US 201203 01400A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0301400 A1 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 C07K 6/8 (2006.01) City, CA (US); Joshua Michael C07K 7/08 (2006.01) Donaldson, Lumberton, NJ (US); C07K L/4 (2006.01) Cindy Zer, Duarte, CA (US); A 6LX 39/395 (2006.01) Krzysztof Bzymek, Pasadena, CA A61R 49/00 (2006.01) (US); Kendra Nicole Avery, CI2N 5/071 (2010.01) Pasadena, CA (US); Jun Xie, C07K 7/06 (2006.01) Duarte, CA (US) C07K 2/00 (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 1 of 79 US 2012/0301400 A1

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MEDTOPES 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 SEQID NO: 1. 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 SEQID 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 SEQID NO: 1, 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: 1, 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 SEQID NO: 1 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 , 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 (scEvs), less thanator about 10M, less than at or about 5uM, or less diabodies, minibodies, and other fragments), and altered than at or about 2 uM, less than at or about 1 uM, 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 Fc 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 biologics (e.g., CovX-bodies). Conjugation to 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 scEvs 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-target 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 tophanat 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, synergy, 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 A1 Nov. 29, 2012

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 SEQID 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 SEQID Kabat numbering, and/or the VH region does not contain an NO: 74. asparagine oralanine 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) 1 (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 (oran FR-L1, 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, 9495,96, 97,98, VH region does not contain an asparagine or alanine at posi or 99% identical to the FR-L1, 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 SEQID 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 CH1, e.g., region comprises at least one CDR that is distinct from the human CL and/or CH1. CDRs of the heavy chain sequence set forth in SEQID 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) 1 (FR-L1), an FR-L2, an amino acid sequence of SEQID 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 SEQID NO: 71 or SEQID NO: 61 (or an FR-L1, 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-L1, 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 forthin SEQID 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 leastator about 75,80, 85,90,91,92, heavy chain FR1 (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 SEQID NO: including a heavy chain CDR 1, 2, and/or 3 and/or a light 72 or SEQID NO: 63 (or an FR-H1, FR-H2, FR-H3, and/or chain CDR1, 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, , abciximab, adalimumab, , ale and/or FR-H4 of the heavy chain of SEQID 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 SEQID NO: imab, , ectumomab, belimumab, benralizumab, 72. , brentuximab, canakinumab, capromab, capro 0016. In some aspects, the provided antibody or fragment mab pendetide, , certolizumab, clivatuZumab has a CDR of the light chain sequence set forth in SEQID NO: tetraxetan, daclizumab, denosumab, eculizumab, edrecolo 61, a CDR of the heavy chain sequence set forth in SEQID mab, efalizumab, , , fanolesomab, NO: 63. Fbta05, fontolizumab, gemtuzumab, , goli 0017. In some aspects, the VL comprises the amino acid mumab, ibritumomab, igovomab, infliximab, , sequence of SEQID NO: 76 and the VH comprises the amino , mepolizumab, muromonab, muromonab-CD3. acid sequence of SEQID NO: 77. natalizumab, , . , 0018. In some aspects, the antibody has a VL region with omalizumab, , palivizumab, , an amino acid sequence comprising a light chain framework ranibizumab, , Satumomab, Sulesomab, ibritumo region (FR) 1 (FR-L1), an FR-L2, an FR-L3, and/oran FR-L4 mab, , tocilizumab, to situmomab, tras of the light chain sequence set forth in SEQID NO: 9 (or an tuzumab, Trbs07, ustekinumab, visilizumab, votumumab, FR-L1, FR-L2, FR-L3, and/or FR-L4 that is at least at or , and/or brodalumab; and/or anrukinzumab, about 75, 80, 85,90, 91, 92,93, 94, 95, 96, 97,98, or 99% bapineuZumab, , , , ino identical to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of SEQ tuZumab, mavrilimumab, , rillotu ID NO: 9); and/or a VH region with an amino acid sequence mumab, Sifalimumab, taneZumab, tralokinumab, tremeli having a heavy chain FR1 (FR-H1), an FR-H2, an FR-H3. mumab, urelumab, the antibody produced by the hybridoma and/oran FR-H4 of the heavy chain sequence set forth in SEQ 1OB5 (see Edelson & Unanue. Curr Opin Immunol, 2000 IDNO: 6 (oran FR-H1, FR-H2, FR-H3, and/or FR-H4 that is August; 12(4):425-31), B6H12.2 (abcam) or otheranti-CD47 at leastator about 75,80, 85,90,91, 92,93, 9495,96, 97,98, antibody (see Chao et al., Cell, 142,699-713, Sep. 3, 2010): or 99% identical to the FR-H1, FR-H2, FR-H3, and/or FR-H4 and/or an antibody or fragment thereofhaving a sequence set of SEQ ID NO: 9). In some examples, the VL region com forth in any of SEQID 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) IIb/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 SEQID NO: 6. kin-6 receptor (IL-6R). IL-2, interleukin-2 receptor a-chain US 2012/0301400 A1 Nov. 29, 2012

