Proteins binding NKG2d, CD16 and EGFR

Abstract

To provide: multispecific binding proteins that bind the NKG2D receptor, CD16 and EGFR; and pharmaceutical compositions and therapeutic methods useful for the treatment of cancer.SOLUTION: In some embodiments, the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 145, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 150. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 145, and the VL comprises an amino acid sequence of SEQ ID NO: 150.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 061,510, filed August 5, 2020, the entire disclosure of which is hereby incorporated by reference herein for all purposes. Sequence Listing

[0002] This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 4, 2021, is named DFY-084WO_SL.txt and is 169,786 bytes in size.

[0003] FIELD OF THEINVENTION The present application relates to multispecific binding proteins that bind to NKG2D, CD16 and epidermal growth factor receptor (EGFR). [Background technology]

[0004] background Despite substantial research efforts, cancer continues to be a significant clinical and economic burden in countries around the world. According to the World Health Organization (WHO), cancer is the second leading cause of death. Among the existing treatment modalities are surgery, radiation therapy, chemotherapy, biological therapy, immunotherapy, hormonal therapy, stem cell transplantation and precision medicine. Despite extensive research in these areas, highly effective and curative solutions have yet to be identified, especially for the most aggressive cancers. Furthermore, many of the existing anti-cancer treatment modalities have significant adverse side effects.

[0005] Cancer immunotherapy is desirable because it is highly specific and can use the patient's own immune system to promote the destruction of cancer cells.Fusion proteins such as bispecific T cell engagers are cancer immunotherapies described in the literature that bind to tumor cells and T cells to promote the destruction of tumor cells.Antibodies that bind to certain tumor-associated antigens and certain immune cells have been described in the literature.See, for example, WO2016 / 134371 and WO2015 / 095412.

[0006] Natural killer (NK) cells are components of the innate immune system and constitute approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells - i.e., via cytolytic granules containing perforin and granzymes, as well as via death receptor pathways. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other leukocytes to target tissues.

[0007] NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited through activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated through their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of some immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein expressed by essentially all natural killer cells, where it serves as an activating receptor. NKG2D is also found on T cells, where it acts as a costimulatory receptor. The ability to modulate NK cell function through NKG2D is useful in a variety of therapeutic situations, including malignancies.

[0008] Mutations resulting in epidermal growth factor receptor (EGFR) overexpression or hyperactivity are associated with many cancers, including non-small cell lung cancer, anal cancer, glioblastoma, and epithelial tumors of the head and neck. These somatic mutations involving EGFR lead to its constant activation, which results in uncontrolled cell division. In glioblastoma, a more specific or less specific mutation of EGFR, called EGFRvIII, is often observed. Mutations, amplifications, or misregulation of EGFR or family members are associated with other solid tumors, including colorectal, renal cell, bladder, cervical, ovarian, pancreatic, and liver cancers. Anti-EGFR monoclonal antibodies, such as cetuximab, panitumumab, necitumumab and zalutumumab, have been developed.However, there remains a need for new useful antibodies and related therapies for use in the treatment of cancer with higher efficacy and reduced adverse effects. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention [Means for solving the problem]

[0010] Abstract The present application provides a multispecific binding protein that binds to the NKG2D receptor, the CD16 receptor, and EGFR on natural killer cells. Such proteins can bind to more than one NK activating receptor and can block the binding of natural ligands to NKG2D. In certain embodiments, the protein can stimulate human NK cells. In some embodiments, the protein can stimulate human and other species of NK cells, such as rodents and cynomolgus monkeys. Also provided are formulations containing any one of the proteins disclosed herein; cells containing one or more nucleic acids expressing the proteins; and methods of using the proteins to enhance tumor cell death.

[0011] Accordingly, one embodiment of the present application is a protein comprising: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds EGFR; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16, wherein the second antigen-binding site is comprised of (i) complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR4) of SEQ ID NOs: 136, 157, and 138, respectively. and a light chain variable domain (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively; or (ii) a VH comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively; and a VL comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 142, respectively.

[0012] In some embodiments, the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 157 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively.

[0013] In some embodiments, the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively.

[0014] In some embodiments, the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 137 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively.

[0015] In some embodiments, the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 142, respectively.

[0016] In some embodiments, the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 145, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 150. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 145, and the VL comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 170, and the VL comprises the amino acid sequence of SEQ ID NO: 171.

[0017] In some embodiments, the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 135, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 150. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 135, and the VL comprises the amino acid sequence of SEQ ID NO:150.

[0018] In some embodiments, the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 145, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 147. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 145, and the VL comprises the amino acid sequence of SEQ ID NO:147.

[0019] Another aspect of the application provides a protein comprising: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds EGFR; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16, wherein the second antigen-binding site comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 135, and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 139, wherein the VH comprises a S62R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 135, and / or the VL comprises a D92R substitution and / or a F87Y substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139.

[0020] In some embodiments, the VH comprises an S62R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 135. In some embodiments, the VL comprises a D92R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139. In some embodiments, the VH comprises an S62R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 135 and the VL comprises a D92R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139.

[0021] In some embodiments of any one of the above aspects, the VL includes an F87Y substitution compared to SEQ ID NO:139, as numbered under the Chothia numbering scheme.

[0022] In some embodiments, the second antigen binding site comprises a single chain variable fragment (scFv), wherein the scFv comprises an amino acid sequence at least 90% identical to or including a sequence selected from the group consisting of SEQ ID NOs: 152, 154, 148 and 158.

[0023] In some embodiments, a protein of the disclosure comprises a polypeptide comprising an amino acid sequence at least 90% identical to or including a sequence selected from the group consisting of SEQ ID NOs: 167, 168, and 166.

[0024] In some embodiments, the second antigen-binding site has a dissociation constant (K) less than or equal to 5 nM as measured by surface plasmon resonance (SPR). D In some embodiments, the second antigen-binding site binds to human EGFR with a dissociation constant (K) less than or equal to 6 nM as measured by surface plasmon resonance (SPR). D ) which binds to rhesus monkey EGFR.

[0025] In some embodiments, the first antigen-binding site that binds to NKG2D is a Fab fragment, and the second antigen-binding site that binds to EGFR is an scFv. In some embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site that binds to EGFR is a Fab fragment. In some embodiments, the protein further comprises an additional antigen-binding site that binds to EGFR. In some embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second and additional antigen-binding sites that bind to EGFR are Fab fragments, respectively. In some embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second and additional antigen-binding sites that bind to EGFR are scFv, respectively. In some embodiments, the amino acid sequences of the second and additional antigen-binding sites are identical.

[0026] In some embodiments, the scFv that binds NKG2D is linked to an antibody constant domain or a portion thereof sufficient to bind to CD16 via a hinge comprising Ala-Ser or Gly-Ser, and the scFv comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, each scFv that binds EGFR is linked to an antibody constant domain or a portion thereof sufficient to bind to CD16 via a hinge comprising Ala-Ser or Gly-Ser, and the scFv comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

[0027] In some embodiments, within the scFvs that bind to NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, within each scFv that binds to EGFR, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, as numbered under the Kabat numbering scheme. In some embodiments, within the scFvs that bind to NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In some embodiments, within each scFv that binds to EGFR, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In some embodiments, the flexible linker comprises (G4S)4 (SEQ ID NO: 119).

[0028] In some embodiments, in the scFvs that bind to NKG2D, the heavy chain variable domain is located at the C-terminus of the light chain variable domain. In some embodiments, in each scFv that binds to EGFR, the heavy chain variable domain is located at the C-terminus of the light chain variable domain. In some embodiments, in the scFvs that bind to NKG2D, the heavy chain variable domain is located at the N-terminus of the light chain variable domain.

[0029] In some embodiments, in each scFv that binds to EGFR, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In some embodiments, the Fab fragment that binds to NKG2D is not located between the antigen binding site and Fc or a portion thereof. In some embodiments, the Fab fragment that binds to EGFR is not located between the antigen binding site and Fc or a portion thereof.

[0030] In some embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 81, 82 and 112, respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In some embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 81, 82 and 97, respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In some embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 95, and the VL of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85. In some embodiments, the VH of the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 95, and the VL of the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 85.

[0031] In some embodiments, the antibody Fc domain comprises a hinge and a CH2 domain. In some embodiments, the antibody Fc domain is a human IgG1 antibody Fc domain. In some embodiments, the antibody Fc domain or portion thereof comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody or to SEQ ID NO:118.

[0032] In some embodiments, at least one polypeptide chain of the antibody Fc domain comprises one or more mutations at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439, numbered according to the EU numbering system, relative to SEQ ID NO: 118. In some embodiments, at least one polypeptide chain of the antibody Fc domain is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366V ... and one or more mutations selected from 66M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0033] In some embodiments, one polypeptide chain of the antibody heavy chain constant region has one or more amino acids selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439, numbered according to the EU numbering system, relative to SEQ ID NO:118. or a plurality of mutations; and the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439, numbered according to the EU numbering system, compared to SEQ ID NO: 118. In some embodiments, one polypeptide chain of the antibody heavy chain constant region comprises a K360E and a K409W substitution, numbered according to the EU numbering system, compared to SEQ ID NO: 118; the other polypeptide chain of the antibody heavy chain constant region comprises a Q347R, D399V and F405T substitution, numbered according to the EU numbering system, compared to SEQ ID NO: 118.

[0034] In some embodiments, one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution, numbered according to the EU numbering system, relative to SEQ ID NO:118; and the other polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution, numbered according to the EU numbering system, relative to SEQ ID NO:118.

[0035] Another aspect of the application provides a protein comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:167; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:165.

[0036] Another aspect of the application provides a protein comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:168; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:165.

[0037] Another aspect of the application provides a protein comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:166; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:165.

[0038] Another aspect of the present application provides a formulation comprising a protein disclosed herein and a pharma- ceutically acceptable carrier.

[0039] Another aspect of the present application provides a cell comprising one or more nucleic acids encoding the protein disclosed herein.In some embodiments, the cell comprises one or more nucleic acids encoding the protein comprising the amino acid sequence of SEQ ID NO:167 or SEQ ID NO:169.

[0040] Another aspect of the present application provides a purified protein as disclosed herein. In some embodiments, the protein is purified using a method selected from the group consisting of centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography and mixed mode chromatography.

[0041] Another aspect of the present application provides a method of enhancing tumor cell death, comprising exposing tumor cells and natural killer cells to an effective amount of a protein disclosed herein or a formulation disclosed herein, wherein the tumor cells express EGFR.

[0042] Another aspect of the present application provides a method of treating cancer, comprising administering an effective amount of the protein or formulation disclosed herein to a patient in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, breast cancer, kidney cancer, colorectal cancer, gastric cancer, brain cancer, glioma, bladder cancer, head and neck cancer, bladder cancer, pancreatic cancer and liver cancer, cervical cancer, ovarian cancer or prostate cancer. In some embodiments, the cancer expresses EGFR.

