Compositions and methods for treating and diagnosing cancers associated with surface K-Ras

Abstract

The present disclosure describes compositions and methods for the treatment and diagnosis of 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

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 533,024, filed August 16, 2023, and U.S. Provisional Application No. 63 / 613,718, filed December 21, 2023.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on August 14, 2024, named "93700-411218-SL.xml", and has a size of 291,716 bytes.

[0005] Invention Field

[0006] This invention relates to compositions for treating and diagnosing cancers associated with the expression of surface K-Ras antigens (including mutated surface K-Ras antigens), and to compositions in methods for treating and diagnosing cancers associated with the expression of surface K-Ras antigens (including mutated surface K-Ras antigens). The invention also relates to antibodies, bispecific antibodies, immune cells having chimeric antigen receptors, and antibody-drug conjugates for treating and diagnosing cancers associated with the expression of surface K-Ras antigens (including mutated surface K-Ras antigens). Background of the Invention

[0008] While there have been extensive experimental and clinical efforts to target Kirsten Rat Sarcoma Viral Oncogene Homolog (K-Ras or KRAS) using mutation-specific covalent small molecule inhibitors (Punekar et al., Nat. Rev. Clin. Oncol. 2022 Oct;19 (10):637-655), there is only limited experimental data describing protein-based technologies that target this oncogenic pathway. This is largely due to the difficulty of delivering protein therapeutics across the cell membrane, where mutated K-Ras is thought to activate downstream signaling pathways in an unregulated manner (PMID: 21924373). Given the well-established dogma that K-Ras is localized in intracellular domains (on the inner leaflet of the cell membrane or in intracellular organelles) and functions exclusively therein (see, e.g., Hancock, Nat Rev Mol Cell Biol. 2003 May;4(5):373-84. doi: 10.1038 / nrm1105. PMID: 12728271.), extracellular surface-presented therapies and diagnostics (e.g., antibody drug conjugates (ADCs) and chimeric antigen receptor T cells (CAR-T cells)) based on mutated K-Ras proteins (other than those targeting small K-Ras peptides presented in HLA molecules) have not been developed in cancers associated with K-Ras expression.

[0009] SUMMARY

[0010] This document discloses therapeutic and diagnostic methods based on extracellular surface presentation of surface K-Ras antigens (including mutated surface K-Ras antigens) and compositions for said methods. For example, this document discloses compositions comprising a binder-therapeutic agent complex, said binder-therapeutic agent complex comprising a binder linked to a therapeutic agent, wherein the binder specifically binds to surface K-Ras antigens expressed on the outer surface of cancer cells, and wherein neither the composition nor the binder-therapeutic agent complex contains an intracellular delivery compound. On one hand, the binder is an antibody or antibody fragment. On the other hand, the binder is an antibody or antibody fragment that selectively binds to surface K-Ras antigens, wherein the surface K-Ras antigen contains a mutation at residues 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. On another hand, 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 G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On another hand, 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 G12D mutation as shown in SEQ ID NO: 295. On yet another hand, 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 G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, 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 G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the binder is an antibody fragment.

[0011] On one hand, the binder is a peptide or protein. On the other hand, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 295. On the other hand, the binder is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the binder is a peptide or protein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the binder is a peptide or protein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the binder is an antibody described in Table 1 or Table 2, or an antibody selected by the method shown in Example 4 or Example 5.

[0012] On the one hand, therapeutic agents are selected from cytotoxic agents, cell growth inhibitors, toxins, or radionuclides. On the other hand, therapeutic agents are selected from DNA damaging agents (alkylating agents), antimetabolites, topoisomerase inhibitors, mitosis inhibitors, antitumor antibiotics, and microtubule disruptors. On the one hand, the therapeutic agent is selected from calicheamicin, saporin, maytansinoid, auristatin, lidamycin, methotrexate, vinblastine, vincristine, pyrrolobenzodiazepines and other benzodiazepine derivatives, duocarmycins, tubulolysins, alpha-amanitin or bouganin protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxins, carboplatin, spliceostatin C, docetaxel, thailanstatin A, or any combination thereof.

[0013] On one hand, the binder is linked to the therapeutic agent via a linker selected from the following: maleimide hexanoyl linker, peptide-based linkers (including but not limited to valine-citrulline linkers), β-glucuronide linkers, 4-( N -maleimide-methyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linker, disulfide linker, or acid-sensitive linker. On one hand, the therapeutic agent is a radionuclide. On the other hand, the radionuclide is a radionuclide that emits β-particles or α-particles. On one hand, the radionuclide is selected from the following radionuclides that emit α-particles: astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227. On one hand, the radionuclide is a radionuclide that emits β-particles. On one hand, the radionuclide that emits β-particles is selected from iodine-131, rhenium-186, yttrium-90, samarium-153, and lutetium-177.

[0014] This article also discloses compositions for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells in a subject, wherein the subject has not been administered an intracellular delivery compound, or wherein the composition is not administered to the subject in conjunction with an intracellular delivery compound, or the composition is not formulated for administration to the subject in conjunction with an intracellular delivery compound, and wherein the cancer cells express surface K-Ras antigen on their outer surface. On one hand, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, cholangial cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. On the other hand, the composition is formulated for administration by intravenous or subcutaneous injection.

[0015] This document also discloses methods for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells in a subject, said methods comprising administering to the subject a therapeutically effective amount of any of the compositions described herein, wherein the subject is not administered an intracellular delivery compound in conjunction with the composition, and wherein the cancer cells express surface K-Ras antigen on their outer surface. On one hand, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. On the other hand, the administration step is performed by intravenous injection or subcutaneous injection.

[0016] This article also discloses methods for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells in a subject, said methods 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 aspect, the means is an antibody. In another aspect, the means 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. In yet another aspect, the means is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. On one hand, the tool is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On another hand, the tool is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 295. On yet another hand, the tool is 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On yet another hand, the tool 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On yet another hand, the tool is an antibody fragment.

[0017] On one hand, the tool is a peptide or protein. On the other hand, the tool is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the tool is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 295. On the other hand, the tool is a peptide or protein that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the tool is a peptide or protein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the tool is a peptide or protein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the tool is an antibody or fragment thereof described in Table 1 or Table 2, or an antibody selected by the method shown in Example 4 or Example 5. On the other hand, the cancer cell is a pancreatic cancer cell, lung cancer cell, or colorectal cancer cell. On the other hand, the administration step is performed by intravenous or subcutaneous injection. On the other hand, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues at the amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0018] On one hand, any of the methods for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or killing cancer cells in a subject, further includes the step of administering an additional therapeutic agent to the subject before or simultaneously with the administration of the composition, wherein the additional therapeutic agent is separate from the therapeutic agent present in the composition, and wherein the administration of the additional therapeutic agent results in an increase in the binding availability of surface K-Ras antigens on the outer surface of the cancer cells. On one hand, the additional therapeutic agent is a K-Ras small molecule inhibitor. On one hand, the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in the surface K-Ras antigen. On one hand, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. On one hand, the surface K-Ras antigen contains a G12D mutation. On one hand, the surface K-Ras antigen contains the sequence shown in SEQ ID NO: 295. On one hand, the additional therapeutic agent is administered to the subject 1 to 14 days before the administration of the composition. On one hand, the additional therapeutic agent is administered to the subject 3 to 7 days before the administration of the composition.

[0019] On one hand, any of the methods for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or killing cancer cells in a subject, further includes the step of administering a therapeutic agent to the subject before or simultaneously with the administration of the composition, wherein the administration of the therapeutic agent results in an increase in the expression of surface K-Ras antigen on the outer surface of the cancer cells. On one hand, the therapeutic agent is a K-Ras small molecule inhibitor. On one hand, the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in the surface K-Ras antigen. On one hand, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. On one hand, the surface K-Ras antigen contains a G12D mutation. On one hand, the therapeutic agent is administered to the subject 1 to 14 days before the administration of the composition. On one hand, the therapeutic agent is administered to the subject 3 to 7 days before the administration of the composition.

[0020] This article also discloses a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain contains a binding agent that specifically binds to surface K-Ras antigen expressed on the outer surface of cancer cells. On one hand, the transmembrane domain is selected from CD3-ζ, CD28, CDE28a, CD4, or combinations thereof. On the other hand, the intracellular domain is selected from CD28, CD27, 4-1BB, OX40, and / or ICOS. On one hand, the binding agent is an antibody or antibody fragment. On the other hand, the binding agent is an antibody or antibody fragment that selectively binds to 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the binder is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, wherein the surface K-Ras antigen contains any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. On another hand, 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 G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On another hand, 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 G12D mutation based on the amino acid sequence shown in SEQ ID NO: 295. On yet another hand, 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 G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, 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 G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the binder is an antibody fragment. On the other hand, the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen has greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen has greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.On one hand, the surface K-Ras antigen shares greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen shares greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen shares greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen shares greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the surface K-Ras antigen shares greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0021] This article also discloses vectors containing nucleotide sequences encoding any of the chimeric antigen receptors described herein.

[0022] This article also discloses immune cells expressing any of the chimeric antigen receptors described herein. On the one hand, the immune cells are cells derived from an individual. On the other hand, the immune cells are T cells derived from an individual. On the other hand, the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof. On the other hand, the immune cells are T cells. On the other hand, the immune cells are CD4+ T cells or CD8+ T cells.

[0023] This document also discloses compositions comprising immune cells expressing chimeric antigen receptors that target surface K-Ras antigens (including mutated surface K-Ras antigens) expressed on the extracellular surface of cancer cells. On one hand, the immune cells are derived from autologous, syngeneic, allogeneic, or xenogeneic sources. On the other hand, the immune cells are T cells, and wherein the T cells are derived from autologous, syngeneic, allogeneic, or xenogeneic sources. On the other hand, the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof. On the other hand, the immune cells are T cells. On the other hand, the immune cells are CD4+ T cells or CD8+ T cells.

[0024] This article also discloses immune cells for treating cancer in an individual and compositions containing immune cells, wherein the cancer comprises cancer cells expressing surface K-Ras antigens on the outer surface of the cancer cells. The immune cells comprise cells derived from the individual suffering from cancer. The immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof. The immune cells are CD4+ T cells or CD8+ T cells. The cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. The subject has been administered an additional therapeutic agent, or the immune cells are used in conjunction with the additional therapeutic agent. The additional therapeutic agent is a K-Ras small molecule inhibitor. The K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in the surface K-Ras antigen. The K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. The surface K-Ras antigen contains a G12D mutation. The therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the immune cells. On the one hand, the treatment agent is administered to the subject 3 to 7 days before the administration of immune cells.

[0025] This article also discloses a method for treating an individual with cancer, said cancer comprising cancer cells expressing surface K-Ras antigens on their outer surface, said method comprising administering to the individual a therapeutically effective amount of immune cells expressing any of the chimeric antigen receptors described herein. On one hand, the immune cells include cells derived from the individual with cancer. On one hand, the immune cells are T cells derived from the individual with cancer. On one hand, the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof. On one hand, the immune cells are T cells. On one hand, the immune cells are CD4+ T cells or CD8+ T cells. On one hand, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0026] This article also describes a method for treating an individual with cancer comprising cancer cells expressing surface K-Ras antigens on their outer surface, the method comprising administering to the individual a therapeutically effective amount of any of the compositions described herein. In one aspect, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0027] On one hand, any of the methods for treating an individual with cancer further includes the step of administering a therapeutic agent to the subject before or simultaneously with the administration of immune cells expressing chimeric antigen receptors, said cancer comprising cancer cells expressing surface K-Ras antigens on the outer surface of cancer cells, and wherein the administration of the therapeutic agent results in an increase in the binding availability of surface K-Ras antigens on the outer surface of cancer cells. On one hand, the therapeutic agent is a K-Ras small molecule inhibitor. On one hand, the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in surface K-Ras antigens. On one hand, the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236. On one hand, the surface K-Ras antigen contains a G12D mutation. On one hand, the therapeutic agent is administered to the subject 1 to 14 days before the administration of immune cells. On one hand, the therapeutic agent is administered to the subject 3 to 7 days before the administration of immune cells.

[0028] This article also discloses a method for diagnosing cancers associated with the expression of surface K-Ras antigen on the outer surface of cells, the method comprising the steps of: (a) obtaining a cell sample from a subject containing a cell population; (b) exposing the cell sample to a reagent capable of binding the surface K-Ras antigen, wherein the cell population is not lysed or otherwise permeabilized to avoid the reagent reacting with intracellular K-Ras; and (c) measuring the presence of the reagent on the outer surface of the cell population present in the cell sample. In one aspect, the surface K-Ras antigen is a mutated surface K-Ras antigen. In another aspect, the reagent is an antibody or antibody fragment that binds to the surface K-Ras antigen. In another aspect, the antibody or antibody fragment contains a label. In another aspect, the label is a fluorescent dye. In another aspect, steps (b) and (c) are performed on a sample previously obtained from the subject.

[0029] On one hand, methods for diagnosing cancers associated with the expression of K-Ras antigen on the outer surface of cells also include the addition of a secondary antibody that binds to an antibody or a portion of an antibody fragment, wherein the secondary antibody is labeled. On one hand, step (c) is performed by applying the cell sample after step (b) to a flow cytometer. On one hand, step (c) is performed by electron microscopy. On one hand, prior to step (b), the cell sample is exposed to a cell surface membrane dye. On one hand, step (c) is performed by confocal microscopy. On one hand, the cell sample is selected from pancreatic cells, colorectal cells, bile duct cells, ovarian cells, endometrial cells, and lung cells.

[0030] This document also discloses a method for diagnosing cancers associated with the expression of surface K-Ras antigen, the method comprising the steps of: (a) obtaining a cell sample from an object containing a cell population; (b) labeling proteins on the extracellular surface of the cells with a first reagent; (c) lysing the cell population to produce a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second reagent, wherein the second reagent selectively binds the first reagent; (e) removing the captured labeled proteins from the surface and removing the first reagent from the captured labeled proteins to produce a cell surface protein sample; (f) exposing the cell surface protein sample to a third reagent capable of selectively binding the surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth reagent, wherein the fourth reagent carries a detectable label and binds to a portion of the third reagent; and (h) measuring the presence of the detectable label. In one aspect, steps (f)-(h) are performed by Western blotting. In another aspect, the methods of steps (b) to (h) are performed on a sample previously obtained from the object.

[0031] On the one hand, any of the methods for diagnosing cancer associated with the expression of surface K-Ras antigen further includes subjecting a subject to one or more of the following steps (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more): (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 the composition, wherein the composition comprises a tool for selectively binding surface K-Ras antigen expressed on the outer surface of cancer cells; (iv) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody; (v) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or antibody fragment that selectively binds surface K-Ras antigen, wherein the surface K-Ras antigen comprises a component based on SEQ ID NO: 1 or SEQ ID NO: 1. (vi) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L; (vii) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L; (viii) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;(ix) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (x) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody fragment; (xi) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is a peptide or protein; (xii) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is a peptide or protein that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. (xiii) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is a peptide or protein that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (xiv) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is a peptide or protein that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (xv) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is a peptide or protein that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. G12V mutation of the amino acid sequence shown in 2; (xvi) Administer a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding to surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is an antibody or fragment thereof described in Table 1 or Table 2, or an antibody selected by the method shown in Example 4 or 5;(xvii) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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); (xviii) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2; (xix) Administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. 2. Having greater than 80% sequence identity; (xx) administering a therapeutically effective amount of the composition, wherein the composition comprises a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the surface K-Ras antigen has greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 or greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2; (xxi) administering to an individual a therapeutically effective amount of immune cells expressing any one of the chimeric antigen receptors described herein; (xxii) administering to an individual a therapeutically effective amount of immune cells expressing chimeric antigen receptors, wherein the immune cells comprise cells derived from an individual with cancer; (xxiii) administering to an individual a therapeutically effective amount of immune cells expressing any one of the chimeric antigen receptors described herein; (xxii) administering to an individual a therapeutically effective amount of immune cells expressing chimeric antigen receptors, wherein the immune cells are T cells derived from an individual with cancer; (xxiv) administering to an individual (xxii) administering a therapeutically effective amount of immune cells expressing any one of the chimeric antigen receptors described herein; (xxii) administering a therapeutically effective amount of immune cells expressing chimeric antigen receptors to an individual, wherein the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof; (xxv) administering a therapeutically effective amount of immune cells expressing any one of the chimeric antigen receptors described herein to an individual; (xxii) administering a therapeutically effective amount of immune cells expressing chimeric antigen receptors to an individual, wherein the immune cells are T cells; (xxvi) administering a therapeutically effective amount of immune cells expressing any one of the chimeric antigen receptors described herein to an individual;(xxvii) administering to an individual a therapeutically effective amount of immune cells expressing a chimeric antigen receptor, wherein the immune cells are CD4+ T cells or CD8+ T cells; (xxviii) administering a therapeutically effective amount of a composition, wherein the composition comprises a tool 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 composition; or (xxix) a combination of the above, if the surface K-Ras antigen is expressed on the outer surface of cells. On the one hand, the therapeutic agent is therapeutically effective in inhibiting the growth or proliferation of cancer cells, or otherwise cytotoxic to cancer cells. On the other hand, the subject is not administered an intracellular delivery compound in combination with the composition.

[0032] This document also discloses therapeutically effective amounts of any of the disclosed compositions for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or killing cancer cells in a subject, wherein the subject has been administered an additional therapeutic agent, and wherein the administration of the additional therapeutic agent stimulates the expression of K-Ras antigen on the outer surface of the cancer cells. On one hand, the subject has not been administered an intracellularly delivered compound in conjunction with the composition. On one hand, the additional therapeutic agent is a K-Ras small molecule inhibitor. On one hand, the additional therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the composition. On one hand, the additional therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the composition. On one hand, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. On one hand, the additional therapeutic agent and the composition are administered to the subject substantially simultaneously.

[0033] This article also discloses bispecific antibodies comprising a first binding domain linked to a second binding domain, wherein the first binding domain selectively binds to surface K-Ras antigen expressed on the outer surface of cancer cells, and wherein the second binding domain selectively binds to antigen expressed on the surface of immune effector cells. In one aspect, the first binding domain comprises a light chain variable region and a heavy chain (HC) variable region (VH). In another aspect, the first binding domain comprises a light chain (LC) variable region (VL) and a constant region (FC), as well as a heavy chain variable region and a constant region. In another aspect, the second binding domain comprises a light chain variable region and a heavy chain variable region. In another aspect, the second binding domain comprises a light chain variable region and a constant region, as well as a heavy chain variable region and a constant region. In another aspect, the surface K-Ras antigen has at least 60% homology with SEQ ID NO: 1 or SEQ ID NO: 2. In another aspect, the first binding domain selectively binds to a region on the surface K-Ras antigen, said region comprising residues 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the first binding domain selectively binds to surface K-Ras antigen, having any one of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L. On another hand, the first binding domain selectively binds to surface K-Ras antigen, having a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On another hand, the first binding domain selectively binds to surface K-Ras antigen, having a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 295. On another hand, the first binding domain selectively binds to surface K-Ras antigen, having a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On another hand, the first binding domain selectively binds to surface K-Ras antigen, having a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. On one hand, the antigens expressed on the surface of immune effector cells are selected from TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3ε, CD3δ, CD3ζ, CD137, CD16, and CD64. On the other hand, the antigen expressed on the surface of immune effector cells is CD3ε. On the other hand, immune effector cells are selected from T cells, neutrophils, macrophages, monocytes, and NK cells.