(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-1beta, prostate spe meditope-enabled antibody, according to Kabat numbering. cific membrane antigen (PSMA), CD3, EpCAM, EGF recep 0030. In some aspecst, the meditope binds to a meditope tor (EGFR), MUC1, 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 SEQID NO: 71 and a heavy chain CD11a, e.g., human CD11a, an integrin, e.g., alpha-V beta-3 comprising an amino acid sequence set forth in SEQID NO: integrin, vitronectin receptor alpha v beta 3 integrin, HER2, 72; to a meditope-enabled antibody having a light chain hav neu, CD3, CD15, CD20 (small and/or large loops), Interferon ing an amino acid sequence set forth in SEQID NO:9 and a gamma, CD33, CA-IX, TNFalpha, CTLA-4, carcinoembry heavy chain comprising an amino acid sequence set forth in onic antigen, IL-5. CD3 epsilon, CAM, Alpha-4-integrin, SEQID NO: 6; and/or to a meditope-enabled antibody having IgE. e.g., IgE Fc region, an RSV antigen, e.g., F protein of a light chain having an amino acid sequence set forth in SEQ respiratory syncytial virus (RSV), TAG-72, NCA-90 (granu ID NO: 68 and/or a heavy chain having an amino acid locyte cell antigen), IL-6, GD2, GD3, IL-12, IL-23, IL-17, sequence set forth in SEQID NO: 70. CTA A16.88, IL 13, interleukin-1 beta, beta-amyloid, IGF-1 0031. In some aspects, the peptide is between 5 and 16 receptor (IGF-1R), delta-like ligand 4 (DLL4), alpha subunit amino acids in length. of granulocyte macrophage colony stimulating factor recep 0032. In some aspects, the peptide has the formula: tor, hepatocyte growth factor, IFN-alpha, nerve growth factor, IL-13, CD326, Programmed cell death 1 ligand 1 (PD-L1, wherein: a.k.a. CD274, B7-H1), CD47, and CD137. 0033 X1=Cys, Gly, B-alanine, 2,3-diaminopropionic 0025. In some aspects, the antibody or fragment has a light acid, 3-azidoalanine, or null; chain having P8, V9 or I9, I10 or L10, Q38, R39, T40, N41 0034 X2=Gln or null: G42, S43, P44, R45, D82, I83, A84, D85, Y86, Y87, G99, 0035 X3=Phe, Tyr, B-B'-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, Gln, 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, I89,Y90, Y91, W103, G104, Q105, G106, T107, L108, V111, T110, Y147, E150, P151, V152, T173, F174, P175, 0036 X4=Asp or Asn; A176, V177, Y185, S186, and/or L187, according to Kabat 0037 X5-Leu: B-B'-diphenyl-Ala; Phe; a non-natural numbering. analog of phenylalanine, tryptophan, or tyrosine; a hydratable 0026. Also provided are complexes containing an anti carbonyl-containing residue; or a boronic acid-containing body orantibodies (e.g., meditope-enabled antibodies) bound residue; to one or more meditope. The antibody orantibody can be any 0038 X6–Seror Cys: of the meditope-enabled antibodies described herein, such as 0039 X7=Thr or Seror 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 X 10-Leu, Gln, Glu, B-B'-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 X11=Lys; and above. Among the provided meditopes are those comprising 0044 X 12-Cys, Gly, 7-aminoheptanoic acid, B-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: 1 or 2 or cyclic peptide derived therefrom. In other 0045 wherein: aspects, the meditope is a peptide of SEQID NO: 1 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 thanator about 10 uM, less than at or about 5uM, 0048 formula I is not SEQID NO: 1 or SEQID NO: 2 or or less than at or about 2 uM, less than at or about 1 uM, 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 X1 and X12, X1 and X11, X3 and tion, NMR, IR, calorimetry titrations; Kinetic exclusion; Cir X11, X4 and X11, or X2 and X12. 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. B-3'-diphenyl-Ala., a branched alkyl, oran 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 Kabat numbering, and/or phenyl Substituent. US 2012/0301400 A1 Nov. 29, 2012

0050. Among the provided meditopes are peptides having C(O)C alkyl, —CH=CH-COC-alkyl, an amino acid selected from the group consisting of the —COH, or —COC-alkyl group; sequences set forth in SEQID NOs: 1, 2, and 15-55, e.g., 1, 2, 0067 R is H, Cisalkyl, C-scycloalkyl, branched 16-18, 23, 29, 31, 32, 36,39, 42,43, 45,46, 51, 52,54, and 55, alkyl, or aryl; e.g., 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45,46, 51, 52, 54, I0068) R' is H or a Cisalkyl, Csalkenyl, Csalky and 55, or a cyclic peptide derived therefrom. nyl, C-scycloalkyl, 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 —N. —NH, -OH, -SH, halogen, oxo, acetal, ketal, —B(OH), boronic ester, phosphonate ester, ortho (X) ester, -CH=CH-CHO, -CH=CH-C(O)Cl. 4alkyl, —CH=CH-COC alkyl, -COH, and —COC alkyl group; and (0069 R is H, -NHR'. or a Calkyl, Cscy cloalkyl, C2-12alkenyl, C2-salkynyl, or aryl group, each optionally substituted with one or more substitu ents selected from the group consisting of —N. —NH, -OH, -SH, oxo, Cacetal, Caketal, —B(OH), boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)Cl. 4alkyl, -CH=CH-COC-alkyl, and —COC. 4alkyl group; or 0070 (b) a C-alkyl substituted with an oxo, acetal, ketal. —B(OH), boronic ester. —SH, —OH, phospho nate ester, ortho ester, —CH=CH-CHO, -CH=CH-C(O)Calkyl, -CH=CH-COC. 4alkyl, or —COC-alkyl group; (0071 R is Calkyl or C-alkylene-R': 0072 wherein R is COH, -CONH2, —CH-NHC (O)NH, or CH-NHC(=NH)NH; 0073 R' is: 0.074 (1) a Cisalkyl 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), boronic ester, phospho I0052 R and R are each, independently, H or phenyl, nate ester, ortho ester, —CH=CH-CHO, optionally substituted with one, two, or three substituents -CH=CH-C(O)Calkyl, -CH=CH-COC. independently selected from Calkyl, -OH, fluoro, 4alkyl, -COC alkyl, -COH, and —CONH chloro, bromo, and iodo; group; or 0053 R is: 0075 (2) a Calkyl group substituted with one or two 0054 (A) Cisalkyl, 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), boronic ester, phospho with one, two, or three substituents independently nate ester, ortho ester, —COC alkyl, -CH=CH selected from —OH, fluoro, chloro, bromo, and iodo; CHO, -CH=CH-C(O)Calkyl, -CH=CH 0.076 n is 0 or 1; COC-alkyl, -COH, and —CONH2 group; or 0.077 p is 0 or 1: 0055 (B) a Calkyl group substituted with: 0078 X is Cisalkylene or Csalkenylene, each carbon 0056 a) one or two phenyl groups, wherein each thereof optionally substituted with —COH, -NH2 or phenyl is optionally substituted with one, two, or NHC(O)R’; 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 0.058 R is Calkyl-OH or - Calkyl-SH: 0080 R is - Calkyl or -CH(R)COH: 0059 R7 is Calkyl-OH or - Calkyl-SH: I0081 wherein R is —H or —Calkyl optionally 0060 m is 0, 1, 2, 3, 4, or 5: substituted with —OH, -SH, or—NH; 0061 R is: or a pharmaceutically acceptable salt thereof. 0062 (a) OH, NR'R'', N(R)C(O)R, or I0082 In one embodiment, a meditope contains a cysteine N(R)C(-NR)R’; that covalently binds to a cysteine in a meditope binding site. 0063 wherein: Such a meditope may be conjugated to any Substance, mol 0064 R is H; ecule or compound, which may be a therapeutic molecule, I0065 R is HorCisalkyl 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. 0.066 —B(OH), —SH, boronic ester, phosphonate I0083. 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 A1 Nov. 29, 2012 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 metalion, 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 organic 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. 0084. Also among the provided meditopes are multivalent one aspect, the meditope-enabled antibody or fragment is meditopes, such as those comprising two or more meditopes bound to the one or more meditope, Such that administration and one or more linker, e.g., where each of the two or more of the meditope-enabled antibody and the one or more medi 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 Fc 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 LM, 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 I0086 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 thenisolating 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 Kabat 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 A1 Nov. 29, 2012

