KRAS-specific antibodies and uses thereof

Abstract

Provided herein are anti-KRas antibodies that bind to mutant KRas-GDP and anti-KRas antibodies that bind to alkylated mutant KRas-GDP, and methods for using the same. Also provided herein are methods for screening for KRas inhibitors and methods for measuring KRas binding to the antibodies described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 018356, filed April 30, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated by reference in its entirety into this specification: Sequence Listing in computer readable form (CRF) (Filename: P35630WO_SEQLIST.TXT, Recorded: March 23, 2021, Size: 60,938 bytes).

[0003] FIELD OF THE INVENTION The present invention relates to KRas-specific antibodies and methods of use thereof. [Background technology]

[0004] KRAS is one of the most frequently mutated oncogenes in cancer (Kranenburg, O., Biochim. Biophys. Acta 2005 1756). KRAS encodes a member of the Ras family of guanosine triphosphate (GTPase) enzymes that function in signal transduction from cell surface receptors to intracellular effector pathways (Pylayeva-Gupta, Y. et al., Nat Rev Cancer 2011 11). Ras GTPases cycle between an active state bound to guanosine 5'-triphosphate (GTP) and an inactive state bound to guanosine 5'-diphosphate (GDP). In cancer, KRAS G12C Oncogenic mutations in KRas, including those in tumor-driven cells, impair its GTPase activity, leading to the accumulation of activated GTP-bound forms of KRas, resulting in constitutive activation of downstream pathways of KRas, promoting proliferation and inhibiting apoptosis (Pylayeva-Gupta, Y. et al. Nat Rev Cancer 2011 11).

[0005] Despite its long-recognized prevalence in cancer, KRas has long been considered a poorly druggable target (McCormick, F. Clin Cancer Res 2015 21:8). G12C There are other alleles of KRAS associated with cancer other than KRAS (Haigis, KM, Trends Cancer 2017 3:10). Thus, there is a need in the art for KRas-specific antibodies that specifically bind to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP). Summary of the Invention

[0006] In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GDP).

[0007] In some embodiments, the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof opens and stabilizes the SWII pocket.

[0009] In some embodiments, the human KRas is KRas G12C , KRas G12V , KRas G12R , KRas Q61H , KRas G12D , and KRas G13D The KRas mutant is selected from the group consisting of:

[0010] In some embodiments, human KRas is KRas G12C , KRas G12V , KRas G12D, and KRas G13D The KRas mutant is selected from the group consisting of:

[0011] In some embodiments, the Kras mutant is Kras G12C is.

[0012] In some embodiments, KRas G12C -GDP, KRas G12C Alkylated with specific covalent inhibitors.

[0013] In some embodiments, the isolated antibody or antigen-binding fragment is an antibody or antigen-binding fragment of KRas alkylated with MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, or JNJ-74699157. G12C -A GDP-binding alkylated conformation-specific KRas antibody.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof stabilizes the SWII pocket of the KRas mutant protein.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9); (ii) a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10); and (iii) CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 12); (ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13); and (iii) CDR-H3 containing the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14) Includes.

[0016] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:15 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:16.

[0017] In some embodiments, the antibody (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO: 1); (ii) a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 2); and (iii) CDR-L3 comprising the amino acid sequence LQDHDYPLT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 4); (ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 5); and (iii) CDR-H3 containing the amino acid sequence GFYVRNWFDP (SEQ ID NO: 6) Includes.

[0018] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:7 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 17); (ii) CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 18); and (iii) CDR-L3 comprising the amino acid sequence QQYYSYPFT (SEQ ID NO: 19); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO: 20); (ii) CDR-H2 comprising the amino acid sequence AISSSGSSTYYADSVKG (SEQ ID NO: 21); and (iii) CDR-H3 containing the amino acid sequence DQGGYGYPGESWFDY (SEQ ID NO: 22) Includes.

[0020] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:23 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:24.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 25); (ii) CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 26); and (iii) CDR-L3 comprising the amino acid sequence QQSYSPPWT (SEQ ID NO: 27); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 28); (ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 29); and (iii) CDR-H3 containing the amino acid sequence AFYSYMDV (SEQ ID NO: 30) Includes.

[0022] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:31 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:32.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 33); (ii) CDR-L2 comprising the amino acid sequence LGSNRAS (SEQ ID NO: 34); and (iii) CDR-L3 comprising the amino acid sequence MQALQTPLT (SEQ ID NO: 35); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 36); (ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 37); and (iii) CDR-H3 containing the amino acid sequence ERTILTGYYGFDY (SEQ ID NO: 38) Includes.

[0024] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:39 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:40.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 41); (ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 42); and (iii) CDR-L3 comprising the amino acid sequence GTWDSSLTGYV (SEQ ID NO: 43); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 44); (ii) CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 45); and (iii) CDR-H3 containing the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 46) Includes.

[0026] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:47 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:48.

[0027] In some embodiments, the antibody (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 81); (ii) a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 82); and (iii) CDR-L3 comprising the amino acid sequence AAWDDSLSGWV (SEQ ID NO: 83); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 84); (ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 85); and (iii) CDR-H3 containing the amino acid sequence SFGPYAFDV (SEQ ID NO: 86) Includes.

[0028] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:87 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:88.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 49); (ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 50); and (iii) CDR-L3 comprising the amino acid sequence GTWDSSLTGWV (SEQ ID NO: 51); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 52); (ii) CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 53); and (iii) CDR-H3 containing the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 54) Includes.

