are not ity of a therapeutic antibody or functional fragment thereof is murine-specific or chimeric antibody-specific. PpL binds to provided herein. Such a method may include administering to -66% of murine and -50% of human IgG Kappa light chains a subject a therapeutically effective amount of a pharmaceu and SpA binds to 12% of murine and 50% of human variable tical composition via any suitable route of administration. heavy chains (Graille et ah, 2002). In contrast, as demon The pharmaceutical composition may include a meditope or strated herein, the interaction of meditopes with meditope- meditope variant in combination with a meditope enabled enabled antibodies is highly specific, which can avoid adverse antibody, a multivalent meditope or meditope variant tether effects, including off-target effects, e.g., avoiding binding of ing entity in combination with a meditope enabled antibody, the therapeutic compound to immunoglobulins endogenous a meditope-enabled therapeutic antibody or functional frag to the subject. ment thereof, a pharmaceutically acceptable carrier, and any [0377] In some embodiments, a meditope administered in combination thereof. The enhanced binding affinity of the combination with a meditope enabled antibody, an antibody - multivalent meditope may be attributed to the multivalent meditope complex, a multivalent tethering agent adminis cross-linking of IgGs bound to the cell surface. Crosslinking tered in combination with a meditope enabled antibody, or a IgGs (through parental murine M425 antibody or using anti- combination thereof may be conjugated to one or more imag IgG IgM) significantly affects signaling, receptor endocytosis ing agent. In one aspect, an imaging agent may include, but is and recycling, and cell death. Thus, multivalent peptides may not limited to a fluorescent, luminescent, magnetic protein, or act synergistically with a therapeutic monoclonal antibody to radionuclide protein, peptide or derivatives thereof (e.g., enhance its therapeutic efficacy. genetically engineered variants). Fluorescent proteins that [0380] In some embodiments, the meditope, alone or as may be expressed by the mRNA component include green part of the tethering entity, may contain a cysteine or other fluorescent protein (GFP), enhanced GFP (EGFP), red, blue, suitable alkylating agent that binds to a Fab cysteine at the yellow, cyan, and sapphire fluorescent proteins, and reef coral binding site, thus creating a cysteine-cysteine interaction. fluorescent protein. Luminescent proteins that may be Alternatively, the meditope may bind to the Fab at an urmatu- US 2012/0301400 Al Nov. 29, 2012 32

ral amino acid (e.g., p-acetylphenylalanine). The Cys medi- example, a small molecule, peptide or scFv associated with tope is conjugated to any substance and directs the conjugate such a meditope is recognized by the meditope-binding site to the IgG. (e.g, framework binding interface) of the CovX antibody. [0381] An antibody-meditope complex may also be used in When these components are combined, the resulting bivalent a method for directing treatment to a particular type of cell or CovX-Body™ possesses the biologic actions of the small population of cells in a disease or condition that can be tar molecule, peptide or scFv while also retaining an extended geted by a therapeutic antibody. Such a method of treatment half-life of the antibody. may include administering a therapeutically effective amount [0386] In some embodiments, the provided meditopes, of a pharmaceutical composition to a subject having the dis meditope-enabled antibodies, and complexes thereof, are ease or condition via any suitable route of administration. The used in treatment, diagnosis or imaging of a disease or con pharmaceutical composition may include a meditope or dition, including any cancer, disease or other condition that meditope variant in combination with a meditope enabled may be treated or targeted using a therapeutic antibody. Can antibody, a multivalent meditope or meditope variant tether cers avoid immune surveillance by actively suppressing the ing entity in combination with a meditope enabled antibody, immune system. One method envisioned for counteracting a meditope-enabled therapeutic antibody or functional frag this immunosuppression is through vaccination using ment thereof. epitopes of antigens that are either uniquely expressed or [0382] In other embodiments, a method for imaging tumors over-expressed by the tumor cells. For example, monoclonal or other tissues is provided. In such methods, an unmodified antibodies (mAbs) that block signaling pathways, sequester therapeutic antibody may be administered to taiget a tumor or growth factor and/or induce an immune response have been other tissue that overexpress the corresponding antigen. Sub successfully implemented in the clinic to treat cancer and sequently, a multivalent meditope tethering entity that is other diseases. labeled with an imaging agent is administered via any suitable [0387] Thus, the diseases and conditions include any can route of administration and will bind to the therapeutic anti cer, as well as other diseases and conditions, such as those bodies that are bound to the target tumor or tissue. See FIG. 8. targeted using therapeutic mAbs, including, but not limited Examples of imaging agents include but are not limited to to, leukemia and lymphomas (which can be treated or imaged radiolabels (e.g, 3H, 14C, 35S, 90Y, 99mTc, 1251,177Lu, 166Ho, using, e.g, meditope-enabled versions of alemtuzumab, bec- and 153Sm), metal or magnetic labels (e.g, gold, iron, gado tumomab, gemtuzumab, FBTA05, ibritumomab tiuzetan, linium), biotin, chelating agents (e.g, 1,4,7,10-tetraazacy- ofatumumab, rituximab, tositumomab), breast cancer (which clododecane-N,N',N",N'"-tetraacetic acid (“DOTA”)) or any can be treated or imaged using, e.g, meditope-enabled ver agent described above. In one embodiment, the imaging agent sions of trastuzumab, adecatumumab, ertumaxomab) pros used with the method described herein is DOTA. tate cancer (which can be treated or imaged using, e.g, medi [0383] There are several advantages over known methods tope-enabled versions of adecatumumab, capromab to the imaging or treatment methods described above. First, pendetide, etaracizumab), colorectal cancer (which can be the bivalent character of a therapeutic monoclonal antibody treated or imaged using, e.g, meditope-enabled versions of enhances the selectivity to the tumor. As discussed further labetuzumab, panitumumab, altumumab pentetate, votu- below, this enhanced selectivity is lost with the use of scFvs mumab), gastrointestinal cancers (which can be treated or and is not entirely compensated for by enhanced affinity. imaged using, e.g, meditope-enabled versions of arcitu- Second, the mass of the labeled multivalent meditope tether mumab, catumaxomab), ovarian cancer (which can be treated ing entity will be below the renal threshold for filtering (less or imaged using, e.g, meditope-enabled versions of abago- than 60 kDa, may be as low as -10 kDa), allowing it to be vomab, catumaxomab, etaracizumab, igovomab, orego- easily filtered out of the body. In contrast, direct labeling of a vomab), lung cancer (which can be treated or imaged using, therapeutic antibody with an imaging agent or other thera e.g, meditope-enabled versions of anatumumab mafenatox), peutic agent is typically avoided because it will circulate for pancreatic cancer (which can be treated or imaged using, e.g, extended periods (days to weeks) in the body. Thus, imaging meditope-enabled versions of clivatuzumab tetraxetan), renal of tumors or other diseased organs is often accomplished cancer (which can be treated or imaged using, e.g, meditope- using less selective scFvs. enabled versions of girentuximab), melanoma cancer (which [0384] In further embodiments, the meditopes may be used can be treated or imaged using, e.g, meditope-enabled ver to join two or more therapeutic molecules to form a pharma sions of etaracizumab, ipilimumab, TRBS07), glioma (which ceutical compound that may be administered, as part of a can be treated or imaged using, e.g, meditope-enabled ver pharmaceutical composition, in a therapeutically effective sions of nimotuzumab), bone metastases (which can be amount to a subject for treatment of cancer, autoimmune treated or imaged using, e.g, meditope-enabled versions of disease or other conditions. The two or more therapeutic denosumab), head and neck cancer (which can be treated or molecules may include, but are not limited to, functional imaged using, e.g, meditope-enabled versions of zalutu- antibody fragments (e.g, F(ab')2 or Fab fragments), peptides mumab), cardiovascular disease (which can be treated or or other small molecules that can taiget tumor or disease- imaged using, e.g, meditope-enabled versions of abcix- specific receptors such as those described above. The thera imab), autoimmune disorders (which can be treated or peutic molecules may be two or more of the same therapeutic imaged using, e.g, meditope-enabled versions of adali- molecule or alternatively, may be two or more different mol mumab, infliximab), rheumatoid arthritis (which can be ecules that target the same tumor or diseased tissue. treated or imaged using, e.g, meditope-enabled versions of [0385] In some embodiments, the pharmaceutical com atlizumab, golimumab, infliximab), transplant rejection pound or composition may include a proprietary antibody, or (which can be treated or imaged using, e.g, meditope-en portion thereof, such as a CovX-Body™. In one example, the abled versions of basiliximab, daclizumab, muromonab- meditopes are used as linkers to join two or more therapeutic CD3), Crohn’s disease (which can be treated or imaged using, molecules to a specially designed CovX antibody. In one e.g, meditope-enabled versions of certolizumab, fontoli- US 2012/0301400 Al Nov. 29, 2012 33

zumab, natalizumab, infliximab, visilizumab), hemoglobin magnetic bead, plate, column, or other solid support. See FIG. uria (which can be treated or imaged using, meditope-enabled 10. In another example, a meditope having the sequence versions of eculizumab), psoriasis (which can be treated or CQFDLSTRRLRCGGGSK was successfully linked through imaged using, e.g., meditope-enabled versions of efalizumab, amine coupling to a solid support, and is useful, for example, infliximab, ustekinumab), multiple sclerosis (which can be in biacore/surface plasmon resonance studies. See FIG. 29. treated or imaged using, e.g., meditope-enabled versions of Such biacore studies in certain embodiments are performed natalizumab, ustekinumab), asthma (which can be treated or by flowing antibody over an amine coupled long meditope imaged using, e.g., meditope-enabled versions of benrali- chip. zumab, mepolizumab, omalizumab), respiratory syncytial [0393] Methods for coupling proteins to solid supports are virus (RSV) (which can be treated or imaged using, e.g., well known to those skilled in the art and can be used in meditope-enabled versions of palivizumab), macular degen connection with this embodiment. Meditope-enabled anti eration (which can be treated or imaged using, e.g., meditope- bodies or cells expressing the same then can be exposed to the enabled versions of ranibizumab), appendicitis (which can be solid support, whereby they are isolated and purified. treated or imaged using, e.g., meditope-enabled versions of [0394] There are several advantages to such purification fanolesomab) and any other condition that may be targeted or methods compared to other available methods. For example, treated with an antibody. The above-listed antibodies and the provided meditopes are easily synthesized and can be related diseases or disorders are examples only and do not readily added to common solid supports (including magnetic limit the platform. beads). Second, the affinity of the meditopes is easily modu [0388] In certain embodiments, one or more meditopes, lated by point mutations, which enables the fine-tuning of the meditope variants, multivalent meditope tethering agents or purification procedure and avoids harsh conditions such as multivalent meditope variant tethering agents may be conju low pH that is commonly used to elute antibodies from Pro gated to one or more imaging agents, therapeutically effective tein A or Protein L. In some embodiments, these ProteinA or agents or compounds in therapeutically effective amounts or Protein Lresins usually contain the full length (e.g., wildtype) both, such that the binding of the meditopes or variants sequence where there are up to 5 domains. One or more of thereof to one or more meditope-enabled antibody with the these domains are the same as Protein L and Protein A being therapeutically effective compound may treat, prevent, diag used herein. In some examples, the meditopes are made biva nose or monitor a disease or condition. Such conjugation of a lent or multivalent (such as those described in Example 7 high affinity and/or multivalent meditope coupled to medi below) for use in extracting meditope-enabled antibodies, tope-enabled mAbs provides a highly versatile platform tech such as intact meditope-enabled antibodies. nology that will significantly improve mAh based therapeu [0395] In some aspects, the provided purification and iso tics and imaging methods to treat and detect disease (see FIG. lation methods have additional advantages over other purifi 8). cation methods using ProteinA or Protein L, including reduc [0389] III. Other Uses and Compositions tion in the high cost associated with the production of Protein [0390] Also provided are methods and compositions for A or Protein L, improvement compared to the limited life stabilization of scFv (single chain Fab variable) fragments. In cycle of these proteins, and avoidance of the risk of introduc general, scFv fragments (e.g., the scFv format of a given Fab) ing extraneous biological material such as bacterial patho bind antigen with a substantially lower affinity than full- gens, associated with use of Protein L or A. length mAbs and other fragments, such as the Fab itself. Such reduced affinity is attributed, at least in part, to absence of the F. Modification and Screening of Meditopes and Fab constant domain that directly affects the orientation, con Meditope-Enabled Antibodies formational fluctuations of the Fv domains, and possibly, to [0396] I. Modification of Meditope-Enabled Antibodies poor linker design. On the other hand, scFv and other smaller [0397] Alsoprovidedaremethods for modifying meditope- fragments can have other advantages, including better tissue, enabled antibodies to alter one or more properties of the e.g., tumor penetration. antibodies, such as binding affinity or specificity with respect [0391] In some embodiments, provided is a method for to the meditope, and/or other properties. Also provided are improving the stability and affinity of scFv fragments. In one modified antibodies produced by the methods and libraries aspect, such methods are carried out by incorporating a medi for producing the same. In one embodiment, residues that line tope into an scFv fragment by facilitating its interaction with the meditope-binding site of a meditope-enabled antibody residues in the VH and VL regions corresponding to residues and/or that are otherwise important for binding of such an within the meditope binding site of the corresponding Fab or antibody to a meditope, are systematically or randomly whole antibody, for example, by introducing a linker between altered (e.g., using degenerate libraries and selection), for the scFv and the meditope, to stabilize the scFv. Also pro example, to enhance and/or change the specificity of the vided are complexes containing the scFv bound to one or meditope or meditope analogs (see, for example, Sheedy et al. more meditopes, and compositions containing the same, and 2007 and Akamatsu et al. 2007, hereby incorporated herein uses thereof in the provided methods. In some aspects, such by reference, for methods of making alterations). In some methods will help to enforce the proper orientation of the aspects, the residues are substituted with natural or non variable domains and thus enhance the affinity (FIG. 9). natural amino acids or both, in order to improve the affinity of [0392] Also provided are isolation and purification meth the meditope interaction and/or alter another property of the ods using the meditopes, including methods for purifying meditope-antibody interaction. The incorporation of a non meditope-enabled antibodies and fragments thereof, and/or natural amino acid in an antibody to generate a bi-specific cells expressing such antibodies, and compositions and kits antibody was described by Hutchins et al. 2011). for use in the same. In one embodiment, such methods are [0398] Residues of meditope-enabled antibodies that make carried out by coupling the meditope (e.g., any of the pro contact with the meditope and/or are otherwise important, vided meditopes) to a solid support, such as a bead, e.g., e.g., line the cavity, for example, residues within 8 A of any US 2012/0301400 Al Nov. 29, 2012 34

atom of a bound meditope, that can be systematically or [0402] In some aspects, an anchor, e.g., a GPI domain, is randomly changed to improve the meditope affinity through coupled to the antibody members of the library, such as to the hydrogen bonding, ionic, electrostatic or steric interaction C-terminus of the antibody (e.g., IgG) heavy chain, to facili can include, but are not limited to, one or more light chain tate selection of the expressed antibodies. The library can be residues (e.g., P8, V9 or 19, HO or L10, S14, E17, Q38, R39, transfected using standard methods and members of the T40, N41 G42, S43, P44, R45, D82,183, A84, D85, Y86,Y87, library expressed. G99, A100, G101, T102, K103, L104, E105, K107, R142, [0403] Mutant antibodies of the library having one or more S162, V163, T164, E165, Q166, D167, S168, orY173 of the desired characteristics then can be selected. In some aspects, light chain, based on Rabat numbering and with reference to members of the library are selected for their ability to bind to cetuximab, meditope-enabled trastuzumab, or meditope-en- an antigen, e.g., to select for mutations that do not affect abled M5 A, or analogous residues in other meditope-enabled antigen binding. In one example, cells expressing antibodies antibodies), and/or one or more heavy chain residues (e.g., of the library that bind to a fluorescently labeled antigen (e.g., Q6, P9, R38, Q39, S40, P41, G42, K43, G44, L45, S84, D86, HER2, EGFR, IGFR, CLTA-4, etc) are selected, e.g., by T87, A88, 189, Y90, Y91, W103, G104, Q105, G106, T107, FACS (FIG. 20). In some aspects (either after or instead of L108, Vl09, T110, Vlll, Y147, E150, P151, V152, T173, antigen-binding selection), cells expressing antibodies of the F174, P175, A176, V177, Y185, 5186, orL187 of the heavy library are selected for another characteristic, such as binding chain, based on Rabat numbering and with reference to cetux to a specific meditope, meditope analog, or other molecule of imab, meditope-enabled trastuzumab, or meditope-enabled interest (e.g., DOTA). In another example, two or more char MSA, or analogous residues in other meditope-enabled anti acteristics are simultaneously selected for. bodies), or a combination thereof. [0404] Selected members of the library, e.g., cells express [0399] For example, in some aspects, one or more of P8, V9 ing the antibodies, are evaluated and characterized, for or 19, HO or L10, Q38, R39, T40, N41 G42, S43, P44, R45, example, to determine the desired mutation or combination of D82, 183, A84, D85, Y86, Y87, G99, A100, G101, T102, mutations for achieving a particular property. In one aspect, R103, L104, E105, R142, S162, V163, T164, E165, Q166, sorted cells are characterized by PCR to identify the resulting Dl 67, 5168, and Yl 73 of the light chain, and/or one or more mutations that facilitate or enhance meditope/analog/small of Q6, P9, R38, Q39, S40, P41, G42, R43, G44, L45, S84, molecule binding. D86, T87, A88, 189, Y90, Y91, W103, G104, Q105, G106, T107, L108, V109, T110, Vlll, Y147, E150, P151, V152, [0405] In some aspects, the methods are carried out in an T173, F174, Pl75, A176, V177, Y185, 5186, andL187 (with iterative fashion, for example, by repeating the mutation, reference to cetuximab, or analogous residues in other medi selection, and characterization process, e.g., multiple times, tope-enabled antibodies) of the heavy chain are mutated. to “evolve/optimize” the binding or other desired character istic^). [0400] Other residues within or in proximity to the medi tope binding site may be mutated to allow a meditope group [0406] II. MeditopeAnalogs and Fragments to hydrate it and bind with high affinity. For example, the [0407] Also provided are meditope analogs, and methods Tyr87 of the light chain residue, the Tyr9 Iof the heavy chain for identifying, producing, and screening such analogs. Thus, residue, or both, may be used to form a hydrate with an in some embodiments, other molecules also bind to meditope aldehyde or boron containing compound originating from a binding sites of meditope-enabled antibodies, with functional meditope analog. In some aspects, as shown in the Examples characteristics similarto those of a meditope. Suchmolecules herein, meditopes do not affect antigen binding and can be are called meditope analogs and include, but are not limited used to deliver drugs, daisy chain/crosslink antibodies, to to, small molecules, aptamers, nucleic acid molecules, pep- increase the efficiency and/or efficacy of therapeutic antibod tibodies and any other substance able to bind to the same ies, and in targeted diagnostic, e.g., imaging, methods, among binding interface as a meditope. In one example, the analog is other uses as described herein. Thus, in some embodiments, a DOTA derivative. the meditope binding site of a provided meditope-enabled [0408] In one embodiment, provided are screening meth antibody is altered, for example, such that it can bind to a ods for identifying such analogs, including small molecule meditope with higher affinity and/or can bind with one or analogs that mimic the meditopes. Such methods include more variant meditopes, meditope analogs, and/or small mol fluorescence polarization assays, diffraction based and NMR ecules such as DOTA or a DOTA derivative (for radioactive based fragment screening, and tethering dynamic combina delivery and/or pre-targeted imaging). torial methods. [0401] In some embodiments, alteration of the residues of [0409] In other embodiments, a fragment-based drug dis the meditope binding site is by systematic alteration. In some covery approach is used to identify fragments, small mol aspects, mutating all sites would involve >206, potentially ecules, such as chemical groups, that bind to the meditopes, >2016, combinations of mutations. Thus, in some aspects, meditope binding sites, and/or meditope-enabled antibodies desired mutations are efficiently identified using a library, in near the meditope binding sites. See, for example, Erlanson, which mutations in the antibody are generated at the DNA Top Curr Chem (2012) 317:1-32. In some examples, the level. In one aspect, the library contains individual members identified fragments are coupled to the meditopes, for that collectively express each individual natural amino acid at example, to improve their affinity or other properties of the one or more positions targeted for mutation. In one example, interaction with the meditope binding site. In other examples, a DNA library is created using degenerate oligonucleotides at they are expanded and/or linked together to generate com sites of interest (e.g., any one or more of the sites described pounds for coupling to meditopes or meditope-enabled anti herein, e.g., Ile89, Thr87, Leul08 and ThrllO of the heavy bodies, or for use as meditope analogs. Methods for fragment chain and Lysl03 and Glul65 light chain), producing a and/or meditope analog discovery include, but are not limited library the members of which collectively encode all 20 natu to, the following. In some examples, the fragments are rally occurring amino acids at these sites. between about or at 100 and about or at 1000 Da. US 2012/0301400 Al Nov. 29, 2012 35

