EGFR ANTIBODY CONJUGATES

Abstract

A maytansinoid is covalently linked through a non-cleavable linker to an EGFR antibody that is a full EGFR antagonist, such as cetuximab or panitumumab. The result is an anti-cancer agent having cytotoxicity that is potentiated in cancer cells but not normal cells. This benefit is not seen with EGFR antibodies that are partial antagonists, or with toxins that are not processed by lysosomes.

Claims

1. A method useful to potentiate the cytotoxicity of an EGFR antibody or an EGFR-binding fragment thereof on EGFR+ disease cells without potentiating the cytotoxicity thereof on normal EGFR+ cells, the method comprising: (i) selecting, for conjugation, an EGFR antibody that is a full EGFR antagonist and competes with cetuximab for binding to EGFR, or an EGFR-binding fragment of said antibody; (ii) selecting, for delivery by the EGFR antibody or fragment thereof, an anti-microtubule toxin; (iii) selecting, for coupling the selected EGFR antibody and the anti-microtubule toxin, a linker; and producing an immunoconjugate that incorporates the linker between the antibody and the toxin, thereby providing an immunoconjugate having a cytotoxicity that is potentiated against EGFR+ disease cells and essentially not potentiated against EGFR+ keratinocyte cells.

2. The method according to claim 1, wherein the selected full antagonist EGFR antibody is cetuximab or panitumumab.

3. The method according to claim 1, wherein the selected anti-microtubule toxin is a maytansinoid.

4. The method according to claim 1, wherein the selected linker is a non-cleavable linker, preferably SMCC.

5. An immunoconjugate comprising (i) a full antagonist EGFR antibody that binds to an EGFR epitope to which cetuximab binds, or an EGFR binding fragment of said antibody or single chain polypeptide based on said antibody, and (ii) an anti-microtubule toxin conjugated therewith, the immunoconjugate having a cytotoxic effect relative to a naked form of said antibody that is (1) potentiated with respect to EGFR+ cancer cells, and (2) substantially unaltered with respect to EGFR+ keratinocytes, wherein the antibody and toxin are conjugated by a linker.

6. The immunoconjugate according to claim 5, wherein the antibody is cetuximab or cetuximab variant comprising one, two or more benign substitutions in the constant region.

7. The immunoconjugate according to claim 5, wherein the antibody is panitumumab.

8. The immunoconjugate according to claim 5, wherein the anti-microtubule toxin is a maytansinoid.

9. The immunoconjugate according to claim 8, wherein the anti-microtubule toxin is DM-1.

10. The immunoconjugate according to claim 5, wherein the linker is a non-cleavable linker.

11. The immunoconjugate according to claim 5, which is cetuximab-SMCC-DM1.

12. The immunoconjugate according to claim 5, which is panitumumab-SMCC-DM1.

13. A pharmaceutical composition comprising: the immunoconjugate of claim 5 in an amount cytotoxic to EGFR+ disease cells, and a pharmaceutically acceptable carrier.

14. A method for producing an anti-cancer composition, comprising the step of combining a pharmaceutically acceptable carrier, and an EGFR antibody that is conjugated to an anti-microtubule toxin to form an immunoconjugate, the immunoconjugate having an effect on cancer cells that is potentiated and an effect on keratinocytes that is essentially not potentiated, relative to the effects of the naked antibody on such cells.

15. (canceled)

16. A method for treating a subject presenting with an EGFR+ disease cell, the method comprising: administering to the subject an amount of an immunoconjugate according claim 5 that is cytotoxic to the EGFR+ disease cell.

17. The method according to claim 16, wherein the EGFR+ disease cell is an EGFR+ cancer cell, a head and neck or colorectal cancer cell.

18. A method for potentiating the effect of a full antagonist EGFR antibody on EGFR+ disease cells without potentiating the effect thereof on normal EGFR+ cells, comprising linking the antibody to an anti-microtubule toxin by a non-cleavable linker.

19. In a method for treating a subject presenting with a tumor that responds to treatment with a full antagonist EGFR antibody, wherein treatment therewith elicits an EGFR antibody-mediated adverse response by keratinocytes, the improvement comprising: treating the subject with the full antagonist EGFR antibody in a form conjugated with an anti-microtubule toxin, whereby the tumor response to treatment with conjugated antibody is enhanced essentially without enhancing the adverse keratinocyte response to treatment, relative to treatment with naked antibody alone.

20. The method according to claim 4, wherein the non-cleavable linker is succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC).

21. The immunoconjugate of claim 10, wherein the non-cleavable linker is succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] These and other aspects of this disclosure are now described in greater detail with reference to the accompanying drawings in which:

[0026] FIG. 1 is a graph showing cytotoxic activity against keratinocytes of naked anti-EGFR antibodies and maytansinoid conjugates thereof. About 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 5 days at 37° C. Viability of the remaining cells was determined by WST-8-based colorimetric assay.

[0027] FIG. 2 is a graph showing cytotoxic activity against cancer cell line of naked anti-EGFR antibodies and maytansinoid conjugates thereof. About 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 5 days at 37° C. Viability of the remaining cells was determined by WST-8-based colorimetric assay.

[0028] FIGS. 1 and 2 herein are reproductions of figures presented in US 2012/0156217.

