USOO9669.108B2

(12) United States Patent (10) Patent No.: US 9,669,108 B2 Williams et al. (45) Date of Patent: Jun. 6, 2017

(54) MEDITOPES AND MEDITOPE-BINDING (56) References Cited ANTIBODIES AND USES THEREOF U.S. PATENT DOCUMENTS (71) Applicant: City of Hope, Duarte, CA (US) 7.754,853 B2 7/2010 Hellendoorn et al. 8,592,149 B2 11/2013 Ramakrishnan (72) Inventors: John C. Williams, Monrovia, CA (US); 8,658,774 B2 2/2014 Williams et al. David A. Horne, Duarte, CA (US); 2004/0001842 A1 1/2004 Michaeli ...... A61K 39,0005 Yuelong Ma, Duarte, CA (US); Heng 2004, OO67503 A1 4, 2004 Tan et all 424,185.1 Wei Chang, Foster City, CA (US); 2005, OO42664 A1 2/2005 Wua etC al.a. Joshua M. Donaldson, Lumberton, NJ 2005, OO69549 A1 3/2005 Herman (US); Cindy Zer, Duarte, CA (US); 2005/026O711 A1 11/2005 Datta et al. Krzysztof Bzymek, Pasadena, CA 2008, OO38260 A1 2/2008 Ponath et al.

(US); Kendra N. Avery Pasadena, CA 39925. A 299 ElC a (US); Jun Xie, Duarte, CA (US) 2009/0202568 A1 8/2009 Eriksson et al. 2010.0068135 A1 3/2010 Rock (73) Assignee: City of Hope,s Duarte,s CA (US) 2010/0076178 A1 3/2010 Ghayur et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. WO WO-96.40210 12/1996 WO WOO 132207 A1 * 5, 2001 ...... A61K 39,385 WO WO-O2/O72832 9, 2002 (21) Appl. No.: 14/453,586 WO WO-2004/101790 11 2004 WO WO-2005/056606 6, 2005 (22) Filed: Aug. 6, 2014 WO WO-2007/O19541 2, 2007 WO WO-2009/062051 5, 2009 (65) Prior Publication Data WO WO 2009099651 A1 * 8, 2009 ...... A61K 31/44 WO WO-2010, 114940 10, 2010 US 2015/OO30535 A1 Jan. 29, 2015 OTHER PUBLICATIONS Related U.S. Application Data Riemer et al., Journal of the National Cancer Institute, 2005, (60) Division- - - of application No. 13/443,804, filed O Apr. 97(22):Carter et 1663-1670.* al., Endocrine-Related Cancer, 2004, 11:659-687. 10, 2012, now Pat. No. 8,962,804, which is a Albanell et al., “, a humanized anti-HER2 monoclonal continuation-in-part of application No. 13/270.207, antibody, for the treatment of breast cancer.” Drugs Today (Barc) filed on Oct. 10, 2011, now Pat. No. 8,658,774. (1999) 35(12):931-946. International Preliminary Report on Patentability for PCT/US2011/ 55656 issued Jun. 4, 2013, 8 pages. (60) Provisional application No. 61/391,558, filed on Oct. Notice of the First Office Action (including translation) for CN 8, 2010, provisional application No. 61/597,708, filed 201280060888.7, mailed Jul. 17, 2015, 24 pages. on Feb. 10, 2012. Smith et al., Sequence of the full-length immunoglobulin kappa chain of mouse myeloma MPC 11, Biochem. J. (1978) 171:337-347. (51) Int. Cl. Supplementary European Search Report for EP 11831753.6, mailed May 12, 2015, 9 pages. C07K 6/8 (2006.01) Supplementary European Search Report for EP 12839868.2, mailed C07K 700 (2006.01) May 12, 2015, 10 pages. A6 IK 47/48 (2006.01) Accardi et al., “Antibodies in single-chain format against tumour A6 IK 38/00 (2006.01) associated antigens: present and future applications.” Curr Med A6 IK 39/00 (2006.01) Chem (2010) 17(17): 1730-1755. A6 IK 5L/It (2006.01) Adams et al., “High affinity restricts the localization and tumor C07K 6/28 (2006.01) penetration of single-chain fiv antibody molecules, Cancer Res C07K 6/32 (2006.01) (2001) 61(12):4750-4755. B825/00 (2011.01) (Continued) (52) U.S. Cl. CPC. A61K 47/48746 (2013.01); A61K 47/48346 Primary Examiner — Hong Sang (2013.01); A61K 47/48669 (2013.01); A61 K (74) Attorney, Agent, or Firm — Sheppard Mullin Richter 51/1087 (2013.01); B82Y5/00 (2013.01); & Hampton LLP C07K 16/18 (2013.01); C07K 16/2863 (2013.01); C07K 16/32 (2013.01); C07K (57) ABSTRACT 2299/00 (2013.01); C07K 23.17/34 (2013.01); Antibodies and meditopes that bind to the antibodies are C07K 2317/40 (2013.01); C07K 231 7/55 provided, as well as complexes, compositions and combi (2013.01); C07K 231 7/567 (2013.01); C07K nations containing the meditopes and antibodies, and meth 2317/622 (2013.01); C07K 2317/92 (2013.01) ods of producing, using, testing, and Screening the same, (58) Field of Classification Search including therapeutic and diagnostic methods and uses. None See application file for complete search history. 8 Claims, 76 Drawing Sheets US 9,669.108 B2 Page 2

(56) References Cited Chung et al., “-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose." N Eng J Med (2008) 358(11): 1109 1117. OTHER PUBLICATIONS Collis et al., “Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable Adams et al., “PHENIX: Building new software for automated loops and the nature of the antigen.” J Mol Biol (2003)325(2):337 354. crystallographic structure determination.” Acta Crystallogr D Biol Colman, “Effects of amino acid sequence changes on antibody Crystallogr (2002) 58:1948-1954. antigen interactions.” Res Immunol (1994) 145:33-36. Adams et al., “Potent and selective inhibitors of the proteasome: Dechant et al., "Complement-dependent tumor cell lysis triggered dipeptidyl boronic acids,” Bioorg Med Chem Lett (1998) 8(4):333 by combinations of epidermal growth factor receptor antibodies.” 338. Cancer Res (2008) 68(13):4998-5003. 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Doppalapudi et al., "Chemical generation of bispecific antibodies.” Bilgicer et al., “A non-chromatographic method for the purification Proc Natl Acad Sci USA (2010) 107(52):22611-22616. of a bivalently active monoclonal IgG antibody from biological Doppalapudi et al., "Chemically programmed antibodies: fluids,” J Am Chem Soc (2009) 131(26):9361-93.67. endothelin receptor targeting CovX-Bodies,” Bioorg Med Chem Bilgicer et al., “A synthetic trivalent hapten that aggregates anti Lett (2007) 17(2):501-506. 2,4-DNP IgG into bicyclic trimers,” J Am Chem Soc (2007) Dornan et al., “Therapeutic potential of an anti-CD79b antibody 129(12):3722-3728. drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non Bokemeyer et al., “Fluorouracil, leucovorin, and oxaliplatin with Hodgkin lymphoma,” Blood (2009) 114(13):2721-2729. and without cetuximab in the first-line treatment of metastatic Du et al., “Structural basis for recognition of CD20 by therapeutic colorectal cancer,” J. 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Ferenczy et al., “Thermodynamics guided lead discovery and opti Burgess et al., “Possible dissociation of the heparin-binding and mization.” Drug Discov Today (2010) 15:919-932. mitogenic activities of heparin-binding (acidic fibroblast) growth Gencoglan et al., “Two cases of acneiform eruption induced by factor-1 from its receptor-binding activities by site-directed inhibitor of epidermal growth factor receptor.” Skin Pharmacol mutagenesis of a single lysine residue.” J Cell Biol (1990) 111(5 Pt Physiol (2007) 2005):260-262. 1):2129-2138. Goodwin et al., “Pretargeted peptide imaging and therapy,” Cancer Butlin et al., “Antibodies with infinite affinity: origins and applica Biother Radiopharm (1999) 14(3):145-152. tions.” Acc Chem Res (2006) 39(10):780-787. 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Chen et al., “MolProbity: all-atom structure validation for Graille et al., “Evidence for plasticity and structural mimicry at the macromolecular crystallography.” Acta Crystallogr D Biol Crystal immunoglobulin light chain-protein L interface.” J Biol Chem logr (2010) 66(Pt 1): 12-21. (2002) 277(49):47500-47506. Chih et al., “Identification of amino acid residues responsible for the Green et al., “Pretargeted radioimmunotherapy for B-cell lympho release of free drug from an antibody-drug conjugate utilizing mas,” Clin Cancer Res (2007) 13(18 Pt 2):5598s-5603s. lysine-succinimidyl ester chemistry.” J Pharm Sci (2011) Guay et al., “Therapeutic utility and medicinal chemistry of 100(7):2518-2525. cathepsin Cinhibitors.” Curr Top Med Chem (2010) 10(7):708–716. Chmura et al., “Antibodies with infinite affinity.” Proc Natl Acad Sci Hansel et al., “The safety and side effects of monoclonal antibod USA (2001) 98(15):8480-8484. ies.” Nat Rev Drug Discov (2010) 9(4):325-338. Cho et al., “Structure of the extracellular region of HER2 alone and Hardegger et al., “Systematic investigation of halogen bonding in in complex with the Herceptin Fab,” Nature (2003) 421 (6924):756 protein-ligand interactions.” Angew Chem Int Ed Engl (2011) T60. 50(1):314-3.18. US 9,669.108 B2 Page 3

(56) References Cited Milo et al., “Chemical and biological evaluation of dipeptidyl boronic acid proteasome inhibitors for use in prodrugs and pro-Soft OTHER PUBLICATIONS drugs targeting solid tumors,” J Med Chem (2011) 54(13):4365 4377. Hartmann et al., “Peptide mimotopes recognized by antibodies Molloy et al., “Therapy: Targeted but not trouble-free: efalizumab cetuximab and induce a functionally equivalent anti and PML.” Nat Rev Rheumatol (2009) 5(8):418-419. EGFR immune response.” Oncogene (2010) 29(32):4517-4527. Morse et al., “EGFR-targeted therapy and related skin toxicity.” Hernandes et al., "Halogen atoms in the modern medicinal chem Semin Oncol Nurs (2006) 22(3):152-162. istry: hints for the drug design.” Curr Drug Targets (2010) Moss et al., “Trastuzumab-induced cardiotoxicity.” Oncol Nurs 11(3):303-314. Forum (2009) 36(6):676-685. Horiuchi et al., “Transmembrane TNF-alpha: structure, function and Mossessova et al., “Ulp1-SUMO crystal structure and genetic interaction with anti-TNF agents,” Rheumatology (Oxford) (2010) analysis reveal conserved interactions and a regulatory element 49(7): 1215-1228. essential for cell growth in yeast,” Mol Cell (2000) 5(5):865-876. Hughes et al., “Fragment based discovery of a novel and selective Muller et al., "Bispecific antibodies for cancer immunotherapy: PI3 kinase inhibitor,” Bioorg Med Chem Lett (2011) 21(21):6586 Current perspectives.” BioDrugs (2010) 24(2):89-98. 6590. Muller et al., “Rigid conformation of an immunoglobulin domain Hutchins et al., “Site-specific coupling and sterically controlled tandem repeat in the A-band of the elastic muscle protein titlin.” J formation of multimeric antibody fab fragments with unnatural Mol Biol (2007) 371(2):469-480. amino acids,” J Mol Biol (2011) 406(4):595-603. Ngo et al., in: The Protein Folding Problem and Tediary Structure International Search Report and Written Opinion for PCT/US2012/ Prediction, Merz et al. (eds.), Birkhauser, Boston, MA (1994) pp. 032938, dated Oct. 17, 2012. 433 and 492-495. International Search Report and Written Opinion for PCT/US2011/ Nicola et al., “Crystal structure of Escherichia coli penicillin 55656, dated May 10, 2012, 8 pages. binding protein 5 bound to a tripeptide boronic acid inhibitor: a role Junutula et al., “Site-specific conjugation of a cytotoxic drug to an for Ser-110 in deacylation.” Biochemistry (2005) 44(23):8207 antibody improves the therapeutic index.” Nat Biotechnol (2008) 8217. 26(8):925-932. Nilson et al., “Purification of antibodies using protein L-binding Kamat et al., “Enhanced EGFR inhibition and distinct epitope framework structures in the light chain variable domain.” J recognition by EGFR antagonistic mAbs C225 and 425.” Cancer Immunol Methods (1993) 164(1):33-40. Biol Ther (2008) 7(5):726-733. Nygaard et al., Uniprot Accession No. F4WN58, dated Jun. 28, Kiessling et al., "Chemical approaches to glycobiology,” Annu Rev 2011, retrieved online from

(56) References Cited Spangler et al., "Combination antibody treatment down-regulates epidermal growth factor receptor by inhibiting endosomal recy OTHER PUBLICATIONS cling.” Proc Natl Acad Sci USA (2010) 107(30): 13252-13257. Stymiest et al., “Synthesis of oxytocin analogues with replacement Scheuer et al., “Strongly enhanced antitumor activity of of Sulfur by carbon gives potent antagonists with increased stabil trastuzumab and combination treatment on HER2 ity.” J Org Chem (2005) 70(20): 7799-7809. positive human xenograft tumor models.” Cancer Res (2009) Tamura et al., “Structural correlates of an anticarcinoma antibody: 69(24):9330-9336. identification of specificity-determining residues (SDRs) and devel Schrag et al., "Cetuximab therapy and symptomatic opment of a minimally immunogenic antibody variant by retention hypomagnesemia,” JNatl Cancer Inst (2005) 97(16): 1221-1224. of SDRs only,” J Immunol (2000) 164(3): 1432-1441. Seeman, “DNA in a material world,” Nature (2003) 421 (6921):427 Teillaud et al., “Engineering of monoclonal antibodies and anti 431. body-based fusion proteins: Successes and challenges,” Expert Opin Shan et al., “Apoptosis of malignant human B cells by ligation of Biol Ther (2005) 5(Suppl 1): S15-S27. CD20 with monoclonal antibodies,” Blood (1998) 91(5): 1644 Thakur et al., "Cancer therapy with bispecific antibodies: Clinical 1652. experience.” Curr Opin Mol Ther (2010) 12(3):340-349. Sharav et al., “Mimotope vaccines for cancer immunotherapy,' Van Cutsem et al., "Cetuximab and as initial treat Vaccine (2007) 25(16):3032-3037. ment for metastatic colorectal cancer." N Engl J Med (2009) Sharkey et al., “Recombinant bispecific monoclonal antibodies 360(14): 1408-1417. prepared by the dock-and-lock strategy for pretargeted radioim Wakankar et al., “Physicochemical stability of the antibody-drug munotherapy,” Semin Nucl Med (2010) 40(3):190-203. conjugate Trastuzumab-DM1: changes due to modification and Sheedy et al., “Isolation and affinity maturation of hapten-specific conjugation processes,” Bioconjug Chem (2010) 21(9): 1588-1595. antibodies.” Biotechnol Adv (2007) 25(4):333-352. Young Jr. et al., “Staphylococcal protein A binding to the Fab Shirasaki et al., “Exploration of orally available calpain inhibitors 2: fragments of mouse monoclonal antibodies,” J Immunol (1984) peptidyl hemiacetal derivatives,” J Med Chem (2006) 49(13):3926 133(6):3163-3166. 3932. International Preliminary Report on Patentability for PCT/US2012/ Shuker et al., “Discovering high-affinity ligands for proteins: SAR 032938, dated Aug. 21, 2013, 13 pages. by NMR,” Science (1996) 274(5292): 1531-1534. First Office Action and Search Report in Chinese Patent Application Skolnick and Fetrow, “From genes to protein structure and function: No. 2011800593.23.2, issued Jul. 6, 2014. novel applications of computational approaches in the genomic era,” Trends Biotechnol (2000) 18(1):34-39. * cited by examiner U.S. Patent Jun. 6, 2017 Sheet 1 of 76 US 9,669,108 B2

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U.S. Patent Jun. 6, 2017 Sheet 32 of 76 US 9,669,108 B2

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U.S. Patent Jun. 6, 2017 Sheet 36 of 76 US 9,669,108 B2

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U.S. Patent Jun. 6, 2017 Sheet 39 Of 76 US 9,669,108 B2

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U.S. Patent Jun. 6, 2017 Sheet 40 of 76 US 9,669,108 B2

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U.S. Patent Jun. 6, 2017 Sheet 43 of 76 US 9,669,108 B2

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40 A1 U.S. Patent Jun. 6, 2017 Sheet 45 of 76 US 9,669,108 B2

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Figure 39 (cont'd)

U.S. Patent Jun. 6, 2017 Sheet 47 of 76 US 9,669,108 B2

Figure 40

SEQ to NO; 15 6 17 18 Data Collection Space group P222 P222 P222 P222 Ce dimensions a, b, c (A) 64.33, 82.84, 64.20, 82.50, 64,02, 82.83, 64.28, 83.25, 22.32 21249 212,10 22.30 8, y () 90.0, 900, 90.0 90,0, 90.0, 90.0 90.0, 900, 90.0 90.0, 90.0, 90.0 Resolution (A) 3000-2.24 32.99-2.53 34-36-2.48 33.9-248 (2.45-2.24) (2.60-2.53) (2.54-2.48) (2.54-2.48) Rmrge 0.070 (0.281) 0.066 (0.334) 0.065 (0.312) 0.072 (0.331) fo() 1905 (5.92) 24.74 (5.2) 24.88 (4.97) 23.52 (4.68) Completeness (%) 99.1 (99.1) 97.6 (91.3) 99.4 (92.6) 99.1 (90.3) Redundancy 5.2 (5.4) 4.9 (4.5) 6.9 (4.27) 4.61 (3.16) Refinement Resolution (A) 2.24 2.53 2.48 2.48 No. Feflections 55,026 37,650 40,641 41,098 Rwork Riree 8,022.6 17.2/23.6 1787.220 174422.9 No. atons Protei 6526 6514 6551 6541 Meditope 174 291 (198) 22 218 Water 507 392 5 469 B-factors Protein Fa 38.6 27.79 25.74. 25.23 Meditope 49.08 43.40 (2751)* 31.78 352 Water 41.78 28.05 28.32 29.65 r. n.S.,d Bond lengths (A) GOO7 O.O.05 O.OOf O.008 Bond angles () 1093 O.903 1.09 1106 Ranachandran favorediatowedfodisallowed 96.9/3.1 iO, 97.6f2.4f00 97.212.710, 1

*3 meditopes in asymmetric unit. Data in parenthesis refer to the meditopes in the meditope binding pocket characteristic for the Wii meditope U.S. Patent Jun. 6, 2017 Sheet 48 of 76 US 9,669,108 B2

Figure 40 (contd)

SEQED NO; 21 22 23 24 Data Collection Space group P222 P222 P222 P222 Cell dimensions a, b, c (A) 64.38, 82,87, 64.24, 83, 4, 64.30, 83.39, 64.46, 83,21, 2300 21.94 22,73 212.44 o, 3, y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90,0, 900, 900 900, 900, 90.0 Resolution (A) 32.65-253 33.5-2.48 33.23-2.49 3496-2.62 (260-2.53) (2.55-2.48) (2.55-2.49) (2.69-2.62) Rarge-F 0.129 (0.637) 0.044 (0.153) 0.056 (0.219) 0.125 (0.470) ifo() 23.46 (7.34) 25.24 (9.23) 9.83 (6.03) 15.21 (3.87) Competeness (%) 99.6 (98.1) 95.1 (72.3) 99.6 (95.4) 98.6 (97.7) Redundancy 3.91 (3.41) 5.2 (4.6) 5.0 (3.66) 4.95 (5.05) Refinement Resolution (A) 2.53 2.48 2.49 262 No. reflections 38,821 39,080 40,816 34,738 Rworld Rree 19, 1124,7 17.9:22.6 17, 22.3 18.4/235 No. atoms Protein 6535 6554 6525 6529 Meditope 2O2 208 198 74. Water 327 493 511 380 B-factors Protein F3 26.57 24.08 24.69 2547 Meditope 316 27.64 30.40 2645 Water 28,48 29,37 29.5 28,75 ..S.d Bond lengths (A) 0.08 0.003 O.OO7 0.002 Bond angles () 185 0.742 113 O,618 Rarachandra favoredfallowedicisallowed 97.212,710.1 96.9;3.1 f.O 97.112.9f).O 96.3f3.7/OO U.S. Patent Jun. 6, 2017 Sheet 49 of 76 US 9,669,108 B2