[0034] This document also discloses methods for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells in a subject, the methods comprising administering a therapeutically effective amount of any bispecific antibody disclosed herein, wherein a surface K-Ras antigen is expressed on the outer surface of the cancer cells. On one hand, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, cholangiocarcinoma cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells. On one hand, the administration step is performed by intravenous or subcutaneous injection. On one hand, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 2: 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). On the other hand, the bispecific antibodies described herein are used for treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or for killing cancer cells in a subject.

[0035] This document also discloses compositions comprising a tool that selectively binds to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is linked to a therapeutic agent, and wherein the composition does not contain an intracellular delivery compound. In one aspect, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0036] This article also discloses a bispecific antibody comprising a first tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells and a second tool for selectively binding to an antigen expressed on the surface of immune effector cells, wherein the first tool is linked to the second tool. On one hand, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0037] This article also discloses a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a tool capable of specifically binding to surface K-Ras antigens expressed on the outer surface of cancer cells. On one hand, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 2: 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). Brief description of the attached diagram

[0039] Figure 1a The design of the R11.1.6-based construct used in Example 1 is described.

[0040] Figure 1b yes Figure 1a The graph shows the growth (left) and viability (right) 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 the paper.

[0041] Figure 1c This is a graphical representation of the growth of lung cancer cell lines (left), colorectal cancer cell lines (middle), and RASless mouse embryonic fibroblast system (MEF) (right) cultured in the presence of R11.1.6 relative to a saline control. The figure shows cell counts on the left and middle, and absorbance of purple formazan on the right, indicating the number of viable cells in the MTT assay. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0042] Figure 1d This is a graphical representation of the growth (left panel) and viability (right panel) of Panc-1 (K-Ras G12D mutant) and MiaPaca-2 (K-Ras G12C mutant) cultured in the presence of R11.1.6, compared to small molecule inhibitors specific to the K-Ras G12D mutation, and in combination of both. Data are plotted relative to tumor cells stained with only anti-His-tagged secondary antibody. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0043] Figure 1eThis is a graphical representation of the growth (left) and viability (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 mutant forms of R11.1.6 (where the codons in the region in contact with the K-Ras mutant are scrambled (scrambled R11.1.6)), the M11.1.2 scaffold protein that binds to mouse serum albumin, and the E11.4.1 protein that binds to human EGFR. Data are plotted against tumor cells stained with anti-His-tagged secondary antibody only. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0044] Figure 1f The graph represents the binding of R11.1.6, disordered R11.1.6, M11.1.2, and E11.4.1 to the surface of live Panc-1 and MiaPaca-2 cancer cells, as defined by the relative median fluorescence intensity (MFI) of anti-His-tagged secondary antibody staining. Data are plotted relative to tumor cells stained with anti-His-tagged secondary antibody only. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0045] Figure 1g This is a graphical representation of the flow cytometry staining of phosphorylated AKT (left panel) and ERK (right panel) with specific antibodies at different time points after adding R11.1.6 to Panc-1 (K-Ras G12D mutant) pancreatic cancer cultures. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0046] Figure 1h It is a graphical representation of the localization of the R11.1.6-based construct by flow cytometry staining with anti-His antibody. All statistics were performed in GraphPad Prism using unpaired t-tests.

[0047] Figure 1i These are high-resolution confocal microscopy images describing the extracellular localization of R11.1.6-based constructs using anti-His antibodies.

[0048] Figure 2a : Using BRAF V600E Rescued RASless MEFs are tested for surface or total binding of anti-K-Ras antibodies.

[0049] Figure 2b : Using BRAF V600E Rescued RASless MEFs are tested for surface or total binding of anti-K-Ras antibodies.

[0050] Figure 2c : Flow cytometry was used to test human anti-K-Ras antibody 1 and BRAF V600E Combinations of G12D mutant versions of K-Ras 4B, PANC-1 or MiaPaca-2 rescued RASless MEFs.

[0051] Figure 2d : Flow cytometry was used to test human anti-K-Ras antibody 2 and BRAF V600E Combinations of G12D mutant versions of K-Ras 4B, PANC-1 or MiaPaca-2 rescued RASless MEFs.

[0052] Figure 2e The binding of human anti-K-Ras antibody 1 to the surface of RASless MEFs rescued with wild-type K-Ras 4A or 4B, or a mutant K-Ras 4B construct was tested using flow cytometry.

[0053] Figure 2f This chart shows representative histograms of surface K-Ras expression as measured by human anti-K-Ras antibody 1 staining in various tumor cell lines. The top plot shows surface staining (black), and the bottom plot shows total staining, as defined by surface + intracellular expression (blue). Solid lines represent human anti-K-Ras antibody 1 staining, and solid histograms represent human IgG isotype controls. Data represent at least one independent experiment.

[0054] Figure 2g These are high-resolution confocal microscopy images depicting the localization of human anti-K-Ras antibody 1 staining on cells.

[0055] Figure 2h Representative histograms of surface K-Ras expression in RASless MEFs, Panc-1, MiaPaca-2, and Capan-2 cell lines are shown for HL-10 and D2H12 antibodies. Solid lines represent anti-K-Ras antibody staining, and solid histograms represent human IgG isotype controls.

[0056] Figure 2i Indicates the use of BRAF V600E RASless MEFs rescued from wild-type K-Ras 4A or 4B or mutant K-Ras 4B constructs were tested in Western blot for specific binding of antibodies to K-Ras (arrow at 21 kDa).

[0057] Figure 2j This indicates three commercially available antibodies that, as confirmed by Western blot analysis, exhibited sensitive and specific binding to either the K-Ras 4B G12D mutant or wild-type K-Ras 4A or 4B. BSA protein assays determined that all blots were loaded with equal amounts of protein, and each blot was then identified.

[0058] Figure 2k Western blot, RT-PCR, and surface flow cytometry analyses of Panc-1 tumor cells after treatment with K-Ras-specific or out-of-order siRNA are shown.

[0059] Figure 2l The image shows the surface expression of K-Ras in Panc-1 cells after eliminating surface MHC class I expression by CRISPR Cas-9 excision of β2 microglobulin, as measured by human anti-K-Ras antibody 1 staining, compared to control parental cells expressing MHC class I. The top panel shows MHC class I expression, and the bottom panel shows a representative histogram of human anti-K-Ras antibody 1 staining. The bottom middle panel shows the fold change in surface staining with human anti-K-Ras antibody 1 relative to the surface staining of the human IgG isotype control.

[0060] Figure 2m This image shows Western blot analysis of K-Ras and cyclin D in bulk cells after biotinylation of proteins on the surface of living cells, compared to proteins that were not pulled down (non-surface / unbiotinylated) or pulled down by streptavidin beads (surface biotinylated). Equal amounts of protein loaded into each lane were identified by BSA proteometry. Data represent at least two experiments for each cell line.

[0061] Figure 3 This is a graphical representation of tumor cell killing, defined as the percentage of cells that survived treatment with multiple concentrations of saponin alone, without a targeting antibody, compared to untreated control cultures. Representative results are shown in triplicate for each group. Arrows point to 4.5 nM saponin. A 4.5 nM concentration of saponin conjugated with a secondary antibody was used in the antibody-drug conjugate (ADC) assay described in Example 3.

[0062] Figure 4 This is a graphical representation of Panc-1 (G12D mutant pancreatic cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of HL-10 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0063] Figure 5 This is a graphical representation of AsPC-1 (G12D mutant pancreatic cell line) cell-specific killing, defined as the percentage of cells treated with HL-10 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0064] Figure 6 This is a graphical representation of LS180 (G12D mutant colorectal cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of HL-10 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0065] Figure 7 This is a graphical representation of SK-LU-1 (G12D mutant pancreatic cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of HL-10 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0066] Figure 8 This is a graphical representation of CAPAN-2 (G12V mutant pancreatic cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of D2H12 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control culture. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0067] Figure 9 This is a graphical representation of SW480 (G12V mutant colorectal cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of D2H12 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control culture. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0068] Figure 10This is a graphical representation of NCI-H2444 (G12V-mutant lung cancer cell line) cell-specific killing, defined as the percentage of cells treated with multiple concentrations of rabbit anti-human K-Ras G12V antibody (clone # D2H12) relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. Representative results are represented in triplicate for each group.

[0069] Figure 11 This is a graphical representation of MiaPaca (G12C mutant pancreatic cell line) cell-specific killing, defined as the percentage of cells treated with various concentrations of HL-10 relative to rabbit IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results are presented in triplicate for each group.

[0070] Figure 12a-12e It is Panc-1 ( Figure 12a AsPC-1 Figure 12b ), MiaPaca-2 ( Figure 12c ) and BXPC3 ( Figure 12d The graph representation of cell-specific killing is defined as the percentage of cells treated with various concentrations of human anti-K-Ras antibody 1 relative to human IgG control antibody in a Fab-Zap assay, compared to untreated control cultures. For some data, the primary antibody was bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Figure 12e This is a control group containing only saponins. Each group has 3 replicates representing the results.

[0071] Figure 13a-13d This is a graphical representation of the specific killing effect of various RASless MEFs, defined as the percentage of cells treated with multiple concentrations of human anti-K-Ras antibody 1 relative to human IgG control antibody, including MRF-BRAF, in a Fab-Zap assay, compared to untreated control cultures. Figure 13a ), MEF (G12D) ( Figure 13b MEF (wild-type KRAS-4A) Figure 13c ) and MEF (KRAS-4B) ( Figure 13d For some data, the primary antibody bound to a secondary antibody containing saponins, and in some data, only the primary antibody was shown. Representative results were obtained from three replicates per group.

[0072] Figure 14Histograms showing K-Ras expression in primary human cells as measured by staining with human anti-K-Ras antibody 1 or human anti-K-Ras antibody 2. Solid lines represent anti-K-Ras antibody staining, and solid histograms represent human IgG isotype controls. Surface (left) or total (right) K-Ras staining was measured in cells from normal or fibrotic lung tissue from a single donor.

[0073] Figure 15a-15c This is a graphical representation of the changes in antibody binding availability of K-Ras on the surface of PANC-1 and MiaPaca cells with and without pretreatment with multiple therapeutic agents (5-FU, MRTX849, and MRTX1133).

[0074] Figure 16a A graphical representation of the survival percentage of PANC-1 cells carrying the G12D K-Ras mutation exposed to surface K-Ras-targeting antibody-drug conjugates, whether pretreated with MRTX1133 or not.

[0075] Figure 16b A graphical representation of the percentage of viable cells compared to untreated, viable control cells is shown. Cells were treated with various combinations of IgG, human anti-K-Ras antibody 1 ADC, RMC-6236, and 5-FU, as indicated. Dashed lines represent cells treated with 10... -7 M saponins treat the maximum level of cytotoxic cells.

[0076] Figure 17 A graphical representation of luminescence in Panc-1 cells expressing luciferase (or without Panc-1 cells for the group “T cells only”) is shown to demonstrate the role of multiple bispecific antibody constructs in T cell-mediated killing.

[0077] Figure 18 A schematic diagram of an exemplary anti-K-Ras-anti-CD3 bispecific antibody construct is provided.

[0078] Figure 19a-19d This is a series of graphical representations showing the results from an ELISA that assesses human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 with those loaded with GDP nucleotides ( Figure 19a ) or GppNHp nucleotides ( Figure 19b K-Ras with a G12D mutation, or loaded with GDP nucleotides ( Figure 19c ) or GppNHp nucleotides ( Figure 19d The combination of wild-type K-Ras.

[0079] Figure 20a-20hIt is a series of graphical representations showing cell-specific killing in various cell lines, defined as the percentage of cells that survived in the Fab-Zap assay, compared to untreated control cultures, after treatment with multiple concentrations of human anti-K-Ras antibody 1, human anti-K-Ras antibody 2, RSV, and saponin-only control.

[0080] Figure 21a-21b These are a series of microscopic images showing the difference between the PA1252PDX mouse model and the control antibody (isotype). Figure 21a ) and PA0787 PDX mouse model ( Figure 21b The results of staining tumor tissue with human anti-K-Ras antibody 1.

[0081] Figure 22a-22d This is a series of graphical representations showing the body weight of Balb / c nude mice with implanted Panc-1 tumor cells and treated with various treatment regimens described in Table 5. Figure 22a and Figure 22c ) and mm 3 The calculated tumor volume ( Figure 22b and Figure 22d ).

[0082] Figure 23 This is a heatmap of HDX-MS experimental results using K-Ras and human anti-K-Ras antibody 1.

[0083] Figure 24 This is a heatmap of HDX-MS experimental results using K-Ras and human anti-K-Ras antibody 2. Invention Details

[0085] Unless otherwise specifically defined herein, all technical and scientific terms shall have the meanings that would be given to them by a person of ordinary skill in the relevant field.

[0086] As used herein, the term "surface K-Ras antigen" refers to a K-Ras peptide or protein expressed on the outer surface of a cell. Surface K-Ras antigens include full-length KRAS4B (SEQ ID NO: 1 - MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM) or KRAS4A (SEQ ID NO: 2 - MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM) and its truncated form expressed on the extracellular surface. The surface K-Ras antigen may also comprise a full-length or truncated K-Ras peptide or protein (including forms KRAS4A and KRAS4B) having a mutation at any of the following amino acids / residues or their corresponding amino acids / residues (based on SEQ ID NO: 1 or SEQ ID NO: 2): 12 (including but not limited to G12A, G12D, G12C, G12V, G12R); 13 (including but not limited to G13D); 61 (including but not limited to Q61H, Q61L) and any other mutations associated with cancer-causing K-Ras peptide or protein forms. For example, K-Ras G12D has the amino acid sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 295).Surface K-Ras antigens may also comprise other forms of K-Ras peptides or proteins capable of being bound by a variety of anti-K-Ras agents, including, for example, antibodies, proteins, and peptides listed in Tables 1 and / or 2. Forms of K-Ras peptides or proteins capable of being bound by a variety of anti-K-Ras agents include, for example, antibodies, proteins, and peptides listed in Tables 1 and / or 2, including, but not limited to, post-translational modified K-Ras. For example, K-Ras post-translational modifications may include, but are not limited to, isopreneation, post-isopreneation, palmitoylation, ubiquitination, phosphorylation, SUMOylation, acetylation, nitrosation, and combinations thereof. The antibodies, proteins, and peptides in Tables 1 and 2 are exemplary structures for selectively binding surface K-Ras antigens and can be used as binding tools or binders in any embodiment of this disclosure targeting 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 are complexed with HLA and expressed on the surface.

[0087] Table 1: Exemplary antibodies, proteins, and peptides that bind to surface K-ras antigens.

[0088]

[0089] Table 2. Exemplary antibodies, proteins, and peptides that bind to surface K-ras antigens.

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] As used herein, the terms "binding agent" or "binding domain" refer to any molecule (peptide, protein, small molecular weight chemical substance, and nucleic acid) or a portion of such a molecule, which, by virtue of their structure, are capable of selectively binding to a target antigen. The terms "selectively binding" or "selectively binding" as used herein mean that the binding agent is capable of binding its target antigen to a population of non-target antigens beyond the target antigen, with an affinity for the target antigen greater than the affinity produced by non-specific binding to non-target antigens (to the extent that any such non-specific binding occurs). Non-limiting examples of binding agents include antibodies, nanobodies, antibody fragments (e.g., scFv), peptides, proteins, and aptamers.

[0096] As used herein, the term “binding availability” when it is related to cell surface antigens should be understood to take into account surface expression, binding chaperone affinity, or a combination thereof.

[0097] As used in this article, “immune effector cells” are cells that participate in the cytolytic immune response, including, for example, T cells, NK cells, monocytes, macrophages, or neutrophils.

[0098] As used herein, the term "cell penetrating peptide" is defined as an amino acid sequence that can lead to the internalization of the peptide and peptide-linked molecules into the cytoplasm of the cell. Examples of cell penetrating peptides are R9 (SEQ ID NO: 3 - RRRRRRRRR-NH2) and penetrating peptide (SEQ ID NO: 4 - RQIKIWFQNRRMKWKK-NH2).

[0099] As used herein, the term "intracellular delivery compound" refers to a compound that promotes or initiates the passage of a material or composition across the cell membrane, wherein the material or composition would not have passed through the cell membrane and been internalized in the absence of the intracellular delivery compound. Examples of intracellular delivery compounds are cell-penetrating peptides, liposomes, nanoparticles, and dendritic macromolecules. In some embodiments, the intracellular delivery compound is a cell-penetrating peptide. In some embodiments, the intracellular delivery compound is not a cell-penetrating peptide.

[0100] 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 in K-Ras function.

[0101] As used herein, the term "antibody" is defined as a protein having a set of immunoglobulin protein domains, commonly referred to as "heavy chains" and "light chains," having definite complementary sites that functionally recognize epitopes on target antigens. The term antibody should be understood to include antibody fragments, mutants, variants, derivatives, or engineered versions of molecules containing functional complementary sites that recognize target epitopes. Antibody fragments also include separate fragments consisting of heavy and light chain variable regions and recombinant single-chain polypeptide molecules, wherein the light and heavy chain variable regions are linked by peptide linkers ("scFv"). Antibody fragments also include F(ab')2, Fab', Fab, Fv, sFv, heavy chain variable antibodies (VHH, also known as nanobodies), single-domain antibodies (e.g., heavy chain variable domains containing heavy chain antibodies), etc.

[0102] Chimeric antibodies are recombinant proteins containing variable domains, including complementarity-determining regions (CDRs) derived from antibodies of a non-human species, while the constant domains of the antibody molecule are derived from those of human antibodies. Humanized antibodies are recombinant proteins in which the CDRs of antibodies from a species are transferred from the variable heavy and light chains of that species' antibody to the variable heavy and light chain domains of humans. Human antibodies generally refer to antibodies obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigen challenge.

[0103] As used herein, the terms “antibody-drug conjugate” and “ADC” refer to antibodies linked to a functional molecule for the purpose of delivering that molecule to a structure (i.e., a cell) containing a target antigen. The functional molecule is often referred to as the “payload” or “warhead”.