[0029] FIG. 3 is a graph showing cytotoxic activity against H226 cancer cell line of naked anti-EGFR antibody cetuximab and maytansinoid conjugates thereof. About 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 72 hours at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0030] FIG. 4 is a graph showing cytotoxic activity against A431 cancer cell line of naked anti-EGFR antibody cetuximab and maytansinoid conjugates thereof. About 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 72 hours at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0031] FIG. 5 is a graph showing the cytotoxic activity of naked anti-EGFR antibody cetuximab and maytansinoid conjugates thereof against normal keratinocyte cell line HaCaT. About 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 5 days at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0032] FIG. 6 is a graph showing the cytotoxic activity of naked anti-EGFR antibody cetuximab and maytansinoid conjugates thereof against primary keratinocytes. 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 72 hours or 5 days at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0033] FIG. 7 is a graph showing the cytotoxic activity of naked anti-EGFR antibody panitumumab and maytansinoid conjugates thereof against primary keratinocytes. 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 72 hours or 5 days at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0034] FIG. 8 is a graph showing the cytotoxic activity of naked anti-EGFR antibody panitumumab and maytansinoid conjugates thereof against primary keratinocytes. 2,000 to 3,000 cells/well were seeded in a flat-bottom 96-well tissue culture plate, and incubated with various concentrations of test article in culture media for 72 hours or 5 days at 37° C. Viability of the remaining cells was determined via ALAMARBLUE® cell viability assay.

[0035] FIGS. 9-13 show results with immunoconjugates that incorporate elements not selected according to the criteria prescribed herein, as discussed in Example 4.

[0036] FIG. 9 shows that panitumumab and cetuximab are very strong EGFR activation blockers and thus will not be potentiated on normal keratinocytes by conjugation, whereas partially antagonistic antibodies such as J2898A is potentiated (see FIGS. 10 and 11).

[0037] FIG. 10 shows that partially antagonistic anti-EGFR antibody by IMGN (J2989A) is potentiated by conjugation (IMGN289) on normal keratinocytes (see also Setiady et al., Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr. 6-10; Washington, D.C. Philadelphia (Pa.): AACR; Cancer Res. 2013; 73(8 Suppl):Abstract nr 5463).

[0038] FIG. 11 shows the toxicity of cetuximab mutant antibody, 6-LC (having reduced affinity to make it partially antagonistic against EGFR), is potentiated by conjugation to payload via non-cleavable linker (6LC-DM1).

[0039] FIG. 12 shows panitumumab, and panitumumab-DM1 effects on keratinocytes, and reveals that conjugation of panitumumab via non-cleavable linker to DM (Avid300-DM1) does not increase its activity against normal cells; 2C9-DM1 is cetuximab-DM1 conjugate used as a control.

[0040] FIG. 13 shows that conjugation of cetuximab to MMAE anti-microtubule payload by a cleavable linker (valine-citrulline) potentiates its toxicity against both normal cells and MDA-MB-468 cancer cells (Cetux 2C9-MMAE), whereas conjugation via non-cleavable linker (SMCC) only (Cetux2C9-DM1) potentiates anti-cancer activity, thus demonstrating a favorable therapeutic window.

DETAILED DESCRIPTION

[0041] The present immunoconjugates are based on antibodies that bind to the human epidermal growth factor receptor (hEGFR), a protein that is presented on the surface of many different cell types including, particularly, skin cells such as keratinocytes. As used herein, the term “hEGFR” refers to any protein that comprises the expressed and processed product of the human her-1 gene, wherein the protein is designated as UniProtKB/Swiss-Prot P00533. The term “EGFR” is used generically herein, and refers to the wild-type protein and all naturally occurring variants thereof. The term “wtEGFR” is used more specifically with reference only to the wild-type form of human EGFR. The term “EGFRvIII” refers to the EGFR variant protein that comprises the expressed and processed product of a variant of the her-1 gene lacking exons 2-7, and thus includes only the polypeptide sequence encoded by exons 1 and 8 of her-1. The term “domain III” is not related to EGFRvIII, and instead refers to a location within EGFR, and represents an extracellular site that is key for EGF ligand binding, and binding of highly antagonistic antibodies cetuximab and panitumumab (Voigt et al., Functional Dissection of the Epidermal Growth Factor Receptor Epitopes Targeted by Panitumumab and Cetuximab, 2012 Nov.; 14(11): 1023-1031).

[0042] The present immunoconjugates comprise an EGFR antibody that is a full antagonist at the EGFR. An EGFR antibody that is a “full antagonist” is an antibody that blocks completely, or nearly so, the transmission of a signal that is stimulated, in the normal course, by the EGF ligand through wtEGFR to the wtEGFR-coupled tyrosine kinase. EGFR antibodies that are full antagonists are particularly EGFR antibodies that bind directly to EGFR domain III. EGFR antibodies having these properties and an EGFR binding affinity of 5 nanomolar (nM) or less are particularly preferred for inclusion in the present immunoconjugates. By this measure, and as shown in FIG. 9, at least the following known antibodies are not suitable for use in the present immunoconjugates: J2898A, INTELLIMAB™ 6-LC (a cetuximab variant taught in WO 2012/100346), nimotuzumab, and matuzumab.

[0043] It is found that when a full antagonist EGFR antibody is selected to deliver a toxic payload, the killing effect is potentiated only on EGFR+ cancer cells and not on EGFR+ normal cells such as keratinocytes. This is not the case when the EGFR antibody is a partial antagonist, i.e., an antibody that allows transmission of some EGF-mediated signal. When conjugated to a toxin, these partial antagonist antibodies show a potentiation of the killing effect at both normal cells that are EGFR+ and cancer cells that are EGFR+.