Figure 40 (cont'd)

SEQ D NO: 25 3. 32 33 Data Collection Space group P222 P222 P222 P222 Ce dirnensions 64 10, 83.16, 64.22. 8253, 64.28, 82,64, 64.16, 82,46, a, b, c (A) 212.32 212.53 212.50 212.05 c., 3, y () 90.0, 90.0, 90.0 900, 90.0, 90.0 900, 90.0, 90.0 900, 90.0, 90.0 33. 4-2.55 33.00-2.60 38.00-255 32.5-2.49 Resolution (A) (2,62-2.55) (2,67-2.60) (2,62-2.55) (2,55-249) Rmrg-F 0.094 (0.374) 0.093 (0.385) 0.085 (0.365) 0.039 (0.163) tfo() 19, 13 (4.60) 16.79 (4.65) 20.27 (4.54) 31,38 (9.58) Completeness (%) 99.4 (94.1) 95.8 (93.7) 96.7 (91.5) 99.6 (96.1) Redundancy 4.33 (3.96) 4.35 (4.36) 5.1 (5.1) 5.01 (3.61) Referent Resolution (A) 2.55 2.6 2.55 2.49 No effections 37701 34,156 36,560 40,268 Rworkfree 178/2.2.5 18.8:23, 8.7/22.7 16.9f22.0 No. atoms Protein 6517 6527 6545 6509 Meditope 190 192 188 174 Water 404 338 332 471 B-factors Protein Fab 25.98 236 27.94 25,70 Meditope 26.64 24.70 2973 53.33 Wate 28.08 22.78 29.40 355 ..S.d Bond lengths (A) 0.008 OOO3 OOO2 O003 Bond angles () 22 0.777 O631 0.77 Ramachandran favorediaowedfodisallowed 96,573.4f0. 97.4f2.6/OO 96.23.8/OO 97.2f 2.81OO U.S. Patent Jun. 6, 2017 Sheet 50 Of 76 US 9,669,108 B2

Figure 40 (cont'd)

SEQ D NO: 34 35 36 37 Data Collection see - page page page page - Ce dinensions 64, 18, 82,66, 63.97, 82.50, 64.0, 82.21, 6404, 82.51, a, b, c (A) 2191 21 188 21.90 21154. ci, 3, () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 900, 90.0, 90.0 33,02-2.50 32.95-250 344-2.5 34.222.48 Resolution (A) (2.56-2.50) (2.62-2.55) (2.58-2.51) (2.55-248) Ringd-F 0.047 (0.177) 0.094 (0.405) 0.046 (0.238) 0.039 (0.144) fo() 30.78 (9.25) 17.76 (3.68) 25.22 (6.50) 30, 13 (8.86) Completeness (%) 99.8 (99.0) 99.0 (90.1) 98.5 (92.3) 99.4 (92.5) Redundancy 6.02 (470) 4.00 (3.70) 4.12 (3.52) 5.77 (4.4) Refinement Resolution (A) 250 2.55 2.5 248 No. reflections 39,848 37,028 38,565 40,290 Rworkfree 16.91225 6.9f24. 9.023.9 7.9,235 No. atoms Protein 6507 6555 6528 6535 Meditope 182 190 218 187 Water 503 285 338 454. B-factors Protein Fa 262 32O2 34,24 26.67 Meditope 40.13 3.842 4705 A8.78 Water 2778 33.79 32.96 3.14. f,ns,d Bond engths (A) 0.004 O005 OOO3 O008 Bond angles () O.876 O.904 O.749 1.12O Ranacharda favoredfallowed/cdisallowed 97.3f2.7/O.O 96.4.13.8f O.O 96.4f3.5.0.1 96.O.f4. Of O.O U.S. Patent Jun. 6, 2017 Sheet 51. Of 76 US 9,669,108 B2

Figure 40 (cont'd)

SEC D NO: 38 39 40 4. Data Collection Space group P222 P22-24 P2212 P2422 Ce dimensions a, b, c (A) 64.05, 83.16, 64.4, 83.19, 64.21, 83,04, 64.34, 82.57, 22,26 22,46 22.26 212.05 ot, 3, y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 900, 90.0, 90.0 900, 90.0, 90.0 Resolution (A) 33.13-2.50 33.16-248 34.40-248 34,29-248 (2.56-2.50) (255-2.48) (2.54-2,48) (2.54-2.48) Rmrg-F 0,084 (0.375) 0.054 (0, 186) 0.051 (0,184) 0.035 (0.147) lfo () 20.51 (4.31) 29.15 (8.34) 30.80 (7.93) 38.15 (11.37) Completeness (%) 99.2 (92.2) 99.3 (93.2) 99.4 (93.6) 98.2 (91.1) Redundancy 6.26 (4.94) 5,79 (4.10) 5.40 (3.86) 6.17 (4.25) Refinement Resolution (A) 2.50 2.48 2.48 2.48 No, refections 39,826 40,872 40,869 40,195 Rworkfree 19.1 f240 8.1123.3 78.22.4 18.6123.8 No. atoms Protein SS24. 8539 6499 6539 Meditope 190 92 192 192 Water 361 485 466 475 B-factors Protei Fa 26.9 19, 5 2004 20,24 Meditope 33.05 26.32 26.6 27.05 Water 28.90 24.3 24.30 2457 r.m.S.d Bond lengths (A) 0.004 0.003 OOO7 O.007 Bond angles () 0.822 O,760 107 1.125 Ranachandian favorediaowedicisatiowed 96.6/34f00 96.8f3, f(0. 97. Of2.9/O. 96.9/30/0.1 U.S. Patent Jun. 6, 2017 Sheet 52 Of 76 US 9,669,108 B2

Figure 40 (cont'd)

SEQED NO: 42 43 46 Data Collection Space group P222 P222 P222 P222 Ceil dimensions 64.06, 82.71, 64. 14, 82.57, 64.28, 83,05, a, b, c (A) 63.95, 82.38, 22.39 21207 22,37 21,54. o, 8, Y () 90.0, 90.0, 90, O 90.0, 900, 90.0 90,0, 90,0, 90.0 90.0, 90.0, 90.0 34.742.48 34,262.55 32.71-2.55 34, 17-2.50 Resolution (A) (2.54-2.48) (2.61-2.55) (2,62-2.55) (2.56-2.50) Reorgi-F 0.045 (0.209) 0.101 (0.482) 0.078 (0.313) 0.066 (0.337) ifo(i) 28.89 (7.15) 20.57 (3.85) 21.03 (4.48) 20.98 (4.11) Completeness (%) 98.7 (89.6) 99.2 (93.0) 97.6 (95.1) 99.1 (90.6) Redundancy 5.85 (4,14) 5.49 (4.89) 4.47 (469) 3.92 (3.01) Refinement Resolution (A) 2.48 255 255 250 No, refections 40,599 37,507 37,078 39,231 Rworkfree 17.2,28 19.8,252 8.222.3 8.2:22.9 No, atons Proteir 6539 6531 6522 6520 Meditope 185 82 190 198 Water 490 338 459 42 B-factors Prote: Fa 234 31.73 23.90 27, 12 Meditope 26.53 39.04 37,29 37.46 Water 24.76 31.88 28.49 29.83 ..S.d Bond lengths (A) O.007 0.004 0.003 0.004 Bond angles () 136 0.755 O.767 O.921 Ramachandran favoredfallowedfodisallowed 97.112.9f.O 96.6/34f0.0 96.5/3.5/O. 96.93. iOO U.S. Patent Jun. 6, 2017 Sheet 53 Of 76 US 9,669,108 B2

Figure 40 (cont'd)

SECR D NO; 49 5 52 53 Data Collection Space group P2242 P222 P222 P222 Ceil dimensions a, b, c (A) 64.24, 82.94, 64. 3, 82.47, 64.27, 82.74, 64.15, 82.75, 224 22.49 22.37 267 o, 3, y () 900, 90.0, 90.0 90,0, 90,0, 90.0 900, 90.0, 90.0 90,0, 90,0, 90.0 Resolution (A) 3438-2.52 34.23-2.48 34.79-249 33.03-250 (2.59-2.52) (2.54-2.48) (2.55-2.49) (2.56-2.50) Rargd-F 0.057 (0,196) 0.087 (0.374) 0.067 (0.308) 0.059 (0.238) Ho() 26.03 (7,14) 18,64 (4.04) 27.6 (5,79) 21,92 (5.96) Competeness (%) 97.4 (91.4) 99.1 (90.3) 99.1 (94.2) 98.4 (91.0) Redundancy 4.60 (3.89) 4.76 (3.19) 454 (3.53) 4.30 (3.12) Refinement Resolution (A) 2.52 2.48 2.49 250 No reflections 38,211 40,649 40,191 39,261 Rworkfree 8.6F22.9 8, 1122.9 7.3f22.9 80/234 No. atons Protein 6587 6544 6529 6549 Meditope 196 197 185 206 Water 363 4.38 454 36 B-factors Protein Fab 23,36 2305 2147 25.78 Meditope 27.69 33.77 27.9 47.45 Water 25.75 2689 25.45 28,54 r.m.S.d Bond engths (A) O.006 O008 O.OO7 0.005 Bond angles () 1079 1164: 1076 0.970 Ramachandra favored faiowedfoisailowed 96.93.110.0 97.3/2.740.O 96.9/3.110.0 96.93.O.O.1 U.S. Patent Jun. 6, 2017 Sheet 54 of 76 US 9,669,108 B2

Figure 40 (cont'd)

SEQ O NO; 54 55 Data Collection Space group P222 P222 Ce dimensions 64,58, 83.06, 64. 18, 82.89, a, b, c (A) 21 183 212,90 o, 3, y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 33.17-248 34,82-2.50 Resolution (A) (2.55-248) (2.57-2.50) Rmrgd-F 0.036 (0.169) 0.064 (0.245) ifo () 34.78 (9.23) 20.15 (5.87) Completeness (%) 98.0 (85.0) 99.6 (96.1) Redundancy 5.47 (4.28) 4.68 (3.53) Refinement Resolution (A) 2.48 2.50 No reflections 40,269 40,034 Rworkfree 19. Of 23.0 168/216 No atons Protein 6577 6569 Meditope 184 94 Water 294 5O1 B-factors Protein Fab 37.76 20,94 Meditope 50, 4 28,28 Water 36,97 25,34 f.m.S.C. Bond lengths (A) 0.004 O.OO7 Bond angles () 0.864. 1,095 Ramachandran favorediatiowed/disallowed 97.1 f2,810.1 96.913, 1100 U.S. Patent Jun. 6, 2017 Sheet 55 Of 76 US 9,669,108 B2

Figure 41 Diphenylalanine, position 3

90; -orrors K. s. 230 nM

... 8 so so 3oo ts zös 8.

Kai 2400 nM U.S. Patent Jun. 6, 2017 Sheet 56 of 76 US 9,669,108 B2

Figure 42 Phe3

Diphenylalanine3. U.S. Patent Jun. 6, 2017 Sheet 57 Of 76 US 9,669,108 B2

Figure 43

-- on 800 10 10 1. treated treated

to to it is is Alexa 488 Alexa 488 meditope-enabled Wild-type trastuzumab trastuzumab U.S. Patent Jun. 6, 2017 Sheet 58 Of 76 US 9,669,108 B2

Figure 44

is' s se so to's is' se s s' Alexa 647-AAPL (WT) U.S. Patent Jun. 6, 2017 Sheet 59. Of 76 US 9,669,108 B2

Figure 45 {x :--SKory 8 Y six-E-SK snRE-SK

WTT+MPL-5K O.tv WTT+MPLK

M-P-5K

U.S. Patent Jun. 6, 2017 Sheet 61 of 76 US 9,669,108 B2

Figure 47A A) light chain nuc leic acid (SEQ D NO 60) and am id (SEQ D NO 61) Sequences

w Y". s W.'

U.S. Patent Jun. 6, 2017 Sheet 62 of 76 US 9,669,108 B2

Figure 47B B) Heavy chain nuc e ic aci (SEQ iD NO 62) and amino acid (SEQ D NO 63) Sequences

- : -

^^ -

U.S. Patent Jun. 6, 2017 Sheet 63 of 76 US 9,669,108 B2

Figure 48 U.S. Patent Jun. 6, 2017 Sheet 64 of 76 US 9,669,108 B2

: 88:888x38x8-88

::8:

::

388 88: 888xistops:-- 880 as editope-fe

388. 88.3:

kix8 888 U.S. Patent Jun. 6, 2017 Sheet 65 of 76 US 9,669,108 B2

Figure 50

White Sticks: 5-dipheylalanine meditope bound to trastuzumab MeMab; Dark Grey Sticks: 5-diphenylalanine meditope bound to cetuximab; Outlined Sticks show Fab residues.

U.S. Patent Jun. 6, 2017 Sheet 66 of 76 US 9,669,108 B2

Figure 51 rastuzumab vs Meditope-Enabied Merab Meditope Enabied Miemab on Cetuximab Thr? Asp85 Asp85

1. Pro/thrao ? Pfefile:3 Glyfasnaf fe83 Asn-ff

U.S. Patent Jun. 6, 2017 Sheet 67 of 76 US 9,669,108 B2

Figure 52

U.S. Patent Jun. 6, 2017 Sheet 68 of 76 US 9,669,108 B2

::::::::::::::::::

::::::::::::::::::: :::::::::::

U.S. Patent Jun. 6, 2017 Sheet 69 of 76 US 9,669,108 B2

Figure 54 Control Meditope-Protein L (10nM)

Meditope enabled rastuzumah-GP 68.9%

Meditope-Protein L

U.S. Patent Jun. 6, 2017 Sheet 73 Of 76 US 9,669,108 B2

Figre $8 meditepe waiiants vs. 8888 ig. 8:::::::::ges at St Kii 8:3

3888 8. U.S. Patent Jun. 6, 2017 Sheet 74 of 76 US 9,669,108 B2

Figure 59 HPC

MaSS Spectru}} * C6H13N 1906, ESI-MS, calculated m/z. 714.94 M--2H, found 714.94 M+2H. yma 1204163 #1-11 RT; 0.00-0.19 AV: 1 NL: 1.25E5 T: FEMS + p NS: Fulms 150.00-2000.00 714.9375 300- z;2 90 80 70 60 50 40 30 566.8892 20- zs? 2-2 1 O.7892 430,941 1382,7390 16546892 z:1 2: 1990.4625 .. as: . ill-tip-ik- Bill lii: zza? - : ; -- : ; ; ; ; ; 1000 1200 14OO 1600 1800 2OOO niz U.S. Patent Jun. 6, 2017 Sheet 75 Of 76 US 9,669,108 B2

Figure 60

yrna; 20328 6 it -40 RT: 0.8-0.74 AV: 40 N. 5.33E3 MP-mono-DOTA f: FiMS + p NSE Futi ms (600.00-2000.00 189.526 100- 2-5

1823,919.5 :e5 MP-di-DOA g s MP s '

; M 828, 27 z:5 1896.9856 48. 25 as . 839.003 1905.3478 1748.684 803,922 zs5 z:5 ze? 1853 96.9426 1766.4538 788.4325 : 2a: 8819234 2-5 1940.1349

Z5 s? se ... r ze? i i ! retres i -- Fair, prever, ; : 1763 1780 800 820 840 88O 88 900 1920 1940 96.O. riz U.S. Patent Jun. 6, 2017 Sheet 76 of 76 US 9,669,108 B2

Figure 61 A treated Cottro

s

i 3.