[0030] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:55 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:56.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 57); (ii) a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 58); and (iii) a CDR-L3 comprising the amino acid sequence NSRDSSGNHWV (SEQ ID NO: 59); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 60); (ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 61); and (iii) CDR-H3 containing the amino acid sequence TNNYGYRYFDY (SEQ ID NO: 62) Includes.

[0032] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:63 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:64.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 65); (ii) a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 66); and (iii) CDR-L3 comprising the amino acid sequence NSRDSTDNHLWV (SEQ ID NO: 67); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 68); (ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 69); and (iii) CDR-H3 comprising the amino acid sequence ATSSGYYYFDY (SEQ ID NO: 70) Includes.

[0034] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:71 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:72.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 73); (ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 74); and (iii) CDR-L3 comprising the amino acid sequence GTWDNSLSVWV (SEQ ID NO: 75); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 76); (ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 77); and (iii) CDR-H3 containing the amino acid sequence GKGIVGWGFFGMDV (SEQ ID NO: 78) Includes.

[0036] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:79 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:80.

[0037] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to human KRas-GDP, wherein the isolated antibody or antigen-binding fragment thereof binds to amino acids W99, K5, L6, V7, S39, D54, L54, Y71, T74, and / or G75 of human KRas.

[0038] In another aspect, the invention provides one or more isolated nucleic acids encoding the light chain variable domain and heavy chain variable domain of a KRas antibody of an antibody or antigen-binding fragment described herein. In another aspect, the invention provides a vector comprising the nucleic acid. In another aspect, the invention provides a host cell comprising the vector.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a detectable label.

[0040] In some embodiments, the invention provides a method for producing an antibody or fragment thereof that binds KRas-GDP, comprising culturing the host cell described above under conditions suitable for expression of a vector encoding the antibody, and recovering the antibody.

[0041] In another aspect, the present invention provides a method for the treatment of KRas G12C -KRas with higher affinity than GTP G12C - a method for screening for antibodies that bind to GDP, comprising: (a) Antibody library i) KRas G12C -GDP, ii) KRas G12C KRas alkylated with specific covalent inhibitors G12C -GDP, and iii) KRas bound to a non-hydrolyzable GTP analog G12C contacting the (b) KRas bound to a non-hydrolyzable GTP analogue G12C Alkylates KRas with higher affinity than G12C -GDP and non-alkylated KRas G12C -Selecting antibodies that bind to GDP The present invention provides a method comprising:

[0042] In some embodiments, the library is a synthetic phage library.

[0043] In another aspect, the invention provides a method for detecting KRas-GDP in a biological sample, comprising contacting the biological sample with a KRas antibody or antigen-binding fragment provided herein.

[0044] In some embodiments, the method further comprises contacting the biological sample with an antibody that binds to KRas-GTP, wherein the amount of KRas-GDP and the amount of KRas-GTP are determined.

[0045] In another aspect, the present invention provides a kit comprising a KRas antibody or antigen-binding fragment thereof described in any one of paragraphs

[0006] to

[0037] conjugated to a detectable label and instructions for detecting the antibody or antigen-binding fragment thereof.

[0046] In another aspect, the present invention provides a method for obtaining an inhibitor of a KRas mutant, comprising contacting an anti-KRas antibody or antigen-binding fragment thereof with a KRas mutant, screening for compounds, and identifying compounds that bind to the KRas mutant bound to the antibody or antigen-binding fragment thereof.

[0047] In some embodiments, the compound comprises a molecule that covalently modifies KRas in the SWII pocket.

[0048] In some embodiments, the compound comprises a covalent inhibitor that alkylates at least one residue within the SWII pocket.

[0049] In some embodiments, the compound comprises a molecule that non-covalently modifies KRas in the SWII pocket.

[0050] In some embodiments, the KRas mutant is KRas G12C , KRas G12V , KRas G12D , KRas G13D , KRas G12R , or KRas Q61H is.

[0051] In one aspect, the invention provides a method for detecting alkylation of KRas, comprising contacting a biological sample with an anti-KRas antibody or antigen-binding fragment, and detecting the antibody or antigen-binding fragment thereof that binds to the alkylated KRas.

[0052] In some embodiments, detection is by Kras G12C This includes the detection of

[0053] In some embodiments, the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

[0054] In another aspect, the invention provides a method for detecting alkylation of KRas in a mammal, comprising administering a KRas antibody or antigen-binding fragment thereof to the mammal and detecting the antibody or antigen-binding fragment thereof that binds to the alkylated KRas.

[0055] In another aspect, the present invention provides a method for detecting alkylation of KRas in a patient treated with a KRas inhibitor, the method comprising: (a) Obtaining a sample from a patient; (b) contacting the sample with an anti-KRas antibody; (c) measuring the amount of KRas bound by the antibody or antigen-binding fragment thereof. Includes.

[0056] In some embodiments, the KRas inhibitor is MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, or JNJ-74699157.

[0057] In some embodiments, the amount of KRas bound by the antibody or antigen-binding fragment thereof determines the dosage of the KRas inhibitor administered to the patient.

[0058] In some embodiments, detection is by Kras G12C This includes the detection of

[0059] In some embodiments, the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

[0060] In some embodiments, the mammal is a human.