ID NO: 1, 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, , 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, , efalizumab, for modifying a meditope, comprising effecting one or more etaracizumab, ertumaxomab, fanolesomab, Fbta05, fontoli Zumab, gemtuzumab, girentuximab, golimumab, ibritumo amino acid substitutions, insertions, or deletions, to a peptide mab, igovomab, infliximab, ipilimumab, labetuZumab, having the sequence set forth in any of SEQ ID NOs: 1, 2, mepolizumab, muromonab, muromonab-CD3, natalizumab, 16-18, 23, 29, 31, 32, 36,39, 42,43, 45,46, 51, 52, 54, and 55, necitumumab, nimotuZumab. ofatumumab, omalizumab, or cyclic peptide derived therefrom, thereby altering the pH oregovomab, palivizumab, panitumumab, ranibizumab, rit dependence of the binding affinity of the peptide for a medi uximab, Satumomab, Sulesomab, ibritumomab, ibritumomab tope-enabled antibody or fragment thereof. Such as any of tiuxetan, tocilizumab, to situmomab, trastuzumab, Trbs07, those described above. In some aspects, the method decreases ustekinumab, visilizumab. Votumumab, Zalutumumab, and the binding affinity of the peptide for the antibody or fragment brodalumab, or is the antibody produced by the hybridoma at a lysosomal pH level. In other aspects, the method 1OB5, or is B6H12.2. increases the binding affinity in a hypoxic environment. 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 SEQID NO: 1. 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) uM, 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, 0097. In some aspects, each of the one or more amino acid 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and/or 187 Substitutions is at a position selected from the group consist of the heavy chain, of the meditope-enabled antibody, accord ing of position 8, 9, 10, 38, 39, 40, 4142, 43, 44, 45, 82.83, ing to Kabat numbering. In some aspects, the modification 84, 85,86, 87,99, 100, 101, 102, 103,104,105,142,162, 163, methods are provided in tandem, to produce modified medi 164, 165, 166, 167, 168, and 173 of the light chain, and/or 6, topes and modified meditope-enabled antibodies that bind to 9,38, 39, 40, 41,42, 43,44, 45, 84, 86, 87, 88, 89,90,91, 103, one another, Such as by making modifications in the antibody 104, 105, 106, 107, 108, 109, 110, 147, 150, 151, 152, 173, based on modifications in a modified meditope, or vice versa. 174, 175, 176, 177, 185, 186, and 187 of the heavy chain, 0103 Also provided are meditope analogs, including according to Kabat 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 I9, I10 or L10, Q38, R39, T40, N41 G42, S43, P44, R45, D82, to any of the meditopes described above. I83, A84, D85, Y86, Y87, G99, A100, G101, T102, K103, L104, E105, R142, S162, V163, T164, E165, Q166, D167, BRIEF DESCRIPTION OF THE DRAWINGS S168, and Y173, according to Kabat numbering and a heavy chain having Q6, P9, R38, Q39, S40, P41, G42, K43, G44, 0104 FIG. 1 shows meditope peptides binding to frame L45, S84, D86, T87, A88, I89,Y90,Y91, W103, G104, Q105, work loops of cetuximab. FIG. 1A: The complex of cetux G106, T107, L108, V111, T110, Y147, E150, P151, V152, imab Fab (light chain is denoted by V, and C.; heavy chain is T173, F174, P175, A176, V177, Y185, S186, and L187, denoted by V, and C) and cyclic COFDLSTRRLKC (de according to Kabat numbering. picted within the shaded area and labeled with the word 0098. Also provided are polynucleotides comprising a “meditope') (SEQ ID NO: 1) 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. 1B (top) shows the libraries comprising the vectors. stick representation of the coFD meditope and (bottom) the 0099. Also provided are screening methods, such as those stick representation of the coYN 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, PK, 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 ch14.18 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 coFD 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 A1 Nov. 29, 2012 bridge from Asp85 of the cetuximab light chain to the guani recycling and/or elicits an immune response. The addition of dinium group of the meditope Arg9 and the backbone amide an antibody Such as which recognizes a different of Leu10. 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 c(RFD and coYN with an immobilized Fab. The receptor (right panel 1). 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 mAb 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 1:1 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 schv-meditope linker. schvs 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}ss). 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 coFD meditope and cetux tope Sequence. imab Fab, in which the coFD 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 coFD meditope coupled to beads could bind to cetux binds at the complementarity determining regions at a signifi imab. Biotinylated coFD 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 1), 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 coFD 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 Scv or off-rate of the multivalent meditope was reduced by at least Fab. The experiments indicate that saturation of the schv 36 fold. (Note the time scales between the two experiments could not beachieved at concentrations as high as 100LLM of are different.) the coFD 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 schv readily binds to the soluble EGFR domain III fragment, cein isothiocyanate (FITC)-labeled meditope dimer (“14 in indicating that the CDR loops were functional. FIG. 13), with an HPLC 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 c(RFD. 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 uM and 60 uM 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 Fc Region of an IgG. In this distinct sites. The top images show Superposition of the example, as the Fc is naturally homodimerized during expres cQFD-Fab complex and the EGFR-Fab complex (1YY8) 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. I0120 FIG. 17 shows the results of a series of exemplary 0110 FIG.7 shows Fab framework binders. 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 A1 Nov. 29, 2012