CetLIXima + Meditope-FC meditope-FC Only US 9,669,108 B2 1. 2 MEDTOPES AND MEDITOPE-BINDING antibodies, compounds and compositions including peptides ANTIBODIES AND USES THEREOF and other molecules, and related methods that address Such needs. PRIORITY CLAIM SUMMARY This application is a divisional of U.S. application Ser. No. 13/443,804, filed Apr. 10, 2012, now U.S. Pat. No. Among the provided embodiments are antibodies, com 8,962,804, which claims the benefit of International Appli pounds and compositions, including peptides and other cation Number PCT/US2011/055656 filed Oct. 10, 2011, molecules for use with the antibodies, as well as methods and U.S. Provisional Patent Application Ser. No. 61/597, 10 and uses thereof and for producing the same, as well as 708, filed Feb. 10, 2012, and is a continuation-in-part of U.S. compounds, compositions, complexes, mixtures, and sys application Ser. No. 13/270,207, filed Oct. 10, 2011, now tems, e.g., kits, containing the same. In some aspects, the U.S. Pat. No. 8,658,774, which claims the benefit of U.S. provided embodiments afford improved efficacy, synergy, Provisional Patent Application Ser. No. 61/391,558, filed 15 specificity, and/or safety, compared with available antibod Oct. 8, 2010, the contents of each of which are incorporated ies and associated compounds, compositions, methods and herein by reference in their entirety. USS. This invention was made with Government support of Provided are antibodies, including meditope-enabled anti NCI Comprehensive Cancer Center Grant number bodies (including fragments thereof), which bind to (i.e., are CA0335752-28. The government has certain rights in this capable of binding to) one or more meditope. In some invention. aspects, the antibodies bind to a meditope that is a cyclic peptide derived from a peptide having an amino acid SUBMISSION OF SEQUENCE LISTING ON sequence of SEQ ID NO: 1. ASCII TEXT FILE In some aspects, the antibodies bind to a meditope that is 25 a cyclic peptide derived from a peptide having an amino acid The content of the following submission on ASCII text sequence of SEQID NO: 2. In some aspects, the antibodies file is incorporated herein by reference in its entirety: a bind to a meditope that is a peptide having an amino acid computer readable form (CRF) of the Sequence Listing (file sequence selected from the group consisting of the name: 706122000110SeqList.txt, date recorded: Aug. 5, sequences set forth in SEQID NO: 1, 2, 16-18, 23, 29, 31, 2014, size: 331,783 KB). 30 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, and 55, or a cyclic peptide derived therefrom, or the sequences set forth in SEQ BACKGROUND ID NOs: 1, 2, and 15-55, or a cyclic peptide derived therefrom, or to any of the meditopes described herein. In Monoclonal antibodies (mAbs) are used in a number of Some cases, the meditope is a cyclic peptide derived from a therapeutic, diagnostic, and research applications. Therapeu 35 peptide of the amino acid sequence set forth in SEQID NO: tic and diagnostic areas include cancer, antiviral treatment, 1 or 2. autoimmune and inflammatory disease, allergy, cardiovas In some aspects, the antibody or fragment binds to the cular disease, osteoporosis, and rheumatology. meditope with a particular affinity, Such as an affinity that is Protein engineering and other efforts have generated equal to or Substantially equal to one of the exemplary 40 antibodies described herein, Such as cetuximab, meditope mAbs with improved efficacy and targeting (e.g., bispecific enabled trastuzumab, meditope-enabled M5A, or other mAbs), improved localization, tissue penetration, and blood exemplified antibody. In some examples, the antibodies bind clearance (e.g., single chain Fab variable fragments (scEvs), to the meditope or meditopes with a dissociation constant of diabodies, minibodies, and other fragments), and altered less than at or about 10 M, less than at or about 5 uM, or immunostimulatory, safety, toxicity, and/or pharmacoki 45 less than at or about 2 uM, less than at or about 1 uM, less netic/pharmacodynamics properties. Such as those contain than at or about 500, 400, 300, 200, 100 nM, or less, such ing modified Fc regions (e.g., through mutation or glycosy as at or about 200 picomolar or less, for example, with such lation). mAbs have been reengineered to permit site-specific a dissociation constant, as measured by a particular tech conjugation of Small molecules for improved delivery (e.g., nique. Such as Surface plasmon resonance (SPR), Isothermal ThioMABs) or to irreversibly bind to their antigen (e.g., 50 Titration calorimetry (ITC), fluorescence, fluorescence infinite affinity mAbs). mAbs have also been developed to polarization, NMR, IR, calorimetry titrations; Kinetic exclu improve the circulation and presentation of bioactive pep sion; Circular dichroism, differential scanning calorimetry, tides and other biologics (e.g., CovX-bodies). Conjugation or other known method, e.g., by SPR. to various agents has allowed targeted immunotherapy and In some aspects, the antibodies include a heavy chain diagnostic methods. Hetero-multimeric scFVs and scFVs or 55 variable (VH) region and/or a light chain variable (VL) mAbs fused to avidin have been developed for pre-targeted region. In some aspects, the VL region has an amino acid therapy and to improve the detection limits for tumor sequence comprising a threonine, serine, or aspartate at imaging. position 40, a residue other than glycine at position 41, Although mAbs can be effective and have advantages and/or an aspartate or asparagine at position 85, according to over Small molecule approaches, existing antibodies and 60 Kabat numbering, and/or comprises an isoleucine or leucine methods have various limitations. These can include adverse at position 10 and isoleucine at position 83, according to side effects resulting from off-target interactions, and/or Kabat numbering, and/or comprises a valine or isoleucine at collateral damage due to, among other things, long circula position 9 and a residue other than glutamine at position 100, tion times of antibody-drug conjugates). There is a need for according to Kabat numbering. In some examples, the amino improved antibodies and associated compounds, including 65 acid sequence of the VL region has a threonine at position those providing improved efficacy, synergy, specificity, and 40, an asparagine at position 41, and an aspartate at position safety, and methods and uses of the same. Provided are 85, according to Kabat numbering. US 9,669,108 B2 3 4 In some aspects, the VH region has an amino acid at or about 75, 80, 85, 90,91, 92,93, 94, 95, 96, 97,98, or sequence comprising a serine or proline at position 40 and 99% identical to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of an isoleucine, tyrosine, methionine, phenylalanine, or tryp SEQ ID NO: 9); and/or a VH region with an amino acid tophan at position 89, according to Kabat numbering. In sequence having a heavy chain FR1 (FR-H1), an FR-H2, an Some examples, the amino acid sequence of the VH region FR-H3, and/or an FR-H4 of the heavy chain sequence set has a serine at position 40 and an isoleucine at position 89. forth in SEQID NO: 6 (or an FR-H1, FR-H2, FR-H3, and/or according to Kabat numbering. FR-H4 that is at least at or about 75, 80, 85,90, 91, 92,93, In some examples, the amino acid sequence of the VL 94, 95, 96, 97, 98, or 99% identical to the FR-H1, FR-H2, region has a valine or isoleucine at position 9, an isoleucine FR-H3, and/or FR-H4 of SEQID NO:9). In some examples, or leucine at position 10, an arginine at position 39, a 10 the VL region comprises at least one complementarity threonine at position 40, an asparagine at position 41, a determining region (CDR) that is distinct from the CDRs of glycine at position 42, a serine at position 43, an isoleucine the VL sequence set forth in SEQID NO: 9; and/or the VH at position 83, an aspartate at position 85, and an alanine at region comprises at least one CDR that is distinct from the position 100, according to Kabat numbering; and/or the CDRs of the VH sequence set forth in SEQ ID NO: 6. amino acid sequence of the VH region has a serine at 15 In some examples, the VL region comprises the amino position 40 and an isoleucine at position 89, according to acid sequence of SEQID NO: 73. In some examples, the VH Kabat numbering. region comprises the amino acid sequence of SEQ ID NO: In some aspects of the provided antibodies, the VL region 74. does not contain a proline at position 40, a glycine at In some aspects, the antibody or fragment has a VL region position 41, and/or a threonine at position 85, according to with an amino acid sequence comprising a light chain Kabat numbering, and/or the VH region does not contain an framework region (FR) 1 (FR-L1), an FR-L2, an FR-L3, asparagine or alanine at position 40 and/or a valine at and/or an FR-L4 of the light chain sequence set forth in SEQ position 89, according to Kabat numbering. ID NO: 68 (or an FR-L1, FR-L2, FR-L3, and/or FR-L4 that In some aspects, the VL region does not contain an serine is at least at or about 75, 80, 85,90, 91, 92,93, 94, 95, 96, at position 10, a proline at position 40, a glycine at position 25 97, 98, or 99% identical to the FR-L1, FR-L2, FR-L3, and/or 41, an phenylalanine at position 83, and/or a threonine at FR-L4 of the light chain of SEQ ID NO: 68). In some position 85, according to Kabat numbering, and/or the VH examples, the VL region comprises at least one complemen region does not contain an asparagine or alanine at position tarity determining region (CDR) that is distinct from the 40 and/or a valine at position 89, according to Kabat CDRs of the light chain sequence set forth in SEQ ID NO: numbering. 30 69; and/or the VH region comprises at least one CDR that is In some aspects, the antibodies (including fragments) distinct from the CDRs of the heavy chain sequence set forth further include one or more constant regions, typically in SEQID NO: 70. human constant region(s). Such as a CL and/or CH1, e.g., In some examples, the VL region comprises the amino human CL and/or CH1. acid sequence of SEQ ID NO: 75. In some aspects, the provided antibody or fragment has a 35 In some aspects, the antibody does not specifically bind to VL region with an amino acid sequence comprising a light the epitope of an EGFR that is specifically bound by chain framework region (FR) 1 (FR-L1), an FR-L2, an cetuximab, does not contain the CDRs of cetuximab, and/or FR-L3, and/or an FR-L4 of the light chain sequence set forth does not compete for antigen binding with cetuximab. In in SEQID NO: 71 or SEQID NO: 61 (or an FR-L1, FR-L2, other aspects, the antibody is cetuximab. FR-L3, and/or FR-L4 that is at least at or about 75, 80, 85, 40 In some aspects, the antibodies or fragments compete for 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% identical to the antigen binding with, specifically bind to the same antigen FR-L1, FR-L2, FR-L3, and/or FR-L4 of the light chain of or epitope as, and/or contain one, more, or all CDRS (or SEQ ID NO: 71 or 61), and in some aspects at least one CDRs comprising at least at or about 75, 80, 85, 90,91, 92, complementarity determining region (CDR) that is distinct 93, 94, 95, 96, 97,98, or 99% identity to the CDRs), e.g., from the CDRs of the light chain sequence set forth in SEQ 45 including a heavy chain CDR 1, 2, and/or 3 and/or a light ID NO: 71; and/or a VH region with an amino acid sequence chain CDR1, 2, and/or 3, of one or more known antibodies, having a heavy chain FR1 (FR-H1), an FR-H2, an FR-H3. including any commercially available antibody, Such as and/or an FR-H4, of the heavy chain sequence set forth in , abciximab, adalimumab, , SEQID NO: 72 or SEQ ID NO: 63 (or an FR-H1, FR-H2, , altumomab, altumomab pentetate, anatu FR-H3, and/or FR-H4 that is at least at or about 75, 80, 85, 50 momab, anatumomab mafenatoX, arcitumomab, atlizumab, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% identical to the basiliximab, , ectumomab, belimumab, benrali FR-H1, FR-H2, FR-H3, and/or FR-H4 of the heavy chain of Zumab, , brentuximab, canakinumab, SEQID NO: 72 or 63), and in some aspects at least one CDR capiromab, capromab pendetide, , certoli that is distinct from the CDRs of the heavy chain sequence Zumab, clivatuZumab tetraxetan, daclizumab, denosumab, set forth in SEQ ID NO: 72. 55 eculizumab, , efalizumab, , ertu In some aspects, the provided antibody or fragment has a maxomab, fanolesomab, Fbta05, fontolizumab, gemtu CDR of the light chain sequence set forth in SEQID NO: 61, Zumab, , golimumab, ibritumomab, igovomab, a CDR of the heavy chain sequence set forth in SEQID NO: infliximab, , , mepolizumab, mur 63. omonab, muromonab-CD3, natalizumab, , In some aspects, the VL comprises the amino acid 60 . , omalizumab, , sequence of SEQ ID NO: 76 and the VH comprises the palivizumab, panitumumab, ranibizumab, rituximab, Satu amino acid sequence of SEQ ID NO: 77. momab, Sulesomab, ibritumomab, , In some aspects, the antibody has a VL region with an tocilizumab, to situmomab, trastuzumab, Trbs07, usteki amino acid sequence comprising a light chain framework numab, visilizumab. Votumumab, , and/or region (FR) 1 (FR-L1), an FR-L2, an FR-L3, and/or an 65 brodalumab; and/or anrukinzumab, bapineuZumab, dalotu FR-L4 of the light chain sequence set forth in SEQ ID NO: Zumab, , , inotuZumab, mavrilim 9 (or an FR-L1, FR-L2, FR-L3, and/or FR-L4 that is at least umab, , rillotumumab, sifalim US 9,669,108 B2 5 6 umab, taneZumab, tralokinumab, tremelimumab, urelumab, dissociation constant, as measured by a particular technique, the antibody produced by the hybridoma 10B5 (see Edelson such as surface plasmon resonance (SPR), Isothermal Titra & Unanue. Curr Opin Immunol, 2000 August; 12(4):425 tion calorimetry (ITC), fluorescence, fluorescence polariza 31), B6H12.2 (abcam) or other anti-CD47 antibody (see tion, NMR, IR, calorimetry titrations; Kinetic exclusion; Chao et al., Cell, 142, 699-713, Sep. 3, 2010); and/or an 5 Circular dichroism, differential scanning calorimetry, or antibody or fragment thereof having a sequence set forth in other known method, e.g., by SPR. any of SEQ ID NOs: 78-124, and/or 125-170. In some aspects, the meditope binding site includes resi In some aspects, the antibody or fragment specifically dues 40, 41, 83, and/or 85 of the light chain of the meditope binds to an antigen selected from the group consisting of enabled antibody, according to Kabat numbering, and/or CA-125, glycoprotein (GP) IIb/IIIa receptor, TNF-alpha, 10 residues 39, 89, 105, and/or 108 of the heavy chain of the CD52, TAG-72, Carcinoembryonic antigen (CEA), interleu meditope-enabled antibody, according to Kabat numbering. kin-6 receptor (IL-6R). IL-2, interleukin-2 receptor a-chain In some aspects, the meditope binds to a meditope (CD25), CD22, B-cell activating factor, interleukin-5 recep enabled antibody having: a light chain comprising an amino tor (CD125), VEGF, VEGF-A, CD30, IL-1beta, prostate acid sequence set forth in SEQID NO: 71 and a heavy chain specific membrane antigen (PSMA), CD3, EpCAM, EGF 15 comprising an amino acid sequence set forth in SEQID NO: receptor (EGFR), MUC1, human interleukin-2 receptor, 72; to a meditope-enabled antibody having a light chain Tac, RANK ligand, a complement protein, e.g., C5, having an amino acid sequence set forth in SEQ ID NO: 9 EpCAM, CD11a, e.g., human CD11a, an integrin, e.g., and a heavy chain comprising an amino acid sequence set alpha-V beta-3 integrin, vitronectin receptor alpha v beta 3 forth in SEQ ID NO: 6; and/or to a meditope-enabled integrin, HER2, neu, CD3, CD15, CD20 (small and/or large antibody having a light chain having an amino acid sequence loops), Interferon gamma, CD33, CA-IX, TNF alpha, set forth in SEQID NO: 68 and/or a heavy chain having an CTLA-4, carcinoembryonic antigen, IL-5, CD3 epsilon, amino acid sequence set forth in SEQ ID NO: 70. CAM, Alpha-4-integrin, IgE, e.g., IgE Fc region, an RSV In some aspects, the peptide is between 5 and 16 amino antigen, e.g., F protein of respiratory syncytial virus (RSV), acids in length. TAG-72, NCA-90 (granulocyte cell antigen), IL-6, GD2, 25 In some aspects, the peptide has the formula: GD3, IL-12, IL-23, IL-17, CTAA16.88, IL13, interleukin-1 beta, beta-amyloid, IGF-1 receptor (IGF-1R), delta-like X1-X2-X3-X4-XS-X6-X7-X8-X9-X1O-X11-X12 (Formula I) ligand 4 (DLL4), alpha Subunit of granulocyte macrophage wherein: colony stimulating factor receptor, hepatocyte growth factor, X1-Cys, Gly, B-alanine, 2,3-diaminopropionic acid, IFN-alpha, nerve growth factor, IL-13, CD326, Programmed 30 B-azidoalanine, or null; cell death 1 ligand 1 (PD-L1, a.k.a. CD274, B7-H1), CD47, X2=Gln or null: and CD137. X3=Phe, Tyr, B-B'-diphenyl-Ala. His, Asp, 2-bromo-L- In some aspects, the antibody or fragment has a light phenylalanine, 3-bromo-L-phenylalanine, or 4-bromo-L- chain having P8, V9 or I9, I10 or L10, Q38, R39, T40, N41 phenylalanine, Asn., Gln, a modified Phe, a hydratable G42, S43, P44, R45, D82, I83, A84, D85, Y86, Y87, G99, 35 carbonyl-containing residue; or a boronic acid-containing A100, G101, T102, K103, L104, E105, R142, S162, V163, residue; T164, E165, Q166, D167, S168, and/or Y173, according to X4-Asp or Asn; Kabat numbering, and/or has a heavy chain having Q6, P9. X5=Leu; B-B'-diphenyl-Ala; Phe; a non-natural analog of R38, Q39, S40, P41, G42, K43, G44, L45, S84, D86, T87, phenylalanine, tryptophan, or tyrosine; a hydratable carbo A88, I89, Y90, Y91, W103, G104, Q105, G106, T107, 40 nyl-containing residue, or a boronic acid-containing residue; L108, V111, T110, Y147, E150, P151, V152, T173, F174, X6=Ser or Cys; P175, A176, V177, Y185, S186, and/or L187, according to X7=Thr or Ser or Cys: Kabat numbering. X8=Arg, a modified Arg, or a hydratable carbonyl or Also provided are complexes containing an antibody or boronic acid-containing residue; antibodies (e.g., meditope-enabled antibodies) bound to one 45 X9=Arg, Ala: or more meditope. The antibody or antibody can be any of X10-Leu, Gln, Glu, B-B'-diphenyl-Ala; Phe, a non-natu the meditope-enabled antibodies described herein, such as ral analog of phenylalanine, tryptophan, or tyrosine; a any of the aforementioned antibodies (including fragments hydratable carbonyl-containing residue, or a boronic acid thereof). The one or more meditope can include any one or containing residue, more of the meditopes described herein, such as those 50 X11=Lys; and described in this section, including monovalent and multi X12-Cys, Gly, 7-aminoheptanoic acid, B-alanine, Valent meditopes, and labeled meditopes, as well as medi diaminopropionic acid, propargylglycine, isoaspartic acid, tope fusion proteins. or null, Also provided are meditopes, e.g., isolated meditopes. wherein: Among the provided meditopes are those described above. 55 the modified Arg has a structure of the formula shown in Among the provided meditopes are those comprising a FIG. 34, peptide that binds to a meditope binding site of a meditope the modified Phe is a Phe with one or more halogen enabled antibody, wherein the peptide is not a peptide of incorporated into the phenyl ring, and SEQ ID NO: 1 or 2 or cyclic peptide derived therefrom. In formula I is not SEQ ID NO: 1 or SEQ ID NO: 2 or a other aspects, the meditope is a peptide of SEQID NO: 1 or 60 cyclic peptide derived therefrom. 