[0043] These and other aspects and advantages of the TriNKET described in this application are explained by the following drawings, detailed description and claims. In an embodiment of the present invention, for example, the following items are provided: (Item 1) (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to EGFR; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; A protein comprising: the second antigen-binding site being (i) a heavy chain variable domain (VH) comprising the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) sequences of SEQ ID NOs: 136, 157 and 138, respectively; and a light chain variable domain (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively; or (ii) a VH comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively; and a VL comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 142, respectively. , including protein. (Item 2) The protein of item 1, wherein the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 157 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively. (Item 3) The protein of item 2, wherein the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively. (Item 4) The protein of item 2, wherein the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 137 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 151, respectively. (Item 5) The protein of item 1, wherein the VH comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 146 and 138, respectively, and the VL comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 140, 141 and 142, respectively. (Item 6) (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to EGFR; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; A protein comprising: the second antigen-binding site comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 135, and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 139; A protein wherein the VH comprises an S62R substitution, as numbered under the Chothia numbering scheme, compared to SEQ ID NO: 135, and / or the VL comprises a D92R substitution and / or an F87Y substitution, as numbered under the Chothia numbering scheme, compared to SEQ ID NO: 139. (Item 7) 7. The protein of item 6, wherein the VH comprises an S62R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 135. (Item 8) 7. The protein of item 6, wherein the VL comprises a D92R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139. (Item 9) 7. The protein of item 6, wherein the VH comprises an S62R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 135, and the VL comprises a D92R substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139. (Item 10) 10. The protein of any one of items 1 to 9, wherein the VL comprises a F87Y substitution as numbered under the Chothia numbering scheme compared to SEQ ID NO: 139. (Item 11) 11. The protein of any one of items 1 to 3 and 6 to 10, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 145 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 150. (Item 12) 12. The protein of any one of items 1 to 3 and 6 to 11, wherein the VH comprises the amino acid sequence of SEQ ID NO: 145 and the VL comprises the amino acid sequence of SEQ ID NO: 150, or the VH comprises the amino acid sequence of SEQ ID NO: 170 and the VL comprises the amino acid sequence of SEQ ID NO: 171. (Item 13) 11. The protein of any one of items 1, 2, 4, 6, 8 and 10, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 135 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 150. (Item 14) 14. The protein of any one of items 1, 2, 4, 6, 8, 10 and 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 135 and the VL comprises the amino acid sequence of SEQ ID NO: 150. (Item 15) 11. The protein of any one of items 1, 5 to 7 and 10, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 145 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 147. (Item 16) The protein according to any one of items 1, 5 to 7, 10 and 15, wherein the VH comprises the amino acid sequence of SEQ ID NO: 145 and the VL comprises the amino acid sequence of SEQ ID NO: 147. The protein according to any one of claims 1 to 16, wherein the second antigen-binding site comprises a single chain variable fragment (scFv), and the scFv comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 152, 154, 148 and 158. 18. The protein of any one of items 1 to 3, 6 to 12 and 17, wherein the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 152. (Item 19) 19. The protein of any one of items 1 to 3, 6 to 12, 17 and 18, wherein the scFV comprises the amino acid sequence of SEQ ID NO: 152. (Item 20) 18. The protein of any one of items 1, 2, 4, 6, 8, 10, 13, 14 and 17, wherein the scFV comprises an amino acid sequence at least 90% identical to SEQ ID NO: 154. (Item 21) The protein of any one of items 1, 2, 4, 6, 8, 10, 13, 14, 17 and 20, wherein the scFV comprises the amino acid sequence of SEQ ID NO: 154. (Item 22) 18. The protein according to any one of items 1, 5 to 7, 10 and 15 to 17, wherein the scFV comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 148. . (Item 23) 23. The protein of any one of items 1, 5 to 7, 10, 15 to 17 and 22, wherein the scFV comprises the amino acid sequence of SEQ ID NO: 148. (Item 24) 24. The protein according to any one of items 1 to 23, wherein the protein comprises a polypeptide comprising an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 167, 168 and 166. (Item 25) 26. The protein according to any one of claims 1 to 3, 6 to 12, 17 to 19 and 24, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 167. 26. The protein of any one of items 1 to 3, 6 to 12, 17 to 19, 24 and 25, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 167. (Item 27) 25. The protein of any one of items 1, 2, 4, 6, 8, 10, 13, 14, 17, 20, 21 and 24, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 168. (Item 28) 28. The protein of any one of items 1, 2, 4, 6, 8, 10, 13, 14, 17, 20, 21, 24 and 27, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 168. (Item 29) 25. The protein of any one of items 1, 5 to 7, 10, 15 to 17 and 22 to 24, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 166. (Item 30) 30. The protein of any one of items 1, 5 to 7, 10, 15 to 17, 22 to 24 and 29, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 166. (Item 31) The second antigen-binding site has a dissociation constant (K) less than or equal to 5 nM as measured by surface plasmon resonance (SPR). D 31. The protein according to any one of items 1 to 30, which binds to human EGFR at the C-terminal end of the cytoplasmic domain. (Item 32) The second antigen-binding site has a dissociation constant (K) less than or equal to 6 nM as measured by surface plasmon resonance (SPR). D 32. The protein according to any one of items 1 to 31, which binds to rhesus monkey EGFR at the C-terminal end of the cytoplasmic domain. (Item 33) 33. The protein of any one of items 1 to 16, 31 and 32, wherein the first antigen-binding site that binds to NKG2D is a Fab fragment and the second antigen-binding site that binds to EGFR is an scFv. (Item 34) 33. The protein of any one of items 1 to 16, 31 and 32, wherein the first antigen-binding site that binds to NKG2D is an scFv and the second antigen-binding site that binds to EGFR is a Fab fragment. (Item 35) 33. The protein of any one of items 1 to 16, 31 and 32, further comprising an additional antigen binding site that binds to EGFR. (Item 36) The first antigen-binding site that binds to NKG2D is an scFv and binds to EGFR. 36. The protein of claim 35, wherein the second and the additional antigen binding sites are each a Fab fragment. (Item 37) 36. The protein of claim 35, wherein the first antigen-binding site that binds to NKG2D is an scFv, and the second and additional antigen-binding sites that bind to EGFR are each scFv. (Item 38) 38. The protein according to any one of items 35 to 37, wherein the amino acid sequences of the second and the additional antigen-binding sites are identical. (Item 39) 39. The protein of any one of items 34 and 36 to 38, wherein the scFv that binds NKG2D is linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or portion thereof sufficient to bind to CD16, and the scFv comprises a heavy chain variable domain and a light chain variable domain. (Item 40) 40. The protein of any one of items 33 and 37 to 39, wherein each scFv that binds EGFR is linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or portion thereof sufficient to bind to CD16, and wherein the scFv comprises a heavy chain variable domain and a light chain variable domain. (Item 41) 41. The protein according to item 39 or 40, wherein the hinge further comprises the amino acid sequence Thr-Lys-Gly. (Item 42) 42. The protein according to any one of items 34 and 36 to 41, wherein within the scFv that binds to NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. (Item 43) 43. The protein according to any one of items 33 and 37 to 42, wherein within each scFv that binds to EGFR, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. (Item 44) 44. The protein according to item 42 or 43, wherein the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, as numbered under the Kabat numbering scheme. (Item 45) 45. The protein of any one of items 34 and 36 to 44, wherein within the scFv that binds to NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. (Item 46) 46. ​​The protein of any one of items 33 and 37 to 45, wherein within each scFv that binds to EGFR, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. (Item 47) 47. The protein according to item 45 or 46, wherein the flexible linker comprises (G4S)4 (SEQ ID NO: 119). (Item 48) 48. The protein of any one of items 34 and 36 to 47, wherein within the scFv that binds to NKG2D, the heavy chain variable domain is located C-terminal to the light chain variable domain. (Item 49) 49. The protein of any one of items 33 and 37 to 48, wherein within each scFv that binds to EGFR, the heavy chain variable domain is located C-terminal to the light chain variable domain. (Item 50) 48. The protein of any one of items 34 and 36 to 47, wherein within the scFv that binds to NKG2D, the heavy chain variable domain is located N-terminal to the light chain variable domain. (Item 51) 51. The protein of any one of items 33, 37 to 48 and 50, wherein within each scFv that binds to EGFR, the heavy chain variable domain is located N-terminal to the light chain variable domain. (Item 52) 52. The protein of any one of items 33, 40-41, 43-44, 46-47, 49 and 51, wherein the Fab fragment that binds to NKG2D is not located between the antigen binding site and the Fc or portion thereof. (Item 53) 51. The protein of any one of items 34, 36, 38-39, 41-42, 44-45, 47-48 and 50, wherein a Fab fragment that binds to EGFR is not located between the antigen binding site and the Fc or portion thereof. (Item 54) 54. The protein of any one of items 1 to 53, wherein the first antigen-binding site that binds to NKG2D comprises a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 81, 82 and 112, respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. (Item 55) 55. The protein of item 54, wherein the first antigen-binding site that binds to NKG2D comprises a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 81, 82 and 97, respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. (Item 56) 56. The protein of item 54 or 55, wherein the VH of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 95 and the VL of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85. (Item 57) 57. The protein of any one of items 54 to 56, wherein the VH of the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 95 and the VL of the first antigen-binding site comprises the amino acid sequence of SEQ ID NO: 85. (Item 58) 58. The protein of any one of the preceding items, wherein the antibody Fc domain comprises a hinge and a CH2 domain. (Item 59) 59. The protein of any one of items 1 to 58, wherein the antibody Fc domain is a human IgG1 antibody Fc domain. (Item 60) 60. The protein according to item 59, wherein the antibody Fc domain or portion thereof comprises an amino acid sequence at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody or to SEQ ID NO: 118. (Item 61) at least one polypeptide chain of the antibody Fc domain comprises the amino acid sequence Q347, Y349, L351, numbered according to the EU numbering system, relative to SEQ ID NO: 118; 61. The protein of item 59 or 60, comprising one or more mutations at one or more positions selected from S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439. (Item 62) at least one polypeptide chain of the antibody Fc domain is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, 62. The protein of any one of items 59 to 61, comprising one or more mutations selected from: T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E. (Item 63) one polypeptide chain of said antibody heavy chain constant region comprises one or more mutations at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439, numbered according to the EU numbering system, compared to SEQ ID NO: 118; 63. The protein of any one of items 59 to 62, wherein the other polypeptide chain of the constant region comprises one or more mutations at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439, numbered according to the EU numbering system, compared to SEQ ID NO: 118. (Item 64) 64. The protein of item 63, wherein one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions, numbered according to the EU numbering system, compared to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions, numbered according to the EU numbering system, compared to SEQ ID NO: 118. (Item 65) 65. The protein of item 63 or 64, wherein one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution, numbered according to the EU numbering system, compared to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution, numbered according to the EU numbering system, compared to SEQ ID NO: 118. (Item 66) (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 167; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 165. Proteins including: (Item 67) (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 165. Proteins including: (Item 68) (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 166; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 164; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 165. Proteins including: (Item 69) A formulation comprising the protein according to any one of the preceding items and a pharma- ceutically acceptable carrier. (Item 70) 69. A cell comprising one or more nucleic acids encoding the protein of any one of items 1 to 68. (Item 71) 70. A method for enhancing tumor cell death, comprising exposing tumor cells and natural killer cells to an effective amount of a protein according to any one of items 1 to 68 or the formulation according to item 69, wherein the tumor cells express EGFR. (Item 72) A method for treating cancer, comprising administering an effective amount of a protein according to any one of items 1 to 68 or a formulation according to item 69 to a patient in need thereof. (Item 73) 73. The method of claim 72, wherein the cancer is a solid tumor. (Item 74) 74. The method according to item 72 or 73, wherein the cancer is lung cancer, breast cancer, kidney cancer, colorectal cancer, gastric cancer, brain cancer, glioma, bladder cancer, head and neck cancer, bladder cancer, pancreatic cancer and liver cancer, cervical cancer, ovarian cancer or prostate cancer. (Item 75) 75. The method of any one of items 72 to 74, wherein the cancer expresses EGFR. (Item 76) A cell comprising one or more nucleic acids encoding a protein comprising the amino acid sequence of SEQ ID NO:167. (Item 77) A cell comprising one or more nucleic acids encoding a protein comprising the amino acid sequence of SEQ ID NO:169. (Item 78) 69. The protein of any one of items 1 to 68, which is a purified protein. (Item 79) 79. The protein of item 78, which has been purified using a method selected from the group consisting of centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography and mixed mode chromatography. [Brief description of the drawings]

[0044] [Figure 1] Figure 1 shows a heterodimeric multispecific binding antibody, such as a trispecific binding protein (TriNKET). Each arm can represent either an NKG2D binding domain or a binding domain corresponding to a tumor-associated antigen. In some embodiments, the NKG2D binding domain and the tumor-associated antigen binding domain can share a common light chain.

[0045] [Figure 2-1]Figures 2A-2E show five exemplary formats of multispecific binding proteins, e.g., trispecific binding proteins (TriNKETs). As shown in Figure 2A, either the NKG2D binding domain or the tumor-associated antigen binding domain can be in scFv format (left arm). An antibody containing an NKG2D-targeting scFv, a tumor-associated antigen targeting Fab fragment, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. An antibody containing a tumor-associated antigen targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerized antibody constant region / domain that binds to CD16 is referred to herein as F3'-TriNKET (Figure 2E). As shown in Figure 2B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. Figures 2C-2D show an antibody with three antigen-binding sites, including two antigen-binding sites that bind tumor-associated antigens and an NKG2D binding site fused to a heterodimerized antibody constant region. These antibody formats are referred to herein as F4-TriNKET. Figure 2C shows that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. Figure 2D shows that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. Figure 2E represents a trispecific antibody (TriNKET) containing a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region / domain ("CD domain") that binds to CD16. The antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerizing mutations on the antibody constant region include K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the constant domain at the opposite position (shown as a triangular lock and key shape in the CD domain). The thick bar between the heavy and light chain variable domains of the Fab fragment represents a disulfide bond. [Figure 2-2] Same as above.

[0046] [Diagram 3] Figure 3 is a diagram depicting TriNKET in the Triomab format, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain, derived from two parent antibodies. The Triomab format can be a heterodimeric construct containing 1 / 2 a rat antibody and 1 / 2 a mouse antibody.

[0047] [Figure 4] Figure 4 is a diagram depicting a KiH Common Light Chain form of TriNKET with knobs-into-holes (KIH) technology. KiH is a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation. TriNKET in KiH format can be a heterodimeric construct with two Fab fragments that bind targets 1 and 2, containing two different heavy chains and a common light chain that pairs with both heavy chains.

[0048] [Diagram 5] Figure 5 is a diagram depicting a dual variable domain immunoglobulin (DVD-Ig™) form of TriNKET that combines the target binding domains of two monoclonal antibodies via a naturally occurring flexible linker resulting in a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of a Fab fragment targeting antigen 1. The DVD-Ig™ form contains a conventional Fc.

[0049] [Figure 6] Figure 6 is a diagram depicting TriNKET in the form of an orthogonal Fab fragment interface (Ortho-Fab), a heterodimeric construct containing two Fab fragments that bind target 1 and target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0050] [Figure 7] FIG. 7 is a diagram depicting TriNKET in 2-in-1 Ig format.

[0051] [Figure 8] Figure 8 depicts the ES form of TriNKET, a heterodimeric construct containing two different Fab fragments that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0052] [Figure 9] Figure 9 is a diagram depicting TriNKET in a Fab arm swapped form: an antibody that swaps Fab fragment arms by exchanging the heavy chain and associated light chain (half molecule) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm swapped form (cFae) is a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0053] [Figure 10] FIG. 10 depicts TriNKET in the SEED Body form, a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0054] [Figure 11] Figure 11 is a diagram depicting the LuZ-Y form of TriNKET, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs that bind targets 1 and 2, fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

[0055] [Figure 12] FIG. 12 is a diagram depicting Cov-X body form of TriNKET.

[0056] [Figure 13] 13A-13B are diagrams depicting a κλ-body form of TriNKET, a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by a heterodimerization mutation, where one Fab fragment targeting antigen 1 contains a kappa LC and the second Fab fragment targeting antigen 2 contains a lambda LC. Figure 13A is an exemplary diagram of one form of κλ-body; Figure 13B is an exemplary diagram of another κλ-body.