with cetuximab in a concentration dependent manner. The top I0127 FIG. 24 shows that an antibody containing HER2 four traces also show that the bivalent, meditope Fc 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 mAb 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 mAb (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, I0128 FIG. 25A shows nucleic acid (SEQID NO: 11) and lead compounds (e.g., those shown herein) have been identi amino acid (SEQID NO: 12) sequences for the 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 (SEQID NO: 13) and amino acid (SEQIDNO: 14) sequences enhance the binding affinity (or alter another property) of a for a light chain of trastuzumab with secretion sequence and mAb 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 mAb 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 I0129 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 mAb remains associ 0.130 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 coFD (meditope 1, SEQ ID NO: 1), heavy chain from the selected cells will identify high affinity cQYN (meditope 2, SEQID NO: 2), and cqYD (meditope mAbs. The method can be repeated to select for higher affin 16, SEQID NO: 16) was determined from pH=4.0 to pH=8. ity meditope or meditope analogs. O 0.124 FIG. 21 shows surface representation of sequence I0131 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-Fic, 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. 0.125 FIG. 22 Illustrates that a meditope-enabled trastu I0132 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 mAb 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 1, SEQ ID NO: 1) 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 1 uM. 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. (0.133 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: 1) and a QYN meditope compared to the wild-type (SEQID NO:7). Differences are (SEQID NO:2) using acetuximab 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: 1) 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 1 (SEQID 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 hydrogenbond 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 L10 are nearly identically sequence as compared to the wild-type (SEQ ID NO:10). positioned, but the hydroxyl group of Y5 in the coYN medi Differences are highlighted in grey. tope prevents R8 from interacting with the Q105 backbone, as US 2012/0301400 A1 Nov. 29, 2012

observed in the coFD meditope (“steric clash'). This rear 0.143 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 B-turn (backbone rotation'). FIG. 30D in Tables 3 and 4. shows individual thermodynamic parameters determined by 014.4 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, SEQID NO: 2) shows a significant change in AH information of the Phe3His mutation in the coFD meditope, though lower affinity. Based on the structure, Gln2 in the a variant meditope was synthesized with B.f3'-diphenylala meditope of SEQ ID NO: 1 was replaced with the D-stere nine at position3 (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 0.136 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 mab 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 0.137 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, B.f3'-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 Leu10 (right) to 0147 FIG. 44 shows results of a study described in form a covalent bond to the hydroxyl groups of Seror 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 (WTT), bound of dose-dependent inhibition of the interaction between to a cqFD (SEQ ID NO: 1) 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 mAb 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 (WTT), bound to a cqFD (SEQID NO: 1) 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 arginine 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 014.9 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), SEQID NO:17 (C), SEQID NO:18 (D), SEQID enabled trastuzumab to a similar degree whether the antibody NO:15 (E), SEQID NO:23 (F), SEQID NO:24 (G), SEQID is pre-bound to cells expressing antigen prior to incubation NO:25 (H), SEQID NO:32 (I), SEQID NO:33 (J), SEQID with the meditope (left panel), or meditope-Protein L- and NO:34 (K), SEQID NO:35 (L), SEQID NO:36 (M), SEQID antibody are pre-mixed and then incubated with cells express NO:37 (N), SEQID NO:38 (O), SEQID NO:39 (P), SEQID ing antigen (right panel). TM meditope-enabled trastu NO:40 (Q) and SEQID NO:41 (R), SEQID NO:42 (S), SEQ Zumab. The percentage of MPL-positive cells relative to no ID NO:43 (T), SEQID NO:44 (U), SEQID NO:46 (V) SEQ treatment control is shown in the legend. IDNO:49 (W), SEQID NO:51(X), SEQID NO:52(Y), SEQ (O150 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 A1 Nov. 29, 2012