[0410] Fluorescence PolarizationAssays antibody binding and identifying peaks that are altered, indi [0411] In some aspects, the methods include fluorescence cates an interaction. These peaks can be pre-assigned to a polarization assays and/or other competition-based assays. In specific compound and thus immediately known or the pools one example, to identify alternative molecules that can bind at can be subdivided and the spectra recollected. After several the meditope site and be used for similar functions, a fluores rounds the exact identity of the compound is known. In these cent marker (e.g., Alexafluor, rhodamine, fluorescein) is con experiments, the precise position of the interaction is not jugated to a meditope, e.g., using a suitable method (e.g., known. The binding site can be determined by NMR or the amines, sulfhydryl, carboxylate, sugars or other known meth fluorescence polarization assay. Alternatively, the Fab frag ods), and allowed to interact with a meditope-enabled anti ment can be labeled with NMR active and inactive nuclei body. In general, interaction between the labeled meditope (e.g., 13C, 15N and 2H), multiple NMR experiments per and meditope-enabled antibody causes a change in the fluo formed to assign the spectrum, and then used with the frag rescence polarization/intensity of the fluorescent tag. Once ment library to identify the binding position. established, test compounds (potential analogs), such as [0417] Virtual Ligand Screening small molecule compounds (MW<1000 Da), are added and [0418] Virtual ligand screening is another method that can equilibrated, e.g., with fluorescent tagged meditope-antibody be used to identify lead meditope analogs, fragments, and complex, and the fluorescence polarization is monitored. Test other compounds. Using crystal structures, standard pro compounds that block the meditope-antibody interaction will grams (e.g., Schroerdinger Glide) can define a “box’ about a alter the fluorescent polarization properties. Accordingly, site of a macromolecule (the meditope binding site) and dock another embodiment is a method of identifying compounds known ligands to this site. Potential lead compounds are that can be optimized and used for target delivery. The ana scored by a select energy function and the top 50 to 200 logs or potential analogs may be screened and further char compounds can be purchased. In our initial studies, approxi acterized, e.g., by crystallography or other methods described mately 100 lead compounds have been identified, and using herein for characterization of the meditopes. crystallography, these lead compounds should be shown to [0412] Diffraction Methods demonstrate that they bind to the meditope site. [0413] Diffraction based methods to identify lead com [0419] Inone embodiment, a method of screening for medi pounds are well established (Shuker et al. 1996; Erlanson et topes or meditope analogs is provided herein. Such a method al. 2001; Hughes et al. 2011). Since cetuximab and other may include, but is not limited to, steps of contacting a library meditope-enabled Fabs diffract beyond 2.5 A, this approach of putative meditopes or small molecules with a meditope- is used in certain embodiments to identify lead compounds or enabled antibody; determining whether the putative medi small molecule fragments that can be coupled to a meditope topes of small molecules bind the meditope-enabled antibody or used as analogs. For example, such compounds include at a framework binding interface; identifying one or more fragments that are then linked together to form synthetic candidate meditopes or meditope analogs; determining bind meditope analogs. In one example, a compound library is ing affinity of the one or more candidates; and identifying one developed to soak into crystals of cetuximab or other medi or more of the candidates as a meditope or meditope analogs tope-enabled antibody. Diffraction data from these soaks are when the binding dissociation constant is less than 0.70 μΜ. collected and data sets analyzed. Such methods can identify In other contexts, a low affinity meditope is desired, such that additional sites on meditope-enabled antibodies that are a minimum dissociation constant is specified, for example, amendable for fragment growth and optimization. 0.70 μΜ. In some examples, the candidate is identified as a [0414] The fragments, can be grown (chemically deriva- meditope or analog thereof if it exhibits binding constant of tized) to enhance their binding and specificity. The fragments less than at or about 10 μΜ, less than at or about 5 μΜ, or less can also be chemically tethered to the meditope. Optimization than at or about 2 μΜ, less than at or about I μΜ, less than at of this chemical coupling can significantly enhance the over or about 500, 400, 300, 200, 100 nm, or less. In some cases, all binding affinity of the meditope for the meditope binding the dissociation constant, such as any of those listed herein, is site. Additionally, analogs found by diffraction methods can that measured using a particular technique, such as SPR, be optimized and used in lieu of the meditope for drug deliv Isothermal Titration calorimetry (ITC), fluorescence, fluores ery, multivalent scaffolding and other functions. Further, cence polarization, NMR, IR, calorimetry titrations; kinetic mutations in the light and heavy chains may be made to exclusion; circular dichroism, differential scanning calorim change the specificity of the ligand (meditope) and that these etry, or other known method. For example, in some cases, the diffraction methods (including fluorescence polarization, analog or meditope exhibits a binding constant of less than at NMR screening, and phage display methods) can be used to or about 10 μΜ, less than at or about 5 μΜ, or less than at or optimized alternative ligands. about 2 μΜ, less than at or about I μΜ, less than at or about [0415] NMR Screening 500,400,300,200,100 nm, or less, as measured by SPR or as [0416] NMR can also be used to identify analogs and frag measured by ITC or as measured by any of these methods. ments (e.g., peptide fragments, non-peptide based small mol [0420] Additionally, methods of screening for novel frame ecules) that can be optimized and used as described herein. In work binding interfaces are also provided, and are described one example, to identify such leads, one dimensional (ID) further in the examples below. spectra of pools containing fragments, e.g., 15 to 20 frag [0421] In another embodiment, provided is a method for ments, are collected. In one embodiment, the fragments used identifying and optimizing fragments useful in connection are non-peptide based small molecules. Next, a meditope- with the meditopes, such as in building meditope analogs enabled antibody is added to each pool and a second ID and/or in coupling to the meditopes to improve their function. spectra collected. Compounds that bind (transiently) to a Also provided are such analogs and fragments and com meditope-enabled antibody undergo rapid magnetization pounds. transfer, resulting in a loss of intensity. Thus, in some aspects, [0422] Having described the invention with reference to the comparing the spectra before and after meditope-enabled embodiments and illustrative examples, those in the art may US 2012/0301400 Al Nov. 29, 2012 36

appreciate modifications to the invention as described and 7, 301-11 (2005)). Two Fabs were placed in the asymmetric illustrated that do not depart from the spirit and scope of the unit with a Matthews Coefficient of 3.26 and solvent content invention as disclosed in the specification. The Examples are of 62.4%. The Z scores (standard deviation of the solution set forth to aid in understanding the invention but are not over the mean) were 27 and 25 for the rotational search and 38 intended to, and should not be construed to limit its scope in and 71 for the translational search. A third Fab fragment could any way. The examples do not include detailed descriptions of not be placed (three Fabs in the asymmetric unit cell produces conventional methods. Such methods are well known to those a reasonable Matthews coefficient of 2.18 at 43% solvent). of ordinary skill in the art and are described in numerous The cQFD and cQYN meditopes were built into the density publications. Further, all references cited above and in the manually through multiple iterations using Coot (Emsley, P & Cowtan, K. Coot: model-building tools for molecular examples below are hereby incorporated by reference in their graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-32 entirety, as if fully set forth herein. (2004)) and Phenix (Adams, P D. et al. PHENIX: building new software for automated crystallographic structure deter Example I mination. Acta Crystallogr D Biol Crystallogr 58, 1948-54 Determination of Meditope-Antibody Fab Crystal (2002)). Structures [0430] Crystallization and Structure Determination [0431] As noted above, cetuximab Fabs were generated and [0423] Meditopes were discovered that bind with high purified and mixed with cQFD meditope (cyclic peptide of specificity and affinity within a central cavity of the Fab SEQ ID NO: I) at a 1:10 ratio; commercial factorials were region and Fab fragment of cetuximab. These meditopes were used to screen for crystal formation. Crystals formed after I bound to cetuximab Fabs and the structures determined crys- day at 20° C. Initial diffraction analysis of these crystals talographically. indicated that the unit cell dimensions and space group were [0424] Materials and Methods similar to those for the cetuximab Fab already deposited in the [0425] Reagents. Protein Data Bank (lYY8.pdb) (Li, S. et al. Structural basis [0426] An antigen binding fragment (Fab) of cetuximab for inhibition of the epidermal growth factor receptor by was obtained by digestion of the cetuximab IgG with immo cetuximab. Cancer Cell 7, 301-11 (2005)). The observation bilized papain (Pierce), followed by reverse purification with that the CDR loops in the deposited structure make extensive Protein A and size exclusion chromatography (SEC) on a crystal contacts could have suggested that the cQFD medi Superdex 75 column (GE Flealthcare). The single chain vari tope was not present in the crystal. Nonetheless, the structure able fragment of cetuximab (scFvC225) was synthesized was solved by molecular replacement and the experimental with a twenty amino acid linker between the light chain and maps were examined to identify unmodeled electron density heavy chain. ScFvC225 and soluble epidermal growth factor consistent with the meditope. The initial Fo-Fc map clearly receptor domain III (sEGFRdIII) were expressed in Sf9 cells indicated an area in the middle of the Fab fragment as a and purified as previously described (Donaldson, J. M., Kari, potential binding site (FIG. I). After an initial round of refine C., Fragoso, R. C., Rodeck, U. & Williams, J. C. Design and ment using the Fab model only, a continuous stretch of development of masked therapeutic antibodies to limit off- unmodeled density consistent with the meditope was target effects: Application to anti-EGFR antibodies. Cancer observed. The meditope was built into the density and the R Biol Ther 8 (2009)). and UFree dropped accordingly. Water molecules were added [0427] Meditopes CQFDLSTRRLKC (cQFD; SEQ ID during refinement using Phenix (Adams, P D. et al. PHENIX: NO: I) and CQYNLSSRALKC (cQYN; SEQ ID NO:2), iso building new software for automated crystallographic struc lated from a phage display biopanning experiment as ture determination. Acta Crystallogr D Biol Crystallogr 58, describedby Riemer5A. B. et ah, Vaccinationwithcetuximab 1948-54 (2002)). The diffraction data and refinement statis mimotopes and biological properties of induced anti-epider tics are given in Table 5 below. mal growth factor receptor antibodies, JNatl Cancer Inst 97, 1663-70 (2005), were synthesized, oxidized and purified at TABLE 5 the City of Hope Synthetic and Polymer Chemistry Core Facility. Diffraction Data and Refinement Statistics for Meditope-Antibody ______Co-Crystals for Meditopes cQFD, cQYN______[0428] Crystallization and Diffraction Data. [0429] Cetuximab Fabs (5 mg/mL) were mixed with cQFD cQYD cQYN and cQYN meditopes at a 1:10 molar ratio and screened for Data collection crystal formation using the Qiagen JCSG Core Suites (IIV) at 20° C. Co-crystals that diffracted beyond 2.2 A were grown in Space group P212121 P212121 100 mM sodium phosphate/citrate, pH 4.5, 2.5 M sodium/ Cell dimensions potassium phosphate and 1.6% w/v mesoerythritol. The crys a, b, c (A) 64.26, 82.59, 211.63 64.16, 82.52, tals were wicked through 14% w/v mesoerythritol and flash 211.88 frozen in liquid nitrogen. Crystallization trials and initial a, β, γ (°) 90.0,90.0, 90.0 90.0,90.0,90.0 screening studies were carried out in the X-ray facility at City Resolution (A) 19.85-2.24 (2.30-2.24)* 29.61-2.20 (2.26-2.20)* of Hope. Diffraction data were collected at the Stanford Syn erge-F 0.078 (0.50) 0.067 (0.26) Hol 17.0 (3.1) 25.5 (6.0) chrotron Radiation Lab, beam lines 9.1 and 11.1. The initial Completeness (%) 97.4 (83.3) 99.9 (100) phases were determined by molecular replacement using the Redundancy 4.2 (3.2) 7.9 (8.2) program Phaser (McCoy, A. J. et al. Phaser crystallographic Refinement software. J Appl Crystallogr 40, 658-674 (2007)) with the Resolution (A) 19.85-2.24 29.61-2.20 unliganded structure of cetuximab (pdb:lYY8—chains A No. reflections 53791 58047 and B) (Li, S. et al. Structural basis for inhibition of the ^workf^free 18.2/22.6 17.4/22.2 epidermal growth factor receptor by cetuximab. Cancer Cell US 2012/0301400 Al Nov. 29, 2012 37

[0438] Characterization of the Meditope-Fab Interface. TABLE 5-continued [0439] Affinity analysis by SPR was performed as previ ously described (Donaldson et ah, 2009; Li et ah, 2005, Diffraction Data and Refinement Statistics for Meditope-Antibody supra). Briefly, scFvC225 or FabC225 (cetuximab Fab) was ______Co-Crystals for Meditopes cQFD, cQYN______immobilized on a CM5 chip using amine chemistry. Peptide cQYD cQYN or sEGFRdIII affinities were assessed by equilibrium meth ods at 20° C. and fit to the equation RU={Rmax*[L]}/{[L] No. atoms *Krf}+K0Jset. SEC was performed using a Superdex 200 Protein 6602 6528 10/300 column (GE Healthcare). The proteins were mixed, Ligan d/ion 200/15 188/15 incubated at room temperature for 20 min and applied to the Water 581 618 column at 4° C. B-factors [0440] The EGFR-expressing MDA-MB-468 cell line was Protein 31.4 32.0 used to test cetuximab binding in the presence of peptide Ligan d/ion 50.3/67.4 64.0/44.2 meditope. Labeled cetuximab (AF488, Invitrogen) was Water 37.5 38.7 added for 20 min with or without 60 μΜ cQFD peptide at 4° R.m.s. deviations C. Labeled MOPS-21 was used as an isotype control. Cell Bond lengths (A) 0.004 0.007 fluorescence was determined using a FACS Calibur instru Bond angles (°) 0.913 1.100 ment (BD Biosciences).

^values in parenthesis are for the highest resolution shell [0441] Analysis of Meditope/Fab Interface [0442] The interface of the binding site between the medi [0432] Based on these observations and as a point of com tope (SEQ ID NO: I) and the cetuximab Fab was determined parison, crystals of the cetuximab Fab bound to the meditope to be formed by all four domains of the IgG Fab (e.g., the cQYN (SEQ ID NO: 2) were produced. As for the meditope variable heavy and light chain domains, the light chain con of SEQ ID NO: I, clear unmodeled electron density was stant region, and the heavy chain CHI). Using the PISA observed in the center of the Fab. Using the first structure, the server, the buried surface area at the cQFD or cQYN medi- differences in sequences were modeled accordingly and mul tope-Fab interface was 904 (±28) A2 and 787 (±42) A2, tiple rounds of refinement were carried out. Representative respectively, and approximately equally distributed between electron density maps of both meditopes are shown in FIG. the light and heavy chains. FIGS. 2, 35, and 36 show the IB. The diffraction data and refinement statistics for the residues and the loops from the Fab that were determined to cQYN-Fab complex also are presented in Table 5, above. contact the meditope. [0443] Both meditopes (SEQ ID NOs: I and 2) were deter Example 2 mined to make multiple hydrogen bonds and hydrophobic contacts with the cetuximab Fab. The amino acid sequences Characterization of the Meditope Binding Site of of cetuximab (murine chimeric IgG) were aligned with a Cetuximab Fab chimeric monoclonal IgG (chi 4.18) used as an isotype con trol in the phage display experiments that originally identified [0433] The meditope binding site of cetuximab was char the meditopes of SEQ ID NOs: I and 2, and trastuzumab (a acterized as discussed below. It was demonstrated that this humanized monoclonal antibody that binds to ErbB2) (see meditope binding site is unique and has not been found to FIG. 2A). The structure of trastuzumab Fab also was super exist in the immunoglobulins examined to date. imposed onto the structure of cetuximab Fab bound to the meditope of SEQ ID NO: I. FIG. 2A shows amino acid [0434] Materials and Methods differences between the residues within the central cavity [0435] In addition to those described in Example I above, meditope-binding interface of the cetuximab Fab (murine the following materials and methods were used. chimera IgG) and chl4.18 and trastuzumab. [0436] Meditopes and Point Mutations. [0444] Superposition of the humanized trastuzumab Fab [0437] As described in Example I, CQFDLSTRRLKC (lNZ8.bdb) on the cetuximab Fab indicated that Arg9 of the (cQFD; SEQ ID NO:I) and CQYNLSSRALKC (cQYN; cQFD meditope binds to a unique cavity created by the SEQ ID NO:2), were synthesized, oxidized and purified at the murine variable light chain. Specifically, Asp85, Ile83 and City of Elope Synthetic and Polymer Chemistry Core Facility. Thr40 of the murine variable light chain of cetuximab, based Alanine point mutations in the cQFD meditope were gener on Rabat numbering, were shown to be important with ated at residues 3 (Phe3 to Ala), 5 (Leu5 to Ala), 8 (Arg8 to respect to binding to the Arg9 residue of the cQFD meditope Ala) and 10 (Leu 10 to Ala) and the mutants produced bacte- (FIG. 2B). Asp85 in the murine framework region of the rially by encoding the peptides at the C-terminus of SMT3 variable light chain was shown to make a salt bridge to the (Mossessova, E. & Lima, C. D. Ulp I-SUMO crystal structure guanidinium group of Arg9 of the cQFD meditope (d^ and genetic analysis reveal conserved interactions and a regu 0/31=2.7 and 2.9 A & Anh2 OZ32=3.0 and 3.1 A). The latory element essential for cell growth in yeast. Mol Cell 5, carboxyl group of Asp85 also was shown to make a hydrogen 865-76 (2000)). The peptides were oxidized by dialysis into bond to the backbone amide nitrogen of LeulO of the cQFD buffer without DTT and purified by SEC to obtain monomers. meditope (Aod2 HN=2.1 and 2.8 A). The hydroxyl group of Before surface plasmon resonance (SPR) analysis, ubiquitin- Thr40 from the light chain also was shown to make a hydro Iike protease (Ulpl) was added to the samples to release the gen bond to guanidinium group of the meditope Arg9 (dOG1 peptides. Each of the biosynthesized mutant peptides con .. ot/i=3.2 A for both). Superimpositionofthe structures also tained an additional serine residue at the N-terminus due to showed that the phenyl ring in Phe83 and the pyrrolidine ring the ULPl cleavage site. Ulpl and SMT3 were run as controls of Pro40 found in the human variable light chain sequence and did not interact with the cetuximab Fab. (corresponding to He and Thr, respectively, in cetuximab) are US 2012/0301400 Al Nov. 29, 2012 38

