US 20120177568A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0177568 A1 WILLIAMS et al. (43) Pub. Date: Jul. 12, 2012

(54) NOVEL MEDITOPES AND RELATED C07K 7/06 (2006.01) MEDITOPE- A6IP37/02 (2006.01) DELIVERY SYSTEMS, SYNTHESIS AND A6IP39/04 (2006.01) THERAPEUTIC USES THEREOF C07K 4/00 (2006.01) C07K L/4 (2006.01) (76) Inventors: John C. WILLIAMS, Monrovia, B82Y5/00 (2011.01) CA (US); David A. Horne, Duarte, CA (US); Yuelong Ma, Duarte, CA (52) U.S. C. ... 424/1.49; 530/300: 530/327: 530/387.3; (US); Heng Wei Chen, Foster City, 530/408; 530/322; 435/188: 530/387.1:530/387.9; CA (US) 424/133.1; 424/9.1; 424/9.6; 435/375; 530/326; 977/773; 977/915;977/927 (21) Appl. No.: 13/270,207 (57) ABSTRACT (22) Filed: Oct. 10, 2011 Meditope variants and methods for their use are provided Related U.S. Application Data herein. A meditope variant as described herein comprises a peptide having a sequence CQFDLSTRRLKC (SEQ ID (60) Provisional application No. 61/391,558, filed on Oct. NO: 1) or CQYNLSSRALKC (SEQID NO:2) that has one or 8, 2010. more modifications at of least one amino acid residue of the sequence. Multivalent meditope variant tethering entities are Publication Classification also provided. Such entities may include two or more medi (51) Int. Cl. topes coupled via a long linker, multivalent scaffold, biotin A6 IK 5L/It (2006.01) streptavidin, or IgGFc domain. Further, methods of treating, C07K 7/08 (2006.01) imaging or diagnosing a disease or condition are provided. C07K 9/00 (2006.01) Such methods may include administering a therapeutically CI2N 9/96 (2006.01) effective amount of a pharmaceutical composition to a Sub C07K I6/44 (2006.01) ject, the pharmaceutical compound comprising an antibody A 6LX 39/395 (2006.01) meditope complex; a multivalent tethering agent in combina A6R 49/00 (2006.01) tion with a monoclonal antibody or functional fragment CI2N 5/09 (2010.01) thereof, or a combination thereof. Patent Application Publication Jul. 12, 2012 Sheet 1 of 44 US 2012/0177568 A1

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Figure 31 SEC ID NO: 515) 616) 717 818) Data Collection Space group P222 P222 P222 P222 Cell dimensions a, b, c (A) 64.33, 82.84, 64.20, 82.50, 64.02, 82.83, 64.28, 83.25, 212.32 212.49 212.10 212.30 o, 3, Y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (A)8 30.00-2.24 (2.45 32.99-2.53 34.36-2.48 (2.54- 33.19-248 2.24) (2.60-2.53) 2.48) (2.54-2.48) 0.070 (0.281) 0.066 (0.334) 0.065 (0.312) 0.072 (0.331) I/o(I) 1905 (5.92) 24.74 (5.21) 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) 6.19 (4.27) 4.61 (3.16) Refinement Resolution (A) 2.24 2.53 2.48 2.48 NO. reflections 55,026 37,650 40,641 41,098 Rwork/Rfree 18.0/22.6 17.2/23.6 17.6/22.O 17.4/22.9 NO. atoms Protein 6526 6514 6551 6541 Meditope 174 291 (198)" 212 218 Water 507 392 511 469 B-factors Protein Fab 38.16 27.79 25.74 25.23 Meditope 49.08 43.40 (27.51)* 31.78 31.52 Water 41.78 28.05 28.32 29.65 r.m.S.C. Bond lengths (A) OOO7 O.OO7 OOO8 Bond angles () 1093 1091 1106 Ramachandran favored/allowed/disallowed 96.9/3.1/O.O 96.8/3.2/O.O 97.6/2.4/O.O 97.2/2.7/0.1

*3 meditopes in asymmetric unit. Data in parenthesis refer to the meditopes in the meditope binding pocket characteristic for the WT meditope Patent Application Publication Jul. 12, 2012 Sheet 37 of 44 US 2012/0177568 A1

Figure 31 (cont.) SEQID NO: 1222 1323 1424) 1525) Data Collection Space group P2212 P222 P2212 P222 Cell dimensions 64.24, 83.14, 64.30,83.39, 64.46, 83.21, 64.10, 83.16, a, b, c (A) 21194 212.73 212.44 212.32 o, 3, Y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0

s 33.15-2.48 33.23-2.49 34.96-2.62 33.14-2.55 Resolution (A) (2.55-2.48) (2.55-2.49) (2.69-2.62) (2.62-2.55) Rmrgd-F 0.044 (0.153) 0.056 (0.219) 0.125 (0.470) 0.094 (0.374) I/o(I) 25.24 (9.23) 19.83 (6.03) 15.21 (3.87) 19.13 (4.60) Completeness (%) 95.1 (72.3) 99.6 (95.4) 98.6 (97.7) 99.4 (94.1) Redundancy 5.2 (4.6) 5.0 (3.66) 4.95 (5.05) 4.33 (3.96) Refinement Resolution (A) 2.48 2.49 2.62 2.55 NO. reflections 39,080 40,816 34,738 377O1 Rwork/Rfree 17.9/22.6 17.1/22.3 184,235 178/225 NO. atOmS Protein 6554 6525 6529 6517 Meditope 208 198 174 190 Water 493 511 38O 4O4 B-factors Protein Fab 24.08 24.69 25.47 25.98 Meditope 27.64 30.40 26.45 26.64 Water 29.37 29.15 28.75 28O8 r.m.S.C. Bond lengths (A) O.OO7 O.OO2 O.OO8 Bond angles () 11 O3 O618 1122 Ramachandran faVOredfallOWed/disallowed 96.9/31/O.O 97.1/2.9/O.O 96.3/3.7/O.O 96.5/34/O. Patent Application Publication Jul. 12, 2012 Sheet 38 of 44 US 2012/0177568 A1

Figure 31 (cont.) SECR D NO: 2131) 2232) 2333) 2434) Data Collection Space group P222 P222 P222 P222 Cell dimensions 64.28, 82.64, 64.16, 82.46, 64.18, 82.66, 63.97, 82.50, a, b, c (A) 212.5O 212.05 211.91 21,188 o, B, Y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (A) 30.00-255 32.51-2.49 33.02-2.50 32.95-2.50 (2.62-2.55) (2.55-2.49) (2.56-2.50) (2.62-2.55) Rmrgd-F 0.085 (0.365) 0.039 (0.163) 0.047 (0.177) 0.094 (0.405) I/O (I) 20.27 (4.54) 31.38 (9.58) 30.78 (9.25) 17.76 (3.68) Completeness (%) 96.7 (91.5) 99.6 (96.1) 99.8 (99.0) 99.0 (90.1) Redundancy 5.1 (5.1) 5.01 (3.61) 6.02 (4.70) 4.00 (3.70) Refinement Resolution (A) 2.55 2.49 2.50 2.55 NO. reflections 36,560 40,266 39,848 37,028 Rwork/Rfree 18.7/22.7 16.9/22.O 16.9/22.5 16.9f21.4 NO. atoms Protein 6545 6509 65O7 6555 Meditope 188 174 182 190 Water 332 471 503 285 B-factors Protein Fab 27.94 25.7O 21.62 32.02 Meditope 2973 53.33 4O13 38.42 Water 29.40 31.55 27.78 33.79 r.m.S.C. Bond lengths (A) O.OO2 0.004 Bond angles () O631 O.876 Ramachandran favored/allOWed/disallowed 96.2/3.8/O.O 97.2/2.8/O.O 97.3/2.7/O.O 96.4/3.6/O.O Patent Application Publication Jul. 12, 2012 Sheet 39 of 44 US 2012/0177568 A1

Figure 31 (cont.) SEC ID NO: 2535) 2636) 2737) 2838) Data Collection Space group P222 P222 P222 P222 Cell dimensions 64.01, 82.21, 64.04, 82.51, 64.05, 83.16, 64. 14, 83.19, a, b, c (A) 211.90 21154 212.26 212.46 o, B, Y. () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (A) 3414-2.51 34.22-2.48 33.13-2.50 33.16-2.48 (2.58-2.51) (2.55-2.48) (2.56-2.50) (2.55-2.48) Rmrgd-F O.046 (0.238) 0.039 (0.144) O.084 (0.375) O.054 (0.186) I/o(I) 25.22 (6.50) 30.13 (8.86) 20.51 (4.31) 29.15 (8.34) Completeness (%) 98.5 (92.3) 99.4 (92.5) 99.2 (92.2) 99.3 (93.2) Redundancy 4.12 (3.52) 5.77 (4.14) 6.26 (4.94) 5.79 (4.10) Refinement Resolution (A) 2.51 2.48 2.50 2.48 NO reflections 38,565 40,290 39,826 40,872 Rwork/Rree 19.023.9 17.9723.5 19.1/240 181/23.3 NO. atoms Protein 6528 6535 6524 6539 Meditope 218 187 190 192 Water 338 454 361 485 B-factors Protein Fab 34.24 26.67 26.19 1915 Meditope 47.05 48.78 33.05 26.02 Water 32.96 31.14 28.90 24.13 r.m.S.C. Bond lengths (A) O.OO8 0.004 Bond angles () 1.12O O.822 Ramachandran favored allowedfodisallowed 96.4/35/O. 96.6/34/O.O 96.8/3.1/O. Patent Application Publication Jul. 12, 2012 Sheet 40 of 44 US 2012/0177568 A1

Figure 31 (cont.) SEC ID NO: 2939) 3040 Data Collection Space group P2212 P222 Cell dimensions a, b, C(A)8 64.21, 83.04, 212.26 64.34, 82.57, 212.05 O, 3, Y () 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (A)8 34.40-2.48 (2.54-2.48) 34.29-2.48 (2.54-2.48) Rmrgd-F 0.051 (0.184) 0.035 (0.147) I/O (1) 30.80 (7.93) 38.15 (11.37) Completeness (%) 99.4 (93.6) 98.2 (91.1) Redundancy 5.40 (3.86) 6.17 (4.25) Refinement Resolution (A) 2.48 2.48 NO. reflections 40,869 40,195 Rwork/Rree 17.8/22.4 18.6/23.8 NO. atOms Protein 6499 6539 Meditope 192 192 Water 466 475 B-factOrS Protein Fab 2004 20.24 Meditope 26.61 27.05 Water 24.30 24.57 r.m.S.C. Bond lengths (A) OOO7 O.OO7 Bond angles () 1.O71 1.125 RamaChandran favored/allowed/disallowed 97.0/2.9/O1 96.9/3. O/O.1 Patent Application Publication Jul. 12, 2012 Sheet 41 of 44 US 2012/0177568 A1

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NOVELMEDITOPES AND RELATED ing the sequence CQFDLSTRRLKC (SEQ ID NO: 1) or MEDITOPE-MONOCLONAL ANTIBODY CQYNLSSRALKC (SEQID NO:2). DELIVERY SYSTEMS, SYNTHESIS AND 0005 Characterization of the meditopes by surface plas THERAPEUTIC USES THEREOF mon resonance indicated that cOFD and coYN bind to Fab with dissociation constants of between approximately 0.70 PRIORITY CLAIM 0.95 and 2-5 uM, respectively. Point mutations designed to disrupt specific interactions further support the structural 0001. This application claims the benefit of U.S. Provi model. FACS analysis revealed that high concentrations of sional Application Ser. No. 61/391,558 filed Oct. 8, 2010, the coFD meditope (60 uM) produced negligible differences which is incorporated herein by reference in its entirety. in the ability of to bind to MDA-MB-468 cells, which over-express the EGFR receptor. Thus, meditope bind BACKGROUND ing does not affectantigenbinding. In addition, the meditopes were conjugated with a fluorescent group and shown to bind 0002 Cancers avoid immune surveillance by actively sup to MDA-MB-468 cells pretreated with cetuximab, but not to pressing the immune system. One method envisioned for MDA-MB-468 cells pretreated with the murine anti-EGFR counteracting this immunosuppression is through vaccina antibody, M425 (FIG. 42). Collectively, these data define a tion using epitopes of antigens that are either uniquely meditope binding interface within the Fab framework and expressed or over-expressed by the tumor cells. For example, demonstrate that the meditope binding does not inhibit anti monoclonal antibodies (mAbs) that block signaling path gen binding (e.g., does not act as an allosteric regulator). ways, sequester growth factor and/or induce an immune 0006. In another embodiment, meditopes (e.g., coYN or response have been Successfully implemented in the clinic to cQFD) or variants thereofhaving the novel binding properties treat cancer and other diseases. Due to their favorable prop described above may be used to add functionality to thera erties and clinical Success, mAbs have been and continue to peutic monoclonal antibodies (“mabs'). In a further embodi be the subject of intense protein engineering efforts. These ment, the meditopes described herein may be exploited to efforts have yielded bispecific mAbs for improved targeting: affect the efficacy of therapeutic Substances Such as mono single chain Fab variable fragments (ScPVs), diabodies, and clonal antibodies and functional fragments thereof or other minibodies for better tumor penetration and blood clearance: Substances such as Small molecules. In yet another embodi and modified Fcs (through mutation or glycosylation) to alter ment, tags or detectable labels may be attached to the medi immunostimulation or improve pharmacokinetic/pharmaco topes for identification of certain types of cells or tissues for dynamic properties. Likewise, mAbs have been reengineered diagnostic or therapeutic use. to permit the site-specific conjugation of small molecules for 0007. In a separate embodiment, a meditope contains a improved delivery (e.g., ThioMABs) or to irreversibly bind to cysteine that covalently binds to a cysteine in the Fab at the their antigen (e.g., infinite affinity mAbs). MAbs have also meditope binding site. The meditope is conjugated to any been developed to improve the circulation and presentation of bioactive peptides and other biologics (e.g., CovXbodies). Substance, molecule or compound, which may be therapeutic Hetero-multimeric scEvs or scEvs or mAbs fused to avidin molecule. Such as a small molecule diagnostic molecule. Such have also been developed for pre-targeted therapy and to as a marker. The “Cys meditope' directs the conjugate to the improve the detection limits for tumor imaging. IgG and binds via a covalent linkage. 0003. Although mAbs can be effective and have some BRIEF DESCRIPTION OF THE DRAWINGS advantages over Small molecule approaches, limitations such as adverse side effects due to off-target interactions or collat 0008 FIG. 1 shows meditope peptides binding framework eral damage due to long circulation times of radionuclide loops of cetuximab. conjugated mAbs indicates that there remains considerable 0009 FIG. 1A: The complex of cetuximab Fab (light room to improve their efficacy, including improved targeting chain denoted by V, and C, heavy chain denoted by V and and Synergy. Therefore, enhancement of antibody and Small C) and cyclic CQFDLSTRRLKC (depicted within the molecule therapeutic efficacy would be useful and desired in shaded area and labeled with the word “meditope') (SEQID the treatment of cancer and other diseases. NO: 1) indicates that the meditope binds to an interface of the Fab framework, which is distinct from the CDR loops of SUMMARY cetuximab. FIG. 1B shows the stick representation of the cQFD meditope and FIG. 1C shows the stick representation 0004 Antibody binding peptides C-QFDLSTRRLK-C of the coYN meditope. The N- and C-terminal cysteines are (cQFD; SEQ ID NO: 1) and C-QYNLSSRALK-C (cGYN: Solvent exposed and display high thermal factors. SEQID NO:2) were shown to have novel mab binding prop 0010 FIG.2 shows certain embodiments of the cetuximab erties. Specifically, coFD and coYN, which are also called Fab binding interface. Cetuximab is a murine chimera and “meditopes were shown to bind to a region of the Fab therefore has a mix of murine Ig variable domains and human framework of the anti-EGFR mAb cetuximab and not to bind constant Ig domains. FIG. 2A shows the corresponding resi the complementarity determining regions (CDRs) that bind dues for the phage negative selection, ch14.18, and the antigen. The binding region on the Fab framework is distinct humanized that make contact with the meditope. from other framework-binding antigens, such as the Superan FIG. 2B, top panel, shows a stereoView of Arg9 of the coFD tigens Staphylococcal protein A (SpA) (Graille et al., 2000) meditope that occupies a distinct pocket encoded by the and Peptostreptococcus magnus protein L (PpI) (Graille et murine sequence of Cetuximab (foreground). Trastuzumab al., 2001), and was previously unknown. Accordingly, a first Fab (1 N87.pdb; background) (Cho et al., 2003) is superim embodiment is a framework binding interface comprising a posed. There is a salt bridge from Asp85 to the guanidinium framework region of a unique murine-human antibody or group of the meditope Arg9 and the backbone amide of functional fragment thereofthat binds a cyclic meditope hav Leu10. FIG. 2B, bottom panel, graphically represents the US 2012/0177568 A1 Jul. 12, 2012