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 SEQID NO: 70). NO: 62) and heavy chain amino acid (SEQID 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), 0.161 FIG. 58 shows surface plasmon resonance (SPR) with structures of the 5-position f3,3'-diphenylalanine medi data for a 5-diphenyl meditope bound to an M5A 8M medi tope (SEQID NO: 18, meditope 18) and cqFD (meditope 1, tope enabled antibody. SEQ ID NO: 1) 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 HPLC 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, NHS-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 B.B'-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 0.165 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 (rIgG) frag (O155 FIG. 52 shows the structure of meditope (position ments, single chain variable fragments (scFV) and single B.B'diphenyl), Protein L (left), Protein A (right) and Fab (grey domain antibodies (e.g., SdAb, SdPV, 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 of immunoglobulins, such as intrabod ies, peptibodies, chimericantibodies, 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-sch V, 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 (O157 FIG. 54 shows that a GPI-linked 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. 1x106 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 CH1 domain of a human IgG2, IgG4, IgG3, and IgG1 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 1 (SEQ ID NO:1, 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) (SEQID NO: 68) compared to the by Kabat et al. (1991), “Sequences of Proteins of Immuno wild-type M5A light chain sequence (SEQ ID NO: 69). logical Interest, 5th Ed. Public Health Service, National Shaded residues represent eight point mutations that were Institutes of Health, Bethesda, Md. (“Kabat numbering introduced in the light chain of the M5A antibody, allowing it scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 US 2012/0301400 A1 Nov. 29, 2012

(“Chothia’ numbering scheme), MacCallum et al., J. Mol. defined by the Kabat, 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 Pet 0171 The term “meditope-enabled' antibody and al., “IMGTunique numbering for immunoglobulin and T cell “meNAb’ refer to an antibody or functional fragment thereof receptor variable domains and Ig Superfamily V-like 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 Plück 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 antibody containing the amino acid residues that interact with variable domains: an automatic modeling and analysis tool.” a bound meditope, which residues include framework region J Mol Biol, 2001 June 8; 3.09(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 Kabat 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 Kabat and Chothia schemes is based the Fab, 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, CH1, 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 Kabat 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 Kabat numbering. 0.174. In some embodiments, the meditope binding site is (0169 Table 1, below, lists the positions of CDR-L1, CDR located within a cavity formed by residues 8, 9, 10, 38,39, 40, L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified 4142,43,44, 45,82,83, 84,85,86,87,99, 100, 101, 102,103, by the Kabat, Chothia, and Contact schemes, respectively. 104, 105,142, 162, 163, 164, 165, 166, 167, 168, and 173 of For CDR-H1, residue numbering is given listed using both the the light chain and 6, 9,38, 39, 40, 41, 42, 43, 44, 45, 84, 86, Kabat and Chothia numbering schemes. It is noted that 87, 88, 89,90,91, 103,104,105,106, 107, 108, 111, 110, 147, because the Kabat 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-H1 loop when the heavy chain of the antibody, according to Kabat number numbered using the Kabat numbering convention varies 1ng. between H32 and H34, depending on the length of the loop. 0.175 With respect to a Fab portion of a meditope-enabled antibody, the meditope binding site includes residues within TABLE 1. the central cavity. The meditope-binding site typically further CDR Kabat Chothia Contact includes constant region residues. 0176 The terms “meditope', as used herein, refers to a CDR-L1 L24--L34 L24--L34 L30--L36 peptide or peptides that binds to a meditope-binding site of a CDR-L2 LSO--L56 LSO--L56 L46--L55 CDR-L3 L89--L97 L89--L97 L89--L96 meditope-enabled antibody, which antibody has a threonine CDR-H1 H31--H3SB H26--H32. . .34 H3O-H3SB at position 40, an asparagine at position 41, and an aspartage (Kabat Numbering") at position 85 of its light chain, according to Kabat number CDR-H1 H31--H3S H26--H32 H3O-H3S (Chothia Numbering) ing, or contains a meditope binding site containing residues CDR-H2 HSO--H6S HS2--HS6 H47-l-HS8 that correspond to those within the meditope-binding site of CDR-H3 H95--H102 H95--H102 H93-H101 cetuximab, meditope-enabled trastuzumab, or meditope-en abled M5A, disclosed herein. Exemplary meditopes include, Kabat 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 coFD and coYN peptides and 'Al-Lazikani 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 encompassa (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 givenantibody or region thereof. Such as a variable region molecule, or compound that is useful in treatment of a disease thereof, should be understood to encompassa (or the specific) or condition. framework region as defined by any of the known Schemes. In 0.178 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 A1 Nov. 29, 2012

therapeutic effect in a subject, Such as preventing or treating modalities. Further, the administration of the two or more a target condition, alleviating symptoms associated with the agents, drugs, treatment regimens, treatment modalities or a condition, producing a desired physiological effect, or allow combination thereofmay 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 (to situmomab) 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 21' 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 0.184 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 1 intracardiac, intradermal, intramuscular, intraperitoneal, to 12 carbon atoms. Representative alkyl groups include, but intrapulmonary, intraspinal, intrasternal, 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-1-butyl, 3-me ous, transmucosal, or transtracheal. thyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 0181 “In combination” or “in combination with, when 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-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-1-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 “C, 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 A1 Nov. 29, 2012