[0104] As used herein, the term "ADC linker" or "linker" refers to the chemical link between an antibody and its payload / warhead. These linkers may include those capable of being cleaved by proteases, pH conditions, disulfide bond reduction, or those that are cleavable extracellularly or intracellularly. Other linkers are cleavable and are broken down by the cell into their active forms. "Linker" also refers to a peptide linked to a polypeptide, such as a peptide used for bispecific antibodies. Peptide linkers can be from about 2 amino acids to about 30 amino acids, and all lengths and intermediate ranges therein.

[0105] As used herein, the term "payload" or "warhead" refers to a molecule conjugated to an antibody in an ADC via a linker. In some embodiments, the ADC payload or warhead is a therapeutic agent.

[0106] As used herein, the term "therapeutic agent" refers to any molecule that causes a therapeutic effect (as defined herein). Particularly suitable therapeutic agents for ADCs include cytotoxic agents, cell growth inhibitors, radionuclides, or toxins. Cytotoxic agents are those that can induce the exhaustion, elimination, and / or killing of target cells, such as microtubule disruptors (e.g., maytansanoids, mertansine, emtansine, soravtansine, ravtansine, auristatins, MMAE (methyl-ouristatin E), MMAF (methyl-ouristatin F), tubulolysin, and taxols), topoisomerase inhibitors (e.g., topotecan, govitecan, deruxtecan, rezetecan), and DNA damaging agents (e.g., exatecan, topoisomerase 1 inhibitor (SN-38), cazidac, antramycin, tesirine, pyroxine, doxorubicin, and pyrrolobenzodiazepine (PBD) dimers). Cell growth inhibitors are agents that can inhibit the growth and / or proliferation of target cells. Radionuclides are nuclides with excess nuclear energy, making them unstable; these include radionuclides that emit beta-particles or alpha-particles. Examples of alpha-particle-emitting radionuclides include astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227. Examples of beta-particle-emitting radionuclides include iodine-131, rhenium-186, yttrium-90, samarium-153, strontium-89, and lutetium-177. When a radionuclide degrades, it can release alpha, beta, or gamma radiation, thereby damaging adjacent cells. It should be understood that many therapeutic agents suitable for delivery via ADCs exist, and the list herein should be considered exemplary rather than a limitation on potential agents.

[0107] The term "therapeutic effect" refers to an improvement in clinical characteristics. Non-limiting examples of clinical characteristics include reduction in tumor size, inhibition or restriction of tumor growth or cancer cell proliferation, reduction or prevention of metastasis, prevention or reduction of cancer recurrence after remission, alleviation of cancer-related symptoms, improvement of patient lifespan, and delay of clinical disease progression. Therefore, a "therapeuticly effective amount" is the amount of the composition of this disclosure that causes the therapeutic effect.

[0108] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T-cell receptor / immune receptor engineered to specifically respond to an antigen. Traditionally, this involves engineering an antibody, or antibody fragment or derivative (i.e., scFv), onto transmembrane and intracellular effector domains via a "spacer" sequence. This allows signal transduction to be induced when the CAR binds to a homologous epitope specifically recognized by the antibody. The transmembrane domain is used to anchor the CAR to the cell membrane of the transduced cell. The intracellular effector domain (i.e., the intracellular domain) participates in the normal signal transduction mechanisms of the transduced cell. CAR expression is achieved by introducing it into transduced T cells via a vector (see below).

[0109] As used herein, the term "vector" refers to a medium used to deliver DNA or protein into cells. Vectors typically contain DNA encoding a desired gene that integrates into the vector's DNA. Vectors can include, but are not limited to, plasmids, viruses, and bacteria. In the case of CAR, the vector typically comprises a virus engineered to introduce CAR genetic material into the desired cells.

[0110] As used herein, “sequence identity [percentage] (%)” relative to a reference polypeptide sequence means the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, and does not consider any conserved substitutions as part of sequence identity. “Conserved substitution” means replacing an amino acid residue with another amino acid residue that has similar properties (e.g., size, charge, and hydrophobicity).

[0111] Antibody drug conjugate

[0112] In one aspect of this disclosure, the use of antibody-drug conjugates (ADCs) for treating cancers associated with the expression of surface K-Ras antigens is provided. ADCs typically comprise three parts: (1) a binding tool (e.g., an antibody) for selectively binding one or more surface K-Ras antigens; (2) a payload or warhead (typically a therapeutic agent); and (3) a linker tool for conjugating the binding tool to the payload.

[0113] The binding agent used in the ADC of this invention should have target specificity for surface K-Ras antigens expressed on target cancer cells but not on healthy cells. This functionality can be achieved using antibodies, antibody fragments, or peptides, such as those listed in Tables 1 and 2. The antibody should have high binding affinity and low immunogenicity and cross-reactivity, while still retaining the property of allowing attachment to the warhead. The antibody can be chimeric, humanized, or fully human, and can be monospecific, bispecific, trispecific, 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 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 shown 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 comprises any of the following mutations based on the amino acid sequence shown 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 comprises any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind to such sequences without any of these mutations, or if it can bind to such sequences without such mutations, its affinity would not be 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 the surface K-Ras antigen, the surface K-Ras antigen having greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage of sequence identity from 70% to 99% with SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2).In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 75% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 96% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater than 97% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the binding agent is an antibody or antibody fragment that binds to the surface K-Ras antigen, the surface K-Ras antigen having greater 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 greater than 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. Binders suitable for the ADC embodiments disclosed herein include those listed in Tables 1 and / or 2 that are suitable for a given cancer expressing a mutation.

[0114] The specific antigens used to develop the antibodies of this invention may include K-Ras regions known to include mutations found in cancer, such as those found at residues 12, 13, and 61. Examples 4 and 5 herein describe specific methods for developing antibodies of this disclosure for use in ADCs.

[0115] The linkers used in the ADCs of this invention must provide a stable interaction between the antibody and the payload to avoid premature release of the payload and unintended off-target effects. When bound to the antibody, the linker should also maintain the payload in an inactive, non-toxic state, but should be able to release the payload after internalization into the cell. Therefore, particularly suitable linkers for conjugating the binder to the payload include cleavable linkers under intracellular conditions, such that cleavage of the linker releases the cytotoxic or cell-growth-inhibiting portion from the antibody within the cell (e.g., in an endosome or lysosomal compartment); or non-cleavable linkers, such that the cell-growth-inhibiting portion or the cytotoxic portion or its derivatives are released from the antibody after degradation (e.g., within a lysosome or via a proteasome). Cleavable linkers may include those cleavable at low pH (i.e., sensitive to hydrolysis when exposed to certain pH conditions), such as acid-labile linkers (e.g., hydrazones, ureas, thioureas, cis-aconitine, orthoesters, acetals, ketals, etc.). These acid-unstable linkers are relatively stable at neutral pH levels, such as in blood and extracellular environments, and relatively unstable at pH 5.5 or lower than the approximate pH of lysosomes. Cleavable linkers may also include those cleaved by lysosomal or endosomal proteases (e.g., cathepsin B) or enzymes (β-glucuronidase), such as peptide linkers (e.g., Val-Ala, Val-citrulline, Gly-Gly-Phe-Gly, Gly-Phe-Leu-Gly, and Phe-Leu) or β-glucuronide linkers. Cleavable linkers can also include those that are cleavable under reducing conditions (e.g., disulfide bonds), such as disulfide linkers (e.g., SATA (N-succinimidyl-S-acetylthioacetate), SMCC (succinimidyl-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid ester), SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SPDP). (N-Succinimidyl-3-(2-pyridyldithio)propionate). Uncleavable linkers may include those that are not easily cleaved by proteases, cleaved at low pH, or released under reducing conditions, such as maleimide-alkylene or maleimide-aryl linkers. In one embodiment, the linker is any one or more of maleimide hexanoyl linkers, peptide-based linkers (including but not limited to valine-citrulline linkers), β-glucuronide linkers, SMCC linkers, disulfide linkers, or acid-sensitive linkers.

[0116] Various therapeutic agents can be administered as payloads in the target ADC. Optionally, such agents can be administered simultaneously, before, or after the administration of the ADC. The therapeutic agents of the ADC of the present invention include, for example, drugs, toxins, oligonucleotides, immunomodulators, hormones, hormone antagonists, enzymes, enzyme inhibitors, radionuclides, and angiogenesis inhibitors (in addition to those listed above). Therapeutic agents also include, for example, cytotoxic drugs (e.g., vinca alkaloids), anthracycline antibiotics (e.g., doxorubicin), 2-PDox or pre-2-PDox, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotic agents, antiangiogenic agents, and pro-apoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, CPT-11, camptothecin, protein body inhibitors, mTOR inhibitors, HDAC inhibitors, and tyrosine kinase inhibitors. Other useful anticancer cytotoxic drugs used for simultaneous or sequential administration or for the preparation of ADCs include nitrogen mustard, alkyl sulfonates, nitrosoureas, triazine, folic acid analogs, COX-2 inhibitors, antimetabolites, pyrimidine analogs, purine analogs, platinum coordination complexes, mTOR inhibitors, tyrosine kinase inhibitors, protein body inhibitors, HDAC inhibitors, camptothecin, hormones, etc.

[0117] Methods of conjugating linkers to antibodies may include random conjugation (e.g., conjugation to surface-exposed lysine residues) or site-specific conjugation (e.g., conjugation to cysteine ​​residues of reduced interchain disulfide bonds, conjugation occurring during glycan remodeling, or conjugation to included non-natural amino acids or specific amino acid sequences). As those skilled in the art will understand, the number of therapeutic agents conjugated to the antibody molecule can vary, such that the collection of ADCs can be heterogeneous in nature, with some antibodies containing one conjugated agent, some containing two conjugated agents, some containing three conjugated agents, etc. (and some not). The degree of heterogeneity depends in particular on the chemicals used to conjugate the therapeutic agents. In one embodiment, the number of therapeutic agents conjugated to a single anti-K-Ras antibody or its antigen-binding fragment can range from one to eight or more components.

[0118] ADCs are typically administered to patients via intravenous injection, but can also be administered subcutaneously. The starting dose for first-in-human clinical trials is usually determined from preclinical pharmacology and nonclinical pharmacokinetic and toxicological data. Typically, the starting dose of an ADC is 1 mg / kg or close to 1 mg / kg, and can be tested up to 20 mg / kg. Typical dosing intervals for ADCs are once weekly, once every two weeks, or once every three weeks. Optimal dosage and dosing intervals are influenced by the antibody, linker, cytotoxic agent, or cell growth inhibitor, as well as the conjugation chemistry used to generate and use the ADC alone. ADCs are typically provided as lyophilized powders, which are reconstituted into solutions (e.g., saline, dextran, or similar solutions) prior to injection.

[0119] In some 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.

[0120] Bispecific antibody

[0121] In another aspect of this disclosure, compositions for treating cancers expressing surface K-Ras antigens include bispecific antibodies. Immune cell redirection bispecific antibodies are a therapeutic approach in which antibodies are engineered to bring immune cells very close to target-expressing cells to stimulate immune cell killing of the target cells. Bispecific antibodies are engineered to be specific to target cells via antibodies, Fab, or scFv (or other antibody fragments) specific to the target antigen, and to immune cells via antibodies, Fab, or scFv (or other antibody fragments) specific to immune cells. Immune cell redirection bispecific antibodies may include T cell redirection bispecific antibodies, natural killer (NK) cell redirection bispecific antibodies, and macrophage redirection bispecific antibodies. Typically, but not exclusively, T cell redirection bispecific antibodies bring T cells very close to target-expressing cells to stimulate T cells to kill target cells via the T cell surface protein CD3.

[0122] The most common forms of bispecific antibodies are single-chain variable fragments (scFv) without the Fc fragment or full-length IgG-like asymmetric antibodies. However, many other forms are currently in use and under development. See Table 3 for the bispecific antibody forms considered in this article.

[0123] Table 3: Exemplary antibody structures and configurations for bispecific and trispecific implementation schemes.

[0124]

[0125]

[0126]

[0127] In one embodiment, the bispecific antibody of this disclosure includes a first binding domain linked to a second binding domain. In one embodiment, the bispecific antibody of this disclosure includes a bispecific antibody selected from the following forms: 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), bispecific antibody, bispecific antibody-Fc (type 1), bispecific antibody-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 fusion.

[0128] The first binding domain contains a first tool for selectively binding surface K-Ras antigens. Structures suitable for selectively binding surface K-Ras antigens include: any Fab region of the antibodies listed in Table 1 and / or Table 2; any scFv construct containing a light chain variable region and a heavy chain variable region of any antibody listed in Table 1 and / or Table 2; any Fab-Fc region of the antibodies listed in Table 1 and / or Table 2; any scFv construct containing a light chain variable region and a heavy chain variable region linked to an Fc region of any antibody 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 Fc mutation similar to those disclosed in Example 13 for the bispecific construct); Figure 18The anti-K-Ras Fab light chain (SEQ ID NO: 27) and Fab heavy chain and Fc portions (SEQ ID NO: 26) shown; scFv, which includes a 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 (including heavy chain CDR1, CDR2, and CDR3 corresponding to SEQ ID NO: 10, 11, and 12, and light chain CDR1, CDR2, and CDR3 corresponding to SEQ ID NO: 14, 15, and 16, respectively); scFv, which includes a light chain variable region of SEQ ID NO: 21 linked to the heavy chain variable region of SEQ ID NO: 17 of human anti-K-Ras antibody 2 (including heavy chain CDR1, CDR2, and CDR3 corresponding to SEQ ID NO: 18, 19, and 20, and light chain CDR1, CDR2, and CDR3 corresponding to SEQ ID NO: 22, 23, and 24, respectively); SEQ The Fab regions of SEQ ID NOs 9 and 13 may or may not have an Fc portion; the Fab regions of SEQ ID NOs 17 and 21 may or may not have an Fc portion. The first binding domain may also include a second tool for binding different regions or epitopes of the surface K-Ras antigen. Structures suitable for binding the surface K-Ras antigen are as described above, except that the second tool must bind to a different region of the surface K-Ras antigen than the first tool. Therefore, any structure corresponding to the first tool cannot be the same structure corresponding to the second tool. It should be understood that the structures identified herein for binding the surface K-Ras antigen can also be used in compositions relating to antibody-drug conjugates in this disclosure when applied to bispecific antibodies.

[0129] The second binding domain contains tools for selectively binding antigens on the surface of immune effector cells. Structures suitable for binding antigens to immune effector cells include... Figure 18 The CD3 scFv-Fc polypeptide (SEQ ID NO: 28) shown selectively binds any Fab construct, scFv construct, or other functional antibody fragment, including functional CD3 antibodies, of TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3δ, CD3ζ, CD137, CD16, and CD64. Exemplary CD3 binding domains (e.g., VH and VL domains) are listed in Table 4. Similar to the first binding domain, the second binding domain may also include a second tool for binding to another antigen on the surface of immune effector cells. Structures suitable for binding antigens on the surface of immune effector cells are as described above, except that the second tool must bind to a different region of the antigen than the first tool. Therefore, any structure corresponding to the first tool cannot be the same structure corresponding to the second tool.

[0130] Table 4: Exemplary antibodies that bind to CD3 antigens.

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Chimeric antigen receptor T cell

[0139] In another aspect of this disclosure, a composition for treating cancers expressing surface K-Ras antigens includes 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.

[0140] The extracellular domain is a binding agent, wherein the binding agent specifically binds to surface K-Ras antigen expressed on the outer surface of cancer cells. In one embodiment, the binding agent is an antibody or antibody fragment. In another embodiment, the binding agent is an antibody or antibody fragment that selectively binds to 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the binding agent is an antibody or antibody fragment that selectively binds to surface K-Ras antigen, wherein the surface K-Ras antigen contains any of the following mutations based on the amino acid sequence shown 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 any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind such a sequence without any of these mutations, or if it can bind such a sequence without such mutations, its affinity would not be 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 greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage of sequence identity from 70% to 99% with SEQ ID NO: 1 or SEQ ID NO: 2. In any of the above embodiments, 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). Binders suitable for the CAR-T cell embodiments disclosed herein include those listed in Tables 1 and / or 2 suitable for a given cancer expressing a mutation.

[0141] In any of the foregoing embodiments, the chimeric antigen receptor may have a transmembrane domain selected from CD3-ζ, CD28, CDE28a, CD4, or combinations thereof.

[0142] In any of the foregoing embodiments, the chimeric antigen receptor may have an intracellular domain selected from CD28, CD27, 4-1BB, OX40 and / or ICOS.

[0143] In some embodiments, immune cells are transformed with a vector that expresses the nucleotide sequence encoding the CAR described herein. The vector can be any vector capable of expressing the CAR protein within immune cells. The vector may also contain control sequences that allow it to replicate and / or be expressed in prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above-described host cells are incubated to sustain them and allow the vector to replicate. It is also understood and known that techniques and conditions allow for the large-scale production of the vector and the production of nucleic acids encoded by the vector and their homologous polypeptides, proteins, or peptides.

[0144] Immune cells can be autologous, syngeneic, allogeneic, or xenogeneic cells. Transfecting immune cells with a vector encoding any of the aforementioned chimeric antigen receptors to generate CAR-T cells or CAR-immune cells will be administered to a subject with cancer expressing the surface K-Ras antigen. In one embodiment, the immune cells are derived from the individual to whom the vector will be transfected. In another embodiment, the immune cells are derived from a different individual. In any of the foregoing embodiments, the immune cells are selected from T cells, natural killer (NK) cells, dendritic cells, or mixtures thereof. In another embodiment, the immune cells are CD4+ T cells or CD8+ T cells.

[0145] In some implementations, CAR-T cell administration will require leukapheresis. Patient blood cells are collected, and the collected cells can be enriched or depleted to obtain the desired cell population. In a preferred embodiment, patient T cells will be isolated. The isolated patient T cells will be expanded, modified with engineered CAR receptors, and infused into the patient using methods known to those skilled in the art.

[0146] Methods of treatment, administration, formulation and dosing

[0147] On one hand, methods are provided for treating subjects suffering from cancers associated with surface K-Ras antigen expression. These methods include administering a therapeutically effective amount of a composition comprising a binder capable of specifically binding to 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 shown 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 any of the following mutations based on the amino acid sequence shown 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 any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind such a sequence without any of these mutations, or if it can bind such a sequence without such mutations, its affinity will not be 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 greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage of sequence identity from 70% to 99% with SEQ ID NO: 1 or SEQ ID NO: 2. Binders suitable for the therapeutic methods disclosed herein include those listed in Tables 1 and / or 2 suitable for a given expressed mutation in cancer, human anti-K-Ras antibody 1, and human anti-K-Ras antibody 2. In any of the above method embodiments, the binder may be linked to another binder that binds to an antigen on the surface of an immune effector cell, as discussed herein with respect to embodiments of compositions comprising bispecific antibodies, and all features and embodiments disclosed therewith are incorporated herein.In any of the above-described method embodiments, the binder may be linked to a therapeutic agent that is cytotoxic to the target cancer cells, as discussed herein with respect to embodiments of compositions comprising antibody-drug conjugates, and all features and embodiments disclosed therewith are incorporated herein. In any of the above-described method embodiments, the binder may be part of a chimeric antigen receptor expressed on immune effector cells, as discussed herein with respect to embodiments of compositions comprising CAR-T cells, and all features and embodiments disclosed therewith are incorporated herein. In any of the above-described embodiments, the method does not include the administration of an intracellular delivery compound.