[0057] [Figure 14] Figure 14 is a diagram depicting an Oasc-Fab heterodimer construct comprising a Fab fragment that binds target 1 and a scFab that binds target 2, both fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0058] [Figure 15] Figure 15 is a diagram depicting DuetMab, a heterodimeric construct containing two different Fab fragments that bind antigens 1 and 2, and an Fc that is stabilized by a heterodimerization mutation. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct light and heavy chain pairing.

[0059] [Figure 16] Figure 16 depicts a Cross mAb, a heterodimeric construct with two different Fab fragments that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation. The CL and CH1 domains, as well as the VH and VL domains, are switched, e.g., CH1 is fused in-line with the VL, and CL is fused in-line with the VH.

[0060] [Figure 17] 17 is a diagram depicting Fit-Ig, a homodimeric construct in which a Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of a Fab fragment that binds antigen 1. The construct contains wild-type Fc.

[0061] [Figure 18] FIG. 18 shows structural modeling of panitumumab with an S62R substitution (under the Chothia numbering scheme) in VH, where the hydrogen bond between this Arg and Asp at position 1 of VL may contribute to stabilizing the VH-VL interface.

[0062] [Figure 19] FIG. 19 shows structural modeling of panitumumab with a F87Y substitution (under the Chothia numbering scheme) in VL, where the hydrogen bond between this Tyr and Gln at position 39 of VH may contribute to stabilizing the VH-VL interface.

[0063] [Figure 20] FIG. 20 shows structural modeling of panitumumab with a D92R substitution (under the Chothia numbering scheme) in the VL, where van der Waals contacts between this Arg (in CDRL3) and Tyr at position 32 of the VL (in CDRL1) may contribute to stabilization of the paratope.

[0064] [Figure 21] FIG. 21 is an SPR sensorgram for titration of EGFR-TriNKET-1 binding to human EGFR.

[0065] [Figure 22] FIG. 22 is an SPR sensorgram for titration of EGFR-TriNKET-2 binding to human EGFR.

[0066] [Diagram 23] FIG. 23 is an SPR sensorgram for titration of EGFR-TriNKET-3 binding to human EGFR.

[0067] [Figure 24] FIG. 24 is an SPR sensorgram for titration of EGFR-TriNKET-4 binding to human EGFR.

[0068] [Diagram 25] Figures 25A, 25B, 25C, and 25D are plots showing thermograms for EGFR-TriNKET-1 (Figure 25A), EGFR-TriNKET-2 (Figure 25B), and EGFR-TriNKET-3 (Figure 25C), respectively, in PBS at pH 7.4, as determined by differential scanning calorimetry (DSC) analysis.

[0069] [Figure 26] Figures 26A and 26B are plots showing thermograms for EGFR-TriNKET-3 (Figure 26A) and EGFR-TriNKET-4 (Figure 26B), respectively, in 20 mM histidine, 250 mM trehalose, 0.01% PS80, pH 6.0, as determined by DSC analysis.

[0070] [Figure 27] FIG. 27 is a plot showing the binding affinity of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), EGFR-TriNKET-2 ("EGFR2"), EGFR-TriNKET-3 ("EGFR3") and panitumumab to EGFR-positive H2172 cancer cells.

[0071] [Figure 28] FIG. 28 is a plot showing NK cell-mediated lysis of EGFR-expressing H2172 cancer cells in the presence of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), EGFR-TriNKET-3 ("EGFR3"), EGFR-TriNKET-4 ("EGFR4"), and cetuximab.

[0072] [Figure 29] FIG. 29 is a plot showing CD8+ T cell-mediated lysis of EGFR-positive 786-0 cancer cells in the presence of a range of concentrations of EGFR-TriNKET-3 ("EGFR3"), EGFR-TriNKET-4 ("EGFR4"), and cetuximab.

[0073] [Diagram 30] FIG. 30 is a plot showing IFN-γ production from NK cells when incubated with EGFR-positive BT-474 cancer cells in the presence of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), EGFR-TriNKET-2 ("EGFR2"), EGFR-TriNKET-3 ("EGFR3"), and cetuximab.

[0074] [Diagram 31] FIG. 31 is a plot showing IFN-γ production from IL-2-activated NK cells when incubated with EGFR-positive BT-474 cancer cells in the presence of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), EGFR-TriNKET-3 ("EGFR3"), and cetuximab.

[0075] [Diagram 32] FIG. 32 is a plot showing the proliferation of EGFR-positive cell lines in the presence of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), EGFR-TriNKET-2 ("EGFR2"), EGFR-TriNKET-3 ("EGFR3"), panitumumab, and cetuximab over a 72 hour period.

[0076] [Diagram 33] FIG. 33 is a plot showing the percentage of EGFR-expressing target cells that were phagocytosed by macrophages as a result of antibody-dependent cellular phagocytosis (ADCP) following a two-hour incubation in the presence of a range of concentrations of EGFR-TriNKET-1 ("EGFR1"), a variant of EGFR-TriNKET-1 with L234A, L235A and P329G (LALAPG) mutations in the Fc domain ("EGFR1-CD16si"), EGFR-TriNKET-3 ("EGFR3"), EGFR-TriNKET-4 ("EGFR4") and cetuximab, as measured by flow cytometry.

[0077] [Figure 34-1]34A-34D are plots showing tumor volume over time in nude mice xenografted with EGFR-positive NCI-H292 cells treated with the indicated isotype control or EGFR-TriNKET. FIG. 34A shows tumor volume in mice treated with 300 μg, 100 μg, or 30 μg of EGFR-TriNKET at the indicated time points. FIG. 34B shows tumor volume in mice treated with 300 μg of isotype control or EGFR-TriNKET at the indicated time points. FIG. 34C shows tumor volume in mice treated with 100 μg of isotype control or EGFR-TriNKET at the indicated time points. FIG. 34D shows tumor volume in mice treated with 30 μg of isotype control or EGFR-TriNKET at the indicated time points. [Figure 34-2] Same as above.

[0078] [Diagram 35] Figures 35A-35B are plots showing tumor volume or body weight over time in nude mice xenografted with EGFR-positive NCI-H292 cells treated with the indicated isotype control, EGFR-TriNKET, or cetuximab. Figure 35A shows tumor volume in mice treated with 100 μg of isotype control, EGFR-TriNKET, or cetuximab at the indicated time points. Figure 35B shows body weight in mice treated with 100 μg of isotype control, EGFR-TriNKET, or cetuximab at the indicated time points. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells, as well as EGFR. In some embodiments, the multispecific binding proteins further comprise an additional antigen binding site that binds to EGFR. The present application also provides pharmaceutical compositions that include such multispecific binding proteins, as well as therapeutic methods that use such multispecific binding proteins and pharmaceutical compositions for purposes such as treating cancer. Various aspects of the multispecific binding proteins described in the present application are presented in the following sections, although aspects of the multispecific binding proteins described in a particular section should not be limited to any particular section.

[0080] To facilitate understanding of this application, several terms and phrases are defined below.

[0081] As used herein, the terms "a" and "an" mean "one or more" and include the plural forms unless the context is inappropriate.

[0082] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly variable regions within the V regions of the heavy and light chains are called "hypervariable regions" and are interposed between more conserved adjacent regions known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, resulting in a "single domain antibody". The antigen-binding site can be present in an intact antibody, an antigen-binding fragment of an antibody that retains the antigen-binding surface, or a recombinant polypeptide such as an scFv that uses a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0083] The term "tumor-associated antigen" as used herein refers to any antigen, including but not limited to, proteins, glycoproteins, gangliosides, carbohydrates, lipids, associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. In certain embodiments of the present disclosure, the term "tumor-associated antigen" refers to EGFR.

[0084] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, ape, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0085] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound described herein) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or to a route of administration. As used herein, the term "treat" includes any effect, such as alleviating, reducing, modulating, improving or eliminating, that results in the improvement of a condition, disease, disorder, or the like, or ameliorating the symptoms thereof.

[0086] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with a carrier, inert or active, that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0087] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0088] As used herein, the term "pharmaceutical acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of the compounds described herein that can provide the compounds described herein or their active metabolites or residues upon administration to a subject. As known to those skilled in the art, the "salts" of the compounds of the present application can be obtained from inorganic or organic acids and inorganic or organic bases. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, can also be used in the preparation of salts useful as intermediates in obtaining the compounds of the present application and their pharmaceutically acceptable acid addition salts.

[0089] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of the formula NW4 + (Wherein, W is C 1~4 Examples of the compounds include those in which the aryl group is an alkyl group.

[0090] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include Na + , NH4 + and NW4 + (Wherein, W is C 1~4 Examples of suitable cations include the anions of the compounds of the present application in complex with suitable cations such as alkyl groups, alkyl cations, and the like.

[0091] For therapeutic use, the salts of the compounds of the present application are contemplated as pharma- ceutically acceptable. However, salts of acids and bases that are non-pharma-ceutically acceptable may also find use, for example, in the preparation or purification of a pharma- ceutically acceptable compound.

[0092] As used herein, EGFR (also known as epidermal growth factor receptor, ErbB-1, or HER1 in humans) refers to the protein with Uniprot accession number P00533 (human), as well as related isoforms and orthologs.

[0093] Throughout this specification, when compositions are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions that consist essentially of or consist of the recited components, and that there are processes and methods according to this application that consist essentially of or consist of the recited processing steps.

[0094] In general, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the variable's preceding definition controls. I. Protein

[0095] The present application provides a multispecific binding protein that binds to the NKG2D and CD16 receptors on natural killer cells and EGFR. The multispecific binding protein is useful in the pharmaceutical compositions and treatment methods described herein. The binding of the multispecific binding protein to the NKG2D and CD16 receptors on natural killer cells enhances the activity of natural killer cells toward the destruction of tumor cells expressing tumor antigens. The binding of the multispecific binding protein to tumor antigen-expressing tumor cells brings these cells closer to natural killer cells, facilitating the direct and indirect destruction of tumor cells by natural killer cells. Multispecific binding proteins that bind to NKG2D, CD16 and other targets are disclosed in International Patent Publication Nos. WO2018148445 and WO2019157366, which are not incorporated herein by reference. Further description of some exemplary multispecific binding proteins is provided below.

[0096] The first component of the multispecific binding protein is an antigen-binding site that binds to NKG2D receptor expressing cells, including but not limited to NK cells, γδ T cells and CD8 +Upon binding to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

[0097] The second component of the multispecific binding protein is an antigen-binding site that binds to EGFR.EGFR-expressing cells can be found in indications such as solid tumors, such as lung cancer, breast cancer, kidney cancer, colorectal cancer, gastric cancer, brain cancer, glioma, bladder cancer, head and neck cancer, bladder cancer, pancreatic cancer and liver cancer, cervical cancer, ovarian cancer or prostate cancer.The antigen-binding site that binds to EGFR comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) derived from panitumumab, with mutations in VH and VL that increase thermal stability and retain affinity to EGFR.

[0098] The third component of the multispecific binding protein is an antibody Fc domain or portion thereof or antigen binding site that binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells and follicular dendritic cells.

[0099] The additional antigen-binding sites of the multispecific binding protein may bind to the same tumor-associated antigen (EGFR). In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second and additional antigen-binding sites that bind to EGFR are each an Fab fragment. In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second and additional antigen-binding sites that bind to EGFR are each an scFv. In certain embodiments, the first antigen-binding site that binds to NKG2D is an Fab fragment, and the second and additional antigen-binding sites that bind to EGFR are each an scFv. In certain embodiments, the first antigen-binding site that binds to NKG2D is an Fab fragment, and the second and additional antigen-binding sites that bind to EGFR are each an Fab fragment.

[0100] Each antigen-binding site may incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g. arranged like an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may be a single domain antibody, e.g. a V like a camelid antibody. H H antibodies or V antibodies like those found in cartilaginous fishes NAR It may be an antibody.

[0101] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having an amino acid sequence identical to the amino acid sequence of a light chain variable domain present in the first antigen-binding site.

[0102] The multispecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain and a second immunoglobulin light chain (Figure 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain polypeptide, a first heavy chain variable domain and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and optionally a first light chain constant domain. The first immunoglobulin light chain together with the first immunoglobulin heavy chain forms an antigen binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain polypeptide, a second heavy chain variable domain and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain comprises a second light chain variable domain and optionally a second light chain constant domain. The second immunoglobulin light chain forms an antigen binding site that binds to EGFR together with the second immunoglobulin heavy chain. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide together can bind to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0103] Another exemplary format involves a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain (e.g., FIG. 2A). In some embodiments, the first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain polypeptide fused, via either a linker or an antibody hinge, to a single-chain variable region fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pair and bind to NKG2D or bind to EGFR. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain polypeptide, a second heavy chain variable domain and a CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. In some embodiments, the second immunoglobulin heavy chain pairs with an immunoglobulin light chain and binds to NKG2D or binds to EGFR, except that if the first Fc domain polypeptide is fused to an scFv that binds to NKG2D, the second immunoglobulin heavy chain pairs with an immunoglobulin light chain that binds to EGFR but not to NKG2D, and vice versa. In some embodiments, the scFv in the first immunoglobulin heavy chain binds to EGFR, and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain bind to NKG2D when paired (e.g., FIG. 2E). In some embodiments, the scFv in the first immunoglobulin heavy chain binds to NKG2D, and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain bind to EGFR when paired. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide together can bind to CD16 (e.g., FIG. 2A).

[0104] Another exemplary format involves a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain and a second immunoglobulin heavy chain (e.g., FIG. 2B). In some embodiments, the first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain polypeptide fused, via either a linker or an antibody hinge, to a single chain variable region fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that are paired and bind NKG2D or that bind EGFR. In some embodiments, the second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain polypeptide fused, via either a linker or an antibody hinge, to a single chain variable region fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pair and bind NKG2D or EGFR, with the proviso that when the first Fc domain polypeptide is fused to an scFv that binds NKG2D, the second Fc domain polypeptide is fused to an scFv that binds EGFR but not NKG2D, and vice versa. In some embodiments, the first Fc domain polypeptide and the second Fc domain polypeptide together are capable of binding CD16 (e.g., FIG. 2B).