Superposition of the Fab fragments indicating Subtle effects bodies (left panel) and blocks receptor signaling, alters due the F to Y in the meditope. Specifically, the hydrophobic endocytosis and receptor recycling and/or elicits an immune groups, F/Y3 L5, and L10 are nearly identically positioned, response. The addition of a multivalent meditope tethers/ but the hydroxyl group of Y5 in the coYN meditope prevents cross-links the therapeutic mAb and can enhance its thera R8 from interacting with the Q111 backbone, as observed in peutic potential (right). The right panel depicts the daisy the coFD meditope (“steric clash”).This rearrangement also chain linkage expected from a trivalent meditope. results in a concomitant change in the backbone residues of the B-turn (backbone rotation'). (0017 FIG. 9 shows scv-meditope linker. scFvs are cre 0011 FIG. 3 is two line graphs showing surface plasmon ated by fusing the light chain variable domain to the heavy resonance (SPR) traces of coFD and cqYN and the Fab chain variable domain (or vice versa) through a 12-20 amino fragment. The cetuximab Fab used for crystallographic stud acid linker (typically GGGSs). ies was coupled to a CM5 chip at low densities. Traces at 0018 FIG. 10 is a gel showing that the meditope selec increasing concentrations of the coFD (FIG. 3A) and coYN tively binds the murine cetuximab. Biotinylated coFD pep (FIG.3B) meditopes are shown. The series of traces are fit to tide was added to avidin coupled beads, thoroughly washed, single binding site model using a simple exponential (e.g., and equilibrated in PBS. Cetuximab was then added to the signal=A*expkon*time). The residuals of the fit are shown beads (lane 1), washed (lanes 2-4), and then eluted (lane 5). below each. The top band is the IgG heavy chain and bottom band is the 0012 FIG. 4 illustrates that the present meditopes do not IgG light chain. bind to the CDRs as was previously hypothesized. FIG. 4A 0019 FIG. 11 is a depiction of the steric mask. The medi demonstrates a 2.0 A crystal structure, wherein the meditope tope can be fused to the N-terminus of the Fab light or heavy binds to middle hole of the cetuximab Fab. The antigen, chain through a flexible linker containing a tumor associated EGFR domain III, binds at the complementarity determining protease site. An intramolecular interaction, as shown, would regions at a significant distance from the meditope binding sterically occlude the antigen binding site. site. FIG. 4B shows gel results on the left-hand side and related size exclusion chromatography results on the right 0020 FIG. 12 shows the off-rate determination for Avidin hand side. Size exclusion experiments of the individual com peptide mask on Cetuximab. The left panel shows that the ponents, Fab, EGFR domain III, and SMT-cOFD meditope, binding kinetics of a biotinylated coFD meditope was mea as well as an admixture of all three, indicate the formation of sured by SPR using immobilized cetuximab IgG. The boxed a heterotrimeric complex and coelute. The non-reducing panel above the Surface plasmon resonance traces depicts a SDS-PAGE gel shows the fraction that eluted first, indicating cartoon of the monovalent meditopes being passed over the the presence of all three components within the new peak (the bivalent IgG. As shown in the right panel, Avidin was satu left-most peak for “complex,” shaded light gray) observed rated with biotinylated meditope and passed over the same from the admixture. FIG. 4C shows the results of a FACS immobilized cetuximab IgG. The off-rate of the multivalent analysis, indicating that the meditope binds to EGFR positive meditope was reduced by at least 36 fold. (Note the time MD-MBA-468 cells only in the presence of cetuximab (ar scales between the two experiments are different.) The boxed rows). The meditope alone or the meditope in the presence of panel above the SPR traces depicts a “bivalent-bivalent inter M425, a murine EGFR antibody, does not bind. FIGS. 4D and action” between the tetravalent meditope and the bivalent 4E show results of Surface plasmon resonance experiments IgG. using a sensor chip coupled with a cetuximab Schv. The 0021 FIG. 13 shows biophysical data obtained for the experiments indicate that saturation of the schv could not be meditopes. FIG. 13A shows a SPR sensogram of the unmodi achieved at concentrations as high as 100 uM of the coFD fied meditope cQFD (SEQID NO:1) and the QYN meditope meditope. The same experiments using the cetuximab Fab (SEQID NO:2) using acetuximab Fab chip. FIG. 13B shows coupled sensor chip indicate full saturation. The dissociation a representative binding isotherm of the meditope (SEQ ID constant from this experiment is 660 nM. Control SPR NO: 1) and Fab (top) and integration (bottom). FIG. 13C experiments show that the cetuximab scFv readily binds shows superposition of the cetuximab Fabs bound to the soluble EGFR domain III fragment, indicating that the CDR unmodified meditope and Phe3Tyr. Oval 1 indicates the Phe/ loops are functional. Tyr position. Oval 2 shows shift in the Arg8 side chain. The 0013 FIG.5 is a graph of fluorescence intensity compared arrow indicates a shift of the backbone that leads to a favor to cell count and shows fluorescently-labeled cetuximab able hydrogen bond network and may account for a favorable binding to MDA-MB468 cells in the presence of c(RFD. change in the enthalpy. FIG. 13D shows individual thermo Cetuximab binds to MDA-MB468 cells without peptide, and dynamic parameters determined by ITC of different meditope in the presence of 6 uM and 60 uM peptide as well as the variants. The Phe/Tyr variant (V2) shows a significant change isotype control. in AH though lower affinity. Based on the structure, GIn2 in 0014 FIG. 6 shows how meditope and EGFR bind to the meditope was replaced with the D-stereomer. ITC analy distinct sites. The top images show Superposition of the sis of this meditope, (SEQID NO:15), revealed a significant cQFD-Fab complex and the EGFR-Fab complex (1YY8) in increase in entropy and loss in enthalpy. stereo. The bottom images show that residues 39-46 of the 0022 FIG. 14 shows the chemical structure of the medi heavy chain are flexible and accommodate the meditope. tope. The circles indicate positions that will be modified to 0015 FIG.7 shows Fab frameworkbinders. Superposition improve the meditope affinity for the Fab. The boxes indicate of Fabs bound to meditope, Protein A, and Protein L indicate cyclization strategies. Click chemistry and olefin metathe that each binds to a unique site on the Fab. sis, middle and right boxes, respectively, are additional routes 0016 FIG. 8 illustrates one mechanism of action for to cyclize the meditope. enhancing tumor therapy. Overexpression of antigen (e.g., (0023 FIG. 15 illustrates the synthesis of lactam (2, SEQ ErbB receptor family) on tumor cells binds to bivalent anti ID NO:31) and fluorescein-labeled lactam (3). US 2012/0177568 A1 Jul. 12, 2012

0024 FIG.16 shows the sites of optimization according to the antigen are labeled with distinct chromophores). The GPI Some embodiments. Ovals indicate cavities that may opti linker ensures the selected mAb remains associated with the mize affinity of the meditope. cell encoding the gene sequence. Sequencing the genes 0025 FIG. 17 shows a modified Arg8 residue that may be encoding the light can heavy chain from the selected cells will used to optimize the meditope. identify high affinity mAbs. The method can be repeated to 0026 FIG. 18 shows the hydrophilic interface in the vicin selected for higher affinity meditope or meditope analogs. ity of Phe3. Halogens incorporated in the phenyl ring may 0037 FIG. 29 shows 3D surface representations of form strong noncovalent bonds to the hydroxyl group of sequence differences. The dark grey regions in the top panel TyrS7 or Tyr94, as well as backbone carbonyls. indicate amino acid differences between cetuximab and the 0027 FIG. 19 illustrates covalent interactions between the fully human trastuzumab framework. Residues inside the box meditope and the backbone framework. A hydratable side have been mutated onto the trastuzumab framework. The chain may be incorporated at Arg8 (left) or Leu10 (right) to CDR loops of trastuzumab have been identified (dark regions: form a covalent bond to the hydroxyl groups of Seror Tyr of bottom panel) and grafted onto the cetuximab framework. the Fab. 0038 FIG.30 shows stick structures foreighteen modified 0028 FIG. 20 illustrates synthesis of dimeric and trimeric meditopes, corresponding to SEQ ID NO:15 (A), SEQ ID meditopes according to Some embodiments. NO:16 (B), SEQID NO:17 (C), SEQID NO:18 (D), SEQID 0029 FIG. 21 illustrates the characterization of a fluores NO:22 (E), SEQID NO:23 (F), SEQID NO:24 (G), SEQID cein isothiocyanate (FITC)-labeled meditope dimer 7. The NO:25 (H), SEQID NO:31 (I), SEQID NO:32 (J), SEQID left panel shows an HPLC trace offinal bivalent meditope and NO:33 (K), SEQID NO:34 (L), SEQID NO:35 (M), SEQID the right panel shows its mass spectrum. NO:36 (N), SEQID NO:37 (O), SEQID NO:38 (P), SEQID 0030 FIG. 22 shows the nucleic acid sequence (SEQ ID NO:39 (Q) and SEQ ID NO:40 (R). The sequence of these NO:3) and the corresponding amino acid sequence (SEQID structures is shown in Tables 1 and 2. NO:4) for a meditope-Fc tethering agent according to some 0039 FIG.31 is a table showing the X-ray diffraction data embodiments. for the structures shown in FIG. 39. 0031 FIG. 23 shows the structure of a bivalent meditope. 0040 FIG. 32 illustrates the results of an MTT assay com The meditope is directly fused to the N-terminus of a peptide paring the efficacy of a bivalent meditope-Fc to that of a linker that is directly fused to the Fc Region of an IgG. The Fc monomeric meditope. FIG. 41A shows that only the medi is naturally homodimeric. Thus the Meditope-Fc construct is tope-Fc, and not the monomeric meditope, can inhibit cell bivalent. growth when combined with cetuximab. FIG. 41B shows that 0032 FIG. 24 shows the results from an exemplary FACS the meditope-Fc enhances the cell-killing capacity of cetux analysis. MDA-MB-468 cells that over express EGFR recep imab. tor were pre-treated with 10 nM of cetuximab for 30 minutes, 0041 FIG.33 illustrates that the meditope binds to unique rinsed, and then treated with either the meditope-Fc construct cetuximab framework, but not to human framework or other or the monomeric meditope at four concentrations. The bot murine-chimera frameworks. The wildtype meditope was tom trace is a negative control (no antibody). Next four traces conjugated to a CM5 chip for Surface plasmon resonance show that a monomeric meditope binds to cells pre-treated studies and cetuximab (meditope binding Fab), trastuzumab with the cetuximab in a concentration dependent manner. The (fully human framework), and (murine-human chi top four traces also show that the bivalent, meditope Fc binds meric framework) were tested concentrations 0.01, 0.05, 0.1, to cells pre-treated with the cetuximab in a concentration 0.5 and 1 uM. Only cetuximab bound to the meditope-conju dependent manner, but with higher affinity (e.g. shifted to the gated chip. Superposition of the molecular structure of tras right). This is predicted and consistent with a multivalent tuzumab (1 N87: Cho et al. 2003) and rituxumab (2OSL; Du interaction. et al. 2007) Fabs on to the meditope bound cetuximab Fab 0033 FIG. 25 is an alternative linker, including a coiled structure further highlights the uniqueness of the framework. coil, which may be used accordance with certain embodi Moreover, this structureal comparison gives a clear indication mentS. of which residues originating from the cetuximab Fab con 0034 FIG. 26 illustrates a fluorescence polarization assay tribute to meditope binding and how they can be grafted onto of dose-dependent inhibition of cetuximab interaction with other Fab framework regions. fluorescein-labeled peptide by unlabeled peptide. The assay 0042 FIG. 34 shows representative surface plasmon reso identified novel Small molecule compounds that can compete nance studies of meditope variants. Top trace—Based on the with the meditope form Ab binding and thus can be developed structural information of the Phe3His mutation in the medi to function in a similar manner as a meditope. As an important tope, a new meditope was synthesized with 3,3'-biphenylala control, we demonstrate here that a non-labeled meditope can nine at position 3. A significant improvement in the binding displace fluorescently-labeled meditope. Equilibration of the affinity for cetuximab was observed, ~4 fold, as observed by displacement at three times points indicates that the assay is Surface plasmon resonance. Bottom trace-aminohexanoic robust and amendable to high throughput screening. acid was used to replace the disulfide bridge of the original 0035 FIG. 27 shows five lead compounds identified from meditope. While the binding affinity was decreased, these the fluorescent polarization screen. data indicate that modifications can be made to the meditope 0036 FIG. 28 illustrates steps to alter and/or enhance the to address potential issues with pharmacokinetics, pharma binding affinity of a mAb for a meditope or compound bind codynamics and toxicity in animal and human studies. It is ing at the meditope site using a directed random library. noted that this combination can be combined with unnatural Specifically, a gene library where codons for mAb residues amino acids at other positions within the meditope. that line the meditope binding site are replaced with NNK 0043 FIG.35 illustrates structural information to improve (where N is any nucleotide and K is a Thymine or Guanosine) meditope affinity. The top panel shows crystal structure of a can be selected using FACs sorting (where the meditope and meditope bound cetuximab. Phenylalanine 3 binds within the US 2012/0177568 A1 Jul. 12, 2012

Fab cavity. Substitution of this position with histidine and (iv) amine reactive by incorporating p-isopropylthiocarbon Subsequent structural analysis indicates the side chain (imi ryl-phenylalanine or p-ethylthiocarbonyl-phenylalanine, (v) dazole) takes on a new conformation (middle panel). Based azide alkyne Huisgen cycloaddition by incorporating p-pro on this observation, 3,3'-biphenylalanine which could place pargyloxyphenylalanine or p-azidophenylalanine or any one phenyl side chain at the original position and one phenyl other Suitable unnatural amino acid known in the art. In one side chain at the imidazole ring was substituted at position 3. embodiment, the meditope may direct a reactive group to an The crystal structure indicates that this substitution mimics unnatural amino acid incorporated into the Fab. Suchs as both side chain conformations. Surface plasmon resonance p-acetylphenylalanine. studies shows that this substitute binds with higher affinity (FIG. 43 top panel). 0047. In addition, the meditope framework binding inter face can be grafted onto other mAbs to generate a meditope DETAILED DESCRIPTION enabled mAb. Because the meditope binding site can be grafted onto other mAbs, the binding interface represents a 0044 An antibody delivery system that selectively deliv broadly useful platform technology that may be used not just ers therapeutic or imaging agents to a target tissue, and meth ods for their use are provided herein. The antibody delivery with cetuximab for EGFR targeted conditions, but with any system may include an antibody framework binding interface monoclonal antibody. The term “meditope-enabled' anti (“framework binding interface' or “binding interface') body, monoclonal antibody or therapeutic antibody refers to bound to an antibody binding molecule (or “meditope') to any antibody that is able to bind a meditope at its framework forman antibody-meditope complex. The antibody-meditope binding interface, including cetuximab. Thus, the platform complex may be further conjugated to one or more additional may be expanded for use in treatment, diagnosis or imaging antibody-meditope complexes, therapeutic agents, imaging of any cancer, disease or other condition that may be treated agents or a combination thereof for use in methods described or targeted using a therapeutic antibody, including, but not further below. limited to, leukemia and lymphomas (which can be treated or 0045. According to some embodiments, an antibody imaged using, e.g., , bectumumab, gemtu framework binding interface that can bind a meditope is Zumab, FBTA05, ibritumomab tiu Zetan, , ritux formed by a framework region, not by a complementarity imab, to situmomab), (which can be treated or determining region (CDR), of an antibody or functional frag imaged using, e.g., trastuzumab, adecatumumab, etrumaX ment thereof. An “antibody or functional fragment thereof as omab) (which can be treated or imaged using, used herein refers to an immunoglobulin molecule that spe e.g., adecatumumab, capiromab pendetide, ), cifically binds to, or is immunologically reactive with a par colorectal cancer (which can be treated or imaged using, e.g., ticular antigen or epitope, and includes both polyclonal and , , altumumab pentetate, Votu monoclonal antibodies. The term “antibody' includes geneti mumab), gastrointestinal cancers (which can be treated or cally engineered or otherwise modified forms of immunoglo imaged using, e.g., arcitumumab, ), ovarian bulins, such as intrabodies, peptibodies, chimeric antibodies, cancer (which can be treated or imaged using, e.g., abago fully human antibodies, humanized antibodies, meditope Vomab, catumaXomab, etaracizumab, igovomab, orego enabled antibodies and heteroconjugate antibodies (e.g., Vomab), lung cancer (which can be treated or imaged using, bispecific antibodies, diabodies, triabodies, tetrabodies, tan e.g., anatumumab mafenatox), pancreatic cancer (which can dem di-scFv, tandem tri-sch v). The term “functional antibody be treated or imaged using, e.g., clivatuZumab tetraxetan), fragment includes antigen binding fragments of antibodies renal cancer (which can be treated or imaged using, e.g., including, but not limited to, fragment antigen binding (Fab) ), cancer (which can be treated or fragment, F(ab') fragments, Fab' fragments, FV fragments, imaged using, etaracizumab, , TRBS07), glioma recombinant IgG (rlgG) fragments, single chain variable (which can be treated or imaged using, e.g., ), fragments (ScPV) and single domain antibodies (e.g., SaAb. bone metastases (which can be treated or imaged using, e.g., sdFv, nanobody) fragments. denosumab), head and neck cancer (which can be treated or 0046 According to some embodiments, the binding inter imaged using, e.g., ), cardiovascular disease face may be exploited or optimized to enhance binding and (which can be treated or imaged using, e.g., abciximab), imaging of antibodies or functional fragments thereof. In a autoimmune disorders (which can be treated or imaged using, separate embodiment, a meditope may contain a cysteine e.g., adalimumab, infliximab), rheumatoid arthritis (which residue that binds to an engineered cysteine in the Fab at the can be treated or imaged using, e.g., atlizumab, golimumab, meditope binding site (e.g., ThioMAbs). The meditope is infliximab), transplant rejection (which can be treated or thereby conjugated to any diagnostic and/or therapeutic Sub imaged using, e.g., basiliximab, daclizumab, muromonab stance, molecule or compound. For example, the Substance CD3), Crohn's disease (which can be treated or imaged using, may be a small molecule diagnostic molecule, such as a e.g., certolizumab, fontolizumab, natalizumab, infliximab, marker. The “Cys meditope' directs the conjugate to the IgG visilizumab), hemoglobinuria (which can be treated or and binds via a covalent linkage. Alternatively, the meditope imaged using, eculizumab), psoriasis (which can be treated or may be conjugated to the Fab to one or more unnatural amino imaged using, e.g., efalizumab, infliximab, ustekinumab), acids that are incorporated into the meditope binding site. multiple Sclerosis (which can be treated or imaged using, e.g., Examples of linkages that may be made with unnatural amino natalizumab, ustekinumab), asthma (which can be treated or acids include (i) stable hydraZone and oxime linkages by imaged using, e.g., benralizumab, mepolizumab, omali incorporation of p-acetylphenylalanine, m-acetylphenylala Zumab), respiratory syncytial virus (RSV) (which can be nine, p-(3-oxobutaboyl)-1-phenylalanin, and p-(2-amino-3- treated or imaged using, e.g., palivizumab), macular degen hydroxyethyl)phenylalanine, (ii) thiol reactive by incorporat eration (which can be treated or imaged using, e.g., ranibi ing phenylselenidylalanine, (iii) a UV crosslinker containing Zumab), appendicitis (which can be treated or imaged using, benzophenone by incorporating p-benzoyl-1-phenylalanine, e.g., fanolesomab) and any other condition that may be tar US 2012/0177568 A1 Jul. 12, 2012