[0148] The amount of the composition embodied in this invention can vary according to the patient's needs, but should be provided in a therapeutically effective amount.

[0149] Some embodiments of the invention include the co-administration of the compositions embodied in the invention with other treatments such as chemotherapy, surgery, radiation therapy, immunomodulators, and other therapeutic agents. Similarly, we have found that treatment of K-Ras-expressing cells with chemotherapy (e.g., 5-FU) and small molecule inhibitors of K-Ras activity leads to increased binding availability of surface K-Ras antigens or increased expression of them (see Examples 11, 12a, and 12b).

[0150] Therefore, in one embodiment, a method of treating, inhibiting, or reducing the proliferation of cancer cells in a subject, or killing cancer cells in a subject, includes administering a therapeutic agent to the subject before or simultaneously with any of the aforementioned compositions embodied in this invention. In this case, the therapeutic agent should be understood as any therapeutic agent that can be separated and isolated from the compositions embodied in this invention. In one specific embodiment, the therapeutic agent is chemotherapy, such as 5-FU. In another specific embodiment, the therapeutic agent is a small molecule inhibitor of K-Ras, including but not limited to MRTX1133 (activity against K-Ras with G12D mutations), MRTX849 (activity against K-Ras with G12C mutations), or RMC-6236 (activity against various GTP-binding RAS proteins, including K-Ras WT and K-Ras G12 mutants). In yet another specific embodiment, multiple therapeutic agents are used. For example, in one specific embodiment, one or more therapeutic agents are chemotherapy and small molecule inhibitors of K-Ras. In another specific embodiment, one or more therapeutic agents are 5-FU chemotherapeutic agents and small molecule inhibitors 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 various GTP-binding RAS proteins, including K-Ras WT and K-Ras G12 mutants). More generally, K-Ras small molecule inhibitors are inhibitors that specifically inhibit the activity of K-Ras forms carrying mutations present in surface K-Ras antigens targeted by the compositions embodied in this invention, including any of the known K-Ras mutations described herein.

[0151] The therapeutic agent is administered to the subject 1 to 21 days prior to application of the composition and on any specific date 1 to 21 days prior to application, as well as any intermediate range in between (e.g., 3 to 7 days, 7 to 14 days, 10 to 21 days, etc.).

[0152] The method or route of administration and dosage for embodiments involving pretreatment or concurrent treatment with additional therapeutic agents will be such that the therapeutic agent is typically administered and given under standard treatment conditions for the indication for which the therapeutic agent is routinely used. In some embodiments, the dosage may be reduced from a dose that is considered indicated or approved.

[0153] The method or route of administration of the compositions embodied in this invention is any suitable method or route determined by a skilled physician / clinician. Non-limiting examples of administration methods include injection, infusion, or implantation. Non-limiting examples of routes of administration include parenteral, intravenous, tumor, arterial, intramuscular, peritoneal, and / or subcutaneous.

[0154] The dosage and frequency of administration can vary depending on the clinical situation. The dosage and frequency can be determined by any physician or clinician in the field. In some cases, a higher or lower dose may be appropriate if an effective dose is administered. The frequency of administration may require only a single dose or multiple doses. For CAR-T administration, the number of cells administered can vary, but a preferred implementation is 10. 4 -10 9 Cells / kg

[0155] The compositions disclosed herein can be used to treat a variety of cancers associated with K-Ras mutation expression or K-Ras overexpression, such as pancreatic cancer (including pancreatic duct adenocarcinoma (PDAC)), lung cancer (including non-small cell lung cancer (NSCLC)), cholangiocarcinoma, ovarian cancer, endometrial cancer, or colorectal cancer.

[0156] It should be understood that any public or implementation method relating to the treatment, suppression or mitigation of cancer is equally applicable to its medical use.

[0157] Diagnosis

[0158] Because the therapeutic compositions and methods of this disclosure rely on the surface expression of K-Ras antigen (surface K-Ras antigen) in cancer cells, rather than the intracellular expression of K-Ras, it is important to first determine whether the cancer of the target cell expresses this specific antigen on its surface. Therefore, this disclosure also provides methods and compositions for diagnosing cancers expressing surface K-Ras antigen. Typically, the diagnostic composition may contain any binding agent or other compound capable of specifically detecting surface K-Ras antigen on cells, and the method employed must be able to distinguish or separate the surface K-Ras antigen from the K-Ras antigen present within the cells. In view of these considerations, those skilled in the art should be able to identify many different diagnostic methods, and therefore the specific embodiments of this disclosure should be understood as exemplary rather than limiting.

[0159] The binding agents described herein for use with ADCs and CAR-T cells can also provide useful reagents for detecting surface K-Ras antigens in cancer cells. However, any reagent that cannot be transported across the cell membrane (or can be modified to not cross the cell membrane or otherwise enter the cell) and is specific for surface K-Ras antigens can be used as a diagnostic instrument. In one embodiment, the binding agent is an antibody or antibody fragment. In another embodiment, the binding agent is an antibody or antibody fragment that selectively binds to surface K-Ras antigens, wherein the surface K-Ras antigen contains a mutation at residues 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the binding agent is an antibody or antibody fragment that selectively binds to surface K-Ras antigens, wherein the surface K-Ras antigen contains any of the following mutations based on the amino acid sequence shown 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 any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, but the binder does not bind such a sequence without any of these mutations, or if it can bind such a sequence without such mutations, its affinity will not be 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 greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or any percentage of sequence identity between 70% and 99% with SEQ ID NO: 1 or SEQ ID NO: 2. Binders suitable for the diagnostic embodiments disclosed herein include those listed in Tables 1 and / or 2 suitable for a given cancer expressing a mutation, including human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2. In any of the above embodiments, the method does not include the administration of an intracellular delivery compound.

[0160] In one embodiment, the diagnostic method of this disclosure involves (a) obtaining a cell sample from an object containing a cell population; (b) exposing the cell sample to a reagent capable of binding a surface K-Ras antigen, wherein the cell population is not lysed or otherwise permeabilized to avoid the reagent reacting with the intracellular form of K-Ras; and (c) measuring the presence of the reagent on the outer surface of the cell population present in the cell sample. In one embodiment, the surface K-Ras antigen is a mutated surface K-Ras antigen. The diagnostic method of the foregoing embodiments can be performed on samples already obtained from an object.

[0161] In one implementation, the step of obtaining a cell sample from an object containing a cell population includes guided biopsy, such as fine-needle aspiration biopsy. In this method, a thin, hollow needle is inserted into the skin and into the target tissue sample. This can be achieved via CT guidance, ultrasound guidance, or tactile sensation. Alternatively, endoscopic procedures such as colonoscopy, upper endoscopy, or endoscopic retrograde cholangiopancreatography (ERCP) can be used to approach the lesion within the gastrointestinal tract. The biopsy needle is used to aspirate a small amount of cell sample (and fluid) for analysis. Furthermore, larger needles can be used to obtain large core needle biopsies that can be used to obtain larger masses of cells. However, the desired outcome of fine-needle biopsy or FNA is to obtain a single-cell suspension in which the cells remain intact to assess cell surface antigen expression.

[0162] In another embodiment, the step of obtaining a cell sample from a solid tumor can be performed by the following steps: (1) cutting the tissue into small pieces (e.g., about 1 mg); (2) suspending the small pieces in a culture medium and then dissociating them (e.g., using a MACS dissociator 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 retrieve single cells; (5) washing the cells to remove digestive enzymes and DNase; and (6) passing the cells through a strainer and resuspending them in an appropriate culture medium. It should be understood that many alternative methods exist for obtaining a cell suspension from a solid tumor or sample, and the embodiments described herein should be considered exemplary rather than limiting.

[0163] Cell samples may include pancreatic cells, lung cells, bile duct cells, ovarian cells, endometrial cells, or colorectal cells.

[0164] The cell sample can then be exposed to a reagent capable of binding to the surface K-Ras antigen. During this step, care should be taken to avoid lysis or subjecting the cells to conditions that allow the reagent to react with the intracellular form of K-Ras. Preferably, the reagent (e.g., the binding agent of this disclosure) should be one that cannot cross the cell membrane without the use of an intracellular delivery compound. In one embodiment, the reagent is an antibody or antibody fragment or other protein or peptide that binds to the surface K-Ras antigen. In a further embodiment, the antibody or antibody fragment contains a label, such as a fluorescent dye. In other embodiments, the method includes applying a first reagent and exposing the sample to a second binding agent that binds to a portion of the first reagent, wherein the second binding agent is labeled.

[0165] The measurement steps can then be performed using methods commonly used in the art to determine the interaction between the reagent and the surface K-Ras antigen, such as applying the cell sample to a flow cytometer or electron microscopy.

[0166] Confocal microscopy can also be used, and will also include an additional step in which the cell sample is exposed to a cell surface membrane dye before the cell sample is exposed to a reagent that reacts with the surface K-Ras antigen.

[0167] In another embodiment, the diagnostic method includes the following steps: (a) obtaining a cell sample from an object containing a cell population; (b) labeling proteins on the extracellular surface of the cells with a first reagent; (c) lysing the cell population to produce a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second reagent, wherein the second reagent selectively binds the first reagent; (e) removing the captured labeled proteins from the surface and removing the first reagent from the captured labeled proteins to produce a cell surface protein sample; (f) exposing the cell surface protein sample to a third reagent capable of selectively binding a surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth reagent, wherein the fourth reagent carries a detectable label and binds to a portion of the third reagent; and (h) measuring the presence of the detectable label. An example of this embodiment includes protein blotting. The diagnostic method of the foregoing embodiments can be performed on samples already obtained from an object.

[0168] When diagnosing cancers expressing surface K-Ras antigens, any of the above-described therapeutic compositions and methods can be used on the patient.

[0169] Exemplary embodiments of the present disclosure

[0170] Implementation Scheme 1: A composition comprising: a binder-therapeutic agent complex containing a binder linked to a therapeutic agent, wherein the binder specifically binds to surface K-Ras antigen expressed on the outer surface of cancer cells, and wherein neither the composition nor the binder-therapeutic agent complex contains an intracellular delivery compound.

[0171] The composition according to embodiment 1, wherein the binding agent is an antibody or an antibody fragment.

[0172] According to the composition of embodiment 1, wherein the binding agent is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen having a mutation at residue 12, 13 or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0173] According to the composition of embodiment 1, wherein the binding agent is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen having any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0174] According to the composition of embodiment 1, wherein the binding agent is an antibody or antibody fragment that selectively binds to a surface K-Ras antigen, the surface K-Ras antigen having a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0175] According to the composition of embodiment 1, wherein the binding agent is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0176] According to the composition of embodiment 1, wherein the binding agent is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0177] The composition according to embodiment 1, wherein the binding agent is an antibody fragment.

[0178] The composition according to embodiment 1, wherein the binding agent is a peptide or protein.

[0179] According to the composition of embodiment 1, wherein the binding agent is a peptide or protein that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0180] The composition according to embodiment 1, wherein the binder is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0181] The composition according to embodiment 1, wherein the binder is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0182] The composition according to embodiment 1, wherein the binder is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0183] The composition according to embodiment 1, wherein the binder is an antibody described in Table 1 or Table 2, or an antibody selected by the method shown in Example 4 or Example 5.

[0184] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the therapeutic agent is selected from cytotoxic agents, cell growth inhibitors, toxins or radionuclides.

[0185] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the therapeutic agent is selected from DNA damaging agents (alkylating agents), antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, and microtubule disruptors.

[0186] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the therapeutic agent is selected from chachiin, saponins, maytansine, olistatin, lidamycin, methotrexate, vincristine, pyrrolobenzodiazepines and other benzodiazepine derivatives, pyroximide, tubulolysin, α-amanitin or bouganin protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxins, carboplatin, sprisstatin C, docetaxel, tyransstatin A, or any combination thereof.

[0187] The composition according to any one of the preceding embodiments of the applicable embodiment 1, wherein the binder is linked to the therapeutic agent via a linker selected from the following: maleimide hexanoyl linker, peptide-based linker (including but not limited to valine-citrulline linker), β-glucuronide linker, SMCC linker, disulfide linker, or acid-sensitive linker.

[0188] The composition according to any one of the preceding embodiments described in embodiment 1, wherein the therapeutic agent is a radionuclide.

[0189] The composition according to any one of the preceding embodiments as described in embodiment 1, wherein the radionuclide is a radionuclide that emits β-particles or a radionuclide that emits α-particles.

[0190] The composition according to any one of the preceding embodiments described in embodiment 1, wherein the radionuclide is selected from the following radionuclides that emit alpha particles: astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223 and thorium-227.

[0191] The composition according to any one of the preceding embodiments as described in embodiment 1, wherein the radionuclide is a radionuclide that emits β-particles.

[0192] The composition according to any one of the preceding embodiments as described in embodiment 1, wherein the radionuclide emitting β-particles is selected from iodine-131, rhenium-186, yttrium-90, samarium-153 and lutetium-177.

[0193] Implementation Scheme 2: A method for treating, inhibiting or reducing the proliferation of cancer cells in a subject or killing cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of Implementation Schemes 1 and 7 and a composition comprising a bispecific antibody according to any one of Implementation Scheme 16, wherein the subject is not administered an intracellular delivery compound in conjunction with the administration of the composition, and wherein surface K-Ras antigen is expressed on the outer surface of the cancer cells.

[0194] According to the method described in Implementation Scheme 2, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0195] The method according to any one of the preceding embodiments as described in embodiment 2, wherein the administration step is performed by intravenous injection or subcutaneous injection.

[0196] Implementation Scheme 3: A method for treating, inhibiting or reducing the proliferation of cancer cells in a subject or killing cancer cells in a subject, the method comprising administering a therapeutically effective amount of a composition, wherein the composition comprises a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells.

[0197] According to the method described in implementation scheme 3, the tool is an antibody.

[0198] According to the method of embodiment 3, the tool 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 residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0199] According to the method of embodiment 3, the tool is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen comprises any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0200] According to the method of embodiment 3, the tool is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0201] According to the method of embodiment 3, the tool is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0202] According to the method of embodiment 3, the tool is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0203] According to the method described in implementation scheme 3, the tool is an antibody fragment.

[0204] The method according to embodiment 3, wherein the tool is a peptide or protein.

[0205] According to the method of embodiment 3, the tool is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0206] According to the method of embodiment 3, the tool is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a mutation at residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0207] According to the method of embodiment 3, the tool is a peptide or protein that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen contains a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0208] According to the method of embodiment 3, the tool is a peptide or protein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0209] According to the method of embodiment 3, the tool is an antibody or fragment thereof described in Table 1 or Table 2, or an antibody selected by the method shown in Example 4 or Example 5.

[0210] According to the method described in Implementation Scheme 3, the cancer cells are pancreatic cancer cells, lung cancer cells, or colorectal cancer cells.

[0211] The method according to any one of the preceding embodiments as described in embodiment 3, wherein the administration step is performed by intravenous injection or subcutaneous injection.

[0212] According to the method of any one of the preceding embodiments applicable according to embodiment 3, wherein the surface K-Ras antigen has a mutated full-length or truncated form of K-Ras at any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0213] Implementation scheme 4: Chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain contains a binding agent that specifically binds to surface K-Ras antigen expressed on the outer surface of cancer cells.

[0214] According to the chimeric antigen receptor of embodiment 4, the transmembrane domain is selected from CD3-ζ, CD28, CDE28a, CD4 or a combination thereof.

[0215] The chimeric antigen receptor according to any one of the preceding embodiments as described in embodiment 4, wherein the intracellular domain is selected from CD28, CD27, 4-1BB, OX40 and / or ICOS.

[0216] The chimeric antigen receptor according to any one of the preceding embodiments described in embodiment 4, wherein the binder is an antibody or an antibody fragment.

[0217] According to embodiment 4, the applicable chimeric antigen receptor of any of the preceding embodiments is wherein 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 residue 12, 13, or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0218] According to embodiment 4, the applicable chimeric antigen receptor of any of the preceding embodiments is wherein the binder is an antibody or antibody fragment that selectively binds to the surface K-Ras antigen, wherein the surface K-Ras antigen comprises any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0219] According to embodiment 4, the applicable chimeric antigen receptor of any one of the preceding embodiments is wherein 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 G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0220] According to embodiment 4, the applicable chimeric antigen receptor of any one of the preceding embodiments is used, wherein 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 G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0221] According to embodiment 4, the chimeric antigen receptor of any one of the preceding embodiments is applicable, wherein 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 G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0222] The chimeric antigen receptor according to any one of the preceding embodiments described in embodiment 4, wherein the binder is an antibody fragment.

[0223] The chimeric antigen receptor according to any one of the preceding embodiments described in embodiment 4, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0224] The chimeric antigen receptor according to any one of the preceding embodiments described in embodiment 4, wherein the surface K-Ras antigen has greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0225] The chimeric antigen receptor according to any one of the preceding embodiments described in embodiment 4, wherein the surface K-Ras antigen has greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0226] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0227] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the surface K-Ras antigen has greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0228] The composition according to any one of the preceding embodiments as described in Embodiment 1, wherein the surface K-Ras antigen has greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0229] The method according to any one of the preceding embodiments as described in embodiment 3, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0230] The method according to any one of the preceding embodiments as described in embodiment 3, wherein the surface K-Ras antigen has greater than 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0231] According to the method of any one of the preceding embodiments applicable according to embodiment 3, the surface K-Ras antigen has greater than 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or greater than 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0232] Implementation Scheme 5: A vector comprising a nucleotide sequence encoding a chimeric antigen receptor applicable to any of the preceding implementation schemes as described in Implementation Scheme 4.

[0233] Implementation Scheme 6: Immune cells expressing the chimeric antigen receptor described in any one of the applicable implementation schemes as described in Implementation Scheme 4.

[0234] The immune cells described in Implementation Scheme 6 are cells derived from an individual.

[0235] The immune cells described in Implementation Scheme 6 are T cells derived from an individual.

[0236] The immune cells according to embodiment 6 are selected from T cells, NK cells, dendritic cells, or mixtures thereof.

[0237] The immune cells described in Implementation Scheme 6 are T cells.

[0238] The immune cells described in Implementation Scheme 6 are CD4+ T cells or CD8+ T cells.

[0239] Implementation Scheme 7: A composition comprising immune cells expressing a chimeric antigen receptor that targets surface K-Ras antigen expressed on the extracellular surface of cancer cells.