positioned such that they would sterically occlude the side “Structure of the extracellular region of F1ER2 alone and in chain of Arg9 in the cQFD meditope. complex with the Flerceptin Fab,” 2003 Feb. 13; 421(6924): [0445] Although the Arg9 side chain in the cQFD meditope 756-60) and rituxumab (20SL; Du et ah, JBiol Chem, 2007 mapped to the differences between the murine andhuman Fab May 18; 282(20):15073-80. Epub 2007 Mar. 29) Fabs on to sequences, the cQYN meditope encodes an alanine at the the meditope bound cetuximab Fab structure further high same position as Arg9 in the cQFD meditope, and thus could lighted the uniqueness of the framework. potentially have bound to the human Fab. Although the hydro [0449] Collectively, these results suggest that the differ gen bond to the guanidinium group of Arg9 of the cQFD ences in the framework region of cetuximab and the control meditope to Asp85 of the light chain was not present for the IgG (as opposed to the differences in the CDRs) accounted for cQYN meditope, the carboxyl group of Asp85 of the light the selection of the meditope to the central cavity of the Fab chain was shown to retain its hydrogen bond to the backbone (Te., cetuximab and the phage display control antibody dif amide nitrogen of LeulO of the cQYN meditope. Differences between the cQFD and cQYN meditopes and their interaction fered in regions other than CDRs). with the cetuximab Fab were determined. Superposition of [0450] Meditopes do not Induce Laige Conformational the structures of the two meditopes (cQFD and cQYN) bound Changes in the Fab to cetuximab Fab (specifically of the Ca atoms of the heavy [0451] Based on the location of the meditope-cetuximab and light Fab chains) showed that the hydrophobic side chains Fab interface, it was determined whether the meditopes per of residues Phe/Tyr3, Leu5 and Leul Oofeach meditope were turbed the relative orientation of the IgG domains to the positioned nearly identically, indicating that interaction of unligated and/or ligated structure. First, the light and heavy these residues with the antibody were important for binding chains within the asymmetric unit cell of both meditope com (FIG. 2B). The phenyl ring was shown to pack against the plexes were compared and then each chain was compared to conserved Gln39 of the heavy chain variable region (Rabat the unligated and EGFR-Iigated structures. The variable numbering); the Leu5 side chain was shown to pack against domains of the light chains bound to either meditope were He 89 and Leu 108 of the heavy chain variable region (Rabat essentially identical to the unliganded structure of cetuximab numbering), as well as the phenyl ring from Phe3/Tyr3 of the (r.m.s.d. average: cQFD, 0.231±0.014 A, cQYN, 0.18±0.01 meditopes. LeulO of the meditopes was shown to bind to an A). Flowever, the variable domains of the heavy chain showed exposed pocket located on the variable light chain. a greater divergence (r.m.s.d. average: cQFD, 0.85±0.01 A, [0446] The backbone traces of the cQFD and cQYN pep cQYN, 0.88±0.01 A). This divergence mainly stemmed from tides, however, did deviate. Specifically, the Arg8 side chain the position of framework region 2 (residues 39-46), since structure of the cQFD meditope is extended and makes a deleting the residues in this region and recalculating the r.m. strong backbone hydrogen bond to the backbone carbonyl of s.d. resulted in a much lower value: cQFD, 0.18±0.01 A; Glnl 05 of the Fab heavy chain Uiv771 0=c=2.6 and 2.8; cQYN, 0.31±0.01 A) (FIG. 6). In addition, this region was Anh2 0=c=2.% and 2.9 A). The hydroxyl group of Tyr3 in also shown to be displaced in the Fab C225-EGFR (cetux the cQYN peptide, however, sterically interferes with the imab Fab-antigen) co-crystal structure, and its relative B-fac- Arg8 side chain (FIG. 2B) and blocks the interaction between tor value suggested that it is flexible (FIG. 6). Finally, the Arg8 of the cQYN meditope and Glnl 05 of the heavy chain. presence of the meditope did not result in significant changes Consistent with this observation, both Arg8 side chains in the to the CDR structure relative to the EGFR bound or unbound cQYN complex are poorly defined in the electron density map structure. Although the backbone of Tyr98 in the heavy chain and takes on at least two different rotamers. (There are two CDR loop 3 of the EGFR-Iiganded structure was shown to be Fab-meditope complexes in the asymmetric unit.) Concomi flipped as compared to the Fab structure bound to either tant with this change, a shift in the backbone hydrogen bond meditope, this flip is also observed in the unliganded cetux pattern was observed. The amide carbonyl of Thr7 in the imab Fab structure (Li et ah, 2005, supra). cQFD meditope makes a hydrogen bond to the amide Asn41 [0452] Contribution of Meditope Residues to Antibody in the cetuximab Fab light chain d^.^ 0=c=21 and 2.8 A). Binding This hydrogen bond shifts to the carbonyl of the Arg8 back [0453] Based on the structure of the cQFD meditope-Fab bone in the amide of backbone of Asn41 in the cQYN peptide complex, as well as the sequence similarity of cQYN to (Ac=o h-n=3 · 0 and 3.1 A). cQFD, several point mutations in the cQFD meditope were [0447] The superposition indicated that the change from generated (Phe3—»Ala, Leu5—»Ala, Arg8—»Ala and Pro40 and Gly41 in the human light chain sequence to Thr Leu 10—» Ala) to characterize the role of these residues to the and Asn in the cetuximab light chain relieves a steric con overall binding affinity of the meditope. To assess binding straint and affords two additional points for forming hydro affinity, the cetuximab Fab fragment was coupled to a CM5 gen bonds to the meditopes. In the cQFD meditope-cetux- chip using standard amine chemistry and surface plasmon imab Fab structure, the amide nitrogen of Fab Asn41 was resonance (SPR) was used to measure the binding properties shown to interact with the carbonyl of Thr7 Uivj7 0=c=2.1 of the various meditopes. The affinities of the synthetic cQFD and 2.8 A) of the meditope and the side chain amide of Fab and cQYN meditopes to the cetuximab Fab were measured to Asn41 was shown to interact with the carbonyl of Ser6 (And2 be 950±30 nM and 3.5±0.1 μΜ, respectively (n=3). The bind ... O=C=3.2 and 3.2 A) of the meditope. In the cQYN medi- ing kinetics were also measured (FIG. 3). The association tope-cetuximab Fab structure, the shift of the backbone constants, modeled as a bimolecular interaction, were 4.2 results in a shift of the hydrogen bond pattern. The amide (±0.1 )x IO4 M“1 s“1 and 1.8 (±0.1 )x IO4 M“1 s“1 for cQFD and nitrogen of Fab Asn41 interacts with carbonyl of Arg8 of cQYN, respectively. The dissociation constants were 2.5 (±0. cQYN Uivj7 (^¢.=3.0 and 3.1 A) but the side chain I)xl0-2 S-1 and 8.6 (±0.1)xl0“2 s_1 for cQFD and cQYN, interaction with Fab Asn41 is lost. respectively. The R73 values based on these measurements, [0448] In addition to these results, superposition of the 430 (±30) nM for cQFD and 3.5 (±0.1) μΜ for cQYN, are in molecular structure oftrastuzumab (1N8Z; Cho et ah, Nature, close agreement with the equilibrium measurements. US 2012/0301400 Al Nov. 29, 2012 39

[0454] Next, the affinity of each mutated cQFD meditope the peptides at the C-terminus of SMT3 (Mossessova et ah, was measured. The point mutation and wild type cQFD medi- 2000). Before surface plasmon resonance (SPR) analysis, topes were generated as C-terminal fusions to SMT3 and ubiquitin-like protease (Ulpl) was added to the samples to cleaved with Ulpl before the analysis. The biologically-pro release the peptides. An N-terminal serine residue remained duced, wild type cQFD meditope bound with an affinity of as a remnant of the protease cleavage site. 770 nM, similar to the synthetically-produced cQFD, [0464] Simultaneous Binding of EGFR and Meditope to whereas the mutations Phe3—»Ala, Leu5—»Ala and Fab Arg8—»Ala significantly reduced the affinity for the cetux- [0465] The cetuximab Fab was incubated with sEGFRdIII imab Fab (Table 6, below). In particular, theArg8—»Ala muta and cQFD and applied to an analytical SEC column. A peak tion resulted in an approximately 183-fold loss in binding at 13.9 mL was observed, indicating the formation of a het- affinity. erotrimeric complex between these three components. Non [0455] In Table 6, WT=cQFD (SEQ ID NO: I), F3A=SEQ reducing SDS-PAGE of the peak showed the presence of all ID NO: 26 with an additional serine before the cysteine, three components (FIG. 4B). In comparison, the individual L5A=SEQ ID NO: 27 with an additional serine before the components eluted at 15.2 mL (Fab C225), 15.6 mL (sEG- cysteine, R8A=SEQ ID NO: 28 with an additional serine FRdIII) and 16.3 mL (SMT-CQFDLSTRRLKC; SEQ ID before the cysteine, and LlOA=SEQ ID NO: 29 with an NO:l). The results showed that the meditopes and EGFR additional serine before the cysteine. could bind to cetuximab simultaneously, indicating that meditope binding did not occlude antigen binding by cetux TABLE 6 imab. [0466] In addition, Forster resonance energy transfer Dissociation constants for “WT” (cQFD) and mutant meditopes Dissociation constants of cOFD mimotooe mutants (FRET) measurements were used to confirm that the medi tope did not affect antigen binding by cetuximab. The fluo Kb AAG rescent signal emitted from Alexfluor 488-labeled cetuximab Ligand (μΜ) (kcal/mol) Fab was quenched upon binding of Alexafluor 555-labeled WT 0.77 _ sEGFRdIII. Titration of labeled cetuximab with labeled sEG- F3A 34 2.3 FRdIII afforded a binding constant of 20 nM, essentially the L5A 57 2.6 same value as was observed using SPR (data not shown). To R8A 141 3.1 test for allostery, labeled cetuximab Fab was incubated with LlOA 2.2 0.63 sEGFRdIII at concentrations ranging from 5 to 1000 nM in the presence of the cQFD meditope (66 μΜ). The experiment [0456] Finally, the Fab of trastuzumab, a humanized thera was performed in triplicate. Similar to the SEC studies, no peutic monoclonal antibody, was coupled to a CM5 chip to substantial change in the binding constant of the Fab-sEG- characterize the affinity of the cQFD and cQYN meditopes to FRdIII interaction was observed. a human framework. Equilibrium measurements revealed [0467] Taken together, these biochemical studies indicate that the dissociation constants for both meditopes exceed 150 that the meditope-Fab interaction does not substantially μΜ. affect antigen binding. These observations are similar to results with Protein L and Protein A, which also bind to the Example 3 Fab framework region without affecting antigen binding to Binding of Meditopes to Regions Other than CDRs Fab. [0468] cQFD Meditope does not Bind to CDR Loops of [0457] Materials and Methods Cetuximab [0458] It was demonstrated that binding of the meditopes [0469] To further confirm that the meditopes did not bind to cQFD and cQYN, and others, to a meditope-enabled antibody CDRs, binding between the cQFD meditope and the scFv (cetuximab), does not affect the ability of the antibody to bind fragment of cetuximab was assessed. In the scFv, the CDR to antigen, meaning that the meditope-enabled antibodies can loops remain intact, but the Fab variable domains are directly bind to antigen and a meditope simultaneously. connected through a short peptide linker, eliminating the Fab [0459] In addition to those described in Examples I and 2 constant domains. In other words, much of the meditope above, the following materials and methods were used. binding pocket is eliminated in the scFv, while the CDRs are [0460] Reagents. minimally affected. SPR demonstrated that EGFR domain III [0461] As described above, the single chain variable frag and the cQFD meditope bound to cetuximab Fab tethered to ment of cetuximab (scFvC225) was synthesized with a a CM5 chip (See FIG. 4D). In addition, EGFR domain III twenty amino acid linker between the light chain and heavy bound with a minimal affinity loss to the scFv tethered to a chain. ScFvC225 and soluble epidermal growth factor recep second CM5 chip. Flowever, relative to Fab binding, the tor domain III (sEGFRdIII) were expressed in Sf9 cells and cQFD meditope did not saturate the scFv at concentrations as purified as previously described (Donaldson et ah, 2009). high as 100 μΜ of meditope. This indicates minimal, if any, [0462] Meditopes and Point Mutations. affinity of the meditope for the CDRs, consistent with the [0463] As described above, CQFDLSTRRLKC (cQFD; crystallographic studies. SEQ ID NO:I) and CQYNLSSRALKC (cQYN; SEQ ID [0470] cqFD Meditope Binding does not Affect Cetuximab NO:2), were cyclized through the oxidation of the cysteines Binding to EGFR-Expressing Cells to create a disulfide bond and purified at the City of Elope [0471] As described above, the cetuximab Fab could bind Synthetic and Polymer Chemistry Core Facility. Alanine to the cQFD meditope and EGFR domain III simultaneously. point mutations in the cQFD meditope were generated at It was further shown that this meditope did not affect cetux residues 3 (Phe3 to Ala), 5 (Leu5 to Ala), 8 (Arg8 to Ala) and imab (full IgG) binding to EGFR-expressing cells. FACS 10 (Leu 10 to Ala) and were produced bacterially by encoding analysis was used to follow the binding of the IgG, as a US 2012/0301400 Al Nov. 29, 2012 40

function of meditope concentration, to MDA MB-468 cells, straint of intramolecular “masked” IgG construct will be sev which overexpress EGFR. Cells were incubated with cetux- ered at the tumor site and permit antibody binding. This imab in the presence of increasing cQFD meditope concen design principle would avoid binding of the intramolecularly trations. Even with meditope concentrations greater than 60 ‘masked’ IgG to healthy tissues and avoid adverse side effects μΜ, no significant changes in cetuximab binding to the cells due to off-target binding. Off-rate determination for avidin- were observed. This observation is consistent with the SEC peptide mask on cetuximab showed that a multivalent medi studies described above, and indicates that the meditope does tope bound with higher affinity than a monovalent meditope, not act as an allosteric regulator of antigen binding. but did not mask. [0472] Simultaneous binding to the Fab of EGFR domain III and the meditope is shown at concentrations significantly Example 4 above the K13 of the meditope Like the cQFD and cQYN meditopes, superantigens SpA and PpL, bind to the Fab Generation of Meditope-Enabled Antibodies framework region and do not affect antigen binding (see Graille, M. et al. Crystal structure of a Staphylococcus aureus [0478] Based on structural information, additional medi Protein A domain complexed with the Fab fragment of a tope-enabled antibodies were generated. human IgM antibody: structural basis for recognition of [0479] A. Generation of Meditope-Enabled Trastuzumab B-cell receptors and superantigen activity. ProcNatlAcad Sci by Mutation USA 97, 5399-404 (2000)); Graille, M. et al. Complex [0480] A meditope-enabled F1ER2-binding antibody was between Peptostreptococcus magnus Protein L and a human generated by mutating trastuzumab. The sequence differ antibody reveals structural convergence in the interaction ences between the heavy and light chain variable region modes of Fab binding proteins. Structure 9, 679-87 (2001); framework regions of a human IgG (trastuzumab-lN8Z.pdb) Young, W. W., Jr., Tamura, Y., Wolock, D. M. & Fox, J. W. compared to those of cetuximab were mapped onto the crystal Staphylococcal Protein A binding to the Fab fragments of structure of cetuximab Fab bound to cQFD meditope. Resi mouse monoclonal antibodies. J Immunol 133, 3163-6 dues in the human framework corresponding to residues lin (1984); Graille, M. et al. Evidence for plasticity and structural ing the meditope binding site in cetuximab were mutated to mimicry at the immunoglobulin light chain-Protein L inter contain the corresponding residues present in cetuximab; face. JBiol Chem 211, 47500-6 (2002)). additionally, S9 and SlOof the light chain, according to Rabat [0473] Meditope Binding in Relation to Other Fab-Binding numbering, were mutated to isoleucine and leucine, respec Proteins tively. FIGS. 23 A and 23C show nucleic acid sequences of a [0474] The meditope binding site on the Fab is distinct from portion of the heavy chain (SEQ ID NO: 5) and the light chain that of other proteins that bind to the framework region of a (SEQ ID NO: 8), respectively, of the mutant (meditope-en Fab. For example, domain D of Protein A, isolated from abled) trastuzumab. FIGS. 23B and 23D show the amino acid Staphylococcus aureus, binds to the framework of heavy sequences of a portion of the heavy chain (SEQ ID NO:6) and chain (Vh) of human IgG; the B1 domain of Protein L, iso light chain (SEQ ID NO: 9), respectively, of the meditope- lated from Peptostreptococcus magnus, binds to the frame enabled trastuzumab. FIGS. 23B and 23D also show a com work region of kappa light chain (Vi) of human IgG; and parison of these amino acid sequences and those of a portion domain II of Protein G, isolated from a Streptococcal strain, of the heavy and light chain of wild type trastuzumab (SEQ binds to the framework of heavy chain (Vc) of human IgG. ID NO:7 and SEQ ID NO:10, respectively). Unlike these interactions, in which the interface is predomi [0481] The nucleic acids encoding the heavy and light nantly confined to a single Fab domain that is solvent-ex- chains of this mutant, meditope-enabled trastuzumab were posed, the meditope-Fab interface was shown to be formed by synthesized using standard methods and subcloned into a all four domains (e.g., the variable and constant domains of standard vector for mammalian expression. The meditope- the heavy and light chains). enabled trastuzumab IgG was purified using standard meth [0475] The buried surface areas at the cQFD and cQYN ods and characterized for the ability to bind to F1ER2 and the meditope-cetuximab Fab interface were shown to be 904 cQFD meditope. (±28) A2 and 787 (±42) A2, respectively, and generally dis [0482] Meditope-Enabled Trastuzumab Binds to Antigen tributed equally between the light and heavy chains. These and Meditope and has Indistinguishable PK/PD Properties as values are roughly similar to those for the interfaces of the Wild-Type Trastuzumab Protein A, L and G domains bound to their Fab domains: 580 [0483] In one study, to characterize antigen binding, wild- A2 (IDEE.pdb), 714 A2 (lHEZ.pdb), and 518 A2 (1QKZ. type trastuzumab and meditope-enabled trastuzumab were pdb), respectively. There are two unique interfaces between labeled with Alexa 647 using standard protocols. A cQFD the variable region of the Fab and Protein L, which buries 646 (SEQ ID NO: I) meditope-Fc fusion protein (produced as A2, and a minor interaction between the constant region of the described in Example 7 and shown in FIGS. 15 and 16) was Fab and Protein G, which buries 184 A2. labeled withAlexa488 using the same protocols. To show that [0476] Steric Mask the meditope-Fc binds to the meditope-enabled trastuzumab [0477] The meditope can be tethered to the N-terminus of and not to the wild-type trastuzumab, SKB R3 cells (0.5xl06), either the light chain or heavy chain N-terminus of a murine which over-express F1ER2, were incubated with labeled wild- chimera or meditope-enabled human mAh through a flexible type and meditope-enabled trastuzumab for 30 minutes. linker (FIG. 11). The N-termini of mAh IgGs are juxtaposed Unbound antibody was washed and the cells were incubated to the antigen binding site and the extension from the N-ter- with the meditope-Fc construct for 30 minutes. Antibody mini through the flexible linker will sterically interfere with binding and meditope binding were analyzed by FACS analy antigen binding. By encoding a tumor specific protease site sis. The FACS data demonstrate that meditope-enabled tras (e.g., MMP9, MMP14, prostate-specific antigen (PSA) serine tuzumab binds to F1ER2 expressed on the SKBR3 cells (e.g., protease or other suitable site) in the linker, the steric con the meditope site can be grafted onto an antibody without loss US 2012/0301400 Al Nov. 29, 2012 41