geted or treated with an antibody. The above-listed antibodies chimera Fab and the selected meditope. In contrast, PpI and and related diseases or disorders are examples only and do not SpA are not murine-specific. PpI binds to ~66% of murine limit the platform. and ~50% of human IgG Kappa light chains and SpA binds 0048. In one embodiment, cyclic antibody binding pep 12% of murine and 50% of human variable heavy chains tides C-QFDLSTRRLK-C (cGFD; SEQ ID NO: 1) and (Graille et al., 2002). C-QYNLSSRALK-C (cGYN: SEQ ID NO:2) can bind the 0.052 Cetuximab is currently used for the treatment of antibody framework binding interface via a non-covalent EGFR-expressing metastatic colorectal cancer and head and binding interaction. This non-covalent nature of the binding neck cancers. Cetuximab recognizes a conformational interaction opens new alternative methods for modifying the epitope (e.g., folded EGFR domain III). A comparison of framework binding interface, the peptide, or both, expanding antibodies that recognize Small molecule antigens, peptides the possibilities for generating highly specific meditope Vari and protein epitopes show that the interfaces tend to possess ants. In one embodiment, the coFD and coYN peptides bind different features (Collis et al., 2003). Small molecules tend to a framework region of the Fab region of cetuximab, and not to be cradled in a small pocket; peptides fit into a linear grove; to the CDR regions, as shown by diffraction and biophysical and proteins contact a large Surface. Thus, the peptides that data as described below. This binding site is distinct from the Successfully recreate the protein-antibody interaction, as with binding sites of other framework-binding antigens such as the cetuximab-EGFR, are generally those with the protein anti Superantigens Staphylococcal protein A (SpA) and Pep gen having an unfolded domain with a large contacting Sur to streptococcus magnus protein L (PpI) (FIG. 7). In addition, face area. For example, trastuzumab recognizes a protein protein A and Protein L will bind to IgGs present in the antigen (conformational epitope), but makes contact prima patient. Thus the interaction is specific. rily with a number of geometrically constrained loops (Du et 0049 X-ray crystallographic analysis has revealed that the al., 2007: Cho et al., 2003). This type of biological recogni peptide binds within the Fab cavity, as defined by the heavy tion is more compatible with peptide mimics. and light chains (see FIGS. 1 and 4A), with a binding constant 0053. The coFD and cqYN meditopes were originally of ~700 nM (see FIGS. 13 A-13B). This interaction can be identified as candidate peptides for binding the CDR region of exploited to, among other things, improve the therapeutic cetuximab. Phage display libraries have been used to identify efficacy of naked mAbs, enhance targeted delivery of imaging peptides that bind the complementarity determining regions or therapeutic agents, and improve mAb-based imaging (CDRs) of established therapeutic antibodies with the pre methods. Sumption that the peptides would mimic the antigenic epitope 0050. The cqFD and cqYN meditopes bind to a chimeric to artificially stimulate an immune response to that antigen framework binding region, Such as that found in cetuximab. (Riemer et al., 2004: Riemer et al., 2005; Li et al., 2006). The term “meditope' (a term combining medius+topos, Latin These peptide mimics, also referred to as mimotopes or anti for middle and Greek for place), as used herein refers to an idiotypes, can be chemically synthesized, eliminating poten antibody binding peptide, such as the coFD and coYN pep tial biological contamination and reducing cost. Vaccines tides or variants thereof, that bind between the Fab light and based on this design, however, have resulted in partial heavy chains of an antibody. Other molecules may also bind responses, and are often followed by later recurrence of dis between the Fab light and heavy chains of an antibody with a ease without loss of the immune response to tumor antigen framework binding functionality similar to that of a medi (Sharav et al., 2007). tope. Such molecules may include, but are not limited to, 0054 Because the coFD and cQYN meditopes bind the Small molecules, aptamers, nucleic acid molecules, peptibod framework and not the CDR region, said meditopes are not ies and any other Substance able to bind the same binding likely candidates for specific cetuximab-like antibody immu interface as a meditope, The framework binding interface nogens for use as a vaccine. Although Reimer et al. observed described herein is distinct and distant from the CDR, which that sera collected from mice immunized with KLH-coupled binds to the antigen epitope (FIG. 4). Additionally, biochemi meditope produced antibodies that bound to A431 cells and cal and cell-based assays have demonstrated that the coFD block their proliferation (Riemer et al., 2005), the studies and coYN cyclic peptides can bind to murine chimeric cetux described herein show that multiple copies of the meditopes imab that is pre-bound to EGFR (FIG. 4). coupled to adjuvant render the vaccine into a promiscuous 0051 Cetuximab is a unique murine-human chimera and crosslinker of murine IgGs (akin to a Superantigen). In Sup several interactions between the coFD and coYN meditopes port of this embodiment, similar germ line or B-cell derived and the cetuximab Fab are specific to this murine-human framework sequences were observed in the mouse light chain chimeric IgG framework residues as determined by anatomic and both mouse and human heavy chains using BLAST. model described herein. Further, the meditopes fail to bind to Thus, when tethered to an adjuvant, the meditope peptides human IgG framework (e.g., trastuzumab), indicating this may function to activate the B-cell population with conserved interaction is specific for this murine-chimeric antibody, but framework regions and produce a generalized immune not specific for other murine chimeras Such as rituximab response as opposed to a specific immune response required (FIG. 33). This supports that the Cetuximab framework is for a vaccine. The reported ELISA competition assays do not highly specific. Superposition of multiple human and murine indicate the specific binding site of EGFR nor the degree to Fabs onto the cetuximab-cyclic peptide structure indicated which the polyclonal serum was specific for the cetuximab that the key interactions between this peptide and murine epitope on EGFR (e.g., the report did not indicate whether or human chimeric Fab are absent in both human-only and not the serum-derived IgG could be blocked by unlabeled murine-only IgG structures. Point mutations of key residues cetuximab). The diffraction and biochemical studies do not within the cyclic peptide reduced its binding affinity for the support using the coYN or cGFD meditopes as a vaccine in Fab, further confirming the high specificity and structural humans. model (data not shown). Thus, the interaction appears to be 0055 Additional meditopes that may be used according to specific to the central cavity of this specific murine-human the embodiments described herein include any small peptide US 2012/0177568 A1 Jul. 12, 2012 that binds to an antibody framework binding interface (i.e., and/or Leu10, and an incorporation of hydratable carbonyl between the Fab light and heavy chains) of cetuximab or any functionality. Each amino acid position may be altered with other therapeutic antibody. For example, in addition to the an unnatural amino acid or chemically conjugated with a cyclic peptides cGFD and coYN, some embodiments include fragment. It is shown herein that that Arg9 mutated to citural one or more variants of c(RFD and coYN. line binds to cetuximab. In addition, the amino and carboxy 0056 Structural and biophysical methods or modifica terminican be extended to make additional contact to the Fab. tions may be used in tandem with chemical synthesis to Further, protein L has been added to the N-terminus of the develop meditope variants that have increased or altered meditope and preliminary data shows that this binds with affinity (e.g., due to changes in pH, as discussed in the much higher affinity. Such modifications are discussed fur examples below) to cetuximab as compared to the unmodified ther in Examples 4 and 5. meditopes, coFD and coYN. Furthermore, conjugation of an 0057 According to the embodiments described herein, the unmodified meditope or a variant meditope onto a multivalent meditope variant peptides may be one or more of the cyclic tethering entity or scaffold may significantly improve the peptides shown in Tables 1 and 2, which were synthesized overall affinity and targeting of the multivalent meditope to a with demonstrated binding affinities to cetuxamab. With the meditope-enabled mAb bound to tumor-associated antigen. exception of SEQID NO:1 and SEQID NO:2, the sequences Modifications to c(RFD and coYN meditopes that may be represent novel structures. The peptides in Table 1 use a used to generate meditope variants with high affinity may disulfide linkage to connect the C and N terminal of each include, but are not limited to, use of one or more of the peptide while the peptides in Table 2 utilize a lactambridge or following: a head-to tail cyclic lactampeptide, a modification method other than disulfide like 3+2] cycloaddition as the of Arg8, a modification of Phe3, a modification of Leu5 COnnectOr.

TABLE 1.

Modification (under SEQ ID NO Sequence lined) Linkage method 1. C-OFDLSTRRLK- C original Disulfide 1 - Cys: 12-Cys

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

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

16 C- QYDLSTRRLK- C Y = Tyrosine Disulfide 1 - Cys: 12-Cys 17 C- OXDLSTRRLK- C X = 3-3-di-phenyl-Ala Disulfide 1 - Cys: 12-Cys

18 C- OFDXSTRRLK- C X = 3-3-di-phenyl-Ala Disulfide 1 - Cys: 12-Cys

19 C- OFDESTRXLK- C F = phenylalanine, X = Disulfide 1 - Cys: 12-Cys citrulline

2O C-OFDESTRRLK- C F = phenylalanine Disulfide 1 - Cys: 12-Cys

21 C- OFDESTRRLK- C E = glutamic acid Disulfide 1 - Cys: 12-Cys

22 C-OFDYSTRRLK- C Y = Tyrosine Disulfide 1 - Cys: 12-Cys

23 C- OFDLSTRROK- C Q = glutamine Disulfide 1 - Cys: 12-Cys

24 C- OFDLSTROLK- C Q = glutamine Disulfide 1 - Cys: 12-Cys

25 C- QYNLSTARLK- C Y = Tyrosine; N = Disulfide 1 - Cys: 12-Cys asparagine; A = alanine

26 C-QADLSTRRLK- C A = alanine Disulfide 1 - Cys: 12-Cys

27 C- OFDASTRRLK- C A = alanine Disulfide 1 - Cys: 12-Cys

28 C- OFDLSTARLK- C A = alanine Disulfide 1 - Cys: 12-Cys

29 C- OFDLSTRRAK- C A = alanine Disulfide 1 - Cys: 12-Cys

3O C- OFDLSTRREK- C E = glutamic acid Disulfide 1 - Cys: 12-Cys

31 C- OFDLSTRRLK- CGGSK Disulfide 1 - Cys: 12-Cys US 2012/0177568 A1 Jul. 12, 2012

TABLE 2 SEQ ID NO Sequence Modification (underlined) Linkage method 32 G-OFDLSTRRLK-G G = Glycine Lactam 1- Gly: 1.2-Gly

33 G-OHDLSTRRLK-G H = hlistidine Lactam 1- Gly: 1.2-Gly 34 G-QNDLSTRRLK-G N = asparagine Lactam 1- Gly: 1.2-Gly 35 G-QODLSTRRLK-G Q = glutamine Lactam 1- Gly: 1.2-Gly

36 G-QXDLSTRRLK-G X = 2-bromo-L-phenylalanine Lactam 1- Gly: 1.2-Gly 37 G-QXDLSTRRLK-G X = 3-bromo-L-phenylalanine Lactam 1- Gly: 1.2-Gly 38 G-QXDLSTRRLK-G X = 4-bromo-L-phenylalanine Lactam 1- Gly: 1.2-Gly 39 G-QFDLSTRXLK-G X = citrulline Lactam 1- Gly: 1.2-Gly

4 O G-QFDLSTXXLK-G X = citrulline Lactam 1- Gly: 1.2-Gly

41 G-OFDLSTXRLK-G X = citrulline Lactam 1- Gly: 1.2-Gly

42 Q-FDLSTRRLK-X X = 7-aminoheptanoic acid Lactam 1-Gln: 11-X

43 X-QFDLSTRRLK-X X = B-alanine Lactam 1-X: 12-X

44 X-QFDLSTRRLK-X X = diaminopropionic acid; X" = Lactam 1-X: 12-X" iso-aspartic acid

45 X-QFDLSTRRLK-X, X = B-alanine; X' = iso Lactam 1-X: 12-X" aspartic acid

46 X-QFDLSTRRLK-X X = diaminopropionic acid; X" Lactam 1-X: 12-X" 3-alanine 47 F-DLSTRRL-K Lactam 1-Phe: 9-Lys

48 C-OFDLSTRRLK- C Disulfide 1 - Cys: 12-Cys; Lactam 4-Asp to 11-Lys

49 Q-YDLSTRRLK-X Y = Tyrosine, X = 7 Lactam 1-Gln: 11-X aminoheptanoic acid SO X-QFDLSTRRLK-X, X = B-Azidoalanine, X' = 3 + 2 cycloaddition Azide-1- propargylglycine X: alkyne-12-X'