[0061] In another aspect, the present invention provides a method for the treatment of KRas G12C KRas in subjects treated with specific covalent inhibitors G12C The present invention provides a method for detecting alkylation of a compound comprising: (a) KRas G12C administering an anti-KRas antibody or antigen-binding fragment to a subject after treatment with a specific covalent inhibitor; and (b) detecting an antibody or antigen-binding fragment thereof that binds to alkylated KRas; Includes.

[0062] In some embodiments, KRas G12C Specific covalent inhibitors are ARS-1952, ARS-853, ARS-1620, MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, or JNJ-74699157.

[0063] In some embodiments, the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

[0064] In one aspect, the present invention provides a method for the treatment of KRas G12C The present invention provides a method for treating a KRas-mediated cancer, the method comprising administering to a patient with such cancer an anti-KRas antibody or antigen-binding fragment thereof.

[0065] In some embodiments, KRas G12C The mediated cancer is NSCLC, colon cancer, or pancreatic cancer.

[0066] In another aspect, the present invention provides a crystallized chaperone comprising an anti-KRas antibody or antigen-binding fragment thereof.

[0067] In another aspect, the invention provides a method for crystallizing KRas, optionally bound to a KRas inhibitor, comprising contacting an anti-KRas antibody or antigen-binding fragment thereof with KRas, and solving the crystal structure of the complex.

[0068] In some embodiments, KRas is KRas G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H is.

[0069] In another aspect, the present invention provides a biosensing surface for measuring the binding of a compound to KRas, wherein: (i) the biosensing surface comprises a hydrogel in which a KRas protein and an anti-KRas antibody or antigen-binding fragment are colocalized; (ii) KRas and the antibody or antigen-binding fragment thereof have sufficient freedom within the hydrogen to engage with each other to form an affinity complex; (iii) the local concentrations of KRas and the antibody or antigen-binding fragment thereof exceed the dissociation affinity constant by at least 10-fold, and the local concentrations promote the formation of an affinity complex; (iv) the percentage of unbound KRas protein and anti-KRas antibody is less than about 50%; (v) a KRas inhibitor compound is injected over the biosensing surface for at least 5 seconds; (vi) Binding of a KRas inhibitor compound to the anti-KRas antibody is measured on at least one sensing channel.

[0070] In some embodiments, the hydrogel is about 10 nm to 500 nm, 10 nm to 300 nm, 10 to 250 nm, or about 10 to 200 nm thick.

[0071] In some embodiments, the present invention provides a biosensing surface for measuring the binding of compounds to KRas that has been biotinylated.

[0072] In some embodiments, the present invention provides a biosensing surface for measuring the binding of compounds to KRas, the biosensing surface being attached to a BIACORE sensor chip.

[0073] In another aspect, the invention provides a method of screening compounds for anti-KRas inhibitor activity, the method comprising measuring binding of the compound to KRas, where KRas binds to an anti-KRas antibody, and binding is measured using a biosensing surface.

[0074] In another aspect, the invention provides a method for measuring binding of a KRas mutant protein to an anti-KRas antibody described herein, the method comprising: (i) contacting a biosensing surface with KRas to form a biosensing surface bound to KRas; (ii) contacting the KRas-bound biosensing surface with an anti-KRas antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is in molar excess relative to the KRas protein; and (iii) detecting the binding and affinity of the antibody or antigen-binding fragment thereof to KRas using surface plasmon resonance; Includes.

[0075] In another aspect, the invention provides a method for measuring binding of a KRas mutant protein to an anti-KRas antibody described herein, the method comprising: (i) contacting a biosensing surface with an anti-KRas antibody or antigen-binding fragment thereof to form a biosensing surface bound to the anti-KRas antibody; (ii) contacting a biosensing surface bound to an anti-KRas antibody with KRas, wherein the antibody or antigen-binding fragment thereof is in molar excess relative to the KRas protein; and (iii) detecting the binding and affinity of the antibody or antigen-binding fragment thereof to KRas using surface plasmon resonance; Includes.

[0076] It should be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. [Brief explanation of the drawings]