of antigen specificity) (FIG. 22A), and that the meditope-Fc diagram’ figure of all three structures superimposed, demon binds to the meditope-enabled trastuzumab, but not to wild- strating no significant differences overall. Some differences type trastuzumab (FIG. 22B). in CDR loops were observed, as expected (given binding of [0484] In another study, wild-type and meditope-enabled these antibodies to different antigens). trastuzumab were labeled with Alexafluor 488 using standard [0490] Additionally, animal studies indicated that the bio protocols. SKBR3 cells (0.5xl06) were trypsinized, washed distribution of the meditope-enabled trastuzumab and wild- once with 0.1% BSA, and incubated with I, 10, or 100 nM type trastuzumab were indistinguishable. wild-type or meditope-enabled trastuzumab, for 30 minutes. [0491] These data demonstrate that antibodies can be effec Unbound antibody was washed twice and analyzed by flow tively meditope-enabled, while retaining other functions, by cytometry. The results are shown in FIG. 43, demonstrating mutating residues to correspond with those in the meditope- that meditope-enabled trastuzumab had a similar affinity for binding interface of cetuximab. antigen-expressing cells as did wild-type trastuzumab. [0492] Meditope-Enabled Trastuzumab Binds to Meditope [0485] In another study, a cQFD(SEQ ID NO: l)meditope- and Antigen, Whether Contacted Sequentially or Pre-Mixed Protein L fusion protein (MPL) was labeled with Alexafluor with Meditope 647 using the same standard protocols. SKBR3 cells were [0493] Another study demonstrated that meditope bound to trypsinized, washed once with 0.1% BSA, and then pre meditope-enabled trastuzumab with similar efficiency bound to wild-type trastuzumab (WT T) or meditope-enabled whether the cells were pre-bound with antibody (sequential) trastuzumab (TM) (by incubation for 30 minutes). The cells or a pre-formed meditope-antibody complex (pre-mixed) was then were washed, followed by another 30 min incubation applied to cells. The results are shown in FIG. 46. In this with 0.1 μΜ or I μΜ MPL (right). Cells were washed twice study, SKBR3 cells were trypsinized, washed once with 0.1% and analyzed by flow cytometry (FACS). Cells not pre-bound BSA, then incubated with 10 nM meditope-enabled trastu antibody (MPL (WT) only) were used as a negative control. zumab (TM) for 30 min, washed, and then incubated for The results are shown in FIG. 44. Histograms demonstrate another 30 min with 4, 20, or 100 nM MPL (left panel). that cells bound to meditope-enabled trastuzumab, but not Alternatively, 10 nM of TM was premixed with 4, 20, or 100 unbound cells or cells bound to wild-type trastuzumab, bound nM of MPL for 30 min on ice, followed by application of the to the meditope-Protein L fusion protein. Dot plots show mixture to the cells for I hr (right panel). Cells were washed percentage of cells in each quadrant relative to the MPL only twice and analyzed by FACS. Percentage of MPL-positive control. cells relative to no-treatment control is shown in the legend. [0486] As shown in FIG. 45, the same results were obtained [0494] B. Generation of Meditope-Enabled HER2-Binding using a meditope-Protein L in which all but one lysine in the Antibody by CDR-Grafting Protein L were mutated to Arg/Asn residues (MPL-5K). [0495] A meditope-enabled HER2-binding antibody was [0487] These data demonstrate that meditope-enabled tras generated by grafting the CDRs of trastuzumab onto cetux tuzumab is able to bind to antigen to the same degree as the imab. wild-type antibody, and that that the meditope-enabled tras [0496] Nucleic acid and amino acid sequences of a heavy tuzumab (but not wild-type trastuzumab) binds to the medi- chain of trastuzumab are shown in FIG. 25 A (SEQ ID NOs: tope. Thus, these data confirm that binding of the meditopes 11 and 12, respectively), with signal sequence and other to the meditope binding site of this antibody did not affect sequences. Nucleic acid and amino acid sequence of a light antigen binding in this study, demonstrating the tripartite chain of trastuzumab are shown in FIG. 25B (SEQ ID NOs: interaction. 13 and 14, respectively), with signal sequence and other [0488] FIG. 50 (top panel) shows stick representations of sequences. the structures of meditope 18 (SEQ ID NO: 18, shown in [0497] While the “boundaries” of the CDR loops of mAbs Table 3, with a β,β'-diphenylalanine at position 5) bound to have been clearly defined by sequence homology structural cetuximab (dark grey sticks), the same meditope (18) bound methods in general, the crystal structure of trastuzumab was to meditope-enabled trastuzumab (white sticks), and wild- superimposed onto cetuximab and the position of each resi type trastuzumab (outline), superimposed. The bottom panel due examined to address potential differences outside of the shows a ribbon cartoon comparing wild-type and meditope- CDR loops that may have a secondary effect on the confor enabled trastuzumab. The results demonstrate that the posi mation of the CDR loops; based on this information, addi tion of residues important for binding with the meditope are in tional modifications beyond modifying the CDRs. The amino nearly identical places in cetuximab and the meditope-en acid sequences of the light and heavy chains of the antibody abled trastuzumab. The right panel of FIG. 50 shows a ribbon designed to contain trastuzumab-like CDRs on a cetuximab- cartoon of wild-type and meditope-enabled trastuzumab, Iike framework were translated into DNA sequences and the demonstrating little difference in structure. genes encoding each were synthesized. The amino acid and [0489] Likewise, FIG. 51, in the upper-left panel, shows a nucleic acid sequences of the resulting heavy and light chains superposition of the structures of trastuzumab and trastu of antibodies containing trastuzumab-like CDRs grafted onto zumab memAb (labeled “Meditope-enabled Memab”) with cetuximab-like framework are shown in FIG. 47. Specifically, certain residues involved in meditope-binding in the medi FIG. 47A shows light chain nucleic acid (SEQ ID NO: 60) tope-enabled antibody illustrated by sticks. The top right and light chain amino acid (SEQ ID NO: 61) sequences of a panel shows a superposition of the structures of meditope- CDR-grafted meditope-enabled trastuzumab (with trastu enabled trastuzumab (memAb) and cetuximab, with the same zumab-like CDRs grafted onto a cetuximab-like framework), residues labeled. As shown, Ile83 takes on two rotamers in the with the signal sequence and other residues shaded. FIG. 47B respective structures, which was determined not to be prob shows heavy chain nucleic acid (SEQ ID NO: 62) and heavy lematic, given that in the meditope bound meditope-enabled chain amino acid (SEQ ID NO: 63) of this antibody. trastuzumab crystal structure, it assumes the same rotamer as [0498] The genes were then subcloned in frame into the observed in cetuximab. The bottom panel is a ‘cartoon/ribbon remaining IgG DNA sequence, confirmed by DNA sequenc US 2012/0301400 Al Nov. 29, 2012 42

ing and placed in individual expression vectors. The resulting cells. Two stable lines were obtained, expressing the medi expression vectors were transfected into NSO cells for co tope-enabled mAh at -5 mg/L. expression of the heavy and light chains. As the expressed [0502] Binding of the meditope-enabled M5A to the M5A full-length CDR-grafted IgG was secreted, the supernatant antigen (CEA) was demonstrated using LS174T cells by was clarified by centifugation, concentrated, and passed over FACS. 500 nM of Alexa Fluor 488 labeled wild-type or medi a Protein A column. IgG was eluted using a low pH solution tope-enabled M5A was incubated with trypsinized cells and 9 and immediately neutralized. SDS-PAGE (poly acrylamide μΜ of Alexa Fluor 647 labeled meditope-Protein L (see gel electrophoresis) was carried out on the full-length CDR- Example 10) for 30 min, room temperature. Cells were grafted IgG, under reducing conditions. The results indicated washed twice with 0.1% BSA and FACS analysis was per two protein bands with apparent masses consistent with the formed. The results are shown in FIG. 57. Given that M5A is light and heavy chain. The position of the bands migrated at a humanized antibody with the same backbone as trastu similar positions compared to wild-type cetuximab. zumab, results showed that the meditope-Protein L bound [0499] To characterize antigen binding, wild-type trastu- readily to the wild-type M5 A. However, an enhanced binding zumab and the trastuzumab CDR-grafted, meditope-enabled was observed with the meditope-enabled M5A (M5A 8M), mAh (memAb) were labeled with Alexafluor 647 using stan which is due to avidity gained from simultaneous binding of dard protocols. As described above, cQFD (SEQ ID NO: I) both the meditope and Protein L. meditope-Fc (produced as described in Example 7 and shown [0503] Additionally, SPR measurements were carried out in FIGS. 15 and 16); Meditope-Fc was labeled with Alex- as described herein using surface plasmon resonance, con afluor 488 using the same protocols. To show that the medi- firming binding of various meditope variants to this medi tope-Fc binds to the trastuzumab CDR-grafted, meditope- tope-enabled antibody, including CQA(diphenyl)DLSTR- enabled mAh and not to the wild-type trastuzumab, SKBR3 RLKC, CQFDA(diphenyl)STRRLKC, meditope 31, and the cells (0.5xl06) which over-express HER2, were incubated cQYN meditope (FIG. 58). with labeled wild-type or CDR-grafted meditope-enabled trastuzumab as produced in this example, for 30 minutes. Example 5 Unbound antibody was washed and the cells were incubated Modification of the Meditope Binding Site with the meditope-Fc construct for 30 minutes. Antibody binding and meditope binding were analyzed by FACS analy [0504] The residues lining the meditope-binding site of one sis. As an important component of the “CDR-grafting.” The ormore of the provided meditope-enabled antibodies, such as results are shown in FIG. 24. The FACS data showed that the cetuximab, are systematically or randomly altered, for trastuzumab CDR-grafted, meditope-enabled mAh bound to example, using degenerate libraries and selection, to enhance HER2 expressed on the SKBR3 cells, demonstrating that and/or change the specificity of the meditope or meditope CDR loops of one antibody (trastuzumab) grafted onto a analogs (see Sheedy et al. 2007 and Akamatsu et al. 2007, for meditope-enabled antibody (cetuximab) framework retain methods of making alterations). Residues at these positions the ability to bind to antigen (HER2) (FIG. 24A). The FACS are substituted with natural or non-natural amino acids or data also showed that the meditope-Fc bound to the trastu both, for example, to improve the affinity of the meditope zumab CDR-grafted, meditope-enabled mAh, but not to interaction and/or alter another property of the meditope- wild-type trastuzumab (FIG. 24B), demonstrating that a antibody interaction. CDR-grafted meditope-enabled antibody binds to meditope. [0505] Residues of meditope-enabled antibodies that make [0500] It is noted that optimizing the production of the contact with the meditope, line the cavity, and/or are other CDR-grafted meditope-enabled trastuzumab would produce wise important (such as residues described for modification more material for more rigorous/quantitative characteriza herein), are mutated. In one example, structural data, such as tion. The low amount of material and thus less rigorous/ those obtained in the studies described above, are used to quantitative characterization (e.g., uncertainty in the final replace residues in the Fab, by mutagenesis, for example, to concentration of the meditope-enabled trastuzumab and its add additional hydrogen bonds, substitute amino acids for labeling with Alexafluor 647) in this study likely accounted unnatural amino acids or alter the hydrophobic interface, for for the apparent reduced affinity. There are art-known meth example, in ways that might better complement meditope ods to optimize antigen binding. The data show that the CDR- binding. (See FIG. 21). grafted meditope-enabled trastuzumab bind the trastuzumab [0506] In one example, individual residues are systemati antigen (i.e., bind to HER2 overexpressing SKBR3 cells) and cally altered, followed by production and characterization of bind to the meditope of SEQ ID NO: I (i.e., antigen-express the mutant antibodies. Inanother example, a library of IgGs is ing cells pretreated with the CDR-grafted meditope-enabled generated at the DNA level using degenerate oligos at sites of trastuzumab bind to the meditope). interest to produce members that collectively encode all 20 naturally-occurring amino acids at the site or sites of interest, C. Generation of Meditope-Enabled CEA-Binding such that the library produces individual members of the Antibody (Meditope-Enabled M5A) by Mutation library having each amino acid substituted at one or more given site. [0501] Additionally, an anti-CEA antibody (M5A) was [0507] A GPI domain was added, e.g., to the C-terminus of meditope-enabled by mutation of residues to correspond to the Ig heavy chain, of the antibodies in the library. In one those of cetuximab. Specifically, as shown with shading in example, the library is transfected using standard methods; FIG. 56, eight point mutations were introduced in the light antibodies (e.g., IgGs) from the library are expressed. chain of the M5A antibody, allowing it to bind to meditopes. [0508] To demonstrate binding to a GPI-Iinked meditope- The wild-type heavy chain sequence and the meditope-en enabled antibody according to these methods, a GPI-Iinked abled light chain sequence were cloned into a Lonza meditope-enabled trastuzumab was produced. FIG. 54 shows glutamine selection expression vector and transfected in NSO that this GPI-Iinked meditope-enabled trastuzumab bound to US 2012/0301400 Al Nov. 29, 2012 43

a meditope-Protein L (MPL), produced as described in nector. For example, meditope 55 is a linear peptide that binds Example 10, below. In this study, Ixl CF 6 cells/sample were within the meditope-binding site. Individual meditopes in used. Cells were removed from plates by gentle pipetting and these tables are discussed below. were washed once with 0.1% BSA (w/v) in PBS. AF647 MPL [0512] As described above, the cyclic peptide cQFD (SEQ was diluted to 10 nM in wash buffer and incubated with cells ID NO: I) and cQYN (SEQ ID NO: 2) were identified and for 30 min at room temperature. Cells were washed twice and co-crystallized with cetuximab Fab, and shown to bind in a then analyzed by FACS. cavity created by the light and heavy chains of the Fab. Bio [0509] Antibodies produced from the library are screened physical and biochemical methods were used to characterize for one or more desired traits. In one example, to select this interaction. Specifically, mutation of Phe3, Leu5, and mutations that do not affect antigen binding, antibodies from Arg8 to alanine reduced the affinity of the meditope for the the library (e.g., cells expressing the antibodies) that bind to a binding interface by 44-183-fold. See Tables 6 and 7. fluorescently labeled antigen to which the antibodies specifi [0513] Meditope Modification and Chemistry Design cally bind (e.g, HER2, EGFR, IGFR, CLTA-4, etc.) are [0514] Based on the structural and thermodynamic data, selected, e.g, by FACS (FIG. 20). In one example, antibodies multiple positions within the provided meditopes were iden selected fortheir antigen-binding capabilities are subjected to tified as targets for modification, e.g, with substitutions and/ another round of selection, for example, to select for mutant or non-natural amino acids, for example, to enhance the over antibodies that bind to a specific meditope, meditope analog, all binding affinity and/or to alter another property. or other molecule of interest. Modifications included generation of head-to-tail cyclic lac [0510] Following selection, the selected antibodies are tam peptides, modification of Arg8, modification of Phe3, characterized to determine the desired combination of muta modification of Leu5, modification of LeulO, and incorpora tions. In one example, following cell sorting, PCR is used to tion of hydratable carbonyl functionality (see FIG. 31). identify the resulting mutations that facilitate or enhance [0515] Modification of Arg8. meditope/analog/small molecule binding. In one example, [0516] Modifications were made to Arg8. Based on struc this process is repeated multiple times to ‘evolve/optimize’ tural data, it was determined that Arg8 of the unmodified the desired characteristic, e.g, binding, pFl dependency, PK, meditope (cQFD; SEQ ID NO: I) is extended, making a and/or PD. hydrogen bond with the heavy chain carbonyl of Q105 of the meditope-enabled antibody heavy chain. The immediate area Example 6 about this residue is hydrophobic, yet solvent-exposed (FIG. 33A). Variant Meditopes [0517] Structural data indicated that incorporation of a [0511] Several variant meditopes were generated. For modified Arg8 residue that maintains the guanidinium func example, certain meditope variants in Tables 3 and 4 (above) tionality for meditope-enabled antibody H-bonding, while were synthesized, with demonstrated binding affinities to simultaneously introducing a hydrophobic arm to partially fill cetuximab. Forthepeptides in Table 3, a disulfide linkage was the cavity, could produce significant gains in binding, due to used to connect the C and N termini (except that meditope 31 entropic increases, as supported by ligand docking calcula contained an additional tail, meaning that the disulfide link tions. age is not between the two terminal residues). Meditopes 26, [0518] A variant meditope was generated containing an 27, 28, and 29 were biosynthesized and thus in this example, n-butyl-arginine at position 8, meditope 54 (SEQ ID NO: 54, contained an additional serine before the first cysteine, Te, shown in Table 4), as follows: position zero. Further, in some embodiments, meditope 31 may optionally include a GGSK linker. For the peptides in Synthesis of Fmoc-N-butyl Arg Derivative 3 Table 4, a lactam bridge, a linkage other than disulfide (such (Meditope 54) as [3+2] cycloaddition), or no linkage was used as the con [0519]

16 US 2012/0301400 Al Nov. 29, 2012 44

[0520] To a stirred solution of 15, above, (23 mg, 0.03 3H), 4.16 (t, J=6.4 Hz, 1H), 3.30-3.02 (m, 4H), 2.92 (s, 2H), mmol) in CH2Cl2 (0.6 mL) were added EDCI (12 mg, 0.06 2.54 (s, 3H), 2.48 (s, 3H), 2.07 (s, 3H), 1.98-1.80 (m, 1H), mmol, 2 equiv) and n-butylamine (4.4 mg, 0.06 mmol, 2 1.76-1.50 (m, 3H), 1.45 (s, 6H), 1.36-1.26 (m, 4H), 0.85 (t, equiv). After 5 min at room temperature (rt), the solvent was J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 171.9, 158.7, removed in vacuum. The residue was purified by silica gel column chromatography (40-50% EtOAc/Hex) to afford the 156.4, 155.0,144.0, 143.8,141.5, 138.5,133.8, 132.4,131.5, product 16, above, (23 mg, 95%). 1H NMR (400 MHz, 128.0, 127.3, 125.2, 124.6, 120.3, 119.6, 117.6, 86.5, 67.4, CDCl3) δ 7.78-7.70 (m, 2H), 7.60-7.54 (m, 2H), 7.42-7.37 66.5, 53.3, 47.3, 43.5, 41.5,41.0, 30.9, 30.7,28.8, 25.4, 20.2, (m, 2H), 7.35-7.27 (m, 2H), 5.95-5.83 (m, 1H), 5.52-5.42 (m, 19.5, 18.2, 13.9, 12.7; HRMS C41H52N4O7S [M+Na]+ calc’d 1H), 5.36-5.22 (m, 2H), 4.61 (d, J=5.4 Hz, 2H), 4.44-4.30 (m, 767.3449. found 767.3455.