[0044] The present immunoconjugates are based more particularly, and in one embodiment, on the hEGFR antibody known as cetuximab, now commercially available from Eli Lilly and Company under the trade name ERBITUX®. Cetuximab is a recombinant, human/mouse chimeric IgG antibody that binds specifically to the extracellular domain of wtEGFR. The amino acid sequences of the CDRs for both the heavy chain of cetuximab (SEQ ID NOS:1-3) and the light chain of cetuximab (SEQ ID NOS:4-6) are listed herein. Also listed are the amino acid sequences of the heavy chain variable region (SEQ ID NO:7) and of the light chain variable region (SEQ ID NO:8) of cetuximab, together with the amino acid sequences of the complete heavy chain (SEQ ID NO:9) and complete light chain (SEQ ID NO:10) of cetuximab.

[0045] Cetuximab variants useful herein are highly antagonistic EGFR-binding agents that compete with cetuximab for binding to human EGFR. Useful cetuximab variants have been mentioned hereinabove, and include fragments of cetuximab comprising the EGFR binding sites of cetuximab, such as all of the light chain and heavy chain CDRs herein recited. Other cetuximab variants useful herein are cetuximab variants that incorporate one, two or more substitutions outside the antigen binding domains, such as in the framework region or in the constant region (Fc). Such substitutions are benign in the sense that they do not reduce cytotoxicity relative to cetuximab per se.

[0046] In another embodiment, the present immunoconjugates are based on the EGFR antibody known as panitumumab, now commercially available and sold under the trade name VECTIBIX®. Panitumumab is a recombinant, fully human IgG2 antibody that binds specifically to the extracellular domain of wtEGFR. The amino acid sequences of the heavy and light chains of panitumumab are listed in U.S. Pat. No. 6,235,883 and U.S. Pat. No. 7,807,798. A useful alternative to panitumumab is an EGFR binding variant thereof that competes with panitumumab for EGFR binding, such as a fragment of panitumumab that incorporates its antigen binding sites but has an otherwise lost or altered constant region.

[0047] The present immunoconjugates can also be based on still other EGFR antibodies provided they show full antagonist activity as defined above, such as EGFR antibodies that bind selectively to domain III of EGFR, and any other EGFR antibodies that compete with EGF and block fully, or nearly so, the transmission of EGF-stimulated downstream signaling.

[0048] In the present immunoconjugates, the full antagonist EGFR antibody, such as cetuximab or panitumumab, is conjugated to an anti-microtubule toxin such as maytansinoid toxin. By “anti-microtubule toxin” is meant an agent having cell toxicity mediated by interference with the microtubule structures important for cell mitosis, such as by inhibiting the formation of tubulin or by inhibiting the organization thereof.

[0049] Included within this toxin family are the maytansinoids and auristatins, and many other agents developed more recently and having the same mechanism of action. The auristatins in particular block cell replication by inhibiting polymerization of tubulin and are thus anti-mitotic. The structure of an auristatin useful herein and known as MMAE, or vedotin, is shown below:

##STR00001##

[0050] There are various forms of maytansinoids that are useful. These are all based on the complex structure of a natural molecule, maytansine.

[0051] Particularly useful are the maytansinoids including DM-1 and DM-4. In a specific embodiment, the toxin coupled to the EGFR MAb is DM-1 having the structure shown infra.

[0052] Also useful as anti-microtubule toxins are dolostatins, auristatins, tubulysins and cryptophycins. Specific examples of useful species within each genus include dolostatin 10, monomethyl dolostatin 10, auristatin E, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F, HTI-286, tubulysin M, as well as the tubulin binders such as tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, colchicine DA, and maytansinoids AP-3, DM-1 and DM-4.

[0053] Conjugates of a full antagonist EGFR antibody such as cetuximab or panitumumab, and an anti-microtubule toxin such as a maytansinoid or auristatin, can be formed using any technique presently known or later developed that couples a linker that is “non-cleavable.” These linkers remain intact and retain the antibody and toxin in covalent association throughout conditions normally encountered following administration to a subject, including extracellular environments. More specifically, non-cleavable linkers result in ADC constructs for which release of the cytotoxic payload is achieved by destruction of the antibody by intracellular lysosomes.

[0054] Methods of linker integration are described, for instance, in U.S. Pat. Nos. 5,208,020; 8,088,387; and 6,441,163. A preferred method is to modify the EGFR antibody, e.g., cetuximab, with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introduce maleimido groups followed by reaction of the modified antibody with a thiol-containing maytansinoid to give a thioether-linked conjugate. The resulting chemical structures are shown below. Conjugates with 1 to 10 drug molecules per antibody molecule result.

##STR00002##

[0055] Other useful forms of non-cleavable linkers include N-Succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), sulfo-MBS and succinimidyl-iodoacetate, as described in the literature, to introduce 1-10 reactive groups (see, Yoshitake et al., Eur. J. Biochem. 110:395-399 (1979); Hashida et al., J. Applied Biochem. 56-63 (1984); and Liu et al., 18:690-697 (1979)). Particularly useful for linking auristatins as anti-microtubule toxins are the non-cleavable maleimidocaproyl linkers described by Doronina et al., in Bioconjugate Chem. 2006 Jan.-Feb.; 17(1):114-24).