0058. In certain embodiments, one or more meditopes, 0060 A multivalent tethering agent may include two or meditope variants, multivalent meditope tethering agents or more coFD and coYN meditopes or any of the other novel multivalent meditope variant tethering agents may be conju meditope variants that are coupled through a long linker and gated to one or more imaging agents, therapeutically effective biotin to streptavidin to create a multivalent meditope tether agents or compounds in therapeutically effective amounts or ing entity. In one embodiment, the multivalent meditope teth both, such that the binding of the meditopes or variants ering entity is a tetravalent meditope tethering agent. The thereof to one or more meditope-enabled antibody with the tetrameric tethering entity is shown by Surface plasmon reso therapeutically effective compound may treat, prevent, diag nance to have enhanced binding to an IgG as compared to the monovalent peptide, which is consistent with a multivalent nose or monitor a disease or condition. Such conjugation of a interaction. The tetravalent meditope also enhances the bind high affinity and/or multivalent meditope coupled to medi ing affinity of cetuximab in EGFR positive cells by FACS tope-enabled mAbs provides a highly versatile platform tech analysis. nology that will significantly improve mab based therapeu 0061 Multivalent meditope scaffolds can be used to scaf tics and imaging methods to treat and detect disease (see FIG. fold or "daisy-chain' meditope-enabled mAbs bound to 8). tumor associated antigen to enhance ligandantagonism, alter 0059. In some embodiments, the murine specific coFD receptor endocytosis, and/or improve an immune response and coYN meditopes or derivatives thereof may be used to through ADCC/CDC (FIG. 8). Monoclonal antibodies tether two or more antibodies or functional fragments thereof. (mAbs) encode two Fab domains coupled to a dimeric Fc. As When used in a tethering method described below, the medi such, these bivalent IgGs preferentially bind to cells that topes may be part of a multivalent tethering agent (also express antigen at high densities. It has been demonstrated referred to as a “meditope scaffold' or “scaffold') to enhance that a second mAb that binds to a unique epitope on the same cancer or tumor therapy and imaging. antigen can cluster the receptors and more effectively kill US 2012/0177568 A1 Jul. 12, 2012 tumor cells. This clustering can be recapitulated through the a variety of organic or inorganic Small molecules commonly use of multivalent meditopes. The use of a multivalent medi referred to as “dyes.” “labels.” or “indicators.” Examples tope avoids necessity of identifying a second mAb and the include fluorescein, rhodamine, acridine dyes, Alexa dyes, associated and significant cost of its development thereof. and cyanine dyes. 0062. The specificity and binding of the meditopes, medi 0066 Enzymes that may be used as an additional imaging tope variants, multivalent meditope tethering agents and mul agent in accordance with the embodiments of the disclosure tivalent meditope variant tethering agents may be used to include, but are not limited to, horseradish peroxidase, alka deliver therapeutic agents, diagnostic agents (e.g., imaging line phosphatase, acid phoshatase, glucose oxidase, B-galac agents), or a combination of thereof for treatment, diagnosis tosidase, B-glucoronidase or B-lactamase. Such enzymes may or imaging a disease or condition when administered in com be used in combination with a chromogen, a fluorogenic bination with one or more meditope-enabled monoclonal compound or a luminogenic compound to generate a detect antibodies. Thus, multivalent meditopes can be used for pre able signal. targeted therapy and imaging as described further below by 0067 Radioactive substances that may be used as an addi administering a meditope-enabled monoclonal antibody tional imaging agent in accordance with the embodiments of before administering the meditopes, meditope variants, mul the disclosure include, but are not limited to, F. P. P. tivalent meditope tethering agents or multivalent meditope 45Ti, 7Sc, 52Fe, Fe, 62Cu, Cu, 67Cu, 7Ga, Ga, 77As, variant tethering agents. Further, the use of multivalent medi 86Y. 8OY. 89s, 897r, 94TC, 94TC, 99mTC, Mo, 105Pd, 105Rh, topes can enhance selectivity and improve tumor detection as '''Ag, In, 123, 1241, 125I, 13 II, 142Pr 14.Pr 14°Pm, 'Sim, has been demonstrated for engineered scFVS or chemically 154-1581Gd, 161Tb, 166Dy, 166Ho, 169Er, 175Lu, 177Lu, 186Re, conjugated mAbs, but avoids potential immunogenicity 188Re, 18°Re, 194Ir, 198Au, Au, 21 At 2. Pb, 212Bi, 212Pb, inherent in these non-human constructs. 'Bi, Ra, and’AC. Paramagnetic ions that may be used 0063. In some embodiments, a meditope administered in as an additional imaging agent in accordance with the combination with a meditope enabled antibody, an antibody embodiments of the disclosure include, but are not limited to, meditope complex, a multivalent tethering agent adminis ions of transition and lanthanide metals (e.g. metals having tered in combination with a meditope enabled antibody, or a atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals combination thereofmay be conjugated to one or more imag include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, ing agent. In one aspect, an imaging agent may include, but is Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. not limited to a fluorescent, luminescent, or magnetic protein, 0068. When the imaging agent is a radioactive metal or peptide or derivatives thereof (e.g., genetically engineered paramagnetic ion, the agent may be reacted with another variants). Fluorescent proteins that may be expressed by the long-tailed reagent having alongtail with one or more chelat mRNA component include green fluorescent protein (GFP), ing groups attached to the longtail forbinding these ions. The enhanced GFP (EGFP), red, blue, yellow, cyan, and sapphire long tail may be a polymer Such as a polylysine, polysaccha fluorescent proteins, and reefcoral fluorescent protein. Lumi ride, or other derivatized or derivatizable chain having pen nescent proteins that may be expressed by the mRNA com dant groups to which may be added for binding to the metals ponent include luciferase, aequorin and derivatives thereof. or ions. Examples of chelating groups that may be used Numerous fluorescent and luminescent dyes and proteins are according to the disclosure include, but are not limited to, known in the art (see, e.g., U.S. Patent Application Publica ethylenediaminetetraacetic acid (EDTA), diethylenetri tion 2004/0067503; Valeur, B., “Molecular Fluorescence: aminepentaacetic acid (DTPA), DOTA, NOTA, NETA, por Principles and Applications. John Wiley and Sons, 2002: phyrins, polyamines, crown ethers, bis-thiosemicarbazones, Handbook of Fluorescent Probes and Research Products, polyoximes, and like groups. The chelate is normally linked Molecular Probes, 9.sup.th edition, 2002; and The Hand to the PSMA antibody or functional antibody fragment by a book A Guide to Fluorescent Probes and Labeling Tech group which enables the formation of a bond to the molecule nologies, Invitrogen, 10th edition, available at the Invitrogen with minimal loss of immunoreactivity and minimal aggre web site; both of which are hereby incorporated by reference gation and/or internal cross-linking. The same chelates, when as if fully set forth herein.) complexed with non-radioactive metals, such as manganese, 0064. In other aspects, the meditope administered in com iron and gadolinium are useful for MRI, when used along bination with a meditope enabled antibody, the antibody with the antibodies and carriers described herein. Macrocy meditope complex, the multivalent tethering agent adminis clic chelates such as NOTA, DOTA, and TETA are of use with tered in combination with a meditope enabled antibody, or a a variety of metals and radiometals including, but not limited combination thereof may be further conjugated to or other to, radionuclides of gallium, yttrium and copper, respectively. wise associated with a non-protein imaging agent or a deliv Other ring-type chelates Such as macrocyclic polyethers, ery vehicle Such as a nanoparticle, radioactive substances which are of interest for stably binding nuclides, such as (e.g., radioisotopes, radionuclides, radiolabels or radiotrac ‘Ra for RAIT may be used. In certain embodiments, chelat ers), dyes, contrast agents, fluorescent compounds or mol ing moieties may be used to attacha PET imaging agent, Such ecules, bioluminescent compounds or molecules, enzymes as an Al-F complex, to a targeting molecule for use in PET and enhancing agents (e.g., paramagnetic ions). In addition, it analysis. should be noted that some nanoparticles, for example quan 0069. In some embodiments, a meditope, an antibody tum dots and metal nanoparticles (described below) may also meditope complex, a multivalent tethering agent, or a com be suitable for use as an imaging agent or a therapeutic agent bination thereof may be conjugated to one or more therapeu (e.g., using hyperthermal and photodynamic therapies as well tic agents. as imaging agents through fluorescence and or MRI contrast). 0070 A “therapeutic agent” as used herein is an atom, 0065. Fluorescent and luminescent substances that may be molecule, or compound that is useful in the treatment of used as an additional imaging agent in accordance with the cancer or other conditions described herein. Examples of embodiments of the disclosure include, but are not limited to, therapeutic agents that may be conjugated to an antibody US 2012/0177568 A1 Jul. 12, 2012

meditope complex, a multivalent tethering agent, a multiva trastuzumab, tretinoin, trimitrexate, alrubicin, Vincristine, lent tethering agent or a combination thereof include, but are vinblastine, vindestine, vinorelbine, Vorinostat, or Zoledronic not limited to, drugs, chemotherapeutic agents, therapeutic acid. antibodies and antibody fragments, toxins, radioisotopes, 0072 Therapeutic antibodies and functional fragments enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic thereof, that may be used as therapeutic agents in accordance agent at the site of the tumor), nucleases, hormones, immu with the embodiments of the disclosure include, but are not nomodulators, antisense oligonucleotides, RNAi molecules limited to, alemtuzumab, , cetuximab, edreco (e.g., siRNA or shRNA), chelators, boron compounds, pho lomab, gemtuzumab, , panitumumab, toactive agents and dyes. The therapeutic agent may also rituximab, to situmomab, and trastuzumab and other antibod ies associated with specific diseases listed herein. include a metal, metal alloy, intermetallic or core-shell nano 0073 Toxins that may be used as therapeutic agents in particle bound to a chelator that acts as a radiosensitizer to accordance with the embodiments of the disclosure include, render the targeted cells more sensitive to radiation therapy as but are not limited to, ricin, abrin, ribonuclease (RNase), compared to healthy cells. Further, the therapeutic agent may DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral include paramagnetic nanoparticles for MRI contrast agents protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, (e.g., magnetite or FesO4) and may be used with other types and Pseudomonas endotoxin. of therapies (e.g., photodynamic and hyperthermal therapies. 0074 Radioisotopes that may be used as therapeutic And imaging (e.g., fluorescent imaging (Au and CdSe)). agents in accordance with the embodiments of the disclosure 0071 Chemotherapeutic agents are often cytotoxic or include, but are not limited to, 'P, Sr, Y. "Tc, Mo, cytostatic in nature and may include alkylating agents, anti 13 II, 'Sim, 77Lu, 18.Re, 213Bi, 223Ra and 225Ac. metabolites, anti-tumorantibiotics, topoisomerase inhibitors, 0075. The meditope-mAb technology allows for a system mitotic inhibitors hormonetherapy, targeted therapeutics and that may be used to generate an antibody-meditope complex immunotherapeutics. In some embodiments the chemothera that may be further conjugated to one or more meditope peutic agents that may be used as therapeutic agents in accor enabled antibody, a therapeutic agent, an imaging agent or a dance with the embodiments of the disclosure include, but are combination thereof. Thus, a set of meditopes or high affinity not limited to, 13-cis-Retinoic Acid, 2-Chlorodeoxyadenos meditope variants, each conjugated to a unique cytotoxic or ine, 5-Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, imaging agent, would allow for the co-administration of a 6-Thioguanine, actinomycin-D, adriamycin, aldesleukin, desired meditope conjugate and meditope-enabled mAb for alemtuzumab, alitretinoin, all-transretinoic acid, alpha inter treatment. The meditope conjugates are an improvement over feron, altretamine, amethopterin, amifostine, anagrelide, the current antibody-drug conjugates, which have drawbacks anastroZole, arabinosylcytosine, arsenic trioxide, amsacrine, Such as a reduced specificity due to chemical modification or aminocamptothecin, aminoglutethimide, asparaginase, aza release of payload. cytidine, bacillus calmette-guerin (BCG), bendamustine, 0076. This platform technology has a broad impact on the mAb delivery field and provides useful methods for the treat bevacizumab, bexarotene, bicalutamide, bortezomib, bleo ment and diagnosis of EGFR-positive cancers including col mycin, buSulfan, calcium leucoVorin, citroVorum factor, orectal and squamous cell carcinoma head and neck cancers capecitabine, canertinib, carboplatin, carmustine, cetuximab, where cetuximab is indicated. Additionally, grafting the chlorambucil, cisplatin, cladribine, cortisone, cyclophospha framework binding interface onto other therapeutic antibody, mide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, allows for the platform technology to be utilized in methods decitabine, denileukin diftitox, dexamethasone, dexasone, for the treatment and diagnosis of several other cancers, dis dexraZoxane, dactinomycin, daunorubicin, decarbazine, doc eases and other conditions as described above. etaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epo 0077. A method for enhancing the binding affinity of a etin alfa, erlotinib, everolimus, exemestane, estramustine, therapeutic antibody or functional fragment thereof is pro etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiri vided herein. Such a method may include administering to a dol, floxuridine, fludarabine, fluorouracil, flutamide, gefi Subject a therapeutically effective amount of a pharmaceuti tinib, gemcitabine, , goserelin, cal composition via any suitable route of administration. The granulocyte-colony stimulating factor, granulocyte macroph pharmaceutical composition may include a meditope or age-colony stimulating factor, hexamethylmelamine, hydro meditope variant in combination with a meditope enabled cortisone hydroxyurea, ibritumomab, interferon alpha, inter antibody, a multivalent meditope or meditope variant tether leukin-2, interleukin-11, isotretinoin, ixabepilone, ing entity in combination with a meditope enabled antibody, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapa a meditope-enabled therapeutic antibody or functional frag tinib, lenalidomide, letrozole, leucovorin, leuprolide, liposo ment thereof, a pharmaceutically acceptable carrier, and any mal Ara-C, lomustine, mechlorethamine, megestrol, mel combination thereof. The enhanced binding affinity of the phalan, mercaptopurine, meSna, methotrexate, multivalent meditope may be attributed to the multivalent methylprednisolone, mitomycin C, mitotane, mitoxantrone, cross-linking of IgGs bound to the cell Surface. Crosslinking nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, IgGs (through parental murine 425 antibody or using anti paclitaxel, pamidronate, pemetrexed, panitumumab, PEG IgG IgM) significantly affects signaling, receptor endocytosis Interferon, pegaspargase, pegfilgrastim, PEG-L-asparagi and recycling, and cell death. Thus, multivalent peptides may nase, pentostatin, plicamycin, prednisolone, prednisone, pro act synergistically with a therapeutic monoclonal antibody to carbazine, raloxifene, rituximab, romiploStim, ralitrexed, enhance its therapeutic efficacy. Sapacitabine, SargramoStim, satraplatin, Sorafenib, Sunitinib, 0078. In some embodiments, the meditope, alone or as Semustine, Streptozocin, tamoxifen, tegafur, tegafur-uracil, part of the tethering entity, may contain a cysteine or other temsirolimus, temozolamide, teniposide, thalidomide, Suitable alkylating agent that binds to a Fab cysteine at the thioguanine, thiotepa, topotecan, toremifene, to situmomab, binding site, thus creating a cysteine-cysteine interaction. US 2012/0177568 A1 Jul. 12, 2012

Alternatively, the meditope may bind to the Fab at an unnatu may be used as a linker to join two or more therapeutic ral amino acid (e.g., p-acetylphenylalanine). The Cys medi molecules to a binding interface orbinding site of a specially tope is conjugated to any substance and directs the conjugate designed CovX antibody. A small molecule, peptide or schv to the IgG. that is associated with a meditope that acts as a CoVX-specific 0079 An antibody-meditope complex may also be used in linker is recognized by its framework binding interface, a method for directing treatment to a particular type of cell or which is the active binding site of the CovX antibody. When population of cells in a disease or condition that can be tar these components are combined, the resulting bivalent CovX geted by a therapeutic antibody. Such a method of treatment BodyTM possesses the biologic actions of the small molecule, may include administering atherapeutically effective amount peptide or schv while also retaining an extended half-life of of a pharmaceutical composition to a subject having the dis the antibody. ease or condition via any Suitable route of administration. The pharmaceutical composition may include a meditope or 0084. In addition to the benefits described above with meditope variant in combination with a meditope enabled respect to therapeutic monoclonal antibodies, meditopes used antibody, a multivalent meditope or meditope variant tether as linkers to tether or bind to therapeutic molecules can be ing entity in combination with a meditope enabled antibody, synthesized and are more cost effective to produce than a meditope-enabled therapeutic antibody or functional frag monoclonal antibodies. For example, although a number of ment thereof. preclinical/clinical trials are investigating the co-administra 0080. In other embodiments, a method for imaging tumors tion of two monoclonal antibodies, the costs of producing and or other tissues is provided. In such methods, an unmodified marketing such a therapeutic is likely to be prohibitive. therapeutic antibody may be administered to target a tumor or I0085. The schv (single chain Fab variable fragment) for other tissue that overexpress the corresponding antigen. Sub mat of the cetuximab Fab binds to the EGFR domain III with sequently, a multivalent meditope tethering entity that is a substantially lower affinity than the Fab itself, which is labeled with an imaging agent is administered via any suitable similar to other scEvs of clinical interest. This is attributed, at route of administration and will bind the therapeutic antibod least in part, to absence of the Fab constant domain that ies that are bound to the target tumor or tissue. See FIG. 8. directly affects the orientation, conformational fluctuations Examples of imaging agents include but are not limited to of the Fv domains, and possibly, to poor linker design. radiolabels (e.g., H, C, S, Y, Tc, I, II, 77Lu, 0086. Therefore, in some embodiments, a method for Ho, and 'Sm), metal or magnetic labels (e.g., gold, iron, improving the stability and affinity of the schvs is provided. gadolinium), biotin, chelating agents (e.g., 1,4,7,10-tetraaza The method comprises incorporating the peptide meditope in cyclododecane-N,N',N',N"-tetraacetic acid (“DOTA)) or the sclv linker, to stabilize the schv. This will help to enforce any agent described above. In one embodiment, the imaging the proper orientation of the variable domains and thus agent used with the method described herein is DOTA. enhance the affinity (FIG.9). 0081. There are several advantages over known methods I0087. The embodiments described above can be applied to to the imaging or treatment methods described above. First, human or humanized antibodies (e.g., trastuzumab) by the bivalent character of a therapeutic monoclonal antibody replacing one or more of the human framework residues (i.e. IgG enhances the selectivity to the tumor. As discussed fur generating one or more point mutations) with their corre ther below, this enhanced selectivity is lost with the use of sponding murine residues. The human residues that are ScFVs and other minibodies and cannot be made up through replaced by corresponding murine residues are found within enhanced affinity. Second, the mass of the labeled multivalent the central Fab cavity of the human framework and are there meditope tetheringentity will be below the renal threshold for fore not exposed to the immune system (e.g., they should not filtering (less than 60 kD, may be as low as ~10 kD), allowing be antigenic). In addition, antigenicity prediction algorithms it to be easily filtered out of the body. In contrast, direct may be further used to indicate that the human sequence with labeling of a therapeutic antibody IgG with an imaging agent the point mutations should not be antigenic. or other therapeutic agent is typically avoided because it will 0088. In some embodiments, the one or more human circulate for extended periods in the body. Thus, imaging of framework residues that are replaced by the corresponding tumors or other diseased organs is often accomplished using murine residues may be selected from light chain framework less selective scEvs. residues 10, 39-43, 83, 85, 100 and 104 (Kabat numbers) I0082 Infurther embodiments, the cqFD and cqYN medi and/or heavy chain framework residue numbers 40, 89 and topes or variants thereof may be used to join two or more 111 (Kabat numbers) (see FIG. 2). In one embodiment, the therapeutic molecules to form a pharmaceutical compound one or more human framework residues that are replaced by that may be administered, as part of a pharmaceutical com the corresponding murine residues are light chain framework position, in a therapeutically effective amount to a Subject for residues including, but not limited to, 40, 41, 83 and 85. treatment of cancer, autoimmune disease or other conditions. Specifically, in one embodiment, light chain framework The two or more therapeutic molecules may include, but are Pro40 is replaced with Thr(P40T) or Ser(P40S), light chain not limited to, functional antibody fragments (e.g., F(ab') or framework Gly41 is replaced with Asn (G41N), light chain Fab fragments), peptides or other Small molecules that can framework residue Phe83 is replaced with Ile (F83I) or Val target tumor or disease-specific receptors such as those (F83V) and light chain framework residue Thr85 is replaced described above. The therapeutic molecules may be two or with Asp (T85D) or Asn (T85N). This meditope-site grafting more of the same therapeutic molecule or alternatively, may (or “murinization' or “mousification') of this Fab cavity be two or more different molecules that target the same tumor within human monoclonal antibodies can be used to create a or diseased tissue. unique handle for meditope binding and used with the tech 0083. In some embodiments, the pharmaceutical com nology previously disclosed. In addition, additional point pound may include the use of proprietary antibody, orportion mutations may be engineered by generating a pharmacophore thereof, such as a CoVX-BodyTM. For example, the meditopes binding model to further enhance the affinity of the meditope. US 2012/0177568 A1 Jul. 12, 2012