[0240] The composition according to embodiment 7, wherein the surface K-Ras antigen is a mutated surface K-Ras antigen.

[0241] The composition according to embodiment 7, wherein the immune cells are derived from autologous, syngeneic, allogeneic, or xenogeneic sources.

[0242] The composition according to embodiment 7, wherein the immune cells are T cells, and wherein the T cells are derived from autologous, syngeneic, allogeneic, or xenogeneic sources.

[0243] The composition according to embodiment 7, wherein the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof.

[0244] The composition according to embodiment 7, wherein the immune cells are T cells.

[0245] The composition according to embodiment 7, wherein the immune cells are CD4+ T cells or CD8+ T cells.

[0246] Implementation Scheme 8: A method for treating an individual with cancer, said cancer comprising cancer cells expressing surface K-Ras antigens on the outer surface of cancer cells, said method comprising administering to the individual a therapeutically effective amount of immune cells expressing the chimeric antigen receptor as described in any one of Implementation Scheme 4.

[0247] According to the method described in embodiment 8, the surface K-Ras antigen is a mutated surface K-Ras antigen.

[0248] According to the method described in embodiment 8, the immune cells comprise cells from an individual suffering from cancer.

[0249] According to the method described in implementation scheme 8, the immune cells are T cells from an individual suffering from cancer.

[0250] According to the method described in embodiment 8, the immune cells are selected from T cells, NK cells, dendritic cells, or a mixture thereof.

[0251] According to the method described in implementation scheme 8, the immune cells are T cells.

[0252] According to the method described in Implementation Scheme 8, the immune cells are CD4+ T cells or CD8+ T cells.

[0253] According to the method described in Implementation Scheme 8, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0254] Implementation Scheme 9: A method for treating an individual with cancer, said cancer comprising cancer cells expressing surface K-Ras antigens on the outer surface of cancer cells, said method comprising administering to the individual a therapeutically effective amount of the composition described in any one of Implementation Schemes 7.

[0255] According to the method described in Implementation Scheme 9, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0256] Implementation Scheme 10: A method for diagnosing cancer associated with surface K-Ras antigen expression on the outer surface of cells, the method comprising the steps of: (a) obtaining a cell sample from an object containing a cell population; (b) exposing the cell sample to a reagent capable of binding surface K-Ras antigen, wherein the cell population is not lysed or otherwise permeabilized to avoid the reagent reacting with the intracellular form of K-Ras; and (c) measuring the presence of the reagent on the outer surface of the cell population present in the cell sample.

[0257] According to the method of embodiment 10, the surface K-Ras antigen is a mutated surface K-Ras antigen.

[0258] According to the method described in embodiment 10, the reagent is an antibody or antibody fragment that binds to the surface K-Ras antigen.

[0259] According to the method of embodiment 10, the antibody or antibody fragment contains a label, and in some embodiments, the label is a fluorescent dye.

[0260] The method according to embodiment 10 further includes the addition of a secondary antibody that binds to a portion of the antibody or antibody fragment, wherein the secondary antibody is labeled.

[0261] According to the method of any one of the applicable embodiments described in embodiment 10, step (c) is performed by applying the cell sample after step (b) to a flow cytometer.

[0262] The method according to any one of the applicable embodiments of embodiment 10, wherein step (c) is performed by electron microscopy.

[0263] The method according to any one of the applicable embodiments of embodiment 10, wherein prior to step (b), the cell sample is exposed to a cell surface membrane dye, and wherein step (c) is performed by confocal microscopy.

[0264] The method according to any one of the applicable embodiments described in embodiment 10, wherein the cell sample is selected from pancreatic cells, colorectal cells, bile duct cells, ovarian cells, endometrial cells, and lung cells.

[0265] Implementation Scheme 11: A method for diagnosing cancer associated with the expression of surface K-Ras antigen, the method comprising the steps of: (a) obtaining a cell sample from an object containing a cell population; (b) labeling proteins on the extracellular surface of the cells with a first reagent; (c) lysing the cell population to produce a cell lysate sample; (d) capturing the labeled proteins by applying the cell lysate sample to a surface coated with a second reagent, wherein the second reagent selectively binds the first reagent; (e) removing the captured labeled proteins from the surface and removing the first reagent from the captured labeled proteins to produce a cell surface protein sample; (f) exposing the cell surface protein sample to a third reagent capable of selectively binding the surface K-Ras antigen; (g) exposing the cell surface protein sample to a fourth reagent, wherein the fourth reagent carries a detectable label and binds to a portion of the third reagent; and (h) measuring the presence of the detectable label.

[0266] According to the method described in implementation scheme 11, steps (f)-(h) are performed by protein blotting.

[0267] The method according to any one of the applicable embodiments of embodiment 10 or 11 further includes subjecting the subject to the method of any one of embodiments 2, 3, 8 or 9 if the mutated form of K-Ras is expressed as a mutated surface K-Ras antigen on the outer surface of the cell.

[0268] The composition according to any one of the applicable embodiments described in Embodiment 1, wherein the therapeutic agent is therapeutically effective in inhibiting the growth or proliferation of cancer cells, or otherwise has cytotoxicity to said cancer cells.

[0269] The method according to any embodiment of embodiment 3, wherein the object is not administered an intracellular delivery compound in conjunction with the administration of the composition.

[0270] Implementation Scheme 12: The method according to any embodiment of Implementation Scheme 2 further includes the step of applying an additional therapeutic agent to the subject before or during the application of the composition, wherein the additional therapeutic agent is separate from the therapeutic agent present in the composition.

[0271] According to the method of embodiment 12, the application of additional therapeutic agents leads to an increased availability of surface K-Ras antigens for binding on the outer surface of cancer cells.

[0272] According to the method described in embodiment 12, the additional therapeutic agent is a K-Ras small molecule inhibitor.

[0273] According to the method of embodiment 12, the additional therapeutic agent is a K-Ras small molecule inhibitor, and wherein the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in surface K-Ras antigens.

[0274] According to the method of embodiment 12, the additional therapeutic agent is a K-Ras small molecule inhibitor, wherein the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0275] The method according to any one of the applicable embodiments of embodiment 12, wherein the surface K-Ras antigen contains a G12D mutation.

[0276] The method according to any one of the applicable embodiments of embodiment 12, wherein the additional therapeutic agent is applied to the subject 1 to 14 days prior to the application of the composition.

[0277] The method according to any one of the applicable embodiments of embodiment 12, wherein the additional therapeutic agent is applied to the subject 3 to 7 days prior to the application of the composition.

[0278] Implementation Scheme 13: The method according to any of the embodiments of Implementation Scheme 3, 9 or 17, further comprising the step of administering a therapeutic agent to the subject before or during the administration of the composition, wherein the administration of the therapeutic agent results in an increase in the expression of surface K-Ras antigen on the outer surface of cancer cells.

[0279] The method according to embodiment 13, wherein the therapeutic agent is a K-Ras small molecule inhibitor.

[0280] According to the method of embodiment 13, the therapeutic agent is a K-Ras small molecule inhibitor, wherein the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in surface K-Ras antigens.

[0281] The method according to embodiment 13, wherein the therapeutic agent is a K-Ras small molecule inhibitor, wherein the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0282] The method according to any applicable embodiment of embodiment 13, wherein the surface K-Ras antigen contains a G12D mutation.

[0283] The method according to any applicable embodiment of embodiment 13, wherein the therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the composition.

[0284] The method according to any applicable embodiment of embodiment 13, wherein the therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the composition.

[0285] Implementation Scheme 14: The method according to any embodiment of Implementation Scheme 8 further includes the step of administering the therapeutic agent to the individual before or simultaneously with the administration of immune cells expressing chimeric antigen receptors.

[0286] According to the method of embodiment 14, the administration of the therapeutic agent leads to an increase in the binding availability of the K-Ras antigen on the outer surface of the cancer cells.

[0287] The method according to embodiment 14, wherein the therapeutic agent is a K-Ras small molecule inhibitor.

[0288] According to the method of embodiment 14, the therapeutic agent is a K-Ras small molecule inhibitor, wherein the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in surface K-Ras antigens.

[0289] The method according to embodiment 14, wherein the therapeutic agent is a K-Ras small molecule inhibitor, wherein the K-Ras small molecule inhibitor is MRTX1133 or RMC-6236.

[0290] The method according to any applicable embodiment of embodiment 14, wherein the surface K-Ras antigen contains a G12D mutation.

[0291] The method according to any applicable embodiment of embodiment 14, wherein the therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the immune cells.

[0292] The method according to any applicable embodiment of embodiment 14, wherein the therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the immune cells.

[0293] Implementation Scheme 15: A therapeutically effective amount of the composition according to any of the embodiments of Implementation Scheme 1 or 7, or a composition comprising the bispecific antibody according to Implementation Scheme 16, for use in treating, inhibiting or reducing the proliferation of cancer cells in a subject, or killing cancer cells in a subject, wherein the subject has been administered an additional therapeutic agent other than the composition before or simultaneously with the composition.

[0294] The composition for the stated use according to embodiment 15, wherein the application of an additional therapeutic agent stimulates the expression of surface K-Ras antigen on the outer surface of cancer cells.

[0295] The composition for the intended use according to embodiment 15, wherein the subject has not yet been administered an intracellular delivery compound in conjunction with the administration of the composition.

[0296] The composition for the stated purpose according to any applicable embodiment of embodiment 15, wherein the additional therapeutic agent is a K-Ras small molecule inhibitor.

[0297] The composition for the intended use according to any applicable embodiment of embodiment 15, wherein the additional therapeutic agent is administered to the subject 1 to 14 days prior to the application of the composition.

[0298] The composition for the intended use according to any applicable embodiment of embodiment 15, wherein the additional therapeutic agent is administered to the subject 3 to 7 days prior to the application of the composition.

[0299] The composition for the stated purpose according to any applicable embodiment of embodiment 15, wherein the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0300] The composition for the intended use according to any applicable embodiment of embodiment 15, wherein the additional therapeutic agent and the composition are applied substantially simultaneously to the subject.

[0301] Implementation Scheme 16: A bispecific antibody comprising: a first binding domain linked to a second binding domain, wherein the first binding domain selectively binds to surface K-Ras antigen expressed on the outer surface of cancer cells, and wherein the second binding domain selectively binds to antigen expressed on the surface of immune effector cells.

[0302] According to the bispecific antibody of embodiment 16, the first binding domain includes a light chain variable region and a heavy chain variable region.

[0303] According to the bispecific antibody of embodiment 16, the first binding domain includes a light chain variable region and a constant region, as well as a heavy chain variable region and a constant region.

[0304] The bispecific antibody according to any embodiment of embodiment 16, wherein the second binding domain comprises a light chain variable region and a heavy chain variable region.

[0305] The bispecific antibody according to any embodiment of embodiment 16, wherein the second binding domain comprises a light chain variable region and a constant region as well as a heavy chain variable region and a constant region.

[0306] The bispecific antibody according to any embodiment of embodiment 16, wherein the surface K-Ras antigen has at least 60% homology with SEQ ID NO: 1 or SEQ ID NO: 2.

[0307] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to a region on the surface K-Ras antigen, the surface K-Ras antigen containing a mutation at residue 12, 13 or 61 based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0308] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen, said surface K-Ras antigen having any of the following mutations based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

[0309] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen, the surface K-Ras antigen having a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0310] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to the surface K-Ras antigen, the surface K-Ras antigen having a G12C mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0311] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to the surface K-Ras antigen, the surface K-Ras antigen having a G12V mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0312] The bispecific antibody according to any embodiment of embodiment 16, wherein the first binding domain selectively binds to a surface K-Ras antigen, the surface K-Ras antigen having a G12D mutation based on the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0313] The bispecific antibody according to any embodiment of embodiment 16, wherein the antigen expressed on the surface of immune effector cells is selected from TCRα, TCRβ, TCRδ, TCRγ, CD3β, CD3γ, CD3ε, CD3δ, CD3ζ, CD137, CD16 and CD64.

[0314] The bispecific antibody according to any embodiment of embodiment 16, wherein the antigen expressed on the surface of immune effector cells is CD3ε.

[0315] The bispecific antibody according to any embodiment of embodiment 16, wherein the immune effector cells are selected from T cells, neutrophils, macrophages, monocytes and NK cells.

[0316] Implementation Scheme 17: A method for treating, inhibiting or reducing the proliferation of cancer cells in a subject or for killing cancer cells in a subject, said method comprising administering a therapeutically effective amount of the bispecific antibody described in any embodiment of Implementation Scheme 16, wherein the surface K-Ras antigen is expressed on the outer surface of the cancer cells.

[0317] According to the method of embodiment 17, the subject is not administered an intracellular delivery compound in conjunction with the administration of the bispecific antibody.

[0318] According to the method described in Implementation Scheme 17, the cancer cells are selected from pancreatic cancer cells, lung cancer cells, bile duct cancer cells, ovarian cancer cells, endometrial cancer cells, or colorectal cancer cells.

[0319] The method according to any embodiment of embodiment 17, wherein the administration step is performed by intravenous injection or subcutaneous injection.

[0320] The method according to any embodiment of embodiment 17, wherein the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0321] Implementation Scheme 18: A composition comprising: a tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells, wherein the tool is linked to a therapeutic agent, and wherein the composition does not contain an intracellular delivery compound.

[0322] The composition according to embodiment 18, wherein the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0323] Implementation Scheme 19: A bispecific antibody comprising: a first tool for selectively binding to a surface K-Ras antigen expressed on the outer surface of cancer cells and a second tool for selectively binding to an antigen expressed on the surface of immune effector cells, wherein the first tool is linked to the second tool.

[0324] According to the bispecific antibody of embodiment 19, the surface K-Ras antigen is a full-length or truncated form of K-Ras and has a mutation at any of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

[0325] Implementation Scheme 20: A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a tool capable of specifically binding to surface K-Ras antigens expressed on the outer surface of cancer cells.

[0326] According to the chimeric antigen receptor of embodiment 20, the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any of the following amino acids / residues based on the amino acid position in SEQ ID NO:1 or SEQ ID NO:2: 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).

[0327] Example

[0328] Example 1: Inhibition of K-Ras signaling from the cell surface provides control over tumor growth

[0329] We utilized the hyperthermophilic archaea sulfurophyte (Folium sulfideum) Sulfolobus solfataricusA charge-neutralizing variant of the Sso7d protein, named R11.1.6 (SEQ ID NO: 5-ATVKFTHQGEEKQVDISKIKWVIRWGQYIWFKYDEDGGAKGWGYVSE KDAPKELLQ MLKKR), binds with high affinity and specificity to switch I of 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. Figure 1a To deliver this antagonistic scaffold intracellularly, we conjugated it to a cell-penetrating peptide (CPP): R9 (R9-R11.1.6) or penetratin (penetratin-R11.1.6). Additionally, we used R11.1.6 as an internal control in the absence of any cell-penetrating peptide. Figure 1b Next, we evaluated the growth and viability of two pancreatic cancer cell lines driven by K-Ras mutations of G12D (Panc-1) or G12C (MiaPaca-2) in vitro. We seeded 20,000 cells / well in DuPont modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) in flat-bottomed 96-well plates and counted the cells after 5 days. Cell viability was calculated based on trypan blue exclusion, where dead cells appeared blue due to absorption of the viability dye, and live cells remained unstained. Viability was measured using an Invitrogen (ThermoFisher, USA) Countess II FL cell viability and cell counter instrument. We noted a G12D mutation-specific inhibition of tumor cell proliferation (…). Figure 1c - Growth (left panel) and viability (right panel). Surprisingly, R11.1.6 altered Panc-1 growth and viability to a similar degree with or without conjugation to cell-penetrating peptides. Similar data were replicated in K-Ras-driven lung and colorectal cancer cell lines. Figure 1d R11.1.6-mediated inhibition of Panc-1 growth and viability is similar to that of the G12D inhibitor MRTX1133 (PMID: 36216931), and the combined use of these two agents enhances the therapeutic effect. Figure 1e ).

[0330] Further evaluation of growth inhibition was performed using control proteins derived from *Leymus sulphureus* with similar molecular weights. These included mutant forms of R11.1.6 (in which codons in the region in contact with the K-Ras mutant are scrambled to prevent binding) (SEQ ID NO: 6 - ATVKFTHQGEEKQVDISKIKFVWRKGYVRIWGYDEDGGWGAGKYVSE KDAPKELLQMLKKR), M11.1.2 (a scaffold protein that binds to mouse serum albumin) (SEQ ID NO: 7 - ATVKYTYRGEEKRVDISKIKWVNRWGQHLAFKYDKGGGAAGYGWVSE KDAPKELLQ MLEKR), and E11.4.1 (which binds to human EGFR) (SEQ ID NO: 8 - ATVKFTYQGEEKQVDISKIMYVIRGGQRIAFGYDEGDGAWGDGIVSE KDAPKELLQMLEKQ). Compared to R11.1.6, none of these control constructs inhibited tumor growth. Figure 1f Although some constructs, such as E11.4.1, are able to bind to the surface of tumor cells ( Figure 1gFurthermore, the inhibition of tumor growth by wild-type R11.1.6 was associated with a typical reduction in signal transduction via the AKT and ERK pathways. We detected lower phosphorylation of AkT (at serine 473) and ERK1 / 2 (threonine 202, tyrosine 204) by fluorescence flow cytometry. We started with 200,000 cells in 96-well V-plates and used BD phosflow buffer set III (BD Biosciences, catalog number 558050) to stabilize and optimize the phosphorylation fraction. Next, we stained cells with either PE-conjugated antiphospho-Akt (ser473, clone D9E) (Cell Signaling Technologies, catalog number 5315S) at a concentration of 0.125 μg / 100 μl or PE-conjugated antiphospho-ERK1 / 2 (ThermoFisher, USA, catalog number 12-9109-42) at a concentration of 0.125 μg / 100 μl. Cells were washed in FACS staining buffer and run on an LSR Fortessa II flow cytometer (BD Biosciences, USA). Acquisitions were performed using FACS DIVA software (BD Biosciences) and analyzed in FlowJo software (Tree Star, OR, USA). This is an important finding because both Akt and ERK1 / 2 are important downstream molecules of K-Ras signaling, as described by small molecule inhibitors of K-Ras (PMID: 12509763). Figure 1a ).