[0056] In a specific embodiment, the immunoconjugate comprises cetuximab linked to DM-1 by an SMCC linker.

[0057] In another specific embodiment, the immunoconjugate comprises panitumumab linked to DM-1 by an SMCC linker.

[0058] Thus, in a general aspect, the disclosure provides a method useful to potentiate the anti-cancer activity of an EGFR antibody without potentiating the effect thereof on normal EGFR+ cells, the method comprising: [0059] (i) selecting, for conjugation, an EGFR antibody that is a full EGFR antagonist; [0060] (ii) selecting, for delivery by the EGFR antibody, an anti-microtubule toxin; [0061] (iii) selecting, for coupling the selected EGFR antibody to the anti-microtubule toxin, a linker that is non-cleavable; and [0062] incorporating the linker between the antibody and the toxin to provide an immunoconjugate having a cytotoxic activity that is potentiated against EGFR+ disease cells but not against normal EGFR+ cells.

[0063] As will be appreciated from the preceding disclosure, the full antagonist EGFR antibody is preferably cetuximab or panitumumab. The anti-microtubule toxin is preferably an auristatin or a maytansinoid, and is most preferably DM-1. The non-cleavable linker is preferably SMCC.

[0064] In another specific embodiment, there is the proviso that the full antagonist EGFR antibody is not cetuximab. In a further specific embodiment, there is the proviso that the full antagonist EGFR antibody is not panitumumab.

[0065] Therapeutic formulations of the conjugate are prepared for therapeutic use directly or for storage by mixing the conjugate having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16.sup.th edition, A. Osol, Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum, albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagines, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0066] The active ingredients to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile membranes.

[0067] Sustained-release preparations may be prepared. Suitable examples of sustained-release include semipermeable matrices of solid hydrophobic polymers containing the conjugate, which matrices are in the form of shapes articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate, and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0068] The conjugates are useful to treat EGFR+ disease cells. Such treatment results in a reduction in the number, size or distribution of such disease cells in subjects presenting with them. In embodiments, the conjugates are used to treat EGFR+ disease cells that are EGFR+ cancer cells and tumors comprising them. Such treatment results preferably in a reduction in the number, size, volume or distribution of such cancer cells and tumors comprising them, or at least in a reduction in the rate at which such disease cells increase in number, size, volume or distribution of such cells and tumors in subjects presenting with them.

[0069] Subjects presenting with EGFR+ cancer cells can be identified with the aid of assays that detect the receptor, as protein or as nucleic acid precursor (DNA or RNA) in physiological samples such as biopsied tissue. A suitable test for EGFR protein is the commercially available and FDA approved Dako EGFR PHARMDX® test kit.

[0070] For the treatment of subjects presenting with EGFR+ cancer cells, the appropriate dosage of the conjugate will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventative or therapeutic purposes, previous therapy, the patients clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

[0071] For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of conjugate is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 500 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0072] It will thus be appreciated that an effective amount of the immunoconjugate is an amount effective alone or as part of a treatment regimen that retards or inhibits the rate of growth or proliferation of EGFR+ disease cells.

[0073] An EGFR+ disease cell is a disease cell that presents EGFR on its surface as detectable, for instance, by EGFR antibody binding, or by detection of intracellular mRNA encoding her-1. Particular EGFR+ disease cells include those having on their surface an abnormally high density and/or activity of EGFR molecules, or the presence of the EGFRvIII variant of EGFR.

[0074] It may be useful to administer the present conjugates by intravenous infusion first as loading dose, followed by maintenance dose, such as at an initial dose of 4 mg/kg over 90 minutes, then 2 mg/kg over 30 minutes, once weekly for as many as 52 weeks, with follow up as required. In the specific case of the panitumumab conjugate, dosing might be based on that utilized for panitumumab per se, which comprises 6 mg/kg given once every two weeks as a one hour infusion.

[0075] The conjugates are useful in the treatment of a variety of cancers, to inhibit the growth or proliferation of EGFR+ cancer cells and tumors comprising them, including hematopoietic cell cancers and solid tumors. Conditions or disorders to be treated include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulva, and thyroid); hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors; leukemias and lymphoid malignancies. In particular embodiments, the antibody or bivalent fragments are used in the treatment of such cancer cells that express EGFRvIII, as determined by the screening assays herein described. In particular embodiments, the cancer cells are EGFR+-presenting cancer cells that include head and neck cancers and especially squamous cell carcinoma of the head and neck, colorectal cancers, gastrointestinal cancers, brain tumors including glioblastomas, and tumors of the lung including non-small-cell lung carcinoma, and of the breast, pancreas, esophagus, kidney, ovary, cervix and prostate. In specific embodiments, the EGFR+ cancer is one for which cetuximab has received FDA marketing approval, such as squamous cell carcinoma of the head and neck and colorectal cancers.

[0076] It will be appreciated that subjects who could benefit from the present method include mammals including humans as well as livestock, and pets.

