Compositions and methods for the treatment and diagnosis of cancers related to surface K-RAS

Abstract

This disclosure describes compositions and methods for treating and diagnosing cancers that express K-Ras on the outer surface of cancer cells, as well as novel antibody-drug conjugates for use in such methods.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority rights to U.S. Provisional Application No. 63 / 533,024, filed on 16 August 2023, and U.S. Provisional Application No. 63 / 613,718, filed on 21 December 2023.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML copy created on 14 August 2024 is named "93700-411218-SL.xml" and has a size of 291,716 bytes.

[0003] The present invention relates to compositions for use in methods for treating and diagnosing cancers associated with the expression of surface K-Ras antigens, including mutant surface K-Ras antigens. The present invention also relates to antibodies, bispecific antibodies, immune cells having chimeric antigen receptors, and antibody-drug conjugates used to treat and diagnosing cancers associated with the expression of surface K-Ras antigens, including mutant surface K-Ras antigens. [Background technology]

[0004] Despite considerable experimental and clinical efforts to target Kirsten rat sarcoma virus oncogene homologs (K-Ras or KRAS) using mutation-specific covalent small molecule inhibitors (Punekar et al., Nat. Rev. Clin. Oncol. 2022 Oct;19(10):637-655), experimental data describing protein-based technologies targeting this oncogenic pathway are limited. This is primarily due to the difficulty in delivering protein-based therapeutics across the cell membrane into cells where mutant K-Ras is thought to activate downstream signaling pathways in an unregulated manner (PMID:21924373). Because it has been accepted as the standard theory that K-Ras localizes and functions only in intracellular regions, such as either the inner lobe of the cell membrane or within intracellular organelles (see, for example, Hancock, Nat Rev Mol Cell Biol. 2003 May;4(5):373-84.doi:10.1038 / nrm1105.PMID:12728271), therapeutic and diagnostic approaches based on the extracellular surface presentation of mutant K-Ras proteins, such as antibody-drug conjugates (ADCs) and chimeric antigen receptor T cells (CAR-T cells), have not been developed in cancers associated with K-Ras expression. [Overview of the project]

[0005] This specification discloses therapeutic and diagnostic approaches based on the extracellular surface presentation of a surface K-Ras antigen, including a mutant surface K-Ras antigen, and compositions for using the same. For example, this specification discloses a composition comprising a conjugate-therapeutic complex, the conjugate being linked to a therapeutic agent, wherein the conjugate specifically binds to a surface K-Ras antigen expressed on the extracellular surface of cancer cells, and neither the composition nor the conjugate-therapeutic complex contains any intracellular delivery compounds. In one embodiment, the conjugate is an antibody or antibody fragment. In one embodiment, the conjugate is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12D mutation described in SEQ ID NO: 295. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the binder is an antibody fragment.

[0006] In one embodiment, the binder is a peptide or protein. In one embodiment, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen containing a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen containing a G12D mutation described in SEQ ID NO: 295. In one embodiment, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen containing a mutation at residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen containing a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen containing a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the binder is an antibody described in Table 1 or Table 2, or an antibody selected by the method described in Example 4 or Example 5.

[0007] In one embodiment, the therapeutic agent is selected from the group consisting of cytotoxic agents, cell proliferation inhibitors, toxins, or radionuclides. In another embodiment, the therapeutic agent is selected from the group consisting of DNA damaging agents (alkylating agents), antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, and microtubule disruptors. In another embodiment, the therapeutic agent is selected from the group consisting of calicheamicin, saporin, mytansinoids, auristatin, ridamycin, methotrexate, vinblastine, vincristine, pyrrolobenzodiazepines and other benzodiazepine derivatives, duocalmycin, tubulicin, α-amanitin or bougainin protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxin, carboplatin, spliceostatin C, docetaxel, tylanstatin A, or any combination thereof.

[0008] In one embodiment, the binder is linked to the therapeutic agent by a linker selected from the group consisting of maleimidocaproyl linker, peptide-based linker (including, but not limited to, valine-citrulline linker), β-glucuronide linker, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, disulfide linker, or acid-sensitive linker. In one embodiment, the therapeutic agent is a radionuclide. In one embodiment, the radionuclide is a β-particle emitting radionuclide or an α-particle emitting radionuclide. In one embodiment, the radionuclide is an α-particle emitting radionuclide selected from the group consisting of astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227. In one embodiment, the radionuclide is a β-particle emitting radionuclide. In one embodiment, the β-particle emitting radionuclide is selected from the group consisting of iodine-131, rhenium-186, yttrium-90, samarium-153, and lutetium-177.

[0009] This specification also discloses compositions for use in treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or in killing cancer cells, wherein the subject has not been administered an intracellular delivery compound, or the composition is not intended for administration to the subject in combination with an intracellular delivery compound, or is not formulated for administration to the subject in combination with an intracellular delivery compound, and the cancer cells express a surface K-Ras antigen on the outer surface of the cancer cells. In one embodiment, the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. In one embodiment, the composition is formulated for administration by intravenous or subcutaneous injection.

[0010] This specification also discloses a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering a therapeutically effective amount of any of the compositions described herein to the subject, wherein the subject is not administered an intracellularly delivered compound in conjunction with the administration of the composition, and the cancer cells express a surface K-Ras antigen on the outer surface of the cancer cells. In one embodiment, the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. In one embodiment, the administration step is carried out by intravenous or subcutaneous injection.

[0011] This specification also discloses a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells. In one embodiment, the means is an antibody. In one embodiment, the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen comprises a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In one embodiment, the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12D mutation described in SEQ ID NO: 295. In one embodiment, the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is an antibody fragment.

[0012] In one embodiment, the means is a peptide or protein. In one embodiment, the means is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12D mutation described in SEQ ID NO: 295. In one embodiment, the means is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a mutation at residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen includes a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the means is an antibody or fragment thereof as described in Table 1 or Table 2, or an antibody selected by the method described in Example 4 or Example 5. In one embodiment, the cancer cells are pancreatic cancer cells, lung cancer cells, or colorectal cancer cells. In one embodiment, the administration step is carried out by intravenous or subcutaneous injection. In one embodiment, the surface K-Ras antigen has a mutation in one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), in full-length or cleaved form of K-Ras.

[0013] In one embodiment, a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, further comprises administering an additional therapeutic agent to the subject before or simultaneously with the administration of the composition, the additional therapeutic agent being distinct from the therapeutic agent present in the composition, and the administration of the additional therapeutic agent increases the binding availability of surface K-Ras antigen on the outer surface of cancer cells. In one embodiment, the additional therapeutic agent is a K-Ras small molecule inhibitor. In one embodiment, the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen. In one embodiment, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. In one embodiment, the surface K-Ras antigen includes the G12D mutation. In one embodiment, the surface K-Ras antigen includes the sequence described in SEQ ID NO: 295. In one embodiment, the additional therapeutic agent is administered to the subject 1 to 14 days before the administration of the composition. In one embodiment, the additional therapeutic agent is administered to the subject 3 to 7 days before the administration of the composition.

[0014] In one embodiment, a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, further comprises administering a therapeutic agent to the subject before or simultaneously with the administration of a composition, thereby increasing the expression of surface K-Ras antigen on the outer surface of cancer cells. In one embodiment, the therapeutic agent is a K-Ras small molecule inhibitor. In one embodiment, the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen. In one embodiment, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. In one embodiment, the surface K-Ras antigen includes the G12D mutation. In one embodiment, the therapeutic agent is administered to the subject 1 to 14 days before the administration of the composition. In one embodiment, the therapeutic agent is administered to the subject 3 to 7 days before the administration of the composition.

[0015] This specification also discloses a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a binder, and the binder specifically binds to a surface K-Ras antigen expressed on the outer surface of cancer cells. In one embodiment, the transmembrane domain is selected from the group consisting of CD3-zeta, CD28, CDE28a, CD4, or a combination thereof. In one embodiment, the intracellular domain is selected from the group consisting of CD28, CD27, 4-1BB, OX40, and / or ICOS. In one embodiment, the binder is an antibody or antibody fragment. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12D mutation described in SEQ ID NO: 295. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes the G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the binder is an antibody fragment. In one embodiment, the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.In one embodiment, the surface K-Ras antigen has more than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or more than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the surface K-Ras antigen has more than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or more than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] This specification also discloses vectors comprising nucleotide sequences encoding any of the chimeric antigen receptors described herein.

[0017] This specification also discloses immune cells expressing any of the chimeric antigen receptors described herein. In one embodiment, the immune cells are cells derived from an organism. In one embodiment, the immune cells are T cells derived from an organism. In one embodiment, the immune cells are selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof. In one embodiment, the immune cells are T cells. In one embodiment, the immune cells are CD4+ T cells or CD8+ T cells.

[0018] Also disclosed herein is a composition comprising an immune cell that expresses a chimeric antigen receptor targeting surface K-Ras antigens including a mutant surface K-Ras antigen, expressed on the extracellular surface of cancer cells. In one aspect, the immune cell is derived from a source that is autologous, syngeneic, allogeneic, or xenogeneic. In one aspect, the immune cell is a T cell derived from a source that is autologous, syngeneic, allogeneic, or xenogeneic. In one aspect, the immune cell is selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof. In one aspect, the immune cell is a T cell. In one aspect, the immune cell is a CD4+ T cell or a CD8+ T cell.

[0019] Also disclosed herein are an immune cell and a composition comprising the immune cell for use in treating cancer in an individual, wherein the cancer comprises cancer cells that express a surface K-Ras antigen on the outer surface of the cancer cells. In one aspect, the immune cell comprises a cell derived from an individual having cancer. In one aspect, the immune cell is selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof. In one aspect, the immune cell is a CD4+ T cell or a CD8+ T cell. In one aspect, the cancer cell is selected from the group consisting of pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. In one aspect, the subject has been administered an additional therapeutic agent, or the immune cell is for co-administration with an additional therapeutic agent. In one aspect, the additional therapeutic agent is a K-Ras small molecule inhibitor. In one aspect, the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen. In one aspect, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. In one aspect, the surface K-Ras antigen comprises a G12D mutation. In one aspect, the therapeutic agent is administered to the subject 1 day to 14 days before the administration of the immune cell. In one aspect, the therapeutic agent is administered to the subject 3 days to 7 days before the administration of the immune cell.

[0020] Also disclosed herein is a method of treating an individual having a cancer comprising cancer cells expressing a surface K-Ras antigen on the outer surface of the cancer cells, the method comprising administering to the individual a therapeutically effective amount of immune cells that express any of the chimeric antigen receptors described herein. In one aspect, the immune cells comprise cells derived from the individual having the cancer. In one aspect, the immune cells are T cells derived from the individual having the cancer. In one aspect, the immune cells are selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof. In one aspect, the immune cell is a T cell. In one aspect, the immune cell is a CD4+ T cell or a CD8+ T cell. In one aspect, the cancer cell is selected from the group consisting of pancreatic cancer cell, lung cancer cell, cholangiocarcinoma cell, ovarian cancer cell, endometrial cancer cell, or colorectal cancer cell.

[0021] Also described herein is a method of treating an individual having a cancer comprising cancer cells expressing a surface K-Ras antigen on the outer surface of the cancer cells, the method comprising administering to the individual a therapeutically effective amount of any of the compositions described herein. In one aspect, the cancer cell is selected from the group consisting of pancreatic cancer cell, lung cancer cell, cholangiocarcinoma cell, ovarian cancer cell, endometrial cancer cell, or colorectal cancer cell.

[0022] In one aspect, any of the methods of treating an individual having a cancer comprising cancer cells that express surface K-Ras antigen on the outer surface of the cancer cells further comprises administering a therapeutic agent to the individual before or concurrently with the administration of the immune cells expressing the chimeric antigen receptor, wherein the administration of the therapeutic agent increases the binding availability of the surface K-Ras antigen on the outer surface of the cancer cells. In one aspect, the therapeutic agent is a small-molecule K-Ras inhibitor. In one aspect, the small-molecule K-Ras inhibitor specifically inhibits the activity of the form of K-Ras having the mutation present in the surface K-Ras antigen. In one aspect, the small-molecule K-Ras inhibitor is MRTX1133 or RMC-6236. In one aspect, the surface K-Ras antigen comprises a G12D mutation. In one aspect, the therapeutic agent is administered to the subject 1 day to 14 days before the administration of the immune cells. In one aspect, the therapeutic agent is administered to the subject 3 days to 7 days before the administration of the immune cells.

[0023] This specification also discloses a method for diagnosing cancer associated with the expression of surface K-Ras antigen on the outer surface of cells, comprising: (a) obtaining a cell sample from a subject including a cell population; (b) exposing the cell sample to a drug capable of binding to the surface K-Ras antigen, wherein the cell population is not cytolyzed or otherwise permeabilized to avoid the drug reacting with intracellular K-Ras; and (c) measuring the presence of the drug on the outer surface of a cell population present in the cell sample. In one embodiment, the surface K-Ras antigen is a variant surface K-Ras antigen. In one embodiment, the drug is an antibody or antibody fragment that binds to the surface K-Ras antigen. In one embodiment, the antibody or antibody fragment includes a label. In one embodiment, the label is a fluorescent dye. In one embodiment, steps (b) and (c) are performed on a sample previously obtained from the subject.

[0024] In one embodiment, a method for diagnosing cancer associated with the expression of surface K-Ras antigen on the outer surface of cells further comprises a secondary antibody conjugated to a portion of an antibody or antibody fragment, wherein the secondary antibody is labeled. In one embodiment, step (c) is carried out by applying the cell sample to a flow cytometer after step (b). In one embodiment, step (c) is carried out by an electron microscope. In one embodiment, the cell sample is exposed to a cell surface membrane dye before step (b). In one embodiment, step (c) is carried out by a confocal microscope. In one embodiment, the cell sample is selected from the group consisting of pancreatic cells, colorectal cells, bile duct cells, ovarian cells, endometrial cells, and lung cells.

[0025] This specification also discloses a method for diagnosing cancer associated with the expression of a surface K-Ras antigen, comprising: (a) obtaining a cell sample from a subject including a population of cells; (b) labeling proteins on the outer surface of the cells with a first agent; (c) lysing the cell population to obtain a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second agent, wherein the second agent selectively binds to the first agent; (e) removing the captured labeled proteins from the surface and removing the first agent from the captured labeled proteins to obtain a cell surface protein sample; (f) exposing the cell surface protein sample to a third agent capable of selectively binding to a surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth agent, wherein the fourth agent has a detectable label and binds to a portion of the third agent; and (h) measuring the presence of the detectable label. In one embodiment, steps (f) to (h) are performed by Western blotting. In another embodiment, the methods of steps (b) to (h) are performed on a sample previously obtained from the subject.

[0026] In one embodiment, any method for diagnosing cancer associated with the expression of surface K-Ras antigen further comprises the steps of subjecting the subject to one or more of the following steps (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more steps): (i) administering a therapeutically effective amount of any of the compositions described herein; (ii) administering a therapeutically effective amount of any of the compositions described herein by intravenous or subcutaneous injection; (iii) administering a therapeutically effective amount of a composition wherein the composition is surface K-Ras expressed on the outer surface of cancer cells. - A step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the means is an antibody; (v) A step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the means is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen is Sequence ID No. 1 or Sequence No. (vi) administering a therapeutically effective amount of a composition comprising a mutation in residue 12, 13, or 61 based on the amino acid sequence described in No. 2, and the means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. (vii) administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, (viii) administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the means is(ix) a step of administering an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, and the composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, (x) a step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to surface K-Ras antigens expressed on the outer surface of cancer cells, wherein the means is an antibody fragment; (xi) a step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to surface K-Ras antigens expressed on the outer surface of cancer cells, wherein the means is a peptide or protein; (xii) a step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to surface K-Ras antigens expressed on the outer surface of cancer cells (xiii) a step of administering a therapeutically effective amount of a composition comprising means for selectively binding to a surface K-Ras antigen, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, and the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 (xiv) Administering a therapeutically effective amount of a composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is a peptide or protein that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, (xv) Administering a therapeutically effective amount of a composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is a peptide or protein that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2,(xvi) a step of administering a therapeutically effective amount of a composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is a peptide or protein that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is an antibody or fragment thereof as described in Table 1 or Table 2. (xvii) a step of administering an antibody selected by the method described in Example 4 or Example 5, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the surface K-Ras antigen has a mutation in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D) (i) and 61 (including, but not limited to, Q61H and Q61L), a step of administering a full-length or cleaved form of K-Ras; (xviii) a step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the surface K-Ras antigen has more than 70% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (xix) a step of administering a therapeutically effective amount of a composition, wherein the composition is a surface K-Ras antigen expressed on the outer surface of cancer cells (xx) A step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the surface K-Ras antigen has more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or more than 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, (xxi) a therapeutically effective amount,(xxii) A step of administering to an individual an effective dose of immune cells expressing any of the chimeric antigen receptors described herein, wherein the immune cells include cells derived from an individual having cancer, (xxiii) A step of administering to an individual an effective dose of immune cells expressing any of the chimeric antigen receptors described herein, (xxii) A step of administering to an individual an effective dose of immune cells expressing a chimeric antigen receptor, wherein the immune cells are T cells derived from an individual having cancer, (xxiv) A step of administering to an individual an effective dose of immune cells expressing any of the chimeric antigen receptors described herein, (xxii) A step of administering to an individual an effective dose of immune cells expressing a chimeric antigen receptor, wherein the immune cells are selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof, (xxv) A step of administering (xxii) a step of administering an effective amount of immune cells expressing any of the chimeric antigen receptors described herein to an individual, wherein the immune cells are T cells, (xxvi) a step of administering a therapeutically effective amount of immune cells expressing any of the chimeric antigen receptors described herein to an individual, (xxvii) a step of administering a therapeutically effective amount of immune cells expressing a chimeric antigen receptor to an individual, wherein the immune cells are CD4+ T cells or CD8+ T cells, (xxviii) a step of administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the subject is not administered an intracellular delivery compound in combination with the administration of the composition, or (xxix) a combination thereof when the surface K-Ras antigen is expressed on the outer surface of cells. In one embodiment, the therapeutic agent is therapeutically effective in inhibiting the growth or proliferation of cancer cells, or, if not, is cytotoxic to cancer cells. In one embodiment, the subject is not administered an intracellularly delivered compound in combination with the administration of the composition.

[0027] This specification also discloses a therapeutically effective amount of any of the compositions disclosed herein for use in treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or in killing cancer cells, wherein the subject has been administered an additional therapeutic agent, the administration of which stimulates the expression of surface K-Ras antigen on the outer surface of the cancer cells. In one embodiment, the subject has not been administered an intracellularly delivered compound in conjunction with the administration of the composition. In one embodiment, the additional therapeutic agent is a K-Ras small molecule inhibitor. In one embodiment, the additional therapeutic agent was administered to the subject 1 to 14 days prior to the administration of the composition. In one embodiment, the additional therapeutic agent was administered to the subject 3 to 7 days prior to the administration of the composition. In one embodiment, the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. In one embodiment, the additional therapeutic agent and the composition are administered to the subject substantially simultaneously.

[0028] This specification also discloses a bispecific antibody comprising a first binding domain linked to a second binding domain, wherein the first binding domain selectively binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the second binding domain selectively binds to an antigen expressed on the surface of immune effector cells. In one embodiment, the first binding domain comprises a light chain variable region and a heavy chain (HC) variable region (VH). In one embodiment, the first binding domain comprises a light chain (LC) variable region (VL) and a constant region (FC), and a heavy chain variable region and a heavy chain constant region. In one embodiment, the second binding domain comprises a light chain variable region and a heavy chain variable region. In one embodiment, the second binding domain comprises a light chain variable region and a light chain constant region, and a heavy chain variable region and a heavy chain constant region. In one embodiment, the surface K-Ras antigen has at least 60% homology to SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the first binding domain selectively binds to a region on the surface K-Ras antigen containing residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the first binding domain selectively binds to a surface K-Ras antigen having one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In another embodiment, the first binding domain selectively binds to a surface K-Ras antigen having the G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the first binding domain selectively binds to a surface K-Ras antigen having the G12D mutation described in SEQ ID NO: 295. In another embodiment, the first binding domain selectively binds to a surface K-Ras antigen having the G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the first binding domain selectively binds to a surface K-Ras antigen having a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the antigen expressed on the surface of immunoeffector cells is selected from the group consisting of TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3ε, CD3δ, CD3ζ, CD137, CD16, and CD64.In one embodiment, the antigen expressed on the surface of immune effector cells is CD3ε. In another embodiment, the immune effector cells are selected from the group consisting of T cells, neutrophils, macrophages, monocytes, and NK cells.