4-(2-methyl-3-butene)-pentenyl, and the like. The term “C. alkenyl, where Xandy 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 D CD plary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, 4-butyl-2-hexy 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), wherein each R group is independently a refers to a monocyclic, aromatic heterocycle having five ring Calkyl, or the two R groups taken togetherform a C-alky 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 -CH(OR), group, but are not limited to, imidazolyl pyrazolyl, triazolyl, tetra wherein each R group is independently a Calkyl, or the two Zolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, R groups taken together form a C2-alkylene. 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) group, by 1.3-cycloaddition reactions such as a Huisgen reaction wherein each R group is independently a Calkyl, or the two between an azide and a propargyl group. R groups taken together form a C2-alkylene. 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 substituents. As used herein, the term “unsubstituted” means 0194 The term “halo' represents chloro, fluoro, bromo, or that the specified group bears no substituents. As used herein, iodo. In some embodiments, halo is chloro, fluoro, or bromo. the term “optionally substituted” means that the specified The term “halogen as used herein refers to fluorine, chlorine, group is unsubstituted or substituted by the specified number bromine, or iodine. of substituents. Where the term "substituted” is used to (0195 The term “ortho ester” refers to a -C(OR) group, describe a structural system, the Substitution is meant to occur wherein each R group is independently a Calkyl, or two of at any Valency-allowed position on the system. the R groups taken together form a C2-alkylene 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(s) that can occurat 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) group, wherein each R group is independently a ents' means 1, 2, 3, 4, or 5 substituents. In another embodi Calkyl, or the two R groups taken togetherform a C-alky ment, one or more Substituent means 1, 2, or 3 Substituents. lene 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, R., or R. 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, 1, 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, —OH refers to a moiety in which an oxygenatom 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 ric or chiral centers and therefore exist in different stereoiso US 2012/0301400 A1 Nov. 29, 2012

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 inorganic 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 orimidazole, 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. 0207 Diastereomeric mixtures may be separated into their amount of a suitable acid or base. Such as an equivalent individual diastereomers on the basis of their physical chemi amount, in a medium Such as one in which the salt precipitates cal differences by methods well known to those skilled in the or in an aqueous medium followed by lyophilization. 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., 1,1'-methylene-bis(2-hydroxy-3-naphthoate)) salts. also be separated by use of chiral HPLC 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 organic 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 arounda 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. Hence, a script. For example, the substructure: pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. 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, organic amines) Such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain halides 0210 where p is 0 or 1, means that the X NH 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 1). 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.) Handbook 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-VCH. 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 & Drug Administration, US 2012/0301400 A1 Nov. 29, 2012

MD, available from FDA). These disclosures are incorpo C-QFDLSTRRLK-C (cGFD; SEQ ID NO: 1) and rated herein by reference thereto. C-QYNLSSRALK-C (cGYN; 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 Protein A (SpA) and Peptostreptococcus magnus vent is water and the solvates are hydrates. Protein L (PpI) (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 H. H. 'C, C, "'C, C. Meditope-Enabled Antibodies and Complexes 'N, O, 7O, P, P, S, F, C1, and II, respectively. Such isotopically labelled compounds are useful in metabolic 0221 Provided are meditope-enabled antibodies that are studies (for example with ''C), reaction kinetic studies (with, capable of binding to one or more meditopes via meditope for example HorH), detection or imaging techniques such binding sites. In some cases, the meditope-enabled antibody as positron emission tomography (PET) or single-photon binds to a cyclic peptide of SEQID NO: 1 or 2 (meditope 1 or emission computed tomography (SPECT) including drug or 2) and/or to one or more variants thereof. Such as meditopes 1. Substrate tissue distribution assays, or in radioactive treat 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, or ment of patients. In particular, an 'F or 'C labeled com 55 (meditopes based on peptides having the sequences set pound may be particularly suitable for PET or SPECT stud forth in SEQID NOs: 1, 2, 16-18, 23, 29, 31, 32, 36,39, 42, ies. Further, substitution with heavier isotopes such as 43, 45, 46, 51, 52, 54, or 55), or in some cases, any of deuterium (i.e., H) may afford certain therapeutic advan meditopes 1, 2, or 15-55. Among the provided meditope tages resulting from greater metabolic stability, for example enabled antibodies are those that bind to a meditope or medi increased in Vivo half-life or reduced dosage requirements. topes with an affinity similar to that of cetuximab. For Isotopically labeled compounds of this invention and pro example, in certain aspects, the antibodies bind to the medi drugs thereof can generally be prepared by carrying out the tope(s) with a dissociation constant of less thanator about 10 procedures disclosed in the schemes or in the examples and uM, less than at or about 5uM, or less than at or about 2 uM, preparations described below by substituting a readily avail less thanator about 1 uM, less thanator about 500, 400, 300, able isotopically labeled reagent for a non-isotopically 200, 100 nM, or less, such as at or about 200 picomolar or less. In some cases, the dissociation constant, such as any of those labeled reagent. listed herein, is that measured using a particular technique, Such as Surface plasmon resonance (SPR), isothermal titra B. Antibodies and Antibody-Binding Substances 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 uM, less than at or about 5uM, or less than at or about 2 generally bind to regions/binding sites of the antibodies and uM, less than at or about 1 uM, 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 , 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: 1 binds to a meditope-binding site within the US 2012/0301400 A1 Nov. 29, 2012

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. 1 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 are replaced with correspond binding site and the coFD (SEQID NO: 1, meditope 1) and ing murine residues; in certain embodiments, they are cQYN (SEQID 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 (CH1 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 Kabat Superposition of the molecular structure of trastuzumab numbering (see Kabat 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 Herceptin 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, (2OSL: Duet al., J Biol Chem, 2007 May 18:282(20): 15073 89 and 105, according to Kabat 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 Kabat num the framework. Superposition of multiple human and murine bering. Also encompassed within the present disclosure are Fabs onto the cetuximab-cyclic peptide (meditope 1) 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 coFD cyclic pep 9, 10,39, 40, 41,42, 43,83, 85, 100, and/or 104, and/or heavy tide (meditope 1) 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 Kabat 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-targeted is replaced with Asn (G41N), light chain framework residue delivery and imaging, and improving mAb-based imaging Phe83 is replaced with Ile (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 SEQID NO: herein, including meditope-enabled trastuzumab and medi 1 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 Kabat 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 A1 Nov. 29, 2012