[0077] [Figure 1A] The crystal structure of Ras is superimposed. [Figure 1B] 1 shows the in vitro phage display selection strategy used to identify alkylated KRasG12C-GDP-specific monoclonal antibodies. [Figure 1C] Enzyme-linked immunosorbent assay (ELISA) data for selected anti-KRas monoclonal antibodies binding to KRasG12C-GDP+GNE1952, non-alkylated KRasG12C-GDP, and negative controls are shown. [Figure 1D] Shown are data from surface plasmon resonance (SPR) analysis of KRas binding to selected anti-KRas antibodies 1A5 (left side of x-axis) and 2H11 (right side of x-axis) upon alkylation with different agents. [Figure 1E] Representative SPR traces of selected anti-KRas antibodies 1A5 and 2H11 against KRasG12C-GDP+GNE1952 and KRasG12C-GDP are shown, with time (seconds) on the x-axis and response units on the y-axis. [Figure 1F] 1 shows the results of epitope binning of selected anti-KRas antibodies. [Figure 1G] 1 shows immunoprecipitation of alkylated KRasG12C-GDP by selected anti-KRas antibodies 1A5 and 2H11 from cells treated with ARS-1620 and non-alkylated KRasG12C-GDP. [Figure 2A]1A5 anti-KRas antibody binding KRasG12C in cells treated with various covalent molecules compared to DMSO control in H1171 KRASG12C mutant cancer cells is shown using immunofluorescence (IF) assays. [Figure 2B] Shown is staining of KRasG12C with 1A5 anti-KRas antibody upon ARS-1620 treatment in H1171 KRASG12C mutant cancer cells with varying time (hours on the y-axis) and dose of ARS-1620 (shown in μM along the x-axis compared to DMSO control). [Figure 2C] Figure 1 shows the lack of observable KRas staining with the 1A5 anti-KRas antibody in HCT116 KRasG13D cells treated with the KRasG12C inhibitor GNE-1952. [Figure 2D] Immunoblot analysis of KRas pathway markers, pERK, and pS6, in response to alkylation-induced KRas inhibition in a bulk population of H1171 KRASG12C mutant cancer cells. Cells were treated with DMSO, 5 μM ARS-853, and / or 50 μg / ml cyclohexamide as indicated. Samples were collected 6, 24, or 48 hours after treatment or washout, as indicated. [Figure 2E] Immunofluorescence assay showing 1A5-bound KRasG12C across different KRASG12C mutant cancer cell models. Cells were treated with 5 μM ARS-1620. The relative amount of KRasG12C expression in each cancer cell model is indicated by a + sign. [Figure 2F] Flow cytometry measurements of 1A5 staining (y-axis) and pS6 staining (x-axis) in H1171 KRASG12C mutant cancer cells treated with increasing doses of ARS-1620 compared to DMSO control (left). [Figure 3A] Differential immunoprecipitation of alkylated and non-alkylated KRasG12C in H1171 KRASG12C mutant cancer cells treated with DMSO or ARS-1620 by selected anti-KRas antibodies 1A5 and 2H11 is shown in comparison to a set of commercially available antibodies. [Figure 3B] ELISA using a set of commercially available antibodies (indicated on the x-axis) on KRasG12C-GDP+GNE1952 is shown in comparison to non-alkylated KRasG12C-GDP, KRasG12C-GMPcP, and NeutrAvidin alone. [Figure 3C] Immunofluorescence using 1A5 anti-KRas antibody (top row) and iDab6 (bottom row) for a dose titration of ARS-1620 is shown. DNA stained with DAPI is shown in blue. The dose of ARS-1620 is indicated in nM in each image. [Figure 4A] Immunohistochemistry using 1A5 anti-KRas antibody on NCI-H358 (high KRasG12C expression) xenografts in female C / B17 SCID mice after 8 and 24 hours of treatment with 50 mg / kg or 200 mg / kg ARS-1620, compared to vehicle-only controls. [Figure 4B] NCI-H2122 (low KRasG12C expression) xenografts in female CRL nude mice after 8 hours of treatment with 50 mg / kg or 200 mg / kg ARS-1620 are shown compared to vehicle-only controls. [Figure 4C] The percentage of NCI-H358 xenograft cells positive for 1A5, as measured by flow cytometry, is shown as a gray bar (left y-axis). Relative expression of pS6 (a KRAS pathway marker) is shown as a black circle (x-axis). Samples were treated with 50 mg / kg or 200 mg / kg ARS-1620 or vehicle-only control for 8 or 24 hours. [Figure 5A] SPR data for KRasWT treated with GNE-1952, ARS-853, or ARS-1620 at concentrations ranging from 1 to 50 μM in the absence (top row) or presence (bottom row) of 2H11 anti-KRas antibody are shown. Time (seconds) is shown on the x-axis, and response units are shown on the y-axis. [Figure 5B]SPR data are shown for KRasG12C or KRasWT treated with GNE-1952 or the "non-warhead" form of GNE-1952 (lacking reactive acrylamide functionality) in the absence (top) or presence (bottom) of 2H11 anti-KRas antibody. Time (seconds) is shown on the x-axis and response units are shown on the y-axis. [Figure 6A] The crystal structure of the anti-KRas antibody:KRasG12C complex is shown. Figure 6A shows 2H11 Fab bound to KRasG12C-GDP (upper structure). The KRas structure is shown as ribbons, SWII (SW2) is labeled, GDP is shown as a stick, Mg2+ is shown as a sphere, and the Cys12 residue is highlighted with a thick stick. 2H11 Fab is shown as a ribbon with a transparent surface. The lower structure in Figure 6A is a surface mapping of the KRas epitope of 2H11, rotated relative to the upper structure. [Figure 6B] The crystal structure of the anti-KRas antibody:KRasG12C complex is shown. Figure 6B shows a close-up of the antibody-antigen contact surface. The complementarity-determining regions (CDRs) that directly contact KRas are shown as ribbons. Dotted lines indicate hydrogen bonds, with SWI, SWII, CDR, GDP, and Cys12 indicated. The anchor, HC.Trp99, is shown as a thick bar. [Figure 6C] Figure 6C shows the crystal structure of the anti-KRas antibody:KRasG12C complex. Figure 6C compares the KRasG12C / 2H11 complex in the presence and absence of GNE-1952. The GNE-1952 compound is shown as a stick figure. The SWII residues in both structures are shown as thin sticks, with Cys12 and His95 indicated. [Figure 6D] Figure 6D shows the crystal structure of the anti-KRas antibody:KRasG12C complex. Figure 6D shows the alignment of the 2H11 anti-KRas antibody bound to KRasG12C-GDP with the DCAI compound bound to KRas. [Figure 6E]Figure 6E shows the crystal structure of the anti-KRas antibody:KRasG12C complex. Figure 6E shows the alignment of the 1A5 anti-KRas antibody bound to KRasG12C-GDP with GNE-1952 KRasG12C-GDP. [Figure 6F] Figure 6F shows the crystal structure of the anti-KRas antibody:KRasG12C complex. Figure 6F shows a comparison of the structures of iDab6 and 2H11 in binding to KRas SWI. [Figure 7] ELISA experiments using the 1A5 and 2H11 anti-KRas antibodies that bind to a panel of KRas-GDP mutants are shown, with KRas genotype (or BSA control) on the x-axis and OD650nm on the y-axis. [Figure 8] An exemplary single-cycle kinetic analysis of a single SWII-binding compound without 2H11 co-capture is shown. Time is shown on the x-axis in seconds, and response is shown on the y-axis in relative units (RU). A single-site pseudo-first-order model was fitted, yielding a k of 3.29 × 10 (1 / Ms), a k of 1.3 (1 / s), and a K of approximately 4 μM. [Figure 9] Figure 1 shows an exemplary single-cycle kinetic analysis of a single SWII-binding compound undergoing 2H11 Fab co-capture. Time is shown in seconds on the x-axis, and response is shown in relative units (RU) on the y-axis. A two-site pseudo-first-order model was fitted to the data, returning interaction constants for the high-affinity site: k(1 / Ms) of 6.6 x 10, k(1 / s) of 0.025, and K(D) of approximately 4 μM. [Figure 10] 2H11-Fab co-cooperativity factor values ​​determined using a co-capture SPR assay for the 11 SWII-binding compounds shown on the x-axis bound to KRasG12V-GDP (gray bars) and KRasG13D-GDP (white bars). [Figure 11A-1]1 shows an alignment of 2H11 with the light chain CDR sequences of antibody variants Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8, with the L1, L2, and L3 regions indicated for both Kabat and Chlothia numbering. CDR-L1 contact residues are indicated. [Figure 11A-2] 1 shows an alignment of 2H11 with the light chain CDR sequences of antibody variants Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8, with the L1, L2, and L3 regions indicated for both Kabat and Chlothia numbering. CDR-L2 and L3 contact residues are indicated. [Figure 11B-1] 1 shows an alignment of the heavy chain CDR sequences of 2H11 and antibody variants Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8, with the H1, H2, and H3 regions indicated for both Kabat and Chlothia numbering. CDR-H1 contact residues are indicated. [Figure 11B-2] 1 shows an alignment of the heavy chain CDR sequences of 2H11 and antibody variants Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8, with the H1, H2, and H3 regions indicated for both Kabat and Chlothia numbering. CDR-H2 and H3 contact residues are indicated. DETAILED DESCRIPTION OF THE INVENTION