0089. The embodiments described herein identify and simultaneous administration (or "coadministration'), admin characterize a novel interface within the Fab framework, and istration of a first agent prior to or after administration of a demonstrates that it does not perturb the binding of Fab to the second agent, as well as in a temporally spaced order of up to antigen or act as an allostericantagonist of EGFR binding. In several days apart. Such combination treatment may also addition, the two peptide meditopes do not bind to the cetux include more than a single administration of any one or more imab CDRs, and thus do not mimic the antigen (EGFR). of the agents, drugs, treatment regimens or treatment modali 0090. A “therapeutically effective amount,” “therapeuti ties. Further, the administration of the two or more agents, cally effective concentration” or “therapeutically effective drugs, treatment regimens, treatment modalities or a combi dose' is the amount of a compound that produces a desired nation thereof may be by the same or different routes of therapeutic effect in a subject, Such as preventing or treating administration. a target condition, alleviating symptoms associated with the 0094. A “therapeutic antibody' may refer to any antibody condition, or producing a desired physiological effect. The or functional fragment thereof that is used to treat cancer, precise therapeutically effective amount is the amount of the autoimmune diseases, transplant rejection, cardiovascular composition that will yield the most effective results in terms disease or other diseases or conditions such as those of efficacy of treatment in a given subject. This amount will described herein. Examples of therapeutic antibodies that vary depending upon a variety of factors, including but not may be used according to the embodiments described herein limited to the characteristics of the therapeutic compound include, but are not limited to murine antibodies, murinized or (including activity, pharmacokinetics, pharmacodynamics, humanized chimera antibodies or human antibodies includ and bioavailability), the physiological condition of the sub ing, but not limited to, Erbitux (cetuximab), ReoPro (abcix ject (including age, sex, disease type and stage, general physi imab), Simulect (basiliximab), Remicade (infliximab); cal condition, responsiveness to a given dosage, and type of Orthoclone OKT3 (muromonab-CD3); Rituxan (rituximab), ), the nature of the pharmaceutically acceptable BeXXar (to situmomab) Humira (adalimumab), Campath (ale carrier or carriers in the formulation, and the route of admin mtuzumab), Simulect (basiliximab), Avastin (bevacizumab), istration. One skilled in the clinical and pharmacological arts Cimzia (certolizumab pegol), Zenapax (daclizumab), Soliris will be able to determine a therapeutically effective amount (eculizumab), Raptiva (efalizumab), Mylotarg (gemtu through routine experimentation, namely by monitoring a Zumab), Zevalin (ibritumomab tiuxetan), Tysabri (natali Subject's response to administration of a compound and Zumab). Xolair (omalizumab), Synagis (palivizumab), adjusting the dosage accordingly. For additional guidance, Vectibix (panitumumab), Lucentis (ranibizumab), and Her see Remington: The Science and Practice of Pharmacy 21 ceptin (trastuzumab). Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott (0095 “Treating” or “treatment” of a condition may refer Williams & Wilkins, Philadelphia, Pa., 2005. to preventing the condition, slowing the onset or rate of devel 0091. A “pharmaceutically acceptable carrier refers to a opment of the condition, reducing the risk of developing the pharmaceutically acceptable material, composition, or condition, preventing or delaying the development of symp vehicle that is involved in carrying or transporting a com toms associated with the condition, reducing or ending Symp pound of interest from one tissue, organ, or portion of the toms associated with the condition, generating a complete or body to another tissue, organ, or portion of the body. For partial regression of the condition, or some combination example, the carrier may be a liquid or Solid filler, diluent, thereof. excipient, solvent, or encapsulating material, or some com 0096. In one embodiment, a method of screening for novel bination thereof. Each component of the carrier must be meditopes or Small molecules with framework binding func “pharmaceutically acceptable' in that it must be compatible tionality similar to that of a meditope is provided herein. Such with the other ingredients of the formulation. It also must be a method may include, but is not limited to, steps of contact Suitable for contact with any tissue, organ, or portion of the ing a library of putative meditopes or Small molecules with a body that it may encounter, meaning that it must not carry a meditope-enabled antibody; determining whether the puta risk of toxicity, irritation, allergic response, immunogenicity, tive meditopes of small molecules bind the meditope-enabled or any other complication that excessively outweighs its antibody at a framework binding interface; identifying one or therapeutic benefits. more candidate meditopes or a small molecules with frame 0092. A “route of administration' may refer to any admin work binding functionality similar to that of a meditope; istration pathway known in the art, including but not limited determining binding affinity of the one or more candidates; to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, and identifying one or more of the candidates as a meditope or transdermal, or vaginal. “Transdermal administration may small molecule with framework binding functionality similar be accomplished using a topical cream or ointment or by to that of a meditope when the binding dissociation constant means of a transdermal patch. “Parenteral refers to a route of is at least 0.70 uM. Additionally, methods of screening for administration that is generally associated with injection, novel framework binding interfaces are also provided, and are including infraorbital, infusion, intraarterial, intracapsular, described further in the examples below intracardiac, intradermal, intramuscular, intraperitoneal, (0097 Having described the invention with reference to the intrapulmonary, intraspinal, intrasternal, intrathecal, intrau embodiments and illustrative examples, those in the art may terine, intravenous, Subarachnoid, Subcapsular, Subcutane appreciate modifications to the invention as described and ous, transmucosal, or transtracheal. illustrated that do not depart from the spirit and scope of the 0093. “In combination” or “in combination with,” as used invention as disclosed in the specification. The Examples are herein, means in the course of treating the same disease or set forth to aid in understanding the invention but are not condition in the a Subject using two or more agents, drugs, intended to, and should not be construed to limit its scope in treatment regimens, treatment modalities or a combination any way. The examples do not include detailed descriptions of thereof (e.g., an antibody in combination with a meditope or conventional methods. Such methods are well knownto those a multivalent tethering agent), in any order. This includes of ordinary skill in the art and are described in numerous US 2012/0177568 A1 Jul. 12, 2012

publications. Further, all references cited above and in the round of refinement using the Fab model only, a continuous examples below are hereby incorporated by reference in their stretch of unmodeled density consistent with the meditope entirety, as if fully set forth herein. was observed. The meditope was built into the density and the R and RFree dropped accordingly. Water molecules were Example 1 added during refinement using Phenix (Adams et al., 2002). The diffraction data and refinement statistics are given in Determination of Crystal Structures Table 3 below. 0098. Materials and Methods 0099 Reagents. The antigenbinding fragment F(ab)" (or “Fab') of cetuximab was obtained by digestion of the IgG with immobilized papain (Pierce), followed by reverse puri Experimental and Refinement Statistics for Co-Crystal Structures fication with protein A and size exclusion chromatography (SEC) on a Superdex 75 column (GE Healthcare). The single chain binding fragment (ScFVC225) of cetuximab was Syn Data collection thesized with a twenty amino acid linker between the light Space group P221.21 P221.21 chain and heavy chain. ScFvC225 and soluble epidermal Cell dimensions growth factor receptor domain III (sEGFRdIII) were a, b, c (A) 64.29, 82.53,211.7 64.16, 82.52,211.9 expressed in Sf9 cells and purified as previously described Cl, f, Y () 90.00, 90.00, 90.00 90.00, 90.00, 90.00 (Donaldson et al., 2009). Resolution (A) 20-2.24 (2.3-2.24) 30-2.20 (2.26-2.20) 0100 Meditopes CQFDLSTRRLKC (cGFD; SEQ ID Rmrga F 0.078 (0.50) 0.067 (0.26) NO: 1) and CQYNLSSRALKC (cGYN; SEQID NO:2), iso fo(I) 17 (3.1) 25.5 (6.0) lated from a phage display as previously described (Riemeret Completeness (%) al. 2005), were synthesized, oxidized and purified at the City fo > 0 91.6 (74.4) 97.0 (92.8) of Hope Synthetic and Polymer Chemistry Core Facility. fo > -3 97.4 (83.3) 99.9 (100) Redundancy 4.2 (3.2) 7.9 (8.2) 0101 Crystallization and Diffraction Data. The Fab frag Refinement ment of cetuximab (5 mg/mL) was mixed with individual meditopes at a 1:10 molar ratio and screened using the Qiagen Resolution (A) 19-2.24 29.6-220 JCSG Core Suites (IIV) at 20° C. Co-crystals that diffracted No. reflections" 53791 S8047 beyond 2.2 A were grown in 100 mM sodium phosphate/ Rork Rfee 18423.4 18.623.0 citrate, pH 4.5, 2.5M sodium/potassium phosphate and 1.6% No. atoms w/v mesoerythritol. The crystals were wicked through 14% Protein 66O2 6529 w/v mesoerythritol and flash frozen in liquid nitrogen. Crys Mimotope 200 188 Water 556 539 tallization trials and initial screening studies were carried out B-factors in the X-ray facility at the City of Hope. Diffraction data were Protein collected at the Stanford Synchrotron Radiation Lab, beam Fab 31.4 31.2 lines 9.1 and 11.1. The initial phases were determined by Mimotope 49.7 62.6 molecular replacement using the program Phaser (McCoy et Water 37.1 37.1 al., 2007) with the unliganded structure of cetuximab (pdb: r.m.S.d. 1YY8-chains A and B) (Li et al., 2005). Two Fabs were Bond lengths (A) O.OO3 O.OO7 placed in the asymmetric unit with a Matthews Coefficient of Bond angles () O.710 1.07 3.26 and solvent content of 62.4%. The Z scores (standard Ramachandran deviation of the solution over the mean) were 27 and 25 for the rotational search and 38 and 71 for the translational favored allowed disallowed 96.70.2.0.1 98.0.2.00.0 search. A third Fab fragment could not be placed (three Fabs *Values in parentheses are for highest-resolution shell with Io(F) > 0. in the asymmetric unit cell produces a reasonable Matthews "FoCF) > 1.99 for and > 1.36 for cGFD or cGYN co-crystals, respectively, coefficient of 2.18 at 43% solvent). The meditopes were built into the density manually through multiple iterations using 0104 Based on these observations and as a point of com Coot (Emsley et al., 2004) and Phenix (Adams et al., 2002). parison, crystals were produced of the cetuximab Fab bound 0102 Crystallization and Structure Determination to the second meditope identified by phage display, coYN. As 0103) To identify the binding site of the meditopes on before, clear unmodeled electron density was observed in the cetuximab, the Fab fragment was generated and purified, center of the Fab. Using the first structure, the differences in mixed with the coFD meditope at a 1:10 ratio and commer sequences were modeled accordingly and multiple rounds of cial factorials were used to screen for crystal formation. Crys refinement were carried out. Representative electron density tals formed after 1 to 3 days at 20° C. Initial diffraction maps of both meditopes are shown in FIGS. 1B and C. analysis of these crystals indicated that the unit cell was similar to the cetuximab Fab already deposited in the Protein Example 2 Data Bank (1 YY8.pdb) (Li et al., 2005), and the crystal Meditopes are Specific to a Murine Framework packing (e.g., the CDRS were excluded) suggested the pep Region of Cetuximab Fab tide was not present at the CDR loops. Nonetheless, the structure was solved by molecular replacement and the 0105. Materials and Methods experimental maps were examined to identify unmodeled 0106. In addition to those described in Example 1 above, electron density consistent with the meditope. The initial the following materials and methods were used. Fo-Fc map clearly indicated an area in the middle of the Fab 0107 Meditopes and point mutations. As described above, fragment as a potential binding site (FIG. 1). After an initial CQFDLSTRRLKC (cQFD; SEQID NO:1) and CQYNLSS US 2012/0177568 A1 Jul. 12, 2012

RALKC (cQYN; SEQID NO:2), were synthesized, oxidized body, differences between the coFD and coYN meditopes and purified at the City of Hope Synthetic and Polymer and their interaction with the cetuximab Fab were deter Chemistry Core Facility. Alanine point mutations in the mined. Superposition of the Ca atoms of the heavy and light cQFD meditope were generated at residues 3 (Phe3 to Ala), 5 Fab chains from the coFD and coYN structures show that the (Leu5 to Ala), 8 (Arg8 to Ala) and 10 (Leu10 to Ala) and were hydrophobic side chains of residues Phe/Tyr3, Leu5 and produced bacterially by encoding the peptides at the C-ter Leu10 from each meditope are positioned nearly identically minus of SMT3 (Mossessova et al., 2000). Before surface (FIG. 2B). However, the backbone traces of the coFD and plasmon resonance (SPR) analysis, ubiquitin-like protease cQYN peptides deviate significantly. Specifically, the Arg8 (Ulp1) was added to the samples to release the peptides. side chain structure of the coFD meditope is extended and 0108 Characterization of the meditope-Fab interface. makes a strong backbone hydrogen bond to the backbone Affinity analysis by SPR was performed as previously carbonyl of GIn 111 of the Fab heavy chain (dNH described (Donaldson et al., 2009; Li et al., 2005). Briefly, sch v0225 or FabC225 was immobilized on a CM5 chip using O=C=2.8 A). The hydroxyl group of Tyr3 in the cqYN pep amine chemistry. Peptide or sGFRdIII affinities were tide, however, sterically interferes with the Arg8 side chain assessed by equilibrium methods at 20° C. and fit to the (FIG. 2B) and blocks the interaction between Arg8 of the equation RU={Rmax*L}/{L*Kd}+Roffset. SEC was cQYN meditope and Asn111 of the heavy chain. Consistent performed using a Superdex 20010/30 column (GE Health with this observation, both Arg8 side chains in the coYN care). The proteins were mixed, incubated at room tempera complex are poorly defined in the electron density map and ture for 20 min and applied to the column at 4°C. takes on at least two different rotamers. (There are two Fab 0109. The MDA-MB-468 cell line was used to test cetux meditope complexes in the asymmetric unit.) Concomitant imab binding in the presence of peptide meditope. Labeled with this change, a shift in the backbone hydrogen bond cetuximab (AF488, Invitrogen) was added for 20 min with or pattern was observed. The amide carbonyl of Thr7 in the without 60 uMcQFD peptide at 4°C. Labeled MOPS-21 was cQFD meditope makes a hydrogen bond to the amide Asn-11 used as an isotope control. Cell fluorescence was determined in the cetuximab Fab light chain (dNH OC=2.7 A). This using a FACS Calibur instrument (BD Biosciences). hydrogen bond shifts to the carbonyl of the Arg8 backbone in 0110 Analysis of Meditope/Fab Interface the amide of backbone of Asn41 in the coYN peptide (dC=O 0111. The interface of the binding site between the medi HN=3.0 A). tope and the Fab identified herein is formed by all four 0114 Collectively, the differences between the cQFD and domains of the IgG (e.g., the variable and constant domains of cQYN (e.g., loss of a salt bridge between R8 and the heavy the heavy and light chains). Using the PISA server, the buried chain Fab) and sequence differences in the Fab light chain surface area at the coFD or cGYN meditope-Fab interface where Arg9 in coFD binds (e.g., the loss of the hydrogen was 904 (+28) A2 and 787 (+42) A2, respectively, and equally bond between Asp85 and the amide of Leu10 for either medi distributed between the light and heavy chains. FIGS. 2 and 4 tope) indicate that the meditopes selected to bind to the show the residues and the loops from the Fab that contacts the murine chimera have substantially weaker interactions with meditope. human Fab frameworks and were removed in the washes 0112 Both meditopes make multiple hydrogen bonds and during the selection. hydrophobic contacts with the cetuximab Fab. FIG. 2A shows 0115 Meditopes do not Induce Large Conformational the subtle differences between the residues of the central Changes in the Fab cavity binding interface of the cetuximab Fab (murine chi 0116 Based on the location of the meditope-Fab interface, mera IgG), the humanized monoclonal IgG used as an isotype it was determined whether the meditopes perturbed the rela control in the phage display experiments (ch14.18) and the tive orientation of the IgG domains to the unligated and/or humanized trastuzumab Fab. Superposition of the humanized ligated structure. First, the light and heavy chains within the trastuzumab Fab on the cetuximab Fab indicated that Arg9 of asymmetric unit cell of both meditope complexes were com the coFD meditope binds to a unique cavity created by the pared and then each chain was compared to the unligated and mouse variable light chain. Specifically, Asp85, 11 e83 and EGFR-ligated structures. The variable domains of the light Thr40 of the mouse variable light chain are important with chains bound to either meditope were essentially identical to respect to binding to the Arg9 residue of the coFD meditope the unliganded structure of cetuximab (r.m.S.d. average: (FIG. 2B). Asp85 in the murine framework makes a salt cQFD, 0.231+0.014 A, cqYN, 0.18+0.01 A). However, the bridge to the guanidinium group of Arg9 of the coFD medi variable domains of the heavy chain showed significantly tope (dNE OD1=2.8 A & dNH/2 OD2=3.0 A). The higher divergence (r.m.s.d. average: cqFD, 0.85+0.01 A. carboxyl group of Asp85 also makes a hydrogen bond to the cQYN, 0.88+0.01 A). It is noted that this divergence stems backbone amide of Leu10 of the cqFD meditope (dOD2 primarily from the position of framework loop 2 (residues HN=2.7A). The hydroxyl group of Thr40 from the light chain 39-46), since deleting the residues in this loop and recalcu also makes a hydrogen bond to guanidinium group of the lating the rim.S.d. produced a much lower value: coFD, meditope Arg9 (dOG1 NH1=3.1 A). The phenyl ring in 0.18+0.01 A: cqYN, 0.31+0.01 A) (FIG. 6). In addition, this Phe83 and the pyrrolidine ring of Pro40 in the human Fab loop is also displaced in the Fab C225-EGFR co-crystal struc sterically occlude the side chain of Arg9. ture, and its relative B-factor value suggests that it is flexible 0113 Although the selection of the Arg9 side chain in the (FIG. 6). Finally, the presence of the meditope does not result cQFD meditope maps to the differences between the murine in significant changes to the CDR structure relative to the and human Fab sequences, it was also noted that the coYN EGFR bound or unbound structure. Although the backbone of meditope encodes an alanine at the same position as Arg9 in Tyr101 in the heavy chain CDR loop 3 of the EGFR liganded the coFD meditope, and thus could potentially bind to the structure is flipped as compared to the Fab structure bound to human Fab. To discern why the coYN meditope was selected either meditope, this flip is also observed in the unliganded despite pretreating the phage library with the hu14.18 anti cetuximab Fab structure (Li et al., 2005). US 2012/0177568 A1 Jul. 12, 2012