NHn-Bu

PhMeNH, Pd(PPh3)4

NHFmoc

[0521] To a stirred solution of 16, above, (23 mg, 0.03 mmol) in THF (0.8 mL) were added N-methylaniline (10 mg, 0.09 mmol, 3 equiv) and Pd(PPh3)4 (2 mg, 0.0015 mmol, 0.05 equiv). After 45 min at rt, the solvent was removed in vacuum. The residue was purified by silica gel column chromatogra phy (MeOH:CH2Cl2:HOAc=25:l:0.1) to afford the product 17, above, (20 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 7.78-7.70 (m, 2H), 7.60-7.54 (m, 2H), 7.40-7.34 (m, 2H), 7.30- 7.24 (m, 2H), 5.95-5.83 (m, 1H), 4.48-4.10 (m, 5H), 3.30- 3.02 (m, 4H), 2.90 (s, 2H), 2.58 (s, 3H), 2.50 (s, 3H), 2.07 (s,3H), 2.00-1.86 (m, 1H), 1.86-1.74 (m, 1H), 1.72-1.60 (m, 2H), 1.45 (s, 6H), 1.30-1.22 (m, 4H), 0.82 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 174.9, 158.9, 156.6, 155.2, 144.0,143.8, 141.5,138.5, 133.3,132.4, 129.3,128.5, 128.0, 127.3, 125.3, 124.8, 120.2, 117.7, 86.6, 67.4, 53.4, 47.4, 43.4, 41.6, 41.1, 31.4,29.9, 28.8, 25.2,20.1, 19.5, 18.2, 13.9, 12.7; HRMS C38H48N4O7S [M+Na]+ calc’d 727.3136. found 727.3144. [0522] See Martin, N. I., and Liskamp, R. M. J Org. Chem. 2008, 73,7849-7851. US 2012/0301400 Al Nov. 29, 2012 45

Synthesis of Arg-Butyl Meditope 54. [0523]

[0524] Additionally, meditope 54 ((SEQ ID NO: 54), in the desired to introduce one or more halogen atom onto the phe above structure) was prepared as according to solid phase nyl ring, which could participate in halogen bonding (rela Fmoc synthesis protocol using Fmoc-N-butyl Arg derivative tively strong non-covalent bonding, similar to a hydrogen 17. FIG. 59 shows an FlPLC trace and mass spectrum of bond but involving the interaction of a halogen such as bro meditope 54. mine or chlorine with an oxygen atom), such as by incorpo [0525] The structure of meditope 54 bound to a cetuximab ration of an ortho-meta- and/or para-bromo phenyl substitu Fab fragment (backside view) is shown in FIG. 48, with ent to favorably place a bromine atom for halogen bonding structures of the 5-position β,β'-diphenylalanine meditope with Tyr87 (light chain), Gln39, and/or Tyr91 (heavy chain) (SEQ ID NO: 18, meditope 18) and cQFD (meditope I, SEQ of a meditope-enabled antibody, respectively. Meditopes 36 ID NO: I) superimposed. As shown in Table 7, below, medi (SEQ ID NO: 36), 37 (SEQ ID NO: 37), and 38 (SEQ ID NO: tope 54 was determined by SPR to bind to cetuximab with an 38) were generated, containing 2-bromo-L-phenylalanine, average Kd of 745.2 nM. 3-bromo-L-phenylalanine, and 4-bromo-L-phenylalanine, [0526] In some examples, this meditope-enabled antibody respectively, in place of the Phe at position 3. These medi binding pocket identified in these studies is explored as topes were co-crystallized with cetuximab Fab. These com described herein for new contacts (meditopes or analogs) that mercially available derivatives were incorporated by SPPS. may increase affinity of a meditope-meditope-enabled anti Structures are shown in FIG. 39. Diffraction data are shown in body interaction. FIG. 40. Affinities of some of these meditopes for the cetux [0527] Modification of Phe3. imab Fab (average Kd values) were determined by SPR and [0528] Various modifications were made to Phe3 of cQFD. are listed in Table 7, below. Structural data demonstrated that the hydroxyl group of the [0530] Additionally, based on the structural information for meditope variant Phe3Tyr cQYN (SEQ ID NO: 2) has an the meditope containing a Phe3His mutation (meditope 33, alteration in the extended conformation of the Arg8 side chain SEQ ID NO: 33), a meditope was synthesized with β,β'- as compared to cQFD (meditope I) (see FIGS. 30C and 35). diphenylalanine at position 3 (meditope 17, SEQ ID NO: 17). SPR demonstrated that the overall affinity of this variant for As shown in FIG. 41, a significant improvement in the bind the cetuximab Fab was reduced. ITC measurements indicated ing affinity for cetuximab was observed by SPR, representing a significant decrease in entropy for this Phe3Tyr cQYN a roughly 4-fold increase compared to cQFD (increase in variant upon binding that was off-set by a favorable increase affinity by a factor of approximately 4 to 5 (-200 nM)). in enthalpy compared to unmodified meditope (SEQ ID NO: [0531] Modification of Leu5 and LeulO. I) (from -2.1 kCal/mol to -7.9 kCal/mol [n=3]) (FIG. 30D). [0532] Modifications were made to Leu5 and LeulO. It was Structural data suggested the formation of a favorable hydro determined that the Leu5 and LeulO side chains make hydrogen bond network, with water bound to the Fab. phobic contacts to the meditope-enabled Fab (FIG. 36, right [0529] It was determined that when bound to a meditope- panel; LeulO). In one example, natural amino acids (Phe/Tyr/ enabled antibody, the hydrophobic phenyl ring of Phe3 was Trp) and/or non-natural analogs (e.g., β,β'-diphenylalanine, surrounded by a fairly polar array of side chain residues of a branched alkyl, extended aromatics such as napthyl, etc.) are meditope-enabled antibody (cetuximab) Fab (FIG. 35). It was systematically introduced via SPPS at one or both of these US 2012/0301400 Al Nov. 29, 2012 46

positions. The observation, above, that introduction of β,β'- [0541] Meditope 55, a linear peptide, was produced and diphenylalanine at position three increased the overall affinity demonstrated to bind to the meditope-enabled antibody, for the cetuximab Fab (see FIGS. 41-42), demonstrated suc- cetuximab, albeit with reduced affinity. cessby introducing such non-natural amino acids inthemedi- [0542] Hydratable Carbonyl Functionality. topes. Accordingly, a β,β'-diphenylalanine was introduced at [0543] In another example, a meditope with hydratable car position 5 and the average affinity of the resulting meditope bonyl capabilities is developed to create a highly selective but (meditope 18, SEQ ID NO: 18) determined by SPR to be 687 irreversible interaction. Several Fab hydroxyl-bearing side nM (see Table 7, below). These data confirm that use of chains in a meditope-enabled antibody that surround the structural biology to identify regions for alteration and muta meditope cavity are exploited through selective trapping, by tion of residues are useful to alter characteristics of the medi- formation of their corresponding hemi-acetal or -ketal, using topes, such as to improve their binding kinetics. In another a hydratable-enabled meditope. For example, Arg8 of the embodiment, the same modification may be made to Leuf 0. meditope extends in proximity to Ser43 of the light chain (3.5 [0533] Alternative Cyclization Strategies and Replacement A) and Tyr91 of the heavy chain (3.8 and 4.0 A) of the of Disulfide Bridge meditope-enabled antibody light chain, according to Rabat [0534] Alternative cyclization strategies were used to numbering (FIG. 36, left panel). In one example, incorpora replace the disulfide bridge in cQFD and other meditopes. As tion of a hydratable carbonyl functionality at the end of Arg8 shown in Table 4, various lactam cyclization strategies were or LeulO of the meditope allows selective formation of a used, including those involving natural and non-natural serine or tyrosine hemi-acetal, which essentially affords irre amino acids, generated based on different starting materials, versible binding. In another example, a residue containing including glycine, β-Ala, 7-aminoheptanoic acid, diamino- boronic acid is integrated into the meditope as an alternative propionic acid, and isoaspartic acid, to produce different lac to a hydratable carbonyl group. Boronic acid plays an impor tam ring sizes (see FIG. 31, left and middle boxes). tant role in the structural activity of bortezamib (Velcade®), [0535] In one example, a 7-aminoheptanoic acid was used which is used to treat multiple myeloma. Representative to replace the disulfide bridge of the original cQFD meditope, examples of such hydratable residues are also shown in FIG. and the affinity of the resulting meditope for the cetuximab 36 or 34, where R=—CH2CHO or —CH2B(OH)2. In some Fab fragment determined by SPR (meditope 42, SEQ ID NO: examples, such analogs are modified using SPPS (Duggan, P 42). The SPR data are shown in FIG. 41, bottom panel. J. and D. A. Offermann (2007). “The Preparation of Solid- Although the binding affinity was decreased compared to Supported Peptide Boronic Acids Derived from 4-Borono-L- cQFD, these data indicate that modifications can be made to phenylalanine and their Affinity for Alizarin,” Australian the meditope to address potential issues with pharmacokinet Journal of Chemistry, 60(11): 829-834. ics, pharmacodynamics and toxicity in animal and human [0544] Other Methods studies. It is noted that alternative linkers can be combined [0545] In some examples, fluorescence polarization assays with unnatural amino acids atotherpositions within the medi are used to identify meditope variants that can displace a tope. given meditope, such as SEQ ID NO: I. In other examples, [0536] Other variant meditopes with lactam linkages were the same technique is used to identify small molecules that generated using glycine, 7-aminoheptanoic acid, iso-aspartic can displace the meditope and then use these small molecules acid, β-alanine, and diaminopropionic acid (see meditopes as templates to further improve the binding affinity of the 42,43, 44, 45, 46, 49, 51, 52, 53, and 54, IistedinTable 4 and meditopes. others). Affinities for some of these meditope variants for [0546] Characterization of Meditopes cetuximab Fab are listed in Table 7. [0547] For characterization, variant meditope peptide Iyo- [0537] An azide alkyne Huisgen cycloaddition was used to philized powders were suspended in 500 pL of 10 mM Tris produce a triazole as a linkage in meditope 50, by incorpo pH 8.0 buffer and dialyzed 3 times into I L OfH2O each time. rating propargyIglycine at position 12 and beta-azidoalanine The final volume after dialysis was carefully measured and at position I and carrying out a cyclization between these absorbance measurements were taken to estimate the concen termini. tration (typically I-IOmM). These stock solutions were used [0538] Additional cyclization strategies, such as ‘click’ to make dilutions into HBS-EP buffer (10 mM Hepes pH 7.4, chemistry and olefin metathesis, also were used (FIG. 31, 150 mM NaCl, 3 mM EDTA and 0.05% v/v surfactant P20) right boxes). For example, head-to-tail lactam peptides were for SPR measurements. SPR measurements were carried out designed and synthesized by solid phase peptide synthesis on the GE Biacore TlOO instrument using a CM5 chip with (SPPS) starting from Fmoc-Asp (Wang resin LL)-Oall (FIG. cetuximab IgG or cetuximab Fab ligand immobilized using 31, lower left box & FIG. 32). One such variant, meditope 32 amine coupling chemistry. Ligands were immobilized at low (SEQ ID NO: 32), with FITC conjugation, was shown to bind levels suitable for kinetic data. Typical kinetics SPR experi to cetuximab in a similar manner but with slightly reduced ments were carried out at a flow rate of 30 pL/min using affinity compared to the unmodified meditope of SEQ ID NO: HBS-EP as both running and regeneration buffer. Kinetic I (meditope I). Other head-to-tail lactam variant meditopes parameters were calculated using the Biacore TlOO evalua produced include meditopes 33-47,49, and 50-54 (see Tables tion software version 2.0.1. 4 and 7). Meditope 48 in Table 4 was engineered with a [0548] Isothermal titration calorimetry experiments were disulfide linkage and a 4:11 lactam linkage. performed in 100 mM Hepes, pH 7.4 at 25° C. using Nano [0539] Meditope 32 was conjugated with fluorescein for ITC calorimeter (TA Instruments). In a typical experiment, FACS analysis. In another example, this strategy is applied to 250 μΐ of protein (Fab or IgG) at 0.03-0.06 mM were loaded conjugate the meditope with DOTA for in vivo PET imaging. into the calorimeter cell (163 μΐ or 185 μΐ) and the titrant [0540] Structural data demonstrated that additional posi (meditope, at 0.3-0.8 mM) was loaded into a 50 μΐ syringe. tions are amendable to cyclization, such as by cyclization The cell solution was stirred at 250 rpm and upon equilibra between residues 3 and 11 or 4 and 11. tion the titrant was added in 2-2.5 μΐ increments. Heat of the US 2012/0301400 Al Nov. 29, 2012 47

reaction was measured and the data was processed using NanoAnalyze software (TA Instruments). Background heat TABLE 7-continued was subtracted by averaging the last four measurements or by subtracting heat of reaction obtained from titration of the Dissociation Constants Determined bv SPR meditope at the same concentration into buffer containing no SEQ ID NO/ protein. Meditope No. Average Kd (nM) [0549] A number of the meditope variants were co-crystal- 22 54820 lized with cetuximab Fab fragment. Meditopes were purified 23 9460 to >95% homogeneity and structurally characterized by mass 24 14180 spectrometry. Peptides were dialyzed in water. Their concen 25 39340 trations were measured, for example, in some cases, by UV- 26 34000 Vis and calibrated with elemental analysis, and diluted 27 57000 28 140000 (>100x) into the appropriate buffer. The structures of some of 29 2200 these meditope variants, corresponding to SEQ ID NOs: 31 102.5 15-18, 22-25 and 31-40, are shown in FIG. 39. 32 5041 33 No binding [0550] Interactions of various meditopes with the medi- observed tope-enabled antibody cetuximab were characterized by 34 No binding X-Ray diffraction. Since the co-crystallization conditions of observed the cetuximab Fab fragment and the meditope of SEQ ID NO: 35 No binding observed I are well-established, diffraction quality crystals were typi 36 1791 cally obtained in I to 3 days, typically I day. Full data sets 37 29130 were collected in 8 to 12 hours with an in-house source 39 8186 (Rigaku007-F1F and an R-Axis IV++) and in less than IOmin 40 No binding observed at the Stanford Synchrotron Radiation Lightsource, which 41 No binding allows for rapid characterization of the interactions of the observed meditope variants with cetuximab. 42 1520 [0551] Diffraction data were collected for various different 43 1619 44 16490 meditope-Fab (cetuximab) complexes, including complexes 45 4634 containing the cQFD (SEQ ID NO: I) and cQYN (SEQ ID 46 5467 NO: 2) meditopes and modified meditopes. X-ray diffraction 48 517.1** data for several meditopes are shown in FIG. 40. Most of 49 23800 50 21002 these co-crystal structures were shown to diffract beyond 2.4 51 433.3 A and be well refined, with R and Riiee less than 20 and 24%, 52 1264 respectively. All of these meditope variants have very good 53 No binding stereochemical values (Molprobity scores above 89tA percen observed 54 745.2 tile). 55 8684 [0552] For SPR measurements, low density and high den sity chips were conjugated with the cetuximab Fab or full **In this study, meditope 48 hydrolyzed to the original meditope. IgG. Each chip was first characterized using a soluble frag [0554] Binding of several meditopes to the meditope-en- ment of the entire extracellular domain of EGFR (residues abled antibody cetuximab was rigorously characterized by 1-621). Similarkinetics and binding affinities were observed ITC, SPR, X-ray diffraction and combinations thereof. ITC as previously reported. Using the low density chips, on and measurements were performed on a TA Instruments off rates were measured for the unmodified meditope and nanoITC, with as little as I -2 mg of peptide permeasurement. determined to be ko„=9.2xl0-4 M_1s_1 andkOJf=9.9xl0_3 s-1, ITC, SPR and X-ray diffraction data, e.g., collectively, pro respectively. With meditopes of SEQ ID NO: I and 2, con vide atomic detail to guide subsequent chemical modifica sistent with ITC, similar values for the Fab-conjugated chip tions and ultimately improve the affinity of the meditopes and the IgG-conjugated chip were observed, demonstrating and/or make other alterations to the meditopes. A calculation that either can be used for binding assessments. based on AG=-RT In Ka shows that the difference between [0553] Table 7, below, lists information on the average dis micromolar and nanomolar affinity of a meditope for cetux sociation constants (K13S), which were determined by SPR, imab results from a change in free energy at 300 K of -4 for several of the meditopes listed in Tables 3 and 4. kCal/mol, which is on the order of a strong hydrogen bond. Thus, the loss of an ordered water molecule from a protein TABLE 7 binding pocket or the reorientation of an amino acid residue- Dissociation Constants Determined by SPR chain may be sufficient to alter binding by orders of magni tude. SEQ ID NO/ [0555] The data in this example demonstrate that a large Meditope No. Average Kd (nM) number of meditope permutations may be systematically and I 898.8 efficiently introduced, indicating that a large number of medi 2 3500 tope variant permutations may be generated, for example, to 15 18300 produce altered meditope-meditope enabled antibody bind 16 2828 ing affinity, pharmacokinetics (PK), pharmacodynamics 17 624.9 18 687 (PD), toxicity, and/or ability to bind or strength of binding 21 11180 under differing conditions, such as pH, e.g., pH dependence, for example, to produce high affinity meditopes. US 2012/0301400 Al Nov. 29, 2012 48