2, or a cyclic peptide derived therefrom. In some aspects, the peptide is a cyclic peptide. Such as In some aspects, the meditope (e.g., the peptide) binds to one in which the cyclization is by disulfide bridge, a the meditope binding site with a dissociation constant of less thioether bridge, a lactam linkage, cycloaddition. In certain than at or about 10 uM, less than at or about 5 uM, or less aspects, where the peptide is one of Formula I, the cycliza than at or about 2 uM, less than at or about 1 uM, less than 65 tion is via a linkage between X1 and X12, X1 and X11, X3 at or about 500, 400, 300, 200, 100 nM, or less, such as at and X11, X4 and X11, or X2 and X12. In some aspects, or about 200 picomolar or less, for example, with such a where the peptide is one of Formula I, thenon-natural amino US 9,669,108 B2 7 8 acid is B-3'-diphenyl-Ala., a branched alkyl, or an extended —B(OH), —SH, boronic ester, phosphonate ester, aromatic. In some aspects, where the peptide is one of ortho ester, -CH=CH-CHO,-CH=CH-C(O) Formula I, each of the one or more halogen is an ortho Calkyl, -CH=CH-COC-alkyl, -COH, or meta-, or para-bromo phenyl Substituent. —COC alkyl group; Among the provided meditopes are peptides having an 5 R is H. Cisalkyl, C-scycloalkyl, branched alkyl, or amino acid selected from the group consisting of the aryl; sequences set forth in SEQID NOs: 1, 2, and 15-55, e.g., 1, R" is H or a Cisalkyl, Csalkenyl, Csalkynyl, 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, C-scycloalkyl, branched alkyl, or aryl group, each and 55, e.g., 16-18, 23, 29, 31, 32, 36,39, 42, 43, 45,46, 51, optionally substituted with one or more substituents 10 selected from the group consisting of —N —NH2, 52, 54, and 55, or a cyclic peptide derived therefrom. —OH, -SH, halogen, oxo, acetal, ketal. —B(OH), In some embodiments, the meditope comprises a com boronic ester, phosphonate ester, ortho ester, pound of Formula (X): -CH=CH-CHO, -CH=CH-C(O)C alkyl, —CH=CH-COC alkyl, —COH, and 15 —COC alkyl group; and (X) R is H.; NHR'. or a Calkyl, Cscycloalkyl, C2-12alkenyl, C2-salkynyl, or aryl group, each optionally substituted with one or more substituents selected from the group consisting of —N —NH2, —OH, -SH, oxo, Cacetal, Caketal. —B(OH), boronic ester, phosphonate ester, ortho ester, -CH=CH-CHO, -CH=CH-C(O)C alkyl, —CH=CH-COC-alkyl, and —COC-alkyl group; or 25 (b) a C- alkyl Substituted with an oxo, acetal, ketal, —B(OH), boronic ester. —SH, —OH, phosphonate ester, ortho ester, -CH=CH-CHO,-CH=CH-C (O)Calkyl, —CH=CH-COC alkyl, O —COC-alkyl group; 30 R is Calkyl or -C-alkylene-R; wherein R is COH, -CONH, -CH-NHC(O)NH2, or CH-NHC(=NH)NH: RO is: (1) a Cisalkyl optionally Substituted with one or more 35 Substituents selected from the group consisting of OXo, wherein: acetal, ketal. —B(OH), boronic ester, phosphonate the center marked with “*” is in the “R” or “S” configura ester, ortho ester, -CH=CH-CHO,-CH=CH-C tion; (O)Calkyl, —CH=CH-COC-alkyl, —CO R and Rare each, independently, H or phenyl, optionally Calkyl, -CO2H, and —CONH2 group; or 40 (2) a Calkyl group Substituted with one or two phenyl substituted with one, two, or three substituents indepen groups, or one naphthyl, imidazole, or indole group, dently selected from Calkyl, -OH, fluoro, chloro, wherein each phenyl is optionally substituted with one, bromo, and iodo; two, or three substituents independently selected from R is: —OH, fluoro, chloro, bromo, and iodo; (A) Cisalkyl, optionally Substituted with one or more 45 n is 0 or 1; Substituents selected from the group consisting of oXo, p is 0 or 1; acetal, ketal. —B(OH), boronic ester, phosphonate X is Cisalkylene or Csalkenylene, each carbon thereof ester, ortho ester, —COC alkyl, —CH=CH-CHO, optionally substituted with —COH, -NH2 or —NHC —CH=CH-C(O)C alkyl, -CH=CH-CO, (O)R’; Calkyl, -CO2H, and —CONH2 group; or 50 wherein one carbon of said alkylene is optionally replaced (B) a Calkyl group Substituted with: with —C(O)NH , a 5-membered heteroaryl ring, or a) one or two phenyl groups, wherein each phenyl is —S—S—; and optionally substituted with one, two, or three sub R” is - Calkyl or -CH(R)COH: stituents independently selected from —OH, fluoro, wherein R is - Hor—Calkyl optionally substituted chloro, bromo, and iodo; or 55 with -OH, -SH, or - NH; b) a naphthyl, imidazole, or indole group; or a pharmaceutically acceptable salt thereof. R is Calkyl-OH or - Calkyl-SH: In one embodiment, a meditope contains a cysteine that R’ is Calkyl-OH or - Calkyl-SH: covalently binds to a cysteine in a meditope binding site. m is 0, 1, 2, 3, 4, or 5: Such a meditope may be conjugated to any Substance, R is: 60 molecule or compound, which may be a therapeutic mol (a) -OH, -NRR, N(R)C(O)R, or N(R)C ecule. Such as a small molecule diagnostic molecule. Such as (—NR)R; a marker. In some aspects, the “Cys meditope' directs the wherein: conjugate to the Ig and binds via a covalent linkage. R is H: Also among the provided meditopes are labeled medi R’ is H or Cisalkyl optionally substituted with one or 65 topes, such as those comprising a meditope (such as any of more Substituents selected from the group consisting those described above) and a therapeutic or diagnostic agent. of oxo, acetal, and ketal, In some aspects, the therapeutic or diagnostic agent is US 9,669,108 B2 10 selected from the group consisting of a metal chelator topes coupled to a therapeutic or diagnostic agent. In one bound to a metal ion, a small molecule, a chemotherapeutic aspect, the meditope-enabled antibody or fragment and one agent, a therapeutic antibody or functional fragment, a toxin, or more meditope are administered sequentially. In another a radioisotope, an enzyme, a nuclease, a hormone, an aspect, they are administered simultaneously. Generally, the immunomodulator, an oligonucleotide, an organic or inor one or more meditope comprises a peptide that binds to a ganic nanoparticle, an RNAi molecule, an siRNA, a chela meditope binding site of the meditope-enabled antibody or tor, a boron compound, a photoactive agent, a dye, fluores fragment. In one aspect, the meditope-enabled antibody or cent or luminescent Substance, an enzyme, an enhancing fragment is bound to the one or more meditope, Such that agent, a radioactive Substance, and a chelator. administration of the meditope-enabled antibody and the one Also among the provided meditopes are multivalent medi 10 or more meditope comprises administering a complex of the topes, such as those comprising two or more meditopes and meditope-enabled antibody and the meditope. In another one or more linker, e.g., where each of the two or more aspect, the meditope-enabled antibody is administered prior meditopes is a peptide that binds to a meditope binding site to administration of the one or more meditope. of a meditope-enabled antibody. Such multivalent meditopes In some aspects, the one or more meditope is coupled to may comprise any of the meditopes described herein, e.g., 15 atherapeutic agent, such as a therapeutic agent selected from above. In one aspect, the two or more meditopes comprise the group consisting of drugs, chemotherapeutic agents, at least three meditopes, or at least four meditopes. In one therapeutic antibodies, toxins, radioisotopes, enzymes, aspect, the one or more linker includes a peptide, a small chelators, boron compounds, photoactive agents, dyes, met chemical scaffold, a biotin-streptavidin, an organic or inor als, metal alloys, and nanoparticles. ganic nanoparticle, a polynucleotide sequence, peptide Also provided are diagnostic methods, such as those nucleic acid, an organic polymer, or an immunoglobulin Fc carried out by administering to a subject Such pharmaceu domain. tical compositions, or any of the meditope-enabled antibod Also among the provided embodiments are meditope ies, meditopes, complexes, and/or other compound enabled antibody-meditope complexes, including those con described herein, e.g., above, and detecting binding of the taining any of the meditope-enabled antibodies and any of 25 administered composition, antibody, meditope, complex, the meditopes described herein, e.g., described above. In and/or compound to a Substance in the Subject, e.g., to an Some cases, the peptide binds to the meditope binding site antigen. In some aspects, the diagnostic method includes with a dissociation constant of less than 10 LM, as measured administering to a Subject one or more meditope-enabled by surface plasmon resonance (SPR), or another affinity as antibody or fragment thereof, e.g., any of those described described above. 30 above. In some aspects, it further includes detecting binding Also provided are methods using the meditopes and/or of the antibody or fragment to an antigen in the Subject. In antibodies, such as any of the above meditopes and/or some aspects, the meditope-enabled antibody or fragment antibodies. For example, provided are methods for purifying and one or more meditope are administered sequentially; in a meditope-enabled antibody or fragment thereof, or a cell other aspects, they are administered simultaneously. In one expressing the meditope-enabled antibody or fragment 35 example, the meditope-enabled antibody or fragment is thereof. In one aspect, Such methods include a step of bound to the one or more meditope, Such that administration contacting a composition containing the meditope-enabled of the meditope-enabled antibody and the one or more antibody, fragment, or cell with a meditope, under condi meditope comprises administering a complex of the medi tions whereby the meditope-enabled antibody or fragment tope-enabled antibody and the meditope. In another binds to the peptide. In some aspect, they further include 40 example, the meditope-enabled antibody is administered then isolating the antibody or fragment or cell. Such meth prior to administration of the one or more meditope. In some ods in Some aspects thereby purify the antibody, fragment, aspects, the meditope or meditopes is a multivalent medi or cell. tope. In some aspects, the meditope is coupled to a diag In some aspects, the meditope is coupled to a solid nostic agent, such as an imaging agent. Such as an imaging Support. In some aspects, the isolation or purification is 45 agent selected from the group consisting of fluorescent effected by a change in pH. Substances, luminescent Substances, dyes, indicators, and Also provided are compositions, e.g., pharmaceutical radioactive Substances. In some cases, the imaging agent is compositions, comprising the meditopes (including multi DOTA. valent and labeled meditopes), meditope-enabled antibodies, Also provided are methods for generating meditope and complexes, and/or other compounds described herein, 50 enabled antibodies or fragments thereof, e.g., based on e.g., above. In one example, the composition includes the template antibodies. In some aspects. Such methods include complex, meditope, and/or meditope-enabled antibody, and effecting one or more amino acid Substitutions in a template a pharmaceutically acceptable carrier. antibody or fragment thereof. In one example, the Substitu Also provided are methods of treatment, Such as those tions include a substitution at position 40, 41, or 85 of the carried out by administering to a Subject Such pharmaceu 55 VL region, and/or position 40 or position 89 of the VH tical compositions, or any of the meditope-enabled antibod region, according to Kabat numbering. ies, meditopes, complexes, and/or other compound In some aspects, the methods generate a meditope-en described herein, e.g., above. In one example, the methods abled antibody or fragment thereof which binds to a medi include administering to the Subject an antibody or fragment tope that is a peptide having an amino acid sequence selected as described above. 60 from the group consisting of the sequences set forth in SEQ In one example, the method of treatment includes admin ID NO: 1, 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, istering to a subject one or more meditope-enabled antibody 51, 52, 54, and 55, or a cyclic peptide derived therefrom. or fragment, e.g., any of those described above. In one In some aspects, the template antibody or fragment is example, the method includes administering to the Subject selected from the group consisting of abagovomab, abcix one or more meditope-enabled antibody or fragment as 65 imab, adalimumab, adecatumumab, alemtuzumab, altu described above, and a meditope, e.g., any of those momab, altumomab pentetate, anatumomab, anatumomab described above, including multivalent meditopes and medi mafenatoX, arcitumomab, atlizumab, basiliximab, bectu US 9,669,108 B2 11 12 momab, ectumomab, belimumab, benralizumab, bevaci Also provided are methods for modifying meditopes and Zumab, brentuximab, canakinumab, capromab, capromab meditope-enabled antibodies, including a method for modi pendetide, catumaXomab, certolizumab, clivatuZumab tet fying a meditope, comprising effecting one or more amino raxetan, daclizumab, denosumab, eculizumab, edrecolomab, acid Substitutions, insertions, or deletions, to a peptide efalizumab, etaracizumab, , fanolesomab, having the sequence set forth in any of SEQ ID NOs: 1, 2, Fbta05, fontolizumab, gemtuzumab, girentuximab, golim 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45, 46, 51, 52, 54, and umab, ibritumomab, igovomab, infliximab, ipilimumab, 55, or cyclic peptide derived therefrom, thereby altering the labetuZumab, mepolizumab, muromonab, muromonab pH-dependence of the binding affinity of the peptide for a CD3, natalizumab, necitumumab, nimotuZumab. ofatu meditope-enabled antibody or fragment thereof. Such as any mumab, omalizumab, oregovomab, palivizumab, panitu 10 of those described above. In some aspects, the method mumab, ranibizumab, rituximab, Satumomab, Sulesomab, decreases the binding affinity of the peptide for the antibody ibritumomab, ibritumomab tiuxetan, tocilizumab, to situ or fragment at a lysosomal pH level. In other aspects, the momab, trastuzumab, Trbs07, ustekinumab, visilizumab, method increases the binding affinity in a hypoxic environ Votumumab, Zalutumumab, and brodalumab, or is the anti ment. body produced by the hybridoma 10B5, or is B6H12.2. 15 Also provided are methods for modifying a meditope In some aspects, the meditope-enabled antibody binds to enabled antibody, Such as those comprising: effecting one or one or more meditopes (e.g., one of SEQ ID NO: 1, 2, or more modifications at position 8, 9, 10, 38, 39, 40, 4142, 43. cyclic peptide derived therefrom), e.g., those described 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, 104, above, with a particular affinity, Such as with a dissociation 105, 142, 162, 163, 164, 165, 166, 167, 168, and/or 173 of constant of less than 10, 5, or 2 (or other number specified the light chain, or 6, 9,38, 39, 40, 41, 42, 43, 44, 45, 84, 86, above) uM, as measured by SPR, or other method described 87, 88, 89,90, 91, 103, 104, 105, 106, 107, 108, 109, 110, above. 147, 150, 151,152, 173, 174, 175, 176, 177, 185, 186, and/or In some aspects, each of the one or more amino acid 187 of the heavy chain, of the meditope-enabled antibody, Substitutions is at a position selected from the group con 25 according to Kabat numbering. In some aspects, the modi sisting of position 8, 9, 10, 38, 39, 40, 41 42, 43, 44, 45, 82, fication methods are provided in tandem, to produce modi 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, 104, 105, 142, fied meditopes and modified meditope-enabled antibodies 162, 163, 164, 165, 166, 167, 168, and 173 of the light chain, that bind to one another, Such as by making modifications in and/or 6, 9,38, 39, 40, 41, 42, 43, 44, 45, 84, 86, 87, 88, 89, the antibody based on modifications in a modified meditope, 90, 91, 103, 104, 105, 106, 107, 108, 109, 110, 147, 150, 30 or vice versa. 151, 152, 173, 174, 175, 176, 177, 185, 186, and 187 of the Also provided are meditope analogs, including those that heavy chain, according to Kabat numbering. In some bind to any of the meditope-enabled antibodies described aspects, the meditope-enabled antibody contains a light above, and/or with binding properties comparable to any of chain having P8, V9 or I9, I10 or L10, Q38, R39, T40, N41 the meditopes described above. G42, S43, P44, R45, D82, I83, A84, D85, Y86, Y87, G99, 35 A100, G101, T102, K103, L104, E105, R142, S162, V163, BRIEF DESCRIPTION OF THE DRAWINGS T164, E165, Q166, D167, S168, and Y173, according to Kabat numbering and a heavy chain having Q6, P9, R38, FIG. 1 shows meditope peptides binding to framework Q39, S40, P41, G42, K43, G44, L45, S84, D86, T87, A88, loops of cetuximab. FIG. 1A: The complex of cetuximab I89, Y90, Y91, W103, G104, Q105, G106, T107, L108, 40 Fab (light chain is denoted by V, and C.; heavy chain is V111, T110, Y147, E150, P151, V152, T173, F174, P175, denoted by V, and C) and cyclic COFDLSTRRLKC A176, V177, Y185, S186, and L187, according to Kabat (depicted within the shaded area and labeled with the word numbering. “meditope') (SEQ ID NO: 1) indicates that the meditope Also provided are polynucleotides comprising a nucleo binds to an interface of the Fab framework, which is distinct tide sequence encoding the meditope-enabled antibodies and 45 from the CDR loops of cetuximab. FIG. 1B (top) shows the meditopes, as well as vectors including the same, and stick representation of the coFD meditope and (bottom) the libraries comprising the vectors. stick representation of the coYN meditope. The N- and Also provided are screening methods. Such as those C-terminal cysteines are solvent exposed and display high including the steps of expressing antibodies or fragments thermal factors. thereof from the libraries, and selecting an antibody or 50 FIG. 2 shows certain embodiments of the cetuximab Fab fragment thereof from among the expressed antibodies or binding interface. Cetuximab is a human-murine chimera fragments. In some cases, the selection is based on a binding and, therefore, has a mix of murine Ig variable domains and affinity, pH tolerance, pH dependence, toxicity, PK, or PD of human constant Ig domains. FIG. 2A shows residues of the selected antibody or fragment thereof, and/or is effected ch14.18, and the humanized trastuzumab, which correspond by contacting the antibodies or fragments thereof with a 55 to those residues of Cetuximab that make contact with the meditope and detecting binding to one or more of the meditope. Sequences shown include QRTNGS (SEQ ID antibodies or fragments thereto. NO:171); IADYY (SEQ ID NO:172); AGTKLE (SEQ ID Also provided are methods for selecting a meditope NO:173); QKPGQS (SEQ ID NO:174); LGVYF (SEQ ID analog or meditope variant, including those comprising: (a) NO:175); QKPGKA (SEQ ID NO:176); FATYY (SEQ ID combining a meditope and a meditope-enabled antibody or 60 NO:177); QGTKVE (SEQ ID NO:178); QSPGKG (SEQID fragment thereof, whereby the meditope non-covalently NO:179); QGTL (SEQ ID NO:180); QNIGKS (SEQ ID binds to the antibody or fragment thereof; and (b) adding a NO:181); QGTS (SEQ ID NO:182): QAPGKG (SEQ ID candidate meditope variant or analog; and (c) measuring NO:183). FIG. 2B shows a stereoView of Arg9 of the coFD displacement of the meditope by the candidate meditope meditope that occupies a distinct pocket encoded by the variant or analog, wherein displacement of the meditope by 65 murine sequence of Cetuximab (foreground). There is a salt the candidate variant or analog identifies it as the meditope bridge from Asp85 to the guanidinium group of the medi variant or analog. tope Arg9 and the backbone amide of Leu10. US 9,669,108 B2 13 14 FIG. 3 shows surface plasmon resonance (SPR) traces of lent meditope (in this particular example, trivalent) tethers/ cQFD and coYN with an immobilized Fab. The cetuximab cross-links the therapeutic mAb and can enhance its thera Fab used for crystallographic studies was coupled to a CM5 peutic potential (right). In addition, the multivalent meditope chip at low densities. Traces at increasing concentrations of (shown here as a trivalent version) can also carry an imaging the cqFD (FIG. 3A) and cqYN (FIG. 3B) meditopes are agent, which can allow for both enhanced therapeutics as shown. The series of traces are fit to a simple 1:1 Langmuir well as imaging. binding model. The residuals of the fit are shown below FIG. 9 shows an scFv-meditope linker. schvs are created each. by fusing the light chain variable domain to the heavy chain FIG. 4 illustrates that the coFD and coYN meditopes do variable domain (or vice versa) through a 12-20 amino acid not bind to the CDRs of cetuximab, as was previously 10 hypothesized. FIG. 4A shows an overlay of two crystal linker (typically (GGGS) (SEQ ID NOs: 184-186). In structures, one of a cetuximab Fab and its antigen, EGFR this example, a portion of the flexible linker sequence can be domain III, the other showing the coFD meditope and replaced with the meditope sequence. cetuximab Fab, in which the coFD meditope binds to the FIG. 10 shows results from an SDS-PAGE showing that central cavity of the cetuximab Fab. The antigen, EGFR 15 the coFD meditope coupled to beads could bind to cetux domain III, binds at the complementarity determining imab. Biotinylated coFD peptide was added to avidin regions at a significant distance from the meditope binding coupled beads, thoroughly washed, and equilibrated in PBS. site. FIG. 4B shows SDS-PAGE results on the left-hand side Cetuximab was then added to the beads (lane 1), washed and the corresponding size exclusion chromatography (lanes 2-4), and then eluted (lane 5). The top band is the IgG results on the right-hand side. Size exclusion experiments of heavy chain and bottom band is the IgG light chain. the individual components, Fab, EGFR domain III, and FIG. 11 is a depiction of an exemplary embodiment of a SMT-cOFD meditope, as well as an admixture of all three, steric mask. In this exemplary embodiment, the meditope is indicate the formation of a hetero-trimeric complex that fused to the N-terminus of the Fab light or heavy chain coeluted. The non-reducing SDS-PAGE gel shows the frac through a flexible linker containing a tumor associated tion that eluted first, indicating the presence of all three 25 protease site, to sterically occlude the antigen binding site. components within the new peak (the left-most peak for FIG. 12 shows SPR-determined binding kinetics for mon “complex,” shaded light gray) observed from the admixture. ovalent (left panel) and tetravalent (right panel) meditopes to FIG. 4C shows the results of a FACS analysis, indicating cetuximab. The panel above the Surface plasmon resonance that the cQFD meditope bound to EGFR positive MD traces depicts a cartoon of the monovalent meditopes being MBA-468 cells only in the presence of cetuximab (arrows). 