[0105] In some embodiments, the single chain variable region fragment (scFv) is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises the amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge connecting the scFv (e.g., the scFv that binds EGFR or the scFv that binds NKG2D) and the antibody heavy chain constant domain comprises the amino acids Ala-Ser. In some embodiments, the hinge connecting the scFv (e.g., the scFv that binds EGFR or the scFv that binds NKG2D) and the antibody heavy chain constant domain comprises the amino acids Gly-Ser. In some other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence provides flexibility in binding to the target antigen and can balance flexibility and optimal geometry.

[0106] In some embodiments, the single chain variable fragment (scFv) comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance the stability of the scFv. For example, the disulfide bridge may be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions being numbered according to Kabat. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker may be used, for example, a (G4S)4 linker ((GlyGlyGlyGlySer)4 (SEQ ID NO: 119). In some embodiments of the scFv, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In some embodiments of the scFv, the heavy chain variable domain is located at the C-terminus of the light chain variable domain.

[0107] The multispecific binding proteins described herein may further comprise one or more additional antigen binding sites. The additional antigen binding site(s) may be fused to the N-terminus of the constant region CH2 domain or the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the additional antigen binding site(s) may take the form of a single chain variable fragment (scFv), optionally disulfide stabilized, resulting in a tetravalent or trivalent multispecific binding protein. For example, the multispecific binding protein comprises a first antigen binding site that binds NKG2D, a second antigen binding site that binds EGFR, an additional antigen binding site that binds EGFR, and a sufficient antibody constant region or portion thereof that binds CD16, or a fourth antigen binding site that binds CD16. Any one of these antigen binding sites may take the form of a Fab fragment or scFv, such as the scFv described above.

[0108] In some embodiments, the additional antigen binding site binds to a different epitope of EGFR than the second antigen binding site. In some embodiments, the additional antigen binding site binds to the same epitope as the second antigen binding site. In some embodiments, the additional antigen binding site comprises the same heavy and light chain CDR sequences as the second antigen binding site. In some embodiments, the additional antigen binding site comprises the same heavy and light chain variable domain sequences as the second antigen binding site. In some embodiments, the additional antigen binding site has the same amino acid sequence as the second antigen binding site. In some embodiments, the additional antigen binding site comprises different heavy and light chain variable domain sequences from the heavy and light chain variable domain sequences of the second antigen binding site. In some embodiments, the additional antigen binding site has a different amino acid sequence from the sequence of the second antigen binding site. In some embodiments, the second antigen binding site and the additional antigen binding site bind different tumor associated antigens. In some embodiments, the second antigen binding site and the additional antigen binding site bind different antigens. Exemplary formats are shown in Figures 2C and 2D. Thus, the multispecific binding protein can provide bivalent engagement of EGFR. Bivalent engagement of EGFR by the multispecific binding protein can stabilize EGFR on the tumor cell surface and enhance NK cell cytotoxicity against tumor cells. Bivalent engagement of EGFR by the multispecific binding protein can confer stronger binding of the multispecific binding protein to tumor cells, thereby promoting a stronger NK cell cytotoxic response against tumor cells, particularly against tumor cells expressing low levels of EGFR.

[0109] Multispecific binding proteins can take additional formats. In some embodiments, the multispecific binding protein is in the form of a Triomab, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain, derived from two parent antibodies.

[0110] In some embodiments, the multispecific binding protein is in KiH format and involves knobs-into-holes (KiH) technology. H The idea behind "knobs-into-holes (KiH)" Fc technology is to engineer the three domains to create either "knobs" or "holes" in each heavy chain to promote heterodimerization. The concept behind "knobs-into-holes (KiH)" Fc technology is to replace small residues with bulky residues (e.g., T366W in the EU numbering system). CH3A The first step was to introduce a "knob" into one of the CH3 domains (CH3A) by replacing the adjacent residues closest to the knob with smaller residues (e.g., T366S / L368A / Y407V CH3B ), and a complementary "hole" face was engineered on the other CH3 domain (CH3B). The "hole" mutations were optimized by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(1):26-35). X-ray crystal structure of KiH Fc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9):1947-57;Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variants with improved affinity for FcγRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the CH3 domain-to-CH3 domain core interface, while knob-knob and hole-hole interfaces disfavor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0111] In some embodiments, the multispecific binding protein is in the dual variable domain immunoglobulin (DVD-Ig™) format, which combines the target binding domains of two monoclonal antibodies via a naturally occurring flexible linker, resulting in a tetravalent IgG-like molecule.

[0112] In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab). In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191-8), structure-based domain design involves the addition of LC and HC domains to only one Fab fragment, without any changes to the other Fab fragment. VH-CH1 Complementary mutations are introduced at the interface.

[0113] In some embodiments, the multispecific binding protein is a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is an ES format, which is a heterodimeric construct containing two different Fab fragments that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in Fc.

[0114] In some embodiments, the multispecific binding protein is in the form of a κλ-body, which is a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by a heterodimerization mutation, where Fab fragment 1 targeting antigen 1 contains a kappa LC and Fab fragment 2 targeting antigen 2 contains a lambda LC. Figure 13A is an exemplary diagram of one form of κλ-body; Figure 13B is an exemplary diagram of another κλ-body.

[0115] In some embodiments, the multispecific binding protein is in the form of a Fab fragment arm exchange (an antibody that exchanges Fab arms by swapping the heavy chain and associated light chain (half molecule) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody).

[0116] In some embodiments, the multispecific binding protein is in the form of a SEED body. The chain exchange engineered domain (SEED) platform is a feature that expands the therapeutic applications of natural antibodies and has been designed to generate asymmetric and bispecific antibody-like molecules. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins in the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while refusing homodimerization of AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

[0117] In some embodiments, the multispecific binding protein is of the LuZ-Y form, in which a leucine zipper is used to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0118] In some embodiments, the multispecific binding protein is in the form of a Cov-X body. In bispecific CovX bodies, two different peptides are linked using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. The pharmacophore is responsible for the functional activity, while the antibody scaffold provides a long half-life and Ig-like distribution. The pharmacophore can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies (Doppalapudi VR et al., PNAS (2010), 107(52);22611-22616).

[0119] In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer, comprising a Fab fragment that binds target 1 and a scFab that binds target 2, fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0120] In some embodiments, the multispecific binding protein is in the DuetMab format, a heterodimeric construct containing two different Fab fragments that bind antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct LC and HC pairing.

[0121] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, a heterodimeric construct with two different Fab fragments that bind targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains, as well as the VH and VL domains, are switched, e.g., CH1 is fused in frame with VL and CL is fused in frame with VH.

[0122] In some embodiments, the multispecific binding protein is in the Fit-Ig format, a homodimeric construct in which a Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of a Fab fragment that binds antigen 1. The construct contains a wild-type Fc.

[0123] The individual components of the multispecific binding proteins are described in more detail below. NKG2D binding site

[0124] Upon binding to the NKG2D and CD16 receptors and EGFR on natural killer cells, the multispecific binding protein can bind to more than one NK activating receptor and block the binding of natural ligands to NKG2D.In some embodiments, the protein can stimulate the NK cells of humans and other species, such as rodents and cynomolgus monkeys.In some embodiments, the protein can stimulate the NK cells of humans and other species, such as cynomolgus monkeys.

[0125] Table 1 lists peptide sequences of heavy and light chain variable domains that can be combined to bind to NKG2D. In some embodiments, the heavy and light chain variable domains are arranged in a Fab format. In some embodiments, the heavy and light chain variable domains are fused together to form a scFv.

[0126] The NKG2D binding sites listed in Table 1 all activate human NK cells, although their binding affinities for NKG2D may differ.

[0127] Unless otherwise indicated, the CDR sequences provided in Table 1 are determined by Kabat numbering. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0128] In certain embodiments, the first antigen-binding site that binds to NKG2D (e.g., human NKG2D) comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the VL of the same antibody disclosed in Table 1. In certain embodiments, the first antigen-binding site is selected from the VH and VL sequences of antibodies disclosed in Table 1, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., Chothia C & The first antigen-binding site comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 as determined by Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art. In certain embodiments, the first antigen-binding site comprises the heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of an antibody disclosed in Table 1.

[0129] In certain embodiments, a first antigen-binding site that binds to NKG2D comprises a heavy chain variable domain derived from SEQ ID NO: 1, e.g., having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 1 and / or by incorporating amino acid sequences identical to the CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3) and CDR3 (SEQ ID NO: 4) sequences of SEQ ID NO: 1. The heavy chain variable domain associated with SEQ ID NO: 1 can be linked with various light chain variable domains to form an NKG2D-binding site. For example, a first antigen binding site incorporating a heavy chain variable domain related to SEQ ID NO:1 may further incorporate a light chain variable domain selected from sequences derived from SEQ ID NOs:5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 46. For example, the first antigen-binding site incorporates a heavy chain variable domain having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO:1, and a light chain variable domain having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to any one of the sequences selected from SEQ ID NOs:5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 46.

[0130] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 26, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 32. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 27 or 28, 29 and 30 or 31, respectively (e.g., SEQ ID NO: 27, 29 and 30, respectively, or SEQ ID NO: 28, 29 and 31, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34 and 35, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29 and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29 and 30, respectively, or SEQ ID NOs: 28, 29 and 31, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34 and 35, respectively.

[0131] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 36, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 42. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 37 or 38, 39 and 40 or 41, respectively (e.g., SEQ ID NO: 37, 39 and 40, respectively, or SEQ ID NO: 38, 39 and 41, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44 and 45, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39 and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39 and 40, respectively, or SEQ ID NOs: 38, 39 and 41, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44 and 45, respectively.

[0132] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 47, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 49. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29 and 48, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34 and 51, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29 and 48, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34 and 51, respectively.

[0133] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 52, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 58. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55 and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55 and 56, respectively, or SEQ ID NOs: 54, 55 and 57, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60 and 61, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55 and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55 and 56, respectively, or SEQ ID NOs: 54, 55 and 57, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60 and 61, respectively.

[0134] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 62, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 68. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 63 or 64, 65 and 66 or 67, respectively (e.g., SEQ ID NO: 63, 65 and 66, respectively, or SEQ ID NO: 64, 65 and 67, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60 and 69, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65 and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65 and 66, respectively, or SEQ ID NOs: 64, 65 and 67, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60 and 69, respectively.

[0135] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 89, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 92. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55 and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55 and 90, respectively, or SEQ ID NOs: 54, 55 and 91, respectively). In certain embodiments, the VL comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44 and 94, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55 and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55 and 90, respectively, or SEQ ID NOs: 54, 55 and 91, respectively); and (b) a VL comprising a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44 and 94, respectively.

[0136] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 70, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 75. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72 and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72 and 73, respectively, or SEQ ID NOs: 115, 72 and 74, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77 and 78, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72 and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72 and 73, respectively, or SEQ ID NOs: 115, 72 and 74, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77 and 78, respectively.

[0137] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 79, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 83 or 84, respectively (e.g., SEQ ID NO: 80, 82 and 83, respectively, or SEQ ID NO: 81, 82 and 84, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82 and 83, respectively, or SEQ ID NOs: 81, 82 and 84, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0138] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 95, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 96 or 97, respectively (e.g., SEQ ID NO: 80, 82 and 96, respectively, or SEQ ID NO: 81, 82 and 97, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82 and 96, respectively, or SEQ ID NOs: 81, 82 and 97, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0139] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 98, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 99 or 100, respectively (e.g., SEQ ID NO: 80, 82 and 99, respectively, or SEQ ID NO: 81, 82 and 100, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82 and 99, respectively, or SEQ ID NOs: 81, 82 and 100, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0140] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 101, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 102 or 103, respectively (e.g., SEQ ID NO: 80, 82 and 102, respectively, or SEQ ID NO: 81, 82 and 103, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82 and 102, respectively, or SEQ ID NOs: 81, 82 and 103, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0141] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 104, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 105 or 106, respectively (e.g., SEQ ID NO: 80, 82 and 105, respectively, or SEQ ID NO: 81, 82 and 106, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82 and 105, respectively, or SEQ ID NOs: 81, 82 and 106, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0142] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 107, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 80 or 81, 82 and 108 or 109, respectively (e.g., SEQ ID NO: 80, 82 and 108, respectively, or SEQ ID NO: 81, 82 and 109, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82 and 108, respectively, or SEQ ID NOs: 81, 82 and 109, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0143] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 110, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 85. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82 and 111, respectively, or SEQ ID NOs: 81, 82 and 112, respectively). In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82 and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82 and 111, respectively, or SEQ ID NOs: 81, 82 and 112, respectively); and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77 and 87, respectively.

[0144] In certain embodiments, the first antigen binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 113 and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 114.

[0145] In certain embodiments, the first antigen binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO:116 and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO:117.