[0077] Other therapeutic regimens may be combined with the administration of the conjugates of the disclosure. For example, the patient to be treated may also receive radiation therapy, such as external beam radiation. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, ed. M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede or follow administration or the conjugate or may be given simultaneously therewith. The conjugate may be combined with any anti-cancer toxins, or any other suitable drug particularly including irinotecan (CPT-11), cisplatin, cyclophosphamide, melphalan, dacarbazine, doxorubicin, daunorubicin, and topotecan, as well as tyrosine kinase inhibitors, particularly including EGFR kinase inhibitors such as AG 1478 ((4-(3-chloroanilino-6,7-dimethoxyquinazoline), gefitinib (IRESSA®), erlotinib (TARCEVA®), lapatinib (TYKERB®), canertinib (PD183805, Pfizer), PKI-166 (Novartis), PD158780 and pelitinib.

[0078] It may also be desirable to administer antibodies or conjugates against other tumor-associated antigens or their ligands, such as antibodies, that bind to the ErbB2 (including trastuzumab marketed as HERCEPTIN®, and pertuzumab marketed as OMNITARG™), ErbB3, ErbB4, or vascular endothelial factor (VEGF), and/or antibodies that bind to EGF or TGFα.

[0079] In another embodiment of the disclosure, an article of manufacture containing conjugate in an amount useful for the treatment of the disorders described herein is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating a cancer condition. The article of manufacture may further compromise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other matters desirable from a commercial and use standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0080] An anti-cancer immunoconjugate according to the disclosure may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Unit doses of the conjugate are suitably 50 mgs, 100 mgs, 150 mgs, 200 mgs, 250 mgs, 300 mgs and 400 mgs. The drug can be formulated in single use vials at a concentration such as 20 mg/mL, for instance, 100 mg in 5 mL vehicle such as 0.9% saline, 200 mg in 10 mL or 400 mg in 20 mL.

[0081] Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, pulmonary, or oral administration.

Example 1—Preparation of Cetuximab-SMCC-DM1 Conjugate (A-H)

[0082] A. Preparation and Measurement of Cetuximab Antibody

[0083] Cetuximab is obtained from the open market, or is produced as described in WO 2012/100346, for conjugation to DM1 using the non-cleavable heterobifunctional cross-linking reagent SMCC.

[0084] Cetuximab antibody was then buffer exchanged into 50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA; pH 6.5 buffer (Buffer A). All buffers in this experiment were tested to be free of endotoxin using a chromogenic Limulus amoebocyte lysate (LAL) method (Cambrex). The concentration of antibody was measured using an extinction coefficient of 1.45 mL/mg/cm at 280 nm and a molecular weight of 145,781 g.

[0085] B. Preparation and Measurement of SMCC Stock Solution

[0086] A 20 mM solution of SMCC (6.69 mg/mL) (Concortis Biosystems Corp.) was prepared in DMSO. The solution was diluted 1/40 in Assay Buffer and the absorbance of the samples was measured at 302 nm. The concentration of the stock solution was calculated using a molar extinction coefficient of 602/M/cm.

[0087] C. Preparation and Measurement of DM1 Stock Solution

[0088] A 10 mM solution of DM1 (free thiol form; Concortis Biosystems Corp.) was prepared in DMA (7.37 mg/mL). The absorbance of dilutions of the stock solution in ethanol was measured at 280 nm. The concentration of stock DM1 was calculated by using a molar extinction coefficient of 5700/M/cm at 280 nm. The concentration of free —SH in the stock DM1 preparation was measured using Elman's reagent (DTNB). Dilutions of the stock solution were prepared in assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO (1/100th volume) was added. The increase in absorbance at 412 nm was measured against a reagent blank and the concentration was calculated using an extinction coefficient of 14150/M/cm. The concentration of —SH resulting from the Elman's assay was used to represent the DM1 stock concentration in calculations for conjugation conditions.

[0089] D. Modification of Cetuximab with SMCC Cross-Linker

[0090] The antibody was modified using a 7.5-fold molar excess of SMCC at 20 mg/mL antibody. The reaction was carried out in Buffer A (95% v/v) with DMSO (5% v/v) for 2 hours at room temperature with stirring.

[0091] E. G25 Chromatography to Remove Excess SMCC

[0092] The cetuximab-SMCC reaction mixture was gel-filtered through a 1.5×4.9 cm pre-packed column of Sephadex G25 resin equilibrated in Buffer A. The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was assayed spectrophotometrically using the extinction co-efficient described above.

[0093] F. Conjugation of Cetuximab-SMCC with DM1

[0094] The modified antibody was reacted with a 1.7-fold excess of DM1 over linker (assuming five linkers per antibody). The reaction was carried out at 10 mg/mL antibody concentration in Buffer A (94% v/v) with DMA (6% v/v). After addition of DM1, the reaction was incubated at room temperature for 16.5 hours with stirring.

[0095] G. Conjugation Purification by G25 Chromatography

[0096] The conjugation reaction mixture was gel-filtered through a 1.5×4.9 cm pre-packed column of Sephadex G25 resin equilibrated in 1× phosphate-buffered saline (PBS), pH 6.5 (Buffer B). The load and elution volumes were according to the manufacturer's instructions (Amersham Biosciences). The number of DM1 molecules linked per mole of cetuximab was determined by measuring absorbance at both 252 nm and 280 nm of the eluted material. The DM1/antibody ratio was found to be 2 and 4. The resulting conjugate was analyzed for binding and cytotoxicity.

[0097] H. Testing of Cetuximab-SMCC-DM1

[0098] The cell lines used in these studies have the following characteristics:

[0099] A431: human epithelial carcinoma of vulva cell line; available from ATCC; plated at 4000 cells/well in DMEM-10% FBS, 100 μl/well in 96-well plate.