[0556] In one example, after characterization by ITC, SPR (FIG. 61A, white arrows). This co-localization, however, was and diffraction methods, the meditope with the highest affin absent in cells which that were not pre-treated with cetuximab ity (or other desired property, e.g., pH dependence) is subse (FIG. 61B). quently modified, e.g., to further improve the desired property [0563] Taken together, these experiments show that the (e.g., overall affinity or pH dependence). meditope binds to EGFR-expressing cells that are pre-treated with cetuximab, that using a multivalent scaffold affords cre Example 7 ates a higher apparent affinity for cetuximab pre-treated cells, and that the apparent binding affinity, in this study, was more Generation of Multivalent Meditopes sensitive. [0564] The ability of a bivalent meditope-Fc to induce cell [0557] Bivalent and other multivalent meditopes were gen death in conjunction with the meditope-enabled antibody, erated, e.g., for use in enhancing selectivity and/or binding cetuximab, was confirmed using an MTT assay. 4000 MDA- affinity by “cross-linking” meditope-enabled antibodies on MB-468 cells were placed in each well of a 96 well plate in 80 the surface of cells expressing antigen. μΐ of medium. 10 μΐ of I μΜ cetuximab was added along with [0558] Bivalent Meditope-Fc 10 μΐ of 0.1, I or 10 μΜ of monovalent meditope (cQFD— [0559] The use of the Fe region to ‘dimerize’ ligands is (control) or bivalent meditope-Fc (two cQFDs) to a final established and described, for example, by Jazayeri J A & concentration of 0.1 μΜ cetuximab and 0.01, 0.1 and I μΜ Carroll G J., “Fc-based cytokines: prospects for engineering meditope or meditope-Fc. Each component was also added superior therapeutics,” BioDrugs, 22(1):11-26 (2008) To gen alone with PBS as control. After a 48-hour incubation, 10 μΐ erate a bivalent meditope, the meditope cQFD (SEQ ID NO: of MTT reagent was added and allowed to incubate for I) was fused to the N-terminus of the Fe region of an IgG another 4 hours. The culture supernatant was then removed, through a flexible peptide linker of 17 amino acids in length, 100 μΐ of MTT crystal dissolving reagent was added, and the comprised of glycine and serines. The length of the linker was plate was read at 630 nm. Neither addition of monovalent chosen to roughly match the distance between the Fabs of an meditope (alone or with cetuximab), or meditope-Fc alone IgG. The nucleic acid and amino acid sequences of the result altered cell growth significantly. Addition of meditope-Fc ing “meditope-Fc” are shown in FIG. 15 (SEQ ID NO: 3 and together with cetuximab, however, inhibited cell growth, as 4, respectively). The structure of the meditope-Fc is shown in shown in FIG. 28A. FIG. 16. [0565] The ability of the multivalent meditope-Fc to [0560] To demonstrate enhanced binding to antigen enhance cell killing by cetuximab to a degree comparable to afforded by the multivalency of the meditope-Fc, 0.5xl06 a second anti-EGFR antibody was demonstrated by MTT MDA-MB-468 cells were labeled with 10 nM cetuximab for assay. The assay compared the enhancement of cetuximab- 30 min at room temperature, washed, then incubated with 0.1, mediated inhibition of antigen-expressing tumor cell growth 0.3,1 and3 μΜ of bivalent meditope-Fc or monomeric medi by meditope-Fc and by M425 (a mouse anti-EGFR antibody). tope for 30 min at room temperature, washed, and then ana 4000 MDA-MB-468 cells were placed in each well of a 96 lyzed by FACS. As shown in FIG. 17, FACS analysis dem well plate in 80 μΐ of medium. 10 μΐ of I μΜ cetuximab was onstrated that the meditope-Fc, corrected for the added, along with 10 μΐ of either 2, 4 or 8 μΜ of meditope-Fc stoichiometry, bound to cells pre-treated with cetuximab with or M425, to a final concentration of 0.1 μΜ cetuximab and higher affinity compared to the meditope monomer. The inter either 0.2, 0.4 or 0.8 μΜ meditope-Fc or M425. cetuximab action was demonstrated to be specific to the meditope-en added with PBS alone was used as control. After a 48-hour abled mAh (cetuximab). These data demonstrate synergy incubation period, 10 μΐ MTT reagent was added and the using meditope-Fc, combined with a meditope enabled mAh, mixture allowed to incubate for an additional 4 hours. The and thus that a bivalent meditope can be substituted for a culture supernatant was removed, 100 μΐ of MTT crystal second antibody to produce syneigistic effects. dissolving reagent added, and the plate read at 630 nm. As [0561] In another study, the meditope-Fc fusion protein shown in FIG. 28B, meditope-Fc and M425 enhanced the was labeled with Alexa488 as described above. MDA-MB- cell-killing capacity of cetuximab to a similar degree. 468 cells were labeled with cetuximab or M425 (a murine [0566] In some examples, the composition of and the dis anti-EGFR antibody) for 30 minutes. Unbound antibody was tance between the Fe and meditope are systematically washed and the cells were incubated with the meditope-Fc explored to optimize affinity and specificity. In one example, (600 nM, 180 nM, or 60 nM) for 30 minutes. Antibody bind each natural or unnatural residue is substituted at any position ing and meditope binding were analyzed by FACS analysis. within the linker, for optimization. In another example, the The FACS data, shown in FIG. 49 demonstrate that the medi- linker is ‘rigidified’ to limit the radius of gyration and to tope-Fc bound to the cells incubated with cetuximab, but not enhance the affinity and specificity of the Fc-meditope. In one to cells incubated with M425 or those not incubated with any example, a coiled coil domain is placed between the meditope antibody, with increasing signal at higher concentrations of and the Fe (FIG. 18). In another example, inert protein meditope-Fc. domains (e.g., immunoglobulin folds) are substituted for the [0562] Similar to the FACS experiments described above, linker. In one example, multiple immunoglobulin folds are MDA-MB-468 cells were treated with Alexa 555-labeled placed between the meditope and the Fe domain. In certain cetuximab, washed, incubated with Alexa 488-labeled medi- examples, the composition of the linker is of human origin, tope-Fc, and washed. The cells were then imaged at 20x by e.g., to mitigate potential antigenicity. microscopy (FIG. 61A). Strong surface-associated staining [0567] Multivalent Scaffolds of Cetuximab was observed. The meditope-FC also localized [0568] To address the receptor constraints on the linker, to the cell surface and appeared to co-localize with cetuximab meditopes were coupled to multivalent scaffolds. (FIG. 61A). Concomitant with this staining, we also observe [0569] Multivalent meditopes were designed to “latch-on” the co-localization of the labeled meditope-Fc was observed to adjacent IgGs to form a “daisy-chain”-like array (see FIG. US 2012/0301400 Al Nov. 29, 2012 49

8). Synthesis of a FITC-Iabeled bivalent meditope was devel In one example, the data are compared with data from anti oped using “Click” chemistry, using compound 2 (SEQ ID body (e.g., cetuximab)-only treated cells and cells treated NO: 32). Templates 4 and 5 of FIG. 13, were used to form bl with inhibitors (e.g., tyrosine kinase inhibitors (AG1478)). and trivalent meditopes, respectively. A 30 A PEG bifunc An increase in cell death as a function of multivalent medi tional arm was incorporated in the synthesis of a FITC-Ia- tope concentration is observed. beled bivalent meditope containing meditopes of SEQ ID [0577] In one example, to further confirm the additive NOs: 32 (meditopes 32), namely compound 13, shown in effects of the multivalent meditope, the non-labeled, monova FIG. 13. As also shown in FIG. 13, a trivalent meditope lent meditope is used to compete with the labeled multivalent (compound 14) also was successfully synthesized. FIG. 14 meditope for the antigen-bound cetuximab. illustrates the characterization of this fluorescein isothiocy anate (FITC)-Iabeledmeditope compound 13. Example 8 [0570] In other examples, differing lengths of polyethylene glycol (PEG) (and other) linkers are used, for example, to Meditope Binding Affinity as a Function of pH optimize binding. In other examples, this synthetic approach is used to incorporate DOTA for radionuclide imaging. The [0578] The composition of the meditope was altered to distance between the CDR regions within an IgG is -130 A. affect the binding affinity as a function of pH. As shown in End-to-end distances of commercially available PEGs extend FIG. 27, the binding affinity of three different meditope vari to 90 A (Pierce), which would exceed the IgG distance. In one ants was measured as a function of buffer pH. The results example, the length of the PEG linker is systematically var demonstrated that cQYD (SEQ ID NO: 16, meditope 16) ied, bearing in mind this constraint. meditope variant had a marked decrease in affinity for the [0571] In some examples, trivalent or higher valency scaf meditope-enabled antibody cetuximab at lower pH. Substi folds are used, with the goal of having more than one antibody tution of the aspartate to asparagine in the cQYN variant “daisy chained”. In some example, different scaffolds and produced a flat pH dependence. The affinity of the cQFD linkers are used to generate high affinity multivalent medi meditope (SEQ ID NO: I) was determined to be slightly topes. In one example, DNA is used as a more rigid scaffold. greater at higher pHs. Collectively, these data demonstrate [0572] Different scaffolds of biological and chemical ori that the affinity of the meditope-memAb interaction can be gin also are used to achieve multivalency. This includes, but is tailored to pH, which is used, for example, to generate medi not limited to, constructing a bivalent or trivalent scaffold, tope variants for the specific release at low pH, such as in using streptavidin or collagen (http://ip.com/patapp/ lysozymes for drug delivery, and/or to bind with higher affin EP2065402A1), strepavidin as a tetravalent scaffold, unique ity in a hypoxic environment, e.g., tumor stroma. scaffolds (http://www.sciencedirect.com/science/article/pii/ S0022283611000283), Origami DNA (http://www.nature. Example 9 com/nnano/joumal/v4/n4/abs/nnano.2009.5.html) and the like. A chemical scaffoldmay also be created using molecules Meditope Analogs, Fragments, and Other Com including, but not limited to, DNA (single strand, duplex, pounds Holliday junctions, aptamers and the like), RNA (single strand, hairpin, stem loop, aptamers and the like), PNA (pep [0579] Screening methods were carried out to identify tide nucleic acids), DNA/PNA duplexes and triplexes for meditope analogs and compounds, such as small molecules, rigidity, inorganic or organic nanoparticles (directly coupled fragments, aptamers, nucleic acid molecules, peptibodies or coupled through organic polymers such as PEG), organic and/or other substances that bind meditope-enabled antibod polymers that can form duplexes with themselves and/or with ies near meditope-binding sites and in some aspects can be DNA or PNA. linked to meditopes, for example, to improve their affinities [0573] Multivalent Meditope Characterization for the meditope-binding sites. [0574] In one example, the multivalent meditopes are char [0580] Fluorescence Polarization Assays: acterized by SPR and ITC, to verify that conjugation to the [0581] To identify compounds, including alternative mol multivalent scaffold does not affect the meditope-IgG inter ecules that could bind at the meditope binding site of medi action. tope-enabled antibodies and thus be used for similar func [0575] In other examples, FACS analysis, cell viability tions, a displacement assay was established. A cyclic assays, and other assays are used. For example, cell viability meditope was synthesized and chemically attached to a fluo assays are used as described above for meditope-Fc to quan rescein, generating a fluorescein-labeled meditope of the fol tify the effect of the multivalent meditope directly on cells lowing sequence: AcCQFDLSTRRLRCGGGSK (SEQ ID that overexpress the antigen recognized by the meditope- NO: 31, cysteines form a disulfide linkage)-Fluorescein. This enabled antibody of choice, such as EGFR when the medi- fluorescently-labeled peptide was then titrated with cetux tope-enabled antibody is cetuximab. For example, MTT, 3-(4, imab and the fluorescence polarization measured. The inter 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, action between the labeled meditope and mAh cause a change is used to quantify the number or percentage of viable cells. In in the fluorescence polarization/intensity of the fluorescent some examples, a shift at far lower concentrations than tag. The dissociation constant, I μΜ, closely matched values observed for the corresponding monovalent meditope and/or obtained from surface plasmon resonance and isothermal a relative increase in percentage of cells that shift, is observed. titration calorimetry. A non-labeled meditope, AcCQFDL [0576] In some examples, for multivalent meditopes that STRRLRCGGGSK (SEQ ID NO: 31), was used to displace demonstrate activity in such assays, Western blot analysis is the fluorescein-label peptide pre-bound to cetuximab. The performed to follow other parameters, such as phosphoryla fluorescence polarization was monitored. Compounds that tion status of EGFR, AKT, and MAPK in the case of antibod blocked the meditope-antibody interaction altered the fluo ies targeting the EGFR signaling pathway, such as cetuximab. rescent polarization properties. As shown in FIG. 37, a sig US 2012/0301400 Al Nov. 29, 2012 50

moid curve indicative of a competition reaction was [0592] In another example, additional analogs found by observed. Accordingly, this method is useful for identifying diffraction methods are optimized and used in lieu of the meditope analogs. meditope for drug delivery, multivalent scaffolding and other [0582] Based on these data, an initial screen to identify functions. small molecules capable of displacing the meditope was car [0593] In another example, mutations in the light and heavy ried out. In this study, 42 lead compounds at concentrations of chains are made to change the specificity of the ligand (medi 50 μΜ were identified from a library of 30,000 small mol tope) and all the above-described methods (including fluo ecule compounds. FIG. 38 shows five such lead compounds. rescence polarization, NMR screening, phage display, and [0583] In another example, these compounds are character diffraction methods are used to optimized alternative ligands. ized further, for example, by crystallography. Example 10 [0584] Diffraction Methods. [0585] Cetuximab Fab was shown to diffract beyond 2.5 A, Meditope-Protein L Fusion as shown above. Established diffraction-based methods [0594] Characterization by diffraction of the cQFD medi- (well-established for identifying lead compounds (Shuker et tope-enabled Fab fragment bound to a meditope demon al. 1996; Erlansonet al. 2001; Flughes et al. 2011)) were used strated that the N- and C-termini of the meditope were juxta to identify candidate compounds, including compounds that posed to the location of bound Protein L, a bacterial protein can be coupled to a meditope, for example, to improve affinity that binds to human IgGs. See FIG. 52, showing the crystal for the meditope binding site. A library of small molecules structure of meditope 18 (5-β,β'-diphenyl), Protein L (left), was developed to soak into crystals of cetuximab. Diffraction Protein A (right) and Fab (grey cartoon) and meditope-en- data from these soaks was collected and several data sets abled trastuzumab Fab. analyzed. In these initial studies, two additional sites were [0595] To generate a meditope that binds to meditope-en- identified on cetuximab that are amendable for fragment abled antibodies with greater affinity via eneigy additivity, a growth and optimization. meditope-Protein L fusion polypeptide was produced. Based [0586] In another example, such fragments (small mol on structural data information, four glycines were introduced ecules that can serve as building blocks for larger entities), to link the C-terminus of the cQFD meditope and the N-ter- such as chemical groups, e.g., imidazole or other chemical minus of Protein L. The coding sequence of a meditope- group, are grown (chemically derivatized) to enhance their Protein L fusion protein is set forth in SEQ ID NO: 56. binding and specificity and/or are chemically tethered to the [0596] The amino acid sequence of an encoded protein, meditope. Optimization of this chemical coupling can signifi including a His6-Smt3 tag (plain text), meditope (under cantly enhance the overall binding affinity. lined), and Protein L (bold) is set forth below and in SEQ ID [0587] NMR Screening: NO: 57: [0588] NMR was used to identify fragments for optimiza tion of meditopes, e.g., by linkageto the meditope, and/or use in lieu of the meditope (i.e., as meditope analogs). To identify (SEQ ID NO: 57) these leads, one dimensional (ID) spectra of pools containing HHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVS

15 to 20 fragments were collected. Cetuximab was added to Dgsseiffkikkttplrrlmeafakrqgkemdslrflydgiriqadqt each pool and second ID spectra were collected. Compounds that bound (transiently) with cetuximab underwent rapid PEDLDMEDND11EAHREQIGGSCOFDT,STRRT,KCGGGGSEVTIKVULI magnetization transfer, resulting in a loss of intensity. The FADGKIQT AEKGT FEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNI spectra were compared before and after addition of cetux imab and altered peaks identified, indicating interactions. KFAG [0589] In one example, these peaks are pre-assigned to a [0597] The amino acid sequence of the meditope-Protein L specific compound, and thus immediately known. Alterna fusion protein, after cleavage of the tag, is set forth below tively, the pools are subdivided and the spectra recollected. (meditope underlined; two cysteins that are cyclized (e.g., by After several rounds, the exact identity of the compound is peroxide or overnight with air) set forth in bold text) SEQ ID known. In these experiments, the precise position of the inter NO: 58: action is not known. The binding site is determined by NMR or the fluorescence polarization assay. Alternatively, the Fab fragment is labeled with NMR active and inactive nuclei (e.g., (SEQ ID NO: 58) 13C, 15N and 2H), followed by multiple NMR experiments SCOFDTiSTRRTiKCGGGGSEVTTKVNLTFADGKTOTAEFKGTFEEATAE performed to assign the spectrum, and use of the fragment library to identify the binding position. Using this procedure, AYRYAALLAKVNGEYTADLEDGGNHMNIKFAG . a set of initial lead compounds has been identified (FIG. 19, [0598] Binding constants for the interaction of this fusion bottom). protein (and separately, the interaction of each individual [0590] Virtual Ligand Screening: component) with meditope-enabled antibody trastuzumab [0591] Virtual ligand screening was used to identify lead were measured by surface plasmon resonance (SPR). The compounds to function as meditopes. Using crystal structure, binding constant for the interaction between Protein L and the standard programs (e.g., Schroerdinger Glide) were used to meditope-enabled trastuzumab was approximately 0.5 μΜ. define a “box’ about a site of the macromolecule (the medi For the interaction between the meditope and the meditope- tope binding site); known ligands were docked to this site. enabled trastuzumab Fab fragment, it was approximately I Potential lead compounds were scored by a select energy μΜ. The binding constant for the interaction between the function. In this study, approximately 100 lead compounds meditope-Protein L fusion protein and the IgG, however, was were identified. 165 pM. FIG. 53 shows surface plasmon resonance (SPR) US 2012/0301400 Al Nov. 29, 2012 51