[0029] This specification also discloses a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering a therapeutically effective amount of one of the bispecific antibodies disclosed herein, wherein a surface K-Ras antigen is expressed on the outer surface of the cancer cells. In one embodiment, the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. In one embodiment, the administration step is carried out by intravenous or subcutaneous injection. In one embodiment, the surface K-Ras antigen has a mutation in one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), in either a full-length or cleaved form of K-Ras. In a further embodiment, the bispecific antibodies described herein are intended for use in treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or in killing cancer cells.

[0030] This specification also discloses a composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is linked to a therapeutic agent, and the composition does not contain an intracellular delivery compound. In one embodiment, the surface K-Ras antigen has a mutation in one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), in either a full-length or cleaved form of K-Ras.

[0031] This specification also discloses a bispecific antibody comprising a first means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells and a second means for selectively binding to an antigen expressed on the surface of immunoeffector cells, wherein the first means is linked to the second means. In one embodiment, the surface K-Ras antigen has a mutation in one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), in either a full-length or cleaved form of K-Ras.

[0032] This specification also discloses a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes means for specifically binding to a surface K-Ras antigen expressed on the outer surface of a cancer cell. In one embodiment, the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H and Q61L), in either a full-length or cleaved form of K-Ras. [Brief explanation of the drawing]

[0033] [Figure 1a] The design of the R11.1.6-based structure used in Example 1 is shown.

[0034] [Figure 1b] This graph shows the growth (left panel) and viability (right panel) of Panc-1 (K-Ras G12D mutant) and MiaPaca-2 (K-Ras G12C mutant) cells cultured in the presence of the R11.1.6-based construct described in Figure 1a.

[0035] [Figure 1c] This graph shows the growth of lung cancer cell lines (left), colorectal cancer cell lines (center), and RASless mouse embryonic fibroblast (MEF) cell lines (right) cultured in the presence of R11.1.6, compared to a saline control. The left and center graphs show cell counts, while the right graph shows the absorbance of purple formazan, which indicates the number of viable cells in the MTT assay. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0036] [Figure 1d] This graph shows the growth (left panel) and viability (right panel) of Panc-1 (K-Ras G12D mutant) and MiaPaca-2 (K-Ras G12C mutant) cells cultured in the presence of R11.1.6, compared to K-Ras G12D mutant-specific small molecule inhibitors and combinations of these two. Data were plotted against staining of tumor cells using only anti-His tagged secondary antibodies. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0037] [Figure 1e] This graph shows the growth (left) and survival rate (right) of Panc-1 (K-Ras G12D mutant) and MiaPaca-2 (K-Ras G12C mutant) cultured in the presence of R11.1.6, compared to various mutant therapeutic proteins based on R11.1.6. Such proteins include a mutant form of R11.1.6 with scrambled codons in the region contacting the K-RAS mutant (scrambled R11.1.6), the M11.1.2 scaffold protein that binds to mouse serum albumin, and E11.4.1 that binds to human EGFR. Data were plotted against staining of tumor cells using only anti-His tagged secondary antibodies. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0038] [Figure 1f] This graph shows the surface binding of R11.1.6, scrambled R11.1.6, M11.1.2, and E11.4.1 to viable Panc-1 and MiaPaca-2 cancer cells, defined by the median relative fluorescence intensity of anti-His tag secondary antibody staining. Data were plotted against staining of tumor cells using anti-His tag secondary antibody only. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0039] [Figure 1g] This graph shows flow cytometry staining of AKT (left panel) and ERK (right panel) at various time points after adding R11.1.6 to Panc-1 (K-Ras G12D variant) pancreatic cancer cultures. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0040] [Figure 1h] This graph shows the localization of R11.1.6-based constructs by flow cytometry staining of anti-His antibodies. ***p<0.001;**p<0.01;*p<0.05;ns p>0.05. All statistics were performed using unpaired t-tests in GraphPad Prism.

[0041] [Figure 1i] This is a high-resolution confocal microscope image illustrating the localization of R11.1.6-based constructs extracellularly after application of an anti-His antibody.

[0042] [Figure 2a]This is a test for surface-bound or fully-bound anti-K-Ras antibodies using RASless MEFS rescued with BRAFV600E.

[0043] [Figure 2b] This is a test for surface-bound or fully-bound anti-K-Ras antibodies using RASless MEFS rescued with BRAFV600E.

[0044] [Figure 2c] This study uses flow cytometry to test human anti-K-Ras antibody 1 for binding to RASless MEFS rescued with BRAFV600E, the G12D variant of K-Ras 4B, PANC-1, or MiaPaca-2.

[0045] [Figure 2d] This study uses flow cytometry to test human anti-K-Ras antibody 2 for binding to RASless MEFS rescued with BRAFV600E, the G12D variant of K-Ras 4B, PANC-1, or MiaPaca-2.

[0046] [Figure 2e] This study uses flow cytometry to test the surface binding of human anti-K-Ras antibody 1 to RASless MEF rescued with wild-type K-Ras 4A or 4B, or a mutant K-Ras 4B construct.

[0047] [Figure 2f] Representative histograms of surface K-Ras expression measured by human anti-K-Ras antibody 1 staining in multiple tumor cell lines are shown. The upper panel shows surface staining (black), and the lower panel shows overall staining defined by both surface and intracellular expression (blue). Solid lines represent human anti-K-Ras antibody 1 staining, and filled histograms represent human IgG isotype controls. The data are representative of at least separate experiments.

[0048] [Figure 2g] This is a high-resolution confocal microscope image illustrating the localization of human anti-K-Ras antibody 1 staining on cells.

[0049] [Figure 2h] Representative histograms of surface K-Ras expression for HL-10 and D2H12 antibodies in RASless MEF, Panc-1, MiaPaca-2, and Capan-2 cell lines are shown. Solid lines represent anti-K-Ras antibody staining, and filled histograms represent human IgG isotype controls.

[0050] [Figure 2i] This represents a test of the specific binding of an antibody to K-Ras (21 kDa arrow) in Western blotting using RASLess MEF rescued with either BRAFV600E, wild-type K-Ras 4A or 4B, or a mutant K-Ras 4B construct.

[0051] [Figure 2j] Western blot analysis revealed three commercially available antibodies that exhibit both high sensitivity and specific binding to either the K-Ras 4B G12D mutant or wild-type K-Ras 4A or 4B. All blots were loaded with equivalent amounts of protein determined by BSA protein assay and identified on each blot.

[0052] [Figure 2k] Western blot, RT-PCR, and surface flow cytometry analyses of Panc-1 tumor cells after treatment with K-Ras-specific or scrambled siRNA are shown.

[0053] [Figure 2l]This figure shows the surface expression of K-Ras, measured by human anti-K-Ras antibody 1 staining, in Panc-1 cells after excision of β2-microglobulin using CRISPR-Cas-9, compared to control parent cells expressing MHC class I. The upper figure shows MHC class I expression, and the lower figure shows a representative histogram of human anti-K-Ras antibody 1 staining. The lower center figure shows the magnification changes comparing human anti-K-Ras antibody 1 surface staining and human IgG isotype control surface staining.

[0054] [Figure 2m] Western blot analyses of K-Ras and cyclin D in bulk cells are shown, comparing proteins that were not pulled down by streptavidin beads (non-surface / non-biotinylated) or were pulled down (surface biotinylated) after biotinylation of surface proteins in live cells. Each lane was loaded with an equivalent amount of protein determined by BSA protein assay. Data are representative of at least two experiments per cell line.

[0055] [Figure 3] This graph represents tumor cell killing, defined as the percentage of surviving cells compared to an untreated control culture, using various concentrations of saporin alone without targeted antibodies. Each group represents a representative value for three replicates. The arrow points to 4.5 nM saporin. This 4.5 nM concentration, conjugated with a secondary antibody, is used in the antibody-drug conjugate (ADC) assay described in Example 3.

[0056] [Figure 4]This graph represents Panc-1 (G12D mutant pancreatic cell line) cell-specific killing, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of HL-10 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0057] [Figure 5] This graph represents cell-specific killing of AsPC-1 (G12D mutant pancreatic cell line) cells, defined as the percentage of viable cells compared to an untreated control culture. The results are from the Fab-Zap assay using HL-10 compared to a rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0058] [Figure 6] This graph represents LS180 (G12D mutant colorectal cell line) cell-specific killing, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of HL-10 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0059] [Figure 7] This graph represents cell-specific killing of SK-LU-1 (G12D mutant pancreatic cell line) cells, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of HL-10 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0060] [Figure 8] This graph represents CAPAN-2 (G12V mutant pancreatic cell line) cell-specific killing, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of D2H12 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0061] [Figure 9] This graph represents SW480 (G12V mutant colorectal cell line) cell-specific killing, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of D2H12 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0062] [Figure 10] This graph represents cell-specific killing of NCI-H2444 (G12V mutant lung cancer cell line), defined as the percentage of viable cells compared to an untreated control culture. It was obtained using various concentrations of rabbit anti-human K-Ras G12V antibody (clone number D2H12) compared to rabbit IgG control antibody in the Fab-Zap assay. Each group represents a representative value for three replicates.

[0063] [Figure 11]This graph represents MiaPaca (G12C mutant pancreatic cell line) cell-specific killing, defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of HL-10 were used in the Fab-Zap assay compared to rabbit IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0064] [Figure 12a] These graphs represent cell-specific killing of Panc-1 (Figure 12a), AsPC-1 (Figure 12b), MiaPaca-2 (Figure 12c), and BXPC3 (Figure 12d), defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of human anti-K-Ras antibody 1 were used in the Fab-Zap assay compared to a human IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing saporin, while in others, only the primary antibody is shown. Figure 12e is the control with saporin alone. Each group represents representative values ​​for three replicates. [Figure 12b] These graphs represent cell-specific killing of Panc-1 (Figure 12a), AsPC-1 (Figure 12b), MiaPaca-2 (Figure 12c), and BXPC3 (Figure 12d), defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of human anti-K-Ras antibody 1 were used in the Fab-Zap assay compared to a human IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing saporin, while in others, only the primary antibody is shown. Figure 12e is the control with saporin alone. Each group represents representative values ​​for three replicates. [Figure 12c]These graphs represent cell-specific killing of Panc-1 (Figure 12a), AsPC-1 (Figure 12b), MiaPaca-2 (Figure 12c), and BXPC3 (Figure 12d), defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of human anti-K-Ras antibody 1 were used in the Fab-Zap assay compared to a human IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing saporin, while in others, only the primary antibody is shown. Figure 12e is the control with saporin alone. Each group represents representative values ​​for three replicates. [Figure 12d] These graphs represent cell-specific killing of Panc-1 (Figure 12a), AsPC-1 (Figure 12b), MiaPaca-2 (Figure 12c), and BXPC3 (Figure 12d), defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of human anti-K-Ras antibody 1 were used in the Fab-Zap assay compared to a human IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing saporin, while in others, only the primary antibody is shown. Figure 12e is the control with saporin alone. Each group represents representative values ​​for three replicates. [Figure 12e] These graphs represent cell-specific killing of Panc-1 (Figure 12a), AsPC-1 (Figure 12b), MiaPaca-2 (Figure 12c), and BXPC3 (Figure 12d), defined as the percentage of viable cells compared to an untreated control culture. Various concentrations of human anti-K-Ras antibody 1 were used in the Fab-Zap assay compared to a human IgG control antibody. In some data, the primary antibody is bound to a secondary antibody containing saporin, while in others, only the primary antibody is shown. Figure 12e is the control with saporin alone. Each group represents representative values ​​for three replicates.

[0065] [Figure 13a]This graph shows cell-specific killing of various RAS-less MEFs, including MRF-BRAF (Figure 13a), MEF (G12D) (Figure 13b), MEF (wild-type KRAS-4A) (Figure 13c), and MEF (KRAS-4B) (Figure 13d), defined as the percentage of viable cells compared to an untreated control culture. The results are obtained using various concentrations of human anti-K-Ras antibody 1 compared to a human IgG control antibody in the Fab-Zap assay. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates. [Figure 13b] This graph shows cell-specific killing of various RAS-less MEFs, including MRF-BRAF (Figure 13a), MEF (G12D) (Figure 13b), MEF (wild-type KRAS-4A) (Figure 13c), and MEF (KRAS-4B) (Figure 13d), defined as the percentage of viable cells compared to an untreated control culture. The results are obtained using various concentrations of human anti-K-Ras antibody 1 compared to a human IgG control antibody in the Fab-Zap assay. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates. [Figure 13c] This graph shows cell-specific killing of various RAS-less MEFs, including MRF-BRAF (Figure 13a), MEF (G12D) (Figure 13b), MEF (wild-type KRAS-4A) (Figure 13c), and MEF (KRAS-4B) (Figure 13d), defined as the percentage of viable cells compared to an untreated control culture. The results are obtained using various concentrations of human anti-K-Ras antibody 1 compared to a human IgG control antibody in the Fab-Zap assay. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates. [Figure 13d]This graph shows cell-specific killing of various RAS-less MEFs, including MRF-BRAF (Figure 13a), MEF (G12D) (Figure 13b), MEF (wild-type KRAS-4A) (Figure 13c), and MEF (KRAS-4B) (Figure 13d), defined as the percentage of viable cells compared to an untreated control culture. The results are obtained using various concentrations of human anti-K-Ras antibody 1 compared to a human IgG control antibody in the Fab-Zap assay. In some data, the primary antibody is bound to a secondary antibody containing a saporin, while in others, only the primary antibody is shown. Each group represents a representative value of three replicates.

[0066] [Figure 14] This figure shows histograms of K-Ras expression in primary human cells, measured by human anti-K-Ras antibody 1 or human anti-K-Ras antibody 2 staining. Solid lines represent anti-K-Ras antibody staining, and filled histograms represent human IgG isotype controls. Cells derived from either normal or fibrous lung tissue of a single donor were measured for surface K-Ras staining (left) or whole K-Ras staining (right).

[0067] [Figure 15a] This graph shows the changes in the antibody-binding availability of K-Ras on the surface of PANC-1 and MiaPaca cells, with and without pretreatment with various therapeutic agents (5-FU, MRTX849, and MRTX1133). [Figure 15b] This graph shows the changes in the antibody-binding availability of K-Ras on the surface of PANC-1 and MiaPaca cells, with and without pretreatment with various therapeutic agents (5-FU, MRTX849, and MRTX1133). [Figure 15c] This graph shows the changes in the antibody-binding availability of K-Ras on the surface of PANC-1 and MiaPaca cells, with and without pretreatment with various therapeutic agents (5-FU, MRTX849, and MRTX1133).

[0068] [Figure 16a] The graph shows the percentage of PANC-1 cells with the G12D K-Ras mutation exposed to a surface K-Ras targeted antibody-drug conjugate, with and without pretreatment with MRTX1133.

[0069] [Figure 16b] The graph shows the percentage of viable cells compared to untreated viable control cells. Cells were treated with various combinations of IgG, human anti-K-Ras antibody 1 ADC, RMC-6236, and 5-FU, as shown. The dotted line indicates the maximum level of cell toxicity achieved by treatment with 10⁻⁷M saporin.

[0070] [Figure 17] The graphs show the luminescence in luciferase-expressing Panc-1 cells (or, in the case of the "T cell alone" group, without Panc-1 cells) to demonstrate the effects of various bispecific antibody constructs against T cell-mediated killing.

[0071] [Figure 18] A schematic diagram of an exemplary anti-K-Ras-anti-CD3 bispecific antibody construct is provided.

[0072] [Figure 19a] This is a series of graphs showing the results from an ELISA evaluating the binding of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 to G12D mutant K-Ras loaded with GDP nucleotide (Figure 19a) or GppNHp nucleotide (Figure 19b), or to wild-type K-Ras loaded with GDP nucleotide (Figure 19c) or GppNHp nucleotide (Figure 19d). [Figure 19b]This is a series of graphs showing the results from an ELISA evaluating the binding of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 to G12D mutant K-Ras loaded with GDP nucleotide (Figure 19a) or GppNHp nucleotide (Figure 19b), or to wild-type K-Ras loaded with GDP nucleotide (Figure 19c) or GppNHp nucleotide (Figure 19d). [Figure 19c] This is a series of graphs showing the results from an ELISA evaluating the binding of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 to G12D mutant K-Ras loaded with GDP nucleotide (Figure 19a) or GppNHp nucleotide (Figure 19b), or to wild-type K-Ras loaded with GDP nucleotide (Figure 19c) or GppNHp nucleotide (Figure 19d). [Figure 19d] This is a series of graphs showing the results from an ELISA evaluating the binding of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 to G12D mutant K-Ras loaded with GDP nucleotide (Figure 19a) or GppNHp nucleotide (Figure 19b), or to wild-type K-Ras loaded with GDP nucleotide (Figure 19c) or GppNHp nucleotide (Figure 19d).

[0073] [Figure 20a] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20b] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20c]This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20d] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20e] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20f] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20g] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay. [Figure 20h] This is a series of graphs showing cell-specific killing in various cell lines, defined as the percentage of viable cells compared to an untreated control culture, using various concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saporin as single controls in the Fab-Zap assay.

[0074] [Figure 21a]These are a series of microscopic images showing the results of staining tumor tissue with human anti-K-Ras antibody 1 in PA1252 PDX mouse models (Figure 21a) and PA0787 PDX mouse models (Figure 21b), compared to a control antibody (isotype). [Figure 21b] These are a series of microscopic images showing the results of staining tumor tissue with human anti-K-Ras antibody 1 in PA1252 PDX mouse models (Figure 21a) and PA0787 PDX mouse models (Figure 21b), compared to a control antibody (isotype).

[0075] [Figure 22a] This is a series of graphs showing the body weight (Figures 22a and 22c) and tumor volume in mm³ (Figures 22b and 22d) of Balb / c nude mice transplanted with Panc-1 tumor cells and treated with various treatment regimens listed in Table 5. [Figure 22b] This is a series of graphs showing the body weight (Figures 22a and 22c) and tumor volume in mm³ (Figures 22b and 22d) of Balb / c nude mice transplanted with Panc-1 tumor cells and treated with various treatment regimens listed in Table 5. [Figure 22c] This is a series of graphs showing the body weight (Figures 22a and 22c) and tumor volume in mm³ (Figures 22b and 22d) of Balb / c nude mice transplanted with Panc-1 tumor cells and treated with various treatment regimens listed in Table 5. [Figure 22d] This is a series of graphs showing the body weight (Figures 22a and 22c) and tumor volume in mm³ (Figures 22b and 22d) of Balb / c nude mice transplanted with Panc-1 tumor cells and treated with various treatment regimens listed in Table 5.

[0076] [Figure 23] This is a heatmap of the results of HDX-MS experiments using K-Ras and human anti-K-Ras antibody 1.

[0077] [Figure 24]This is a heatmap of the results of HDX-MS experiments using K-Ras and human anti-K-Ras antibody 2. [Modes for carrying out the invention]

[0078] Unless otherwise defined herein, all technical and scientific terms shall have the meanings assigned by those skilled in the art in the relevant field.