0117 Contribution of Meditope Residues to Interface 0.124 Meditopes and point mutations. As described above, 0118 Based on the structure of the coFD meditope-Fab CQFDLSTRRLKC (cQFD; SEQID NO:1) and CQYNLSS complex, as well as the sequence similarity of c(RYN to RALKC (cGYN; SEQID NO:2), were synthesized, oxidized cQFD, several point mutations in the coFD meditope were and purified at the City of Hope Synthetic and Polymer generated (Phe3->Ala, Leu5->Ala, Arg8->Ala and Chemistry Core Facility. Alanine point mutations in the Leu10->Ala) to characterize the role of these residues to the cQFD meditope were generated at residues 3 (Phe3 to Ala), 5 overall binding affinity of the meditope. To assess binding (Leu5 to Ala), 8 (Arg8 to Ala) and 10 (Leu10 to Ala) and were affinity, the Fab fragment was coupled to a CM5 chip using produced bacterially by encoding the peptides at the C-ter standard amine chemistry. Next, the affinity of the synthetic minus of SMT3 (Mossessova et al., 2000). Before surface cQFD and coYN meditopes to the cetuximab Fab was mea plasmon resonance (SPR) analysis, ubiquitin-like protease sured. The coFD meditope bound to the Fab with an affinity (Ulp1) was added to the samples to release the peptides. of 950+30 nM whereas the cqYN meditope bound with an 0.125 Simultaneous Binding of EGFR and Meditope to affinity of 3.5-0.1 uM (n=3). The binding kinetics were also Fab measured (FIG. 3). The association constants, modeled as a 0.126 The diffraction data conflict with the hypothesis that bimolecular interaction, were 4.2 (+0.1)x104 M-1s-1 and 1.8 these peptides are effective as vaccines. Specifically, the (+0.1)x104 M-1s-1 for cGFD and cqYN, respectively. The atomic model shows that the meditopes do not directly bind to dissociation constants were 2.5 (+0.1)x10-2 S-1 and 8.6 (+0. the CDRs, and thus do not mimic an antigen epitope. How 1)x10-2s-1 for cGFD and cqYN, respectively. The KD val ever, serum collected from mice inoculated with the coYN ues based on these measurements, 430 (+30) nM for cGFD meditope blocked cell proliferation (Riemer et al., 2005). and 3.5 (+0.1) pM for cGYN, are in close agreement with the Therefore, to test whether or not the meditope occludes anti equilibrium measurements. gen binding, the cetuximab Fab was incubated with EGFR 0119) Next, the affinity of each mutated coFD meditope domain III and coFD and applied to an analytic SEC column. was measured. The point mutation and wildtype coFD medi A peak at 13.9 mL was observed, and non-reducing SDS topes were generated as C-terminal fusions to SMT3 and PAGE of the peak showed the presence of all three compo cleaved with Ulp1 before the analysis. The biologically-pro nents (FIG. 4B). The individual components eluted at 15.2 duced, wildtype coFD meditope bound with an affinity of mL (Fab C225), 15.6 mL (sEGFRdIII) and 16.3 mL (SMT 770 nM, similar to the synthetically-produced coFD, CQFDLSTRRLKC; SEQID NO:1). whereas the mutations Phe3->Ala, Leu5->Ala and 0127. In addition, it was determined whether or not the Arg8->Ala significantly reduced the affinity for the Fab meditope could bind to the schv of cetuximab. In the scFv, the (Table 4, below). In particular, the Arg8->Ala mutation CDR loops remain intact, but the Fab variable domains are resulted in a 140-fold loss in binding affinity. directly connected through a short peptide linker, eliminating the Fab constant domains. In other words, the meditope bind ing pocket is eliminated in the schv, while the CDRs are minimally affected. SPR demonstrated that EGFR domain III Dissociation constants of cQFD minotope mutants and the coFD meditope bound to cetuximab Fab tethered to KD AAG a CM5 chip (See FIG. 4B). In addition, EGFR domain III Ligand (IM) (kcal/mol) bound with a minimal affinity loss to the scFv tethered to a WT 0.77 second CM5 chip. However, relative to Fab binding, the F3A 34 2.3 cQFD meditope did not saturate the sclvat concentrations as LSA 57 2.6 high as 100 uM of meditope. This indicates minimal, if any, R8A 141 3.1 affinity of the meditope for the CDRs, consistent with the L10A 2.2 O.63 crystallographic Studies. I0128 Meditope Does not Affect Cetuximab Binding to 0120 Finally, the Fab of trastuzumab, a humanized thera EGFR-Expressing Cells peutic monoclonal antibody, was coupled to a CM5 chip to I0129. Although the Fab could bind to the meditope and characterize the affinity of the coFD and coYN meditopes to EGFR domain III simultaneously, it was determined whether a human framework. Equilibrium measurements revealed the meditope could affect cetuximab binding to EGFR-ex that the dissociation constants for either meditope exceed 150 pressing cells as a full IgG. To test this, FACS analysis was uM. used to follow the binding of the IgG, as a function of medi tope concentration, to MDA MB-468 cells, which overex Example 3 press EGFR. Cells were incubated with cetuximab in the presence of increasing c(OFD meditope concentrations. Even Cetuximab Fab Binds Meditopes and EGFR Simul with meditope concentrations greater than 60 LM, no signifi taneously cant changes in cetuximab binding to the cells were observed (FIG. 5). This observation is consistent with the SEC studies 0121 Materials and Methods described above, and indicates that the meditope does not act 0122. In addition to those described in Examples 1 and 2 as an allosteric regulator of antigen binding. above, the following materials and methods were used. 0.130. Simultaneous binding to the Fab of EGFR domain 0123 Reagents. The single chain binding fragment III and the meditope is shown at concentrations significantly (scFvC225) of cetuximab was synthesized with a twenty above the K of the meditope. Like the cqFD and cqYN amino acid linker between the light chain and heavy chain. meditopes, superantigens SpA and PpI, bind to the Fab SclvC225 and soluble epidermal growth factor receptor framework region and do not affect antigen binding (Graille domain III (sEGFRdIII) were expressed in Sf9 cells and et al., 2000; Graille et all., 2001: Graille et al., 2002;Younget purified as previously described (Donaldson et al., 2009). al., 1984). US 2012/0177568 A1 Jul. 12, 2012

0131. In addition, the meditope chemical coupled to a the affinity of the meditope for the binding interface by Solid Support can be used to purify the murine chimera cetux 10-140-fold. In addition, a cetuximab scFv that binds to imab, which represents a novel method to purify murine EGFR, but lacks the Fab cavity, could not bind to the medi chimera IgGs as well as meditope-grafted gGs. See FIG. 10. tope. The meditope then binds cetuximab that is already There are several of advantages to this purification approach. bound to the antigen, as shown in by FRET, SPR and FACS First, the meditope is easily synthesized and can be readily analysis studies described in Example 5 below (also see FIG. added to common solid Supports (including magnetic beads). 13). Second, the affinity of the meditopes is easily modulated by 0.136. Since this observation, many meditope variants point mutations, which enables the fine-tuning of the purifi have been generated to establish structure-activity relation cation procedure and avoids harsh conditions such as low pH ships and to improve overall affinity for the Fab. To date, a that is commonly used to elute antibodies. Finally, as number of meditope variants have been co-crystallized with described herein, the meditopes can be made bivalent or the cetuximab Fab, and the binding affinity of each variant has multivalent (such as those described in Example 5 below) and been characterized by SPR and ITC. The structure of these used to extract intact murine or mousified human IgGs. The meditope variants, which correspond to SEQID NOs: 15-18, use of a peptide would also have additional advantages over 22-25 and 31-40, are shown in FIG. 30. Most of these struc current purification methods that use Protein A or Protein L, tures diffract beyond 2.4 A and are well refined with R and including the high cost associated with the production of RFree less than 20 and 24%, respectively (see FIG. 31). All Protein A or Protein L, limited life cycle and, potential intro have very good stereochemical values (Molprobity scores duction extraneous biological material Such as bacterial above 80%). Therefore, a large number of meditope permu pathogens. tations may be systematically and efficiently addressed to (0132 Steric Mask produce a large number of meditope permutations with the 0133. The meditope can be tethered to the N-terminus of goal of producing a high affinity meditope. either the light chain or heavy chain N-terminus of a murine I0137 Meditope Chemistry Design chimera or mousified human mAb through a flexible linker 0.138 Based on the structures and thermodynamic data (FIG. 11). The N-termini of mAb IgGs are juxtaposed to the obtained in the Examples above, multiple positions within the antigen binding site and the extension from the N-termini meditope that can be modified with non-natural amino acids through the flexible linker will sterically interfere with anti to enhance the overall binding affinity of the monovalent gen binding. By encoding a tumor specific protease site (e.g., fragment have been identified. Modifications that may be MMP9, MMP14, prostate-specific antigen (PSA) serine pro used to enhance binding affinity include, but are not limited tease or other suitable site) in the linker, the steric constraint to, a head-to-tail cyclic lactam peptide, modification of Arg8, of intramolecular masked IgG construct will be severed at modification of Phe3, modification of Leu5 and/or Leu10, the tumor site and permitantibody binding. This design prin and incorporation of hydratable carbonyl functionality (FIG. ciple would avoid binding of the intramolecularly masked 14). IgG to healthy tissues and avoid adverse side effects due to 0.139 Head-to-tail cyclic lactam peptides. Various meth off-target binding. The off-rate determination for Avidin-pep ods for cyclization of a meditope may be used to address in tide mask on Cetuximab is shown in FIG. 12. This figure vivo stability and to enable chemoselective control for sub shows that a mutivalent meditope binds with higher affinity sequent conjugation chemistry. For example, a head-to-tail than a monovalent meditope, but does not mask. lactam peptide was designed and synthesized by Solid phase peptide synthesis (SPPS) starting from Fmoc-Asp (Wang Example 4 resin LL)-Oall (FIG. 14, lower left box & FIG. 15). This Optimization of Meditopes Binding to Cetuximab variant, meditope V3, bound to cetuximab in a similar manner but with slightly reduced affinity compared to the unmodified 0134. A series of modifications to the meditope, including meditope. the incorporation of D-amino acids, non-natural amino acids 0140. The head-tail meditope platform may be advanta and different cyclization strategies to optimize the meditope geous for several reasons. First, the lactam is expected to be Fab binding affinity will be investigated (see FIG. 15). Each more stable in vivo. Second, this allows for the introduction of meditope analog may be purified and structurally character a single reactive amine functionality (e.g., Lys11) for Subse ized, and its interaction with the Fab may be analyzed by quent meditope conjugation. For example, the V3 meditope Surface plasmon resonance (SPR), isothermal titration calo variant was conjugated with fluorescein for FACS analysis rimetry (ITC), diffraction methods or a combination thereof. and note that this strategy can be applied to DOTA for future The data gleaned from this synthetic and biophysical in vivo PET imaging (FIG. 17). Further, head-tail meditope approach will be used for Subsequent modifications to opti synthesis affords the material in high yield. The structural mize binding in a reiterative manner. data indicates additional positions are amendable to cycliza 0135 Biochemical and Structural Data. As described tion, including between residues 3 and 11. Each meditope above, cyclic peptides cGFD and coYN were identified by may be characterized by ITC, SPR and diffraction methods phage display with the intention of identifying peptides that and the meditope with the highest affinity is subsequently bind to cetuximab CDR regions, thereby mimicking an EGFR modified to improve the overall affinity. Briefly, these char epitope, as part of a vaccination strategy. In an effort to acterizations are calculated as follows. rationally design tumor-activated, pro-mAbs, c0FR and 0141 Peptidelyophilized powders were suspended in 500 cQYR were co-crystallized with cetuximab Fab, and unex uL of 10 mM Tris pH 8.0 buffer and dialyzed 3 times into 1 L pectedly, the meditope bound in the cavity created by the light of HO each time. The final volume after dialysis was care and heavy chains of the Fab. Many biophysical and biochemi fully measured and absorbance measurements were taken to cal methods were used to characterize this interaction. Spe estimate the concentration (typically 1-10 mM). These stock cifically, mutation of Phe3, Leu5, and Arg8 to alanine reduced solutions were used to make dilutions into HBS-EP buffer (10 US 2012/0177568 A1 Jul. 12, 2012

mM Hepes pH 7.4, 150 mM. NaCl, 3 mM EDTA and 0.05% phenyl ring that could participate inhalogen bonding. Ahalo V/v surfactant P20) for SPR measurements. SPR measure gen bond is a relatively strong non-covalent bond, similar to ments were carried out on the GE Biacore T100 instrument a hydrogen bond but involving the interaction of a halogen using a CM5 chip with Cetuximab IgG or Cetuximab Fab Such as bromine or chlorine with an oxygen atom. For ligand immobilized using amine coupling chemistry. Ligands instance, incorporation of an ortho-, meta-, and/or para were immobilized at low levels suitable for kinetic data. Typi bromo phenyl substituent would favorably place a bromine cal kinetics SPR experiments were carried out at a flow rate of atom for halogen bonding with Tyr87 (light chain), GIn38, 30 uI/min using HBS-EP as both running and regeneration and/or Tyr94 (heavy chain) of the Fab, respectively. Many of buffer. Kinetic parameters were calculated using the Biacore these derivatives are commercially available and may be T100 Evaluation software version 2.0.1. incorporated by SPPS. 0142. All isothermal titration calorimetry experiments 0147 Modification of Leu5 and Leu10. Both of these side were performed in 100 mM Hepes, pH 7.4 at 25°C. using chains make hydrophobic contacts to the Fab (FIG. 19, right Nano ITC calorimeter (TA Instruments). In a typical experi panel; Leu10). To improve the affinity, the amount of surface ment 250 LA of protein (Fab or IgG) at 0.03-0.06 mM were area that can be buried can be extended by incorporating loaded into the colorimeter cell (163 ul or 185 ul) and the non-natural amino acids. This may be illustrated by system titrant (meditope, at 0.3-0.8 mM) was loaded into a 50 ul atically introducing natural (Phe/Tyr/Trp) and non-natural syringe. The cell solution was stirred at 250 rpm and upon analogs (e.g., 3.3-Diphenyl-L-alanine, branched alkyl, equilibration the titrant was added in 2-2.5 ul increments. extended aromatics such as napthyl, etc.) via SPPS at both Heat of the reaction was measured and the data was processed positions. For example, at position three increases the overall using Nano Analyze software (TA Instruments). Background affinity by a factor of approximately 4 to 5 (200 nM). See heat was Subtracted by averaging the last four measurements FIGS. 34-35. This also supports that mutation of residues and or by subtracting heat of reaction obtained from titration of the use of structural biology to identify regions may be used the meditope at the same concentration into buffer containing to improve the binding kinetics of the meditope. no protein. 0148 Hydratable carbonyl functionality. A meditope that 0143 Various lactam cyclization strategies can be used has hydratable carbonyl capabilities may be developed to based on different starting materials including B-Ala, 7-ami create a highly selective but irreversible interaction. Several noheptanoic acid, etc to produce different lactam ring size Fab hydroxyl-bearing side chains that surround the meditope (FIG. 14, left and middle boxes). If necessary, additional cavity may be exploited through selective trapping as their cyclization strategies such as click chemistry and olefin corresponding hemi-acetal or -ketal using a hydratable-en metathesis may be used (FIG. 14, right boxes). abled meditope. For example, Arg8 of the meditope extends 0144) Modification of Arg8. In the unmodified meditope, in proximity to Ser43 (3.5 A) and Tyr)4 (3.8 A) of the Fab Arg8 is extended, making a hydrogen bond with the heavy (FIG. 19, left panel). Incorporation of a hydratable carbonyl chain carbonyl of Asn111. The immediate area about this functionality at the end of Arg8 or Leu10 would allow for residue is hydrophobic yet solvent exposed (FIG.16A). Thus, selective formation of a serine or tyrosine hemi-acetal. Such the incorporation of a modified Arg8 residue that maintains a covalent adduct would essentially afford irreversible bind the guanidinium functionality for Fab H-bonding while ing. In addition, residues containing boronic acid may also be simultaneously introducing a hydrophobic arm to fill the cav integrated into the meditiope as an alternative to a hydratable ity may produce significant gains in binding due to entropic carbonyl group. Boronic acid plays an important role in the increases. This is supported by ligand docking calculations. structural activity of bortezamib (Velcade(R), which is used to For example, modified Arg8 (FIG. 17) represents a class of treat multiple myeloma. Representative examples of hydrat compounds that contain the requisite ambivalent functional able residues are also shown in FIG. 19 where R=CH-CHO ity. or —CH2B(OH). The analogs proposed in this study may be 0145 Modification of Phe3. Using structural data, it was modified using SPPS (52). observed that the hydroxyl group of the meditope variant 0149 Meditope-Cetuximab Binding Characterization Phe3TyrcCRYN alters the extended conformation of the Arg8 0150. Each monovalent meditope is purified to >95% side chain (see FIGS. 13C and 18). The overall affinity of this homogeneity and structurally characterized by mass spec variant for the cetuximab Fab, as determined by SPR, is trometry. Further, all peptides are dialyzed in water, their reduced. ITC measurements indicated a significant decrease concentrations measured by UV-Vis and calibrated with in entropy for this analog upon binding that was off-set by a elemental analysis, and diluted (>100x) into the appropriate favorable increase in enthalpy compared to unmodified medi buffer. Binding to cetuximab is rigorously characterized by tope (from -2.1 kCal/mol to -7.9 kCal/mol n=3) (FIG. ITC, SPR, X-ray diffraction or a combination thereof. ITC 13D). Structural data suggest the formation of a favorable measurements may be performed on a TA Instruments hydrogen bond network, with water bound to the Fab; how nanoITC, which requires only 1-2 mg of peptide per mea ever, more analogs are necessary to understand the structural Surement. In preliminary studies, the unmodified meditope basis of these entropic and enthalpic changes. While binds with similar enthalpy and entropy to the cetuximab Fab enthalpy-driven optimization has proven Successful in many fragment or the fully intact IgG. Therefore, binding measure Small-molecule approaches in drug design, there are also ments are carried out using the full IgG. For SPR measure opportunities in this particular system for engineering ments, low density and high density chips were conjugated increases in entropy as well. Consequently, approaches that with the cetuximab Fab and full IgG. Each chip was first result both in enthalpic and entropic gains in meditope characterized using a soluble fragment of the entire extracel designs may be used to optimize binding. lular domain of EGFR (residues 1-621). Similar kinetics and 0146 For example, the hydrophilic phenyl ring of Phe3 is binding affinities were observed as previously reported. surrounded by a fairly polar array of side chain residues of the Using the low density chips, on and off rates were measured Fab (FIG. 18). Therefore, halogens may be introduced on the for the unmodified meditope and determined to be k=9.2x US 2012/0177568 A1 Jul. 12, 2012