data for a meditope-Protein L fusion (MPL): The top panel [0606] I. Accardi, L., and Di Bonito, P (2010) Antibodies shows the traces of MPL being added to the meditope-en- in single-chain format against tumour-associated antigens: abled trastuzumab at concentrations up to 10 nM. The fit data present and future applications, Curr Med Chem 17, 1730- indicates a binding affinity of 165 pM. The bottom panel 1755. shows the traces of Protein L (only) added at the same con [0607] 2. Adams, G. P, Schier, R., McCall, A. M., Sim centrations to the meditope-enabled trastuzumab, showing mons, H. H., Horak, E. M., Alpaugh, R. K., Marks, J. D., that there was no binding at this concentration in this study. and Weiner, L. M. (2001) Cancer Res 61, 4750-4755. This result demonstrated far stronger binding between the [0608] 3. Adams, J., Behnke, M., Chen, S., Cruickshank5A. fusion protein and meditope-enabled antibody compared to A., Dick, L. R., Grenier, L., Klunder, J. M., Ma, Y T., the individual interactions, indicating improved affinity via Plamondon, L., and Stein, R. L. (1998) Potent and selective additivity. inhibitors of the proteasome: dipeptidyl boronic acids, [0599] Point mutations were created in Protein L and the Bioorg Med Chem Lett 8, 333-338. meditope to ensure specificity. As expected, in both cases, the [0609] 4. Adams, P D., Grosse-Kunstleve, R. W., Hung, L. binding constant was reduced. W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. [0600] To facilitate conjugation of various molecules, such J., Sacchettini, J. C., Sauter, N. K., and Terwilliger, T. C. as therapeutic and diagnostic agents, e.g., cytotoxins, radio (2002) Acta Crystallogr D Biol Crystallogr 58,1948-1954. nucleotides, for example, DOTA, proteins, solid supports, [0610] 5. Adessi, C., and Soto, C. (2002) Converting a and other molecules, e.g., for drug delivery, imaging, or puri peptide into a drug: strategies to improve stability and fication, all lysines in the Protein L-meditope fusion protein bioavailability, Curr Med Chem 9, 963-978. but one were mutated to arginine or another residue. The [0611] 6. Akamatsu, Y, Pakabunto, K., Xu, Z., Zhang, Y, crystal structure shown in FIG. 52 was used to identify all and Tsurushita, N. (2007) Whole lysines on Protein L and the meditope that, if conjugated, [0612] 7. IgG surface display on mammalian cells: Appli would sterically occlude the meditope-Protein L/meditope- cation to isolation of neutralizing chicken monoclonal anti- enabled antibody interaction. Based on this information, the IL-12 antibodies, J Immunol Methods 327, 40-52. meditope-Protein L fusion (MPL) having the sequence set [0613] 8. Alley, S. C, Okeley, N. M, and Senter, P D. forth in SEQ ID NO: 59 (SCQFDLSTRRL- (2010) Antibody-drug conjugates: targeted drug delivery RCGGGGSEVTIRVNLIFADGNIQTAE- for cancer, Curr Opin Chem Biol 14, 529-537. FRGTFEEATAEAYRYAALLARV NGEYTADLEDGGN- [0614] 9. Auffinger, P, Hays, E A., Westhof, E., and Ho, P HMNIKFAG) was produced, in which certain lysines in S. (2004) Halogen bonds in biological molecules, Proc Protein L/meditope (shown in black in FIG. 52—all but one NatlAcadSci USA 101, 16789-16794. lysine) were mutated to Arg or Asn. In the sequence shown [0615] 10. Beck, A., Wurch, T., Bailly, C., and Corvaia, N. above, the residues mutated from lysine to arginine/aspar- (2010) Strategies and challenges for the next generation of agine are shown in bold text. The lysine that was not mutated therapeutic antibodies, Nat Rev Immunol 10, 345-352. (underlined above) is pointed into the solvent. Thus, in the [0616] 11. Beck, A., Wagner-Rousset, E., Bussat, M. C., resulting meditope-Protein L fusion protein, the N-terminal Lokteff, M., Klinguer-Hamour, C., Haeuw, J. F., Goetsch, amine and epsilon amine were left for conjugation, the latter L., Wurch, T., Van Dorsselaer, A., and Corvaia, N. (2008) being solvent exposed and likely more reactive. Trends in glycosylation, glycoanalysis and glycoengineer- [0601] In another example, this unique lysine residue in this ing of therapeutic antibodies and Fc-fusion proteins, Curr mutant is used to PEGylate the MPL, e.g., to address potential Pharm Biotechnol 9, 482-501. antigenicity. [0617] 12. Bilgicer, B., Moustakas, D. T., and Whitesides, [0602] MPL-DOTA-NHS Conjugates G. M. (2007) A synthetic trivalent hapten that aggregates anti-2,4-DNP IgG into bicyclic trimers, J Am Chem Soc [0603] MPL having the sequence set forth in SEQ ID NO: 129, 3722-3728. 59 was conjugated to DOTA-NHS in a reaction carried out [0618] 13. Bilgicer B, Thomas S W 3rd, Shaw B F, Kauf with various ratios (0.5:1,2:1, 10:1, 30:1, 60:1, 120:1, 240:1) man G K, Krishnamurthy V M, Estroff L A, Yang J, Whi of starting material (DOTA-NHS:MPL) in phosphate buffer tesides G M., A non-chromatographic method for the puri (80 mM) for 10 minutes at a pH=l 0, which favors conjugation fication of a bivalently active monoclonal IgG antibody to lysine as compared to N-terminal conjugation. The mono- from biological fluids. J. Am. Chem. Soc. 2009 JUL 8; DOTA-conjugate product increases with increasing DOTA- 131(26):9361-7. NHS. A significant amount of MPL-mono-DOTA-MPL was [0619] 14. Bokemeyer, C., Bondarenko, I., Makhson, A., formed at a 60:1 ratio, and a significant amount of both Hartmann, J. T., Aparicio, J., de Braud, F., Donea, S., MPL-mono-DOTA and MPL-di-DOTA formed at a 120:1 Ludwig, H., Schuch, G., Stroh, C., Loos, A. 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SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 170

<210> SEQ ID NO I <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: I

Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys 15 10

<210> SEQ ID NO 2 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 2 US 2012/0301400 Al Nov. 29, 2012 55

-continued

Cys Gln Tyr Asn Leu Ser Ser Arg Ala Leu Lys Cys 15 10

<210> SEQ ID NO 3 <211> LENGTH: 747 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide-Fc fusion protein

<400> SEQUENCE: 3 gggggctccg gttcaggctc gggcggttca tcgggaggag ggggagggga acctaagtca 60 tgcgataaga cgcacacctg tcctccatgc ccagcccccg agttgcttgg tgggccctca 120 gtattcctct tccctccaaa acccaaagac accttgatga tttcccgcac gccggaagtc 180 acgtgtgtgg tcgtggatgt gagccatgag gatcccgagg tgaagttcaa ttggtacgtg 240 gatggagtag aggtacacaa cgcgaaaacg aagcccaggg aggaacagta caattccaca 300 tatcgcgtgg tgtccgtgct tactgtgttg catcaagact ggctgaatgg gaaggagtat 360 aagtgcaaag tatcaaacaa ggcgctgcct gctccaatcg aaaagaccat ctcgaaggcg 420 aaaggacaac ccagagaacc ccaagtctac acgcttccgc cctcgcggga tgagctcacc 480 aaaaaccagg tatccctcac ttgtttggta aaaggattct acccgtcgga cattgcagtc 540 gagtgggagt cgaatgggca gccggaaaac aactacaaaa caacaccgcc cgtcttggac 600 tccgatggtt cgttctttct ctattcgaag ctcaccgtag acaagtcgag gtggcagcag 660 ggcaacgtct tttcgtgctc agtgatgcat gaggcccttc acaatcacta tacgcagaaa 720 agcctgagcc tgtcaccggg gaagtaa 747

<210> SEQ ID NO 4 <211> LENGTH: 261 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide-Fc fusion protein

<400> SEQUENCE: 4

Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys Gly Gly Gly Ser I 5 10 15

Gly Ser Gly Ser Gly Gly Ser Ser Gly Gly Gly Gly Gly Glu Pro Lys 20 25 30

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35 40 45

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 50 55 60

Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 65 70 75 80

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 85 90 95

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 100 105 HO

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 115 120 125

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala US 2012/0301400 Al Nov. 29, 2012 56

-continued

130 135 140

Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gln Pro Arcj Glu Pro 145 150 155 160

Gln Val Tyr Thr Leu Pro Pro Ser .Arg Asp Glu Leu Thr Lys Asn Gln 165 170 175

Val Ser Leu Thr Cys Leu Val Lys <31y Phe Tyr Pro Ser Asj) He Ala 180 185 190

Val Glu Trf> Glu Ser Asn Gly Gln :Pro Glu Asn Asn Tyr Lys Thr Thr 195 200 205

Pro Pro Val. Leu Asp Ser Asp Gly ;Ser Phe Phe Leu Tyr Sei: Lys Leu 210 215 220

Thr Val Asj> Lys Ser Arg Trp Gln <31n Gly Asn Val Phe Sei: Cys Ser 225 230 235 240

Val Met Hie> Glu Ala Leu His Asn ]His Tyr Thr Gln Lys Sei: Leu Ser 245 250 255

Leu Ser Pro Gly Lys 260

<210> SEQ ID NO 5 <211> LENGTH: 1410 <212 > TYPE: DNA <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 5 atgaaatgct cgtgggtgat CtttttCCtt atggcggtag taaccggagt aaactccgag 60 gtccagctcg tcgaatccgg tggcggcttg gtgcagccgg gtgggtcgtt gcgactgtcg 120 tgcgcagcgt cggggtttaa catcaaagac acctatatcc actgggtgag gcaatcgccc 180 ggaaaggggc tcgaatgggt agccagaatc taccctacga atggttatac tcgatatgcg 240 gactccgtga aaggaagatt caccatcagc gcagatacgt ccaaaaacac tgcatacctc 300 cagatgaata gccttcgggc ggaggacacg gcgatctact actgtagccg gtggggtggg 360 gacgggttct atgcgatgga ctactgggga caggggacgc ttgtaacggt cagctcggcg 420 tcaacaaagg gacctagcgt gtttcccttg gctccctcat cgaaatcaac gtccggtggg 480 acggcggcat tggggtgtct tgtcaaggac tatttccccg agcccgtgac agtctcgtgg 540 aactcgggtg cccttacaag cggcgtacat acgtttcccg ccgtgctcca atcatccgga 600 ctgtattccc tttcatccgt cgtgactgtg ccgtcctcgt cactcggaac gcaaacttac 660 atttgcaatg tcaaccacaa accgtcaaat acaaaggtcg ataagaaggt cgagccaaag 720 tcgtgtgata agacccacac atgccctccc tgtccagcgc cggagctgtt gggagggcct 780 tcagtgttcc tcttcccgcc caaacccaag gacaccctga tgattagccg cacacccgag 840 gtgacgtgtg tcgtcgtcga tgtctcacat gaggacccgg aggtaaagtt caactggtac 900 gtggatggag tcgaagtgca caacgcaaaa acaaaacctc gggaagagca gtacaatagc 960 acgtacagag tagtcagcgt gctcaccgtg ctgcaccagg attggctcaa tggaaaggag 1020 tacaagtgta aagtgtcgaa taaggcgctg cctgccccca tcgaaaagac aatttccaaa 1080 gctaaagggc aaccccgcga gccgcaagta tacaccctcc caccctcgcg cgatgaactg 1140 accaagaacc aggtgtcatt gacgtgtctc gtcaagggct tctatccgag cgacattgca 1200 gtagaatggg aaagcaacgg acagccggaa aacaactaca agactacacc gcctgtcctt 1260 US 2012/0301400 Al Nov. 29, 2012 57

-continued gattcggatg gttccttctt tctttactca aaacttacag tcgacaaatc gaggtggcag 1320 cagggaaatg tgttttcgtg cagcgtgatg cacgaggcct tgcataatca ctatacacag 1380 aagtcgttgt cactgtcgcc gggaaagtaa 1410

<210> SEQ ID NO 6 <211> LENGTH: 184 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly I 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn He Lys Asp Thr 20 25 30

Tyr He His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Arg He Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala He Tyr Tyr Cys 85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 HO

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser 180

<210> SEQ ID NO 7 <211> LENGTH: 184 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly I 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn He Lys Asp Thr 20 25 30

Tyr He His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Arg He Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 US 2012/0301400 Al Nov. 29, 2012 58

-continued

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 HO

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser 180

<210> SEQ ID NO 8 <211> LENGTH: 705 <212> TYPE: DNA <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 8 atggagacag acacgctttt gctttgggtg ttgttgttgt gggtccccgg ttcgacgggg 60 gatattcaga tgacccagtc accgatcctt ctctcggcga gcgtggggga tagagtaacg 120 atcacgtgta gagcgtccca agacgtcaac acagctgtcg cgtggtatca gcagcggaca 180 aatggatcgc cgaggctcct gatctacagc gcatcatttc tctattcggg agtcccctcc 240 cgattttccg gatcgcgcag cggtactgac ttcaccctca cgatttcctc ccttcaaccg 300 gaagatatcg ctgattacta ctgtcagcag cactatacaa cacctcccac tttcggagca 360 gggacaaaag tggagattaa gcgcactgta gcagccccct cggtctttat cttccctcct 420 agcgacgaac aattgaagtc agggaccgcc tcggtggtat gcctgcttaa caacttttac 480 ccacgggaag ccaaagtaca gtggaaggtg gataatgcgc tccagagcgg aaactcccaa 540 gagagcgtga cagaacagga ctcgaaggat tcgacgtact cactcagctc aacgctgacc 600 ctgtcgaaag cggactatga gaaacacaag gtctacgcgt gcgaggtgac ccatcagggc 660 ctgagctccc ccgtaactaa gtcattcaac cggggtgaat gctaa 705

<210> SEQ ID NO 9 <211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 9

Asp He Gln Met Thr Gln Ser Pro He Leu Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr He Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30

Val Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu He 35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gln Pro 65 70

Glu Asp He Ala Asp Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 US 2012/0301400 Al Nov. 29, 2012 59

-continued

Thr Phe Gly Ala Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala 100 105 HO

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

<210> SEQ ID NO 10 <211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 10

Asp He Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly I 5 10 15

Asp Arg Val Thr He Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu He 35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gln Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala 100 105 HO

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

<210> SEQ ID NO 11 US 2012/0301400 Al Nov. 29, 2012 60

-continued

<211> LENGTH: 1440 <212> TYPE: DNA <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 11 tcactatagg ggaagcttgc cgccaccatg aaatgctcgt gggtgatctt tttccttatg 60 gcggtagtaa ccggagttaa ctccgaggtc cagctcgtcg aatccggtgg cggcttggtg 120 cagccgggtg ggtcgttgcg actgtcgtgc gcagcgtcgg ggtttaacat caaagacacc 180 tatatccact gggtgaggca agcgcccgga aaggggctcg aatgggtagc cagaatctac 240 cctacgaatg gttatactcg atatgcggac tccgtgaaag gaagattcac catcagcgca 300 gatacgtcca aaaacactgc atacctccag atgaatagcc ttcgggcgga ggacacggcg 360 gtctactact gtagccggtg gggtggggac gggttctatg cgatggacta ctggggacag 420 gggacgcttg taacggtcag ctcggctagc acaaagggac ctagcgtgtt tcccttggct 480 ccctcatcga aatcaacgtc cggtgggacg gcggcattgg ggtgtcttgt caaggactat 540 ttccccgagc ccgtgacagt ctcgtggaac tcgggtgccc ttacaagcgg cgtacatacg 600 tttcccgccg tgctccaatc atccggactg tattcccttt catccgtcgt gactgtgccg 660 tcctcgtcac tcggaacgca aacttacatt tgcaatgtca accacaaacc gtcaaataca 720 aaggtcgata agaaggtcga gccaaagtcg tgtgataaga cccacacatg ccctccctgt 780 ccagcgccgg agctgttggg agggccttca gtgttcctct tcccgcccaa acccaaggac 840 accctgatga ttagccgcac acccgaggtg acgtgtgtcg tcgtcgatgt ctcacatgag 900 gacccggagg taaagttcaa ctggtacgtg gatggagtcg aagtgcacaa cgcaaaaaca 960 aaacctcggg aagagcagta caatagcacg tacagagtag tcagcgtgct caccgtgctg 1020 caccaggatt ggctcaatgg aaaggagtac aagtgtaaag tgtcgaataa ggcgctgcct 1080 gcccccatcg aaaagacaat ttccaaagct aaagggcaac cccgcgagcc gcaagtatac 1140 accctcccac cctcgcgcga tgaactgacc aagaaccagg tgtcattgac gtgtctcgtc 1200 aagggcttct atccgagcga cattgcagta gaatgggaaa gcaacggaca gccggaaaac 1260 aactacaaga ctacaccgcc tgtccttgat tcggatggtt CCttCtttCt ttactcaaaa 1320 cttacagtcg acaaatcgag gtggcagcag ggaaatgtgt tttcgtgcag cgtgatgcac 1380 gaggccttgc ataatcacta tacacagaag tcgttgtcac tgtcgccggg aaagtaatga 1440

<210> SEQ ID NO 12 <211> LENGTH: 477 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 12

Ser Leu Gly Lys Leu Ala Ala Thr Met Lys Cys Ser Trp Val He Phe 15 10 15

Phe Leu Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Val 20 25 30

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arc3 Leu Ser 35 40 45

Cys Ala Ala Ser Gly Phe Asn He Lys Asp Thr Tyr He His Trp Val 50 55 60

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg He5 Tyr Pro 65 70 75 80 US 2012/0301400 Al Nov. 29, 2012 61

-continued

Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 85 90 95

He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 100 105 HO

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly 115 120 125

Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 130 135 140

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 165 170 175

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180 185 190

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 195 200 205

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 210 215 220

Thr Gln Thr Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 225 230 235 240

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 245 250 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu 275 280 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 325 330 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 340 345 350

Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys 355 360 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 370 375 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 385 390 395 400

Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gln 405 410 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 420 425 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 US 2012/0301400 Al Nov. 29, 2012 62

-continued

<210> SEQ ID NO 13 <211> LENGTH: 730 <212> TYPE: DNA <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 13 ataggggaag cttgccgcca ccatggagac agacacgctt ttgctttggg tgttgttgtt 60 gtgggtcccc ggttcgaccg gtgatattca gatgacccag tcaccgtcat ccctttcggc 120 gagcgtgggg gatagagtaa cgatcacgtg tagagcgtcc caagacgtca acacagctgt 180 cgcgtggtat cagcagaagc caggaaaagc gccgaagctc ctgatctaca gcgcatcatt 240 tctctattcg ggagtcccct cccgattttc cggatcgcgc agcggtactg acttcaccct 300 cacgatttcc tcccttcaac cggaagattt tgctacttac tactgtcagc agcactatac 360 aacacctccc actttcggac aggggacaaa agtggagatt aagcggaccg tagcagcccc 420 ctcggtcttt atcttccctc ctagcgacga acaattgaag tcagggaccg cctcggtggt 480 atgcctgctt aacaactttt acccacggga agccaaagta cagtggaagg tggataatgc 540 gctccagagc ggaaactccc aagagagcgt gacagaacag gactcgaagg attcgacgta 600 ctcactcagc tcaacgctga ccctgtcgaa agcggactat gagaaacaca aggtctacgc 660 gtgcgaggtg acccatcagg gcctgagctc ccccgtaact aagtcattca accggggtga 720 atgctaatga 730

<210> SEQ ID NO 14 <211> LENGTH: 240 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 14

Gly Lys Leu Ala Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val 15 10 15

Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp He Gln Met Thr Gln 20 25 30

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He Thr 35 40 45

Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln 50 55 60

Lys Pro Gly Lys Ala Pro Lys Leu Leu He Tyr Ser Ala Ser Phe Leu 65 70 75 80

Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp 85 90 95

Phe Thr Leu Thr He Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 100 105 HO

Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125

Lys Val Glu He Lys Arg Thr Val Ala Ala Pro Ser Val Phe He Phe 130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln US 2012/0301400 Al Nov. 29, 2012 63

-continued

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240

<210> SEQ ID NO 15 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 2 <223> OTHER INFORMATION: Xaa =D-glutamine

<400> SEQUENCE: 15

Cys Xaa Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys 15 10

<210> SEQ ID NO 16 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 16

Cys Gln Tyr Asp Leu Ser Thr Arg Arg Leu Lys Cys 15 10

<210> SEQ ID NO 17 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 3 <223 > OTHER INFORMATION: Xaa = beta-beta'-di-phenyl-Ala