[0331] Surprisingly, even in the absence of cell-penetrating peptides, and even assuming that mutant and wild-type K-Ras signaling originates only from different intracellular locations, inhibition was significant. However, we suggest that this small 7 kDa scaffold protein and the disordered cell membrane of malignantly transformed cells may allow cytoplasmic penetration even in the absence of cell-penetrating peptides. To evaluate this possibility, we investigated the cellular localization of the R11.1.6 construct by flow cytometry and confocal microscopy. For flow cytometry experiments, cells were first stained with a Live / Dead far redfixable staining kit (ThermoFisher, USA, catalog number L10120). Specifically, 0.2–0.4 × 10⁻⁶ cells were stained with a wide range of 10⁻⁶ cells. 6Cells were seeded in V-shaped plates, and 100 μl of dye (1:1000 dilution of stock concentration according to the manufacturer's protocol) was added to each well. Cells were incubated at room temperature for 15 minutes. Next, cells were washed with PBS containing 2.5% FBS and 0.1% sodium azide (FACS buffer). Cells were then surface-stained by adding 100 μl / well of a 10 μg / ml anti-His PE-labeled antibody (mouse anti-human antibody, Biolegend, CA, USA, catalog #362603). Cells were washed twice with FACS buffer and fixed with Cytofix buffer (BD Biosciences, catalog #554655). The same antibody and reagents were used for intracellular staining, except that cells were permeabilized with BD perm / wash reagent (BD Biosciences, catalog #554723) before adding the PE anti-His antibody. Cells were collected using an LSR Fortessa II flow cytometer with FACS DIVA software (BD Biosciences, USA) and analyzed using FlowJo (TreeStar, USA). For confocal microscopy experiments, cell surface staining was performed in a similar manner to flow cytometry. After staining, cells were carefully plated on 25 mm × 75 mm slides (1 mm thick) and mounted with Prolong Gold Antifade reagent with DAPI (CellSignaling Technologies, USA, Catalog No. 8961). Cells were imaged using a Nikon A1 rotating disk microscope. Analysis was performed using ImageJ software (developed and licensed by NIH). Since all constructs contain polyhistidine tags (His tags), the analysis was performed accordingly. Figure 1h We tested this using fluorescently labeled anti-His-tagged antibodies. For this purpose, Panc-1 (G12D-mutant K-Ras) or MiaPaca-2 (G12C-mutant K-Ras) cell lines were cultured with R9-R11.1.6, penetratin-R11.1.6, or R11.1.6 (without cell-penetrating peptides) for 24 or 96 hours, and their subcellular locations were determined by staining with fluorescently labeled secondary antibodies that recognize the His tag. Figure 1hAs shown, compared to the saline control (defined as 1), there was no significant R11.1.6 presence within tumor cells. However, in Panc-1, the increased binding of R11.1.6 to the tumor cell surface was significant, while the binding to the tumor cell surface was not significant in MiaPaca-2. Considering that R11.1.6 without the cell-penetrating peptide can inhibit tumor cell growth, flow cytometry (…) Figure 1i or high-resolution confocal microscopy ( Figure 1f Surprisingly, none of the constructs crossed the cell membrane; instead, they bound to the cell surface. This binding appears to be mutation-specific, as very few constructs were observed on the surface of MiaPaca-2 cells carrying the G12C mutation that does not bind R11.1.6. Figure 1h , Figure 1i , Example 2: Functional K-Ras is expressed on the extracellular surface of malignant and non-transformed cells In summary, our data suggest that the inhibition of tumor cell growth and viability appears to be mediated by the mutation-specific binding of K-Ras antagonists to the cell surface. This is surprising because K-Ras have been thought to specifically target intracellular cell membranes and subcellular organelles (PMID: 21924373) and have never been described on the cell surface.

[0332] Figure 2a

[0333] K-Ras has been defined as an intracellular protein with no known surface expression. No K-Ras on cell surfaces has been reported in the scientific literature. Because the specificity and sensitivity of available anti-K-Ras antibodies have previously been questioned (PMID: 28951536), we conducted extensive validation work on a variety of commercially available and publicly described domain-specific antibodies for our research. For this purpose, we utilized the RASless mouse embryonic fibroblast system (MEFs), in which MEFs lacking all RAS proteins are rescued via lentiviral transduction of wild-type RAS, mutant RAS, or a V600E mutant variant of BRAF, thus allowing survival and proliferation without all endogenous Ras proteins (PMID: 33977488 and cancer.gov / research / key-initiatives / ras / ras-central / blog / 2017 / rasless-mefs-drug-screens). We tested the antibodies by flow cytometry according to the protocol described in Example 1. Most of the tested antibodies showed some nonspecific binding and were associated with BRAF. V600E Rescue of RASless MEFs Responses ( Figure 2c-b). Some antibodies can detect surface K-Ras antigens on cell surfaces in a sensitive and specific manner, and preferentially bind to the G12D mutant version. For example, human anti-K-ras antibody 1 (intact heavy chain (HC) - SEQ ID NO: 9 - EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI HC CDR1 - SEQ ID NO: 10 - GFTSRHPG; HC CDR2 - SEQ ID NO: 11 - ISHDGSKK; HC CDR3 - SEQ ID NO: 12 - ATSLYSSMDL; Intact light chain (LC) - SEQ ID NO: 13 - QSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTV 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 - EVQLLEPGGGVVQPGRSLRLSCTNSGFSFSGYAMHWVRQAPGKGLEWVAVISFDGSHKYYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCASGGNYYGSGTIVSHGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG; HC CDR1 - SEQ ID NO: 18 - GFSFSGYA; HC CDR2 - SEQ ID NO: 19 - ISFDGSHK; HCCDR3 - SEQ ID NO: 20 - ASGGNYYGSGTIVSHGMDV; complete LC - SEQ ID NO: 21 - QSVLTQPASVSGSPGQSITISCTGTSNDIGAYNYVSWYQQHPGKAPKLMIYDVNNRPSGVPDRFSGSKSGNMASLTISGLQAEDDADYYCSSYTSSSTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS; LC CDR1 - SEQ IDNO: 22 - SNDIGAYNY; LC CDR2 - SEQ ID NO: 23 - DVN;LC CDR3 (SEQ ID NO: 24-SSYTSSSTLVV) staining was clearly visible on the surfaces of K-Ras-rescued MEFs as well as Panc-1 and MiaPaca-2. Figure 2e -d). Further characterization of binding to 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 2f Additional G12D K-Ras tumor cell lines also showed improvement after surface staining with human anti-K-Ras antibody 1. Figure 2g All staining was performed on ice for 30 minutes in V-bottom 96-well plates at an antibody concentration of 10 μg / ml and a volume of 100 μl. Surface staining of live Panc-1 cells with human anti-K-Ras antibody 1 was also observed by high-resolution confocal microscopy. Figure 1i ). Used in Example 1 Figure 2h Confocal microscopy was performed under the same conditions.

[0334] Surface staining was also observed using antibodies against G12V-mutated K-Ras (clone D2H12) and G12D-mutated K-Ras (clone HL10). Figure 2i ). Using BRAF V600E No obvious staining was observed on the rescued RASless MEFs or MiaPaca-2 (a cancer cell line with a K-Ras G12C mutation).

[0335] A group of antibodies showed resistance to BRAF V600E The reactivity of rescued MEFs, or the presence of multiple nonspecific bands in addition to the 22 kDa K-Ras band ( Figure 2j Only rabbit anti-G12D mutant K-Ras (clone D8H7, CellSignaling Technology), mouse anti-K-Ras (clone 4E8, Iowa Hybridoma Bank), and mouse anti-K-Ras (clone 3B10-2F2, Sigma Aldridge) showed sensitivity and specificity against G12D mutant K-Ras 4B, PanK-Ras, and K-Ras 4B splice variants, respectively, via Western blotting. Figure 2kFor Western blotting experiments, cells were lysed in situ at 4°C for 30 min 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 mixture of protease inhibitors. The resulting cell lysates were clarified by centrifugation at 18,800 g. After washing three times with cell lysis buffer, proteins were resolved on a 10% Tris-glycine SDS-PAGE gel (Bio-Rad) and transferred to an Immobilon-P PVDF membrane. The membrane was then blocked for 1 h at 20°C with PBS containing 3% BSA (0.1% Tween 20). Proteins were then detected with the appropriate primary antibody, followed by a suitable secondary antibody conjugated to horseradish peroxidase. Horseradish peroxidase activity was detected using Pierce-enhanced chemiluminescent substrates (Thermo Fisher Scientific) according to the manufacturer's instructions. Chemiluminescence signals were obtained using the ChemiDoc MP imaging system and analyzed using Image Lab 5.1 software (both from Bio-Rad).

[0336] For the K-Ras knockdown assay, we silenced K-Ras gene expression using in vitro siRNA technology. Following the manufacturer's protocol, cells were trypsinized and plated in 500 μl 24-well plates, with a final siRNA concentration of 25 nM in the DharmaFECT™ transfection reagent. In the Panc-1 cell line, K-Ras protein was knocked down using siRNAs (Dharmacon™ Reagents, ON-TARGETplus Human SMARTpool K-RAS siRNA) targeting multiple splice variants of K-Ras mRNA, and the downregulation was confirmed by RT-PCR and intracellular K-Ras proteoblotting. In siRNA-treated cell lines, surface staining of K-Ras by human anti-K-Ras antibody 1 was also reduced. Figure 2l ).

[0337] Previously, it has been described that peptide fragments of mutant and wild-type K-Ras can be presented on the cell surface against their own MHC class I background (PMID: 36099883, PMID: 27959684, PMID: 34272369). We created an MHC class I deficient Panc-1 cell line by CRISPR-Cas-9 excision of β-2 microglobulin and noted that such mutants showed the same staining with human anti-K-Ras antibody 1 as parental cell lines with sufficient MHC class I (PMID: 36099883, PMID: 27959684, PMID: 34272369). Figure 2m ).

[0338] In various cell lines, proteins on cells exposed to the extracellular environment were treated with reactive biotin esters (Pierce Cell Surface Protein Biotinylation and Separation Kit, #A44390) and separated from cell extracts using streptavidin beads based on an established method (PMID:19341246). Similar to the flow cytometry data described above, K-Ras was detected in the surface fraction by Western blot analysis using a K-Ras-specific antibody, although in much smaller amounts than inside the cell membrane. Example 3: Validation of antibody drug conjugates (ADC) for selective killing of cancer cell lines by using mutation specific antibodies ).

[0339] In summary, our data show that both tumors and normal tissues can express K-Ras on their surfaces, and that the binding of such surface K-Ras to protein therapy antagonists can improve cancer growth in a mutation-specific manner.

[0340] mAbs Sci Rep

[0341] 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 method involves an antibody conjugated via a linker to a cytotoxic payload targeting a target antigen expressed on the surface of cancer cells. Such a method reduces systemic exposure, thus reducing toxicity, and provides a significant opportunity for targeted killing of cancer cells (e.g., reviewed in Drago et al. Nat RevClin Oncol. 2021 Jun;18(6):327-344 doi.org / 10.1038 / s41571-021-00470-8). For an ADC to function as a selective agent on target cells, it needs to: a) selectively recognize tumor antigens, and b) be able to kill target cells at concentrations that are non-toxic to cells not recognized by the antibody. The use of mutated surface K-Ras antigens as targets for ADCs has never been explored.

[0342] Saponins are 30 kDa ribosome inhibitors that cannot cross cell membranes without assistance (unless at extremely high concentrations). However, when conjugated with internalizing antibodies, as previously described (DOI: 10.3390 / toxins14030184), they can be used to precisely and specifically kill target cells with little or no off-target toxicity (see, e.g., Lund et al., Figures 3-11 , 6(4), 1038-1050, doi.org / 10.4161 / mabs.28207). Saponins have been used as ADC agents targeting tumor-expressed surface molecules, such as breast cancer-associated Her-2, malignant melanoma-associated CSPG-4, breast cancer-associated EGFR and FRα (see, for example, Hoffmann et al.). Figure 3 10, 8869 (2020), doi.org / 10.1038 / s41598-020-65860-x), thus a validated warhead for ADCs. The commercially available Fab-ZAP assay (Advanced Targeting Systems Inc.) allows for the precise delivery of saponins by a secondary antibody linked to a primary antibody specific for tumor surface antigens, enabling the testing of the efficacy of antibodies against surface antigens for ADCs. For the experiments described below, we used the Fab-ZAP Rabbit Kit [IT-57, KIT-57] (atsbio.com / products / it57 / ), which consists of a goat anti-rabbit monovalent antibody and a chemical conjugate of a ribosome-inactivating protein (i.e., saponins). The antibody used in this kit is an affinity-purified polyclonal antibody against the heavy and light chains of rabbit IgG.

[0343] To investigate the potential of using mutant K-Ras expressed on the cell surface (referred to as mutant surface K-Ras antigen) as a target for ADCs, we 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 mutant surface K-Ras as described in Example 2. Specifically, we used Panc-1 (pancreatic cancer cell line carrying the K-Ras G12D mutation), Capan-2 (pancreatic cancer cell line carrying the K-Ras G12V mutation), AsPC-1 (pancreatic cancer cell line carrying the K-Ras G12D mutation), LS180 (colon cancer cell line carrying the K-Ras G12D mutation), SW480 (colon cancer cell line carrying the K-Ras G12V mutation), Capan-2 (pancreatic cancer cell line carrying the K-Ras G12V mutation), and two lung cancer cell lines (SK-LU-1 carrying the K-Ras G12D mutation and NCI-H2444 carrying the K-Ras G12V mutation). Following the manufacturer's instructions in the Fab-ZAP kit, serial dilutions of the antibody or an unrelated rabbit IgG isotype control antibody were pre-incubated with 4.5 nM FabZAP reagent before being added to the designated cell lines. Cells were seeded into 96-well round-bottom plates according to the manufacturer's protocol, with a total volume of 100 μl per well (including Fab-Zap and different concentrations of antibody or corresponding controls). After incubation at 37°C in a 5% CO2 incubator for 5 days, the percentage of viable cells was determined using XTT assay according to the described protocol (Advanced Targeting Systems Inc. Cytotoxicity Assay Protocol) compared to control wells containing only 4.5 nM FabZAP. Briefly, after incubation, each well was supplemented with 50 μl of XTT reagent to bring the total volume to 150 μl. The plates were then incubated at 37°C for at least 30 minutes, and the absorbance was read at 450 nm using a plate reader. This assay relies on detecting the number of remaining viable cells as measured by cellular metabolism via the colorimetric molecules in the chromogenic reagent on the day of chromogenic development. The mean absorbance in untreated wells is depicted as 100%, with 3 replicates in each treatment group, and is expressed as a percentage relative to the control. See below. Figure 3 As shown, cytotoxicity data were analyzed by comparing well readings (target antibody and Fab-ZAP) with well readings (Fab-ZAP only and saponin only) of control wells (expressed as percentages). GraphPad Prism software was used to analyze the data and plot survival % as a function of primary antibody concentration.

[0344] Saponins alone do not induce cell death at concentrations below 100 nM. Figure 4 Therefore, no significant toxicity was observed at the 4.5 nM concentration used in the ADC assay without a target. Figure 4 (The arrow in the image). To determine whether the mutant K-Ras-specific targeting antibody could be used for an ADC in the presence of other non-toxic 4.5 nM saponin targeting reagents, we next incubated Panc-1 (K-Ras G12D mutant) with variable concentrations of rabbit anti-human G12D mutant K-Ras antibody conjugated with Fab-Zap reagent and noted concentration-specific death (…). Figure 4 Under the same conditions, rabbit IgG conjugated to the Fab-Zap control did not cause killing ( Figure 3 No significant cytotoxicity was observed when using a Fab-Zap conjugate anti-G12V mutant K-Ras-specific antibody in this K-Ras G12D mutant tumor, or when using an anti-G12D mutant K-Ras antibody alone. Figure 5 As mentioned above, saponins alone do not exhibit cytotoxicity until concentrations reaching 100 nm. Figure 6 For other K-Ras G12D-mutated cancers, such as the AsPC-1 pancreatic cancer cell line (…), Figure 7 ), LS180 colorectal cancer cell line ( Figure 8 ) and SK-LU-1 lung cancer cell line ( Figure 9 Similar results are evident for G12V-mutated cancers, such as Capan-2 pancreatic cancer. Figure 10 ), SW480 colorectal cancer ( Figure 11 ) and NCI-H2444 lung cancer cell line ( Figure 12a Similar results were observed with the use of K-Ras G12V-specific antibodies conjugated with Fab-Zap, but anti-K-Ras G12D mutant-specific antibodies did not induce ADC-induced cell death in these K-Ras G12V-expressing tumors. Furthermore, in the K-Ras G12C-expressing pancreatic cancer cell line MiaPaca-2, the use of K-Ras G12D-specific antibodies conjugated with Fab-Zap did not lead to ADC-induced cell death. Figure 12b ).

[0345] In another experiment, human anti-K-Ras antibody 1 was used in Panc-1 ( Figure 12c ) and ASPC-1 ( Figure 12d Similar results were shown in cancers with the K-Ras G12D mutation. For cancers with the G12C mutation (e.g., Mia-Paca-2), similar results were observed. Figure 12eAnd for wild-type cancers (such as MXPC3) Example 4: Development of novel antibodies against surface expressed K-Ras Similar results were observed, but the anti-K-Ras G12D-mutant specific antibody did not induce ADC-induced cell death in these K-Ras G12V-expressing tumors. The saponin control showed cytotoxicity only in Panc-1, ASPC-1, Mia-Paca-2, and BXPC3 cell lines. Example 5: Panning of cell surface expressed K-Ras using a human antibody library Finally, to demonstrate specificity, RASlessless MEFs rescued with BRAF, (G12D) K-Ras 4B, K-Ras-4A, or K-Ras 4B were tested in the alternative Fab-Zap assay with human anti-K-Ras antibody 1 as described above, except that a second human Fab-Zap reagent was used (atsbio.com / products / it51 / ). No increase in cytotoxicity was observed with isotype IgG + FabZap or human anti-K-Ras antibody 1 alone. Human anti-K-Ras antibody 1 + FabZap did not affect the survival of MEF-BRAF or MEF-K-Ras-4A, but produced cytotoxicity in MEF-(G12D) K-Ras 4B and MEF-K-Ras-4B (Figure 13).

[0346] In summary, our data suggest that using surface-binding antibodies specific to mutated forms of surface K-Ras antigens could provide a viable approach to generating tumor-killing ADCs without off-target killing of cells that do not express the same mutated form of surface K-Ras antigen.

[0347] Example 6: Proof of concept for development of CAR-T cells targeting K-Ras

[0348] To develop K-Ras-specific antibodies binding to GDP or GTP, mice will be immunized with either GDP-binding K-Ras or GppNHp-binding K-Ras (GppNHp is a non-hydrolyzable analog of GTP). Prior to immunization, the K-Ras protein will be loaded with either nucleotide. As another strategy for developing mutation-specific antibodies, a peptide containing a G12D mutation spanning amino acids 5–17 of K-Ras (Ac-KLVVVGADGVGKSC-amide) (SEQ ID NO: 13) will be used to immunize additional mice. To minimize responses to wild-type K-Ras binding, some mice will first be attenuated with wild-type K-Ras and treated with cyclophosphamide to kill wild-type K-Ras-responsive B cells. All mice will then be intraperitoneally immunized with a protein or peptide mixed with complete or incomplete Freund's adjuvant. The resulting spleen cells and / or lymph nodes from the immunized mice will be fused with NS1 myeloma cells to form hybridomas. Subsequent antibody clones will be screened by ELISA and flow cytometry. The ELISA will detect their binding to wild-type K-Ras bound to GDP, wild-type K-Ras bound to GppNHp, (G12D) K-Ras bound to GDP, and (G12D) K-Ras bound to GppNHp. The flow cytometry will detect their binding to K-Ras expressed on the surface of AsPC1 (G12D homozygous), SU.86.86 (G12D homozygous), and / or BxPC3 (K-Ras wild-type) cells. Preferred antibody clones will recognize only the K-Ras antigen used to immunize mice. Antibody clones will be screened again before fusion, after fusion, and after subsequent subcloning.