[0078] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein, including patent applications and patent publications, are incorporated herein by reference in their entirety.

[0079] For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0080] As used herein, "KRas" refers to the human KRas protein. In some embodiments, the human KRas comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the KRas protein is a mutant (e.g., a "mutant KRas" or "KRas mutant"). In some embodiments, the mutant KRas comprises one or more mutations relative to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the Kras mutant is an oncogenic mutant. In some embodiments, the KRas protein is a naturally occurring KRas mutant. In some embodiments, the KRas protein is a KRas G12C (i.e., KRas with a cysteine ​​substitution at position 12). In some embodiments, the KRas protein is KRas G12V , KRas G12R , KRas Q61H , or KRas G13DAs used herein, "KRas-GDP" refers to KRas bound to guanosine 5'-diphosphate (GDP). In some embodiments, KRas-GDP is inactive KRas. In some embodiments, inactive KRas is unable to bind to and allosterically activate the kinase activity of RAF kinases, such as c-Raf. In some embodiments, inactive KRas does not activate effector pathways downstream of KRas. In some embodiments, inactive KRas does not activate mitogen-activated protein (MAP) kinase cascades. In some embodiments, inactive KRas does not activate signaling cascades that promote proliferation. In some embodiments, inactive KRas does not activate signaling cascades that suppress apoptosis. In some embodiments, inactive KRas does not activate signaling cascades that promote transcription of the glucose transporter GLUT1.

[0081] As used herein, "KRas-GTP" refers to KRas bound to guanosine 5'-triphosphate (GTP). In some embodiments, KRas-GTP is active KRas. In some embodiments, active KRas can bind to and allosterically activate the kinase activity of a RAF kinase, such as c-Raf. In some embodiments, active KRas activates an effector pathway downstream of KRas. In some embodiments, active KRas activates a mitogen-activated protein (MAP) kinase cascade. In some embodiments, active KRas activates a signaling cascade that promotes proliferation. In some embodiments, active KRas activates a signaling cascade that suppresses apoptosis. In some embodiments, active KRas activates a signaling cascade that promotes transcription of the glucose transporter GLUT1.