[0079] As used herein, the term “surface K-Ras antigen” means a K-Ras peptide or protein expressed on the outer surface of a cell. The surface K-Ras antigen is the full-length KRAS4B (SEQ ID NO: 1-MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTK This includes CVIM) or KRAS4A (SEQ ID NO: 2-MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM) and their cleavage forms. The surface K-Ras antigen may further include full-length or cleaved K-Ras peptides or proteins (including morphologies KRAS4A and KRAS4) having any of the following amino acids / residues or their corresponding amino acids / residues (based on SEQ ID NO: 1 or 2): 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R); 13 (including, but not limited to, G13D); 61 (including, but not limited to, Q61H and Q61L), and any other mutations associated with cancer-causing forms of K-Ras peptides or proteins.For example, K-Ras G12D has the amino acid sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 295). The surface K-Ras antigen may further comprise other forms of K-Ras peptides or proteins that can be conjugated by various anti-K-Ras agents, including, for example, the antibodies, proteins, and peptides listed in Table 1 and / or Table 2. For example, forms of K-Ras peptides or proteins that can be conjugated by various anti-K-Ras agents, including, for example, the antibodies, proteins, and peptides listed in Table 1 and / or Table 2, include, but are not limited to, post-translationally modified K-Ras. For example, post-translational modifications of K-Ras include, but are not limited to, prenylation, post-prenylation, palmitoylation, ubiquitination, phosphorylation, SUMOylation, acetylation, nitrosylation, and combinations thereof. The antibodies, proteins, and peptides in Tables 1 and 2 are exemplary structures for selective binding to surface K-Ras antigens and may be used as conjugates or binders in any embodiment of the disclosure relating to antibody-drug conjugates, chimeric antigen receptors, and bispecific antibodies. As used herein, the term “surface K-Ras antigen” does not include K-Ras-derived peptide-human leukocyte antigen (HLA) (major histocompatibility complex-MHC molecule) complexes, or individual K-Ras-derived peptides that complex with HLA and are expressed on the surface. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0080] As used herein, the terms “binding agent” or “binding domain” refer to any molecule (peptides, proteins, low molecular weight chemicals, and nucleic acids) or a portion of such a molecule that, by its structure, can selectively bind to a target antigen. As used herein, the terms “selectively binding” or “selective binding” mean that the binding agent can bind to the target antigen within a population of non-target antigens that exceeds the target antigen, with an affinity for the target antigen greater than the affinity resulting from nonspecific binding to the non-target antigen (to the extent that such nonspecific binding occurs). Non-exclusive examples of binding agents include antibodies, nanobodies, antibody fragments (e.g., scFv), peptides, proteins, and aptamers.

[0081] As used herein, the term “binding availability” should be understood to take into account surface expression, binding partner affinity, or a combination thereof, when relating to cell surface antigens.

[0082] As used herein, “immune effector cells” refers to cells involved in cytolytic immune responses, including, for example, T cells, NK cells, monocytes, macrophages, or neutrophils.

[0083] As used herein, the term “cell penetrating peptide” is defined as the amino acid sequence that can result in the internal translocation of the peptide, and any molecule bound to it, into the cytoplasm of a cell. Examples of cell penetrating peptides are R9 (SEQ ID NO: 3-RRRRRRRRR-NH2) and penetratin (SEQ ID NO: 4-RQIKIWFQNRRMKWKK-NH2).

[0084] As used herein, the term “intracellular delivery compound” refers to a compound that facilitates or initiates the passage of a substance or composition across the cell membrane that would not normally pass through or be internally transported across the cell membrane in the absence of the intracellular delivery compound. Examples of intracellular delivery compounds include cell membrane permeable peptides, liposomes, nanoparticles, and dendrimers. In some embodiments, the intracellular delivery compound is a cell membrane permeable peptide. In some embodiments, the intracellular delivery compound is not a cell membrane permeable peptide.

[0085] As used herein, the terms "switch 1" and "switch 2" refer to regions in the K-Ras protein that undergo conformational changes and are known to be important for K-Ras function.

[0086] As used herein, the term “antibody” is defined as a protein having a set of immunoglobulin protein domains, commonly referred to as “heavy” and “light” chains, which have a defined paratope that functionally recognizes an epitope on a target antigen. The term “antibody” should be understood to encompass antibody fragments, variants, variations, derivatives, or engineered versions of molecules containing a functional paratope that recognizes a target epitope. Antibody fragments also include isolated fragments consisting of variable regions of the heavy and light chains, as well as recombinant single-chain polypeptide molecules ("scFv") in which the variable regions of the light and heavy chains are linked by a peptide linker. Antibody fragments also include F(ab')2, Fab', Fab, Fv, sFv, heavy-chain variable antibodies (VHH, also called nanobodies), single-domain antibodies (e.g., those containing the heavy-chain variable domain of a heavy-chain antibody), and the like.

[0087] A "chimeric antibody" is a recombinant protein that contains a variable domain containing the complementarity-determining region (CDR) of an antibody derived from a non-human species, while the constant domain of the antibody molecule is derived from the constant domain of a human antibody. A humanized antibody is a recombinant protein in which the CDR of an antibody derived from a certain species has been transplanted from the heavy chain and light chain variable chain of that species into the heavy chain and light chain variable domain of a human antibody. Human antibodies generally refer to antibodies obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigen challenge.

[0088] As used herein, the terms “antibody-drug conjugate” and “ADC” refer to an antibody conjugated to a target antigen-containing structure (i.e., a cell) for the purpose of delivering the functional molecule to that structure. The functional molecule is often referred to as the “payload” or “warhead.”

[0089] As used herein, the terms “ADC linker” or “linker” refer to the chemical linkage between an antibody and a payload / warhead. These linkers may include the ability to be cleaved by proteases, pH conditions, or reduction of disulfide bonds, and the ability to be cleaved either extracellularly or intracellularly. Other linkers are non-cleavable and are degraded by cells into their active form. “Linker” also refers to peptides that link to polypeptides, such as those used in bispecific antibodies. Peptide linkers can range from approximately 2 amino acids to approximately 30 amino acids, as well as any length and intermediate ranges between them.

[0090] As used herein, the terms “payload” or “warhead” refer to a molecule conjugated to the antibody of the ADC via a linker. In some embodiments, the ADC payload or warhead is a therapeutic agent.

[0091] As used herein, the term “therapeutic agent” refers to any molecule that produces a therapeutic effect (as defined herein). Therapeutic agents particularly suitable for use in ADCs include cytotoxic agents, cell proliferation inhibitors, radionuclides, or toxins. Cytotoxic agents are agents that can induce depletion, removal, and / or death of target cells, such as microtubule disruptors (e.g., meitansanoids, meltansine, emtansine, sorabtansine, rabtansine, auristatin, MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), tubulinin, and taxol), topoisomerase inhibitors (e.g., topotecan, govitecan, deruxtecan, rezetecan), and DNA damaging agents (e.g., exatecan, topoisomerase 1 inhibitor (SN-38), calicheamicin, anthramycin, tecilin, duocalmycin, doxorubicin, and pyrrolobenzodiazepine (PBD)-dimers). Cell growth inhibitors are drugs that can inhibit the growth and / or proliferation of target cells. Radionuclides are radionuclides that have excess nuclear energy and are therefore unstable, and include beta-emitting or alpha-emitting radionuclides. Examples of alpha-emitting radionuclides include astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227. Examples of beta-emitting radionuclides include iodine-131, rhenium-186, yttrium-90, samarium-153, strontium-89, and lutetium-177. When radionuclides decay, they may emit alpha, beta, or gamma radiation that can damage nearby cells. It should be understood that there are many therapeutic agents suitable for delivery via ADCs, and that the enumeration herein should be considered illustrative and not limited to potential agents.

[0092] The term "therapeutic effect" refers to an improvement in clinical characteristics. Non-limiting examples of clinical characteristics include reduction in tumor size, inhibition or limitation of tumor growth or proliferation of cancer cells, reduction or prevention of metastasis, prevention or reduction of cancer recurrence after remission, alleviation of cancer-related symptoms, improvement of patient life expectancy, and delay of clinical disease progression. Accordingly, "therapeutic effective dose" is the amount of the composition of this disclosure that produces a therapeutic effect.

[0093] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificial T cell receptor / immune receptor engineered to respond specifically to an antigen. Conventionally, this involves incorporating an antibody, or antibody fragment or derivative (i.e., scFv), into a transmembrane domain and an intracellular effector domain via a “spacer” sequence. This allows signal transduction to be induced when the CAR binds to a congeneral epitope specifically recognized by the antibody. The transmembrane domain works to fix the CAR to the cell membrane of the transduced cell. The intracellular effector domain (i.e., the intracellular domain) is involved in the normal signal transduction mechanism of the transduced cell. T cells are transduced to express a CAR by vector introduction (see below).

[0094] As used herein, the term “vector” refers to a medium used to deliver DNA or proteins into cells. A vector typically has DNA encoding a desired gene, which is incorporated into the vector DNA. Examples of vectors include, but are not limited to, plasmids, viruses, and bacteria. In the case of CARs, the vector typically contains a virus engineered to introduce the CAR gene component into the desired cells.

[0095] As used herein, "[percent](%) sequence identity" to a reference polypeptide sequence means the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences have been aligned to obtain maximum percentage sequence identity and gaps have been introduced as necessary, without considering any conservative substitutions as part of the sequence identity. A "conservative substitution" means replacing an amino acid residue with another amino acid residue that has similar properties such as size, charge, and hydrophobicity. Antibody-drug conjugates

[0096] One aspect of the present disclosure provides the use of an antibody-drug conjugate (ADC) for treating cancers associated with the expression of surface K-Ras antigens. An ADC generally comprises three parts: (1) a conjugate means (e.g., an antibody) for selectively conjugating one or more surface K-Ras antigens; (2) a payload or warhead (generally a therapeutic agent); and (3) a linker means for conjugating the conjugate means to the payload.

[0097] The binding means used in the ADC of the present invention should have target specificity for a surface K-Ras antigen that is expressed on target cancer cells but not on healthy cells. This function can be carried out using antibodies, antibody fragments, or peptides, such as those listed in Tables 1 and 2. The antibodies should have high binding affinity and low immunogenicity and cross-reactivity while still retaining properties that enable linkage to the warhead. The antibodies may be chimeric, humanized, or fully human, and may be monospecific, bispecific, tripspecific, or multispecific. In one embodiment, the binder is an antibody or antibody fragment. In one embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains mutations in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1, 12D, 12C, 12V, 12R, 13D, 1Q61H, and 1Q61L. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1, 12D, 12C, 12V, 12R, 13D, 1Q61H, and 1Q61L, but the binder does not bind to antigens of such sequences that do not have any of those mutations, or, if it can bind to such sequences that do not have such mutations, it binds with an affinity that is not considered clinically relevant or therapeutically effective: 12A, 12D, 12C, 12V, 12R, 13D, 1Q61H, and 1Q61L.In any of the above embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 70% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage of sequence identity between 70% and 99% to SEQ ID NO: 1 or SEQ ID NO: 2 (for example, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 75% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 96% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 97% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 98% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.Suitable binders for use in the embodiments of ADC disclosed herein include those listed in Table 1 and / or Table 2, which are applicable to a given cancer expression mutation.

[0098] The specific antigens used to develop the antibodies of the present invention may encompass regions of K-Ras known to contain mutations found in cancer, such as those found at residues 12, 13, and 61. Examples 4 and 5 of this specification describe specific methods for developing the antibodies of this disclosure for use in ADCs.

[0099] The linker used in the ADC of the present invention needs to provide a stable interaction between the antibody and the payload to avoid premature release of the payload and unintended off-target effects. The linker should also maintain the payload in an inert, non-toxic state when bound to the antibody, but should be able to release the payload upon intracellular translocation. Therefore, particularly suitable linkers for conjugating a conjugate to a payload include linkers that can be cleaved under intracellular conditions such that cleavage of the linker releases a cytotoxic or cell proliferation inhibitory portion from the antibody within the cell (e.g., in an endosomal or lysosomal compartment), or non-cleaving linkers such that the cell proliferation inhibitory or cytotoxic portion, or a derivative thereof, is released from the antibody after antibody degradation (e.g., in a lysosomal or via a proteasome). Cleaving linkers may include those that can be cleaved at low pH (i.e., sensitive to hydrolysis when exposed to a specific pH environment), such as acid-unstable linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.). Such acid-unstable linkers are relatively stable at neutral pH, for example, in blood and the extracellular environment, and relatively unstable at pH 5.5 or below (approximate pH of lysosomes). Scleavage linkers may also include those cleaved by lysosomal or endosomal proteases (e.g., cathepsin B) or enzymes (β-glucuronidase), such as peptidyl linkers (e.g., Val-Ala, Val-citrulline, Gly-Gly-Phe-Gly, Gly-Phe-Leu-Gly, and Phe-Leu) or β-glucuronide linkers.Cleavage linkers also include those that can be cleaved under reducing conditions (e.g., disulfide bonds), such as disulfide linkers (e.g., SATA (N-succinimidyl-S-acetylthioacetate), SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SPDP (N-succinimidyl-3-(2 -Pyridyl dithio)propionate may also be included. Non-cleaving linkers may include those that are not readily cleaved by proteases, are not cleaved at low pH, or are not released under reducing conditions, such as maleimide-alkylene linkers or maleimide-aryl linkers. In one embodiment, the linker is one or more of the following: maleimide caproyl linker, peptide-based linker (including, but not limited to, valine-citrulline linker), β-glucuronide linker, SMCC linker, disulfide linker, or acid-sensitive linker.

[0100] A wide variety of therapeutic agents can be administered as payloads in the target ADC. Alternatively, such agents may be administered simultaneously with, before, or after the administration of the ADC, or separately. Examples of therapeutic agents for the ADC of the present invention include drugs, toxins, oligonucleotides, immunomodulators, hormones, hormone antagonists, enzymes, enzyme inhibitors, radionuclides, and angiogenesis inhibitors (in addition to those listed above). Therapeutic agents further include cytotoxic agents, e.g., vinca alkaloids, anthracyclines, e.g., doxorubicin, 2-PDox or pro-2-PDox, gemcitabine, epipodophyllotoxin, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotic agents, anti-angiogenic agents, and apoptosis-promoting agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecan, proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, and tyrosine kinase inhibitors. Other useful anticancer cytotoxic agents for simultaneous or sequential administration, or for the preparation of ADCs, include nitrogen mustard, alkyl sulfonates, nitrosourea, triazenes, folic acid analogs, COX-2 inhibitors, antimetabolites, pyrimidine analogs, purine analogs, platinum-coordinated complexes, mTOR inhibitors, tyrosine kinase inhibitors, proteosome inhibitors, HDAC inhibitors, camptothecin, and hormones.

[0101] Methods for conjugating linkers to antibodies may include random, stochastic conjugation (e.g., conjugation to surface-exposed lysine) or site-specific conjugation (e.g., conjugation of reduced interchain disulfide bonds to cysteine, conjugation occurring during glycan remodeling, or conjugation to contained non-natural amino acids or specific amino acid sequences). As will be understood by those skilled in the art, the number of therapeutic agents conjugated to antibody molecules can vary, resulting in an inherently heterogeneous ADC assembly, where some antibodies contain one conjugated agent, some two, some three, and so on (and some antibodies contain none). The degree of heterogeneity depends, among other things, on the chemical action used to conjugate the therapeutic agents. In one embodiment, the number of therapeutic agents conjugated to individual anti-K-Ras antibodies or their antigen-binding fragments may range from 1 to 8 or more.

[0102] ADCs are typically administered to patients via intravenous injection, but can also be administered via subcutaneous injection. The starting dose for first-line human clinical trials is usually determined by preclinical pharmacological data, as well as nonclinical pharmacokinetic and toxicity data. Typically, the starting dose of ADC is 1 mg / kg or near 1 mg / kg and may be tested up to 20 mg / kg. Typical dosing intervals for ADCs are once every 1, 2, or 3 weeks. Optimal dose and dosing intervals are influenced by the antibodies, linkers, cytotoxic or cell proliferation inhibitors, and conjugation chemistry used to construct individual ADCs. Typically, ADCs are supplied as lyophilized powders that are reconstituted into a solution (e.g., saline, dextrose, or similar solution) before injection.

[0103] In certain embodiments, the ADC and one or more other antibodies may be administered sequentially or simultaneously as separate antibodies. In alternative embodiments, the antibody or antibody fragment may be administered as a single bispecific or multispecific antibody. bispecific antibody

[0104] In another aspect of this disclosure, a composition for treating cancer expressing a surface K-Ras antigen comprises a bispecific antibody. Immune cell redirecting bispecific antibodies are a therapeutic modality that uses a designed antibody to bring immune cells closer to target-expressing cells to stimulate the killing of target cells by the immune cells. Bispecific antibodies are engineered to have specificity to target cells via an antibody, Fab, or scFv (or other antibody fragment) that is specific to the target antigen, and specificity to immune cells via an antibody, Fab, or scFv (or other antibody fragment) that is specific to the immune cell. Immune cell redirecting bispecific antibodies may include T cell redirecting bispecific antibodies, natural killer (NK) cell redirecting bispecific antibodies, and macrophage redirecting bispecific antibodies. Typically, but not limited to, T cell redirecting bispecific antibodies bring T cells closer to target-expressing cells to stimulate the killing of target cells by T cells via the T cell surface protein CD3.

[0105] The most common bispecific antibody formats are single-chain variable fragments (scFv) without Fc fragments or full-length IgG-like asymmetric antibodies. However, many other formats are currently in use and under development. See Table 3 for the bispecific antibody formats discussed herein. [Table 3-1] [Table 3-2]

[0106] In one embodiment, the bispecific antibody of this disclosure comprises a first binding domain linked to a second binding domain. In one embodiment, the bispecific antibodies of the present disclosure include bispecific antibodies having a format selected from the group consisting of bispecific IgG (type 1), bispecific IgG (type 2), bispecific IgG (type 3), bispecific IgG (type 4), bispecific IgG (type 5), bispecific IgG (type 6), IgG-scFv (type 1), IgG-scFv (type 2), IgG-scFv (type 3), IgG-scFv (type 4), Fv-IgG, Fab-IgG (type 1), Fab-IgG (type 2), Fab-IgG (type 3), diabody, diabody-Fc (type 1), diabody-Fc (type 2), tandem dAb (type 2), tandem dAb-Fc, triple dAb (type 2), triple dAb (type 3), tandem scFv, tandem scFv-scFc, heterodimer Fab / scFv-Fc, and scFv-TCR fusions.

[0107] The first binding domain includes a first means for selectively binding to the surface K-Ras antigen. Suitable structures for selective binding to surface K-Ras antigen include: any scFv construct containing any of the Fab regions of the antibodies listed in Table 1 and / or Table 2, the light chain variable region and heavy chain variable region of any of the antibodies listed in Table 1 and / or Table 2; any scFv construct containing any of the Fab-Fc regions of the antibodies listed in Table 1 and / or Table 2, the light chain variable region and heavy chain variable region linked to the Fc region of any of the antibodies listed in Table 1 and / or Table 2 (in each case, having or not having a "hole" or "knob" mutation in the Fc region, including any of the Fc mutations similar to those disclosed for the bispecific construct of Example 13); the anti-K-Ras Fab light chain (SEQ ID NO: 27) and Fab heavy chain and Fc portions (SEQ ID NO: 26) shown in Figure 18; and an scFv containing the light chain variable region of SEQ ID NO: 13 linked to the heavy chain variable region of SEQ ID NO: 9 of human anti-K-Ras antibody 1 (heavy chain CDR corresponding to SEQ ID NOs: 10, 11, and 12, respectively). Examples include light chain CDRs 1, 2, and 3 (corresponding to SEQ ID NOs. 14, 15, and 16), scFv (including light chain variable region of SEQ ID NOs. 21 linked to heavy chain variable region of SEQ ID NOs. 17 of human anti-K-Ras antibody 2 (corresponding to heavy chain CDRs 1, 2, and 3 corresponding to SEQ ID NOs. 18, 19, and 20, respectively, and light chain CDRs 1, 2, and 3 corresponding to SEQ ID NOs. 22, 23, and 24), Fab regions of SEQ ID NOs. 9 and 13 with or without an Fc portion, and Fab regions of SEQ ID NOs. 17 and 21 with or without an Fc portion. The first binding domain may also include a second means for binding to a different region or epitope of the surface K-Ras antigen. A suitable structure for binding to the surface K-Ras antigen is as described above, except that the second means must bind to a different region of the surface K-Ras antigen than the first means. Therefore, no structure corresponding to the first means can be the same structure corresponding to the second means. As applied to bispecific antibodies, it should be understood that the structures identified herein for binding to surface K-Ras antigens can also be used in compositions of this disclosure, including antibody-drug conjugates.