30 The meditope alone or the meditope in the presence of passed over the bivalent IgG. As shown in the right panel, M425, a murine EGFR antibody, did not bind. FIG. 4D avidin was saturated with biotinylated meditope and passed shows results of Surface plasmon resonance experiments over the same immobilized cetuximab IgG. The off-rate of using a sensor chip coupled with a cetuximab Schv or Fab. the multivalent meditope was reduced by at least 36 fold. The experiments indicate that saturation of the schv could 35 (Note the time scales between the two experiments are not be achieved at concentrations as high as 100 uM of the different.) cOFD meditope. The same experiments using the cetuximab FIG. 13 illustrates the synthesis of dimeric and trimeric Fab coupled sensor chip indicate full saturation. The disso meditopes (G-QFDLSTRRLK-G, SEQID NO:32, in either ciation constant from this experiment is 660 nM. Control orientation) according to some embodiments, for meditopes SPR experiments (bottom panel) show that the cetuximab 40 containing lactam linkages. schv readily binds to the soluble EGFR domain III fragment, FIG. 14 illustrates the characterization of a fluorescein indicating that the CDR loops were functional. isothiacyanate (FITC)-labeled meditope dimer (“14 in FIG. FIG. 5 is a graph of fluorescence intensity compared to 13), which contains two meditopes of SEQ ID NO: 32 cell count and shows fluorescently-labeled cetuximab bind (shown in both orientations), with an HPLC trace of final ing to MDA-MB-468 cells in the presence of c(RFD. Cetux 45 bivalent meditope and its mass spectrum. imab binds to MDA-MB-468 cells without meditope, and in FIG. 15 shows the nucleic acid sequence (SEQ ID NO:3) the presence of 6 uM and 60 uM meditope. As expected, the and the corresponding amino acid sequence (SEQID NO:4) Figure shows that there was no binding of the isotype IgG for a meditope-Fc fusion protein according to Some embodi control to EGFR on MDA-MB468 cells. mentS. FIG. 6 shows how meditope and EGFR bind to distinct 50 FIG. 16 shows an exemplary bivalent meditope. The sites. The top images show superposition of the coFD-Fab meditope is directly fused to the N-terminus of a peptide complex and the EGFR-Fab complex (1YY8) in stereo. The linker that is directly fused to the Fc Region of an IgG. In bottom images show that residues 39-46 of the heavy chain this example, as the Fc is naturally homodimerized during are flexible and accommodate the meditope. expression, the end product from the meditope-Fc construct FIG. 7 shows Fab framework binders. Superposition of 55 is bivalent. Fabs bound to meditope, Protein A, and Protein L indicate FIG. 17 shows the results of a series of exemplary FACS that each binds to a unique site on the cetuximab Fab. analyses monitoring binding of monovalent and bivalent FIG. 8 illustrates a mechanism of action in one embodi meditopes to cetuximab pretreated EGFR-expressing cells. ment for enhancing tumor therapy. In the exemplified MDA-MB-468 cells that over-express EGFR were pre embodiment, bivalent antibodies bound to antigen (e.g., 60 treated with 10 nM of cetuximab for 30 minutes, rinsed, and ErbB receptor family) overexpressed on tumor cells (left then treated with either the meditope-Fc construct or the panel) and blocks receptor signaling, alters endocytosis and monomeric meditope at four concentrations. The bottom receptor recycling and/or elicits an immune response. The trace is a negative control (no antibody). The next four traces addition of an antibody Such as matuZumab which recog show that a monomeric meditope binds to cells pre-treated nizes a different domain of EGFR in combination with 65 with cetuximab in a concentration dependent manner. The cetuximab can, in Some cases be more effective due to daisy top four traces also show that the bivalent, meditope Fc chaining of the Surface receptor (right panel 1). A multiva binds to cells pre-treated with cetuximab in a concentration US 9,669,108 B2 15 16 dependent manner, but with higher affinity (i.e., more shifted while the meditope-Fc bound to the HER2-CDR grafted, to the right). This is predicted and consistent with a multi meditope-enabled mAb (top three traces peak shifted right Valent interaction. in concentration dependent manner), it did not bind to FIG. 18 is a depiction of an alternative meditope-Fc wild-type trastuzumab (second from bottom) or to the nega linker, a coiled coil, which may be used in accordance with 5 tive control (bottom trace). certain embodiments. FIG. 25A shows nucleic acid (SEQID NO: 11) and amino FIG. 19: Fragment screening by NMR. Representative acid (SEQ ID NO: 12) sequences for the heavy chain of a NMR spectra of a fragment pool before (top trace) and after trastuzumab with secretion sequence and engineered restric (bottom trace) cetuximab addition. Using this method, lead tion sites (underlined). FIG. 25B shows nucleic acid (SEQ compounds (e.g., those shown herein) have been identified 10 ID NO: 13) and amino acid (SEQID NO: 14) sequences for and the binding site is being determined using diffraction a light chain of trastuzumab with secretion sequence and studies. engineered restriction sites (underlined). Shaded areas indi FIG. 20 illustrates exemplary steps to alter and/or enhance cate exemplary residues that in some aspects could be the binding affinity (or alter another property) of a mAb for altered to enhance or alter the specificity of the meditope, or a meditope or compound binding at the meditope site using 15 could participate in binding interactions with a modified a directed random library. Specifically, a gene library where epitope. codons for mAb residues that line the meditope binding site FIG. 26 illustrates examples of sequence and structural are replaced with NNK (where N is any nucleotide and K is alignment of the IgG and IgE Fab domains (SEQ ID NOs: a thymidine or guanosine) can be selected using FACS 188-189, respectively), with shaded areas indicating certain sorting (where the meditope and the antigen are labeled with residues corresponding to residues near a meditope binding distinct fluorophores). In another embodiment, the codons site. are substituted with NNN, where N is any nucleotide. The FIG. 27 is a graph showing Surface plasmon resonance GPI linker can ensure the selected mAb remains associated (SPR) studies. The binding affinity of different meditopes to with the cell transfected with vectors encoding the gene cetuximab cqFD (meditope 1, SEQ ID NO: 1), cqYN sequences. Sequencing the genes encoding the light and 25 (meditope 2, SEQID NO: 2), and cqYD (meditope 16, SEQ heavy chain from the selected cells will identify high affinity ID NO: 16) was determined from pH=4.0 to pH=8.0. mAbs. The method can be repeated to select for higher FIG. 28 illustrates the results of an MTT assay comparing affinity meditope or meditope analogs. the efficacy of a bivalent meditope-Fc to that of a monomeric FIG. 21 shows surface representation of sequence differ meditope to inhibit MDA-MB-468 cell growth. FIG. 28A ences of cetuximab and trastuzumab. The dark grey regions 30 shows results of a study in which a meditope-Fic, but not a in the top panel indicate amino acid differences between monomeric meditope, inhibited cell growth when combined cetuximab and the fully humanized trastuzumab framework. with cetuximab. FIG. 28B shows that the meditope-Fc Certain residues inside the box have been mutated onto the enhances the cell-killing capacity of cetuximab, similar to trastuzumab framework. The CDR loops of trastuzumab the combination of M425 and cetuximab. have been identified (dark regions; bottom panel) and 35 FIG. 29 illustrates binding of a meditope to a cetuximab grafted onto the cetuximab framework. meditope binding site (“Cetuximab Binding as shown in FIG. 22 Illustrates that a meditope-enabled trastuzumab Figure), but not to a fully humanized framework (“Trastu binds to a meditope-Fc fusion protein. The meditope binding Zumab No binding as shown in Figure) or another murine site was created on trastuzumab, a humanized mAb that chimera framework (“Rituximab No binding as shown in binds to the tumor antigen, HER2. FACS analysis in the top 40 Figure). The cqFD meditope (meditope 1, SEQ ID NO: 1) panel shows that meditope-enabled trastuzumab binds to was conjugated to a CM5 chip for Surface plasmon reso SKBR3 cells that overexpress HER2 (top two traces). In the nance studies and cetuximab (meditope binding Fab), tras bottom graph, the meditope-Fc binds to meditope-enabled tuZumab (fully humanized framework), and rituximab (mu trastuzumab (top two traces—peak shifted right), but not to rine-human chimeric framework) were tested at wild-type trastuzumab (second from bottom) or to the nega 45 concentrations of 0.01, 0.05, 0.1, 0.5 and 1 uM. Only tive control (bottom trace). cetuximab bound to the meditope-conjugated chip. FIG. 23A shows the nucleic acid (SEQ ID NO:5) FIG. 30 shows biophysical data obtained for the medi sequence of a meditope-enabled trastuzumab heavy chain topes. FIG. 30A shows a SPR sensogram of an unmodified sequence. Signal peptide sequence is shaded in grey. FIG. meditope cQFD (SEQID NO:1) and a QYN meditope (SEQ 23B shows the amino acid (SEQ ID NO:6) sequence of a 50 ID NO:2) using a cetuximab Fab chip. FIG. 30B shows a meditope-enabled trastuzumab heavy chain sequence as representative binding isotherm of the meditope (SEQ ID compared to the wild-type (SEQID NO:7). Differences are NO: 1) and cetuximab Fab (top) and integration (bottom). highlighted in grey. After the amino acids shown in FIG. FIG. 30C shows superposition of both meditopes. Oval 1 23B, there are no differences remaining in the human indicates the Phe/Tyr position. The arrow indicates a shift of sequence of the heavy chain shown. FIG. 23C shows the 55 the backbone that leads to a favorable hydrogen bond nucleic acid (SEQ ID NO:8) sequence of a meditope network and may account for a favorable change in the enabled trastuzumab light chain sequence. Signal peptide enthalpy. The hydrophobic groups, F/Y3 L5, and L10 are sequence is shaded in grey. FIG. 23D shows the amino acid nearly identically positioned, but the hydroxyl group of Y5 (SEQ ID NO:9) sequence of a meditope-enabled trastu in the coYN meditope prevents R8 from interacting with the Zumab light chain sequence as compared to the wild-type 60 Q105 backbone, as observed in the coFD meditope (“steric (SEQ ID NO:10). Differences are highlighted in grey. clash'). This rearrangement also results in a concomitant FIG. 24 shows that an antibody containing HER2-binding change in the backbone residues of the B-turn (“backbone CDR loops grafted on cetuximab-like framework binds to rotation'). FIG. 30D shows individual thermodynamic HER2 and a meditope-Fc. In FIG. 24A, FACS analysis parameters determined by ITC of different meditope vari shows that HER2-CDR grafted meditope-enabled mAb 65 ants. The Phe3Tyr variant (meditope 2, SEQ ID NO: 2) binds to SKBR3 cells, which overexpresses HER2 (top three shows a significant change in AH though lower affinity. traces at different concentrations). FIG. 24B shows that Based on the structure, Gln2 in the meditope of SEQID NO: US 9,669,108 B2 17 18 1 was replaced with the D-stereomer. ITC analysis of this binding affinity for cetuximab of ~4 fold, was observed by meditope, (SEQ ID NO:15), revealed a significant increase Surface plasmon resonance Aminohexanoic acid was used to in entropy and loss in enthalpy. replace the disulfide bridge of the original meditope (bottom FIG. 31 shows the chemical structure of a meditope trace). While the binding affinity was decreased, these data according to Some embodiments. The circles indicate posi indicate that modifications can be made to meditopes to tions that can be modified, e.g., to improve meditope affinity address potential issues with pharmacokinetics, pharmaco for the Fab. The boxes indicate cyclization strategies. dynamics and toxicity in animal and human studies. It is Click chemistry and olefin metathesis, top and bottom far noted that this combination can be combined with unnatural right boxes, respectively, are additional routes to cyclize the amino acids at other positions within a meditope. meditope. 10 FIG. 42 illustrates structural information that can be FIG. 32 illustrates the synthesis of a meditope containing useful, e.g., to improve meditope affinity. The top panel a lactam linkage (2, SEQ ID NO: 32) and a fluroescein shows a crystal structure of a meditope bound cetuximab, labeled meditope containing a lactam linkage (SEQID NO: showing Phe3 bound within the Fab cavity of cetuximab. 32) (3). Substitution of this position with histidine and subsequent FIG. 33, in panels (A) and (B), shows the sites of 15 structural analysis indicates the side chain (imidazole) takes optimization according to some embodiments. Ovals indi on a new conformation (middle panel). Based on this cate cavities in the Fab region of the mAb that may be observation, Bf3'-diphenylalanine which could place one exploited to optimize the affinity of a meditope. phenyl side chain at the original position and one phenyl side FIG. 34 shows a modified Arg8 residue that may be used chain at the imidazole ring was Substituted at position 3. The to optimize meditope binding parameters, and may be used crystal structure indicates that this substitution mimics both in accordance with the embodiments described herein side chain conformations. Surface plasmon resonance stud (R-alkyl, substituted alkyl, aromatic or NHR", where ies shows that this substitute binds with higher affinity (FIG. R"-alkyl, substituted alkyl, or aromatic). 41, top panel). FIG. 35 shows the hydrophilic interface in the vicinity of FIG. 43 shows results of a study demonstrating that a Phe3 of a meditope. Halogens incorporated in the phenyl 25 meditope-enabled trastuzumab provided herein has a similar ring may form strong noncovalent bonds to the hydroxyl binding affinity for antigen as wild-type trastuzumab, as group of cetuximab Tyr87 or Tyr91, as well as to backbone described in Example 4. carbonyls. FIG. 44 shows results of a study described in Example 4, FIG. 36 illustrates covalent interactions between a medi demonstrating that HER2-expressing cells pre-bound with tope and the backbone of cetuximab. A hydratable side chain 30 meditope-enabled trastuzumab (TM), but not those pre may be incorporated at Arg8 (left) or Leu10 (right) to form bound with wild-type trastuzumab (WTT), bound to a a covalent bond to the hydroxyl groups of Seror Tyr of the cQFD (SEQ ID NO: 1) meditope-Protein L fusion (MPL). Fab. Control cells, without pre-bound antibody (MPL (WT) only) FIG. 37 illustrates a fluorescence polarization assay of also did not bind to MPL. dose-dependent inhibition of the interaction between cetux 35 FIG. 45 shows results of a study described in Example 4, imab and a fluorescein-labeled peptide by unlabeled peptide. demonstrating that HER2-expressing cells (SKBR3) pre The assay identified Small molecule compounds that could bound with meditope-enabled trastuzumab (TM), but not compete with the meditope for mAb binding and thus can be those pre-bound with wild-type trastuzumab (WTT), bound developed to function in a similar manner as a meditope. As to a cqFD (SEQ ID NO: 1) meditope-Protein L fusion in a control, it was demonstrated that a non-labeled meditope 40 which all but one lysine of Protein L were mutated to could displace the fluorescently-labeled meditope. Equili arginine and asparagine (MPL-5K). Control cells, not pre bration of the displacement at three times points indicates bound with any antibody (MPL-5K only) also did not bind that the assay is robust and amendable to high throughput to MPL-5K. Screening. FIG. 46 shows results of a study described in Example 4, FIG. 38 shows five lead compounds identified from the 45 demonstrating that meditope binds to meditope-enabled fluorescence polarization screen. trastuzumab to a similar degree whether the antibody is FIG. 39 shows stick structures for eighteen modified pre-bound to cells expressing antigen prior to incubation meditopes, corresponding to SEQ ID NO:22 (A), SEQ ID with the meditope (left panel), or meditope Protein NO:16 (B), SEQ ID NO:17 (C), SEQ ID NO:18 (D), SEQ L—and antibody are pre-mixed and then incubated with ID NO:15 (E), SEQID NO:23 (F), SEQID NO:24 (G), SEQ 50 cells expressing antigen (right panel). TM meditope-en ID NO:25 (H), SEQID NO:32 (I), SEQID NO:33 (J), SEQ abled trastuzumab. The percentage of MPL-positive cells ID NO:34 (K), SEQ ID NO:35 (L), SEQ ID NO:36 (M), relative to no-treatment control is shown in the legend. SEQID NO:37 (N), SEQID NO:38 (O), SEQID NO:39 (P), FIG. 47A shows light chain nucleic acid (SEQID NO: 61) SEQID NO:40 (Q) and SEQID NO:41 (R), SEQID NO:42 and light chain amino acid (SEQ ID NO: 61) sequences of (S), SEQID NO:43 (T), SEQID NO:44 (U), SEQID NO:46 55 a CDR-grafted meditope-enabled trastuzumab (with trastu (V) SEQ ID NO:49 (W), SEQ ID NO:51 (X), SEQ ID Zumab-like CDRs grafted onto a cetuximab-like frame NO:52 (Y), SEQ ID NO:53 (Z), SEQ ID NO:54 (AA), and work), with the signal sequence and other residues shaded. SEQ ID NO:55 (BB). The sequence of these structures is FIG. 47B shows heavy chain nucleic acid (SEQID NO: 62) shown in Tables 3 and 4. and heavy chain amino acid (SEQ ID NO: 63) of this FIG. 40 is a table showing the X-ray diffraction data for 60 antibody. the structures shown in FIG. 39 and additional meditopes in FIG. 48 shows the structure of meditope 54 (see Table 4) Tables 3 and 4. bound to a cetuximab Fab fragment (backside view), with FIG. 41 shows representative Surface plasmon resonance structures of the 5-position f3,3'-diphenylalanine meditope studies of meditope variants. Based on the structural infor (SEQID NO: 18, meditope 18) and coFD (meditope 1, SEQ mation of the Phe3His mutation in the coFD meditope, a 65 ID NO: 1) superimposed. variant meditope was synthesized with B.B'-diphenylalanine FIG. 49 shows the results of a study in which an at position 3 (top trace). A significant improvement in the Alexa488-labeled meditope-Fc fusion protein (600 nM, 180 US 9,669,108 B2 19 20 nM, or 60 nM) was incubated with MDA-MB-468 cells, FIG. 61A shows MDA-MB-468 cells that were pre pre-labeled with cetuximab or M425 (a mouse anti-EGFR incubated with Alexa 555-cetuximab and then incubated antibody) for 30 minutes, and antibody binding and medi with Alexa 488-meditope-Fc. DAPI was used as counter tope binding analyzed by FACS analysis. stain. Images were taken with an Olympus AX70 Automatic FIG. 50 (top panel) shows stick representations of the Upright Microscope. White arrows indicate positive medi structures of meditope 18 (SEQ ID NO: 18, shown in Table tope-Fc staining. FIG. 61B shows quantification of medi 3, with a B.B'-diphenylalanine at position 5) bound to tope-Fc-positive cells. Bar graph showed shows percentage cetuximab (dark grey Sticks), the same meditope (18) bound of meditope-Fc-positive cells of total cells from cetuximab to meditope-enabled trastuzumab (white sticks), and wild pre-treated or untreated samples counted from two fields. type trastuzumab (outline), Superimposed. The bottom panel 10 shows a ribbon cartoon comparing wild-type and meditope DETAILED DESCRIPTION enabled trastuzumab. A. Definitions FIG. 51, in the upper-left panel, shows a superposition of An “antibody,” as used herein, refers to an immunoglobu the structures of trastuzumab and trastuzumab memab (in 15 lin molecule that specifically binds to, or is immunologically this figure, the trastuzumab memab is labeled as “Meditope reactive with an antigen or epitope, and includes both enabled Memab') with certain residues involved in medi polyclonal and monoclonal antibodies, as well as functional tope-binding in the meditope-enabled antibody illustrated by antibody fragments, including but not limited to fragment Sticks. The top right panel shows a Superposition of the antigen binding (Fab) fragments, F(ab')2 fragments, Fab' structures of meditope-enabled trastuzumab (memab) and fragments, FV fragments, recombinant IgG (rIgG) frag cetuximab, with the same residues labeled. The bottom ments, single chain variable fragments (scFV) and single panel shows a “cartoon/ribbon diagram” of all three of these domain antibodies (e.g., SdAb, SdPV, nanobody) fragments. structures Superimposed. The term “antibody” includes genetically engineered or FIG. 52 shows the structure of meditope (position 3f.f3' otherwise modified forms of immunoglobulins, such as diphenyl), Protein L (left), Protein A (right) and Fab (grey 25 intrabodies, peptibodies, chimeric antibodies, fully human cartoon). Lysines in Protein L and the meditope that were antibodies, humanized antibodies, meditope-enabled anti mutated in MPLS described herein are shown in black. bodies and heteroconjugate antibodies (e.g., bispecific anti FIG. 53 shows surface plasmon resonance (SPR) data for bodies, diabodies, triabodies, tetrabodies, tandem di-scFv, a meditope-Protein L fusion (MPL): Top panel shows the tandem tri-sch v). Unless otherwise stated, the term “anti traces of MPL being added to the meditope-enabled trastu 30 body’ should be understood to encompass functional anti Zumab at concentrations up to 10 nM. The fit data indicates body fragments thereof. a binding affinity of 165 pM. The bottom panel shows the The terms "complementarity determining region,” and traces of Protein L (only) added at the same concentrations “CDR,” are known in the art to refer to non-contiguous to the meditope-enabled trastuzumab, showing that there is sequences of amino acids within antibody variable regions, not binding at this concentration. 35 which confer antigen specificity and binding affinity. In FIG. 54 shows that a GPI-linked meditope-enabled tras general, there are three CDRs in each heavy chain variable tuzumab binds to a meditope-Protein L (MPL) fusion pro region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in tein. 1x106 cells/sample were used. Cells were removed each light chain variable region (CDR-L1, CDR-L2, CDR from plates by gentle pipetting and were washed once with L3). “Framework regions” and “FR are known in the art to 0.1% BSA (w/v) in PBS. AF647-labeled-MPL was diluted to 40 refer to the non-CDR portions of the variable regions of the 10 nM in wash buffer and incubated with cells for 30 min at heavy and light chains. In general, there are four FRS in each RT. Cells were washed twice and then analyzed by FACS. heavy chain variable region (FR-H1, FR-H2, FR-H3, and FIG. 55A shows a sequence comparison between the CH1 FR-H4), and four FRS in each light chain variable region domain of a human IgG2, IgG4, IgG3, and IgG1 (SEQ ID (FR-L1, FR-L2, FR-L3, and FR-L4). NOs: 64-67). FIG.55B shows the differences between these 45 The precise amino acid sequence boundaries of a given sequences mapped onto the co-crystal structure of cetux CDR or FR can be readily determined using any of a number imab and meditope 1 (SEQ ID NO:1, cqFD). of well-known schemes, including those described by Kabat FIG. 56 shows the amino acid sequence of the light chain et al. (1991), “Sequences of Proteins of Immunological of a meditope-enabled (“medi') anti-CEA antibody (medi Interest, 5th Ed. Public Health Service, National Institutes tope-enabled M5A) (SEQ ID NO: 68) compared to the 50 of Health, Bethesda, Md. (“Kabat’ numbering scheme), wild-type M5A light chain sequence (SEQ ID NO: 69). Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” Shaded residues represent eight point mutations that were numbering scheme), MacCallum et al., J. Mol. Biol. 262: introduced in the light chain of the M5A antibody, allowing 732-745 (1996), “Antibody-antigen interactions: Contact it to bind to meditopes (for reference the heavy chain M5A analysis and binding site topography, J. Mol. Biol. 262, sequence is set forth in SEQ ID NO: 70). 55 732-745. (Contact numbering scheme), Lefranc M Petal. FIG. 57 shows results of FACS analysis, demonstrating “IMGT unique numbering for immunoglobulin and T cell binding of the meditope-enabled M5A antibody (M5A 8M) receptor variable domains and Ig Superfamily V-like to the M5A antigen (CEA) on LS174T cells. domains.” Dev Comp Immunol. 2003 January; 27(1):55-77 FIG. 58 shows surface plasmon resonance (SPR) data for (“IMGT' numbering scheme), and Honegger A and Plück a 5-diphenyl meditope bound (CQFDA(diphenyl) 60 thun A, “Yet another numbering scheme for immunoglobu STRRLKC) (SEQ ID NO:18) to an M5A 8M meditope lin variable domains: an automatic modeling and analysis enabled antibody. tool.” J Mol Biol, 2001 Jun. 8:309(3):657-70, (AHo num FIG. 59 illustrates the characterization of meditope 54, bering scheme). with an HPLC trace and its mass spectrum. The boundaries of a given CDR or FR may vary depend FIG. 60 illustrates the mass spectrum of DOTA-NHS 65 ing on the scheme used for identification. For example, the MPL conjugates formed from a starting material ratio of Kabat scheme is based structural alignments, while the 120:1 (DOTA-NHS:MPL). Chothia scheme is based on structural information. Num US 9,669,108 B2 21 22 bering for both the Kabat and Chothia schemes is based In some embodiments, the meditope binding site includes upon the most common antibody region sequence lengths, residues 40, 41, 83, and 85 of the light chain of a meditope with insertions accommodated by insertion letters, for enabled antibody, according to Kabat numbering, and/or example, “30a, and deletions appearing in Some antibodies. residues 39, 89, 105, and 108 of the heavy chain of the The two schemes place certain insertions and deletions 5 meditope-enabled antibody, according to Kabat numbering. (“indels’) at different positions, resulting in differential In some embodiments, the meditope binding site is numbering. The Contact scheme is based on analysis of located within a cavity formed by residues 8, 9, 10, 38, 39, complex crystal structures and is similar in many respects to 40, 41 42, 43, 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, the Chothia numbering scheme. 102, 103, 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, Table 1, below, lists the positions of CDR-L1, CDR-L2. 10 CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by and 173 of the light chain and 6, 9, 38, 39, 40, 41, 42, 43, the Kabat, Chothia, and Contact schemes, respectively. For 44, 45, 84, 86, 87, 88, 89,90, 91, 103, 104, 105, 106, 107, CDR-H1, residue numbering is given listed using both the 108, 111, 110, 147, 150, 151, 152, 173, 174, 175, 176, 177, Kabat and Chothia numbering schemes. It is noted that 185, 186, and 187 of the heavy chain of the antibody, because the Kabat numbering scheme places insertions at 15 according to Kabat numbering. H35A and H35B, the end of the Chothia CDR-H1 loop when With respect to a Fab portion of a meditope-enabled numbered using the Kabat numbering convention varies antibody, the meditope binding site includes residues within between H32 and H34, depending on the length of the loop. the central cavity. The meditope-binding site typically fur ther includes constant region residues. TABLE 1. The terms “meditope', as used herein, refers to a peptide or peptides that binds to a meditope-binding site of a CDR Kabat Chothia Contact meditope-enabled antibody, which antibody has a threonine CDR-L1 L24--L34 L24--L34 L3 O-L36 at position 40, an asparagine at position 41, and an aspartage CDR-L2 LSO--L56 LSO--L56 L46--L55 at position 85 of its light chain, according to Kabat num CDR-L3 L89--L97 L89--L97 L89--L96 25 CDR-H1 H31--H3SB H26--H32 . . . 34 H3O-H3SB bering, or contains a meditope binding site containing resi (Kabat Numbering") dues that correspond to those within the meditope-binding CDR-H1 H31--H3S H26--H32 H3O-H3S site of cetuximab, meditope-enabled trastuzumab, or medi (Chothia Numbering) CDR-H2 HSO--H6S HS2--HS6 H47-l-HS8 tope-enabled M5A, disclosed herein. Exemplary meditopes CDR-H3 H95--H102 H95--H102 H93-H101 include, but are not limited to, the coFD and coYN peptides 30 and variants thereof (“meditope variants' or “variant medi Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD topes”), as well as multivalent and labeled meditopes. Other 'Al-Lazikani et al., (1997) JMB 273,927-948 molecules may also bind to meditope binding sites of Thus, unless otherwise specified, a “CDR' or “comple meditope-enabled antibodies, with functional characteristics mentary determining region,” or individual specified CDRs 35 similar to those of a meditope. Such molecules, meditope (e.g., “CDR-H1, CDR-H2), of a given antibody or region analogs, may include, but are not limited to, Small mol thereof, such as a variable region thereof, should be under ecules, aptamers, nucleic acid molecules, peptibodies and stood to encompassa (or the specific) complementary deter any other substance able to bind to the same meditope mining region as defined by any of the known schemes. binding site as a meditope. Likewise, unless otherwise specified, a “FR' or “framework 40 A “therapeutic agent, as used herein, is an atom, mol region,” or individual specified FRS (e.g., “FR-H1, FR-H2). ecule, or compound that is useful in treatment of a disease of a given antibody or region thereof. Such as a variable or condition. region thereof, should be understood to encompassa (or the A “therapeutically effective amount,” “therapeutically specific) framework region as defined by any of the known effective concentration” or “therapeutically effective dose' schemes. In some instances, the scheme for identification of 45 is the amount of a compound that produces a desired a particular CDR, FR, or FRS or CDRs is specified, such as therapeutic effect in a Subject, such as preventing or treating the CDR as defined by the Kabat, Chothia, or Contact a target condition, alleviating symptoms associated with the method. In other cases, the particular amino acid sequence condition, producing a desired physiological effect, or of a CDR or FR is given. allowing imaging or diagnosis of a condition that leads to The term “meditope-enabled' antibody and “meNAb” 50 treatment of the disease or condition. The precise therapeu refer to an antibody or functional fragment thereof that is tically effective amount is the amount of the composition able to bind to a meditope, via a meditope binding site. that will yield the most effective results in terms of efficacy Examples of meditope-enabled antibodies include, but are of treatment in a given Subject. This amount will vary not limited to, cetuximab and others described herein. A depending upon a variety of factors, including, but not “meditope binding site' is a region of the meditope-enabled 55 limited to, the characteristics of the therapeutic compound antibody containing the amino acid residues that interact (including activity, pharmacokinetics, pharmacodynamics, with a bound meditope, which residues include framework and bioavailability), the physiological condition of the sub region (FR) residues of the heavy and light chains. With ject (including age, sex, disease type and stage, general reference to a Fab fragment or a Fab portion of an antibody, physical condition, responsiveness to a given dosage, and the meditope binding site is located within the central cavity 60 type of medication), the nature of the pharmaceutically of the Fab fragment or portion. acceptable carrier or carriers in the formulation, and the The “central cavity, with respect to the three-dimen route of administration. One skilled in the clinical and sional structure of a Fab, refers to the internal cavity of the pharmacological arts will be able to determine a therapeu Fab, lined by portions of the heavy and light chain variable tically effective amount through routine experimentation, and constant regions. The central cavity thus is lined by 65 namely by monitoring a Subject's response to administration residues of the VH, VL, CH1, and CL regions and does not of a compound and adjusting the dosage accordingly. For include the antigen binding site. additional guidance, see Remington: The Science and Prac US 9,669,108 B2 23 24 tice of Pharmacy 21 Edition, Univ. of Sciences in Phila opment of the condition, reducing the risk of developing the delphia (USIP), Lippincott Williams & Wilkins, Philadel condition, preventing or delaying the development of symp phia, Pa., 2005. toms associated with the condition, reducing or ending A “pharmaceutically acceptable carrier” refers to a phar symptoms associated with the condition, generating a com maceutically acceptable material, composition, or vehicle plete or partial regression of the condition, or some combi that is involved in carrying or transporting a compound of nation thereof. interest from one tissue, organ, or portion of the body to As used herein, the terms “including.” “containing,” and another tissue, organ, or portion of the body. For example, “comprising are used in their open, non-limiting sense. the carrier may be a liquid or solid filler, diluent, excipient, As used herein, the singular forms “a,” “an,” and “the Solvent, or encapsulating material, or some combination 10 include plural referents unless the context clearly dictates thereof. Each component of the carrier must be “pharma otherwise. ceutically acceptable' in that it must be compatible with the To provide a more concise description, Some of the other ingredients of the formulation. It also must be suitable quantitative expressions given herein are not qualified with for contact with any tissue, organ, or portion of the body that the term “about'. It is understood that, whether the term it may encounter, meaning that it must not carry a risk of 15 "about is used explicitly or not, every quantity given herein toxicity, irritation, allergic response, immunogenicity, or any is meant to refer to the actual given value, and it is also other complication that outweighs its therapeutic benefits. meant to refer to the approximation to such given value that A “route of administration' may refer to any administra would reasonably be inferred based on the ordinary skill in tion pathway known in the art, including but not limited to the art, including equivalents and approximations due to the aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, experimental and/or measurement conditions for Such given transdermal, or vaginal. “Transdermal administration may value. be accomplished using a topical cream or ointment or by As used herein, “alkyl refers to a saturated, straight- or means of a transdermal patch. “Parenteral refers to a route branched-chain hydrocarbon group having from 1 to 12 of administration that is generally associated with injection, carbon atoms. Representative alkyl groups include, but are including infraorbital, infusion, intraarterial, intracapsular, 25 not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl intracardiac, intradermal, intramuscular, intraperitoneal, 1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1- intrapulmonary, intraspinal, intrasternal, intrathecal, intra butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1- uterine, intravenous, Subarachnoid, Subcapsular, Subcutane pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2- ous, transmucosal, or transtracheal. pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl “In combination' or “in combination with,' when used 30 1-butyl, 3.3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, herein in the context of multiple agents, therapeutics, or isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, treatments, means in the course of treating the same disease and the like, and longer alkyl groups, such as heptyl, octyl, or condition in a subject administering two or more agents, and the like. The term "Calkyl,” where x and y are drugs, treatment regimens, treatment modalities or a com integers, refers to an alkyl with X-y carbon atoms. bination thereof (e.g., an antibody in combination with a 35 As used herein, an “alkenyl refers to a straight- or meditope or a multivalent tethering agent), in any order. This branched-chain hydrocarbon group having one or more includes simultaneous administration (or “coadministra double bonds therein and having from 2 to 12 carbon atoms. tion”), administration of a first agent prior to or after Illustrative alkenyl groups include, but are not limited to, administration of a second agent, as well as in a temporally ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadi spaced order of up to several days apart. Such combination 40 enyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2- treatment may also include more than a single administra butenyl, 4-(2-methyl-3-butene)-pentenyl, and the like. The tion of any one or more of the agents, drugs, treatment term "Calkenyl,” where X and y are integers, refers to an regimens or treatment modalities. Further, the administra alkenyl with X-y carbon atoms. tion of the two or more agents, drugs, treatment regimens, The term “alkylenyl or “alkylene' refers to a divalent treatment modalities or a combination thereof may be by the 45 alkyl group. The term “alkenylene' or “alkenylene' refers to same or different routes of administration. a divalent alkenyl group. A “therapeutic antibody' may refer to any antibody or As used herein, “alkynyl refers to a straight- or functional fragment thereof that is used to treat cancer, branched-chain hydrocarbon group having one or more autoimmune diseases, transplant rejection, cardiovascular triple bonds therein and having from 2 to 10 carbon atoms. disease or other diseases or conditions such as those 50 Exemplary alkynyl groups include, but are not limited to, described herein. Examples of therapeutic antibodies that ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpro may be used according to the embodiments described herein pynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, 4-butyl-2- include, but are not limited to murine antibodies, murinized hexynyl, and the like. or humanized chimera antibodies or human antibodies "Aryl means a mono-, bi-, or tricyclic aromatic group, including, but not limited to, Erbitux (cetuximab), ReoPro 55 wherein all rings of the group are aromatic. For bi- or (abciximab), Simulect (basiliximab), Remicade (inflix tricyclic systems, the individual aromatic rings are fused to imab); Orthoclone OKT3 (muromonab-CD3); Rituxan one another. Exemplary aryl groups include, but are not (rituximab), Bexxar (to situmomab) Humira (adalimumab), limited to, phenyl, naphthalene, and anthracene. Campath (alemtuzumab), Simulect (basiliximab), Avastin The term “boronic ester refers to a substituent —B(OR) (bevacizumab), Cimzia (certolizumab pegol), Zenapax (da 60 wherein each R group is independently a Calkyl, or the clizumab), Soliris (eculizumab), Raptiva (efalizumab), two R groups taken together form a Calkylene. Mylotarg (gemtuzumab), Zevalin (ibritumomab tiuxetan), The term “acetal' refers to a —CH(OR) group, wherein TySabri (natalizumab). Xolair (omalizumab), Synagis each R group is independently a Calkyl, or the two R (palivizumab), VectibiX (panitumumab), Lucentis (ranibi groups taken together form a C2-alkylene. Exemplary Zumab), and Herceptin (trastuzumab). 65 acetal groups include dimethylacetal or diethylacetal, or a “Treating or “treatment of a condition may refer to cyclic acetal. The term “ketal' refers to a —C(OR)— preventing the condition, slowing the onset or rate of devel group, wherein each R group is independently a Calkyl, US 9,669,108 B2 25 26 or the two R groups taken together form a C-alkylene. used to describe a structural system, the Substitution is meant Exemplary ketals include dimethylketal or diethylketal, or a to occur at any Valency-allowed position on the system. cyclic ketal. As used herein, the expression “one or more substituents' The term “halo represents chloro, fluoro, bromo, or iodo. denotes one to maximum possible number of Substitution(s) In some embodiments, halo is chloro, fluoro, or bromo. The that can occur at any Valency-allowed position on the term “halogen' as used herein refers to fluorine, chlorine, system. In a certain embodiment, “one or more Substituents' bromine, or iodine. means 1, 2, 3, 4, or 5 substituents. In another embodiment, The term "ortho ester” refers to a C(OR) group, one or more substituent means 1, 2, or 3 Substituents. wherein each R group is independently a Calkyl, or two Any atom that is represented herein with an unsatisfied of the R groups taken together form a C-alkylene. 10 valence is assumed to have the sufficient number of hydro The term “oxo” means an=O group and may be attached gen atoms to satisfy the atom's Valence. to a carbon atom or a Sulfur atom. When any variable, such as alkyl, R, or R, appears in The term "phosphonate ester refers to a P(O)(OR) more than one place in any formula or description provided group, wherein each R group is independently a Calkyl, herein, the definition of that variable on each occurrence is or the two R groups taken together form a C-alkylene. 15 independent of its definition at every other occurrence. As used herein, the term “cycloalkyl refers to a saturated Numerical ranges, as used herein, are intended to include or partially saturated, monocyclic, fused polycyclic, bridged sequential whole numbers. For example, a range expressed polycyclic, or spiro polycyclic carbocycle having from 3 to as “from 0 to 4” or “0-4” includes 0, 1, 2, 3 and 4. 15 ring carbon atoms. A non limiting category of cycloalkyl When a multifunctional moiety is shown, the point of groups are saturated or partially saturated, monocyclic car attachment to the core is indicated by a line or hyphen. For bocycles having from 3 to 6 carbon atoms. Illustrative example, —OH refers to a moiety in which an oxygen atom examples of cycloalkyl groups include, but are not limited is the point of attachment of the hydroxyl group to the to, the following moieties: remainder of the molecule. Any formula given herein is intended to represent com 25 pounds having structures depicted by the structural formula as well as certain variations or forms. For example, com pounds of any formula given herein may have asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All Stereoisomers, including optical isomers, 30 enantiomers, and diastereomers, of the compounds of the general formula, and mixtures thereof, are considered to fall within the scope of the formula. Furthermore, certain struc tures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. All Such isomeric 35 forms, and mixtures thereof, are contemplated herein as part of the present invention. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more tautomeric or atropisomeric forms, and mixtures thereof. 40 Diastereomeric mixtures may be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers may be separated by 45 converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary Such as a chiral alcohol or Mosher's acid chloride, or formation of a mixture of diaste reomeric salts), separating the diastereomers and converting 50 (e.g., hydrolyzing or de-salting) the individual diastereomers As used herein, the term “5-membered heteroaryl refers to the corresponding pure enantiomers. Enantiomers may to a monocyclic, aromatic heterocycle having five ring also be separated by use of chiral HPLC column. The chiral atoms that are selected from carbon, oxygen, nitrogen, and centers of compounds of the present invention may be sulfur. Examples of 5-membered heteroaryl groups include, designated as “R” or “S” as defined by the IUPAC 1974 but are not limited to, imidazolyl pyrazolyl, triazolyl, tet 55 razolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, iso Recommendations, or by “D' or “L” designations consistent with the peptide literature. thiazolyl pyrrolyl, and thiadiazolyl. Particular examples of As used herein, a box around a portion of a structure, 5-membered heteroaryls include those that may be formed drawn with a subscript, indicates that the structural fragment by 1.3-cycloaddition reactions such as a Huisgen reaction that appears within the box is repeated according to the between an azide and a propargyl group. 60 As used herein, the term "substituted” means that the subscript. For example, the substructure: specified group or moiety bears one or more Suitable Sub stituents. As used herein, the term “unsubstituted” means that the specified group bears no Substituents. As used - Fragment H herein, the term “optionally substituted” means that the 65 specified group is unsubstituted or Substituted by the speci fied number of substituents. Where the term "substituted” is US 9,669,108 B2 27 28 where X is 0, 1, or 2, indicates the fragment is absent from Exemplary basic salts include ammonium salts, alkali the structure, or is —Fragment—, or is metal salts such as sodium, lithium, and potassium salts, —Fragment Fragment—. For example, within formula alkaline earth metal salts such as calcium and magnesium (X), the following substructure: 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. dim 10 ethyl, diethyl, and dibutyl Sulfates), long chain halides (e.g. decyl, lauryl, and Stearyl chlorides, bromides and iodides), where p is 0 or 1, means that the —X NH group aralkyl halides (e.g. benzyl and phenethyl bromides), and within the box is absent from the structure (p is 0), or is others. present once (p is 1). 