[0082] As used herein, an "anti-KRas antibody" is one that binds to human KRas-GDP with sufficient specificity and affinity to be useful for detecting KRas-GDP, detecting alkylated KRas-GDP, and / or stabilizing KRas-GDP. In one embodiment, the extent of binding of an anti-KRas antibody to an unrelated KRas protein is less than about 10% of the binding of the antibody to Kras, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to KRas has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, e.g. 10 -9 ~10 -13 Dissociation constant (K D )

[0083] As used herein, an antibody that "stabilizes KRas-GDP" refers to an antibody that can bind to KRas-GDP and lock KRas in a GDP-bound state preferentially over a GTP-bound state. In some embodiments, antibodies that stabilize KRas-GDP are also referred to as CLAMPs (i.e., "Conformation Locking Antibodies for Molecular Probe discovery").

[0084] "Affinity" refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., KRas, KRas-GDP, and / or alkylated KRas antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative exemplary embodiments for measuring binding affinity are described herein. In some embodiments, affinity is measured using a surface plasmon resonance (SPR) assay. In some embodiments, affinity is measured using an SPR assay using a BIACORE®-T200, BIACORE®-S200, BIACORE®-8k, BIACORE®-2000, or BIACORE®-3000 instrument. In some embodiments, affinity is measured by enzyme-linked immunosorbent assay (ELISA).

[0085] As used herein, a first molecule has a dissociation constant (K) for binding to a second molecule. D ) is lower than that of the third molecule, the molecule binds to the second molecule with "higher affinity" than the third molecule.

[0086] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0087] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called an "Fab" fragment, each with a single antigen-binding site, and a remaining "Fc" fragment, named for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0088] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during the production of the monoclonal antibody preparation. Such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0089] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0090] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by one constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0091] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0092] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0093] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0094] A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 913242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0095] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0096] A "variable region" or "variable domain" is a domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6 thed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880887 (1993); Clarkson et al., Nature 352:624628 (1991).

[0097] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a naturally occurring four-chain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the "complementarity-determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J.Mol.Biol.196:901-917(1987).)

[0098] Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 of VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining regions," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of CDRs referred to as abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:16191633 (2008)). Unless otherwise indicated, HVR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0099] The term "detecting" is used in the broadest sense and includes both qualitative and quantitative measurements of a target molecule. In one embodiment, the detection methods described herein are used to simply confirm the presence of KRas, KRas-GDP, and / or alkylated KRas in a biological sample. In another embodiment, the methods are used to test whether KRas, KRas-GDP, and / or alkylated KRas is present in a sample at detectable levels. In yet another embodiment, the methods can be used to quantify the amount of KRas, KRas-GDP, and / or alkylated KRas in a sample, and even to compare levels of KRas, KRas-GDP, and / or alkylated KRas from different samples.

[0100] A "biological sample" refers to any biological material that may contain KRas, KRas-GDP, and / or alkylated KRas. The sample can be a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum, and plasma, ascites, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, and other components of the body that may contain KRas, KRas-GDP, and / or alkylated KRas. In various embodiments, the sample is a bodily sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is from a clinical patient or a patient treated with a therapeutic KRas antibody. In some embodiments, the biological sample is from a clinical patient or a patient treated with a KRas alkylating agent. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient.

[0101] The term "capture reagent" refers to a reagent (e.g., an antibody) or mixture of such reagents that binds to a target of interest (e.g., KRas, KRas-GDP, and / or alkylated KRas) and, under appropriate conditions, can bind to and capture the target of interest (e.g., KRas, KRas-GDP, and / or alkylated KRas) in a biological sample such that the complex of the capture reagent and the target of interest (e.g., KRas, KRas-GDP, and / or alkylated KRas) can be separated from the remainder of the sample. In certain embodiments, the capture reagent is immobilized or immobilizable.

[0102] The "Fab" fragment contains the heavy-chain variable domain and the light-chain variable domain, and also contains the light-chain constant domain and the first heavy-chain constant domain (CH1). Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the heavy-chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which one or more cysteine ​​residues in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0103] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0104] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0105] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0106] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the invention.

[0107] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0108] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0109] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:7987 (2007).

[0110] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0111] An "isolated nucleic acid encoding an anti-Kras antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-KRas antibody, including one or more such nucleic acid molecules in a single vector or separate vectors, and wherein such one or more nucleic acid molecules are present in one or more locations within a host cell.

[0112] The term "package insert" is used to refer to instructions customarily included in commercial packaging for a therapeutic product, which contain information about the use, directions for use, dosage, administration, concomitant therapy, contraindications and / or precautions for use of such therapeutic product.

[0113] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0114] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (alternatively, given amino acid sequence A can be said to have or contain a particular % amino acid sequence identity with or against given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues matched by the sequence alignment program ALIGN-2 as identical in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0115] A "therapeutically effective amount" is at least the minimum concentration required to achieve measurable improvement or prevention of a particular disorder. The therapeutically effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.

[0116] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain any additional ingredients that are toxic and cannot be tolerated by the subject to whom the formulation is administered.

[0117] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0118] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be carried out prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or palliation of the condition, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease, e.g., cancer, or to slow the progression of a disease.

[0119] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0120] As used herein, a "KRas inhibitor" refers to a compound that inhibits KRas G12C In the case of KRas G12C KRas refers to a covalent inhibitor that specifically alkylates at the Cys12 residue. G12D or KRas G13D As used herein in connection with KRas inhibitors, they can refer to covalent inhibitors (e.g., molecules that covalently bind to Asp12 or Asp13) or non-covalent inhibitors that specifically bind to a given KRas mutant described herein. G12V , KRas G12R , KRas G12D , KRas G13D , and KRas Q61H As used herein in this context, a KRas inhibitor can refer to a non-covalent inhibitor that specifically binds to a given KRas mutant described herein.