[0108] The second binding domain includes means for selectively binding to an antigen on the surface of an immune effector cell. Suitable structures for binding to an antigen on an immune effector cell include any Fab construct, scFv construct, or other functional antibody fragment, such as a functional CD3 antibody, that selectively binds to the CD3 scFv-Fc polypeptide, TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3δ, CD3ζ, CD137, CD16, and CD64, as shown in Figure 18 (SEQ ID NO: 28). Exemplary CD3 binding domains (e.g., VH domain and VL domain) are shown in Table 4. Similar to the first binding domain, the second binding domain may also include a second means for binding to a different antigen on the surface of an immune effector cell. Suitable structures for binding to an antigen on the surface of an immune effector cell are as described above, except that the second means must bind to a different antigen region than the first means. Therefore, any structure corresponding to the first means cannot be the same structure corresponding to the second means. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] Chimeric antigen receptor T cells

[0109] In another aspect of this disclosure, a composition for treating cancer expressing a surface K-Ras antigen comprises engineered immune cells expressing a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0110] The extracellular domain is a binder that specifically binds to the surface K-Ras antigen expressed on the outer surface of cancer cells. In one embodiment, the binder is an antibody or antibody fragment. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D (SEQ ID NO: 295), G12C, G12V, G12R, G13D, Q61H, and Q61L. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind to antigens of such sequences that do not have any of those mutations, or, if it can bind to such sequences that do not have such mutations, it binds with an affinity that is not considered clinically relevant or therapeutically effective: G12A, G12D (SEQ ID NO: 295), G12C, G12V, G12R, G13D, Q61H, and Q61L. In any of the above embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 70% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage between 70% and 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In any of the embodiments described above, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having the sequence K-Ras G12D (SEQ ID NO: 295). Suitable binders for use in the embodiments of CAR-T cells disclosed herein include those listed in Table 1 and / or Table 2, which are applicable to a given cancer expression mutation.

[0111] In any of the embodiments described above, the chimeric antigen receptor may have a transmembrane domain selected from the group consisting of CD3-zeta, CD28, CDE28a, CD4, or a combination thereof.

[0112] In any of the embodiments described above, the chimeric antigen receptor may have an intracellular domain selected from the group consisting of CD28, CD27, 4-1BB, OX40, and / or ICOS.

[0113] In certain embodiments, immune cells are transformed with a vector expressing a nucleotide sequence encoding a CAR as described herein. The vector may be any vector capable of expressing a CAR protein within immune cells. The vector may further include regulatory sequences that enable replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for incubating such host cells to maintain all of the above host cells and enable replication of the vector. Techniques and conditions that enable large-scale production of the vector, as well as the production of nucleic acids encoded by the vector and their homologous polypeptides, proteins, or peptides, are also understood and known.

[0114] Immune cells may be autologous, syngeneic, allogeneic, or heterogeneic cells. CAR-T cells or CAR-immune cells are obtained by transfecting immune cells with a vector encoding one of the aforementioned chimeric antigen receptors, and these are administered to subjects with cancer expressing the surface K-Ras antigen. In one embodiment, the immune cells originate from the individual to which the vector is transfected. In another embodiment, the immune cells originate from different individuals. In any of the embodiments described above, the immune cells are selected from T cells, natural killer (NK) cells, dendritic cells, or a mixture thereof. In another embodiment, the immune cells are CD4+ T cells or CD8+ T cells.

[0115] In some embodiments, administration of CAR-T cells requires leukocyte apheresis. Blood cells from the patient can be collected, and the collected cells can be concentrated or removed to obtain a desired population of cells. In preferred embodiments, the patient's T cells are isolated. The isolated patient T cells are cultured in large quantities, modified with engineered CAR receptors, and injected into the patient by methods known to those skilled in the art. Methods of treatment, administration, formulation, and drug delivery

[0116] In one embodiment, a method is provided for treating subjects having cancer associated with the expression of a surface K-Ras antigen. These methods include administering a therapeutically effective amount of a composition comprising a conjugate that specifically binds to the surface K-Ras antigen expressed on the outer surface of cancer cells. In one embodiment, the conjugate is an antibody or antibody fragment. In another embodiment, the conjugate is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the conjugate is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind to antigens of such sequences that do not have any of those mutations, or, if it can bind to such sequences that do not have such mutations, it binds with an affinity that is not considered clinically relevant or therapeutically effective: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In any of the above embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 70% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage between 70% and 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. Suitable binders for use in embodiments of the treatment methods disclosed herein include those listed in Table 1 and / or Table 2, human anti-K-Ras antibody 1, and human anti-K-Ras antibody 2, which are applicable to a given cancer expression mutation.In any of the embodiments of the above methods, the conjugate may be linked to another conjugate that binds to an antigen on the surface of an immune effector cell, as discussed herein with respect to embodiments of a composition comprising a bispecific antibody, and all features and embodiments disclosed herein are incorporated herein by reference. In any of the embodiments of the above methods, the conjugate may be linked to a therapeutic agent that is cytotoxic to target cancer cells, as discussed herein with respect to embodiments of a composition comprising an antibody-drug conjugate, and all features and embodiments disclosed herein are incorporated herein by reference. In any of the embodiments of the above methods, the conjugate may be part of a chimeric antigen receptor expressed on an immune effector cell, as discussed herein with respect to embodiments of a composition comprising CAR-T cells, and all features and embodiments disclosed herein are incorporated herein by reference. In any of the above embodiments, the method does not involve the administration of an intracellular delivery compound.

[0117] The amount of the composition embodied in this invention may vary depending on the patient's needs, but should be provided in a therapeutically effective amount.

[0118] Certain embodiments of the present invention involve co-administration of the compositions embodied in the present invention with other treatments such as chemotherapy, surgery, radiotherapy, immunomodulators, and other therapeutic agents. Similarly, the inventors have found that treating K-Ras expressing cells with chemotherapy (e.g., 5-FU) and small molecule inhibitors having K-Ras activity results in increased binding availability of the surface K-Ras antigen or, in other ways, increased expression (see Examples 11, 12a, and 12b).

[0119] Accordingly, in one embodiment, a method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, includes administering a therapeutic agent to the subject before or simultaneously with the administration of any of the aforementioned compositions embodied in the present invention. In this case, the therapeutic agent should be understood to be distinct from and separate from any therapeutic agent that may be associated with the compositions embodied in the present invention. In a particular embodiment, the therapeutic agent is a chemotherapeutic agent, for example, 5-FU. In another particular embodiment, the therapeutic agent is a small molecule inhibitor of K-Ras, including but not limited to MRTX1133 (activity against G12D mutant K-Ras), MRTX849 (activity against G12C mutant K-Ras), or RMC-6236 (activity against multiple GTP-bound RAS proteins, including K-RAS WT and K-RAS G12 mutants). In another particular embodiment, multiple therapeutic agents are used. For example, in a particular embodiment, one or more therapeutic agents are a chemotherapeutic agent and a small molecule inhibitor of K-Ras. In another specific embodiment, one or more therapeutic agents are 5-FU chemotherapeutic agents and K-Ras small molecule inhibitors, including but not limited to MRTX1133 (activity against G12D mutant K-Ras), MRTX849 (activity against G12C mutant K-Ras), or RMC-6236 (activity against multiple GTP-bound RAS proteins, including K-RAS WT and K-RAS G12 mutants). More generally, K-Ras small molecule inhibitors specifically inhibit the activity of forms of K-Ras having mutations present on surface K-Ras antigens targeted by the compositions embodied in the present invention, including one of the known K-Ras mutations described herein.

[0120] The therapeutic agent is administered to the subject 1 to 21 days prior to the administration of the composition, as well as on any specific day 1 to 21 days prior to the administration and any intermediate range between those days (e.g., 3 to 7 days, 7 to 14 days, 10 to 21 days, etc.).

[0121] For embodiments involving pretreatment or concurrent treatment with additional therapeutic agents, the method or route of administration and dosage are in a manner in which such therapeutic agents are normally administered and dose-set under standard treatment conditions for indications in which they are routinely used. In some embodiments, the dose may be reduced from what is considered the indicated or approved dose.

[0122] The method or route of administration for the compositions embodied in the present invention may be any appropriate method or route, as determined by a skilled physician / clinician. Non-limiting examples of methods of administration include injection, infusion, or transplantation. Non-limiting examples of routes of administration include parenteral, intravenous, tumor, artery, muscle, peritoneum, and / or subcutaneous.

[0123] Dosage and frequency of administration may vary depending on the clinical condition. Dosage and frequency of administration may be determined by any physician or clinician in the art. In some cases, a higher or lower dose may be appropriate if an effective dose is administered. The frequency of administration may be a single dose or multiple doses may be required. In the case of CAR-T administration, the number of cells administered may vary, but a preferred embodiment is 10 4 ~10 9 It is cells / kg.

[0124] The compositions of this disclosure can be used to treat various cancers associated with the expression of mutations in K-Ras or K-Ras overexpression, such as pancreatic cancer (including pancreatic ductal adenocarcinoma (PDAC)), lung cancer (including non-small cell lung cancer (NSCLC)), cholangiocarcinoma, ovarian cancer, endometrial cancer, or colorectal cancer.

[0125] Any disclosure or embodiment relating to methods for treating, inhibiting, or suppressing cancer should be understood to be equally applicable to such medical uses. diagnosis

[0126] Since the therapeutic compositions and methods of this disclosure rely on the surface expression of the K-Ras antigen on cancer cells (surface K-Ras antigen) as opposed to the intracellular expression of K-Ras, it is important to first determine whether the cancer of interest expresses this particular antigen on its surface. Accordingly, this disclosure further provides methods and compositions for diagnosing cancers that express surface K-Ras antigen. Generally, the diagnostic compositions may contain any binder or other compound that can specifically detect surface K-Ras antigen on cells, and the method used must be able to distinguish or separate the surface K-Ras antigen from the K-Ras antigen present inside cells. With these considerations in mind, a wide variety of diagnostic methods should be identifiable by those skilled in the art, and therefore, the specific embodiments of this disclosure should be understood to be illustrative and not limiting.

[0127] The conjugates described herein for use with ADCs and CAR-T cells may also be useful agents for detecting surface K-Ras antigens in cancer cells. However, any agent that cannot be transported across the cell membrane (or can be modified to not cross the cell membrane or to enter cells in any other way) and that is specific to surface K-Ras antigens may be useful as a diagnostic tool. In one embodiment, the conjugate is an antibody or antibody fragment. In another embodiment, the conjugate is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the conjugate is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In another embodiment, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind to antigens of such sequences that do not have any of those mutations, or, if it can bind to such sequences that do not have such mutations, it binds with an affinity that is not considered clinically relevant or therapeutically effective: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. In any of the above embodiments, the binder is an antibody or antibody fragment that binds to a surface K-Ras antigen having more than 70% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage between 70% and 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. Suitable binders for use in the diagnostic embodiments disclosed herein include those listed in Table 1 and / or Table 2, applicable to a given cancer expression mutation, such as human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2.In any of the embodiments described above, the method does not involve the administration of an intracellular delivery compound.

[0128] In one embodiment, the diagnostic method of the present disclosure includes (a) obtaining a cell sample from a subject including a cell population; (b) exposing the cell sample to a drug capable of binding to a surface K-Ras antigen, wherein the cell population has not been cytolyzed or otherwise permeabilized to avoid the drug reacting with K-Ras in its intracellular form; and (c) measuring the presence of the drug on the outer surface of the cell population present in the cell sample. In one embodiment, the surface K-Ras antigen is a mutant surface K-Ras antigen. The diagnostic methods of the embodiments described above may be performed on a sample already obtained from a subject.

[0129] In one embodiment, the step of obtaining a cell sample from a subject containing a cell population includes a guided biopsy such as fine-needle aspiration biopsy. In this method, a thin, hollow needle is inserted into the target tissue sample by penetrating the skin. This may be done by CT guidance, ultrasound guidance, or palpation. Furthermore, the lesion can be accessed within the gastrointestinal tract by using endoscopic procedures such as colonoscopy, upper endoscopy, or endoscopic retrograde cholangiopancreatography. This biopsy needle is used to aspirate a small sample of cells (and fluids) to be used for analysis. Furthermore, a larger needle may be used to obtain a larger core needle biopsy specimen which can be used to obtain a larger cell mass. However, the desired outcome of this fine-needle biopsy or FNA is to obtain a single-cell suspension in which the cells remain intact for evaluation of cell surface antigen expression.

[0130] In another embodiment, the process of obtaining a cell sample from a solid tumor can be carried out by the following steps: (1) cutting the tissue into small sections (e.g., about 1 mg); (2) suspending the sections in a culture medium and subsequently dissociating them (e.g., using a MACS dissociation device or another device capable of producing a single-cell suspension); (3) digesting the dissociated sample in a culture medium supplemented with collagenase and DNase; (4) repeating the dissociation in step 2 to collect single cells; (5) washing the cells to remove digestive enzymes and DNase; and (6) passing the cells through a strainer and resuspending them in a suitable culture medium. Many alternative methods exist for obtaining cell suspensions from solid tumors or samples, and it should be understood that the embodiments described herein are illustrative and not limiting.

[0131] Cell samples may include pancreatic cells, lung cells, bile duct cells, ovarian cells, endometrial cells, or colorectal cells.

[0132] Next, the cell sample can be exposed to a drug capable of binding to the surface K-Ras antigen. Care should be taken in this step to avoid cell lysis or, otherwise, to avoid exposing the cells to conditions that would cause the drug to react with K-Ras in its intracellular form. Preferably, the drug (e.g., the binder of this disclosure) should be a drug that cannot cross the cell membrane without the use of an intracellular delivery compound. In one embodiment, the drug is an antibody or antibody fragment or another protein or peptide that binds to the surface K-Ras antigen. In a further embodiment, the antibody or antibody fragment includes a label, such as a fluorescent dye. In another embodiment, the method comprises applying a first drug and exposing the sample to a second binder that binds to a portion of the first drug, the secondary binder being labeled.

[0133] The measurement step can then be carried out by methods commonly used in the art to determine the interaction between the drug and the surface K-Ras antigen, for example, by applying a cell sample to a flow cytometer or electron microscope.

[0134] Confocal microscopy can also be used, which further involves an additional step of exposing the cell sample to a cell surface membrane dye before exposing it to a drug that reacts with the surface K-Ras antigen.

[0135] In another embodiment, the diagnostic method includes: (a) obtaining a cell sample from a subject including a cell population; (b) labeling proteins on the outer surface of the cells with a first agent; (c) lysing the cell population to obtain a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second agent, wherein the second agent selectively binds to the first agent; (e) removing the captured labeled proteins from the surface and removing the first agent from the captured labeled proteins to obtain a cell surface protein sample; (f) exposing the cell surface protein sample to a third agent capable of selectively binding to a surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth agent, wherein the fourth agent has a detectable label and binds to a portion of the third agent; and (h) measuring the presence of the detectable label. An example of this embodiment includes a Western blot. The diagnostic method of the above-described embodiment can be performed on a sample already obtained from the subject.

[0136] In diagnosing cancers that express surface K-Ras antigen, any of the aforementioned therapeutic compositions and methods can be used on patients. Exemplary embodiments of the present disclosure

[0137] Embodiment 1: A composition comprising a binder-therapeutic agent complex, wherein the binder specifically binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, and neither the composition nor the binder-therapeutic agent complex contains intracellular delivery compounds.

[0138] The composition according to Embodiment 1, wherein the binder is an antibody or an antibody fragment.

[0139] The composition according to Embodiment 1, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen having a mutation in residue 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0140] The composition according to Embodiment 1, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen having one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0141] The composition according to Embodiment 1, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen having a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0142] The composition according to Embodiment 1, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0143] The composition according to Embodiment 1, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0144] The composition according to Embodiment 1, wherein the binder is an antibody fragment.

[0145] The composition according to Embodiment 1, wherein the binder is a peptide or a protein.

[0146] The composition according to Embodiment 1, wherein the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0147] The composition according to Embodiment 1, wherein the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0148] The composition according to Embodiment 1, wherein the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0149] The composition according to Embodiment 1, wherein the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0150] The composition according to Embodiment 1, wherein the binder is an antibody listed in Table 1 or Table 2, or an antibody selected by the method described in Example 4 or Example 5.

[0151] The composition according to any of the aforementioned embodiments applicable to Embodiment 1, wherein the therapeutic agent is selected from the group consisting of cytotoxic agents, cell proliferation inhibitors, toxins, or radionuclides.

[0152] The composition according to any of the aforementioned embodiments applicable to Embodiment 1, wherein the therapeutic agent is selected from the group consisting of DNA damaging agents (alkylating agents), antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, and microtubule disruptors.

[0153] The composition according to any of the above-described embodiments applicable to Embodiment 1, wherein the therapeutic agent is selected from the group consisting of calicheamicin, saporin, mytansinoid, auristatin, ridamycin, methotrexate, vinblastine, vincristine, pyrrolobenzodiazepine and other benzodiazepine derivatives, duocalmycin, tubulisin, α-amanitin or bougainin protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxin, carboplatin, spliceostatin C, docetaxel, tylanstatin A, or any combination thereof.

[0154] The composition according to any of the above-described embodiments applicable to Embodiment 1, wherein the binder is linked to the therapeutic agent by a linker selected from the group consisting of a maleimidocaproyl linker, a peptide-based linker (including, but not limited to, a valine-citrulline linker), a β-glucuronide linker, an SMCC linker, a disulfide linker, or an acid-sensitive linker.

[0155] A composition according to any of the above embodiments applicable to Embodiment 1, wherein the therapeutic agent is a radionuclide.

[0156] The composition according to the above embodiment applicable to Embodiment 1, wherein the radionuclide is a β-particle emitting radionuclide or an α-particle emitting radionuclide.

[0157] The composition according to the above embodiment applicable to Embodiment 1, wherein the radionuclide is an alpha-particle emitting radionuclide selected from the group consisting of astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227.

[0158] The composition according to the above embodiment applicable to Embodiment 1, wherein the radionuclide is a β-particle emitting radionuclide.

[0159] The composition according to the aforementioned embodiment applicable to Embodiment 1, wherein the β-particle emitting radionuclide is selected from the group consisting of iodine-131, rhenium-186, yttrium-90, samarium-153, and lutetium-177.

[0160] Embodiment 2: A method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering to a subject a therapeutically effective amount of a composition comprising the composition described in any of Embodiments 1 to 7 and a bispecific antibody described in any of Embodiment 16, wherein the subject has not been administered an intracellular delivery compound in conjunction with the administration of the composition, and surface K-Ras antigen is expressed on the outer surface of the cancer cells.

[0161] The method according to Embodiment 2, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0162] The method according to any of the above embodiments applicable to Embodiment 2, wherein the administration step is carried out by intravenous injection or subcutaneous injection.

[0163] Embodiment 3: A method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering a therapeutically effective amount of a composition, wherein the composition comprises means for selectively binding to surface K-Ras antigens expressed on the outer surface of cancer cells.

[0164] The method according to Embodiment 3, wherein the means is an antibody.

[0165] The method according to Embodiment 3, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a mutation in residue 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0166] The method according to Embodiment 3, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0167] The method according to Embodiment 3, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0168] The method according to Embodiment 3, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0169] The method according to Embodiment 3, wherein the means is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0170] The method according to Embodiment 3, wherein the means is an antibody fragment.

[0171] The method according to Embodiment 3, wherein the means is a peptide or a protein.

[0172] The method according to Embodiment 3, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0173] The method according to Embodiment 3, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0174] The method according to Embodiment 3, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0175] The method according to Embodiment 3, wherein the means is a peptide or protein that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen includes a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0176] The method according to Embodiment 3, wherein the means is an antibody or fragment thereof listed in Table 1 or Table 2, or an antibody selected by the method described in Example 4 or Example 5.

[0177] The method according to Embodiment 3, wherein the cancer cells are pancreatic cancer cells, lung cancer cells, or colorectal cancer cells.

[0178] The method according to any of the above embodiments applicable to Embodiment 3, wherein the administration step is carried out by intravenous injection or subcutaneous injection.

[0179] The method according to any of the aforementioned embodiments applicable to Embodiment 3, wherein the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), and is in full-length or cleaved form of K-Ras.

[0180] Embodiment 4: A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain contains a binder, and the binder specifically binds to a surface K-Ras antigen expressed on the outer surface of a cancer cell.

[0181] The chimeric antigen receptor according to Embodiment 4, wherein the transmembrane domain is selected from the group consisting of CD3-zeta, CD28, CDE28a, CD4, or a combination thereof.

[0182] A chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the intracellular domain is selected from the group consisting of CD28, CD27, 4-1BB, OX40, and / or ICOS.