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 1OO. 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 ascetuximab, 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 analanine at position 100; and the VH region has 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% identity 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 oralanine 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) 1 (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 SEQID NO: 71 position 10, a proline at position 40, a glycine at position 41, (or an FR-L1, 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, or 99% according to Kabat numbering, and/or the VH region does not identical to the FR-L1, 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 I9, I10 or L10, Q38, R39, T40, N41 G42, S43, sequence having a heavy chain FR1 (FR-H1), an FR-H2, an P44, R45, D82, I83, A84, D85, Y86, Y87, G99, A100, G101, FR-H3, and/or an FR-H4, of the heavy chain sequence set T102, K103, L104, E105, R142, S162, V163, T164, E165, forth in SEQID NO: 72 (oran FR-H1, FR-H2, FR-H3, and/or Q166, D167, S168, and Y173, 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-H1, FR-H2, FR-H3, G42, K43, G44, L45, S84, D86, T87, A88, I89, Y90, Y91, and/or FR-H4 of SEQID NO: 72), and in some aspects at least W103, G104, Q105, G106, T107, L108, V109, T110, V111, one CDR that is distinct from the CDRs of the heavy chain Y147, E150, P151, V152, T173, F174, P175, A176, V177, sequence set forth in SEQID 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) 1 (FR-L1), an FR-L2, an FR-L3, and/oran FR-L4 of the ing one or more complementarity determining region (CDR) light chain sequence set forth in SEQID NO: 9 (oran FR-L1, (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-L1, FR-L2, FR-L3, and/or FR-L4 of SEQID 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 SEQID 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 FR1 (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 SEQID NO: 5,558,864 and 8,133,982; Kettleborough et al., “Humaniza 6 (or an FR-H1, 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, or 99% importance of framework residues on loop conformation.” identical to the FR-H1, 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 SEQID 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) 1 (FR-L1), an FR-L2, an FR-L3, and/oran FR-L4 of the 0233. In some embodiments, the meditope-enabled anti light chain sequence set forth in SEQID NO: 68 (oran FR-L1, 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-L1, FR-L2, FR-L3, and/or FR-L4 of SEQIDNO: 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 forthin SEQID 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 FR1 (FR-H1), an FR-H2, an FR-H3, and/or an US 2012/0301400 A1 Nov. 29, 2012

FR-H4, of the heavy chain sequence set forth in SEQID NO: or antigen-binding regions thereof, and/or antibodies that 70 (oran FR-H1, 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, or 99% 0242 Table 2 lists CAS(R) Registry Numbers (see www. identical to the FR-H1, 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 SEQID 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) 1 (FR-L1), an FR-L2, an FR-L3, and/oran FR-L4 of the abagovomab 79.2921-10-9 light chain sequence set forth in SEQID NO: 61 (oran FR-L1, abciximab 43653-53-6 adalimumab 331731-18-1 FR-L2, FR-L3, and/or FR-L4 that is at leastator about 75, 80, adecatumumab SO3605-66-1 85, 90,91, 92,93, 94, 95, 96, 97,98, or 99% identical to the alemtuzumab 216SO3-57-O FR-L1, FR-L2, FR-L3, and/or FR-L4 of SEQIDNO: 61), and indium (111 In) S6586-92-4 altumomab pentetate in some aspects at least one CDR that is distinct from the arcitumomab 54361-48-5 CDRs of the light chain sequence set forth in SEQID NO: 61; arcitumumab 54361-48-5 and/or a VH region with an amino acid sequence having a atlizumab 375823-41-9 basiliximab 52923-56-3 heavy chain FR1 (FR-H1), an FR-H2, an FR-H3, and/or an bectumomab S8318-63-9 FR-H4, of the heavy chain sequence set forth in SEQID NO: belimumab 356547-88-1 63 (oran FR-H1, FR-H2, FR-H3, and/or FR-H4 that is at least benralizumab O4451 1-01-4 at or about 75,80, 85,90,91, 92,93, 94, 95, 96, 97,98, or 99% bevacizumab 21 6974-75-3 brentuximab 914.088-09-8 identical to the FR-H1, FR-H2, FR-H3, and/or FR-H4 of SEQ canakinumab 914613-48-2 ID NO: 63), and in some aspects at least one CDR that is capiromab pendetide 45464-28-4 distinct from the CDRs of the heavy chain sequence set forth capiromab S1763-64-3 in SEQID NO: 63. catumaxomab SO9077-98-9 certolizumab 428863-SO-7 0239. In some embodiments, the meditope-enabled anti certolizumab 428863-SO-7 body has one or more CDRs distinct from the CDRs set forth cetuximab 2O5923-56-4 in SEQID 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 21968S-SO-4 other than cetuximab, does not specifically bind to an EGFR, edrecolomab 1S6S86-89-9 efalizumab 214745-43-4 binds to an antigen other than EGFR, and/or does not specifi etaracizumab 892S53-42-3 cally bind to the epitope on EGFR that is specifically bound etrumaxomab SO9077-99-0 by cetuximab. fanolesomab 225239-31-6 0241. In some examples, the meditope-enabled antibody FBTAOS Lymphomun. FBTA05 fontolizumab 326859-36-3 is generated based on a template antibody that is selected gemtuzumab 22O578-59-6 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 Infliximab 170277-31-3 benralizumab, bevacizumab, brentuximab, canakinumab, ipilimumab 4772O2-00-9 capiromab, capromab pendetide, catumaxomab, certoli labetuzumab 219649-07-7 Zumab, clivatuZumab tetraxetan, daclizumab, denosumab, mepolizumab 196O78-29-2 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 ofatumumab 679818-59-8 infliximab, ipilimumab, labetuZumab, mepolizumab, omalizumab 24.2138-07-4 muromonab, muromonab-CD3, natalizumab, necitumumab oregovomab 213327-37-8 nimotuZumab. ofatumumab, omalizumab, oregovomab, palivizumab 188O39-54-5 palivizumab, panitumumab, ranibizumab, rituximab, Satu panitumumab 339177-26-3 momab, Sulesomab, ibritumomab, ibritumomab tiuxetan, ranibizumab 347396-82-1 tocilizumab, to situmomab, trastuzumab, Trbs07, usteki rituximab 174722-31-7 Satumomab 13895S-26-7 numab, visilizumab, Votumumab, Zalutumumab, Sulesomab 167747-19-5 brodalumab, anrukinzumab, bapineuZumab, dalotuZumab, tiuxetan (ibritumomab) 174722-31-7 demcizumab, ganitumab, inotuZumab, mavrilimumab, mox tocilizumab 375823-41-9 etumomab pasudotox, rillotumumab, Sifalimumab, tan to situmomab 192391-48-3 eZumab, tralokinumab, tremelimumab, the antibody pro trastuzumab 18O288-69-1 ustekinumab 815610-63-O duced by the hybridoma 10B5, B6H12.2, and urelumab, votumumab 148.189-70-2 fragments thereof, antibodies having the CDRs and/or anti Zalutumumab 667901-13-5 gen-binding regions thereof, and/or antibodies that compete brodalumab 1174.395-19-7 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 64.8895-38-9 125-170, fragments thereof antibodies having the CDRs and/ US 2012/0301400 A1 Nov. 29, 2012 19