[0121] "AMG-510" structure: JPEG0007822322000001.jpg50170 and refers to the compound having the chemical name 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

[0122] MRTX-849 has the following structure: JPEG0007822322000002.jpg61170 and refers to the compound having the chemical name 2-((S)-4-(7-(8-chloronaphthalen-1-yl)-2-((S)-1-methylpyrrolidin-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl)-1-(2-fluoroacryloyl)piperazin-2-yl)acetonitrile.

[0123] "ARS-1620" has the following structure: JPEG0007822322000003.jpg49170 and refers to the compound having the chemical name (R)-1-(4-(6-chloro-8-fluoro-7-(2-fluoro-6-hydroxyphenyl)quinazolin-4-yl)piperazin-1-yl)prop-2-en-1-one.

[0124] "ARS-853" has the following structure: JPEG0007822322000004.jpg31170 and refers to the compound having the chemical name 1-(3-(4-((4-chloro-2-hydroxy-5-(1-methylcyclopropyl)phenyl)glycyl)piperazin-1-yl)azetidin-1-yl)prop-2-en-1-one.

[0125] "GNE-1952" has the following structure: JPEG0007822322000005.jpg53170 and refers to the compound having the chemical name (R)-1-(4-(6-chloro-7-(5-methyl-1H-indazol-4-yl)quinazolin-4-yl)piperazin-1-yl)prop-2-en-1-one.

[0126] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0127] The term "about" as used herein refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to that value or parameter itself.

[0128] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0129] II. Compositions and Methods A. Anti-Kras antibody i. Human KRas protein In one aspect, the present disclosure provides antibodies that interact with or otherwise bind to an epitope-like region within the human KRas protein. The KRas protein is a 21-kilodalton monomeric GTPase that is part of the RAS / MAPK signaling pathway. KRas is a proto-oncogene and the most frequently mutated oncogene in human cancers (Haigis, KM, Trends Cancer 2017 3:10).

[0130] In some embodiments, human KRas is variously referred to as CK-RAS, cK-ras protein, cK-ras2 protein, c-Kirsten-ras protein, cellular c-Ki-ras2 proto-oncogene, K-ras p21 protein, KI-RAS, Kirsten rat sarcoma viral oncogene homolog, KRAS1, PR310 c-K-ras oncogene, RASK2, RASK_HUMAN, transforming protein p21, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, NS, NS3, OES, CFC2, RALD, K-Ras, KRAS1, KRAS2, K-RAS2A, K_RAS2B, and K-RAS4B.

[0131] Human KRas mRNA has two splice isoforms that result in two variants of the KRas protein. The variant called "KRas isoform b" is the predominant variant and consists of five exons. Isoform b lacks exon 4a and terminates at exon 4b. The second variant (isoform a) is a rare variant that consists of six exons, including exon 4a, and terminates at exon 4a. In this disclosure, the term "KRas" refers to isoform b unless otherwise specified.

[0132] The amino acid sequence of human KRas isoform b is shown below as SEQ ID NO:90. MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM

[0133] The amino acid sequence of human KRas isoform a is shown below as SEQ ID NO:89. MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM

[0134] KRas mutations and their association with various phenotypes, including cancer phenotypes, have been previously reported (see Online Mendelian Inheritance in Man entry number 190070). Multiple mutators of human KRas have been classified as pathogenic by an expert panel of the FDA's Recognition of Public Human Genetic Variant Databases, including the coding sequence variants D153V, G60R, T58I, P34L, Q22R, V14I, and K5N. In some embodiments, the KRas is a mutant KRas having a mutation corresponding to G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, Q61R, A146T, A146P, A146V, or A146T. In some embodiments of the present disclosure, additional mutators of KRas are used, including, for example, KRas G12C , KRas G12D , KRas G13D , KRas G13C , KRas G12V , KRas G12R , and KRas Q61H Includes:

[0135] In some embodiments, KRas couples cell surface receptors to intracellular effector pathways by cycling between "on" and "off" conformations conferred by GTP and GDP binding, respectively. In some embodiments, KRas is bound to GDP; in these embodiments, it is referred to as KRas-GDP, or inactive KRas. In other embodiments, KRas is bound to GTP; in these embodiments, it is referred to as KRas-GTP, or active KRas. The transition between these two states is regulated by guanine nucleotide exchange factors (GEFs), which promote Ras protein activation by stimulating GDP for GTP exchange, and by GTPase-activating proteins (GAPs), which accelerate Ras-mediated GTP hydrolysis (Pylayeva-Gupta, Y. et al. Nat Rev Cancer 2011 11). In some embodiments, oncogenic mutations in KRas disrupt its ability to transition between KRas-GDP and KRas-GTP. In some embodiments, oncogenic substitutions at residues G12 and G13 sterically hinder the formation of van der Waals bonds between KRas and GAP, thus disrupting the proper orientation of the catalytic glutamine (Q61) in RAS, resulting in a marked attenuation of GTP hydrolysis (Pylayeva-Gupta, Y. et al. Nat Rev Cancer 2011 11; Scheffzek K, et al. Science 1997 277). As a result, in some embodiments, KRas is constitutively active.