[0146] The multispecific binding proteins are capable of binding to NKG2D-expressing cells, including, but not limited to, NK cells, γδ T cells and CD8 + Upon binding to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

[0147] The multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells and follicular dendritic cells. The proteins described in this disclosure may have a concentration of from 2nM to 120nM, e.g., from 2nM to 110nM, from 2nM to 100nM, from 2nM to 90nM, from 2nM to 80nM, from 2nM to 70nM, from 2nM to 60nM, from 2nM to 50nM, from 2nM to 40nM, from 2nM to 30nM, from 2nM to 20nM, from 2nM to 10nM, about 15nM, about 14nM, about 13nM, about 12nM, about 11nM, about 10nM, about 9nM, about 8nM, about 7nM, about 6nM, about 5nM, about 4.5nM, about 4nM, about 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 0.5nM to about 1nM, about 1nM to about 2nM, about 2nM to about 3nM, about 3nM to 4nM, about 4nM to about 5nM, about 5nM to about 6nM, about 6nM to about 7nM, about 7nM to about 8nM, about 8nM K between about 9nM, about 9nM to about 10nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM or about 8nM to about 10nM D In some embodiments, the NKG2D binding site binds to NKG2D with an affinity of 10 to 62 nM. D It binds to NKG2D. EGFR binding site

[0148] In one aspect, the present disclosure provides a multispecific binding protein that binds to the NKG2D and CD16 receptors on natural killer cells and EGFR, the EGFR binding site comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) derived from panitumumab, with mutations in the VH and VL. The mutations contemplated individually or in combination in the VH and VL sequences described in the present disclosure include S62R in VH, D92R in VL, and / or F87Y in VL under the Chothia numbering scheme. It has been discovered that these mutations increase the thermal stability of the antigen binding site and retain its affinity for EGFR. Table 2 lists some exemplary sequences of heavy chain variable domains, light chain variable domains, and scFv sequences that can bind to EGFR. The CDR sequences are identical under Chothia numbering as shown. The bolded residues indicate the mutated residues, and the italicized sequences indicate the polypeptide linker. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0149] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises an additional arginine (R) residue at the C-terminus of the VL (e.g., SEQ ID NO: 139, 147, or 150). In certain embodiments, the second antigen-binding site comprises an scFv comprising a VL amino acid sequence that contains an additional arginine (R) residue at the C-terminus of the VL amino acid sequence.

[0150] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises one or more mutations selected from S62R in VH, D92R in VL, and F87Y in VL under the Chothia numbering scheme, compared to the sequence of panitumumab. In certain embodiments, the second antigen-binding site that binds EGFR (e.g., human EGFR) comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the VH of EGFR-binder 1 disclosed in Table 1, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the VL of EGFR-binder 1 disclosed in Table 1. In certain embodiments, the second antigen-binding site that binds EGFR (e.g., human EGFR) comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the VH of EGFR-binder 2, EGFR-binder 3 or EGFR-binder 4 disclosed in Table 2, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the VL of EGFR-binder 2, EGFR-binder 3 or EGFR-binder 4 disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises the VH and VL sequences of EGFR-binder 2, EGFR-binder 3 or EGFR-binder 4 disclosed in Table 2, as determined by Kabat (Kabat et al. al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see, MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745) or any other CDR determination method known in the art. In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3 of EGFR-binder 2, EGFR-binder 3 or EGFR-binder 4 disclosed in Table 2.

[0151] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises the mutations S62R in VH and F87Y in VL under the Chothia numbering scheme compared to the sequence of panitumumab. In certain embodiments, the second antigen-binding site that binds to EGFR comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 135 and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 139. In certain embodiments, the second antigen binding site that binds EGFR comprises a heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 145 and a light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 147.In certain embodiments, the second antigen-binding site comprises the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of EGFR-binder 2 disclosed in Table 2, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 146 and 138, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 146 and 138, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 142, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 148 or 149. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 148 or 149. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 148.In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO:149.

[0152] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises the following mutations under the Chothia numbering scheme compared to the sequence of panitumumab: S62R in VH, F87Y in VL, and D92R in VL. In certain embodiments, the second antigen-binding site that binds to EGFR comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 135 and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 139. In certain embodiments, the second antigen-binding site that binds EGFR comprises a heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 145 and a light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 150. In certain embodiments, the second antigen-binding site that binds EGFR comprises a heavy chain variable domain (VH) that comprises SEQ ID NO: 170 and a light chain variable domain (VL) that comprises SEQ ID NO: 171. In certain embodiments, the second antigen-binding site is selected from the group consisting of the VH and VL sequences of EGFR-binder 3 disclosed in Table 2, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 146, and 138, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 151, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 146 and 138, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 151, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 152 or 153. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 152 or 153. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 152. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 153.

[0153] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises the following mutations under the Chothia numbering scheme in VL: F87Y, and D92R in VL, compared to the sequence of panitumumab. In certain embodiments, the second antigen-binding site that binds to EGFR comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 135, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 139. In certain embodiments, the second antigen binding site that binds EGFR comprises a heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 135 and a light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 150. In certain embodiments, the second antigen-binding site is selected from the VH and VL sequences of EGFR-binder 4 disclosed in Table 2, as described by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745) or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 138, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 151, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 138, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 151, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 154 or 155. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 154. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 155.

[0154] In certain embodiments, the amino acid sequence of the second antigen-binding site comprises the following mutations under the Chothia numbering scheme as compared to the sequence of panitumumab: F87Y in VL, D92R in VL, and optionally S62R in VH. In certain embodiments, the second antigen-binding site that binds to EGFR comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 135, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 139. In certain embodiments, the second antigen binding site that binds EGFR comprises a heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 156 and a light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 150. In certain embodiments, the second antigen-binding site is selected from the consensus VH and VL sequences disclosed in Table 2, including those of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 157, and 138, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 151, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 136, 157 and 138, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 151, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 158 or 159. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO: 158. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence identical to SEQ ID NO:159.

[0155] Alternatively, novel antigen-binding sites capable of binding to EGFR can be identified by screening for binding to the amino acid sequence of EGFR, its isoforms, its variants, its mature extracellular fragments, or fragments containing domains of EGFR. [Table 3-1] [Table 3-2] [Table 3-3]

[0156] In some embodiments, novel antigen-binding sites capable of binding to EGFR can be identified by screening for binding to the amino acid sequences defined by SEQ ID NOs: 160-163, variants thereof, mature extracellular fragments thereof, or fragments containing domains of EGFR.

[0157] In each of the foregoing embodiments, it is contemplated herein that scFv, VH and / or VL sequences that bind EGFR may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5 or 10 amino acid substitutions, deletions or additions) in the framework regions of the VH and / or VL without affecting their ability to bind EGFR. For example, it is contemplated herein that scFv, VH and / or VL sequences that bind EGFR may contain cysteine ​​heterodimerization mutations to promote the formation of disulfide bridges between the VH and VL of the scFv.

[0158] In certain embodiments, the second antigen-binding site of the multispecific binding protein disclosed herein has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM. D In certain embodiments, the antigen-binding site of the present application binds to human EGFR or an extracellular domain thereof with an affinity of less than or equal to 4 nM. DIn certain embodiments, the antigen-binding site of the present application binds to human EGFR or an extracellular region thereof with an affinity of less than or equal to about 2.0 nM, 2.1 nM, 2.2 nM, 2.3 nM, 2.4 nM, 2.5 nM, 2.6 nM, 2.7 nM, 2.8 nM, 2.9 nM, 3.0 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, 4.0 nM, 4.1 nM, 4.2 nM, 4.3 nM, 4.4 nM, 4.5 nM, 4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM, or 5.0 nM. D In certain embodiments, the antigen-binding site of the present application binds to human EGFR or an extracellular region thereof with an affinity greater than or equal to a K value in the range of about 1.0-3.5 nM, 1.0-4.0 nM, 1.0-4.5 nM, 1.0-5.0 nM, 1.5-3.5 nM, 1.5-4.0 nM, 1.5-4.5 nM, 1.5-5.0 nM, 2.0-3.5 nM, 2.0-4.0 nM, 2.0-4.5 nM, 2.0-5.0 nM, 2.5-3.5 nM, 2.5-4.0 nM, 2.5-4.5 nM, 2.5-5.0 nM, 3.0-3.5 nM, 3.0-4.0 nM, 3.0-4.5 nM, or 3.0-5.0 nM. D It binds to human EGFR or its extracellular domain at

[0159] In certain embodiments, the second antigen-binding site of the multispecific binding protein disclosed herein has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM. D In certain embodiments, the antigen-binding site of the present application binds to rhesus monkey EGFR or an extracellular domain thereof with an affinity greater than or equal to a K value in the range of about 1-10 nM, 1-6 nM, 2-10 nM, 2-6 nM, 3-10 nM, 3-6 nM, 4-10 nM, 4-6 nM, 5-10 nM, or 5-6 nM. D It binds to rhesus monkey EGFR or its extracellular domain at

[0160] In certain embodiments, the second antigen-binding site of the multispecific binding protein disclosed herein has a higher thermal stability than the corresponding antigen-binding site having the VH and VL sequences of SEQ ID NOs: 135 and 139, where the second antigen-binding site does not include a G44C mutation in VH and a G100C mutation in VL. In certain embodiments, the second antigen-binding site of the multispecific binding protein disclosed herein has a higher thermal stability than the corresponding antigen-binding site having the amino acid sequences of SEQ ID NOs: 143 and 144, where the second antigen-binding site is in a scFv format in a VL-VH or VH-VL orientation, respectively. Methods for measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), e.g., as disclosed in Example 3 below. In certain embodiments, when the second antigen-binding site is in scFv format in the VL-VH orientation, the melting temperature of the second antigen-binding site (e.g., Tonset or Tm1 of TriNKET in F3' format measured by DSC) is at least 1°C, 2°C, 3°C, 4°C, 5°C, or 6°C higher than the corresponding melting temperature of an scFv having the amino acid sequence of SEQ ID NO: 143. In certain embodiments, when the second antigen-binding site is in scFv format in the VH-VL orientation, the melting temperature of the second antigen-binding site (e.g., Tonset or Tm1 of TriNKET in F3' format measured by DSC) is at least 1°C, 2°C, 3°C, 4°C, 5°C, or 6°C higher than the corresponding melting temperature of an scFv having the amino acid sequence of SEQ ID NO: 144. Fc domain

[0161] In the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mainly concentrated on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406 (6793):267-273). Based on the known domain, mutations can be selected to enhance or reduce the binding affinity to CD16, for example, by using a phage display library or a yeast surface display cDNA library, or mutations can be designed based on the known three-dimensional structure of the interaction. Thus, in certain embodiments, the antibody Fc domain or a portion thereof comprises a hinge and a CH2 domain.

[0162] The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. The promotion of preferential assembly of heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480 and US14 / 830336. For example, the CH3 domain based on human IgG1 can be mutated by incorporating different pairs of amino acid substitutions in these two chains that allow the first and second polypeptides to selectively heterodimerize with each other. All amino acid substitution positions exemplified below are numbered according to the EU index as in Kabat.

[0163] In one scenario, the amino acid substitutions in a first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T) or valine (V), resulting in the larger amino acid substitution (protrusion) fitting into the surface of the smaller amino acid substitution (pocket). For example, one polypeptide can incorporate a T366W substitution and the other can incorporate three substitutions including T366S, L368A and Y407V.

[0164] The antibody heavy chain variable domain of the present application can be optionally linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region, including the hinge, CH2 and CH3 domains, with or without the CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region or IgG4 constant region. In one embodiment, an antibody Fc domain or portion thereof sufficient to bind CD16 has the wild type human IgG1 Fc sequence: The constant region comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 118). In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region of another mammal, such as a rabbit, dog, cat, mouse or horse.

[0165] In some embodiments, the antibody constant domain linked to the scFv or Fab fragment is capable of binding to CD 16. In some embodiments, the protein incorporates a portion of an antibody Fc domain (e.g., a sufficient portion of an antibody Fc domain that binds to CD 16), where the antibody Fc domain comprises a hinge and CH2 domain (e.g., the hinge and CH2 domain of a human IgG1 antibody), and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0166] One or more mutations can be incorporated into the constant region relative to the human IgG1 constant region, for example at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, Q347R, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L366K, T366W, T366S, L366K, T366W, T366K ... 68E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T411D, T411E, K439D and K439E.

[0167] In certain embodiments, mutations that can be incorporated into the CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171 and / or V173. In certain embodiments, mutations that can be incorporated into the Cκ of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174 and / or T164.

[0168] Alternatively, the amino acid substitutions can be selected from the set of substitutions shown in Table 4 below. [Table 4]

[0169] Alternatively, the amino acid substitutions can be selected from the set of substitutions shown in Table 5: [Table 5]

[0170] Alternatively, the amino acid substitutions can be selected from the set of substitutions shown in Table 6 below. [Table 6]

[0171] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 7. [Table 7]

[0172] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 8, in which the position(s) shown in the column for the first polypeptide are replaced by any known negatively charged amino acid, and the position(s) shown in the column for the second polypeptide are replaced by any known positively charged amino acid. [Table 8]

[0173] Alternatively, at least one amino acid substitution can be selected from the following set in Table 9, where the position(s) shown in the example first polypeptide are replaced by any known positively charged amino acid, and the position(s) shown in the example second polypeptide row are replaced by any known negatively charged amino acid. [Table 9]

[0174] Alternatively, the amino acid substitutions can be selected from the following set in Table 10. [Table 10]

[0175] Alternatively, or in addition, the structural stability of the heteromultimeric protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain to form an artificial disulfide bridge within the interface of the two polypeptides.

[0176] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0177] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368 and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0178] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411.

[0179] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405 and T411.

[0180] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411.

[0181] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407.

[0182] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Q347, Y349, K360 and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405.

[0183] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, K360, Q347 and K409.

[0184] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409 and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of D356, E357 and D399.

[0185] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0186] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409.

[0187] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399.

[0188] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution.

[0189] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a S354C substitution.

[0190] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by Q347R, D399V and F405T substitutions.

[0191] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by Q347R, D399V and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions.

[0192] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A and Y407V substitutions.

[0193] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366S, T368A and Y407V substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution.

[0194] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L and T394W substitutions.

[0195] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A and Y407V substitutions. Exemplary Multispecific Binding Proteins

[0196] Listed below are examples of TriNKETs that contain an antigen-binding site that binds EGFR and an antigen-binding site that binds NKG2D, each linked to an antibody constant region, where the antibody constant region contains a mutation that allows heterodimerization of the two Fc chains.