10 'M' sec' and k-9.9x10 sec', respectively. Consis geometry of the bivalent/trivalent/multivalent receptor. The tent with ITC, similar values for the Fab-conjugated chip and geometry of the receptor places strict constraints on the the IgG-conjugated chip were observed. linker, but it also ensures specificity, which is an important 0151. Finally, each meditope-cetuximab interaction may goal for targeted delivery. be characterized by diffraction methods. In preliminary stud 0156 To address the receptor constraints on the linker, the ies, diffraction data was collected for 20 different meditope unmodified or optimized meditopes obtained in Example 4 Fab complexes, including the original c(RFD and coYN may be coupled to a multivalent scaffold. To accomplish this, meditopes and modified meditopes. Since the co-crystalliza the linker is optimized. Because tumor cells have high antigen tion conditions of the Fab and meditope are now well-estab density, a multivalent meditope should “latch-on' to adjacent lished, diffraction quality crystals are typically obtained in 1 IgGs to form a "daisy-chain’-like array (FIG. 8). While an to 3 days. Full data sets are collected in 8 to 12 hours with an intramolecular association of a bivalent meditope and IgG is in-house source (Rigaku 007-HF and an R-Axis IV++) and in possible, the C2 symmetry of the IgG would place severe 10 min at the Stanford Synchrotron Radiation Lightsource, geometrical constraints on the linker for Such an interaction. which allows for rapid characterization of the interactions of A trivalent or higher valency scaffold ensures that more than the meditope variants with cetuximab. one antibody would be "daisy chained. By including a third 0152 Collectively, ITC, SPR and X-ray diffraction data meditope arm, the lifetime of the initial encounter of a triva provide the atomic detail necessary to guide Subsequent lent meditope to antigen-bound antibody will increase. This, chemical modifications and ultimately improve the affinity of in turn, will increase the probability that an additional arm the meditope. A simple calculation based on AG=-RT ln Ka will bind to a neighboring antigen-bound antibody, thus sta shows that the difference between micromolar and nanomolar bilizing the overall complex. affinity results from a change in free energy at 300 K of ~4 (O157 Scaffold Synthesis kCal/mol, which is on the order of a strong hydrogen bond. 0158 Synthesis of a FITC-labeled bivalent meditope was Thus, the loss of an ordered water molecule from a protein developed based on “Click' chemistry using (compound 2 or binding pocket or the reorientation of an amino acid residue SEQID NO:31) (FIG. 20). The use oftemplates 4 and 5 (FIG. chain may be sufficient to alter binding by orders of magni 20) allows for the formation of both bi- and trivalent medi tude. topes, respectively. This synthesis represents an exciting 0153. At least two alternative approaches may be used to advance since the present discovery describes the chemistry improve the affinity of the meditope-Fab interaction. First, developed for the preparation of multivalent meditopes and the structural data obtained in the studies described above allows focus on investigating differing length polyethylene may be used to replace residues in the Fab, by mutagenesis, to glycol (PEG) (and other) linkers for optimal binding. The add additional hydrogen bonds, Substitute amino acids for synthetic approach is also amenable to DOTA incorporation unnatural amino acids or alter the hydrophobic interface that for radionuclide imaging. For example, a 30 A PEG bifunc might better complement meditope binding. (See FIGS. 29 tional arm has been incorporated in the synthesis of a FITC and 29). Second, fluorescence polarization assays may be labeled divalent meditope, namely compound 13 (FIG. 20). used to identify Small molecules that can displace the medi The distance between the CDR regions within an IgG is ~130 tope, and use these Small molecules as templates to further A. Therefore, the length of the PEG linker may be systemati improve the binding affinity. cally varied to ensure this approach is optimal. End-to-end distances of commercially available PEGs extend to 90 A Example 5 (Pierce), which would exceed the IgG distance. 0159 Multivalent Characterization Generation of Multivalent Meditopes 0160 Each multivalent meditope is characterized by SPR 0154 Combinations of monoclonal antibodies (mAbs) and ITC to ensure that conjugation to the multivalent scaffold that recognize unique epitopes on the same antigen (e.g., does not affect the meditope-IgG interaction. These measure EGFR) have been shown to enhance cell death and inhibit ments, however, are limited in their effectiveness in determin tumor growth. While the precise mechanism of this enhanced ing mutlivalency since the IgG is not bound to the Surface of cell death remains debated (immunological response versus a tumor. Instead, FACS analysis and cell viability assays may receptor down regulation versus enhanced ligand antago be used to quantify the effect of the multivalent meditope nism), previous studies indicate that both Mabs should be directly on cells that overexpress EGFR. multivalent (e.g., full IgG or F(ab)') to achieve enhanced cell 0.161 For FACS analysis, cell lines that overexpress death. As such, a multivalent meditope may be substituted as EGFR (MDA-MB-468 and A431) are incubated with cetux a second antibody as shown below. Therefore, the meditopes imab at various concentrations (1 nM to 100 nM). Next, describe herein may be tethered to a scaffold to create a cetuximab-treated cells are incubated with the labeled multi multivalent meditope for enhanced selectivity and binding valent meditope (FIG. 24; Meditope-Fc) at increasing con affinity. centrations (0.1 nM to 1 LM) and analyze the binding char 0155 Specificity and affinity are often achieved through acteristics using a CyAn FACS sorter. A shift at far lower multivalency. This can be expressed as AG, AG1+AG2 concentrations than observed for the monovalent meditope AG for a bivalent ligand, which is equivalent to (FIG. 24) and/or an increase in percentage of cells that shift KKK/K. In the case where the linker makes no may be observed. To further confirm the additive effects contribution to the free energy (K-1), the apparent affin expected for the multivalent meditope, the non-labeled, ity of the bivalent ligand for the bivalent target is the product monovalent meditope may be used to compete with the of the monomeric binding constants. Thus, significant gains labeled multivalent meditope for the antigen-bound cetux in affinity can be achieved (e.g., for a meditope with K-1 imab. uM, the affinity of a theoretical bivalent meditope is 1 pM. 0162 The efficacy of the multivalent meditope at enhanc Such gains, however, are rarely seen, primarily due to the ing cetuximab-mediated cell death using cell viability assays US 2012/0177568 A1 Jul. 12, 2012

may also be measured. Briefly, MDA-MB-468 and A431 cell of bivalent Meditope-Fc or monomeric Meditope for 30 min, lines are plated and treated with varying concentrations of room temperature, washed and analyzed by FACS. As shown cetuximab and multivalent meditopes. As a control, a in FIG. 24, FACS analysis indicates that the meditope-Fc. monovalent meditope is used, which produces similar results corrected for the stoichiometry, binds cells pre-treated with as cetuximab alone. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- cetuximab with significantly higher affinity. Furthermore, we diphenyltetrazolium bromide, may be used to quantify the demonstrate that this interaction is specific to the meditope number of viable cells. For multivalent meditopes that dem enabled mAb (cetuximab). This data indicates that the medi onstrate activity, Western blot analysis may be performed to tope-Fc combined with a meditope enabled mAb is synergis follow the phosphorylation status of EGFR as well as AKT tic and can be substituted for a second antibody. and MAP which are part of the EGFR signaling pathway. 0168 Different scaffolds of biological and chemical ori These data are then compared with data from cetuximab-only gin may also be used to achieve multivalency. This includes, treated cells and cells treated with tyrosine kinase inhibitors but is not limited to, constructing a bivalent or trivalent scaf (AG 1478). Collectively, this should cause an increase in cell fold, using streptavidin or collagen (http://ip.com/patapp/ death as a function of multivalent meditope concentration. EP2065402A1), strepavidin as a tetravalent scaffold, unique (0163 MTT Assay scaffolds (http://www.sciencedirect.com/science/article/pii/ 0164. The effect of the monomeric meditope or the biva S002228361 1000283), Origami DNA (http://www.nature. lent meditope-Fc on inducing cell death in conjunction with com/nnano/journal/v4/na/abs/nnano.2009.5.html) and the cetuximab was investigated using an MTT assay. 4000 MDA like. A chemical scaffold may also be created using molcules MB-468 cells were placed in each well of a 96 well plate in 80 including, but not limited to, DNA (single strand, duplex, ul of medium. 10ul of 1 uM cetuximab is added along with 10 Holliday junctions, aptamers and the like), RNA (single ul of 0.1, 1 or 10 uM of meditope or meditope-Fc to a final Strand, hairpin, stem loop, aptamers and the like), PNA (pep concentration of 0.1 uM cetuximab and 0.01, 0.1 and 1 uM tide nucleic acids), DNA/PNA duplexes and triplexes for meditope or meditope-Fc. Each component was also added rigidity, nanoparticles (directly coupled or coupled through alone with PBS as control. After a 48-hour incubation, 10 ul organic polymers such as PEG), organic polymers that can of MTT reagent was added and let incubate for another 4 form duplexes with themselves and/or with DNA or PNA. For hours. The culture supernatant was then removed, 100 ul of example, in FIGS. 20 and 21, a trivalent meditope was suc MTT crystal dissolving reagent was added, and the plate was cessfully synthesized. read at 630 nm. Meditope or meditope-Fc alone did not alter 0169. In addition, the composition of and the distance cell growth significantly, but only meditope-Fc and not the between the Fc and meditope can be systematically explored monomeric meditope could inhibit cell growth together with to optimize affinity and specificity. In one embodiment, each cetuximab (FIG. 32A). natural or unnatural residue can be substituted at any position 0.165. To show that a multivalent meditope has efficacy in within the linker for optimization. In addition, a linker enhancing cell killing akin to the effect of a second anti between 2 to 100 or more residues is possible (e.g., can be EGFR antibody in addition to cetuximab, the effect of tumor generated by current and future DNA synthesizers and cell growth inhibition by either cetuximab with medito-Fc or inserted between the meditope and the Fc regions. The linker cetuximab with M425 was compared. 4000 MDA-MB-468 may also be rigidified to limit the radius of gyration and to cells were placed in each well of a 96 well plate in 80 ul of enhance the affinity and specificity of the Fc-meditope. For medium. 10 ul of 1 uM cetuximab is added along with 10 ul example, a coiled coil domain may be placed between the of either 2, 4 or 8 uM of meditope-Fc or M425 to a final meditope and the Fc (FIG. 25). Alternatively, inert protein concentration of 0.1 uM cetuximab and either 0.2,0.4 or 0.8 domains (e.g., immunoglobulin folds) may be substituted for uM meditope-Fc or M425. Cetuximab added along with PBS the linker. Multiple immunoglobulin folds can be placed was used as control. After a 48-hour incubation, 10ul of MTT between the meditope and the Fc domain. In certain embodi reagent was added and let incubate for another 4 hours. The ments, the composition of the linker is of human origin to culture supernatant was then removed, 100 ul of MTT crystal mitigate potential antigenicity. dissolving reagent was added, and the plate was read at 630 nm. Meditope-Fc could be seen enhancing the cell-killing Example 6 capacity of cetuximab, although not as yet potent as M425 (FIG.32B). Generation of Meditope Variants 0166 As an alternate approach, different scaffolds and 0170 Screening methods may be used to identify small linkers may be used to generate high affinity multivalent molecule analogs that mimic the meditopes described herein. meditopes. For example, DNA may be used as a scaffold for This includes fluorescence polarization assays, diffraction the meditopes to create a more rigid scaffold. based and NMR based fragment screening, and tethering 0167. The meditope (wildtype) was fused to the N-termi dynamic combinatorial methods. nus of the Fc region of an IgG through a flexible peptide linker 0171 Fluorescence polarization assays: To identify alter (“meditope-Fc') (FIG. 23: SEQ ID NO:3-4). The sequence native molecules that can bind at the meditope site and be and structure of the meditope-Fc are shown in FIGS. 22 and used for similar functions, a fluorescent marker (e.g., Alex 23, respectively. The use of the Fc region to dimerize ligands afluor, rhodamine, fluorescein) may be conjugated to the is been well established (http://www.ncbi.nlm.nih.gov/ original meditope using a Suitable method (e.g., amines, Sulf pubmed/18215087). In this example, a linker 17 amino acids hydryl, carboxylate, Sugars or other known methods) and long consisting of glycine and serines was chosen, but a linker allowed to interact with the Fab or mAb. The interaction of any Suitable length may be used. To demonstrate enhanced between the labeled meditope and mAb causes a change in the binding due to multivalency, 0.5x10 MDA-MB-468 cells fluorescence polarization/intensity of the fluorescent tag. were labeled with 10 nM Cetuximab for 30 min at room Once established, small molecule compounds (MW<1000 temperature, washed, then incubated with 0.1.0.3, 1 and 3 uM Dal) are added and equilibriated with fluorescent tagged US 2012/0177568 A1 Jul. 12, 2012

meditope-antibody complex and the fluorescence polariza action is not known. The binding site can be determined by tion is monitored. Compounds that block the meditope-anti NMR or the fluorescence polarization assay. Alternatively, body interaction will alter the fluorescent polarization prop the Fab fragment can be labeled with NMR active and inac erties. Accordingly, another embodiment is a method of tive nuclei (e.g., 'C, N and H), multiple NMR experi identifying compounds that can be optimized and used for ments performed to assign the spectrum, and then used with target delivery. the fragment library to identify the binding position. Using 0172 Methods this procedure, a set of initial lead compounds has been iden 0173 The cyclic meditope was synthesized and chemi tified (FIG. 29, bottom). cally attached to a fluorescein having the following sequence: 0177 Virtual ligand screening: Virtual ligand screening is CQFDLSTRRLKCGGSK-Fluorescein (SEQ ID:57: cys another method that can be used to identify lead compounds teines form a disulfide). The fluorescent-labeled peptide was to function as a meditope. Using our crystal structure, stan thentitrated with cetuximab and the fluorescence polarization dard programs (e.g., Schroerdinger Glide) can define a “box was measured. The dissociation constant, 1 LM, closely about a site of a macromolecule (the meditope binding site) matched values obtained from Surface plasmon resonance and dock known ligands to this site. Potential lead com and isothermal titration calorimetry. Next, a non-labeled pounds are scored by a select energy function and the top 50 meditope, CQFDLSTRRLKCGGSK (SEQID NO:57), was to 200 compounds can be purchased. In our initial studies, used to displace the fluoroscein-label peptide pre-bound to approximately 100 lead compounds have been identified, and Cetuximab (FIG. 26). A sigmoid curve indicative of a com using crystallography, these lead compounds should be petition reaction was observed. Based on these data, an initial shown to demonstrate that they bind to the meditope site. screen to identify a small molecule capable of displacing the meditope was initiated. In these preliminary studies, 42 lead REFERENCES compounds at concentrations of 50 uM were identified from a library of 30,000 small molecule compounds (see Tables 1 0.178 All references below and cited in the specification and 2 above). FIG. 27 shows five such lead compounds. above are hereby incorporated by reference in their entirety, Further characterization of these compounds is underway as if fully set forth herein. (e.g., crystallography) (0179 1. Accardi, L., and Di Bonito, P. (2010) Antibodies 0.174 Diffraction methods to identify meditope analogs. in single-chain format against tumour-associated antigens: Diffraction based methods to identify lead compounds are present and future applications, Curr Med Chem 17, 1730 well established (Shuker et al. 1996; Erlanson et al. 2001; 1755. Hughes et al. 2011). Since cetuximab Fab diffracts beyond 0180 2. Adams, G. P. Schier, R., McCall, A. M., Sim 2.5 A, this approach is viable to identify lead compounds or mons, H. H., Horak, E. M., Alpaugh, R. K., Marks, J. D., Small molecule fragments that can be coupled to a meditope. and Weiner, L. M. (2001) Cancer Res 61, 4750-4755. In combination with these methods, a library of small mol 0181 3. 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Opti (2002) Acta CrystallogrDBiol Crystallogr58, 1948-1954. mization of this chemical coupling can significantly enhance 0183) 5. Adessi, C., and Soto, C. (2002) Converting a the overall binding affinity. Additional analogs found by dif peptide into a drug: strategies to improve stability and fraction methods can be optimized and used in lieu of the bioavailability, Curr Med Chem 9,963-978. meditope for drug delivery, multivalent scaffolding and other 0.184 6. Akamatsu, Y., Pakabunto, K., Xu, Z., Zhang, Y., functions. Further, mutations in the light and heavy chains and Tsurushita, N. (2007) Whole may be made to change the specificity of the ligand (medi 0185 7. 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SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 4 O