[0183] A chimeric antigen receptor according to any of the above embodiments applicable to Embodiment 4, wherein the binder is an antibody or an antibody fragment.

[0184] The chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a mutation in residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0185] The chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0186] The chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0187] The chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0188] The chimeric antigen receptor according to any of the aforementioned embodiments applicable to Embodiment 4, wherein the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, and the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0189] A chimeric antigen receptor according to any of the above embodiments applicable to Embodiment 4, wherein the binder is an antibody fragment.

[0190] A chimeric antigen receptor according to any of the above embodiments applicable to Embodiment 4, wherein the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0191] A chimeric antigen receptor according to any of the above embodiments applicable to Embodiment 4, wherein the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0192] A chimeric antigen receptor according to any of the above embodiments applicable to Embodiment 4, wherein the surface K-Ras antigen has more than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0193] A composition according to any of the above embodiments applicable to Embodiment 1, wherein the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0194] A composition according to any of the above embodiments applicable to Embodiment 1, wherein the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0195] The composition according to any of the above embodiments applicable to Embodiment 1, wherein the surface K-Ras antigen has more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or more than 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0196] The method according to any of the above embodiments applicable to Embodiment 3, wherein the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0197] The method according to any of the above embodiments applicable to Embodiment 3, wherein the surface K-Ras antigen has more than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0198] The method according to any of the above embodiments applicable to Embodiment 3, wherein the surface K-Ras antigen has more than 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or more than 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0199] Embodiment 5: A vector comprising a nucleotide sequence encoding a chimeric antigen receptor as described in any of the applicable embodiments of Embodiment 4.

[0200] Embodiment 6: Immune cells expressing a chimeric antigen receptor as described in any applicable embodiment of Embodiment 4.

[0201] The immune cells described in Embodiment 6, which are cells derived from an individual.

[0202] The immune cells described in Embodiment 6, which are T cells derived from an individual.

[0203] The immune cells according to Embodiment 6, selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof.

[0204] The immune cell described in Embodiment 6 is a T cell.

[0205] The immune cell according to Embodiment 6, which is a CD4+ T cell or a CD8+ T cell.

[0206] Embodiment 7: A composition comprising immune cells expressing a chimeric antigen receptor that targets a surface K-Ras antigen expressed on the extracellular surface of cancer cells.

[0207] The composition according to Embodiment 7, wherein the surface K-Ras antigen is a mutant surface K-Ras antigen.

[0208] The composition according to Embodiment 7, wherein the immune cells are derived from an autologous, syngeneic, allogeneic, or xenogeneic source.

[0209] The composition according to Embodiment 7, wherein the immune cells are T cells derived from an autologous, syngeneic, allogeneic, or xenogeneic source.

[0210] The composition according to Embodiment 7, wherein the immune cells are selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof.

[0211] The composition according to Embodiment 7, wherein the immune cells are T cells.

[0212] The composition according to Embodiment 7, wherein the immune cells are CD4+ T cells or CD8+ T cells.

[0213] Embodiment 8: A method of treating an individual having cancer comprising cancer cells expressing a surface K-Ras antigen on the outer surface of the cancer cells, comprising administering to the individual a therapeutically effective amount of immune cells expressing the chimeric antigen receptor according to any one of the embodiments of Embodiment 4.

[0214] The method according to Embodiment 8, wherein the surface K-Ras antigen is a mutant surface K-Ras antigen.

[0215] The method according to Embodiment 8, wherein the immune cells comprise cells derived from the individual having cancer.

[0216] The method according to Embodiment 8, wherein the immune cells are T cells derived from the individual having cancer.

[0217] The method according to Embodiment 8, wherein the immune cells are selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof.

[0218] The method according to Embodiment 8, wherein the immune cells are T cells.

[0219] The method according to Embodiment 8, wherein the immune cells are CD4+ T cells or CD8+ T cells.

[0220] The method according to Embodiment 8, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0221] Embodiment 9: A method for treating an individual having cancer, comprising administering to the individual a therapeutically effective amount of the composition described in any embodiment of Embodiment 7, wherein the cancer cells express a surface K-Ras antigen on the outer surface of the cancer cells.

[0222] The method according to Embodiment 9, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0223] Embodiment 10: A method for diagnosing cancer associated with the expression of surface K-Ras antigen on the outer surface of cells, comprising: (a) obtaining a cell sample from a subject including a cell population; (b) exposing the cell sample to a drug capable of binding to surface K-Ras antigen, wherein the cell population has not been cytolyzed or otherwise permeabilized to avoid the drug reacting with K-Ras in its intracellular form; and (c) measuring the presence of the drug on the outer surface of a cell population present in the cell sample.

[0224] The method according to Embodiment 10, wherein the surface K-Ras antigen is a mutant surface K-Ras antigen.

[0225] The method according to Embodiment 10, wherein the drug is an antibody or antibody fragment that binds to a surface K-Ras antigen.

[0226] The method according to Embodiment 10, wherein the antibody or antibody fragment comprises a label, and in some embodiments, the label is a fluorescent dye.

[0227] The method according to Embodiment 10, further comprising a secondary antibody bound to a portion of an antibody or antibody fragment, wherein the secondary antibody is labeled.

[0228] The method according to any applicable embodiment of Embodiment 10, wherein step (c) is performed by applying the cell sample to a flow cytometer after step (b).

[0229] The method according to any applicable embodiment of Embodiment 10, wherein step (c) is carried out by an electron microscope.

[0230] The method according to any applicable embodiment of Embodiment 10, wherein, prior to step (b), the cell sample is exposed to a cell surface membrane dye, and step (c) is performed by a confocal microscope.

[0231] The method according to any applicable embodiment of Embodiment 10, wherein the cell sample is selected from the group consisting of pancreatic cells, colorectal cells, bile duct cells, ovarian cells, endometrial cells, and lung cells.

[0232] Embodiment 11: A method for diagnosing cancer associated with the expression of a surface K-Ras antigen, comprising: (a) obtaining a cell sample from a subject including a cell population; (b) labeling proteins on the outer surface of the cells with a first agent; (c) lysing the cell population to obtain a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second agent, wherein the second agent selectively binds to the first agent; (e) removing the captured labeled proteins from the surface and removing the first agent from the captured labeled proteins to obtain a cell surface protein sample; (f) exposing the cell surface protein sample to a third agent capable of selectively binding to a surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth agent, wherein the fourth agent has a detectable label and binds to a portion of the third agent; and (h) measuring the presence of the detectable label.

[0233] The method according to embodiment 11, wherein steps (f) to (h) are performed by Western blotting.

[0234] The method according to any applicable embodiment of embodiment 10 or 11, further comprising the step of subjecting a subject to the method according to any one of embodiments 2, 3, 8, or 9, when a mutant form of K-Ras is expressed as a mutant surface K-Ras antigen on the outer surface of a cell.

[0235] The composition according to any applicable embodiment of embodiment 1, wherein the therapeutic agent is therapeutically effective to inhibit the growth or proliferation of cancer cells, or is otherwise cytotoxic to cancer cells.

[0236] The method according to any one of embodiments of embodiment 3, wherein the subject is not administered an intracellular delivery compound in combination with administration of the composition.

[0237] Embodiment 12: The method according to any one of embodiments of embodiment 2, further comprising the step of administering an additional therapeutic agent to the subject prior to or concurrently with administration of the composition, wherein the additional therapeutic agent is separate from the therapeutic agent present in the composition.

[0238] The method according to embodiment 12, wherein administration of the additional therapeutic agent increases the binding availability of surface K-Ras antigen on the outer surface of cancer cells.

[0239] The method according to embodiment 12, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor.

[0240] The method according to embodiment 12, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen.

[0241] The method according to embodiment 12, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0242] The method according to any of the applicable embodiments of Embodiment 12, wherein the surface K-Ras antigen contains the G12D mutation.

[0243] The method according to any applicable embodiment of Embodiment 12, wherein an additional therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the composition.

[0244] The method according to any applicable embodiment of Embodiment 12, wherein an additional therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the composition.

[0245] Embodiment 13: The method according to any one of Embodiments 3, 9, or 17, further comprising the step of administering a therapeutic agent to a subject before or simultaneously with the administration of a composition, wherein the administration of the therapeutic agent increases the expression of surface K-Ras antigen on the outer surface of cancer cells.

[0246] The method according to Embodiment 13, wherein the therapeutic agent is a K-Ras small molecule inhibitor.

[0247] The method according to Embodiment 13, wherein the therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen.

[0248] The method according to Embodiment 13, wherein the therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0249] The method according to any of the applicable embodiments of Embodiment 13, wherein the surface K-Ras antigen contains the G12D mutation.

[0250] The method according to any of the applicable embodiments of Embodiment 13, wherein the therapeutic agent is administered to the subject one to fourteen days prior to the administration of the composition.

[0251] The method according to any of the applicable embodiments of Embodiment 13, wherein the therapeutic agent is administered to the subject 3 to 7 days before administration of the composition.

[0252] Embodiment 14: The method according to any embodiment of Embodiment 8, further comprising the step of administering a therapeutic agent to an individual before or simultaneously with the administration of immune cells expressing a chimeric antigen receptor.

[0253] The method according to Embodiment 14, wherein the binding availability of surface K-Ras antigen on the outer surface of cancer cells is increased by administration of a therapeutic agent.

[0254] The method according to Embodiment 14, wherein the therapeutic agent is a K-Ras small molecule inhibitor.

[0255] The method according to Embodiment 14, wherein the therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen.

[0256] The method according to Embodiment 14, wherein the therapeutic agent is a K-Ras small molecule inhibitor, and the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0257] The method according to any of the applicable embodiments of Embodiment 14, wherein the surface K-Ras antigen contains the G12D mutation.

[0258] The method according to any applicable embodiment of Embodiment 14, wherein the therapeutic agent is administered to the subject 1 to 14 days before the administration of immune cells.

[0259] The method according to any applicable embodiment of Embodiment 14, wherein the therapeutic agent is administered to the subject 3 to 7 days before the administration of immune cells.

[0260] Embodiment 15: A composition comprising a therapeutically effective amount of the composition according to any one of Embodiments 1 or 7 or the bispecific antibody of Embodiment 16 for use in treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or in killing cancer cells, wherein the subject is administered an additional therapeutic agent in addition to the composition, either before or simultaneously with the composition.

[0261] The composition for use according to Embodiment 15, wherein the administration of an additional therapeutic agent stimulates the expression of surface K-Ras antigen on the outer surface of cancer cells.

[0262] The composition for use according to Embodiment 15, wherein the subject is not administered an intracellular delivery compound in combination with the administration of the composition.

[0263] A composition for use according to any of the applicable embodiments of Embodiment 15, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor.

[0264] A composition for use according to any applicable embodiment of Embodiment 15, wherein an additional therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the composition.

[0265] A composition for use according to any applicable embodiment of Embodiment 15, wherein an additional therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the composition.

[0266] A composition for use according to any applicable embodiment of Embodiment 15, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0267] A composition for use according to any of the applicable embodiments of Embodiment 15, wherein additional therapeutic agents and compositions are administered to the subject substantially simultaneously.

[0268] Embodiment 16: A bispecific antibody comprising a first binding domain linked to a second binding domain, wherein the first binding domain selectively binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the second binding domain selectively binds to an antigen expressed on the surface of immune effector cells.

[0269] The bispecific antibody according to Embodiment 16, wherein the first binding domain includes a light chain variable region and a heavy chain variable region.

[0270] The bispecific antibody according to Embodiment 16, wherein the first binding domain comprises a light chain variable region and a light chain constant region, and a heavy chain variable region and a heavy chain constant region.

[0271] A bispecific antibody according to any embodiment of Embodiment 16, wherein the second binding domain includes a light chain variable region and a heavy chain variable region.

[0272] A bispecific antibody according to any embodiment of Embodiment 16, wherein the second binding domain comprises a light chain variable region and a light chain constant region, and a heavy chain variable region and a heavy chain constant region.

[0273] A bispecific antibody according to any embodiment of Embodiment 16, wherein the surface K-Ras antigen has at least 60% homology to SEQ ID NO: 1 or SEQ ID NO: 2.

[0274] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a region on a surface K-Ras antigen containing residues 12, 13, or 61 based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0275] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen having one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0276] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen having a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0277] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen having a G12C mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0278] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen having a G12V mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0279] A bispecific antibody according to any embodiment of Embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen having a G12D mutation based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0280] A bispecific antibody according to any embodiment of Embodiment 16, wherein the antigen expressed on the surface of immune effector cells is selected from the group consisting of TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3ε, CD3δ, CD3ζ, CD137, CD16, and CD64.

[0281] A bispecific antibody according to any embodiment of Embodiment 16, wherein the antigen expressed on the surface of immune effector cells is CD3ε.

[0282] A bispecific antibody according to any embodiment of Embodiment 16, wherein the immune effector cells are selected from the group consisting of T cells, neutrophils, macrophages, monocytes, and NK cells.

[0283] Embodiment 17: A method for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells, comprising administering a therapeutically effective amount of a bispecific antibody described in any embodiment of Embodiment 16, wherein the surface K-Ras antigen is expressed on the outer surface of the cancer cells.

[0284] The method according to Embodiment 17, wherein the subject is not administered an intracellular delivery compound in combination with the administration of a bispecific antibody.

[0285] The method according to Embodiment 17, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0286] The method according to any of the embodiments of Embodiment 17, wherein the administration step is carried out by intravenous injection or subcutaneous injection.

[0287] The method according to any embodiment of Embodiment 17, wherein the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H and Q61L), and is in full-length or cleaved form of K-Ras.

[0288] Embodiment 18: A composition comprising means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the means is linked to a therapeutic agent, and the composition does not contain an intracellular delivery compound.

[0289] The composition according to Embodiment 18, wherein the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H and Q61L), and is in full-length or cleaved form of K-Ras.

[0290] Embodiment 19: A bispecific antibody comprising a first means for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, and a second means for selectively binding to an antigen expressed on the surface of immune effector cells, wherein the first means is linked to the second means.

[0291] The bispecific antibody according to Embodiment 19, wherein the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H and Q61L), and is in full-length or cleaved form of K-Ras.

[0292] Embodiment 20: A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes means for specifically binding to a surface K-Ras antigen expressed on the outer surface of a cancer cell.

[0293] The chimeric antigen receptor according to Embodiment 20, wherein the surface K-Ras antigen has mutations in one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, and G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H and Q61L), in either the full-length or cleaved form of K-Ras. [Examples]

[0294] Example 1: Inhibition of K-Ras signaling from the cell surface controls tumor growth.

[0295] The inventors utilized a charge-neutralizing variant of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus, known as R11.1.6 (SEQ ID NO: 5-ATVKFTHQGEEKQVDISKIKWVIRWGQYIWFKYDEDGGAKGWGYVSEKDAPKELLQMLKKR), which binds with high affinity and specificity to switch I of the G12D mutant K-Ras (see, for example, Traxlmayr et al., J Biol Chem. 2016 Oct 21;291(43):22496-22508.doi:10.1074 / jbc.M116.741314). To deliver this antagonistic scaffold into cells, the inventors conjugated it to either a cell membrane permeable peptide (CPP): R9 (R9-R11.1.6) or penetratin (penetratin-R11.1.6). Furthermore, as an internal control, R11.1.6 was used in the absence of the cell membrane permeable peptide (Figure 1a). Next, the growth and viability of two pancreatic cancer cell lines driven by either the G12D (Panc-1) or G12C (MiaPaca-2) mutant K-Ras were evaluated in vitro. 20,000 cells / well were seeded in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in flat-bottom 96-well plates, and cells were counted after 5 days of culture. Cell viability was calculated based on the trypan blue exclusion method, in which dead cells turned blue upon uptake of the viability indicator dye, and live cells were not stained. Survival rates were measured using Countess II FL cell viability and cell counters provided by Invitrogen (ThermoFisher USA). The inventors focused on the inhibition of tumor cell proliferation in a G12D mutation-specific manner (Figure 1b - growth in the left panel, survival rate in the right panel). Surprisingly, R11.1.6 altered Panc-1 growth and survival rates to a similar extent, regardless of whether it was bound to a cell membrane-permeable peptide. Similar data were reproduced in K-Ras-driven lung cancer and colorectal cancer cell lines (Figure 1c).The R11.1.6-mediated inhibition of Panc-1 growth and survival was similar to that of the G12D inhibitor MRTX1133 (PMID:36216931), and the combination of these two drugs enhanced the therapeutic effect (Figure 1d).

[0296] Further evaluation of growth inhibition was performed using control sulfolobus solfatalicus-derived proteins of similar molecular weight. These included a variant form of R11.1.6 (in which the codons in the region in contact with the K-RAS variant are scrambled to prevent binding) (SEQ ID NO: 6-ATVKFTHQGEEKQVDISKIKFVWRKGYVRIWGYDEDGGWGAGKYVSEKDAPKELLQMLKKR), M11.1.2 (a scaffold protein that binds to mouse serum albumin) (SEQ ID NO: 7-ATVKYTYRGEEKRVDISKIKWVNRWGQHLAFKYDKGGGAAGYGWVSEKDAPKELLQMLEKR), and E11.4.1 (which binds to human EGFR) (SEQ ID NO: 8-ATVKFTYQGEEKQVDISKIMYVIRGGQRIAFGYDEGDGAWGDGIVSEKDAPKELLQMLEKQ). Compared to R11.1.6, none of the control constructs were able to inhibit tumor growth, even though some constructs, such as E11.4.1, could bind to the surface of tumor cells (Figure 1f) (Figure 1e). Furthermore, inhibition of tumor growth by wild-type R11.1.6 correlated with a standard decrease in signaling via the AKT and ERK pathways. We detected decreased phosphorylation of Akt (at serine 473 residues) and ERK1 / 2 (threonine 202, tyrosine 204) by phosphorylation flow cytometry. Starting with 200,000 cells in a 96-well V-bottom plate, BD phosflow buffer set III (BD biosciences, catalog no. 558050) was used to stabilize and optimize the phosphorylation moiety. Next, cells were stained with PE conjugate antiphosphorylated Akt (ser473, clone D9E) (Cell Signaling Technologies, catalog number 5315S) at a concentration of 0.125 ug / 100 ul or PE conjugate antiphosphorylated ERK1 / 2 (ThermoFisher USA, catalog number 12-9109-42) at a concentration of 0.125 ug / 100 ul. Cells were washed in FACS staining buffer and run on an LSR Fortessa II flow cytometer (BD Biosciences, USA).Data was acquired using FACS DIVA software (BD Biosciences) and analyzed using FlowJo software (Tree Star, OR, USA). This was an important finding because both Akt and ERK1 / 2 are important downstream molecules of K-Ras signaling, similar to those described for K-Ras small molecule inhibitors (PMID: 12509763) (Figure 1g).