[0197] TriNKETs are contemplated in F3 format, i.e., the antigen binding site that binds EGFR is a Fab and the antigen binding site that binds NKG2D is a scFv. All TriNKETs shown below are in F3' format, i.e., the antigen binding site that binds EGFR is a scFv and the antigen binding site that binds NKG2D is a Fab. In each TriNKET, the scFv may contain Cys substitutions in the VH and VL regions to promote the formation of disulfide bridges between the VH and VL of the scFv.

[0198] The VH and VL of scFv can be connected via a linker, such as a peptide linker. In certain embodiments, the peptide linker is a flexible linker. With regard to the amino acid composition of the linker, the peptide is selected for its properties of providing flexibility, not interfering with the structure and function of other domains of the protein of the present application, and being resistant to cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, the VL is linked to the VH at the N-terminus or C-terminus via a (GlyGlyGlyGlySer)4 ((G4S)4) linker (SEQ ID NO: 119).

[0199] The length of a linker (e.g., a flexible linker) can be "short," e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, or "long," e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues long.

[0200] In certain embodiments, the linker is (GS) n (SEQ ID NO: 120), (GGS) n (SEQ ID NO: 121), (GGGS) n (SEQ ID NO: 122), (GGSG) n (SEQ ID NO: 123), (GGSGG)n (SEQ ID NO: 124) and (GGGGS) n (SEQ ID NO: 125), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker comprises or consists of an amino acid sequence selected from SEQ ID NOs: 119 and 126-134 listed in Table 11. [Table 11]

[0201] In F3'-TriNKET, the EGFR-binding scFv is linked to the N-terminus of Fc via a Gly-Ser linker. The Ala-Ser or Gly-Ser linker is included in the elbow hinge region sequence to balance flexibility and optimal geometry. In certain embodiments, the additional sequence Thr-Lys-Gly can be added to the Ala-Ser or Gly-Ser of the hinge at the N-terminus or C-terminus.

[0202] As used herein to describe these exemplary TriNKETs, Fc includes the hinge, CH2 and CH3 of an antibody. In each exemplary TriNKET, the Fc domain polypeptide linked to the scFv includes the mutations Q347R, D399V and F405T, and the Fc domain polypeptide linked to the Fab includes the matching mutations K360E and K409W to form a heterodimer. The Fc domain polypeptide linked to the scFv further includes an S354C substitution in the CH3 domain, which forms a disulfide bond with the Y349C substitution on the Fc linked to the Fab. These substitutions are in bold in the sequences described in this subsection.

[0203] For example, the TriNKET described in this disclosure is EGFR-TriNKET-2.EGFR-TriNKET-2 comprises (a) the EGFR-scFv-2 (VL-VH) sequence provided in Table 2 in the orientation of VH located C-terminal to VL, linked to an Fc domain polypeptide, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, where the CH1 domain is connected to an Fc domain polypeptide.EGFR-TriNKET-2 comprises three polypeptides:EGFR-scFv-2 (VL-VH)-Fc (SEQ ID NO: 166), A49MI-VH-CH1-Fc (SEQ ID NO: 164), and A49MI-VL-CL (SEQ ID NO: 165). EGFR-scFv-2(VL-VH)-Fc (SEQ ID NO: 166). Bold residues indicate mutated residues in the Fc domain. [ka] A49MI-VH-CH1-Fc (SEQ ID NO: 164). Bold residues indicate mutated residues in the Fc domain, and underlined sequences indicate CDR sequences. [ka] A49MI-VL-CL (SEQ ID NO: 165). The underlined sequences indicate the CDR sequences. [ka]

[0204] EGFR-scFv-2(VL-VH)-Fc (SEQ ID NO: 166) represents the complete sequence of an EGFR-binding scFv linked to an Fc domain polypeptide via a hinge comprising Gly-Ser. The Fc domain polypeptide linked to the scFv comprises Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH-CH1-Fc, as described below. The scFv comprises the heavy chain variable domain of EGFR-binder 2 of Table 2 connected to the C-terminus of the light chain variable domain of EGFR-binder 2 of Table 2 via a (G4S)4 linker (SEQ ID NO: 119), where the scFv further comprises substitutions of Cys in the VH and VL regions at G44 and S100, respectively, thereby facilitating the formation of disulfide bridges between the VH and VL of the scFv.

[0205] A49MI-VH-CH1-Fc (SEQ ID NO: 164) represents the heavy chain portion of the Fab fragment, including the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of NKG2D-binding A49MI, connected to the Fc domain. The Fc domain polypeptide in A49MI-VH-CH1-Fc includes a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution in the Fc polypeptide in EGFR-scFv-2(VL-VH)-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in EGFR-scFv-2(VL-VH)-Fc.

[0206] A49MI-VL-CL (SEQ ID NO: 165) represents the light chain portion of a Fab fragment, containing the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of the NKG2D-binding A49MI.

[0207] In another example, the TriNKET described in this disclosure is EGFR-TriNKET-3. EGFR-TriNKET-3 comprises (a) the EGFR-scFv-3 (VL-VH) sequence provided in Table 2 in the orientation of VH located C-terminal to VL, linked to an Fc domain polypeptide, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, where the CH1 domain is connected to an Fc domain polypeptide. EGFR-TriNKET-3 comprises three polypeptides: EGFR-scFv-3 (VL-VH)-Fc (SEQ ID NO: 167), A49MI-VH-CH1-Fc (SEQ ID NO: 164), and A49MI-VL-CL (SEQ ID NO: 165). EGFR-scFv-3(VL-VH)-Fc (SEQ ID NO: 167). Bold residues indicate mutated residues in the Fc domain. [ka]

[0208] EGFR-scFv-3(VL-VH)-Fc (SEQ ID NO: 167) represents the complete sequence of an EGFR-binding scFv linked to an Fc domain via a hinge comprising Gly-Ser. The Fc domain polypeptide linked to the scFv comprises Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution to form a disulfide bond with a Y349C substitution in A49MI-VH-CH1-Fc, as described below. The scFv comprises the heavy chain variable domain of EGFR-binder 3 of Table 2 connected to the C-terminus of the light chain variable domain of the EGFR-binder 3 clone of Table 2 via a (G4S)4 linker (SEQ ID NO: 119), where the scFv further comprises substitutions of Cys in the VH and VL regions at G44 and S100, respectively, thereby facilitating the formation of disulfide bridges between the VH and VL of the scFv.

[0209] In another example, the TriNKET described in this disclosure is EGFR-TriNKET-4. EGFR-TriNKET-4 comprises (a) the EGFR-scFv-4 (VL-VH) sequence provided in Table 2 in the orientation of VH located C-terminal to VL, linked to an Fc domain polypeptide, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, where the CH1 domain is connected to an Fc domain polypeptide. EGFR-TriNKET-4 comprises three polypeptides: EGFR-scFv-4 (VL-VH)-Fc (SEQ ID NO: 168), A49MI-VH-CH1-Fc (SEQ ID NO: 164), and A49MI-VL-CL (SEQ ID NO: 165). EGFR-scFv-4(VL-VH)-Fc (SEQ ID NO: 168). Bold residues indicate mutated residues in the Fc domain. [ka]

[0210] EGFR-scFv-4(VL-VH)-Fc (SEQ ID NO: 168) represents the complete sequence of an EGFR-binding scFv linked via a Gly-Ser-containing hinge to an Fc domain polypeptide. The Fc domain polypeptide linked to the scFv contains Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, as described below. The scFv comprises the heavy chain variable domain of an EGFR-binder 4 clone of Table 2 connected to the C-terminus of the light chain variable domain of an EGFR-binder 4 clone of Table 2 via a (G4S)4 linker (SEQ ID NO: 119), where the scFv further comprises substitutions of Cys in the VH and VL regions at G44 and S100, respectively, thereby facilitating the formation of disulfide bridges between the VH and VL of the scFv.

[0211] In certain embodiments, the TriNKET described in this disclosure is identical to one of the exemplary TriNKETs described above that contains an EW-RVT Fc mutation, except that the Fc domain polypeptide linked to the NKG2D-binding Fab fragment contains substitutions of Q347R, D399V, and F405T, and the Fc domain polypeptide linked to the EGFR-binding scFv contains matching substitutions K360E and K409W to form heterodimers. In certain embodiments, the TriNKET described in this disclosure is identical to one of the exemplary TriNKETs described above that contains an EW-RVT Fc mutation, except that the Fc domain polypeptide linked to the NKG2D-binding Fab fragment contains "hole" substitutions of T366S, L368A, and Y407V, and the Fc domain polypeptide linked to the EGFR-binding scFv contains a "knob" substitution of T366W to form heterodimers. It is identical to one of the exemplary TriNKETs described above that includes an Fc mutation.

[0212] One of skill in the art will recognize that during production and / or storage of a protein, an N-terminal glutamic acid (E) or glutamine (Q) may cyclize to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Thus, in some embodiments in which the N-terminal residue of a polypeptide's amino acid sequence is E or Q, the corresponding amino acid sequence in which E or Q is replaced with pyroglutamic acid is also contemplated herein.

[0213] Those skilled in the art will also recognize that during production and / or storage of a protein, the C-terminal lysine (K) of the protein may be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed in proteins that contain an Fc domain at their C-terminus. Thus, in some embodiments in which the C-terminal residue of a polypeptide's amino acid sequence (e.g., an Fc domain sequence) is K, the corresponding amino acid sequence with the K removed is also contemplated herein.

[0214] The above multispecific binding protein can be produced by using recombinant DNA technology well known to those skilled in the art.For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector; the first, second and third expression vectors can be stably transfected together into host cells to produce a multimeric protein.

[0215] To achieve the highest yield of multispecific binding protein, different ratios of the first, second and third expression vectors can be investigated to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy or Clonepix.

[0216] Clones can be cultured under conditions suitable for bioreactor scale-up and sustained expression of the multispecific binding proteins. Multispecific binding proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. II. Characterization of Multispecific Binding Proteins

[0217] The multispecific binding proteins described herein comprise an NKG2D binding site, a tumor-associated antigen binding site that binds EGFR, and an antibody Fc domain or portion thereof sufficient to bind CD 16, or an antigen binding site that binds CD 16. In some embodiments, the multispecific binding protein contains an additional antigen binding site that binds the same tumor-associated antigen (EGFR), as exemplified by the F4-TriNKET format (e.g., Figures 2C and 2D).

[0218] In some embodiments, the multispecific binding proteins exhibit similar thermal stability to the corresponding monoclonal antibody, i.e., the monoclonal antibody that contains the same EGFR antigen binding site as that incorporated into the multispecific binding protein.

[0219] In some embodiments, the multispecific binding protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and cells expressing EGFR, such as certain tumor cells. Binding of the multispecific binding protein to NK cells can enhance the activity of NK cells toward the destruction of tumor cells expressing EGFR. It has been reported that NK cells exhibit stronger cytotoxicity against stressed target cells (see Chan et al., (2014) Cell Death Differ. 21(1):5-14). Without wishing to be bound by theory, it is emphasized that when NK cells bind to a population of cells by TriNKET, the NK cells can selectively kill stressed target cells (e.g., malignant cells and cells in the tumor microenvironment). This mechanism may contribute to the increased specificity and reduced toxicity of TriNKET, allowing for the selective elimination of stressed cells even when EGFR expression is not limited to the desired target cells.

[0220] In some embodiments, the multispecific binding protein binds to EGFR with similar affinity as the corresponding anti-EGFR monoclonal antibody (i.e., a monoclonal antibody that contains the same EGFR binding site as that incorporated into the multispecific binding protein). In some embodiments, the multispecific binding protein binds to EGFR with similar affinity as the corresponding monoclonal antibody (i.e., a monoclonal antibody that contains the same EGFR binding site as that incorporated into the multispecific binding protein). In some embodiments, the multispecific binding protein binds to EGFR with similar affinity to the corresponding anti-EGFR monoclonal antibody (i.e., a monoclonal antibody that contains the same EGFR binding site as that incorporated into the multispecific binding protein). + T cells) are more effective at mediating the killing of EGFR-expressing tumor cells.

[0221] In certain embodiments, the multispecific binding proteins described herein that contain a binding site for EGFR activate primary human NK cells when co-cultured with cells expressing EGFR. NK cell activation is indicated by increased CD107a degranulation and IFN-γ cytokine production. Moreover, compared to the corresponding anti-EGFR monoclonal antibodies, the multispecific binding proteins show superior activation of human NK cells in the presence of cells expressing EGFR.

[0222] In some embodiments, a multispecific binding protein described herein that contains a binding site for EGFR enhances the activity of resting and / or IL-2-activated human NK cells when co-cultured with cells expressing EGFR.

[0223] In some embodiments, the multispecific binding proteins provide advantages in targeting tumor cells that express intermediate and low levels of EGFR compared to corresponding monoclonal antibodies that bind EGFR.

[0224] In some embodiments, the multispecific binding proteins disclosed herein that are monovalent to EGFR, e.g., in the F3 or F3' format, reduce or delay skin-related toxicity compared to the corresponding anti-EGFR mAb that is bivalent.

[0225] In some embodiments, the bivalent F4 format of TriNKET (i.e., containing an additional antigen binding site where TriNKET binds to EGFR) improves the activity of TriNKET binding to EGFR, in effect stabilizing the expression and maintenance of high levels of TriNKET on the surface of tumor cells. In some embodiments, F4-TriNKET mediates more potent tumor cell killing than the corresponding F3-TriNKET or F3'-TriNKET. III. Therapeutic Uses

[0226] The present application provides methods for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat a variety of cancers that express EGFR.