<21 Os SEQ ID NO 1 &211s LENGTH: 12 US 2012/0177568 A1 Jul. 12, 2012 23

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: unmodified coFD meditope <4 OOs, SEQUENCE: 1 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 2 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: unmodified coYN meditope <4 OOs, SEQUENCE: 2 Cys Glin Tyr Asn Lieu. Ser Ser Arg Ala Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 3 &211s LENGTH: 867 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: meditope-fc tethering agent

<4 OOs, SEQUENCE: 3 acacccaa.gc tiggctagoga cac catgaag titagctggg to atcttct t t ctgatggca 6 O gtcgtgacag gagtgaattic gtgcc agttt gacctgtcaa Ctcggcgact gaaatgcggit 12 O gggggctic.cg gttcaggctic gigg.cggttca togggaggag gaggagggga acctaagttca 18O tgcgataaga cqcacacctg. tcct c catgc C cagc.ccc.cg agttgcttgg toggc cct ca 24 O gtattoctict tcc ctic caaa acc caaagac accttgatga titt cocqcac gcc.ggaagtic 3OO acgtgtgtgg togtggatgt gagcc atgag gatcc.cgagg taagttcaa ttggtacgtg 360 gatggagtag agg tacacaa cqcgaaaacg aagcc caggg aggalacagta caatticcaca 42O tatic gcgtgg tt cogtgct tactgttgttg catcaag act ggctgaatgg gaaggagtat 48O aagtgcaaag tat caaacaa ggcgctgcct gct coaatcg aaaagac cat Ctcgaaggcg 54 O aaagga caac C cagagaacc C caagttctac acgct tcc.gc cct cqcggga tigagctic acc 6OO aaaaac cagg tat coct cac ttgtttggta aaaggattct accc.gtcgga cattgcagtic 660 gagtgggagt caatgggca gcc.ggaaaac alactacaaaa Calacaccgcc cqtcttggac 72 O tccgatggitt C9ttctitt Ct c tatt.cgaag ct caccgtag acaagtic gag gtggcagcag 78O ggcaacgt.ct titt cqtgctic agtgatgcat gaggc cc titc acaat cact a tacgcagaaa 84 O agcctgagcc tt Caccggg galagtaa 867

<210s, SEQ ID NO 4 &211s LENGTH: 288 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: meditope-Fo tethering agent

<4 OOs, SEQUENCE: 4 Thr Pro Llys Lieu Ala Ser Asp Thr Met Lys Cys Ser Trp Val Ile Phe 1. 5 1O 15 US 2012/0177568 A1 Jul. 12, 2012 24

- Continued Phe Leu Met Ala Val Val Thr Gly Val Asin Ser Cys Glin Phe Asp Leu 2O 25 3O Ser Thr Arg Arg Lieu Lys Cys Gly Gly Gly Ser Gly Ser Gly Ser Gly 35 4 O 45 Gly Ser Ser Gly Gly Gly Gly Gly Glu Pro Llys Ser Cys Asp Llys Thr SO 55 6 O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 65 70 7s 8O Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 85 90 95 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 1OO 105 11 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 115 12 O 125 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 13 O 135 14 O Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 145 150 155 160 Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 1.65 17O 17s Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 18O 185 19 O Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val Ser Lieu. Thir Cys 195 2OO 2O5 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 21 O 215 22O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 225 23 O 235 24 O Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 245 250 255 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 26 O 265 27 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 27s 28O 285

<210s, SEQ ID NO 5 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gln is D-glutamine

<4 OOs, SEQUENCE: 5 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 6 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence US 2012/0177568 A1 Jul. 12, 2012 25

- Continued <4 OOs, SEQUENCE: 6 Cys Glin Tyr Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 7 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is beta-beta-di-phenyl-Ala

<4 OO > SEQUENCE: 7 Cys Glin Xaa Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 8 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is beta-beta-di-phenyl-Ala <4 OOs, SEQUENCE: 8 Cys Glin Phe Asp Xaa Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Xaa is citrulline

<4 OOs, SEQUENCE: 9 Cys Glin Phe Asp Phe Ser Thr Arg Xaa Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 10 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 10 Cys Glin Phe Asp Phe Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence US 2012/0177568 A1 Jul. 12, 2012 26

- Continued

<4 OOs, SEQUENCE: 11 Cys Glin Phe Asp Glu Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 12 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 12 Cys Glin Phe Asp Tyr Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 13 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 13 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Gln Lys Cys 1. 5 1O

<210s, SEQ ID NO 14 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 14 Cys Glin Phe Asp Lieu. Ser Thr Arg Glin Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 15 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 15 Cys Glin Tyr Asn Lieu. Ser Thr Ala Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 16 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 16 Cys Glin Ala Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 17 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2012/0177568 A1 Jul. 12, 2012 27

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 17 Cys Glin Phe Asp Ala Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 18 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 18 Cys Glin Phe Asp Lieu. Ser Thr Ala Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 19 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 19 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Ala Lys Cys 1. 5 1O

<210s, SEQ ID NO 2 O &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 2O Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Glu Lys Cys 1. 5 1O

<210s, SEQ ID NO 21 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 21 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys Gly Gly Ser Lys 1. 5 1O 15

<210s, SEQ ID NO 22 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 22 Gly Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 23 &211s LENGTH: 12 US 2012/0177568 A1 Jul. 12, 2012 28

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 23 Gly Gln His Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 24 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence <4 OOs, SEQUENCE: 24 Gly Glin Asn Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 25 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OOs, SEQUENCE: 25 Gly Glin Glin Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 26 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is 2-bromo-L-phenylalanine

<4 OOs, SEQUENCE: 26 Gly Glin Xaa Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 27 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is 3-bromo-L-phenylalanine

<4 OOs, SEQUENCE: 27 Gly Glin Xaa Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1. 5 1O

<210s, SEQ ID NO 28 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2012/0177568 A1 Jul. 12, 2012 29

- Continued OTHER INFORMATION: modified meditope variant sequence FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: Xaa is 4-bromo-L-phenylalanine

SEQUENCE: 28 Gly Glin Xaa Asp Lieu. Ser Thr Arg Arg Lieu Lys Gly 1.

SEQ ID NO 29 LENGTH: 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: modified meditope variant sequence FEATURE: NAMEAKEY: misc feature LOCATION: (9) ... (9) OTHER INFORMATION: Xaa is citrulline

SEQUENCE: 29 Gly Glin Phe Asp Lieu. Ser Thr Arg Xaa Lieu Lys Gly 1.

SEQ ID NO 3 O LENGTH: 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: modified meditope variant sequence FEATURE: NAMEAKEY: misc feature LOCATION: (8) ... (9) OTHER INFORMATION: Xaa is citrulline

SEQUENCE: 3 O Gly Glin Phe Asp Lieu. Ser Thr Xaa Xala Lieu Lys Gly 1.

SEQ ID NO 31 LENGTH: 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: modified meditope variant sequence FEATURE: NAMEAKEY: misc feature LOCATION: (8) ... (8) OTHER INFORMATION: Xaa is citrulline

SEQUENCE: 31 Gly Glin Phe Asp Lieu. Ser Thr Xaa Arg Lieu Lys Gly 1.

SEQ ID NO 32 LENGTH: 11 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: modified meditope variant sequence FEATURE: NAMEAKEY: misc feature LOCATION: (11) ... (11) OTHER INFORMATION: Xaa is 7-aminoheptanoic acid

SEQUENCE: 32 US 2012/0177568 A1 Jul. 12, 2012 30

- Continued Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 33 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is beta-alanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Xaa is beta-alanine

<4 OOs, SEQUENCE: 33 Xaa Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 34 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAME/KEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is diaminopropionic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Xaa is iso-aspartic acid <4 OOs, SEQUENCE: 34 Xaa Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 35 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is beta-alanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Xaa is iso-aspartic acid <4 OOs, SEQUENCE: 35 Xaa Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 36 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) US 2012/0177568 A1 Jul. 12, 2012 31

- Continued <223> OTHER INFORMATION: Xaa is diaminopropionic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Xaa is beta-alanine

<4 OOs, SEQUENCE: 36 Xaa Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 37 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence

<4 OO > SEQUENCE: 37 Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys 1. 5

<210s, SEQ ID NO 38 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence < 4 OO > SEQUENCE: 38 Cys Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Cys 1. 5 1O

<210s, SEQ ID NO 39 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa is 7-aminoheptanoic acid <4 OOs, SEQUENCE: 39 Glin Tyr Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O

<210s, SEQ ID NO 4 O &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: modified meditope variant sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is beta-Azidoalanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Xaa is propargylglycine

<4 OOs, SEQUENCE: 4 O Xaa Glin Phe Asp Lieu. Ser Thr Arg Arg Lieu Lys Xaa 1. 5 1O US 2012/0177568 A1 Jul. 12, 2012 32

1) A meditope variant comprising a peptide having one or conjugated to one or more therapeutic Substances, imaging more modifications of at least one amino acid residue of the Substances or a combination thereof. sequence CQFDLSTRRLKC (SEQID NO: 1) or CQYNLSS 13) The composition of claim 12, wherein the therapeutic RALKC (SEQID NO:2). agent is one or more of a small molecule, a chemotherapeutic agent, a therapeutic antibody or functional fragment, a toxin, 2) The meditope variant of claim 1, wherein the one or a radioisotope, an enzyme, a nuclease, a hormone, an immu more modifications are single, double, or triple point muta nomodulator, an oligonucleotide, an RNAi molecule, a chela tions of SEQ ID NO: 1 or SEQID NO: 2. tor, a boron compound, a photoactive agent, a dye or a com 3) The meditope variant of claim 1, wherein the one or bination thereof. more modifications are selected from one or more of head-to 14) The composition of claim 12, wherein the imaging tail lactam cyclic peptides, a linear peptide, a modification of Substance is one or more of a fluorescent or luminescent Arg8, a modification of Phe3, a modification of Leu5, a modi Substance, an enzyme, an enhancing agent, a radioactive Sub fication of Leu10, an incorporation of an unnatural amino stance, a chelator, or a nanoparticle. acid, a shortening or lengthening of a bond, an incorporation 15) The composition of claim 12, wherein the therapeutic of an alkylator, and an incorporation of hydratable carbonyl substance is associated with a CovX-BodyTM. functionality. 16) A method of purifying a monoclonal antibody or func 4) The meditope variant of claim 1 selected from one of tional fragment thereof, comprising: SEQID NO:5 through SEQID NO:40. coupling a meditope variant to a solid Support; 5) A multivalent meditope variant tethering entity compris contacting the coupled meditope variant with a solution ing two or more meditopes coupled via a long linker, multi containing or Suspected of containing the monoclonal Valent scaffold, biotin-streptavidin, a nanoparticle, a poly antibody or functional fragment thereof such that the nucleotide sequence, peptide nucleic acid, an organic monoclonal antibody or functional fragment thereof polymer, or IgGFc domain. binds the meditope. 6) The multivalent meditope tethering entity of claim 5, 17) The method of claim 16, wherein the meditope variant wherein the two or more meditopes comprise two or more comprises a peptide having a sequence of any of SEQ ID peptides, each having a sequence CQFDLSTRRLKC (SEQ NO:5-40. ID NO: 1) or CQYNLSSRALKC (SEQID NO:2) that has one 18) The method of claim 16, wherein the monoclonal anti or more modifications of at least one amino acid residue of the body is a meditope-enabled therapeutic antibody selected Sequence. from , abciximab, adalimumab, adecatumumab, 7) The multivalent meditope tethering entity of claim 5, alemtuzumab, atlizumab, altumumab pentetate, anatumumab wherein the multivalent meditope tethering entity is a biva mafenatoX, arcitumumab, basiliximab, bectumumab, ben lent, trivalent or tetravalent meditope tethering entity used to ralizumab, capromab pendetide, catumaxomab, certoli tether two or more therapeutic antibodies or functional frag Zumab, cetuximab, clivatuZumab tetraxetan, daclizumab, ments thereof. denosumab, eculizumab, efalizumab, etaracizumab, etru 8) The multivalent meditope tethering entity of claim 5, maxomab, fanolesomab, FBTA05, fontolizumab, gemtu wherein the multivalent meditope tethering entity enhances Zumab, girentuximab, golimumab, ibritumomab, igovomab, binding stability and/or efficacy of a meditope-enabled anti infliximab, ipilimumab, labetuZumab, mepolizumab, muromonab-CD3, natalizumab, nimotuzumab. ofatumumab, body or functional fragment thereof. omalizumab, , palivizumab, panitumumab, 9) The multivalent meditope tethering entity of claim 8. ranibizumab, rituximab, tiu Zetan, to situmomab, trastu wherein the meditope-enabled antibody is a meditope-en Zumab, TRBS07, ustekinumab, visilizumab, votumumab, abled therapeutic antibody selected from the group of abago Zalutumumab or functional fragment thereof. Vomab, abciximab, adalimumab, adecatumumab, alemtu 19) A method of treating, imaging or diagnosing a disease Zumab, atlizumab, altumumab pentetate, anatumumab or condition comprising administering a therapeutically mafenatoX, arcitumumab, basiliximab, bectumumab, ben effective amount of a pharmaceutical composition to a Sub ralizumab, capromab pendetide, catumaxomab, certoli ject, the pharmaceutical composition comprising an anti Zumab, cetuximab, clivatuZumab tetraxetan, daclizumab, body-meditope complex; a multivalent tethering agent in denosumab, eculizumab, efalizumab, etaracizumab, etru combination with a monoclonal antibody or functional frag maxomab, fanolesomab, FBTA05, fontolizumab, gemtu ment thereof; or a combination thereof, wherein the antibody Zumab, girentuximab, golimumab, ibritumomab, igovomab, meditope complex and the multivalent tethering agent com infliximab, ipilimumab, labetuZumab, mepolizumab, prise a meditope variant. muromonab-CD3, natalizumab, nimotuzumab. ofatumumab, 20) The method of claim 19, wherein the antibody-medi omalizumab, oregovomab, palivizumab, panitumumab, tope complex; the multivalent tethering agent in combination ranibizumab, rituximab, tiu Zetan, to situmomab, trastu with a monoclonal antibody or functional fragment thereof. Zumab, TRBS07, ustekinumab, visilizumab, votumumab, or a combination thereof is conjugated to one or more diag Zalutumumab or functional fragment thereof. nostic or imaging agents for diagnosing or imaging the dis 10) The multivalent meditope tethering entity of claim 5, ease or condition, one or more therapeutic agents for treating further comprising an imaging agent, a therapeutic agent, a the disease, or a combination thereof. nanoparticle, or both. 21) The method of claim 20, wherein the diagnostic or 11) The multivalent meditope tethering entity of claim 5, imaging agent is one or more of a fluorescent or luminescent wherein the imaging agent, therapeutic agent or both is a Substance, an enzyme, an enhancing agent, a radioactive Sub metal chelator bound to a metalion. stance, or a nanoparticle. 12) A composition comprising a meditope or meditope 22) The method of claim 20, wherein the therapeutic agent variant of one or more of SEQ ID NOS: 1, 2, and 5-40 is one or more of a small molecule, a chemotherapeutic agent, US 2012/0177568 A1 Jul. 12, 2012

a therapeutic antibody or functional fragment, a toxin, a labetuZumab, mepolizumab, muromonab-CD3, natalizumab, radioisotope, an enzyme, a nuclease, a hormone, an immu nimotuZumab. ofatumumab, omalizumab, oregovomab, nomodulator, an oligonucleotide, a nanoparticle, an RNAi palivizumab, panitumumab, ranibizumab, rituximab, molecule, a chelator, a boron compound, a photoactive agent, tiuzetan, to situmomab, trastuzumab, TRBS07, ustekinumab, Visilizumab. Votumumab, Zalutumumab or functional frag a dye or a combination thereof. ment thereof. 23) The method of claim 19, wherein the antibody-medi 25) The method of claim 24, wherein the monoclonal anti tope complex; the multivalent tethering agent in combination body is a meditope-enabled monoclonal antibody. with a monoclonal antibody or functional fragment thereof. 26) The method of claim 19, wherein the multivalent teth or a combination thereof enhances the treatment, diagnostic ering agent is administered after administering a therapeuti or imaging efficacy of a therapeutic antibody alone. cally effective amount of cetuximab to the subject or at the 24) The method of claim 19, wherein the antibody-medi same time as administering a therapeutically effective tope complex comprises a meditope and a meditope-enabled amount of cetuximab to the Subject. monoclonal antibody, wherein the meditope-enabled mono 27) A method of targeting cells overexpressing a cancer clonal antibody or functional fragment thereof is a therapeu biomarker, comprising administering atherapeutic amount of tic antibody selected from abagovomab, abciximab, adali a meditope-enabled monoclonal antibody or a functional mumab, adecatumumab, alemtuzumab, atlizumab, fragment thereof in combination with a meditope variant or a altumumab pentetate, anatumumab mafenatoX, arcitu multivalent tethering agent to a population of cells overex mumab, basiliximab, bectumumab, benralizumab, capromab pressing or Suspected of overexpressing the biomarker. pendetide, catumaxomab, certolizumab, cetuximab, clivatu 28) The method of 27, wherein the cancer biomarker is Zumab tetraxetan, daclizumab, denosumab, eculizumab, EGFR and the meditope-enabled monoclonal antibody is efalizumab, etaracizumab, etrumaXomab, fanolesomab, cetuximab. FBTA05, fontolizumab, gemtuzumab, girentuximab, goli mumab, ibritumomab, igovomab, infliximab, ipilimumab,