[0297] Surprisingly, inhibition was observed even in the absence of the cell membrane permeable peptide, despite the assumption that both mutant and wild-type K-Ras signal from only various intracellular locations. However, the inventors hypothesized that the small size of this scaffold protein (7 kDa) and the disorder of the cell membrane in malignant transformed cells could enable cytoplasmic permeability even in the absence of the cell membrane permeable peptide. To evaluate this possibility, the cellular location of the R11.1.6 construct was investigated by flow cytometry and confocal microscopy. For the flow cytometry experiment, cells were first stained with a live / dead far-red fixation-capable staining kit (ThermoFisher USA, catalog number L10120). Specifically, 0.2–0.4 × 10⁻⁶ cells were stained. 6Cells were seeded in a V-bottom plate, and 100 μl of dye (1:1000 dilution from stock concentration according to the manufacturer's protocol) was added to each well. The cells were incubated at room temperature for 15 minutes. Next, the cells were washed with PBS (FACS buffer) containing 2.5% FBS and 0.1% sodium azide. Then, the cells were surface-stained by adding 100 μl / well of anti-His PE-labeled antibody (mouse anti-human antibody, BioLegend, CA, USA catalog no. 362603) at a concentration of 10 μg / ml. The cells were washed twice with FACS buffer and fixed with cytofix buffer (BD biosciences catalog no. 554655). For intracellular staining, the same antibodies and reagents were used, except that the cells were permeabilized with BD perm / wash reagent (BD Biosciences, catalog no. 554723) before adding the PE anti-His antibody. Cells were acquired using an LSR Fortessa II flow cytometer with FACS DIVA software (BD Biosciences USA) and analyzed using FlowJo (Tree Star USA). For confocal microscopy experiments, cells were surface-stained in the same manner as for flow cytometry. After staining, cells were carefully spread onto 25 mm × 75 mm slides (1 mm thick) and mounted using Prolong Gold anti-bleeding reagent containing DAPI (Cell signaling Technologies, USA, catalog number 8961). Cells were imaged using a Nikon A1 spinning disk microscope. Analysis was performed using ImageJ software (developed and licensed by the NIH). Since all constructs contained polyhistidine tags (His tags) (Figure 1a), these were tested using fluorescently labeled anti-His tag antibodies. For this purpose, Panc-1 (G12D mutant K-Ras) or MiaPaca-2 (G12C mutant K-Ras) cell lines were cultured for 24 or 96 hours with either R9-R11.1.6, penetratin-R11.1.6, or R11.1.6 (without cell membrane permeable peptide), and their intracellular location was determined by staining with a fluorescently labeled secondary antibody that recognizes the His tag.As shown in Figure 1h, R11.1.6 was not detected inside tumor cells compared to the physiological saline control (defined as 1). However, increased binding of R11.1.6 to the tumor cell surface was observed in Panc-1 cancer cells, but not in MiaPaca-2 cancer cells. Given that R11.1.6, which does not contain a cell membrane-permeable peptide, was able to inhibit tumor cell growth, it is surprising that neither construct passed through the cell membrane in either flow cytometry (Figure 1h) or high-resolution confocal microscopy (Figure 1i), but rather bound to the cell surface. Since almost no construct was observed on the surface of MiaPaca-2 cells with the G12C mutation that does not bind to R11.1.6, this binding appeared to be mutation-specific (Figures 1f, 1h, and 1i). In summary, our data suggest that the inhibition of tumor cell growth and survival is mediated by mutation-specific binding of K-Ras antagonists to the cell surface. This was a surprising result. This is because K-Ras is thought to be localized only to the cell membrane and intracellular organelles (PMID:21924373), and its presence on the cell surface had not been previously described. Example 2: Functional K-Ras is expressed on the extracellular surface of both malignant and non-transformed cells.

[0298] K-Ras is defined as an intracellular protein that does not exhibit known surface expression. No reports of K-Ras on the cell surface have been published in the scientific literature. Given the previous doubts regarding both the specificity and sensitivity of available anti-K-Ras antibodies (PMID:28951536), the inventors conducted extensive validation of several commercially available, publicly reported antibodies for research use. For this purpose, a RAS-less mouse embryonic fibroblast system (MEF) was utilized. In this system, MEFs lacking all RAS proteins were rescued by lentiviral transduction with either wild-type RAS, mutant RAS, or the V600E mutant variant of BRAF, enabling survival and proliferation in the absence of all endogenous Ras proteins (PMID:33977488 and cancer.gov / research / key-initiatives / ras / ras-central / blog / 2017 / rasless-mefs-drug-screens). The inventors tested the antibodies by flow cytometry according to the protocol described in Example 1. Most of the antibodies tested showed some nonspecific binding, and BRAF V600ERescue RASless MEF was reacted (Figures 2a-2b). Several antibodies were able to detect surface K-Ras antigens on the cell surface in a highly sensitive and specific manner and preferentially bound to the G12D mutant version. For example, human anti-K-Ras antibody 1 (full heavy chain (HC)-SEQ ID NO: 9-EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;HC CDR1-SEQ ID NO: 10-GFTSRHPG;HC CDR2-SEQ ID NO: 11-ISHDGSKK;HC CDR3-SEQ ID NO: 12-ATSLYSSMDL;Complete Light Chain (LC)-SEQ ID NO: 13-QSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;LC CDR1-SEQ ID NO: 14-NSNIGKNY;LC CDR2-SEQ ID NO: 15-EDN;LC CDR3-SEQ ID NO: 16-AAWDDKFGVHWV) and Human Anti-K-Ras Antibody 2 (Complete HC-SEQ ID NO: 17-EVQLLEPGGGVVQPGRSLRLSCTNSGFSFSGYAMHWVRQAPGKGLEWVAVISFDGSHKYYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCASGGNYYGSGTIVSHGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;HC CDR1-SEQ ID NO: 18-GFSFSGYA;HC CDR2-SEQ ID NO: 19-ISFDGSHK;HC CDR3-SEQ ID NO: 20-ASGGNYYGSGTIVSHGMDV;Complete LC-SEQ ID NO: 21-QSVLTQPASVSGSPGQSITISCTGTSNDIGAYNYVSWYQQHPGKAPKLMIYDVNNRPSGVPDRFSGSKSGNMASLTISGLQAEDDADYYCSSYTSSSTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;LC CDR1-SEQ ID NO: 22-SNDIGAYNY;LC CDR2-SEQ ID NO: 23-DVN;LC CDR3-SEQ ID NO: 24-SSYTSSSTLVV) staining was observed on the surface of K-Ras rescue MEFs as well as Panc-1 and MiaPaca-2 cells (Figures 2c-2d). Further characterization of the binding of human anti-K-Ras antibody 1 using RASless MEFs showed binding to K-Ras4A, K-Ras4B, and G12D K-Ras4B, but not to G12V K-Ras4B (Figure 2e). Additional G12D K-Ras tumor cell lines also showed surface staining with human anti-K-Ras antibody 1 (Figure 2f). All staining was performed in V-bottom 96-well plates on ice for 30 minutes at an antibody concentration of 10 ug / ml and a volume of 100 ul. Surface staining of viable Panc-1 cells with human anti-K-Ras antibody 1 was also observed by high-resolution confocal microscopy (Figure 2g). Confocal microscopy was performed using the same conditions as in Figure 1i of Example 1.

[0299] Surface staining was also observed using anti-G12V mutant K-Ras (clone D2H12) and anti-G12D mutant K-Ras (clone HL10) antibodies (Figure 2h). BRAF V600E In RASless MEFS cells rescued using either the MiaPaca-2 or K-Ras G12C mutant cancer cell line, no staining was observed.

[0300] The antibody panel is BRAF V600EThey showed reactivity to rescued MEFs or exhibited multiple nonspecific bands in addition to a 22kDa K-Ras band (Figure 2i). Only rabbit anti-G12D mutant K-Ras (clone D8H7, Cell Signaling Technology), mouse anti-K-Ras (clone 4E8, Iowa Hybridoma Bank), and mouse anti-K-Ras (clone 3B10-2F2 Sigma Aldridge) showed both sensitivity and specificity to G12D mutant K-Ras 4B, pan-K-Ras, and the 4B splice variant of K-Ras, respectively, as determined by Western blotting (Figure 2j). In Western blotting experiments, cells were lysed in situ at 4°C for 30 minutes by adding 10× cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, and a protease inhibitor cocktail. The resulting cell lysates were clarified by centrifugation at 18,800 g. After washing three times with cell lysis buffer, proteins were separated on a 10% Tris-glycine SDS-PAGE gel (Bio-Rad) and transferred to an Immobilon-P PVDF membrane. The membrane was then blocked at 20°C for 1 hour with 3% BSA in PBS containing 0.1% Tween 20. Proteins were then detected using their respective primary antibodies, followed by appropriate secondary antibodies conjugated to horseradish peroxidase. Horseradish peroxidase activity was detected using a Pierce-enhanced chemiluminescent substrate (Thermo Fisher Scientific) according to the manufacturer's instructions. The chemiluminescent signals were acquired using the ChemiDoc MP Imaging System and analyzed with the Image Lab 5.1 software application (both from Bio-Rad).

[0301] In K-Ras knockdown experiments, in vitro siRNA technology was used to silence K-RAS gene expression. Cells were trypsin-treated and seeded in 24-well plates at a final siRNA concentration of 25 nM in DharmaFECT® transfection reagent, 500 μl per well, according to the manufacturer's protocol. Knockdown of the K-Ras protein in the Panc-1 cell line using siRNA targeting multiple splice variants of K-Ras mRNA (Dharmacon® Reagents, ON-TARGETplus Human SMARTpool K-RAS siRNA) resulted in downregulation of intracellular K-Ras in both RT-PCR and Western blotting. Surface staining of K-Ras with human anti-K-Ras antibody 1 was also reduced in siRNA-treated cell lines (Figure 2k).

[0302] It has been previously reported that peptide fragments of both mutant and wild-type K-Ras can be presented on the cell surface in the context of self-MHC class I (PMID:36099883, PMID:27959684, PMID:34272369). MHC class I-deficient Panc-1 cell lines were created by CRISPR-Cas-9 excision of β2-microglobulin. Staining of such mutants with human anti-K-Ras antibody 1 was identical to that of parental cell lines with sufficient MHC class I (Figure 2l).

[0303] Proteins on cells exposed to the extracellular environment were treated with reactive biotin ester (Pierce Cell Surface Protein Biotinylation and Isolation kit, #A44390) in multiple cell lines and isolated from cell extracts using streptavidin beads based on an established methodology (PMID:19341246). Similar to the flow cytometry data described above, K-Ras was detected in the surface fraction, albeit in much smaller amounts than within the cell membrane, by Western blot analysis using a K-Ras-specific antibody (Figure 2m).

[0304] In summary, our data demonstrate that both tumors and normal tissues can express K-Ras on their surfaces, and that such surface K-Ras can be mitigated in a mutation-specific manner by conjugating them to a protein therapeutic antagonist. Example 3: Validation of antibody-drug conjugates (ADCs) for selective killing of cancer cell lines using mutation-specific antibodies.

[0305] The use of antibody-drug conjugates (ADCs) for selective tumor killing is an established method for delivering cytotoxic payloads to cells expressing defined surface targets. This approach involves antibodies conjugated to a cytotoxic payload via a linker directed to a target antigen expressed on the surface of cancer cells. Such methodologies reduce systemic exposure, thereby mitigating toxicity and offering significant potential for targeted killing of cancer cells (see, e.g., Drago et al. Nat Rev Clin Oncol. 2021 Jun;18(6):327-344 doi.org / 10.1038 / s41571-021-00470-8). To act as a selective ADC agent against target cells, the ADC (including targeted antibodies and toxins) must a) selectively recognize the tumor antigen and b) be able to kill target cells at concentrations that are not toxic to cells not recognized by the antibody. The use of mutant surface K-Ras antigens as targets for ADCs has not been investigated to date.

[0306] Saporins are 30 kDa ribosome inhibitors that cannot cross the cell membrane without assistance (except at extremely high concentrations). However, as previously described (DOI:10.3390 / toxins14030184), when saporins are conjugated to internally distributed antibodies, they can be used to precisely and specifically kill target cells with little or no off-target toxicity (see, e.g., Lund et.al., mAbs, 6(4), 1038-1050, doi.org / 10.4161 / mabs.28207). Saporin has been used as an ADC reagent to target tumor surface molecules such as breast cancer-related Her-2, malignant melanoma-related CSPG-4, breast cancer-related EGFR, and FRα (see, e.g., Hoffmann et.al. Sci Rep10,8869(2020), doi.org / 10.1038 / s41598-020-65860-x), and is therefore a validated warhead for ADCs. The commercially available Fab-ZAP assay (Advanced Targeting Systems Inc.) allows for testing the usefulness of antibodies as ADCs against surface antigens by accurately delivering saporin based on a saporin-conjugated secondary antibody that reacts to a primary antibody specific to the tumor surface antigen. In the experiments described below, the inventors utilized the Fab-ZAP rabbit kit [IT-57, KIT-57] (atsbio.com / products / it57 / ), which consists of a chemical conjugate of a goat anti-rabbit monovalent antibody and saporin, a ribosome-inactivating protein. The antibodies used in this kit are affinity-purified polyclonal antibodies against both the heavy and light chains of rabbit IgG.

[0307] To investigate the potential of using mutant K-Ras expressed on the cell surface, known as mutant surface K-Ras antigen, as a target for ADCs, the inventors used rabbit anti-human K-Ras G12D antibody (GenTex, clone HL-10) and rabbit anti-human K-Ras G12V antibody (Cell Signaling Technology, clone D2H12), as well as tumor cell lines expressing the surface mutant K-Ras described in Example 2. Specifically, Panc-1 (pancreatic cancer with K-RAS G12D mutation), Capan-2 (pancreatic cancer with K-RAS G12V mutation), AsPC-1 (pancreatic cell line with K-RAS G12D mutation), LS180 (colon cancer cell line with K-RAS G12D mutation), SW480 (colon cancer cell line with K-RAS G12V mutation), Capan-2 (pancreatic cancer cell line with K-RAS G12V mutation), and two lung cancer cell lines (SK-LU-1 with K-RAS G12D mutation and NCI-H2444 with K-RAS G12V mutation). Serial dilutions of antibodies or unrelated rabbit IgG isotype control antibodies were pre-incubated with 4.5 nM FabZAP reagent and then added to the specified cell lines in the Fab-ZAP kit according to the manufacturer's instructions. Cells were seeded in a round-bottom 96-well plate according to the manufacturer's protocol, with a total volume of 100 μl per well (containing Fab-Zap and different concentrations of antibodies or their respective controls). After incubation at 37°C for 5 days in a 5% CO2 incubator, the percentage of viable cells was determined using the XTT assay according to the protocol described (Advanced Targeting Systems Inc.'s cytotoxicity assay protocol) compared to control wells using 4.5 nM FabZAP alone. Briefly, after the completion of incubation, 50 μl of XTT reagent was added to each well to a total volume of 150 μl. The plate was then incubated at 37°C for a minimum of 30 minutes, and the absorbance at 450 nm was read using a plate reader. This assay is based on detecting the number of remaining viable cells on the day of color development, measured by the cellular metabolism of colorimetric molecules in the colorimetric reagent.The average absorbance of the untreated wells is shown as 100%, and the three replicates in each treatment group are presented as percentages relative to the control. As shown in Figures 3 to 11 below, cytotoxicity data are analyzed by comparing the well readings of the treated wells (target antibody and Fab-ZAP) with the readings of the control (Fab-ZAP alone and saporin alone) and expressing the results as percentages. The data were analyzed using GraphPad Prism software, and survival % was graphed as a function of primary antibody concentration.

[0308] Saporin alone did not induce cell death at concentrations below 100 nM (Figure 3), and therefore, no toxicity was observed at the 4.5 nM concentration used in the ADC assay without a targeting agent (arrow in Figure 3). To determine whether mutant K-Ras-specific target antibodies could be used in ADCs in the presence of a 4.5 nM saporin-targeting reagent, which is normally nontoxic, Panc-1 (K-Ras G12D mutant) was incubated with various concentrations of rabbit anti-human G12D mutant K-Ras antibody conjugated to Fab-Zap reagent, and concentration-specific death was observed (Figure 4). Under the same conditions, rabbit IgG conjugated to Fab-Zap control did not result in death (Figure 4). In this K-Ras G12D mutant tumor, no cytotoxicity was observed when using an anti-G12V mutant K-Ras specific antibody conjugated to Fab-Zap, or when using an anti-G12D mutant K-Ras antibody alone (Figure 4). As mentioned above, saporin alone did not show cytotoxicity until a concentration of 100 nM was reached (Figure 3). Similar results were observed in other K-Ras G12D mutant cancers, such as the AsPC-1 pancreatic cancer cell line (Figure 5), the LS180 colorectal cancer cell line (Figure 6), and the SK-LU-1 lung cancer cell line (Figure 7). Similar results were observed with the use of K-Ras G12V-specific antibodies conjugated to Fab-Zap in G12V-mutated cancers such as Capan-2 pancreatic cancer (Figure 8), SW480 colorectal cancer (Figure 9), and NCI-H2444 lung cancer cell line (Figure 10). However, anti-K-Ras G12D mutation-specific antibodies did not induce ADC-induced cell death in these K-Ras G12V-expressing tumors. Furthermore, the use of MiaPaCa-2, a pancreatic cancer cell line expressing K-Ras G12C, did not induce ADC-induced cell death with K-Ras G12D-specific antibodies conjugated to Fab-Zap (Figure 11).

[0309] Another experiment using human anti-K-Ras antibody 1 showed similar results in K-Ras G12D mutant cancers of Panc-1 (Figure 12a) and ASPC-1 (Figure 12b). Similar results were observed in G12C mutant cancers, e.g., Mia-Paca-2 (Figure 12c), and wild-type cancers, e.g., MXPC3 (Figure 12d), but the anti-K-Ras G12D mutation-specific antibody did not induce ADC-induced cell death in these K-Ras G12V-expressing tumors. Saporin alone as a control showed killing in Panc-1, ASPC-1, Mia-Paca-2, and BXPC3 cell lines (Figure 12e). Finally, to demonstrate specificity, RASless MEF cells rescued with BRAF, (G12D)K-Ras 4B, K-Ras-4A, or K-Ras 4B were tested in a surrogate Fab-Zap assay using human anti-K-Ras antibody 1 as described above, except that human secondary Fab-Zap reagent (atsbio.com / products / it51 / ) was used. Isotype IgG + FabZap or human anti-K-Ras antibody 1 alone did not show increased cytotoxicity. Human anti-K-Ras antibody 1 + FabZap did not affect the survival of MEF-BRAF or MEF-K-Ras-4A, but it did cause cytotoxicity in MEF-(G12D)K-Ras 4B and MEF-K-Ras-4B (Figure 13).

[0310] In summary, our data suggest that using surface-bound antibodies specific to variant forms of the surface K-Ras antigen may provide a viable method for creating ADCs that are selective for tumor killing without off-target killing of cells that do not express the same variant form of the surface K-Ras antigen on the cell surface. Example 4: Development of a novel antibody against surface-expressed K-Ras

[0311] Antibodies specific to K-Ras bound to either GDP or GTP are developed by immunizing mice with GDP-bound K-Ras or GppNHp-bound K-Ras (GppNHp is a non-hydrolyzable analog of GTP). Prior to mouse immunization, either nucleotide is loaded into the K-Ras protein. As a further strategy for developing mutant-specific antibodies, additional mice are immunized with a peptide containing the G12D mutation and spanning positions 5-17 of K-Ras (Ac-KLVVVGADGVGKSC-amide) (SEQ ID NO: 13). To minimize the response binding to wild-type K-Ras, some mice are first subjected to subtractive immunization with wild-type K-Ras and treated with cyclophosphamide to kill B cells responsive to wild-type K-Ras. Then, all mice are intraperitoneally immunized with the protein or peptide mixed with a complete or incomplete Freund's adjuvant. Splenocytes and / or lymph nodes obtained from immunized mice are fused with NS1 myeloma cells to form hybridomas. Subsequent antibody clones are screened by ELISA for binding to GDP-bound wild-type K-Ras, GppNHp-bound wild-type K-RAS, GDP-bound (G12D)K-Ras, and GppNHp-bound (G12D)K-Ras, and by flow cytometry for binding to surface-expressed K-Ras on AsPC1 (G12D homozygous), SU.86.86 (G12D homozygous), and / or BxPC3 (K-Ras wild-type) cells. Preferred antibody clones recognize only the K-Ras antigen used to immunize mice. Antibody clones are screened again before fusion, after fusion, and after subsequent subcloning. Example 5: Panning of cell surface-expressed K-Ras using a human antibody library

[0312] Another method for developing antibodies specific to mutant K-Ras, including K-Ras with mutations such as G12D, G12V, G12C, G13D, and G12R, is to pan existing libraries of human polyclonal antibodies. Two existing libraries of human polyclonal antibodies were used, one containing antibody sequences from naive human donors and the other containing antibody sequences from human donors who were autoimmune patients. These antibodies were cloned, manipulated into single-stranded variable fragments, and introduced into phage display libraries that can be panned and screened to obtain mutant K-Ras specific antibodies that bind to surface K-Ras antigens expressed on the cell surface.