<400> SEQUENCE: 17

Cys Gln Xaa Asp Leu Ser Thr Arg Arg Leu Lys Cys 15 10

<210> SEQ ID NO 18 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 5 <223 > OTHER INFORMATION: Xaa = beta-beta'-di-phenyl-Ala

<400> SEQUENCE: 18

Cys Gln Phe Asp Xaa Ser Thr Arg Arg Leu Lys Cys 15 10 US 2012/0301400 Al Nov. 29, 2012 64

-continued CN O Λ V SEQ ID NO 19 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 9 <223 > OTHER INFORMATION: Xaa = citrulline

Λ O V SEQUENCE: 19

Cys Gln Phe Asp Phe Ser Thr Arg Xaa Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 20 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 20

Cys Gln Phe Asp Phe Ser Thr Arg Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 21 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 21

Cys Gln Phe Asp Glu Ser Thr Arg Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 22 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 22

Cys Gln Phe Asp Tyr Ser Thr Arg Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 23 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 23

Cys Gln Phe Asp Leu Ser Thr Arg Arg Gln Lys Cys I 5 10 CN O Λ V SEQ ID NO 24 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide US 2012/0301400 Al Nov. 29, 2012 65

-continued

<400> SEQUENCE: 24

Cys Gln Phe Asp Leu Ser Thr Arg Gln Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 25 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 25

Cys Gln Tyr Asn Leu Ser Thr Ala Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 26 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 26

Cys Gln Ala Asp Leu Ser Thr Arg Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 27 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 27

Cys Gln Phe Asp Ala Ser Thr Arg Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 28 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 28

Cys Gln Phe Asp Leu Ser Thr Ala Arg Leu Lys Cys I 5 10 CN O Λ V SEQ ID NO 29 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 29

Cys Gln Phe Asp Leu Ser Thr Arg Arg Ala Lys Cys I 5 10 CN O Λ V SEQ ID NO 30 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : US 2012/0301400 Al Nov. 29, 2012 66

-continued

<223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 30

Cys Gln Phe Asp Leu Ser Thr Arg Arg Glu Lys Cys 15 10

<210> SEQ ID NO 31 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: I <223 > OTHER INFORMATION: Xaa = N-acetylcysteine

<400> SEQUENCE: 31

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Arg Cys Gly Gly Gly Ser 15 10 15

Lys

<210> SEQ ID NO 32 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 32

Gly Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Gly 15 10

<210> SEQ ID NO 33 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 33

Gly Gln His Asp Leu Ser Thr Arg Arg Leu Lys Gly 15 10

<210> SEQ ID NO 34 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 34

Gly Gln Asn Asp Leu Ser Thr Arg Arg Leu Lys Gly 15 10

<210> SEQ ID NO 35 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide

<400> SEQUENCE: 35

Gly Gln Gln Asp Leu Ser Thr Arg Arg Leu Lys Gly US 2012/0301400 Al Nov. 29, 2012 67

-continued

I 5 10 CN O Λ V SEQ ID NO 36 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 3 <223 > OTHER INFORMATION: Xaa = 2-bromo-L-phenylalanine

Λ O V SEQUENCE: 36

Gly Gln Xaa Asp Leu Ser Thr Arg Arg Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 37 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 3 <223 > OTHER INFORMATION: Xaa = 3-bromo-L-phenylalanine

Λ O V SEQUENCE: 37

Gly Gln Xaa Asp Leu Ser Thr Arg Arg Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 38 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 3 <223 > OTHER INFORMATION: Xaa = 4-bromo-L-phenylalanine

Λ O V SEQUENCE: 38

Gly Gln Xaa Asp Leu Ser Thr Arg Arg Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 39 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 9 <223 > OTHER INFORMATION: Xaa = citrulline

Λ O V SEQUENCE: 39

Gly Gln Phe Asp Leu Ser Thr Arg Xaa Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 40 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : US 2012/0301400 Al Nov. 29, 2012 68

-continued

<223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 8, 9 <223 > OTHER INFORMATION: Xaa = citrulline

Λ O V SEQUENCE: 40

Gly Gln Phe Asp Leu Ser Thr Xaa Xaa Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 41 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 8 <223 > OTHER INFORMATION: Xaa = citrulline

Λ O V SEQUENCE: 41

Gly Gln Phe Asp Leu Ser Thr Xaa Arg Leu Lys Gly I 5 10 CN O Λ V SEQ ID NO 42 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

Λ O V SEQUENCE: 42

Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 43 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I, 12 <223 > OTHER INFORMATION: Xaa = beta-alanine

Λ O V SEQUENCE: 43

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 44 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I <223 > OTHER INFORMATION: Xaa = diaminopropionic acid <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 12 US 2012/0301400 Al Nov. 29, 2012 69

-continued

<223 > OTHER INFORMATION: Xaa = iso-aspartic acid

Λ O V SEQUENCE: 44

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 45 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I <223 > OTHER INFORMATION: Xaa = beta-alanine <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 12 <223 > OTHER INFORMATION: Xaa = iso-aspartic acid

Λ O V SEQUENCE: 45

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 46 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I <223 > OTHER INFORMATION: Xaa = diaminoproprionic acid <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 12 <223 > OTHER INFORMATION: Xaa = beta-alanine

Λ O V SEQUENCE: 46

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 47 <211> LENGTH: 9 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 47

Phe Asp Leu Ser Thr Arg Arg Leu Lys I 5 CN O Λ V SEQ ID NO 48 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 48

Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys 15 10 US 2012/0301400 Al Nov. 29, 2012 70

-continued CN O Λ V SEQ ID NO 49 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

Λ O V SEQUENCE: 49

Gln Tyr Asp Leu Ser Thr Arg Arg Leu Lys Xaa 15 10 CN O Λ V SEQ ID NO 50 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I <223 > OTHER INFORMATION: Xaa = beta-Azidoalanine <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 12 <223 > OTHER INFORMATION: Xaa = propargylglycine

Λ O V SEQUENCE: 50

Xaa Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 51 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 2 <223 > OTHER INFORMATION: Xaa = beta-beta'-di-phenyl-Ala <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

Λ O V SEQUENCE: 51

Gln Xaa Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 52 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: I <223 > OTHER INFORMATION: Xaa = D-glutamine <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

<400> SEQUENCE: 52 US 2012/0301400 Al Nov. 29, 2012 71

-continued

Xaa Phe Asp Leu Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 53 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 2, 4 <223 > OTHER INFORMATION: Xaa = beta-beta'-di-phenyl-Ala <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

Λ O V SEQUENCE: 53

Gln Xaa Asp Xaa Ser Thr Arg Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 54 <211> LENGTH: 11 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 7 <223 > OTHER INFORMATION: Xaa = n-butyl-arginine <220> FEATURE :

Λ to to V NAME/KEY: VARIANT <222 > LOCATION: 11 <223 > OTHER INFORMATION: Xaa = 7-aminoheptanoic acid

Λ O V SEQUENCE: 54

Gln Phe Asp Leu Ser Thr Xaa Arg Leu Lys Xaa I 5 10 CN O Λ V SEQ ID NO 55 <211> LENGTH: 12 <212 > TYPE : PRT <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed peptide

Λ O V SEQUENCE: 55

Ser Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Ser I 5 10 CN O Λ V SEQ ID NO 56 <211> LENGTH: 594 <212 > TYPE: DNA <213 > ORGANISM: Artificial Sequence <220> FEATURE : <223 > OTHER INFORMATION: synthetically constructed SMT3-peptide-Protein L fusion

Λ O V SEQUENCE: 56 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagcatg 60 tcggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag 120 actcacatca atttaaaggt gtccgatgga tcttcagaga tcttcttcaa gatcaaaaag 180 accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac 240 US 2012/0301400 Al Nov. 29, 2012 72

-continued tccttaagat tcttgtacga cggtattaga attcaagctg atcagacccc tgaagatttg 300 gacatggagg ataacgatat tattgaggct cacagagaac agattggtgg atcctgccag 360 tttgatctga gcacccgccg tctgaaatgc ggtggcggtg gatccgaagt taccatcaaa 420 gtcaacctga ttttcgcaga cggcaaaatc cagacggctg aatttaaagg cacgttcgaa 480 gaagctacgg cggaagcata tcgttacgcg gccctgctgg cgaaagtgaa cggcgaatac 540 acggcagacc tggaagacgg cggcaatcac atgaacatca aattcgctgg ctaa 594

<210> SEQ ID NO 57 <211> LENGTH: 197 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed His6-SMT3-peptide-Protein L fusion

<400> SEQUENCE: 57

His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His I 5 10 15

Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu 20 25 30

Val Lys Pro Glu Val Lys Pro Glu Thr His He Asn Leu Lys Val Ser 35 40 45

Asp Gly Ser Ser Glu He Phe Phe Lys He Lys Lys Thr Thr Pro Leu 50 55 60

Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp 65 70 75 80

Ser Leu Arg Phe Leu Tyr Asp Gly He Arg He Gln Ala Asp Gln Thr 85 90 95

Pro Glu Asp Leu Asp Met Glu Asp Asn Asp He He Glu Ala His Arg 100 105 HO

Glu Gln He Gly Gly Ser Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu 115 120 125

Lys Cys Gly Gly Gly Gly Ser Glu Val Thr He Lys Val Asn Leu He 130 135 140

Phe Ala Asp Gly Lys He Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu 145 150 155 160

Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val 165 170 175

Asn Gly Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn 180 185 190

He Lys Phe Ala Gly 195

<210> SEQ ID NO 58 <211> LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide-Protein L fusion

<400> SEQUENCE: 58

Ser Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys Gly Gly Gly 15 10 15 US 2012/0301400 Al Nov. 29, 2012 73

-continued

Gly Ser Glu Val Thr He Lys Val Asn Leu He Phe Ala Asp Gly Lys 20 25 30

He Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu 35 40 45

Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Lys Val Asn Gly Glu Tyr Thr 50 55 60

Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn He Lys Phe Ala Gly 65 70 75 80

<210> SEQ ID NO 59 <211> LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetically constructed peptide-Protein fusion

<400> SEQUENCE: 59

Ser Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Arg Cys Gly Gly Gly I 5 10 15

Gly Ser Glu Val Thr He Arg Val Asn Leu He Phe Ala Asp Gly Asn 20 25 30

He Gln Thr Ala Glu Phe Arg Gly Thr Phe Glu Glu Ala Thr Ala Glu 35 40 45

Ala Tyr Arg Tyr Ala Ala Leu Leu Ala Arg Val Asn Gly Glu Tyr Thr 50 55 60

Ala Asp Leu Glu Asp Gly Gly Asn His Met Asn He Lys Phe Ala Gly 65 70 75 80

<210> SEQ ID NO 60 <211> LENGTH: 726 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetic light chain

<400> SEQUENCE: 60 aagcttgccg ccaccatgga gacagacacg cttttgcttt gggtgttgtt gttgtgggtc 60 cccggttcga ccggtaccgg tgacatcttg ttgacgcagt cccccgtcat tctgagcgtg 120 tcccccggag agcgggtatc gttttcctgc cgagcctcgc aagatatcaa cactgcgatt 180 gcatggtatc aacagcgcac aaacgggtcg ccgagactgc tcatctactc agcctcgttc 240 ctttatagcg gtgtgccttc gaggttctcg ggatcacggt caggaacgga ttttacactc 300 agcatcaatt ccgtggaatc agaggacatt gcggactact attgtcagca gcactacacc 360 acaccaccga ccttcggcgc tgggacgaaa gtcgaaatca agcggaccgt agcagccccc 420 tcggtcttta tcttccctcc tagcgacgaa caattgaagt cagggaccgc ctcggtggta 480 tgcctgctta acaactttta cccacgggaa gccaaagtac agtggaaggt ggataatgcg 540 ctccagagcg gaaactccca agagagcgtg acagaacagg actcgaagga ttcgacgtac 600 tcactcagct caacgctgac cctgtcgaaa gcggactatg agaaacacaa ggtctacgcg 660 tgcgaggtga cccatcaggg cctgagctcc cccgtaacta agtcattcaa ccggggtgaa 720 tgctaa 726 US 2012/0301400 Al Nov. 29, 2012 74

-continued

<210> SEQ ID NO 61 <211> LENGTH: 241 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetic light chain

<400> SEQUENCE: 61

Lys Leu Ala Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu I 5 10 15

Leu Leu Trp Val Pro Gly Ser Thr Gly Thr Gly Asp He Leu Leu Thr 20 25 30

Gln Ser Pro Val He Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe 35 40 45

Ser Cys Arg Ala Ser Gln Asp He Asn Thr Ala He Ala Trp Tyr Gln 50 55 60

Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu He Tyr Ser Ala Ser Phe 65 70 75 80

Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 85 90 95

Asp Phe Thr Leu Ser He Asn Ser Val Glu Ser Glu Asp He Ala Asp 100 105 HO

Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Ala Gly 115 120 125

Thr Lys Val Glu He Lys Arg Thr Val Ala Ala Pro Ser Val Phe He 130 135 140

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150 155 160

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240

Cys

<210> SEQ ID NO 62 <211> LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetic heavy chain

<400> SEQUENCE: 62 aagcttgccg ccaccatgaa atgctcgtgg gtgatctttt tccttatggc ggtagtaacc 60 ggagttaact cccaagtaca gcttaagcag tcgggtccgg ggctggtgca gccatcgcag 120 tcgttgtcaa tcacatgcac ggtgtcggga ttcaacatta aagacaccta tatccactgg 180 gtccgacaat cccctggtaa agggctggag tggctcggtc ggatctaccc cacgaacgga 240 tacaccaggt ataacacgcc ctttacatcg agactttcaa ttaacgcgga taatagcaag 300 US 2012/0301400 Al Nov. 29, 2012 75

-continued aatcaggtgt tcttcaagat gaatagcctc cagtcaaatg acactgcaat ctactactgt 360 gcccgctggg gaggggatgg cttttatgcg atggactatt gggggcaggg aactttggtc 420 acagtaagct ccgctagcac aaagggacct agcgtgtttc ccttggctcc ctcatcgaaa 480 tcaacgtccg gtgggacggc ggcattgggg tgtcttgtca aggactattt ccccgagccc 540 gtgacagtct cgtggaactc gggtgccctt acaagcggcg tacatacgtt tcccgccgtg 600 ctccaatcat ccggactgta ttccctttca tccgtcgtga ctgtgccgtc ctcgtcactc 660 ggaacgcaaa cttacatttg caatgtcaac cacaaaccgt caaatacaaa ggtcgataag 720 aaggtcgagc caaagtcgtg tgataagacc cacacatgcc ctccctgtcc agcgccggag 780 ctgttgggag ggccttcagt gttcctcttc ccgcccaaac ccaaggacac cctgatgatt 840 agccgcacac ccgaggtgac gtgtgtcgtc gtcgatgtct cacatgagga cccggaggta 900 aagttcaact ggtacgtgga tggagtcgaa gtgcacaacg caaaaacaaa acctcgggaa 960 gagcagtaca atagcacgta cagagtagtc agcgtgctca ccgtgctgca ccaggattgg 1020 ctcaatggaa aggagtacaa gtgtaaagtg tcgaataagg cgctgcctgc ccccatcgaa 1080 aagacaattt ccaaagctaa agggcaaccc cgcgagccgc aagtatacac cctcccaccc 1140 tcgcgcgatg aactgaccaa gaaccaggtg tcattgacgt gtctcgtcaa gggcttctat 1200 ccgagcgaca ttgcagtaga atgggaaagc aacggacagc cggaaaacaa ctacaagact 1260 acaccgcctg tccttgattc ggatggttcc ttctttcttt actcaaaact tacagtcgac 1320 aaatcgaggt ggcagcaggg aaatgtgttt tcgtgcagcg tgatgcacga ggccttgcat 1380 aatcactata cacagaagtc gttgtcactg tcgccgggaa agtaa 1425

<210> SEQ ID NO 63 <211> LENGTH: 460 <212 > TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223 > OTHER. INFORMATION: synthetic heavy chain

<400> SEQUENCE: 63

Lys Leu Ala. Ala Thr Met Lys Cys Ser Trp Val He Phe Phe Leu Met I 5 10 15

Ala Val Val Thr Gly Val Asn Ser Gln Val Gln Leu Lys Gln. Ser Gly 20 25 30

Pro Gly Leu. Val Gln Pro Ser Gln Ser Leu Ser He Thr Cys Thr Val 35 40 45

Ser Gly Phe■ Asn He Lys Asp Thr Tyr He His Trp Val Arg Gln Ser 50 55 60

Pro Gly Lys Gly Leu Glu Trp Leu Gly Arg He Tyr Pro Thr Asn Gly 65 7C) 75 80

Tyr Thr Arg Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser He■ Asn Ala 85 90 95

Asp Asn Ser Lys Asn Gln Val Phe Phe Lys Met Asn Ser Leu. Gln Ser 100 105 HO

Asn Asp Thr Ala He Tyr Tyr Cys .Ala Arg Trp Gly Gly Asp■ Gly Phe 115 120 125

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys US 2012/0301400 Al Nov. 29, 2012 76

-continued

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 210 215 220

Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270

Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys 275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350

Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly 355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400

Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460

<210> SEQ ID NO 64 <211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 15 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 US 2012/0301400 Al Nov. 29, 2012 77

-continued

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Thr Val

<210> SEQ IE) NO 65 <211> LENGTH: 9E5 <212 > TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 65

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg I 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Arg Val

<210> SEQ IE) NO 66 <211> LENGTH: 9E5 <212 > TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 66

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg I 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Arg Val

<210> SEQ ID NO 67 <211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens

<400> SEQUENCE: 67

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys US 2012/0301400 Al Nov. 29, 2012 78

-continued

I 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80

Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Lys Val

<210> SEQ ID NO 68 <211> LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetic light chain

<400> SEQUENCE: 68

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro I 5 10 15

Gly Ser Thr Gly Asp He Gln Leu Thr Gln Ser Pro Val He Leu Ser 20 25 30

Ala Ser Val Gly Asp Arg Val Thr He Thr Cys Arg Ala Gly Glu Ser 35 40 45

Val Asp He Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Thr 50 55 60

Asn Gly Ser Pro Lys Leu Leu He Tyr Arg Ala Ser Asn Leu Glu Ser 65 70 75 80

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr 85 90 95

Leu Thr He Ser Ser Leu Gln Pro Glu Asp He Ala Asp Tyr Tyr Cys 100 105 HO

Gln Gln Thr Asn Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val 115 120 125

Glu He Lys Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro 130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Ala Ala Ser Val Val Cys Leu Leu 145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

<210> SEQ ID NO 69 US 2012/0301400 Al Nov. 29, 2012 79

-continued

<211> LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: synthetic light chain

<400> SEQUENCE: 69

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro I 5 10 15

Gly Ser Thr Gly Asp He Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30

Ala Ser Val Gly Asp Arg Val Thr He Thr Cys Arg Ala Gly Glu Ser 35 40 45

Val Asp He Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro 50 55 60

Gly Lys Ala Pro Lys Leu Leu He Tyr Arg Ala Ser Asn Leu Glu Ser 65 70 75 80

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr 85 90 95

Leu Thr He Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 100 105 HO