[0100] H226: lung squamous cell carcinoma cell line; available at ATCC; plated at 4000 cells/well in RPMI-10% FBS, 100 μl/well in 96-well culture plate.

[0101] MDA-MB-468: mammary gland/breast; derived from metastatic site; pleural effusion; available from ATCC; plated at 4000 cells/well in ATCC-formulated Leibovitz's L-15 Medium (Catalog No. 30-2008) with added fetal bovine serum to a final concentration of 10%; 100 μl/well in 96-well plate.

[0102] HaCaT: in vitro spontaneously transformed keratinocytes from histologically normal skin; available from Chinese Center for Type Culture Collection of Wuhan University; plated at 2000 cells/well in DMEM-10% FBS, 100 μl/well in 96-well culture plate.

[0103] HEKa: Normal Human Primary Epidermal Keratinocytes, adult; available from PromoCell GmbH; plated at 4000 cells/well in EPILIFE® media 100 μl/well, EPILIFE® medium #MEP1500CA, INVITROGEN® +HKGS human keratinocyte growth supplement (#S-001-5).

[0104] Binding studies showed that the conjugation of antibody to DM1 did not affect the K.sub.D; both naked cetuximab antibody and cetuximab-SMCC-DM1 conjugate had the same binding affinity to EGFR by surface plasmon resonance (˜0.3 nM, Table 1). Evaluation of the in vitro cytotoxicity of the sample showed that the cetuximab-SMCC-DM1 conjugate is significantly more cytotoxic compared to cetuximab against cancer cell lines (FIGS. 3 and 4), but that, surprisingly, cetuximab-SMCC-DM1 and naked cetuximab have the same cytotoxic activity against keratinocytes (FIGS. 5 and 6).

TABLE-US-00001 TABLE 1 effects of conjugation to DM1 on cetuximab KD Sample # of experiments EGFR ecd KD (nM) Commercial cetuximab 3 0.27 +/− 0.02 Cetuximab-SMCC-DM1 3 0.28 +/− 0.02

[0105] The effect of DM1 conjugation on different EGFR MAbs (huML66 and huEGFR-7R) is demonstrated in ImmunoGen's published U.S. patent application 2012/0156217, and FIGS. 1 and 2 herein are reproductions of figures appearing in that application. More particularly, as shown in FIG. 1, conjugation of anti-EGFR antibodies huML66-SMCC-DM1 and huEGFR-7R to maytansinoids to create huML66-SMCC-DM1 and huEGFR-7R-SMCC-DM1 immunoconjugates increases cytotoxic activity against normal keratinocytes. Similarly, and as expected, conjugation of anti-EGFR antibodies huML66 and huEGFR-7R to maytansinoids to create huML66-SMCC-DM1 and huEGFR-7R-SMCC-DM1 immunoconjugates significantly potentiates cytotoxic activity of the antibodies against H226 cancer cell line (FIG. 2).

[0106] As shown in FIGS. 3 and 4, DM1 conjugation of anti-EGFR antibody cetuximab (HC-LC) to create cetuximab-SMCC-DM1 immunoconjugate (HC-LC/DM1) significantly potentiates cytotoxic activity of the antibody against cancer cells, as expected and shown in FIG. 3. As shown in FIG. 4, and also as expected, conjugation of anti-EGFR antibody cetuximab (HC-LC) to maytansinoids to create cetuximab-SMCC-DM1 immunoconjugate (HC-LC/DM1) significantly potentiates cytotoxic activity of the antibody against cancer cells.

[0107] However, and quite surprisingly, as shown in FIGS. 5 and 6, conjugation of anti-EGFR antibody cetuximab (HC-LC) to maytansinoids to create cetuximab-SMCC-DM1 immunoconjugate (HC-LC/DM1) did not increase cytotoxicity against the keratinocytes as is reflected in substantially the same IC.sub.50 values for these agents (IC.sub.50 of 0.8269 for conjugate vs. IC.sub.50 of 0.4324 for naked antibody; FIG. 5, HaCaT—normal human keratinocytes cell line and IC.sub.50 of 2.210 for conjugate vs. IC.sub.50 of 0.9348 for naked antibody; FIG. 6, HEKa—primary human keratinocyte cells). As used herein, IC.sub.50 refers to the concentration of drug required to kill 50% of cells.

[0108] The measure of whether cytotoxicity is increased or not is determined above by comparing IC.sub.50 values produced using the ALAMARBLUE® cell viability assay (each experiment performed in triplicate in a 96-well plate). A difference within one log order is deemed insignificant and is considered to reveal no change in cytotoxicity, whereas a difference greater than one log order reveals a significant change in cytotoxicity.