[0313] Several different strategies are used to select antibodies that recognize either GDP or GTP-bound mutant K-Ras. In all strategies, antibodies that bind to wild-type K-Ras protein bound to either of the nucleotides being screened are first removed from the library. The antibody library is then panned for antibodies that bind to mutant K-Ras bound to either GDP or GppNHp. After the initial removal / selection round, up to three further screening rounds may be used. Each subsequent screening round employs removal of antibodies that bind to wild-type K-Ras and selection of mutant K-Ras antibodies as proteins or as surface K-Ras antigens expressed on the cell surface. After the initial selection round, panning is performed in the presence of excess competing wild-type K-Ras protein or cells expressing wild-type K-Ras to further minimize the selection of phages that bind to wild-type K-Ras. To enrich antibodies that bind to specific K-Ras epitopes, some panning rounds involve elution with specific proteins containing the desired epitopes. These specific proteins include antibodies specific to mutant K-Ras such as R11.1.6 and K-Ras G12D. Example 6: Proof of concept for the development of CAR-T cells targeting K-Ras

[0314] The use of chimeric antigen receptor (CAR) T cells for selective tumor killing is an established method for targeting T cells to tumor cells expressing defined surface targets. To investigate the potential of surface K-Ras as a target for CAR T cells, human anti-K-Ras antibody 1 and rabbit anti-human K-Ras G12D antibody (HL-10 antibody) are manipulated to form single-chain antibody fragments. The single-chain antibody fragments contain an 18-amino acid "linker" of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 25) used to link the light and heavy chains. scFv is inserted into a second-generation CAR cassette containing a GM-CSF signaling peptide, a "hinge" region, CD28 transmembrane and costimulatory domains, and a CD3ζ activating domain. The resulting scFv-CAR gene is introduced into a lentiviral vector. Target T cells are then activated, transduced with the scFv-CAR gene, and cultured in large quantities.

[0315] Two measures of CAR-T cell activity are evaluated: cytokine secretion and cell killing. To measure cytokine secretion, expanded cultured anti-K-Ras CAR-T cells are incubated in a 1:1 ratio with cells expressing K-Ras on their surface. After 16 hours of incubation, the cell supernatant is removed. The amount of secreted IL-2 and IFN-γ in the supernatant is measured by ELISA. To measure cell killing, adherent target cells are grown to confluence, and then 10 5 Add individual CAR-T cells. Co-culture the cells for 2-3 days and measure the impedance of the cell layer over time. The total cell lysis rate is calculated from the change in impedance over the entire experimental period. Example 7: Isolation of cell samples for diagnostic testing of surface K-Ras expression in patient samples

[0316] Human tissue derived from the cancerous site, along with adjacent normal tissue, was preserved immediately after the surgical procedure. The tissue was processed immediately according to the following protocol to isolate single cells. Briefly, each tissue was cut into very small sections using a sterile blade. The tissue was then resuspended in RPMI1640 complete medium (containing 10% FBS, 1× penicillin-streptomycin) and dissociation was initiated using a MACS dissociation device (Milteny Biotech) with 2 cycles of 1 minute each. Next, the tissue was digested in digestion medium consisting of IMDM medium supplemented with collagenase type 2 (0.5 mg / ml) and DNase (0.5 ul / sample). The total volume of digestion medium used was 7 ml for each 1 mg tissue size. Next, the tissue immersed in the digestion medium was shaken at 850 rpm for 35 minutes at 37°C.

[0317] After digestion, the tissue was again subjected to a gentle MACS dissociation apparatus to dissociate the digested tissue and collect single cells. Next, the cells were washed twice with 1×PBS to remove digestive enzymes and DNase. The cells were passed through a 70 μM strainer and resuspended in RPMI complete medium. Example 8: Diagnostic test of surface K-Ras expression by flow cytometry

[0318] As described in Example 1, single-cell suspensions obtained from patients can be used for flow cytometry analysis. Flow cytometry analysis of surface K-Ras from primary human tissue was performed using human anti-K-Ras antibody 1 and human K-Ras antibody 2 (Figure 14). In this example, cells obtained from fibrous lung tissue showed increased K-Ras surface expression compared to cells from normal lung tissue. Example 9: Diagnostic test of surface K-Ras expression by Western blotting

[0319] After obtaining patient samples using the method of Example 1, surface K-Ras expression can be analyzed by Western blot analysis. Cell surface proteins can be labeled and then isolated using the Pierce® Cell Surface Biotinylation and Isolation Kit (ThermoFisher Scientific, catalog number: A44390). First, cells are labeled with EZ-Link Sulfo-NHS-SS-biotin, an amine-reactive biotinylation reagent capable of thiol cleavage. The cells are then lysed, and the labeled proteins can be captured with NeutrAvidin agarose. The flow-through can then be recovered from the agarose column. This corresponds to the non-biotinized fraction, which is intracellular protein after cell lysis. The NeutrAvidin agarose-bound biotinylated protein fraction can be eluted using a reducing buffer containing a protease inhibitor cocktail. Dithiothreitol (DTT) is used for elution to reduce the disulfide bond in the biotin label, resulting in the release of the bound protein without the biotin label.

[0320] The eluted surface biotinylated protein fraction and intracellular flow-through protein fraction can then be used for Western blotting with different K-Ras antibodies (see Example 2, Figure 2m). Proteins can be eluted in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, and a protease inhibitor cocktail. The procedures for gel electrophoresis and Western blotting are known to those skilled in the art. Example 9: Diagnostic test of surface K-Ras expression using electron microscopy

[0321] Viable Panc-1 cells can be pre-labeled with either an anti-KRAS antibody or an IgG control, and then stained with a gold-labeled secondary antibody. The cells are then post-fixed in 2% osmium tetroxide in Sorensen buffer for 1 hour, dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 90%, 100%), and embedded in Epon / Araldite resin. The samples are then stained with a pre-mixed solution of uranyl acetate and lead citrate and examined at 200V using an F20 electron microscope equipped with a field emission gun (FEG) electron source. Negative staining data is then acquired using a 4k×4k Ultra Scan CCD camera powered by a Gatan GIF Quantum with DualEELS and high-speed spectral imaging. Example 10: Diagnostic test of surface K-Ras expression using confocal microscopy

[0322] Surface KRAS identification by confocal microscopy was performed using a combination of anti-KRAS antibody and cell surface membrane dye using the CellBrite® Steady Membrane Staining Kit (Biotium Inc., Fremont, CA). Briefly, living cells were labeled with CellBrite® Steady 488 dye (green fluorescence). After labeling the cells with 1X dye in the cell culture medium, they were incubated in a 37°C incubator for 30 minutes. Subsequently, the cells were immediately washed twice with culture medium supplemented with 10% FBS to quench the unbound dye. Next, the cells were subjected to surface K-Ras staining with human anti-K-Ras antibody 1 for 30 minutes on ice, followed by secondary anti-human PE (red fluorescence) antibody staining, similarly on ice for 30 minutes. Up to this staining step, the cells were confirmed to be healthy and viable. Finally, the cells were washed, layered onto slides, and mounted with ProLong® Gold Antifade mounting solution containing DAPI (ThermoFisher Scientific, catalog number: P36931).

[0323] Images were acquired using a spinning disk confocal microscope (Zeiss LSM 880) equipped with Airyscan, and acquired in multiplex mode with ZEISS Airyscan 2 for efficient wide-field super-resolution imaging. Unless otherwise noted, the Z slice was set to 0.2 microns. For high-resolution imaging, a Plan-Apochromat 40x / 1.3 objective lens was used, or for confocal modality performance, a 63X 1.4NA Plan-Apochromat objective lens was used with a 568 nm excitation filter and a 570-615 nm emission filter. Confocal images were deconvolved using contrained iterative deconvolution routines (Zen software version 2.3, Carl Zeiss, Obeköchen, Germany).

[0324] Confocal microscopy images showed clear KRAS staining when stained with anti-KRAS antibody (Figure 2g, left panel), but no red staining was observed with the control antibody (right panel). Membrane dyes and DAPI functioned as cellular markers to distinguish between surface and intracellular regions. Example 11: Increase in surface K-Ras availability by combination of treatments

[0325] The inventors investigated the effects on K-Ras surface expression after treating tumor cell lines with a chemotherapeutic agent (fluorouracil (5-FU)) or mutation-specific K-Ras small molecule inhibitors (G12D-specific MRTX1133, G12C-specific MRTX849).

[0326] For 5-FU treatment, 100,000 PANC-1 cells were seeded in 24-well replication plates. After growing the cells for 24 hours, 10 μM 5-FU was added to the culture medium. At 0, 72, and 120 hours, the cells were detached using TryPLE express and resuspended in PBS (pH 7.2) and a buffer containing 5% FCS. The cells were stained with human K-Ras antibody 1. All staining was performed in V-bottom 96-well plates on ice for 30 minutes at an antibody concentration of 10 μg / ml and a volume of 100 μg. Surface K-Ras staining was quantified by measuring the MFI of mock-treated or 5-FU-treated cells. The magnification change was calculated by normalizing against cells stained with an isotype control. As shown in Figure 15a, treatment with 5-FU increased the availability of surface K-Ras in Panc-1 cells, and the amount of available K-Ras increased with longer treatment duration. Treatment with 5-FU chemotherapy, and potentially other chemotherapy, increased the availability of surface K-Ras.

[0327] In treatment with mutation-specific K-Ras small molecule inhibitors, 100,000 PANC-1 cells were seeded in one set of 24-well replication plates, and 100,000 MiaPaca-2 cells were seeded in a second set of plates. After growing the cells for 24 hours, 10 nM MRTX1133 or 10 nM MRTX849 was added to the culture medium. At 0, 72, and 120 hours, the cells were detached using TryPLE express and resuspended in PBS (pH 7.2) and a buffer containing 5% FCS. The cells were stained with human K-Ras antibody 1. All staining was performed in V-bottom 96-well plates on ice for 30 minutes at an antibody concentration of 10 ug / ml and a volume of 100 ul. Surface K-Ras staining was quantified by measuring the MFI of sham-treated cells or cells treated with either MRTX1133 or MRTX849. The magnification change was calculated by normalizing against cells stained with isotype controls. As shown in Figures 15b-15c, treatment with MRTX1133 increased the availability of surface K-Ras on PANC-1 cells but not on MiaPaca-2 cells. As shown in Figures 15a-15c, treatment with MRTX849 increased the availability of surface K-Ras on MiaPaca-2 cells but not on PANC-1 cells. This result indicates that treatment with mutation-specific small molecule inhibitors affects the availability of K-Ras on the cell surface only if the appropriate K-Ras mutation is present in the cell. Similar to treatment with 5-FU, the amount of available K-Ras increased with longer duration of treatment with the appropriate mutation-specific K-Ras small molecule inhibitor. In summary, several different methods increase the availability of surface K-Ras. Example 12a: Increased K-Ras surface availability due to K-Ras small molecule treatment enhances killing by surface K-Ras targeted antibody-drug conjugates (ADCs).

[0328] In this example, the inventors, as in Example 3, used a commercially available human Fab-ZAP assay (Advanced Targeting Systems Inc.) to test whether increased surface K-Ras availability by MRTX1133 enhances killing by surface K-Ras-targeted ADCs. The experiments described below utilized the Fab-ZAP human kit [IT-51, KIT-51] (atsbio.com / products / it51 / ), which consists of a chemical conjugate of a goat anti-rabbit monovalent antibody and a saporin, a ribosome-inactivating protein. The antibody used in this kit is an affinity-purified polyclonal antibody against both the heavy and light chains of rabbit IgG.

[0329] One experiment tested whether increased surface K-Ras availability by the small molecule inhibitor MRTX1133 enhanced killing by surface K-Ras-targeted ADCs. For a description of MRTX1133, see, for example, Wang et al., Journal of Medicinal Chemistry 2022 65(4), 3123-3133. DOI:10.1021 / acs.jmedchem.1c01688. PANC-1 cells were seeded at 100,000 cells / well in 24-well replication plates. After growing the cells for 24 hours, 10 nM MRTX1133 was added to the culture medium. At 0, 72, and 120 hours, serial dilutions of the antibody, pre-incubated with 4.5 nM FabZAP reagent, were added to the PANC-1 cell line in a Fab-Zap kit according to the manufacturer's instructions. After incubation at 37°C for 5 days in a 5% CO2 incubator, the percentage of viable cells was determined using the XTT assay according to the protocol described (Advanced Targeting Systems Inc.'s cytotoxicity assay protocol) compared to control wells treated with 4.5 nM FabZAP alone. Briefly, after incubation was complete, 50 μl of XTT reagent was added to each well to a total volume of 150 μl. The plate was then incubated at 37°C for at least 30 minutes, and the absorbance at 450 nm was read using a plate reader. This assay is based on detecting the number of viable cells remaining on the day of color development, measured by the cellular metabolism of colorimetric molecules in the colorimetric reagent. The mean absorbance of the untreated well is shown as 100%, and the three replicates in each treatment group are presented as percentages relative to the control. As shown in Figure 16a, cytotoxicity data are analyzed by comparing the well readings of the treated wells with the readings of the control (IgG alone) and expressing them as percentages. The data was analyzed using GraphPad Prism software, and the survival percentage was graphed.

[0330] Saporin alone does not cause cell death at concentrations below 100 nM (Figure 3), therefore no toxicity is observed at the 4.5 nM concentration used in ADC assays without a targeting agent. For comparison, the maximum cell killing obtained with 100 µM free saporin is indicated by a dotted line. Both MRTX1133 alone, and the anti-K-Ras antibody alone with Fab-ZAP, were capable of killing cells (Figure 16a). When MRTX1133 was combined with the anti-Kras antibody plus Fab-ZAP, the effect was greater than either agent alone (Figure 16a). This indicates that the combination of a small molecule inhibitor and a surface K-Ras targeting ADC increases tumor cell killing. Example 12b: Chemotherapeutic agents enhance killing by surface K-Ras targeted antibody drug conjugates (ADC)

[0331] In this example, we used the commercially available Fab-ZAP assay (Advanced Targeting Systems Inc.) described in Example 12a to test whether increased surface K-Ras availability by RMC-6236 enhances killing by surface K-Ras targeted ADC. One combination was an ADC of human anti-K-Ras antibody 1 with the chemotherapeutic agent fluorouracil (5-FU), and a second combination was an ADC of human anti-K-Ras antibody 1 with the small molecule inhibitor designated RMC-6236. For a description of RMC-6236, see, for example, Arbour K et al., Ann Oncol 2023;34:S458.

[0332] PANC-1 cells were seeded at 10,000 cells per well in 96-well replicate plates. After allowing the cells to grow for 24 hours, they were treated with 30 nM RMC-6236, 10 µM 5-FU, or a combination of RMC-6236 and 5-FU. Twenty-four hours after seeding PANC-1 cells, treatment groups were treated with a fixed concentration 10 pre-incubated with a fixed concentration of 4.5 nM FabZAP reagent, according to the manufacturer's instructions for the Fab-Zap kit. -8 M human K-Ras antibody 1 or fixed concentration 10 -8Each cell was administered one of the M-grade IgGs. After incubation at 37°C for 5 days in a 5% CO2 incubator, the percentage of viable cells surviving the treatment was determined using the XTT assay according to the protocol described (Advanced Targeting Systems Inc.'s cytotoxicity assay protocol), compared to a control well treated with Fab-ZAP alone. Briefly, after incubation was complete, 50 μl of XTT reagent was added to each well to a total volume of 150 μl. The plate was then incubated at 37°C for at least 30 minutes, and the absorbance at 450 nm was read using a plate reader. This assay is based on detecting the number of surviving cells remaining on the day of color development, measured by the cellular metabolism of colorimetric molecules in the colorimetric reagent. The mean absorbance of the control (PANC-1 cells treated with IgG) is shown as 100%, and the three replicates in each treatment group are presented as percentages relative to the control. Cytotoxicity data were analyzed by comparing the well readings of the treated wells with the readings of the control (IgG monotherapy) and expressing the results as a percentage. The data was analyzed using GraphPad Prism software, and survival percentages were graphed.

[0333] For comparison, 10 -7 The dotted line indicates the maximum cell killing with free saponins of M. Treatment with human K-RAS antibody 1 + FabZap, RMC-6236, and a combination of human K-Ras antibody 1 + FabZap and RMC-6236 killed PANC-1 cells to a degree equivalent to the maximum killing achieved with high concentrations of free saponins (Figure 16B). However, treatment of PANC-1 cells with RMC-6236 and human K-Ras antibody 1 + FabZap, or with 5-FU and human K-Ras antibody 1 + FabZap, was more effective than either drug alone (Figure 16B).

[0334] In summary, these results indicate that the combination of small molecule inhibitors and chemotherapeutic agents with surface K-Ras-targeted ADCs increased tumor cell killing. Example 13: Surface K-Ras-targeted bispecific antibody increases T cell killing in surface K-Ras-expressing cells.

[0335] To demonstrate whether a bispecific antibody is sufficient to induce T cell-mediated killing of surface K-Ras-expressing cells, the inventors designed an asymmetric bispecific antibody (anti-K-Ras antibody 1 × CD3). As shown in Figure 18, the bispecific antibody consists of the Fab-Fc region of human anti-K-Ras antibody 1 (anti-K-Ras Fab HC-Fc / anti-K-Ras Fab LC) (Fc inactivated by N297G and T366S / L368A / Y407V) and the scFv of a CD3-specific antibody (anti-K-Ras Fab) linked to the inactivated Fc (N297G and T366W). HC-FC-EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 26) and anti-K-Ras Fab LC-QSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (Sequence ID 27) and CD3 OKT3scFv-FC-DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMIWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVS SVEGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKG GGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28)) was also designed using the Fab region of the human anti-RSV antibody and the same CD3 OKT3 scFv-Fc domain (RSV Fab) disclosed above.HC-Fc-QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFFYFDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 29) and RSV It was prepared using Fab LC-DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 30)).

[0336] To test target cell killing, we used Panc-1 luciferase-expressing cell lines (Panc-1L). These cells constitutively express luciferase, and luciferase activity can be used as a surrogate for the number of viable target cells. Importantly, T cells co-cultured with target cells do not express luciferase and therefore do not contribute to luciferase signaling. Thus, luciferase expression becomes a measure of target cell viability only. Briefly, 50,000 Panc-1L cells were seeded at a volume of 100 μl in each well of a 96-well plate optical polymerase black plate (ThermoFisher #165305). T cells were isolated from human PBMCs and added at a ratio of 1:1 (50,000 cells) or 2:1 (100,000 cells) at a volume of 100 μl, so that the total volume of the wells was 200 μl. Each bispecific agent (human anti-K-Ras antibody 1×CD3 bispecific agent and control IgG(RSV)×CD3 bispecific agent) was divided into 10 -7 ~10 -10 The medium was added to the wells within the M concentration range. Each group was seeded into five replication wells. One group received no T cells (Panc-1L alone), and one group received T cells alone without Panc-1L as a control. The cells were cultured on plates in a 5% CO2 incubator at 37°C for 72 hours and then analyzed. On the day of analysis, 100 μl of medium was removed from all wells, and 100 μl of reagent D-luciferin (1× concentration) was added. The wells were thoroughly mixed, and after 5 minutes, luminescence was measured by reading the plate with a Tecan Spark Multimode Microplate reader. The luminescence of each well was recorded. The intensity was proportional to the number of viable cells in each well. The inventors used GraphPad prism for statistical analysis and data display.

[0337] As shown in Figure 17, co-culture of targeted Panc-1L and human T cells with human anti-K-Ras antibody 1×CD3 bispecificity results in a concentration-dependent decrease in viable cells, as measured by luciferase expression. In contrast, co-culture of targeted Panc-1L and human T cells with control IgG×CD3 bispecificity did not result in any change in cell viability compared to co-culture of targeted Panc-1L and human T cells without the bispecific antibody. The highest number of viable cells was observed with Panc-1L cells alone, which, as expected, indicates that human T cells kill target cells at low levels when co-cultured. In summary, this indicates that incubation of target cells and T cells with surface K-Ras targeted bispecificity leads to a dose-dependent increase in target cell killing. Example 13: Evaluation of antibody binding to K-Ras antigen

[0338] To demonstrate antibody binding to K-Ras, an enzyme-linked immunosorbent assay (ELISA) was used. To capture biotinylated proteins, plates were coated overnight at 4°C with 1 ug / ml neutraavidin (neutralite avidin or deglycosylated chicken triavidin). Plates were blocked with 1% BSA / PBS for 60 minutes. Plates were loaded with 1 ug / ml biotinylated (G12D)K-Ras-GDP, (G12D)K-Ras-GppNHp, K-Ras-GDP, K-Ras-GppNHp, or BSA for 60 minutes. Purified antibodies were tested for K-Ras binding at concentrations of 1–0.001 ug / ml for 60 minutes. Antibody detection was performed using anti-human IgG-HRP and TMB staining. Plates were washed three times with PBST during incubation. The mean values ​​of the two sets of measurements are shown along with their standard deviations.