[0227] Therapeutic methods can be characterized according to the cancer being treated. The cancer being treated can be characterized according to the presence of specific antigens expressed on the surface of the cancer cells.

[0228] Cancer characterized by the expression of EGFR includes, but is not limited to, solid tumor cancer.For example, in certain embodiments, cancer is head and neck cancer, colorectal cancer, non-small cell lung cancer, glioma, renal cell carcinoma, bladder cancer, cervical cancer, ovarian cancer, pancreatic cancer or liver cancer.In certain embodiments, cancer is lung cancer (including but not limited to small cell lung cancer or lung adenocarcinoma), breast cancer, invasive ductal carcinoma of the breast, kidney cancer, conventional glioblastoma multiforme, colon cancer, colon adenocarcinoma, gastric cancer, brain cancer, glioblastoma, bladder, head and neck cancer, ovarian cancer or prostate cancer.

[0229] It is contemplated that the proteins, conjugates, cells and / or pharmaceutical compositions described herein can be used to treat a variety of cancers, including but not limited to cancers in which the cancer cells or cells in the cancer microenvironment express EGFR.

[0230] In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is breast cancer, ovarian cancer, esophageal cancer, bladder cancer, or gastric cancer, salivary gland duct cancer, lung adenocarcinoma, or aggressive forms of uterine cancer, such as uterine serous endometrial cancer. In some other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, gastric cancer, testicular cancer, or uterine cancer. In still other embodiments, the cancer is angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary tract cancer, or uterine cancer. tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract cancer cancer), bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, yolk sac tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, eye and orbit cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric pyloric cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic Adenomatous polyposis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell tumor, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer), oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma or Wilms' tumor.

[0231] In certain other embodiments, the cancer is a non-Hodgkin's lymphoma, such as B-cell lymphoma or T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0232] The cancer to be treated can be characterized according to the presence of specific antigens expressed on the surface of cancer cells.In certain embodiments, in addition to EGFR, cancer cells can express one or more of the following: CD2, CD19, CD38, CD40, CD52, CD30, CD70, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, TROP2, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1. IV. Combination Therapy

[0233] Another aspect of the present application provides combination therapy. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0234] Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drofluridone, tetracycline ... These include genil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma (IFN-γ), colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variations of the above agents that may exhibit differential binding to their cognate receptors or increased or decreased serum half-lives.

[0235] Another class of drugs that can be used as part of combination therapy in the treatment of cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4 and (vii) TIM3.Ipilimumab, a CTLA4 inhibitor, has been approved by the US Food and Drug Administration for the treatment of melanoma.

[0236] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0237] Further categories of anti-cancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors + 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, (ii) an inhibitor selected from a cytotoxic agent, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0238] The proteins of the present application may also be used as an adjunct to surgical removal of the primary tumor.

[0239] The amounts and relative timing of administration of the multispecific binding protein and additional therapeutic agent can be selected to achieve a desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need of such administration, the combined therapeutic agent or one or more pharmaceutical compositions containing the therapeutic agents can be administered in any order, such as sequentially, jointly, together, simultaneously, etc. Furthermore, for example, the multispecific binding protein can be administered during the time that the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa. V. Pharmaceutical Compositions

[0240] The present disclosure also features a pharmaceutical composition that contains a therapeutically effective amount of the protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for suitable formulation. Suitable formulations for use in the compositions disclosed herein can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief overview of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0241] In certain embodiments, the composition may be a drug delivery formulation. The intravenous drug delivery formulation described herein may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing a tube and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and may be contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, 12, 27, or 45 vials of the freeze-dried formulation are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored as about 250 mg / vial.

[0242] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be between 3 and 11, e.g., between 5 and 9 or between 6 and 8, and in certain embodiments, between 7 and 8, e.g., between 7 and 7.5. The resulting composition in solid form may be packaged into a number of single-dose units, each containing a fixed amount of one or more of the above-mentioned agents. The composition in solid form may also be packaged in a container in flexible amounts.

[0243] In certain embodiments, the present disclosure provides an extended shelf life formulation comprising a protein described in this disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0244] In certain embodiments, an aqueous formulation is prepared comprising the protein described in the present disclosure in a pH buffer solution. The buffer of the present application may have a pH ranging from about 4 to about 8, for example, from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Intermediate ranges up to the pH recited above are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination of the values ​​recited above as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate buffers (e.g., sodium acetate), succinate buffers (such as sodium succinate), gluconate buffers, histidine buffers, citrate buffers, and other organic acid buffers.

[0245] In certain embodiments, the formulation comprises a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range can be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or from about pH 5.0 to about 5.2. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / mL citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / mL), and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system comprises about 1 to about 1.5 mg / mL citric acid, about 0.25 to about 0.5 mg / mL sodium citrate, about 1.25 to about 1.75 mg / mL disodium phosphate dihydrate, about 0.7 to about 1.1 mg / mL sodium dihydrogen phosphate dihydrate, and about 6.0 to about 6.4 mg / mL sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0246] A polyol may be included in the formulation, which may act as a tonicifier and stabilize the antibody. The polyol is added to the formulation in an amount that may vary with respect to the desired tonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may vary with respect to the molecular weight of the polyol. For example, a small amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain embodiments, the polyol that may be used in the formulation as a tonicifier is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the mannitol concentration may be about 7.5 to about 15 mg / mL. In certain embodiments, the mannitol concentration may be about 10 to about 14 mg / mL. In certain embodiments, the mannitol concentration may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0247] A detergent or surfactant may be added to the formulation. Exemplary surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the surfactant polysorbate 80 or Tween® 80. Tween® 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0248] In embodiments, the protein products described herein are formulated as liquid formulations. The liquid formulations may be provided at 10 mg / mL concentration in USP / Ph Eur Type I 50R vials closed with rubber stoppers and sealed with aluminum crimp seal closures. The stoppers may be made of elastomers in compliance with USP and Ph Eur. In certain embodiments, the vials may be filled with 61.2 mL of protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulations may be diluted with 0.9% saline solution.

[0249] In certain embodiments, the liquid formulations described herein can be prepared as a 10mg / mL concentration solution combined with a stabilizing level of sugar.In certain embodiments, the liquid formulations can be prepared in an aqueous carrier.In certain embodiments, the stabilizer can be added in an amount that is not more than the amount that can cause undesirable or inappropriate viscosity for intravenous administration.In certain embodiments, the sugar can be a disaccharide, such as sucrose.In certain embodiments, the liquid formulation can also include one or more of a buffer, a surfactant, and a preservative.

[0250] In certain embodiments, the pH of the liquid formulation can be adjusted by adding a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0251] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvest / cell clarification, purification, bulk / drug storage, and sample analysis. Deamidation is the loss of NH3 from a protein to form a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a mass loss of 17 Daltons from the parent peptide. Subsequent hydrolysis results in a mass gain of 18 Daltons. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is typically detectable as a mass gain of 1 Dalton. Deamidation of asparagine results in aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain affects the rate of deamidation. Gly and Ser following Asn in the protein sequence result in a higher susceptibility to deamidation.

[0252] In certain embodiments, the liquid formulations described herein may be stored under conditions of pH and humidity to prevent deamidation of the protein product.

[0253] Aqueous carriers for the purposes of this specification are those that are pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0254] Preservatives may be added to the formulations herein, if desired, to reduce bacterial action. The addition of a preservative may, for example, facilitate the manufacture of a multi-use (multiple dose) formulation.

[0255] Intravenous (IV) delivery may be the route of administration in certain instances, for example, when a patient is hospitalized after transplantation receiving all medications via the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the injectable diluted pharmaceutical product is isotonic and suitable for administration by intravenous infusion.

[0256] In certain embodiments, salts or buffer components may be added in amounts between 10 mM and 200 mM. The salts and / or buffers are pharma- ceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate buffer, a carbonate buffer, a citrate buffer, in which case sodium, potassium or ammonium ions may serve as counterions.

[0257] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the manufacture of a multi-use (multiple dose) formulation.

[0258] Aqueous carriers for the purposes of this specification are those that are pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0259] The protein described herein may be in a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also comprise one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0260] The amount of sucrose or maltose useful for stabilizing a lyophilized pharmaceutical product can be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be between 1:2 and 1:5.

[0261] In certain embodiments, the pH of the formulation may be adjusted by adding a pharma- ceutically acceptable acid and / or base before lyophilization. In certain embodiments, the pharma-ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharma-ceutically acceptable base may be sodium hydroxide.

[0262] Prior to lyophilization, the pH of the solution containing the protein described herein may be adjusted to between 6 and 8. In certain embodiments, the pH range for the lyophilized pharmaceutical product may be between 7 and 8.

[0263] In certain embodiments, salts or buffer components may be added in amounts between 10 mM and 200 mM. The salts and / or buffers are pharma- ceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate buffer, a carbonate buffer, a citrate buffer, in which case sodium, potassium or ammonium ions may serve as counterions.

[0264] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present application may contain such bulking agents.

[0265] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple dose) formulation.

[0266] In certain embodiments, lyophilized pharmaceuticals can be reconstituted with aqueous carriers.Aqueous carriers for the purposes of this specification are those that are pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing liquid formulations after lyophilization.Exemplary aqueous carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0267] In certain embodiments, the lyophilized pharmaceutical products disclosed herein are reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

[0268] In certain embodiments, the lyophilized protein product disclosed herein is reconstituted in about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0269] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient.

[0270] The specific dose may be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, the patient's dose may be adapted to the approximate weight or surface area of ​​the patient. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and health of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those skilled in the art, especially in light of the dosage information and the assays disclosed herein. Dosages can also be determined through the use of known assays to determine dosages to be used in conjunction with appropriate dose-response data. Dosages for individual patients can be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex in the patient can be measured to ascertain whether dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, and their dosages, are likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., J. Med. 2003; 2006; 2009). al., Clinica Chimica Acta 308: 33-41, 2001).

[0271] In general, the dosage based on body weight is about 0.01 μg to about 100 mg per kg of body weight, e.g., about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0. 1 μg / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0 .1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 100 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0272] Doses may be given once or more daily, weekly, monthly or yearly, or even once every 2-20 years. One of skill in the art can easily estimate the repetition rate of administration based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the compositions described herein can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by perfusion through a catheter or by direct intralesional injection. Administration can be once or more daily, once or more weekly, once or more monthly, or once or more yearly.

[0273] The above description describes multiple aspects and embodiments of the present application. The present application specifically contemplates all combinations and permutations of the aspects and embodiments. EXAMPLES

[0274] The following examples are illustrative only and are not intended to limit in any way the scope or content of the multispecific binding proteins described in this application. Example 1 Selection of EGFR binding interfaces using SPR and DCS analysis

[0275] This example was designed to develop an EGFR binding site derived from panitumumab with improved thermal stability over panitumumab. Briefly, 25 constructs were designed, each containing a single point mutation selected based on the crystal structure of panitumumab Fab in complex with the D3 domain of EGFR (PDB ID: 5SX4). Except for the three constructs that did not express well, EGFR binding sites were generated to evaluate the effect of the mutations on binding affinity and thermal stability. Each scFv construct also contained a G44C substitution in the VH and a G100C substitution in the VL. The kinetics and affinity for human and rhesus EGFR were evaluated using surface plasmon resonance (SPR). The results are shown in Table 12. Given that the effect for each mutation was minimal, those that caused a significant reduction in affinity were removed from consideration. The thermal stability of the eight constructs was further evaluated by differential scanning fluorimetry (DSF) using the method described in Example 3. The results are shown in Table 13. [Table 12-1] [Table 12-2] [Table 13]

[0276] Based on these results, the following mutations were determined to most effectively improve / retain affinity and thermostability: Heavy chain: S62R (under the Chothia numbering scheme) Light chain: F87Y, D92R (under the Chothia numbering scheme)

[0277] Structural modeling of these mutations is shown in Figures 18-20. As shown in Figure 18, the S62R mutation in the VH chain of panitumumab was modeled to lead to an additional hydrogen bond with D1 of VL, contributing to the stability of the VH / VL interface. As shown in Figure 19, the F87Y mutation in the VL chain of panitumumab was modeled to lead to an additional hydrogen bond with Q39 of VH, contributing to the stability of the VH / VL interface. As shown in Figure 20, the D92R mutation in CDR3 of the VL chain of panitumumab, originally designed to improve affinity (for binding to N449 of EGFR), was unexpectedly modeled to form an additional van der Waals contact with Y32 of CDRL1, stabilizing the paratope.

[0278] The combined effects of the mutations on affinity and thermostability were then assessed using SPR and differential scanning calorimetry (DSC), respectively. The results from the SPR analysis are shown in Table 14, and the results from the DSC analysis are shown in Table 15. [Table 14-1] [Table 14-2] [Table 15] *-H represents a mutation in the heavy chain; -L represents a mutation in the light chain; PANI represents the panitumumab framework. **An additional peak was observed between peak 1 and peak 2.

[0279] PANI-H_S62R-L_F87Y-L_D92R (hereinafter "EGFR-scFv-3") and PANI-H_S62R-L_F87Y (hereinafter "EGFR-scFv-2") in Tables 14 and 15 demonstrated the best affinity and thermal stability. The L:G100C and H:G44C mutations promote disulfide bonds to enhance pairing of the VL and VH chains. Example 2 Surface plasmon resonance analysis of TriNKET

[0280] This example was designed to evaluate the binding affinity of a particular panitumumab-derived EGFR binding site to EGFR. Four TriNKETs containing scFvs derived from panitumumab and its variants were constructed, namely EGFR-TriNKET-1, EGFR-TriNKET-2, EGFR-TriNKET-3 and EGFR-TriNKET-4 (see the "Exemplary Multispecific Binding Proteins" subsection above). The kinetics and affinity of the EGFR-binding constructs for recombinant human and rhesus EGFR were measured by SPR using a Biacore 8K instrument. Samples were bound to anti-hFc IgG was captured on a chip, and a range of concentrations of human or rhesus recombinant EGFR-His was injected over the captured test article. Experiments were performed at physiological temperature of 37° C. Data was analyzed using Biacore 8K Insight evaluation software (GE Healthcare).