[0349] Example 7: Isolation of cell samples for diagnostic testing of surface K-Ras expression in patient samples

[0350] Another approach to developing antibodies specific to mutated K-Ras (including K-Ras with mutations such as G12D, G12V, G12C, G13D, and G12R) is to pan-pick pre-existing libraries of human polyclonal antibodies. Two existing libraries of human polyclonal antibodies are used, one containing antibody sequences from a natural human donor and the other from a human donor from an autoimmune patient. These antibodies are cloned, engineered into single-chain variable fragments, and introduced into a phage display library that can be panned and screened for mutated K-Ras-specific antibodies that bind to surface K-Ras antigens expressed on the cell surface.

[0351] Several different strategies will be used to select antibodies that recognize mutant K-Ras binding to GDP or GTP. In all strategies, the library will initially be depleted of antibodies binding to wild-type K-Ras protein (when binding any nucleotide being screened). Antibodies binding to mutant K-Ras binding to GDP or GppNHp in the antibody library will then be panned. Up to three additional rounds of screening can be used after the first round of depletion / selection. Each round of successive screening will utilize the depletion of antibodies binding to wild-type K-Ras and the selection of mutant K-Ras antibodies, either as proteins or as surface K-Ras antigens expressed on the cell surface. After the first round of selection, panning will be performed in the presence of an overabundance of competitive wild-type K-Ras protein or cells expressing wild-type K-Ras to further minimize the selection of phages binding to wild-type K-Ras. To enrich antibodies binding to specific K-Ras epitopes, several rounds of panning with specific proteins containing the desired epitopes are performed. These specific proteins will include R11.1.6 and antibodies specific to mutated K-Ras (e.g., K-Ras G12D).

[0352] Example 8: Diagnostic testing of surface K-Ras expression by flow cytometry

[0353] Chimeric antigen receptor (CAR) T cells are used for selective tumor killing using a pre-established method of targeting T cells with a defined surface target on tumor cells. To investigate the potential of surface K-Ras as a CAR T cell target, human anti-K-Ras antibody 1 and rabbit anti-human K-Ras G12D antibody (HL-10 antibody) were engineered into single-chain antibody fragments. The single-chain antibody fragments contained an 18-amino acid linker, GSTSGGSGKPGSGEGSTKG (SEQ ID NO: 25), for linking the light and heavy chains. scFv was inserted into a second-generation CAR cassette containing a GM-CSF signal peptide, a hinge region, a CD28 transmembrane and co-stimulatory domain, and a CD3ζ activation domain. The resulting scFv-CAR gene was introduced into a lentiviral vector. Target T cells were then activated, transduced, and amplified using the scFv-CAR gene.

[0354] Two measurements were used to assess CAR-T cell activity: cytokine secretion and cell killing. To measure cytokine secretion, expanded anti-K-Ras CAR-T cells were incubated with cells expressing K-Ras on their surface at a 1:1 ratio. After 16 hours of incubation, the cell supernatant was removed. The levels of secreted IL-2 and IFN-γ in the supernatant were measured by ELISA. To measure cell killing, 10... 5 Before culturing CAR-T cells, adherent target cells are grown to confluence. Cells are co-cultured for 2-3 days, and the impedance of the cell layers is measured over time. Total cell lysis is calculated by the change in impedance over the experimental period.

[0355] Figure 14

[0356] Human tissue from cancerous sites, along with adjacent normal tissue, was preserved immediately after surgery. The tissue was immediately processed to isolate single cells according to the following protocol. Briefly, each tissue was cut into very small pieces using a sterile blade. The tissue was then resuspended in RPMI 1640 complete medium (containing 10% FBS and 1X penicillin-streptomycin) and run for two cycles (1 minute each) in a MACS dissociator (Milteny Biotech) to initiate dissociation. The tissue was then digested in a digestion medium consisting of IMDM medium supplemented with type II collagenase (0.5 mg / ml) and DNase (0.5 μL / sample). For each tissue size of 1 mg, the total digestion medium volume used was 7 ml. Next, the tissue immersed in the digestion medium was incubated at 37°C with shaking at 850 rpm for 35 min.

[0357] Following digestion, the tissue was run again in a mild MACS dissociator to break down the digested tissue and re-obtain single cells. The cells were then washed twice in 1X PBS to remove any digestive enzymes and DNases. The cells were then passed through a 70 μm filter and resuspended in RPMI complete medium.

[0358] Example 9: Diagnostic testing of surface K-Ras expression by western blot

[0359] As shown 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 tissues was performed using human anti-K-Ras antibody 1 and human K-Ras antibody 2. Figure 2m In this embodiment, cells obtained from fibrotic lung tissue showed increased K-Ras surface expression compared to cells from normal lung tissue.

[0360] Example 9: Diagnostic testing of surface K-Ras expression by electron microscopy

[0361] Once patient samples have been obtained using the method of Example 1, Western blot analysis of surface K-Ras expression proteins can be performed. Cell surface proteins can be labeled using the Pierce™ Cell Surface Biotinylation and Separation Kit (ThermoFisherScientific, catalog number: A44390) and then separated. Cells are first labeled with EZ-Link Sulfo-NHS-SS-Biotin (a thiol-cleavable amine-reactive biotinylating agent). Cells are then lysed, and labeled proteins are captured with NeutrAvidin agarose. A flow-through can then be collected from the agarose column, representing the unbiotinylated fraction, which is intracellular protein after cell lysis. The NeutrAvidin agarose-bound biotinylated protein fraction can be eluted using a reduction buffer containing a mixture of protease inhibitors. Dithiothreitol (DTT) is used to elute to reduce the disulfide bonds in the biotinylated label, resulting in the release of unlabeled bound proteins.

[0362] Then, using the eluted surface biotinylated protein fractions and intracellularly flowing protein fractions, Western blotting can be performed with different K-Ras antibodies (see Example 2). Example 10: Diagnostic testing of surface K-Ras expression by confocal microscopy 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 mixture of protease inhibitors. The performance of gel electrophoresis and protein blotting is known to those skilled in the art.

[0363] Figure 2g

[0364] Live Panc-1 cells were pre-labeled with anti-KRAS antibody or IgG control, followed by staining with gold-labeled secondary antibody. Cells were then post-fixed for 1 h in Sorensen's buffer containing 2% osmium tetroxide, dehydrated in a series of increasing ethanol solutions (30%, 50%, 70%, 90%, 100%), and embedded in Epon / Araldite resin. Samples were then stained in a premixed solution of uranyl acetate and lead citrate and examined at 200V using an F20 electron microscope with a field emission gun (FEG) electron source. Data collection for negative staining was then performed using a Gatan GIFQuantum with DualEELS and high-speed spectral imaging, employing a 4k×4k superscan CCD camera.

[0365] Example 11: Increasing surface K-Ras availability by treatment combinations

[0366] Identification of surface KRAS by confocal microscopy was performed using the CellBrite® Steady Membrane Staining Kit (Biotium Inc. Fremont, CA) using a combination of anti-KRAS antibody and cell surface membrane dye. In short, live cells were labeled with CellBrite® Steady 488 dye (green fluorescence). Cells were labeled with the 1X dye in cell culture medium and then incubated at 37°C for 30 minutes. Immediately afterwards, the cells were washed twice with medium supplemented with 10% FBS to quench any unbound dye. Next, the cells were stained with human anti-K-Ras antibody 1 on ice for 30 minutes, followed by staining with anti-human PE (red fluorescence) secondary antibody on ice for 30 minutes. Cells were kept healthy and viable until this staining step. Finally, the cells were washed, mounted on slides, and sealed with ProLong™ Gold Antifade mounting solution containing DAPI (ThermoFisher Scientific, catalog number: P36931).

[0367] Images were acquired using a rotating disk confocal microscope equipped with an Airyscan (Zeiss LSM 880) and imaged in multiple modes using a ZEISS Airyscan 2 for effective super-resolution imaging over a large field of view. Unless otherwise specified, Z-slices were set to 0.2 μm. For high-resolution imaging, a Plan-Apochromat 40x / 1.3 objective was used, or for confocal modal performance, a 63x 1.4NA Plan-Apochromat objective was used, with a 568 nm excitation and 570–615 nm emission filters. Confocal images were deconvolved using a constrained iterative deconvolution routine n (Zen software version 2.3, Carl Zeiss, Obekochchen, Germany).

[0368] Confocal microscopy images reveal the differences in KRAS staining when stained with anti-KRAS antibody (left image). Figure 15a The control antibody showed no visible red staining (right image). Membrane dyes and DAPI were used as cell markers to distinguish between surface and intracellular regions.

[0369] Figures 15b-15c

[0370] We examined the effects on K-Ras surface expression after treating tumor cell lines with chemotherapy agents (fluorouracil (5-FU)) or mutation-specific K-Ras small molecule inhibitors (G12D-specific - MRTX1133, G12C-specific - MRTX849).

[0371] For 5-FU treatment, 100,000 PANC-1 cells were seeded into 24-well replicates. Cells were allowed to grow for 24 hours, and then 10 μM 5-FU was added to the culture medium. At 0, 72, and 120 hours, cells were dislodged using TrypLEexpress and resuspended in buffer containing PBS (pH 7.2), 5% FCS. Cells were stained with human K-Ras antibody 1. All staining was performed on ice for 30 minutes at an antibody concentration of 10 μg / ml and a volume of 100 μl. Surface K-Ras staining was quantified by measuring the MFI of cells treated with either the simulated treatment or those treated with 5-FU. Fold change was calculated by normalizing cells stained with isotype controls. Figures 15a-15c As shown, treatment with 5-FU increased the availability of K-Ras on the surface of Panc-1 cells, with longer treatment times resulting in a greater amount of available K-Ras. Treatment with 5-FU chemotherapy and potentially other chemotherapy therapies further increased the availability of surface K-Ras.

[0372] To treat with a mutation-specific K-Ras small molecule inhibitor, 100,000 PANC-1 cells were seeded into a set of 24-well replicates, and 100,000 MiaPaca-2 cells were seeded into a second set of plates. Cells were allowed to grow for 24 hours, and then 10 nM MRTX1133 or 10 nM MRTX849 was added to the culture medium. At 0, 72, and 120 hours, cells were detached and resuspended in a buffer containing PBS (pH 7.2), 5% FCS. Cells were stained with human K-Ras antibody 1. All staining was performed on ice for 30 minutes at an antibody concentration of 10 μg / ml and a volume of 100 μl for 96-well V-bottom plates. Surface K-Ras staining was quantified by measuring the MFI of cells treated with the simulant treatment or those treated with MRTX1133 or MRTX849. Fold change was calculated by normalizing cells stained with the isotype control. Example 12a: Increased K-Ras surface availability by K-Ras small molecule treatment enhances surface K-Ras targeting by antibody drug conjugates (ADC) killing As shown, treatment with MRTX1133 increased the availability of K-Ras on the surface of PANC-1 cells (but not MiaPaca-2 cells). Figure 15a-15cAs shown, treatment with MRTX849 increased the availability of K-Ras on the surface of MiaPaca-2 cells (but not PANC-1). The results indicate that treatment with mutation-specific small molecule inhibitors only affects the availability of K-Ras on the cell surface when appropriate K-Ras mutations are present in the cells. Similar to treatment with 5-FU, longer treatment with appropriate mutation-specific K-Ras small molecule inhibitors resulted in a greater amount of available K-Ras. In summary, several different methods increased the availability of surface K-Ras.

[0373] Figure 15a-15c Figure 15a-15c Example 12a: Increased K-Ras surface availability by K-Ras small molecule treatment enhances surface K-Ras targeting by antibody drug conjugates (ADC) killing

[0374] In this embodiment, we used a commercially available human Fab-ZAP assay (Advanced Targeting Systems Inc.) similar to that in Example 3 to test whether increasing surface K-Ras availability with MRTX1133 would enhance the killing effect of surface K-Ras-targeted ADCs. For the experiments described below, we used the Fab-ZAP human kit [IT-51, KIT-51] (atsbio.com / products / it51 / ), which consists of a goat anti-rabbit monovalent antibody and a chemical conjugate of a ribosome-inactivating protein (i.e., saponins). The antibody used in this kit is an affinity-purified polyclonal antibody against the heavy and light chains of rabbit IgG.

[0375] In one experiment, we tested whether increasing surface K-Ras availability with the small molecule inhibitor MRTX1133 would enhance the killing effect of 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 replicates. Cells were allowed to grow for 24 hours, and then 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 according to the manufacturer's instructions in the Fab-Zap kit. After incubation at 37°C in a 5% CO2 incubator for 5 days, the percentage of viable cells compared to control wells with only 4.5 nM FabZAP was determined using XTT assay according to the protocol described (Advanced Targeting Systems Inc. Cytotoxicity Assay Protocol). Briefly, after incubation, each well was replenished with 50 μl of XTT reagent to bring the total volume to 150 μl. The plates were then incubated at 37°C for at least 30 minutes, and the absorbance was read at 450 nm using a plate reader. This assay relies on detecting the number of remaining viable cells on the day of color development, as measured by cellular metabolism via the colorimetric molecules in the colorimetric reagent. The average absorbance in the untreated wells is depicted as 100%, with three replicates in each treatment group, and is expressed as a percentage relative to the control. Figure 16a As shown, cytotoxicity data were analyzed by comparing well readings from the treated wells with well readings from the control wells (IgG only) (expressed as a percentage). GraphPad Prism software was used to analyze the data and plot survival percentages.

[0376] Saponins alone do not induce cell death at concentrations below 100 nM. Figure 3 Therefore, no significant toxicity was observed at the 4.5 nm concentration used in the ADC assay without a target. For comparison, the maximum cell-killing effect of 100 μM free saponins is indicated by a dashed line. MRTX1133 alone or anti-K-Ras antibody alone + Fab-ZAP were both capable of killing cells (…). Figure 16a When MRTX1133 is combined with anti-Kras antibody and Fab-Zap, the effect is greater than either reagent alone. Figure 16a This indicates that the combination of small molecule inhibitors with surface K-Ras-targeted ADCs increases tumor cell killing.

[0377] Example 12b: Chemotherapeutic agents enhance the killing of surface K-Ras targeted antibody drug conjugates (ADCs) by antibodies hurt

[0378] In this embodiment, we used the commercially available Fab-ZAP assay (Advanced Targeting Systems Inc.) described in Example 12a to test whether the increased surface K-Ras availability with RMC-6236 would enhance the killing effect of surface K-Ras-targeted ADCs. One combination is an ADC of human anti-K-Ras antibody 1 with the chemotherapeutic agent fluorouracil (5-FU), and a second combination is an ADC of human anti-K-Ras antibody 1 with a small molecule inhibitor called RMC-6236. For a description of RMC-6236, see, for example, Arbour K et al., Ann Oncol 2023;34:S458.

[0379] PANC-1 cells were seeded at 10,000 cells / well in 96-well replicates. After 24 hours of growth, cells were treated with 30 nM RMC-6236, 10 μM 5-FU, or a combination of RMC-6236 and 5-FU. 24 hours after seeding, the treatment groups received a fixed concentration of 10 μM RMC-6236. -8 M human K-Ras antibody 1, or a fixed concentration of 10 -8 IgG from M was pre-incubated with a fixed concentration of 4.5 nM Fab-ZAP reagent according to the manufacturer's instructions in the Fab-ZAP kit. After incubation for 5 days at 37°C in a 5% CO2 incubator, the percentage of viable cells surviving the treatment was determined using an XTT assay according to the protocol described (Advanced Targeting Systems Inc. Cytotoxicity Assay Protocol), compared to control wells treated solely with Fab-ZAP. Briefly, after incubation, each well was supplemented with 50 μl of XTT reagent to bring the total volume to 150 μl. The plate was then incubated at 37°C for at least 30 minutes, and the absorbance was read at 450 nm using a plate reader. This assay relies on detecting the number of remaining viable cells as measured by cellular metabolism via the chromogenic molecules in the chromogenic reagent on the day of colorimetric development. The mean absorbance in the control (PANC-1 cells treated with IgG) is plotted as 100%, with 3 replicates in each treatment group, and is expressed as a percentage relative to the control. Cytotoxicity data were analyzed by comparing well readings from the treatment wells with well readings from the control wells (IgG only) (expressed as a percentage). GraphPad Prism software was used to analyze the data and plot survival percentages.

[0380] For comparison, use 10 -7The maximum cytotoxicity of free saponins is indicated by the dashed line. Treatment of PANC-1 cells with human K-Ras antibody 1 + FabZap, RMC-6236, and combinations of human K-Ras antibody 1 + FabZap and RMC-6236 is equivalent to the maximum cytotoxicity achieved with high concentrations of free saponins (Figure 16B). However, treatment of PANC-1 cells with combinations of RMC-6236 and human K-Ras antibody 1 + FabZap or 5-FU and human K-Ras antibody 1 + FabZap was more effective than either agent alone (Figure 16B).

[0381] In summary, these results show that the combination of small molecule inhibitors and chemotherapeutic agents with surface K-Ras-targeting ADCs increases tumor cell killing.

[0382] Example 13: Bispecific antibodies targeting surface K-Ras increase T cell killing of surface K-Ras expressing cells

[0383] To demonstrate whether bispecific antibodies are sufficient to induce T cell-mediated killing of surface K-Ras-expressing cells, we designed an asymmetric bispecific antibody (anti-K-Ras antibody 1 × CD3). Figure 18As shown, the bispecific antibody was designed as follows: the Fab-Fc region of human anti-K-Ras antibody 1 (anti-K-Ras Fab HC-Fc / anti-K-Ras Fab LC) (Fc is inactivated by N297G and T366S / L368A / Y407V) and the scFv of a CD3-specific antibody linked to the inactivated Fc (N297G and T366W) (anti-K-Ras Fab HC-FC-). EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26) and anti-K-Ras Fab LC-QSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 27) and CD3 OKT3 scFv-FC-DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMIWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28)). The control bispecific (control IgG × CD3) also uses the Fab region of the human anti-RSV antibody and the same CD3 OKT3 scFv-Fc domain disclosed above (RSV Fab HC-Fc -Composed of QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:29) and RSV Fab LC - DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)).