[0339] Both human anti-K-Ras antibodies 1 and 2 bound to wild-type K-Ras loaded with GDP or GppNHp-loaded nucleotides at low levels (Figures 19c-19d). Similarly, human anti-K-Ras antibody 2 bound to (G12D)K-Ras at low levels regardless of the loaded nucleotide. In contrast, human anti-K-Ras antibody 1 bound to (G12D)K-Ras (loaded with either nucleotide) at high levels, indicating preferential binding of human anti-K-Ras antibody 1 to G12D mutant K-Ras (Figures 19a-19d). Example 14: Validation of antibody-drug conjugates (ADCs) for selective killing of cancer cell lines using mutation-specific antibodies.

[0340] To investigate the potential of using mutant K-Ras expressed on the cell surface as an ADC target, the inventors used human anti-K-Ras antibody 1 (Antibody 1) and human anti-K-Ras antibody 2 (Antibody 2), as well as tumor cell lines expressing surface mutant K-Ras. Specifically, Panc-1 K-Ras in the Fab-ZAP assay G12D (Pancreatic cancer with K-RAS G12D mutation), LoVo K-Ras G13D MiaPaca-2 K-Ras G12C (Same as Mia-Paca-2), A549 K-Ras G12S CFPAC-1 K-Ras G12V BXPC3 K-Ras WT HEIC6 K-Ras WT (Non-malignant cell lines), and HUVEC K-Ras WT (Non-malignant cell line). An antibody specific to RSV was used as a negative control for K-Ras binding.

[0341] Serial dilutions of the antibody were pre-incubated with 4.5 nM FabZAP reagent and then added to the specified cell line in the Fab-ZAP kit according to the manufacturer's instructions. Cells were seeded into a round-bottom 96-well plate according to the manufacturer's protocol, with a total volume of 100 μl per well (containing FabZap and different concentrations of antibody or their respective controls). After incubation at 37°C for 5 days in a 5% CO2 incubator, the percentage of viable cells was determined using the XTT assay according to the protocol described (Advanced Targeting Systems Inc.'s cytotoxicity assay protocol) compared to a control well using 4.5 nM FabZAP alone. Briefly, after incubation was complete, 50 μl of XTT reagent was added to each well to a total volume of 150 μl. The plate was then incubated at 37°C for a minimum of 30 minutes, and the absorbance at 450 nm was read using a plate reader. This assay is based on detecting the number of remaining viable cells on the day of color development, measured by the cellular metabolism of colorimetric molecules in the colorimetric reagent. The average absorbance of the untreated wells is shown as 100%, and the three replicates in each treatment group are presented as percentages relative to the control. Example 15: Evaluation of in vivo staining of tumor tissue with anti-K-Ras antibody.

[0342] To demonstrate specific recognition of surface K-Ras in in vivo tumors, tumor fragments from patient-derived xenograft (PDX) models PA0787 and PA1252 (both derived from resected human pancreatic cancer expressing the G12D Kras mutation) (Crown Bio) were inoculated into nude Balb / c mice lacking T cells and B cells, enabling human tumor growth and engraftment. Tumor fragments (2-3 mm in diameter) from primary human tumor xenograft models were subcutaneously inoculated into the upper right / lower flank of each mouse to induce tumor formation. The tumors grew to approximately 500 mm. 3 Once the condition was reached, the mice were injected with antibodies as described below.

[0343] The tumor is 500 mm 3At this point, mice were randomized and injected via tail vein at a dose of 10 ul / g with either human anti-K-Ras antibody 1 or isotype control antibody at a dose of 10 mg / kg. After 30 minutes, the mice were euthanized, tumors were collected, and frozen in optimal cutting temperature (OCT) freezing medium for immunohistochemical evaluation.

[0344] Tumors were sectioned into 5 μm sections using a standard dermatome and processed for immunohistochemistry. Briefly, frozen sections were fixed in cold acetone / methanol (1:1 dilution) for 5 minutes. After washing the sections in PBS for 15 minutes, the sections were blocked in 10% normal goat serum at room temperature for 45 minutes and incubated with secondary goat anti-human IgG (Alexa fluro 488 Invitrogen A-11013) at room temperature for 45 minutes. ProLong® Gold anti-fading reagent containing DAPI (catalog P36931 ThermoFisher Scientific) was added, and coverslides were placed on top. Images were acquired using a Leica Microsystems DM6B-Z.

[0345] As shown in Figures 21a and 21b, both PDX models PA0787 and PA1252 showed better tumor staining with human anti-Kras antibody 1 compared to staining with isotype control antibodies. Therefore, in vivo treatment with anti-K-Ras antibody specifically stained G12D mutant K-Ras-containing pancreatic cancer. Example 16: Validation of antibody-drug conjugates (ADCs) for in vivo selective killing of cancer cell lines using mutation-specific antibodies.

[0346] To test the ability of anti-K-Ras antibodies to control K-Ras mutant tumors in vivo, anti-K-Ras antibody 1 and isotype control antibodies were conjugated to deruxtecan (DXd). DXd is a protease-cleavable linker conjugated to a derivative of exatecan that acts as a topoisomerase I inhibitor. DXd targeted to cancer cells by antibodies as an ADC is used in the FDA-approved ENHERTU® (fam-trastuzumab-deruxtecan-nxki), which is a conjugation of anti-Her-2 antibody and deruxtecan.

[0347] DXd was conjugated to the interchain cysteine ​​of the antibody by stochastic maleimide conjugation. The antibody was first reduced using 10 × molar equivalents of 10 mM TCEP with shaking at 37°C for 90 minutes. The reduced antibody was then conjugated with 10 × molar equivalents of 10 mM DXd at room temperature for 2 hours. The conjugated antibody was evaluated for drug-to-antibody ratio (DAR) by reducing 10 μg of crude conjugate with DTT at 37°C for 30 minutes. The final DAR of the conjugated antibody was approximately 8. The polymeric state of the conjugated antibody was evaluated by size exclusion chromatography and confirmed to be monomeric. The conjugated antibody was purified from unconjugated DXd by dialyzing with PBS (pH 5.6) for 48 hours using a 10K molecular weight cutoff (MWCO) membrane.

[0348] Next, we used a pancreatic cancer cell line-derived xenograft model in which the human tumor cell line PANC-1 was transplanted into Balb / c nude mice. Nude mice are naturally occurring mutant mice (nude) lacking the thymus and hair. The absence of the thymus in nude mice results in a T cell deficiency, which leads to immunodeficiency in nude mice, making them able to accept foreign tissues such as human tumor cells. 5 × 10⁶ mice 6 Individual Panc-1 cells were subcutaneously injected. The average tumor size was approximately 200 mm. 3Randomization was initiated when the target number of mice was reached. Sixty mice were enrolled in the study. All animals were randomly assigned to one of six study groups of 10 mice each. Randomization was performed using the "matched distribution" method. The experiment was conducted as outlined in Table 5. [Table 5] * ROA = Route of administration, iv = Intravenous, ip = Peripheral cavity

[0349] As shown in Figures 22a–22d, anti-Kras antibody 1 conjugated to DXd ADC slowed tumor growth more effectively than mice treated with isotype-ADC and saline controls (group 1 vs. 2 vs. 3 vs. 6) (Figures 22c–22d). Surprisingly, even mice treated with anti-K-Ras antibody 1 showed suppression of tumor growth more effectively than isotype-treated mice (Figures 22a–22b). Gemcitabine treatment slowed tumor growth more effectively than either ADC or antibody treatment, but gemcitabine-treated mice exhibited distress based on weight loss. On the other hand, ADC or antibody-treated mice gained weight at a similar rate to saline-treated controls (Figures 22a–22d). Example 17. Use of HDX-MS to determine antibody sites that interact with K-Ras.

[0350] Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to determine the K-Ras region(s) that interacts with human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 and is therefore accessible on the cell surface. The experiment was performed using the Trajan Leap robotic platform and Waters Cyclic IMS MS. The experiment was divided into two phases: peptide mapping and HDX. In peptide mapping, only free proteins were used to identify peptides for HDX analysis, while HDX required both free proteins and mAb-binding proteins, and differences in deuterium incorporation at each amino acid residue were compared.

[0351] For peptide mapping, the K-Ras G12D sample was diluted to 6 μM in 20 mM phosphate buffer and 150 mM NaCl (pH 7.4) ("H2O-based buffer"), and 6.5 μL of the protein solution was mixed with 43.5 μL of H2O-based buffer. At the end of the reaction, 45 μL of the sample was mixed with 45 μL of pre-dispensed 100 mM phosphate buffer (pH 2.4). 80 μL of the quenched sample was injected into the sample loop, and the quenched protein was digested using a pepsin column (Affipro) at a flow rate of 0.200 mL / min for 210 seconds, followed by trapping and desalting, and then separated through an analytical C18 column at 0.035 mL / min. The K-Ras G12D sample contained isolated polypeptides. The polypeptide had an N-terminal protein tag (MGSHHHHHHHHGSENLYFQGGS-sequence number 293) and a C-terminal protein tag (KLLHHILDAQKMVWNH-sequence number 294) linked to amino acid residues 2-189 of K-Ras G12D, which has sequence number 295.

[0352] In the HDX experiment, 6 μM K-Ras G12D was analyzed in both its free and monoclonal antibody (mAb)-bound states. For the bound state analysis, 12 μM intact mAbs of each type were incubated with K-Ras G12D. The same autosampler volume as in the peptide mapping experiment was used, but the H2O buffer was replaced with D2O buffer (20 mM phosphate buffer, 150 mM NaCl, pD 7.4). Three different labeling time points (2 min, 10 min, and 60 min) were used for both the free and mAb-bound states. Triple samples were collected at all time points. The same flow rate was used for liquid chromatography. Peptide libraries were prepared using PLGS, and deuterium uptake analysis was performed using DynamX 3.0.

[0353] Before switching to deuterium-containing buffer, K-Ras G12D / antibody conjugates were first prepared using human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 in 20 mM phosphate buffer and 150 nM NaCl (pH 7.4). The K-Ras G12D / antibody conjugates were exposed to deuterium for 2 minutes, 10 minutes, and 30 minutes, followed by deuterium exchange quenching. The labeled K-Ras G12D / antibody conjugates were then digested and analyzed by bottom-up mass spectrometry.

[0354] As shown in Table 6 for human anti-K-Ras antibody 1 and Table 7 for human anti-K-Ras antibody 2, HDX-MS analysis of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 showed a significant decrease in deuterium uptake in several portions of KRas G12D. Very similar regions were identified as potential interaction sites between human anti-K-Ras antibody 1 and K-Ras, or between human anti-K-Ras antibody 2 and K-Ras. The decrease in deuterium uptake in these regions suggested that one or more of these regions were directly bound by the antibody, and therefore accessible when KRas is found on the cell surface. [Table 6] [Table 7]

[0355] The HDX-MS-identified regions of K-Ras G12D that interact with either human anti-K-Ras 1 or human anti-K-Ras 2 include the sequences QLIQNHFVDE, SAMRDQY, AINNTKSFED, and KTRQGVDDAF.

[0356] To identify the accessible region of KRas G12D on the cell surface, similar experiments were completed using the K-Ras G12D / peptide R11.1.6 complex. As described in Example 1, R11.1.6 is a small cell membrane-permeable peptide that can bind to surface K-Ras. The crystal structure of KRas G12D (GppNHp) interacting with peptide R11.1.6 was downloaded from PDB(5UFQ). To identify high-affinity non-covalent inhibitors against K-Ras oncogenic mutations, the crystal structure was elucidated by Kauke et al. Sci Rep. 2017 Jul 19;7(1):5831.doi:10.1038 / s41598-017-05889-7. The interaction residues between K-Ras G12D (chain A) and R11.1.6 (chain C) were determined using PyMol and the InterfaceResidues.py script (pymolwiki.org / index.php / InterfaceResidues). The K-Ras G12D residues involved in the interaction with R11.1.6 include 3 GLU, 5 LYS, 6 LEU, 7 VAL, 36 ILE, 37 GLU, 38 ASP, 39 SER, 40 TYR, 41 ARG, 54 ASP, 56 LEU, 62 GLU, 63 GLU, 64 TYR, 66 ALA, 67 MET, 70 GLN, 71 TYR, 74 THR, and 75 GLY. This suggests that the K-Ras G12D region, including residues 3–7, 36–41, 54–56, and 62–74, is accessible on the cell surface for interaction with the antibody.

[0357] The foregoing descriptions of specific embodiments of this disclosure are presented for illustrative and explanatory purposes only. These exemplary embodiments have been selected and described to best illustrate the principles and practical applications of the present invention, thereby enabling those skilled in the art to best utilize the subject matter and various embodiments by making modifications suitable for their specific intended use. While some features may be described for specific embodiments and not for all other embodiments, it should be understood that such features are not necessarily limited to the specific embodiments described and may apply to other embodiments as those skilled in the art may intend based on this disclosure as a whole.

Claims

1. A composition comprising a binder-therapeutic agent complex containing a binder linked to a therapeutic agent, wherein the binder specifically binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, and neither the composition nor the binder-therapeutic agent complex contains intracellular delivery compounds.

2. The composition according to claim 1, wherein the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

3. The composition according to claim 1 or 2, wherein the therapeutic agent is selected from the group consisting of a cytotoxic agent, a cell proliferation inhibitor, a toxin, or a radionuclide.

4. The composition according to claim 1 or 2, wherein the therapeutic agent is selected from the group consisting of DNA damaging agents (alkylating agents), antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, and microtubule disruptors.

5. The composition according to claim 1 or 2, wherein the therapeutic agent is selected from the group consisting of calicheamicin, saporin, mytansinoid, auristatin, ridamycin, methotrexate, vinblastine, vincristine, pyrrolobenzodiazepine and other benzodiazepine derivatives, duocalmycin, tubulisin, α-amanitin or bougain protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxin, carboplatin, spliceostatin C, docetaxel, tylanstatin A, or any combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the binder is linked to the therapeutic agent by a linker selected from the group consisting of a maleimidocaproyl linker, a peptide-based linker (including, but not limited to, a valine-citrulline linker), a β-glucuronide linker, an SMCC linker, a disulfide linker, or an acid-sensitive linker.

7. A composition according to any one of claims 1 to 6, for use in treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or in killing cancer cells, wherein the subject has not been administered an intracellular delivery compound, or the composition is not intended for administration to the subject in combination with an intracellular delivery compound, or is not formulated for administration to the subject in combination with an intracellular delivery compound, and the cancer cells express a surface K-Ras antigen on the outer surface of the cancer cells.

8. The composition for use according to claim 7, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

9. A composition for use according to claim 7 or 8, formulated for administration by intravenous or subcutaneous injection.

10. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain contains a binder, and the binder specifically binds to a surface K-Ras antigen expressed on the outer surface of cancer cells.

11. The chimeric antigen receptor according to claim 10, wherein the transmembrane domain is selected from the group consisting of CD3-zeta, CD28, CDE28a, CD4, or a combination thereof.

12. The chimeric antigen receptor according to claim 10 or 11, wherein the intracellular domain is selected from the group consisting of CD28, CD27, 4-1BB, OX40, and / or ICOS.

13. The chimeric antigen receptor according to any one of claims 10 to 12, wherein the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, and the surface K-Ras antigen comprises one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

14. The chimeric antigen receptor according to any one of claims 10 to 13, wherein the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:

2.

15. The composition according to any one of claims 1 to 6, wherein the surface K-Ras antigen has more than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO:

2.

16. An immune cell expressing the chimeric antigen receptor according to any one of claims 10 to 14.

17. The immune cells according to claim 14, selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof.

18. An immune cell according to claim 16 or 17 for use in treating cancer in an individual, wherein the cancer comprises cancer cells expressing a surface K-Ras antigen on the outer surface of the cancer cells.

19. The immune cells for use according to claim 18, comprising cells derived from the individual having the cancer.

20. Immune cells for use according to claim 18 or 19, selected from the group consisting of T cells, NK cells, dendritic cells, or mixtures thereof.

21. The immune cells for use according to any one of claims 18 to 20, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

22. The composition according to any one of claims 1 to 6, wherein the therapeutic agent is therapeutically effective in inhibiting the growth or proliferation of the cancer cells, or, if not, is cytotoxic to the cancer cells.

23. The composition for use according to any one of claims 7 to 9, wherein the subject is administered an additional therapeutic agent, or the composition is intended for co-administration with an additional therapeutic agent, and the additional therapeutic agent is separate from the therapeutic agent present in the composition.

24. The composition for use according to claim 23, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor.

25. The composition for use according to claim 24, wherein the K-Ras small molecule inhibitor specifically inhibits the activity of a variant of K-Ras present in the surface K-Ras antigen.

26. The composition for use according to claim 24, wherein the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

27. The composition for use according to any one of claims 23 to 26, wherein the additional therapeutic agent is administered to the subject one to fourteen days prior to the administration of the composition.

28. The composition for use according to any one of claims 23 to 26, wherein the additional therapeutic agent is administered to the subject three to seven days prior to the administration of the composition.

29. Immune cells for use according to any one of claims 18 to 21, wherein the subject is being administered an additional therapeutic agent, or the immune cells are intended for co-administration with an additional therapeutic agent.

30. The immune cells for use according to claim 29, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor.

31. The K-Ras small molecule inhibitor specifically inhibits the activity of a form of K-Ras having a mutation present in the surface K-Ras antigen, according to claim 30, for use in immune cells.

32. The immune cells for use according to claim 30, wherein the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

33. The immune cells for use according to claim 32, wherein the surface K-Ras antigen comprises the G12D mutation.

34. Immune cells for use according to any one of claims 29 to 32, wherein the therapeutic agent is administered to the subject one to fourteen days prior to the administration of the immune cells.

35. Immune cells for use according to any one of claims 29 to 32, wherein the therapeutic agent is administered to the subject three to seven days prior to the administration of the immune cells.

36. A bispecific antibody comprising a first binding domain linked to a second binding domain, A bispecific antibody in which the first binding domain selectively binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, and the second binding domain selectively binds to an antigen expressed on the surface of immune effector cells.

37. The bispecific antibody according to claim 36, wherein the first binding domain includes a light chain variable region and a heavy chain variable region.

38. The bispecific antibody according to claim 36, wherein the first binding domain comprises a light chain variable region and a light chain constant region, and a heavy chain variable region and a heavy chain constant region.

39. The bispecific antibody according to any one of claims 36 to 38, wherein the second binding domain includes a light chain variable region and a heavy chain variable region.

40. The bispecific antibody according to any one of claims 36 to 38, wherein the second binding domain comprises a light chain variable region and a light chain constant region, and a heavy chain variable region and a heavy chain constant region.

41. The bispecific antibody according to any one of claims 36 to 40, wherein the surface K-Ras antigen has at least 60% homology to SEQ ID NO: 1 or SEQ ID NO:

2.

42. The bispecific antibody according to any one of claims 36 to 40, wherein the first binding domain selectively binds to a surface K-Ras antigen having one of the following mutations based on the amino acid sequence described in SEQ ID NO: 1, 12D, 12C, 12V, 12R, 13D, Q61H, and Q61L.

43. The bispecific antibody according to any one of claims 36 to 42, wherein the antigen expressed on the surface of immune effector cells is selected from the group consisting of TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3ε, CD3δ, CD3ζ, CD137, CD16, and CD64.

44. A bispecific antibody according to any one of claims 36 to 43, for use in treating, inhibiting, or reducing the proliferation of cancer cells in a target, or in killing cancer cells, wherein the cancer cells express a surface K-Ras antigen on the outer surface of the cancer cells.

45. The bispecific antibody for use according to claim 44, wherein the cancer cells are selected from the group consisting of pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

46. A bispecific antibody for use according to claim 44 or 45, formulated for administration by intravenous or subcutaneous injection.

47. A bispecific antibody for use according to any one of claims 44 to 46, wherein the surface K-Ras antigen has mutations in any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 12 (including, but not limited to, G12A, G12D, G12C, G12V, G12R), 13 (including, but not limited to, G13D), and 61 (including, but not limited to, Q61H, Q61L), being either a full-length or cleaved form of K-Ras.

Images