[0281] Binding sensorgrams from the SPR analysis are shown in Figures 21-24. Figure 21 shows the SPR analysis for titration of EGFR-TriNKET-1, Figure 22 shows the SPR analysis for titration of EGFR-TriNKET-2, Figure 23 shows the SPR analysis for titration of EGFR-TriNKET-3, and Figure 24 shows the SPR analysis for titration of EGFR-TriNKET-1. Parameters calculated from these analyses are shown in Table 16. The results demonstrated that the mutations of interest had no substantial effect on the affinity of the multispecific binding protein for EGFR. [Table 16] Example 3 Differential scanning calorimetry (DSC) analysis of TriNKET

[0282] This example was designed to assess the thermal stability of TriNKET constructs using DSC analysis. Briefly, test articles were buffer exchanged into a buffer of choice using Thermo Scientific Zeba Spin Desalting Columns. The eluate was then diluted to 0.5 mg / mL in the same buffer. 325 μL of sample was loaded into a 96-well deep-well plate with the corresponding buffer blank and analyzed using a Microcal PEAQ-DSC instrument (Malvern Panalytical). The temperature was ramped from 20-25 °C to 100 °C at a rate of 60 °C / h. A buffer background was run in triplicate prior to the analytes. The most representative buffer blank was subtracted from each analyte scan prior to analysis. Data was fitted using the DSC analysis software using a non-two-state model to determine the T onset and T m reported s.

[0283] EGFR-TriNKET-1 was used to obtain a baseline melting curve and identify inflection points, specifically the T-onset (the temperature at which the molecule begins to melt) and Tm1, which are related to scFv stability. EGFR-TriNKET-2, EGFR-TriNKET-3 and EGFR-TriNKET-4 were then analyzed using the same method.

[0284] Table 17 and Figures 25A-25D show the DSC analysis results from the test in PBS, pH 7.4 buffer. Figure 25A shows the DSC analysis for EGFR-TriNKET-1 in PBS, pH 7.4 buffer. Figure 25B shows the DSC analysis for EGFR-TriNKET-2 in PBS, pH 7.4 buffer. Figure 25C shows the DSC analysis for EGFR-TriNKET-3 in PBS, pH 7.4 buffer. Figure 25D shows the DSC analysis for EGFR-TriNKET-4 in PBS, pH 7.4 buffer. Table 18 and Figures 26A and 26B show the DSC analysis results from the test in 20 mM histidine, 250 mM trehalose, 0.01% PS80, pH 6.0. Figure 26A shows the DSC analysis for EGFR-TriNKET-3 in 20 mM histidine, 250 mM trehalose, 0.01% PS80, pH 6.0 buffer. Figure 26B shows the DSC analysis for EGFR-TriNKET-3 in 20 mM histidine, 250 mM trehalose, 0.01% PS80, pH 6.0 buffer. DSC analysis revealed that EGFR-TriNKET-3 (Figure 25C) had improved thermal stability compared to EGFR-TriNKET-2 (Figure 25B), which in turn had improved thermal stability compared to EGFR-TriNKET-1 (Figure 25A). The results also showed similar thermal stability of EGFR-TriNKET-3 (Figure 26A) and EGFR-TriNKET-4 (Figure 26B). However, EGFR-TriNKET-4 was more prone to degradation under accelerated thermostability studies (40° C. for 4 weeks, data not shown), highlighting the benefit of the S62R heavy chain mutation on stability. [Table 17] [Table 18] Example 4 Assessment of TriNKET binding to EGFR-positive cells

[0285] This example was designed to evaluate the binding affinity of EGFR-targeted TriNKET to EGFR expressed on the cell surface. The H2172 human cancer cell line derived from non-small cell lung cancer was used. Briefly, H2172 cells were incubated with EGFR-TriNKET-1, EGFR-TriNKET-2, EGFR-TriNKET-3 or panitumumab for 0.5 hours at 4°C. After incubation, the binding affinity of TriNKET and panitumumab to EGFR was evaluated. + Binding patterns to cells were detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and fold MFI relative to secondary only control was reported.

[0286] Figure 27 shows binding to EGFR-positive cells after incubation with EGFR-TriNKET-1, EGFR-TriNKET-2, EGFR-TriNKET-3 or panitumumab. These EGFR-targeted TriNKETs bound to cells at sub-nM concentrations and with similar or higher maximum binding than panitumumab. Example 5 Cytotoxicity assays of primary human NK cells or CD8+ cells

[0287] This example was designed to evaluate the ability of EGFR-targeted TriNKET to mediate immune effector cell cytotoxicity against EGFR-expressing cancer cells. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell or CD8+ cell isolation. NK cells were isolated using a magnetic bead negative selection technique, and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were left overnight in culture medium without supplemented cytokines and used in cytotoxicity assays the following day. CD8+ were isolated using a magnetic bead negative selection technique, and the purity of CD8+ cells was typically >90% CD3+CD8+. Isolated CD8+ T cells were incubated in medium with IL-15 for 6-13 days for expansion.

[0288] For cytotoxicity assays, human cancer cell lines expressing EGFR were harvested from culture, the cells were washed with HBS, and then added to growth medium for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The labeling of target cells was performed according to the manufacturer's instructions. After labeling, cells were washed three times with HBS and resuspended in culture medium at 0.5–1.0 × 10 5 100 μL of BATDA-labeled cells were added to each well of a 96-well plate. Multiple wells were reserved for spontaneous release from target cells and multiple wells were prepared by the addition of 1% Triton-X for maximum lysis of target cells. Monoclonal antibodies (e.g., cetuximab) or TriNKET against EGFR (e.g., EGFR-TriNKET-1, EGFR-TriNKET-3, and EGFR-TriNKET-4) were diluted in culture medium and 50 μL of diluted mAb or TriNKET was added to each well. Resting NK cells were harvested from the culture, the cells were washed, and depending on the desired effector-to-target (E:T) ratio, 10 μL of BATDA-labeled cells were added to each well of a 96-well plate. Multiple wells were reserved for spontaneous release from target cells and multiple wells were prepared by the addition of 1% Triton-X for maximum lysis of target cells. Monoclonal antibodies (e.g., cetuximab) or TriNKET against EGFR (e.g., EGFR-TriNKET-1, EGFR-TriNKET-3, and EGFR-TriNKET-4) were diluted in culture medium and 50 μL of diluted mAb or TriNKET was added to each well. Resting NK cells were harvested from the culture, the cells were washed, and 10 μL of BATDA-labeled cells were added to culture medium depending on the desired effector-to-target (E:T) ratio. 5 ~2.0×10 6 The NK cells were resuspended at 100 cells / mL. 50 μl of the NK cell suspension was added to each well of the plate for a total culture volume of 200 μl. The plates were incubated at 37°C with 5% CO2 for 2-3 hours before the assay.

[0289] After 2-3 hours of incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 200g for 5 minutes. 20 μL of culture supernatant was transferred to a new microplate provided by the manufacturer and 200 μL of room temperature europium solution was added to each well. Plates were protected from light and incubated on a plate shaker at 250 rpm for 15 minutes and then read using either a Victor 3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis=((experimental release−spontaneous release) / (maximum release−spontaneous release))×100%.

[0290] As shown in Figures 28 and 29, lysis of human NK and CD8+ T cells from H2172 cells (Figure 28) and 786-0 cells (Figure 29), respectively, in the presence of EGFR TriNKET or the anti-EGFR mAb cetuximab was observed within 2 hours of incubation. In both systems, across multiple donors (data not shown), EGFR-TriNKET had sub-nM EC50 values. Compared to cetuximab, which had low to non-existent activity, TriNKET provided greater maximum specific lysis and potency. Example 6 Assessment of NK IFN-gamma production mediated by TriNKET

[0291] This example was designed to evaluate the ability of EGFR-targeted TriNKET to activate NK cells in the presence of EGFR-expressing cancer cells. Briefly, BT-474 EGFR-expressing cancer cells served as target cells. Isolated NK cells (derived from multiple human donors) were rested overnight either in culture medium without supplemented cytokines or in culture medium with recombinant human IL-2. Resting NK cells were incubated with target cells at an effector cell (NK cell) to target cell ratio (E:T ratio) of 2:1 for 24 hours, while IL-2-activated NK cells were incubated with target cells at an E:T ratio of 1:1 for 24 hours. After incubation, IFN-γ secretion from NK cells was analyzed using an IFN-γ ELISA kit according to the manufacturer's instructions, Mesoscale. Plates were read using either a Victor 3 or SpectraMax i3X instrument.

[0292] Figures 30 and 31 show the resulting IFN-gamma production from resting NK cells (Figure 30) and IL-2-activated NK cells (Figure 31), respectively, after incubation with BT-474 cells in the presence of EGFR-TriNKET or anti-EGFR mAb cetuximab. In both cases, EGFR-TriNKET mediated higher production of IFN-gamma by NK cells compared to cells treated with cetuximab. Example 7 EGFR cell proliferation assay

[0293] This example was designed to evaluate the effect of EGFR TriNKET on the proliferation of EGFR-expressing human cancer cell line H292 in the absence of effector cells. Briefly, EGFR-TriNKET and anti-EGFR-mAbs cetuximab and panitumumab were diluted and incubated with H292 cells for 72 hours. After incubation, cell proliferation was measured using Cell-titer Glo according to the manufacturer's instructions.

[0294] Figure 32 shows H292 cell proliferation in the presence of EGFR-TriNKET or anti-EGFR mAb. Cells treated with anti-EGFR mAb showed lower proliferation than those treated with EGFR-TriNKET. This was due to the difference in valency between TriNKET and mAb - TriNKET is monovalent, while mAb is bivalent - and demonstrated the benefit of using lower valency TriNKET, given the association of EGFR targeting with skin-related toxicity observed in mAb. Example 8 ADCP activity assay

[0295] This example was designed to evaluate the antibody-dependent cell phagocytosis (ADCP) activity of macrophages when incubated with EGFR-expressing target cells in the presence of EGFR-TriNKET. Briefly, PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for monocyte isolation. Monocyte cells were isolated using a negative selection technique with magnetic beads, and the purity of isolated monocyte cells was typically >90% CD14. Monocytes were differentiated into macrophages by incubation in GM-CSF and IL-4 for 6 days.

[0296] Macrophages were harvested, labeled with CellTrace Violet, and incubated with CFSE-labeled target cells (8:1) and molecules for 2 hours. Cells were stained and prepared for FACS analysis. Phagocytosis was assessed as follows: % phagocytosis = % target+ and effector+ of parent gate "target+" It was calculated as follows.

[0297] As shown in Figure 33, macrophages incubated with EGFR-expressing H292 cells in the presence of EGFR-TriNKET-1, EGFR-TriNKET-3 and EGFR-TriNKET-4 exhibited higher ADCP activity than those incubated in the presence of the anti-EGFR mAb cetuximab. The results also showed that the ADCP activity of EGFR-TriNKET-1 was lost when the ability of TriNKET to bind to CD16 was abrogated by the L234A, L235A and P329G (LALAPG) mutations in the Fc domain. Example 9 Antitumor efficacy of EGFR-TriNKET in vivo

[0298] This example was designed to test whether EGFR-TriNKET induces antitumor function in vivo. Briefly, nude mice were treated with 4×10 6 NCI-H292 tumor cells were injected subcutaneously. The tumor size averaged approximately 90 mm 3 At volume (day 7 post-inoculation), mice were randomized into different treatment groups: Mice were treated intraperitoneally (IP) twice weekly with hIgG1 isotype, EGFR-TriNKET or cetuximab.

[0299] EGFR-TriNKET significantly reduced NCI-H292 tumor growth in mice administered 300 μg / dose or 100 μg / dose compared to isotype-treated animals (p<0.01; Figures 34A-C). Figure 34A shows the average tumor volume of individual mice treated with 300 μg, 100 μg, 30 μg of EGFR-TriNKET, or hIgG1 isotype control. Figure 34B shows the tumor volume of individual mice treated with 300 μg of EGFR-TriNKET, or hIgG1 isotype control. Figure 34C shows the tumor volume of individual mice treated with 100 μg of EGFR-TriNKET, or hIgG1 isotype control. Figure 34D shows the tumor volume of individual mice treated with 30 μg of EGFR-TriNKET, or hIgG1 isotype control. As shown in Figures 34A, 34B, and 34C, treatment of mice with 300 μg and 100 μg of EGFR-TriNKET significantly reduced tumor volume compared to the hIgG1 isotype control. As shown in Figure 34D, treatment of mice with 30 μg / dose of EGFR-TriNKET significantly delayed tumor progression compared to the hIgG1-treated control group.

[0300] In addition to EGFR-TriNKET dose titration in vivo, the antitumor efficacy mediated by EGFR-TriNKET was compared to cetuximab at an equimolar dose of 100 μg cetuximab. As shown in Figure 35A, treatment of individual mice with EGFR-TriNKET was as potent as cetuximab in inducing antitumor responses in vivo.

[0301] Notably, the EGFR-TriNKET and cetuximab regimen appeared to be well tolerated by NCI-H292 tumor-bearing mice, with no clinical observations or effects on body weight (Figure 35B). Incorporation by Reference

[0302] Unless otherwise stated, the entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated herein by reference for all purposes. Equivalent

[0303] The present application may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the present application described herein. The scope of the present application is accordingly indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

[Claim 1] The invention described in this specification.

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