[0384] To test target cell killing, a cell line expressing Panc-1 luciferase (Panc-1L) was used. These cells constitutively express luciferase, and luciferase activity can be used as a representative of the number of viable target cells. Importantly, T cells co-cultured with the target cells do not express luciferase and therefore do not contribute to luciferase signaling. This allows luciferase expression to be used solely as a measure of target cell viability. Briefly, 50,000 Panc-1L cells were seeded at 100 μl per well in a 96-well 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 100 μl per well, for a total well volume of 200 μl. The corresponding bispecific antibodies (human anti-K-Ras antibody 1 × CD3 bispecific and control IgG (RSV) × CD3 bispecific) were added at a concentration of 10 -7 Up to 10 -10 Cells were plated in wells M. Each group was plated in 5 replicates. One group had no T cells added (Panc-1L only), and one group had T cells alone without Panc-1L as a control. Cells were cultured in the plates at 37°C in a 5% CO2 incubator for 72 hours before analysis. On the day of analysis, 100 μl of culture medium was removed from all wells and 100 μl of reagent D-fluorescein (1X concentration) was added. The wells were thoroughly mixed, and after 5 minutes, luminescence was measured by reading the titration plate in a Tecan Spark multimode microtiter plate reader. The luminescence of each well was recorded, and the intensity was proportional to the number of viable cells in each well. Statistical analysis and data representation were performed using GraphPad Prism.

[0385] like Figure 17 As shown, co-culturing target Panc-1L cells and human T cells with human anti-K-Ras antibody 1 × CD3 bispecific antibody resulted in a concentration-dependent decrease in viable cell viability, as measured by luciferase expression. Conversely, co-culturing target Panc-1L cells and human T cells with control IgG × CD3 bispecific antibody did not result in changes in cell viability compared to co-culturing target Panc-1L cells and human T cells without bispecific antibody. Individual PANC-1L cells exhibited the highest viable cell count, indicating that human T cells killed target cells at a low level during co-culturing, as expected. This together demonstrates that incubation of target cells and T cells with surface K-Ras-targeting bispecific antibody leads to a dose-dependent increase in target cell killing.

[0386] Example 13: Evaluation of antibodies binding to K-Ras antigens

[0387] To demonstrate antibody binding to K-Ras, an enzyme-linked immunosorbent assay (ELISA) was used. The plate was coated overnight at 4°C with 1 μg / ml neutral avidin (neutralite avidin or deglycosylated chicken avidin) to capture the biotinylated protein. The plate was blocked with 1% BSA / PBS for 60 min. The plate was then loaded with biotinylated (G12D) K-Ras-GDP, (G12D) K-Ras-GppNHp, K-Ras-GDP, K-Ras-GppNHp, or BSA at 1 μg / ml for 60 min. K-Ras binding of the purified antibody was tested at concentrations from 1 to 0.001 μg / ml for 60 min. Antibody detection was performed using anti-human IgG-HRP and TMB staining. The plate was washed three times with PBST between incubations. The mean of repeated measurements and their standard deviations are shown.

[0388] Human anti-K-Ras antibodies 1 and 2 bind at low levels to wild-type K-Ras loaded with nucleotides of GDP or GppNHp. Figure 19c-19d Similarly, human anti-K-Ras antibody 2 binds to (G12D) K-Ras at low levels, regardless of the loaded nucleotide. Conversely, human anti-K-Ras antibody 1 binds to (G12D) K-Ras at high levels (loaded with any nucleotide), demonstrating that human anti-K-Ras antibody 1 preferentially binds to G12D-mutated K-Ras. Figure 19a-19d ).

[0389] Example 14: Validation of antibody drug conjugates (ADCs) for selective killing of cancer cell lines by using mutation specific antibodies Example 15: Evaluation of in vivo staining of tumor tissue by anti-K-Ras antibodies

[0390] To investigate the potential of using mutant K-Ras expressed on the cell surface as a target for ADCs, we 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-1K-Ras was used in the Fab-ZAP assay. G12D (Pancreatic cancer carrying the 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 line) and HUVEC K-Ras WT(Non-malignant cell line). RSV-specific antibody was used as a negative control for K-Ras binding.

[0391] Following the manufacturer's instructions in the Fab-ZAP kit, serially diluted antibodies were pre-incubated with 4.5 nM FabZAP reagent and then added to the designated cell lines. Cells were seeded in round-bottom 96-well plates according to the manufacturer's protocol, with a total volume of 100 μl per well (including FabZAP and different concentrations of antibody or corresponding controls). After incubation at 37°C in a 5% CO2 incubator for 5 days, the percentage of viable cells was determined using XTT assay according to the described protocol (Advanced Targeting Systems Inc. Cytotoxicity Assay Protocol) compared to control wells containing only 4.5 nM FabZAP. Briefly, after incubation, each well was replenished with 50 μl of XTT reagent to bring the total volume to 150 μl. The plates were then incubated at 37°C for at least 30 minutes, and the absorbance was read at 450 nm using a plate reader. This assay relies on detecting the number of remaining viable cells as measured by cell metabolism via the chromogenic molecules within the chromogenic reagent on the day of colorimetric development. The average absorbance in the untreated wells is depicted as 100%, with 3 replicates in each treatment group, and is expressed as a percentage relative to the control.

[0392] Figure 21a-21b

[0393] To demonstrate the specific recognition of surface K-Ras in in vivo tumors, tumor blocks 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 Balb / c nude mice lacking T and B cells, allowing for the growth and transplantation of human tumors. Primary human tumor xenograft model tumor blocks (2–3 mm in diameter) were subcutaneously inoculated into the right upper / lower flank of each mouse for tumor development. Once the tumor reached approximately 500 mm... 3 Mice were then injected with the antibodies described below.

[0394] Once the tumor reaches 500 mm 3 Mice were randomized and injected via tail vein with human anti-K-Ras antibody 1 or an isotype control antibody at a dose of 10 mg / kg, at a volume of 10 μL / g. Thirty minutes later, the mice were euthanized, tumors were harvested, and frozen in optimal cutting temperature (OCT) cryogenic medium for immunohistochemical evaluation.

[0395] Tumor sections were cut into 5 μm pieces using a standard dermabrasion knife and subjected to immunohistochemistry. Briefly, frozen sections were fixed for 5 minutes with cold acetone / methanol (1:1 dilution) solution. After washing sections in PBS for 15 minutes, they were blocked with 10% normal goat serum at room temperature for 45 minutes and incubated with a second goat anti-human IgG (Alexa fluro 488 Invitrogen A-11013) at room temperature for 45 minutes. Prolong containing DAPI was added before placing coverslips on top. TM Gold Antifade reagent (catalog p36931, ThermoFisher Scientific). Images obtained using Leica Microsystems DM6B-Z.

[0396] like Example 16: Validation of antibody drug conjugates (ADCs) for selective killing of cancer cell lines in vivo by using mutation specific antibodies As can be seen, 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 pancreatic cancer containing G12D-mutated K-Ras.

[0397] Figure 22a-22d Figure 22c-22d

[0398] To investigate the ability of anti-K-Ras antibodies to provide control over K-Ras-mutant tumors in vivo, we conjugated anti-K-Ras antibody 1 and an isotype control antibody to delutecan (DXd). DXd is a protease-cleavable linker attached to a derivative of ixotecan, which acts as a topoisomerase I inhibitor. The FDA-approved ENHERTU® (delutecan (fam-trastuzumab-deruxtecan-nxki)) (a conjugate of anti-Her-2 antibody and delutecan) has been used to target cancer cells with DXd via the antibody as an ADC.

[0399] DXd was conjugated to the interchain cysteine ​​residues of the antibody via random maleimide conjugation. The antibody was first reduced by shaking at 37°C for 90 minutes with 10 mM TCEP (10 x mol equivalents). The reduced antibody was then conjugated with 10 mM DXd (10 x mol equivalents) at room temperature for 2 hours. The drug-to-antibody ratio (DAR) of the conjugated antibody was evaluated by reducing 10 μg of the 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, confirming it as a monomer. The conjugated antibody was purified by dialyzing unconjugated DXd against PBS (pH 5.6) for 48 hours using a 10K molecular weight cutoff (MWCO) membrane.

[0400] Next, we utilized a cell line-derived xenograft model of pancreatic cancer, in which the human tumor cell line PANC-1 was implanted into Balb / c nude mice. Nude mice are naturally occurring mutant mice lacking both a thymus and hair (nude). The absence of a thymus in nude mice leads to T-cell deficiency, resulting in immunodeficiency and enabling them to accept foreign tissues such as human tumor cells. Mice were subcutaneously injected with 5 × 10⁶ cells… 6 1 Panc-1 cells. When the average tumor size reaches approximately 200 mm. 3 Randomization began at a certain time. Sixty mice were included in the study. All animals were randomly assigned to six study groups, with 10 mice in each group. Randomization was performed based on a "matched distribution" method. The experiment was conducted as outlined in Table 5.

[0401]

[0402] like Figure 22a-22b As shown, anti-Kras antibody 1 conjugated to DXd ADC slowed tumor growth in mice treated with isotype ADC and saline control (Group 1 vs. Group 2 vs. Group 3 vs. Group 6). Figure 22a-22d Surprisingly, even mice treated with anti-K-Ras antibody 1 showed reduced tumor growth compared to mice treated with the same type. Example 17. Determination of antibody sites interacting with K-Ras using HDX-MS. Although gemcitabine treatment slowed tumor growth compared to ADC or antibody treatment, mice treated with gemcitabine showed distress based on weight loss, while mice treated with ADC or antibodies gained weight at a rate similar to that of the saline-treated controls. ​ ).

[0403] ​

[0404] To determine the K-Ras regions that interact with human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 and are therefore accessible on the cell surface, we utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS). Experiments were performed using a Trajan hopping robot platform and Waters Cyclic IMS MS. The experiments were divided into two phases: peptide mapping and HDX. Peptide mapping uses only the free state of the protein and identifies peptides for HDX analysis, while HDX requires both free and mAb-bound proteins and compares differences in deuterium uptake at each amino acid residue.

[0405] For peptide mapping, the K-Ras G12D sample was diluted to 6 μM in 20 mM phosphate buffer, 150 mM NaCl, pH 7.4 (“H2O-based buffer”), and 6.5 μL of protein solution was mixed with 43.5 μL of H2O-based buffer. At the end of the reaction, 45 μL of sample was mixed with 45 μL of pre-split 100 mM phosphate buffer (pH 2.4). 80 μL of quenched sample was injected into a sample loop, and the quenched protein was digested for 210 s using a Pepsin column (Affipro) at a flow rate of 0.200 mL / min before collection and desalting, followed by separation via an analytical C18 column at 0.035 mL / min. The K-Ras G12D sample contained the separated peptides. The polypeptide has an N-terminal protein tag (MGSHHHHHHHHGSENLYFQGGS - SEQ ID NO: 293) and a C-terminal protein tag (KLLHHILDAQKMVWNH - SEQ ID NO: 294), which is coupled to amino acid residues 2-189 of K-Ras G12D with SEQ ID NO: 295.

[0406] For HDX experiments, 6 μM of K-Ras G12D was analyzed in both free and monoclonal antibody (mAb) bound states. In bound state measurements, 12 μM of each intact mAb was incubated with K-Ras G12D. The autosampler used the same volume as in the peptide mapping experiments, but the H2O buffer was replaced with D2O buffer (20 mM phosphate buffer, 150 mM NaCl, pD 7.4). Three different labeled time points (2 min, 10 min, and 60 min) were used in both free and mAb bound states. All time points were collected in triplicate. The same flow rate was used for liquid chromatography. PLG was used to prepare the peptide library, and Dynamic X 3.0 was used for deuterium uptake analysis.

[0407] First, K-Ras G12D / antibody complexes of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 were prepared in 20 mM phosphate buffer, 150 nM NaCl, pH 7.4, and then exchanged into a deuterium-containing buffer. The K-Ras G12D / antibody complexes were exposed to deuterium for 2 min, 10 min, and 30 min, followed by quenching the deuterium exchange. The labeled K-Ras G12D / antibody complexes were then digested and analyzed by bottom-up mass spectrometry.

[0408] HDX-MS analysis of human anti-K-Ras antibody 1 and human anti-K-Ras antibody 2 revealed significantly reduced deuterium incorporation in several moieties of KRas G12D, as shown in Table 6 for human anti-K-Ras antibody 1 and Table 7 for human anti-K-Ras antibody 2. 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 reduced deuterium uptake in these regions indicates that one or more of these regions bind directly to the antibody and are therefore accessible when KRas is found on the cell surface.

[0409] Table 6. Deuterium-incorporated regions in K-ras G12D complexed with human anti-K-ras antibody 1.

[0410]

[0411] Table 7. Deuterium-incorporated regions in K-Ras G12D complexed with human anti-K-ras antibody 2.

[0412]

[0413] The regions identified by HDX-MS of K-Ras G12D interacting with human anti-K-Ras antibody 1 or human anti-K-Ras antibody 2 include the sequences QLIQNHFVDE, SAMRDQY, AINNTKSFED, and KTRQGVDDAF.

[0414] To confirm the identity of the accessible KRas G12D region on the cell surface, a similar experiment was performed using the K-Ras G12D / peptide R11.1.6 complex. As described in Example 1, R11.1.6 is a small cell-penetrating peptide that binds to surface K-Ras. The crystal structure of KRas G12D (GppNHp) interacting with peptide R11.1.6 was downloaded from PDB (5UFQ). The crystal structure was resolved by Kauke et al. Sci Rep. 2017 Jul 19;7(1):5831. doi: 10.1038 / s41598-017-05889-7 and used to identify a high-affinity, non-covalent inhibitor of K-Ras oncogenic mutants. The interaction residues between K-RasG12D (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 interacting with R11.1.6 included: 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 indicates that the region of K-Ras G12D including residues 3–7, 36–41, 54–56, and 62–74 is accessible on the cell surface to interact with the antibody.

[0415] For purposes of illustration and description, the above description of specific embodiments of this disclosure has been provided. Exemplary embodiments were chosen and described to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to best utilize the subject matter and various embodiments, with modifications suitable for the particular intended use. It should be understood that although certain features may be described only in specific embodiments and not in all other embodiments, these features should not be limited to the specific embodiments described and can be applied to other embodiments that a person of ordinary skill in the art would contemplate based on the overall nature of this disclosure.

Claims

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

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

3. The composition according to any one of claims 1-2, wherein the therapeutic agent is selected from cytotoxic agents, cell growth inhibitors, toxins or radionuclides.

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

5. The composition according to any one of claims 1-2, wherein the therapeutic agent is selected from chachiin, saponins, maytansine, olistatin, lidamycin, methotrexate, vincristine, pyrrolobenzodiazepines and other benzodiazepine derivatives, pyroximin, tubulolysin, α-amanitin or bouganin protein toxin, doxorubicin, etoposide, fluorouracil, gemcitabine, paclitaxel, cisplatin, cyclophosphamide, amatoxins, carboplatin, spritzitine C, docetaxel, tylansstatin A, or any combination thereof.

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

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

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

9. The composition for the purpose according to any one of claims 7-8, wherein the composition is 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 comprises a binding agent, wherein the binding agent is specifically capable of binding to surface K-Ras antigen expressed on the outer surface of cancer cells.

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

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

13. The chimeric antigen receptor according to claims 10-12, wherein the binding agent is an antibody or antibody fragment that selectively binds the surface K-Ras antigen, wherein the surface K-Ras antigen comprises any one of the following mutations based on the amino acid sequence shown 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 claims 10-13, wherein the surface K-Ras antigen has greater than 70% sequence identity with SEQ ID NO:1 or SEQ ID NO:

2.

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

2.

16. Immune cells that express the chimeric antigen receptor as described in any one of claims 10-14.

17. The immune cell of claim 14, wherein the immune cell is selected from T cells, NK cells, dendritic cells, or mixtures thereof.

18. The immune cells of claim 16 or 17 for use in treating cancer in an individual, wherein the cancer comprises cancer cells expressing surface K-Ras antigens on the outer surface of the cancer cells.

19. The immune cells for the purpose of claim 18, wherein the immune cells comprise cells derived from an individual suffering from the cancer.

20. The immune cells for the said purpose according to claim 18 or 19, wherein the immune cells are selected from T cells, NK cells, dendritic cells, or mixtures thereof.

21. The immune cells for the purpose according to any one of claims 18-20, wherein the cancer cells are selected from 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 1-6, wherein the therapeutic agent is therapeutically effective in inhibiting the growth or proliferation of the cancer cells, or is cytotoxic to the cancer cells.

23. The composition for the purpose according to any one of claims 7-9, wherein the object has been given an additional therapeutic agent, or wherein the composition is given simultaneously with an additional therapeutic agent, and the additional therapeutic agent is separate from the therapeutic agent present in the composition.

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

25. The composition for the purpose according to claim 24, wherein the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in the surface K-Ras antigen.

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

27. The composition for the purpose according to any one of claims 23-26, wherein the additional therapeutic agent is administered to the subject 1 to 14 days prior to the application of the composition.

28. The composition for the purpose according to any one of claims 23-26, wherein the additional therapeutic agent is applied to the subject 3 to 7 days prior to the application of the composition.

29. The immune cells for the purpose according to any one of claims 18-21, wherein the subject has been administered an additional therapeutic agent, or wherein the immune cells are administered simultaneously with the additional therapeutic agent.

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

31. The immune cells for the stated purpose according to claim 30, wherein the K-Ras small molecule inhibitor specifically inhibits the activity of K-Ras forms carrying mutations present in the surface K-Ras antigen.

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

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

34. The immune cells for the purpose according to any one of claims 29-32, wherein the therapeutic agent is administered to the subject 1 to 14 days prior to the administration of the immune cells.

35. The immune cells for the purpose according to any one of claims 29-32, wherein the therapeutic agent is administered to the subject 3 to 7 days prior to the administration of the immune cells.

36. Bispecific antibodies, which include: A first binding domain is connected to a second binding domain, wherein the first binding domain selectively binds to surface K-Ras antigen expressed on the outer surface of cancer cells, and wherein the second binding domain selectively binds to antigen expressed on the surface of immune effector cells.

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

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

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

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

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

2.

42. The bispecific antibody according to any one of claims 36-40, wherein the first binding domain selectively binds the surface K-Ras antigen, the surface K-Ras antigen having any one of the following mutations based on the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2: G12A, G12D, G12C, G12V, G12R, G13D, Q61H, and Q61L.

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

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

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

46. ​​The bispecific antibody for the purpose according to any one of claims 44-45, wherein the bispecific antibody is formulated for administration by intravenous or subcutaneous injection.

47. The bispecific antibody for the said use according to any one of claims 44-46, wherein the surface K-Ras antigen is a full-length or truncated form of K-Ras with a mutation at any one of the following amino acids / residues based on the amino acid position in SEQ ID NO: 1 or SEQ ID NO: 2: 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).

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