Example 2—Evaluation in Primates

[0109] Cynomolgus monkey species has been previously demonstrated to be a valuable and highly predictive model for evaluating anti-EGFR antibodies toxicities, including dermatologic side-effects. The high level of correlation between Cynomolgus monkey and human toxicity data for EGFR-targeting antibodies is in part due to high homology between the monkey and human EGFR receptors that results in very similar K.sub.D values for the antibodies:

TABLE-US-00002 TABLE 2 Binding affinity of cetuximab and panitumumab to human and Cynomolgus monkey EGFR is consistent across the species. Apparent antibody affinity defined as the antibody concentration (picomolar, pM) required to achieve half-maximal binding in ELISA with recombinant extracellular domains of human EGFR (huEGFR), and Cynomolgus monkey EGFR (cyEGFR). Adapted from Koefoed et al., MABs, 2011 November-December; 3(6): 584-595. Apparent affinity, ELISA assay Domain IIIB Binding Antigens cetuximab panitumumab human sEGFR (pM) 12 32 cynomolgus sEGFR (pM) 14 32

[0110] The cetuximab-SMCC-DM1 immunoconjugate was tested in primates particularly to identify any significant potentiation of antibody side-effects by the conjugated toxin on normal cells. In particular, close attention was paid to dermatologic side-effects that are qualitatively different and/or significantly exacerbated relative to what is expected with the naked fully antagonistic anti-EGFR antibody.

[0111] The study initially made use of two Cynomolgus macaques, all males that were sedated with ketamine and anesthetized with isoflurane to facilitate IV administration of the antibody-drug conjugate administered at 10 mg/kg. Animals were then observed for 5 days prior to sacrifice for general signs of acute toxicity, and examined further.

[0112] The detailed histopathology analysis revealed no severe dermatologic toxicities or other severe and qualitatively new dermatologic side-effects other than those that are expected as a result of naked cetuximab treatment. Also, no other severe on-target toxicities were observed in these animals demonstrating safety of ADCs of this disclosure and absence of significant exacerbation of the naked antibody toxicities.

[0113] Subsequent to successful completion of the acute primate study, repeat-dose safety study has been initiated in additional animals. The ADC was administered once every three weeks at 10 mg/kg dose over four cycles. This dose and schedule was selected based on dosing schedules commonly used with other ADCs in primates and humans (K. A. Poon et al., Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability, Toxicol. Appl. Pharmacol. 2013 Dec. 1; 273(2):298-313; FDA Package Insert for KADCYLA®, accessed Feb. 24, 2014: on the World Wide Web at accessdata.fda.gov/drugsatfda_docs/label/2013/1254271bl.pdf).

[0114] With the exception of reversible liver enzyme elevations, which is an expected class side-effect of ADCs, no other severe and/or qualitatively new dermatologic or other on-target side-effects other than those that are already expected as a result of naked cetuximab treatment (cetuximab Cynomolgus monkey data available on the World Wide Web at accessdata.fda.gov/drugsatfda_docs/bla/2004/125084_ERBITUX_PHARMR_P3.PDF) have been observed in the ongoing study to date. Severe skin toxicity and toxic epidermal necrolysis previously reported with anti-CD44v6 ADC (which also uses SMCC-DM1 payload technology and targets an antigen expressed by normal keratinocytes) has not been observed in this study (Tijink et al., Clin. Cancer Res., 2006, 12:6064). These safety data further validates in vitro results that on-target toxicities of fully antagonist naked anti-EGFR antibodies is not significantly potentiated as a result of conjugation to the cytotoxic payloads.

Example 3—Preparation of Panitumumab-SMCC-DM1 Conjugate (A-H)

[0115] A DM-1-conjugated panitumumab was prepared essentially as described above for the cetuximab counterpart. More particularly,

[0116] A. Preparation and Measurement of Panitumumab Antibody

[0117] Panitumumab is obtained from the open market, or is produced as described in U.S. Pat. No. 6,235,883 or U.S. Pat. No. 7,807,798 for conjugation to DM1 using the non-cleavable heterobifunctional cross-linking reagent SMCC.

[0118] Panitumumab antibody was then buffer exchanged into 50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA; pH 6.5 buffer (Buffer A). All buffers in this experiment were tested to be free of endotoxin using a chromogenic Limulus amoebocyte lysate (LAL) method (Cambrex). The concentration of antibody was measured using an extinction coefficient of 1.45 mL/mg/cm at 280 nm and a molecular weight of 145,781 g.

[0119] B. Preparation and Measurement of SMCC Stock Solution

[0120] A 20 mM solution of SMCC (6.69 mg/mL) (Concortis Biosystems Corp.) was prepared in DMSO. The solution was diluted 1/40 in assay buffer and the absorbance of the samples was measured at 302 nm. The concentration of the stock solution was calculated using a molar extinction coefficient of 602/M/cm.

[0121] C. Preparation and Measurement of DM1 Stock Solution

[0122] A 10 mM solution of DM1 (free thiol form; Concortis Biosystems Corp.) was prepared in DMA (7.37 mg/mL). The absorbance of dilutions of the stock solution in ethanol was measured at 280 nm. The concentration of stock DM1 was calculated by using a molar extinction coefficient of 5700/M/cm at 280 nm. The concentration of free —SH in the stock DM1 preparation was measured using Elman's reagent (DTNB). Dilutions of the stock solution were prepared in assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO (1/100th volume) was added. The increase in absorbance at 412 nm was measured against a reagent blank and the concentration was calculated using an extinction coefficient of 14150/M/cm. The concentration of —SH resulting from the Elman's assay was used to represent the DM1 stock concentration in calculations for conjugation conditions.

[0123] D. Modification of Panitumumab with SMCC Cross-Linker

[0124] The antibody was modified using a 7.5-fold molar excess of SMCC at 20 mg/mL antibody. The reaction was carried out in Buffer A (95% v/v) with DMSO (5% v/v) for 2 hours at room temperature with stirring.