15 Additionally, acids and bases which are generally consid The compounds of the invention can form pharmaceuti ered suitable for the formation of pharmaceutically useful cally acceptable salts, which are also within the scope of this salts from pharmaceutical compounds are discussed, for invention. A “pharmaceutically acceptable salt” refers to a example, by P. Stahl et al., Camille G. (eds.) Handbook of salt of a free acid or base of a compound described herein Pharmaceutical Salts. Properties, Selection and Use. (2002) that is non-toxic, is physiologically tolerable, is compatible Zurich: Wiley-VCH. S. Berge et al. Journal of Pharmaceu with the pharmaceutical composition in which it is formu tical Sciences (1977) 66(1) 1-19; P. Gould, International J. lated, and is otherwise suitable for formulation and/or of Pharmaceutics (1986) 33 201-217: Anderson et al. The administration to a subject. Reference to a compound herein Practice of Medicinal Chemistry (1996), Academic Press, is understood to include reference to a pharmaceutically New York; and in The Orange Book (Food & Drug Admin 25 istration, MD, available from FDA). These disclosures are acceptable salt of said compound unless otherwise indicated. incorporated herein by reference thereto. Compound salts include acidic salts formed with inor Additionally, any compound described herein is intended ganic and/or organic acids, as well as basic salts formed with to refer also to any unsolvated form, or a hydrate, Solvate, or inorganic and/or organic bases. In addition, where a given polymorph of Such a compound, and mixtures thereof, even compound contains both a basic moiety, Such as, but not 30 if such forms are not listed explicitly. “Solvate” means a limited to, a pyridine or imidazole, and an acidic moiety, physical association of a compound of the invention with such as, but not limited to, a carboxylic acid, one of skill in one or more solvent molecules. This physical association the art will recognize that the compound may exist as a involves varying degrees of ionic and covalent bonding, Zwitterion (“inner salt); such salts are included within the including hydrogen bonding. In certain instances the Solvate term “salt as used herein. Salts of the compounds of the 35 will be capable of isolation, for example when one or more invention may be prepared, for example, by reacting a Solvent molecules are incorporated in the crystal lattice of a compound with an amount of a suitable acid or base. Such crystalline solid. “Solvate” encompasses both solution as an equivalent amount, in a medium Such as one in which phase and isolatable solvates. Suitable solvates include those the salt precipitates or in an aqueous medium followed by formed with pharmaceutically acceptable solvents such as lyophilization. 40 water, ethanol, and the like. In some embodiments, the Exemplary salts include, but are not limited, to sulfate, solvent is water and the solvates are hydrates. citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, Any formula given herein is also intended to represent bisulfate, phosphate, acid phosphate, isonicotinate, lactate, unlabeled forms as well as isotopically labeled forms of the salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, compounds. Isotopically labeled compounds have structures bitartrate, ascorbate. Succinate, maleate, gentisinate, fumar 45 depicted by the formulas given herein except that one or ate, gluconate, glucuronate, Saccharate, formate, benzoate, more atoms are replaced by an atom having a selected glutamate, methanesulfonate (“mesylate'), ethanesulfonate, atomic mass or mass number. Examples of isotopes that can benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., be incorporated into compounds of the invention include 1,1'-methylene-bis(2-hydroxy-3-naphthoate)) salts. A phar isotopes of hydrogen, carbon, nitrogen, oxygen, phospho maceutically acceptable salt may involve the inclusion of 50 rous, fluorine, chlorine, and iodine, such as H. H. 'C, C, another molecule Such as an acetate ion, a Succinate ion or 14C, 15N, 18O, 17O, 3 IP 32P 35S, 18F 36C1, and 125I, other counterion. The counterion may be any organic or respectively. Such isotopically labelled compounds are use inorganic moiety that stabilizes the charge on the parent ful in metabolic studies (for example with 'C), reaction compound. Furthermore, a pharmaceutically acceptable salt kinetic studies (with, for example H or H), detection or may have more than one charged atom in its structure. 55 imaging techniques such as positron emission tomography Instances where multiple charged atoms are part of the (PET) or single-photon emission computed tomography pharmaceutically acceptable salt can have multiple counte (SPECT) including drug or substrate tissue distribution rions. Hence, a pharmaceutically acceptable Salt can have assays, or in radioactive treatment of patients. In particular, one or more charged atoms and/or one or more counter ion. an 'F or ''C labeled compound may be particularly suitable Exemplary acid addition salts include acetates, ascor 60 for PET or SPECT studies. Further, substitution with heavier bates, benzoates, benzenesulfonates, bisulfates, borates, isotopes such as deuterium (i.e., H) may afford certain butyrates, citrates, camphorates, camphorsulfonates, fumar therapeutic advantages resulting from greater metabolic ates, hydrochlorides, hydrobromides, hydroiodides, lactates, stability, for example increased in vivo half-life or reduced maleates, methanesulfonates, naphthalenesulfonates, dosage requirements. Isotopically labeled compounds of this nitrates, oxalates, phosphates, propionates, Salicylates, suc 65 invention and prodrugs thereof can generally be prepared by cinates, Sulfates, tartarates, thiocyanates, toluenesulfonates carrying out the procedures disclosed in the schemes or in (also known as tosylates.) and the like. the examples and preparations described below by substi US 9,669,108 B2 29 30 tuting a readily available isotopically labeled reagent for a titrations; kinetic exclusion; circular dichroism, differential non-isotopically labeled reagent. scanning calorimetry, or other known method. For example, B. Antibodies and Antibody-Binding Substances in some cases, the analog or meditope exhibits a binding Provided herein are antibodies, including monoclonal constant of less than at or about 10 uM, less than at or about antibodies (mAbs), and fragments, e.g., functional frag 5uM, or less than at or about 2 uM, less than at or about 1 ments, thereof, and compounds and compositions, including uM, less than at or about 500, 400, 300, 200, 100 nm, or less, peptides, such as meditopes, and meditope analogs, that bind as measured by SPR or as measured by ITC or as measured to the antibodies. The Substances, e.g., meditopes, generally by any of these methods. bind to regions/binding sites of the antibodies and fragments In some examples, the meditope-binding site is a struc other than (e.g., separate from) the complementarity deter 10 tural feature of the monoclonal antibody, cetuximab, a mining regions (CDRS), typically to residues within the human-murine chimeric antibody used for the treatment of framework regions (FRS) and/or constant regions of the EGFR-expressing metastatic colorectal cancer and head and antibodies and fragments. Also provided are complexes, neck cancers. Thus, in Some cases, the meditope-binding site compounds, and compositions containing the antibodies and contains residues corresponding to those within the medi Substances, e.g., meditopes, as well as methods and uses of 15 tope binding site of cetuximab. In a study reported herein, the same, including therapeutic, diagnostic, research, and X-ray crystallographic analysis has revealed that the peptides other uses of the antibodies and Substances, and methods for of SEQID NO: 1 binds to a meditope-binding site within the producing the same. central cavity of the cetuximab Fab fragment, defined by In certain aspects, the provided embodiments are based on various residues of the heavy and light chains (see FIGS. 1 the discovery described herein that certain peptides, and 4A), with a binding constant of ~700 nM (see FIGS. C-QFDLSTRRLK-C (cQFD; SEQ ID NO: 1) and C-QYN 30A-30B). LSSRALK-C (cQYN: SEQID NO: 2), non-covalently bind Several interactions between cetuximab's meditope bind to a murine-human chimeric antibody, cetuximab, by inter ing site and the coFD (SEQ ID NO: 1, meditope 1) and acting with residues outside the complementarity determin cQYN (SEQID NO: 2, meditope 2) meditopes are based on ing regions (CDRS), including residues in the framework 25 particular structural features of cetuximab, particularly and constant region. Also as described herein, the ability to within the framework and constant regions of the central bind to these peptides was based on certain aspects specific cavity of the Fab fragment. The regions of cetuximab that to cetuximab and in the studies herein binding was not constitute the meditope binding site are unique and appear to observed, for example, to fully human antibodies, murine be the result of the chimeric nature of this antibody. Spe antibodies, or other chimeric antibodies). As demonstrated 30 cifically, the Fab variable regions (FVs) are murine and the herein, these meditopes are capable of binding the chimeric Fab constant regions (CH1 and CL) are human. The engi antibody simultaneously with its antigen. See FIG. 4. As neering of this Fab produced a combination of residues not demonstrated herein, the binding site for these meditopes on found in a sequence alignment of murine and human chi cetuximab also are distinct from the binding sites of other meric antibodies. For example, data herein show that the framework-binding antigens such as the Superantigens 35 meditopes did not bind to fully human IgG framework (e.g., Staphylococcal Protein A (SpA) and Peptostreptococcus trastuzumab), to other chimeric antibodies such as rituximab magnus Protein L (PpI) (FIG. 7). In certain aspects, the (FIG. 29), or to mouse antibodies, confirming that the provided embodiments build upon this discovery, for cetuximab meditope-binding site is highly specific. In addi example, by modifying other mabs to allow their binding to tion to these results, Superposition of the molecular structure these and other meditopes (i.e., "meditope-enabling) and 40 of trastuzumab (1N8Z: Cho et al., Nature, "Structure of the generating variant meditopes and/or altered meditope-en extracellular region of HER2 alone and in complex with the abled antibodies, for example, those having altered proper Herceptin Fab.” 2003 Feb. 13; 421 (6924):756-60) and ties, including improved binding affinity, toxicity, PK/PD, rituxumab (2OSL, Du et al., J Biol Chem, 2007 May and/or pH dependence. 18:282(20): 15073-80. Epub 2007 Mar. 29) Fabs on to the C. Meditope-Enabled Antibodies and Complexes 45 meditope bound cetuximab Fab structure further highlighted Provided are meditope-enabled antibodies that are the uniqueness of the framework. Superposition of multiple capable of binding to one or more meditopes via meditope human and murine Fabs onto the cetuximab-cyclic peptide binding sites. In some cases, the meditope-enabled antibody (meditope 1) structure indicated that the key interactions binds to a cyclic peptide of SEQ ID NO: 1 or 2 (meditope between this peptide and murine-human chimeric Fab are 1 or 2) and/or to one or more variants thereof. Such as 50 absent in both human-only and murine-only IgG structures meditopes 1, 2, 16-18, 23, 29, 31, 32, 36, 39, 42, 43, 45,46, compared in this study. Point mutations of key residues 51, 52, 54, or 55 (meditopes based on peptides having the within the coFD cyclic peptide (meditope 1) reduced its sequences set forth in SEQID NOs: 1, 2, 16-18, 23, 29, 31, binding affinity for the cetuximab Fab, further confirming 32, 36,39, 42, 43, 45, 46, 51, 52, 54, or 55), or in some cases, the high specificity and structural model. Thus, the interac any of meditopes 1, 2, or 15-55. Among the provided 55 tion appears to be specific to the central cavity of this meditope-enabled antibodies are those that bind to a medi specific murine-human chimera Fab and the selected medi tope or meditopes with an affinity similar to that of cetux tope. imab. For example, in certain aspects, the antibodies bind to In certain embodiments, the unique interactions between the meditope(s) with a dissociation constant of less than at the meditopes and the cetuximab binding site are exploited or about 10 uM, less than at or about 5 uM, or less than at 60 to generate additional meditope-enabled antibodies. Medi or about 2 LM, less than at or about 1 uM, less than at or tope-enabled antibodies are useful, for example, to improve about 500, 400, 300, 200, 100 nM, or less, such as at or about antibody and cell purification methods, improving the thera 200 picomolar or less. In some cases, the dissociation peutic efficacy of mAbs, enhancing targeted delivery of constant, such as any of those listed herein, is that measured imaging or therapeutic agents, including in pre-targeted using a particular technique, such as Surface plasmon reso 65 delivery and imaging, and improving mAb-based imaging nance (SPR), isothermal titration calorimetry (ITC), fluo methods, and in Some aspects are broadly applicable to any rescence, fluorescence polarization, NMR, IR, calorimetry monoclonal antibody. US 9,669,108 B2 31 32 In some embodiments, the meditope-enabled antibodies Thus, among the provided meditope-enabled antibodies are generated by modifying an antibody other than cetux are antibodies having one or more modifications, typically imab (sometimes referred to as the template antibody). Such amino acid Substitutions, at residues that correspond to as an antibody having one or more CDRs distinct from those positions within the meditope binding site of cetuximab or of cetuximab, to confer the ability to bind to one or more of 5 other meditope-enabled antibody, such as those described 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 human or or a mouse antibody. In one a VL region with a threonine, serine, or aspartate at position aspect, the modifications include Substituting residues 40, a residue other than glycine at position 41, and an 10 aspartate or asparagine at position 85, according to Kabat within the central cavity of the Fab fragment, typically numbering, for example, an antibody with a VL region within the framework regions (FRs) of the heavy and light having a threonine at position 40, an asparagine at position chain variable regions and/or the constant regions to render 41, and an aspartate at position 85. In some embodiments, the template antibody meditope-enabled. For example, the antibody has a VH region with a serine at position 40 and where the template antibody is a human or humanized 15 an isoleucine at position 89 and/or a VH region with a serine antibody, the modifications generally include Substitutions at or proline at position 40 and an isoleucine, tyrosine, methio residues within the heavy and light chain variable region nine, phenylalanine, or tryptophan at position 89, according FRs. In some embodiments, such residues are replaced with to Kabat numbering. In some embodiments, the VL region the corresponding residue present in cetuximab, or compa has an isoleucine or leucine at position 10, and/or an rable amino acid. Thus, in certain embodiments, residues isoleucine at position 83. In some embodiments, the VL within the FRs of a human or humanized antibody are region has a valine or isoleucine at position 9 and/or a replaced with corresponding murine residues; in certain residue other than glutamine at position 100. embodiments, they are replaced by other residues, such as In some examples, the VL region has a valine or isoleu those having similar functional groups or moieties for inter cine at position 9, an isoleucine or leucine at position 10, an acting with the meditopes. Typically, the residues replaced 25 arginine at position 39, a threonine at position 40, an by corresponding murine (or other) residues are found asparagine at position 41, a glycine at position 42, a serine within the central Fab cavity, and thus are not exposed to the at position 43, an isoleucine at position 83, an aspartate at immune system. As such, in Some embodiments, introducing position 85, and an alanine at position 100; and the VH these amino acid Substitutions in a human or humanized region has a serine at position 40 and an isoleucine at antibody do not increase or do not substantially increase the 30 position 89, according to Kabat numbering. antigenicity of the modified template antibody, in the context In some examples, the VL region does not contain a of delivery to a human subject. In addition, antigenicity proline at position 40, a glycine at position 41, or a threonine prediction algorithms may be further used to indicate that at position 85, according to Kabat numbering, and/or the VH the human sequence with the point mutations should not be region does not contain an asparagine or alanine at position antigenic. 35 40 or a valine at position 89, according to Kabat numbering. In some embodiments, the one or more residues that are In some examples, the VL region does not contain an serine replaced, are selected from light chain framework residues at position 10, a proline at position 40, a glycine at position 10, 39-43, 83, 85, 100 and 104, according to Kabat num 41, an phenylalanine at position 83, or a threonine at position bering (see Kabat E. A., et al. (1991) Sequences of Proteins 85, according to Kabat numbering, and/or the VH region of Immunological Interest, Fifth Edition. NIH Publication 40 does not contain an asparagine or alanine at position 40 or No. 91-3242, incorporated herein by reference in its a valine at position 89, according to Kabat numbering. entirety), and/or heavy chain framework residue numbers In some aspects, the antibody has a light chain having P8, 40, 89 and 105, according to Kabat numbering (see FIG. 2). V9 or 19, I10 or L10, Q38, R39, T40, N41 G42, S43, P44, In general, unless otherwise specified, amino acid positions R45, D82, I83, A84, D85, Y86, Y87, G99, A100, G101, in the heavy or light chain of an antibody refer to Kabat 45 T102, K103, L104, E105, R142, S162, V163, T164, E165, numbering. Also encompassed within the present disclosure Q166, D167, S168, and Y173, according to Kabat number are residues in other antibodies corresponding to the resi ing, and/or a heavy chain having Q6, P9, R38, Q39, S40, dues of cetuximab described herein, such as those within the P41, G42, K43, G44, L45, S84, D86, T87, A88, I89, Y90, cetuximab meditope-binding site. In some embodiments, Y91, W103, G104, Q105, G106, T107, L108, V109, T110, residues in the template antibody corresponding to light 50 V111, Y147, E150, P151, V152, T173, F174, P175, A176, chain residues 9, 10, 39, 40, 41, 42, 43, 83, 85, 100, and/or V177, Y185, S186, and L187, according to Kabat number 104, and/or heavy chain residues 40, 89, and/or 105, are 1ng. replaced, for example, with amino acids present at those In other embodiments, the meditope-enabled antibodies positions within cetuximab. are generated via CDR grafting, typically by modifying one In one embodiment, the one or more residues replaced are 55 or more complementarity determining region (CDR) (e.g., light chain framework residues including, but not limited to, one or more of CDRs 1-3) of the heavy and/or light chain of 40, 41, 83 and 85, according to Kabat numbering. In one a meditope-enabled antibody, such as any of the meditope embodiment, light chain residue 40 is replaced with threo enabled antibodies described herein, to replace them with nine; light chain residue 41 is replaced with asparagine, light other CDRs, such as CDRs of existing or new antibodies. chain residue 83 is replaced with isoleucine or valine, and/or 60 CDR grafting is standard practice for producing humanized light chain residue 85 is replaced with aspartate. In a monoclonal antibodies, e.g., by grafting CDRS of an anti particular example, light chain framework Pro40 is replaced body generated in a non-human species, such as mouse, onto with Thr(P40T) or Ser(P40S), light chain framework Gly41 a human antibody framework. See U.S. Pat. Nos. 5,558,864 is replaced with Asn (G41N), light chain framework residue and 8,133,982; Kettleborough et al., “Humanization of a Phe83 is replaced with Ile (F83I) or Val (F83V) and light 65 mouse monoclonal antibody by CDR-grafting: the impor chain framework residue Thr85 is replaced with Asp (T85D) tance of framework residues on loop conformation.” Protein or Asn (T85N). Eng., 4:773-783 (1991). Thus, in certain embodiments, the US 9,669,108 B2 33 34 antigen specificity of a meditope-enabled antibody is altered least one CDR that is distinct from the CDRs of the heavy by grafting the CDRS of preexisting or newly-generated chain sequence set forth in SEQ ID NO: 6. antibodies of interest. Also among the provided meditope In some embodiments, the VL region has an amino acid enabled antibodies are such CDR-grafted meditope-enabled sequence comprising a light chain framework region (FR) 1 antibodies. (FR-L1), an FR-L2, an FR-L3, and/or an FR-L4 of the light In some embodiments, the meditope-enabled antibodies chain sequence set forth in SEQ ID NO: 68 (or an FR-L1, are generated, using one of the antibodies disclosed herein FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75, (e.g., cetuximab, meditope-enabled trastuzumab, or medi 80, 85, 90,91, 92,93, 94, 95, 96, 97,98, or 99% identical tope-enabled M5A (anti-CEA) antibody) as a template to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of SEQID NO: sequence, and carrying out one or more known antibody 10 68), and in some aspects at least one CDR that is distinct engineering methods to alter it, for example, to alter its from the CDRs of the light chain sequence set forth in SEQ antigen-binding characteristics, producing a meditope-en ID NO: 68; and/or a VH region with an amino acid sequence abled antibody with distinct characteristics. Known antibody having a heavy chain FR1 (FR-H1), an FR-H2, an FR-H3. engineering methods typically employed to alter antigen and/or an FR-H4, of the heavy chain sequence set forth in 15 SEQ ID NO: 70 (or an FR-H1, FR-H2, FR-H3, and/or binding and other properties include various in vitro ran FR-H4 that is at least at or about 75, 80, 85,90, 91, 92,93, domization, affinity maturation, and selection methods, 94, 95, 96, 97, 98, or 99% identical to the FR-H1, FR-H2, including error-prone PCR, spiked PCR, site-directed muta FR-H3, and/or FR-H4 of SEQ ID NO: 70), and in some genesis, phage display and other selection methods. Also aspects at least one CDR that is distinct from the CDRs of provided are constructs, libraries, and expression systems, the heavy chain sequence set forth in SEQ ID NO: 70. including GPI-linked expression systems, for carrying out In some embodiments, the VL region has an amino acid Such methods. sequence comprising a light chain framework region (FR) 1 Thus, in certain embodiments, the provided meditope (FR-L1), an FR-L2, an FR-L3, and/or an FR-L4 of the light enabled antibody has a light chain and/or heavy chain chain sequence set forth in SEQ ID NO: 61 (or an FR-L1, variable region with the framework region or regions (FRs) 25 FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75, of a meditope-enabled antibody, Such as cetuximab, a medi 80, 85, 90,91, 92,93, 94, 95, 96, 97,98, or 99% identical tope-enabled trastuzumab, or a meditope-enabled M5A (or to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of SEQID NO: FR(s) with at least at or about 75, 80, 85,90,91, 92,93, 94, 61), and in some aspects at least one CDR that is distinct 95, 96, 97, 98, or 99% identity to the FR(s) of such an from the CDRs of the light chain sequence set forth in SEQ antibody). In some aspects, such an antibody has one or 30 ID NO: 61; and/or a VH region with an amino acid sequence more CDRs that are distinct from the CDRs of that medi having a heavy chain FR1 (FR-H1), an FR-H2, an FR-H3. tope-enabled antibody. and/or an FR-H4, of the heavy chain sequence set forth in For example, in some embodiments, the VL region has an SEQ ID NO: 63 (or an FR-H1, FR-H2, FR-H3, and/or amino acid sequence comprising a light chain framework FR-H4 that is at least at or about 75, 80, 85,90, 91, 92,93, region (FR) 1 (FR-L1), an FR-L2, an FR-L3, and/or an 35 94, 95, 96, 97, 98, or 99% identical to the FR-H1, FR-H2, FR-L4 of the light chain sequence set forth in SEQ ID NO: FR-H3, and/or FR-H4 of SEQ ID NO: 63), and in some 71 (or an FR-L1, FR-L2, FR-L3, and/or FR-L4 that is at least aspects at least one CDR that is distinct from the CDRs of at or about 75, 80, 85, 90,91, 92,93, 94, 95, 96, 97,98, or the heavy chain sequence set forth in SEQ ID NO: 63. 