tor, hepatocyte growth factor, IFN-alpha, nerve growth factor, TABLE 2-continued IL-13, PD-L1, 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 1OOS389-60-5 interest, Such as cancer or other disease. demcizumab (OMP 1292.853-12-3 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 mavrilimumab 1085.337-57-0 human constant regions. In some aspects, the heavy chain moxetumomab 102O748-57-5 constant region includes a CH1, or a portion thereof. In some moxetumomab 102O748-57-5 aspects, the light chain constant region includes a CL, or a pasudotox portion thereof. In some embodiments, the portion of the rillotumumab 8725 14-65-3 constant region is Sufficient to render the antibody capable of Sifallimumab 1143S03-67-6 tanezumab 88O266-57-9 binding to the meditope, e.g., with the requisite binding affin tralokinumab 1044515-88-9 ity. In some aspects, the constant regions are the constant tremelimumab 745O13-59-6 regions of cetuximab or trastuzumab. Thus, in Some aspects, urelumab 934823-49-1 the heavy chain constant regions are one or more human IgG1 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, IgG1, IgG2, IgG3, IgG4, IgEs, IgA1, IgA2, Ig|D, 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 benralizumab, bevacizumab, brentuximab, canakinumab, ated by mutation of residues in other IgGs, such as human, capiromab, 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. Ig|D, 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 (CH1) of a human IgG1, 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 CH1, compared to the IgG1 CH1, tocilizumab, to situmomab, trastuzumab, Trbs07, usteki mapped onto the co-crystal structure of cetuximab and the numab, visilizumab, Votumumab, Zalutumumab, the anti cQFD meditope (meditope 1, SEQID NO: 1). 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 antigenorepitope as Such applicable to isotypes other than the IgG1 of cetuximab. As antibodies, and/or compete for binding with such antibodies another example, the sequence and structural alignment of an to their antigens. IgG1 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) IIb/IIIa receptor, TNF-alpha, CD52, TAG-72, create a uniquehandle 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-1beta, prostate specific mem antibodies. brane antigen (PSMA), CD3, EpCAM, EGF receptor 0248. As described below in section F, also provided are (EGFR), MUC1, human interleukin-2 receptor, Tac, RANK methods for modifying the meditope-enabled antibodies, for ligand, a complement protein, e.g., C5, EpCAM, CD11a, e.g., example, to alter various properties of the meditope-enabled human CD11a, 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 CD15, 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 Fc 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 CTA A16.88, IL 13, interleukin-1 beta, beta-amyloid, IGF-1 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 A1 Nov. 29, 2012 20 fied antibodies and portions thereof, such as a CovX-BodyTM. cQFD and coYN 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, c0FD 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 RNA molecules, encoding the meditope-enabled antibodies, modification of Arg8, a modification of Phe3, a modification and vectors and libraries containing the same, as well as of Leu5, a modification of Leu10, change to the mode of methods for their use, including selection and expression peptide cyclization, and/or an incorporation of hydratable methods and methods for generating transgenic animals carbonyl functionality at one or more positions, and one or using Such constructs. more amino acid deletions or additions. In the case of c(RYN. suitable modifications may include one or more of the fol D. Meditopes lowing: a modification of Arg8, a modification of Leu5, a modification of Leu10, 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 enabled antibodies, and complexes and compositions con functionality at one or more positions, and one or more dele taining the same, and methods and uses thereof. In certain tions or additions. Certain amino acid positions within the embodiments, the meditopes include meditopes 1 and 2 meditope may be deleted or replaced with a different natural (cQFD (SEQID NO: 1) and cqYN (SEQID 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: 1 has been (2004); J Immunol 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 coYN meditopes bind to sites within cetuximab amino acids beyond (i.e., 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 Lhas been added to the C-terminus for use as a vaccine. of the coFD 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 1, 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 1 or 2 (meditope of SEQ ID NO: 1 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 coYN 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, c0FD and binding interface (i.e., between the Fab light and heavy cOYN. 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 cGFD and coYN. 0256 Meditope variants include, but are not limited to, some embodiments include one or more variants ofcCFD and variants incorporating modifications to meditopes, e.g., cQYN.

TABLE 3 SEQ ID Meditope NO Number Sequence Modification (red) Linkage method

1. 1. C-OFDLSTRRLK- C original Disulfide 1 - Cys: 12-Cys

2 2 C- QYNLSSRALK- C original Disulfide 1 - Cys: 12-Cys

15 15 C-qFDLSTRRLK- C q = D-glutamine Disulfide 1 - Cys: 12-Cys