[0125] E. G25 Chromatography to Remove Excess SMCC

[0126] The panitumumab-SMCC reaction mixture was gel-filtered through a 1.5×4.9 cm pre-packed column of SEPHADEX® G25 resin equilibrated in Buffer A. The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was assayed spectrophotometrically using the extinction co-efficient described above.

[0127] F. Conjugation of Panitumumab-SMCC with DM1

[0128] The modified antibody was reacted with a 1.7-fold excess of DM1 over linker (assuming five linkers per antibody). The reaction was carried out at 10 mg/mL antibody concentration in Buffer A (94% v/v) with DMA (6% v/v). After addition of DM1, the reaction was incubated at room temperature for 16.5 hours with stirring.

[0129] G. Conjugation Purification by G25 Chromatography

[0130] The conjugation reaction mixture was gel-filtered through a 1.5×4.9 cm pre-packed column of SEPHADEX® G25 resin equilibrated in 1× phosphate buffered saline (PBS), pH 6.5 (Buffer B). The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The number of DM1 molecules linked per mole of panitumumab was determined by measuring absorbance at both 252 nm and 280 nm of the eluted material. The DM1/antibody ratio was found to be 2 and 4. The resulting conjugate was analyzed for binding and cytotoxicity.

[0131] H. Testing of Panitumumab-SMCC-DM1

[0132] The cell lines used in these studies have the following characteristics: [0133] MDA-MB-468: mammary gland/breast; derived from metastatic site; pleural effusion; available from ATCC; plated at 4000 cells/well in ATCC-formulated Leibovitz's L-15 Medium (Catalog No. 30-2008) with added fetal bovine serum to a final concentration of 10%; 100 μl/well in 96-well plate. [0134] HaCaT: in vitro spontaneously transformed keratinocytes from histologically normal skin; available from Chinese Center for Type Culture Collection of Wuhan University; plated at 2000 cells/well in DMEM-10% FBS, 100 μl/well in 96-well culture plate.

[0135] In vitro studies with keratinocytes demonstrate that conjugation of panitumumab to an anti-microtubule toxin via non-cleavable linker does not potentiate the toxicity of the antibody. The resulting ADC has a similar safety profile on keratinocytes as the naked panitumumab, as well as another ADC based on a fully antagonistic antibody, cetuximab. On MDA-MB-468 cancer cells, significant potentiation of anticancer activity of panitumumab is observed as a result of conjugating the antibody to anti-microtubule toxin via non-cleavable linker. Similar to toxicity observations, panitumumab-based ADC was similarly active as cetuximab-based ADC.

Example 4—Results with Partial Antagonist EGFR MAb Conjugates

[0136] The effect of selecting immunoconjugate components different from those recommended herein is shown in the accompanying drawings. FIG. 9 shows that a partial antagonist EGFR antibody, designated J2989A has an activity that is potentiated on normal keratinocytes when conjugated to DM-1. FIG. 10 shows, similarly, that another partial antagonist antibody, designated 6-LC (a substitution variant of cetuximab having lower relative affinity for EGFR) has an activity that is also potentiated at normal keratinocytes when conjugated to DM-1. FIG. 11 shows that the effect of panitumumab of keratinocytes is not potentiated on keratinocytes when conjugated to DM1, while the anti-cancer effect of the conjugate is potentiated dramatically (FIG. 12). In addition, FIG. 13 shows that conjugation of cetuximab to MMAE by a cleavable linker (valine-citrulline) potentiates its toxicity against normal cells and MDA-MB-468 cancer cells, whereas conjugation via non-cleavable linker (SMCC) potentiates anti-cancer activity.

[0137] Thus, in these studies, only fully antagonistic anti-EGFR antibodies were not potentiated on normal cells by payload conjugation via non-cleavable linkers, whereas toxicity against normal cells of partial antagonist EGFR antibodies was potentiated by the conjugation. Moreover, the effect on this activity profile of the cleavable vs. non-cleavable linkers was subsequently tested, while maintaining payload mechanism of action the same (i.e., anti-microtubule agent). The antibodies were conjugated via cleavable linker to anti-microtubule payload, MMAE as described by Doronina et al., Nature Biotechnology, Nov. 7, 2003, Vol. 21:778-784. Cleavable linker data demonstrate that when the fully antagonistic antibodies are conjugated to their payloads via cleavable linkers, their toxicity against normal cells is potentiated. Thus, a safe anti-EGFR ADC should incorporate a strongly antagonistic anti-EGFR antibody linked to an anti-microtubule payload by a non-cleavable linker.

[0138] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

TABLE-US-00003 Referenced Sequences SEQ ID No. Description 1 Cetuximab heavy chain CDR1 NYGVH 2 Cetuximab heavy chain CDR2 VIWSGGNTDYNTPFTS 3 Cetuximab heavy chain CDR3 ALTYYDYEFAY 4 Cetuximab light chain CDR1 RASQSIGTNIH 5 Cetuximab light chain CDR2 ASESIS 6 Cetuximab light chain CDR3 QQNNNWPTT 7 Cetuximab heavy chain variable region (V.sub.H) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA 8 Cetuximab light chain variable region (V.sub.L) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK 9 Cetuximab complete heavy chain QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWE SNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG K 10 Cetuxitnab complete light chain DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSC SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSC TASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV ACEVTHQGLSSPVTKSFNRGE