99% identical to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of In some embodiments, the meditope-enabled antibody has SEQID NO: 71), and in some aspects at least one CDR that 40 one or more CDRs distinct from the CDRs set forth in SEQ is distinct from the CDRs of the light chain sequence set ID NO: 6, 7, 9, 10, 12, 14, 61, 63, 68, 69, 70, 71, and/or 72. forth in SEQID NO: 71; and/or a VH region with an amino In some embodiments, the meditope is an antibody other acid sequence having a heavy chain FR1 (FR-HD, an than cetuximab, does not specifically bind to an EGFR, FR-H2, an FR-H3, and/or an FR-H4, of the heavy chain binds to an antigen other than EGFR, and/or does not sequence set forth in SEQID NO: 72 (or an FR-H1, FR-H2, 45 specifically bind to the epitope on EGFR that is specifically FR-H3, and/or FR-H4 that is at least at or about 75, 80, 85, bound by cetuximab. 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% identical to the In some examples, the meditope-enabled antibody is FR-H1, FR-H2, FR-H3, and/or FR-H4 of SEQID NO: 72), generated based on a template antibody that is selected from and in some aspects at least one CDR that is distinct from the among abagovomab, abciximab, adalimumab, adecatu CDRs of the heavy chain sequence set forth in SEQID NO: 50 mumab, alemtuzumab, altumomab, altumomab pentetate, 72. anatumomab, anatumomab mafenatoX, arcitumomab, atli In some embodiments, the VL region has an amino acid Zumab, basiliximab, bectumomab, ectumomab, belimumab, sequence comprising a light chain framework region (FR) 1 benralizumab, bevacizumab, brentuximab, canakinumab, (FR-L1), an FR-L2, an FR-L3, and/or an FR-L4 of the light capiromab, capromab pendetide, catumaXomab, certoli chain sequence set forth in SEQ ID NO: 9 (or an FR-L1, 55 Zumab, clivatuZumab tetraxetan, daclizumab, denosumab, FR-L2, FR-L3, and/or FR-L4 that is at least at or about 75, eculizumab, edrecolomab, efalizumab, etaracizumab, ertu 80, 85, 90,91, 92,93, 94, 95, 96, 97,98, or 99% identical maxomab, fanolesomab, Fbta05, fontolizumab, gemtu to the FR-L1, FR-L2, FR-L3, and/or FR-L4 of SEQID NO: Zumab, girentuximab, golimumab, ibritumomab, igovomab, 9), and in some aspects at least one CDR that is distinct from infliximab, ipilimumab, labetuZumab, mepolizumab, mur the CDRs of the light chain sequence set forth in SEQ ID 60 omonab, muromonab-CD3, natalizumab, necitumumab NO: 9; and/or a VH region with an amino acid sequence nimotuZumab. ofatumumab, omalizumab, oregovomab, having a heavy chain FR1 (FR-H1), an FR-H2, an FR-H3. palivizumab, panitumumab, ranibizumab, rituximab, Satu and/or an FR-H4, of the heavy chain sequence set forth in momab, Sulesomab, ibritumomab, ibritumomab tiuxetan, SEQID NO: 6 (or an FR-H1, FR-H2, FR-H3, and/or FR-H4 tocilizumab, to situmomab, trastuzumab, Trbs07, usteki that is at least at or about 75, 80, 85,90,91, 92,93, 94, 95, 65 numab, visilizumab, Votumumab, Zalutumumab, 96, 97,98, or 99% identical to the FR-H1, FR-H2, FR-H3, brodalumab, anrukinzumab, bapineuZumab, , and/or FR-H4 of SEQ ID NO: 6), and in some aspects at demcizumab, ganitumab, inotuZumab, mavrilimumab, mox US 9,669,108 B2 35 36 etumomab pasudotox, rillotumumab, Sifalimumab, tan TABLE 2-continued eZumab, tralokinumab, tremelimumab, the antibody pro duced by the hybridoma 10B5, B6H12.2, and urelumab, Antibody CAS Registry number fragments thereof, antibodies having the CDRs and/or anti brodalumab 174395-19-7 gen-binding regions thereof, and/or antibodies that compete anrukinzumab 910649-32-0 for binding with Such antibodies; and/or antibodies having a bapineuZumab 64.8895-38-9 dalotuzumab OOS389-60-5 sequence set forth in any of SEQ ID NOs: 78-124, and/or demcizumab (OMP- 292.853-12-3 125-170, fragments thereof antibodies having the CDRs 21M18) and/or antigen-binding regions thereof, and/or antibodies ganitumab 905703-97-1 that compete for binding with such antibodies. 10 inotuzumab 635715-01-4 mavrilimumab O85337-57-0 Table 2 lists CAS(R) Registry Numbers for certain anti moxetumomab O2O748-57-5 bodies. moxetumomab O2O748-57-5 pasudotox TABLE 2 rillotumumab 8725 14-65-3 15 Sifallimumab 143SO3-67-6 Antibody CAS Registry number tanezumab 88O266-57-9 tralokinumab O44-515-88-9 abagovomab 79.2921-10-9 tremelimumab 745O13-59-6 abciximab 43653-53-6 urelumab 934823-49-1 adalimumab 331731-18-1 necitumumab 9068OS-06-9 adecatumumab SO3605-66-1 alemtuzumab 216SO3-57-O indium (111 In) S6586-92-4 In other examples, the template antibody is selected from altumomab pentetate among: abagovomab, abciximab, adalimumab, adecatu arcitumomab 54361-48-5 arcitumumab 54361-48-5 mumab, alemtuzumab, altumomab, altumomab pentetate, atlizumab 375823-41-9 anatumomab, anatumomab mafenatoX, arcitumomab, atli basiliximab 52923-56-3 25 Zumab, basiliximab, bectumomab, ectumomab, belimumab, bectumomab S8318-63-9 belimumab 356547-88-1 benralizumab, bevacizumab, brentuximab, canakinumab, benralizumab O44511-01-4 capiromab, capromab pendetide, catumaXomab, certoli bevacizumab 21 6974-75-3 Zumab, clivatuZumab tetraxetan, daclizumab, denosumab, brentuximab 914.088-09-8 canakinumab 914613-48-2 30 eculizumab, edrecolomab, efalizumab, etaracizumab, ertu capiromab pendetide 45464-28-4 maxomab, fanolesomab, Fbta05, fontolizumab, gemtu capiromab S1763-64-3 Zumab, girentuximab, golimumab, ibritumomab, igovomab, catumaxomab SO9077-98-9 certolizumab 428863-SO-7 infliximab, ipilimumab, labetuZumab, mepolizumab, mur certolizumab 428863-SO-7 omonab, muromonab-CD3, natalizumab, necitumumab, cetuximab 2O5923-56-4 35 nimotuZumab. ofatumumab, omalizumab, oregovomab, clivatuzumab tetraxetan 943976-23-6 daclizumab 152923-56-3 palivizumab, panitumumab, ranibizumab, rituximab, Satu denosumab 615258-40-7 momab, Sulesomab, ibritumomab, ibritumomab tiuxetan, eculizumab 21968S-SO-4 tocilizumab, to situmomab, trastuzumab, Trbs07, usteki edrecolomab 1S6S86-89-9 efalizumab 214745-43-4 numab, visilizumab, Votumumab, Zalutumumab, the anti 40 etaracizumab 892S53-42-3 body produced by the hybridoma 10B5, and brodalumab, or etrumaxomab SO9077-99-0 tiuZetan. In some Such examples, the one or more CDRS are fanolesomab 225239-31-6 CDRs present in these template antibodies, and/or the anti FBTAOS Lymphomun/FBTA05 bodies specifically bind to the same antigen or epitope as fontolizumab 326859-36-3 gemtuzumab 22O578-59-6 Such antibodies, and/or compete for binding with Such girentuximab 916138-87-9 45 antibodies to their antigens. golimumab 476181-74-5 Thus, in some cases, the meditope-enabled antibodies ibritumomab 174722-31-7 (including fragments thereof) specifically binds to an anti igovomab 171656-50-1 Infliximab 170277-31-3 gen selected from the group consisting of CA-125, glyco ipilimumab 4772O2-00-9 protein (GP) IIb/IIIa receptor, TNF-alpha, CD52, TAG-72, labetuzumab 219649-07-7 50 Carcinoembryonic antigen (CEA), interleukin-6 receptor mepolizumab 196O78-29-2 muromonab-CD3 140608-64-6 (IL-6R), IL-2, interleukin-2 receptor a-chain (CD25), CD22, natalizumab 189261-10-7 B-cell activating factor, interleukin-5 receptor (CD125), nimotuzumab 828933-61-3 VEGF, VEGF-A, CD30, IL-1beta, prostate specific mem ofatumumab 679818-59-8 brane antigen (PSMA), CD3, EpCAM, EGF receptor omalizumab 24.2138-07-4 55 oregovomab 213327-37-8 (EGFR), MUC1, human interleukin-2 receptor, Tac, RANK palivizumab 188O39-54-5 ligand, a complement protein, e.g., C5, EpCAM, CD11a, panitumumab 339177-26-3 e.g., human CD11a, an integrin, e.g., alpha-V beta-3 integrin, ranibizumab 347396-82-1 vitronectin receptor alpha v beta 3 integrin, HER2, neu, rituximab 174722-31-7 Satumomab 13895S-26-7 CD3, CD15, CD20 (small and/or large loops), Interferon Sulesomab 167747-19-5 60 gamma, CD33, CA-IX, TNF alpha, CTLA-4, carcinoem tiuxetan (ibritumomab) 174722-31-7 bryonic antigen, IL-5, CD3 epsilon, CAM, Alpha-4-integrin, tocilizumab 375823-41-9 IgE. e.g., IgE Fc region, an RSV antigen, e.g., fusion protein to situmomab 192391-48-3 of respiratory syncytial virus (RSV), TAG-72, NCA-90 trastuzumab 18O288-69-1 ustekinumab 815610-63-0 (granulocyte cell antigen), IL-6, GD2, GD3, IL-12, IL-23, votumumab 148.189-70-2 65 IL-17, CTA A16.88, IL13, interleukin-1 beta, beta-amyloid, Zalutumumab 667901-13-5 IGF-1 receptor (IGF-1R), delta-like ligand 4 (DLL4), alpha Subunit of granulocyte macrophage colony stimulating fac US 9,669,108 B2 37 38 tor receptor, hepatocyte growth factor, IFN-alpha, nerve bodies, modified to allow their binding to one or more of the growth factor, IL-13, PD-L1, CD326, CD47, and CD137. In provided meditopes, e.g., with an affinity as described Some examples, the meditope-enabled antigen binds to herein. another antigen identified as a target in a disease or condition Also provided are complexes containing one or more of interest, Such as cancer or other disease. meditope bound to a meditope-enabled antibody, such as any The meditope-enabled antibodies generally further of the antibodies described herein. include a constant region, typically a heavy and light chain Also provided are nucleic acids, such as cDNA and RNA constant region, which generally are human or partially molecules, encoding the meditope-enabled antibodies, and human constant regions. In some aspects, the heavy chain vectors and libraries containing the same, as well as methods constant region includes a CH1, or a portion thereof. In some 10 for their use, including selection and expression methods aspects, the light chain constant region includes a CL, or a and methods for generating transgenic animals using Such portion thereof. In some embodiments, the portion of the COnStructS. constant region is sufficient to render the antibody capable of D. Meditopes binding to the meditope, e.g., with the requisite binding Also provided are meditopes, including variant, modified, affinity. In some aspects, the constant regions are the con 15 and multivalent meditopes, that bind to meditope-enabled stant regions of cetuximab or trastuzumab. Thus, in some antibodies, and complexes and compositions containing the aspects, the heavy chain constant regions are one or more same, and methods and uses thereof. In certain embodi human IgG1 constant regions; in some aspects, the light ments, the meditopes include meditopes 1 and 2 (cCFD chain constant regions are kappa constant chain regions. In (SEQ ID NO: 1) and cqYN (SEQ ID NO: 2)), which were other examples, the constant regions can include those of originally identified as candidate peptides for binding to the other isotypes, including human (or other organism, e.g., CDR region of cetuximab, as described by Riemer, et al. murine or chicken) IgG1, IgG2, IgG3, IgG4, IgEs, IgA1, (2004); J Immunol 173, 394-401; Riemer, et al. (2005); J IgA2, Ig|D, or IgM, and can include kappa or lambda Natl Cancer Inst 97, 1663-1670. As demonstrated herein, constant regions. Thus, among the provided meditope-en the coFD and coYN meditopes bind to sites within cetux abled antibodies are those generated by mutation of residues 25 imab distinct from the cetuximab CDRs, and thus were not in other IgGs, such as human, murine, or chicken, or other likely candidates for specific cetuximab-like antibody immunoglobulins. In other words, the meditope-enabling immunogens for use as a vaccine. methods provided herein may be used for any antibody, Nucleic acids encoding the meditopes, including medi including IgA, IgE, Ig|D, and IgM and from any organism tope variants and multivalent meditopes, as well as vectors, that produces antibodies including but not limited to 30 cells, libraries, and other systems containing the same, also chicken, murine, rat, bovine, rabbit, primates, and goat. are provided. For example, the sequences of the first constant region I. Variant Meditopes (CH1) of a human IgG1, IgG2, IgG3, and IgG4, are com Among the provided meditopes are meditope variants pared in FIG.55A. FIG.55B shows the sequence differences (also called variant meditopes), having one or more modi in the IgG2-4 CH1, compared to the IgG1 CH1, mapped 35 fications, e.g., structural modifications, as compared to onto the co-crystal structure of cetuximab and the coFD meditope 1 or 2 (meditope of SEQ ID NO: 1 or 2), and meditope (meditope 1, SEQ ID NO: 1). As shown in the methods for producing the same. In some embodiments, Figure, the residues that are different among these isotypes cQFD and coYN meditopes are used as starting points in the are not within the meditope-binding region of cetuximab, design of meditope variants. In some aspects, the meditope confirming that the meditope-enabling technology is appli 40 variants are designed to have altered properties, such as cable to isotypes other than the IgG1 of cetuximab. As increased or altered affinity, altered pH dependence, or another example, the sequence and structural alignment of different affinities under different physiological conditions an IgG1 and an IgE Fab domain indicates residues on the for one or more of the provided meditope-enabled antibod IgE near the meditope binding site (FIG. 26). ies, including cetuximab and other antibodies described The provided methods for meditope-site grafting of the 45 herein, e.g., as compared to the unmodified meditopes, Fab cavity within monoclonal antibodies can be used to cQFD and coYN. Meditope variants are designed and create a unique handle for meditope binding and used with produced using various chemical and biophysical methods. the technology previously disclosed and for newly-gener Meditope variants include, but are not limited to, variants ated antibodies. In certain embodiments, the meditope bind incorporating modifications to meditopes, e.g., c0FD and ing site can be created on pre-existing and all future mono 50 cQYN and others described herein. Suitable modifications clonal antibodies. include, but are not limited to, any peptide modification As described below in section F, also provided are meth known in the art, such as, but not limited to, modifications ods for modifying the meditope-enabled antibodies, for to the manner and/or position of peptide cyclization, modi example, to alter various properties of the meditope-enabled fications to one or more amino acid components of the cyclic antibodies, including aspects of the interaction with the 55 peptide, or adding or deleting one or more amino acid from meditopes, including affinity, avidity, pH-dependence, as the cyclic peptide. In a particular example, c0FD may be well as other aspects, including pharmacokinetics (PK) and altered with one or more of the following modifications: a pharmacodynamics (PD) of the antibodies. Thus, also modification of Arg8, a modification of Phe3, a modification among the provided meditope-enabled antibodies are those of Leu5, a modification of Leu10, change to the mode of modified antibodies generated according to those methods, 60 peptide cyclization, and/or an incorporation of hydratable e.g., by generating a pharmacophore binding model, includ carbonyl functionality at one or more positions, and one or ing antibodies having any one or more of the modifications more amino acid deletions or additions. In the case of described in section F, below. cQYN, suitable modifications may include one or more of Also among the antibodies used as template antibodies to the following: a modification of Arg8, a modification of generate the meditope-enabled antibodies are modified anti 65 Leu5, a modification of Leu10, change to the mode of bodies and portions thereof, such as a CovX-BodyTM. Thus, peptide cyclization, and/or an incorporation of hydratable among the provided meditope-enabled antibodies are CovX carbonyl functionality at one or more positions, and one or US 9,669,108 B2 39 40 more deletions or additions. Certain amino acid positions For example, in some aspects, the meditope is one of within the meditope may be deleted or replaced with a meditopes 1, 2, or 15-55, further including a serine before different natural amino acid or an unnatural amino acid, or the first residues, i.e., at position Zero. The meditopes listed the meditope may be chemically conjugated with a frag in Table 3 employ a disulfide linkage to connect the C and ment. It is shown herein that a meditope in which Arg9 of 5 N termini (except that meditope 31 contained an additional SEQ ID NO: 1 has been mutated to citrulline binds to tail, meaning that the disulfide linkage is not between the cetuximab. In addition, the amino and carboxy termini can two terminal residues); for the peptides in Table 4, a lactam be extended with further amino acids beyond (i.e., in addi bridge, a linkage other than disulfide (such as 3+2] cycload tion to) the cyclic portion of the meditope variant in order to dition), or no linkage is used (e.g., an acyclic or linear make additional contact to the Fab. For example, Protein L. 10 variant). Additional meditopes that may be used according has been added to the C-terminus of the coFD meditope and to the embodiments described herein include any meditope preliminary data shows that this binds with much higher as defined herein peptide that binds to an antibody frame affinity. Such modifications are discussed further in work binding interface (i.e., between the Fab light and heavy Examples 6 and 7. chains) of cetuximab or any other therapeutic antibody. For In some embodiments, the meditopes include those listed 15 example, in addition to the cyclic peptides cGFD and coYN. in Tables 3 and 4, as well as such meditopes with additional some embodiments include one or more variants of c(RFD amino acids, such as those up to 16 amino acids in length. and cqYN. TABLE 3 SEQ ID Meditope NO Number Sequence Modification (red) Linkage method 1. 1. C-QFDLSTRRLK-C original Disulfide 1 - Cys: 12-Cys 2 2 C-QYNLSSRALK-C original Disulfide 1 - Cys: 12-Cys 15 15 C-dFDLSTRRLK-C q = D-glutamine Disulfide 1 - Cys: 12-Cys 16 16 C-QYDLSTRRLK-C Y = tyrosine Disulfide 1 - Cys: 12-Cys 17 17 C-QXDLSTRRLK-C X = B-B'-di-phenyl-Ala Disulfide 1 - Cys: 12-Cys 18 18 C-QFDXSTRRLK-C X = B-f'-di-phenyl-Ala Disulfide 1 - Cys: 12-Cys 19 19 C-QFDFSTRXLK- C F = phenylalanine, Disulfide 1 - Cys: 12-Cys X = citrulline 2O 2O C-QFDFSTRRLK- C F = phenylalanine Disulfide 1 - Cys: 12-Cys 21 21 C-QFDESTRRLK- C E = glutamic acid Disulfide 1 - Cys: 12-Cys 22 22 C-QFDYSTRRLK-C Y = tyrosine Disulfide 1 - Cys: 12-Cys 23 23 C-QFDLSTRRQK-C Q = glutamine Disulfide 1 - Cys: 12-Cys 24 24 C-QFDLSTRQLK-C Q = glutamine Disulfide 1 - Cys: 12-Cys 25 25 C-QYNLSTARLK-C Y = tyrosine; N = Disulfide 1 - Cys: 12-Cys asparagine; A = alanine 26 26 C-QADLSTRRLK-C A = alanine Disulfide 1 - Cys: 12-Cys 27 27 C-QFDASTRRLK-C A = alanine Disulfide 1 - Cys: 12-Cys 28 28 C-QFDLSTARLK-C A = alanine Disulfide 1 - Cys: 12-Cys 29 29 C-QFDLSTRRAK-C A = alanine Disulfide 1 - Cys: 12-Cys 3O 3O C-QFDLSTRREK- C E = glutamic acid Disulfide 1 - Cys: 12-Cys 31 31 Acc-QFDLSTRRLR- AcC-N-acetylcysteine Disulfide 1-ACCys: 12-Cys CGGGSK R = arginine

TABLE 4

SEQ ID NO Sequence Modification (red) Linkage method 32 32 G-QFDLSTRRLK-G G = glycine Lactam 1-Gly: 12- Gly 33 33 G-QHDLSTRRLK-G H = hlistidine Lactam 1-Gly: 12- Gly 34 34 G-QNDLSTRRLK-G N = asparagine Lactam 1-Gly: 12- Gly 35 35 G-QQDLSTRRLK-G Q = glutamine Lactam 1-Gly: 12- Gly