[0136] KRas has an allosteric pocket that is only revealed in the GDP-bound state (Ostream, JMet al., Nature 2013 28 503:7477). This pocket is known as the switch II pocket, S-IIP, or SWII. KRas, an example of a KRas mutator, G12CKRas has been targeted by covalent attachment of inhibitors to the Cys12 residue. These inhibitors stabilize the opening of the SWII pocket (Ostream, JMet al., Nature 2013 28 503:7477; Patricelli, MP, et al., Cancer Discov. 2016 6; Lito, P., et al., Science 2016 351). The mechanism of action of such SWII covalent binders (also known as SWII ligands) is thought to be through transient stabilization of the SWII pocket by initial weak binding to the pocket followed by alkylation of Cys12 (Patricelli, MP, et al., Cancer Discov. 2016 6). This allows KRas to be activated. G12C -GDP was locked in an inactive state, inhibiting tumor growth in preclinical models and showing promising clinical activity (Patricelli, MP, et al., Cancer Discov. 2016 6; Fakih, M. et al., J Clin Oncol 2019 37).

[0137] ii. Anti-Kras antibody Described herein are anti-KRas antibodies or antigen-binding fragments thereof that bind to human KRas protein. In some embodiments, the anti-KRas antibody binds to the KRas protein, wherein the KRas protein comprises the amino acid sequence SEQ ID NO: 90. In some embodiments, the anti-KRas antibody binds to human KRas, wherein the antibody binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP). In some embodiments, the anti-KRas antibody has a lower dissociation constant (K ) than KRas-GTP. D In some embodiments, the anti-KRas antibody binds to KRas-GDP with a lower dissociation constant (K) than KRas-GTP at 25°C. D In some embodiments, the anti-KRas antibody binds to KRas-GDP with a lower dissociation constant (K) than KRas-GTP, as determined by surface plasmon resonance. DIn some embodiments, the anti-KRas antibody binds to KRas-GDP with a lower dissociation constant (K) than KRas-GTP, as determined by surface plasmon resonance at 25° C. D In some embodiments, the anti-KRas antibody binds to KRas-GDP with a lower dissociation constant (K) than the anti-KRas antibody binds to KRas-GTP. D ) binds to KRas-GDP. In some embodiments, the anti-KRas antibody binds to KRas-GDP with greater specificity than it binds to KRas-GTP. In some embodiments, the anti-KRas antibody binds to KRas-GDP with greater strength than it binds to KRas-GTP. In some embodiments, the anti-KRas antibody binds to KRas-GDP with a higher association constant or affinity constant than KRas-GTP. In some embodiments, the anti-KRas antibody binds to KRas-GDP preferentially over KRas-GTP.

[0138] In some embodiments, the anti-KRas antibody exhibits no detectable binding to KRas GTP. In some embodiments, the anti-KRas antibody has at least 10-fold, at least 100-fold, at least 1000-fold, or at least 10,000-fold greater affinity for KRas-GDP compared to KRas-GTP. In some embodiments, the anti-KRas antibody has 10- to 10,000-fold greater affinity for KRas-GDP compared to KRas-GTP. In some embodiments, the anti-KRas antibody has 10- to 1,000,000-fold greater affinity for KRas-GDP compared to KRas-GTP.

[0139] In some embodiments, the anti-KRas antibody binds to human KRas, where the antibody binds to KRas bound to GTP (KRas-GTP) with higher affinity than to KRas bound to GDP (KRas-GDP). In some embodiments, the anti-KRas antibody has a lower dissociation constant (K) than KRas-GDP. D In some embodiments, the anti-KRas antibody binds to KRas-GTP with a lower dissociation constant (K) than KRas-GDP at 25°C.D In some embodiments, the anti-KRas antibody binds to KRas-GTP with a lower dissociation constant (K) than KRas-GDP, as determined by surface plasmon resonance. D In some embodiments, the anti-KRas antibody binds to KRas-GTP with a lower dissociation constant (K) than KRas-GDP, as determined by surface plasmon resonance at 25° C. D In some embodiments, the anti-KRas antibody binds to KRas-GTP with a lower dissociation constant (K) than the anti-KRas antibody binds to KRas-GDP. D ) binds to KRas-GTP. In some embodiments, the anti-KRas antibody binds to KRas-GTP with greater specificity than it binds to KRas-GDP. In some embodiments, the anti-KRas antibody binds to KRas-GTP with greater strength than it binds to KRas-GDP. In some embodiments, the anti-KRas antibody binds to KRas-GTP with a higher association or affinity constant than KRas-GDP. In some embodiments, the anti-KRas antibody binds to KRas-GTP preferentially over KRas-GDP.

[0140] In some embodiments, the anti-KRas antibody binds to inactive KRas with higher affinity than to active KRas. In some embodiments, the anti-KRas antibody binds to inactive KRas with higher stability than it binds to active KRas. In some embodiments, the anti-KRas antibody binds to inactive KRas with lower K than it binds to active KRas. D In some embodiments, the anti-KRas antibody binds to inactive KRas with greater specificity than to active KRas. In some embodiments, the anti-KRas antibody binds to inactive KRas with greater specificity than to active KRas. In some embodiments, the anti-KRas antibody binds to inactive KRas with greater strength than to active KRas. In some embodiments, the anti-KRas antibody binds to inactive KRas preferentially over active KRas.

[0141] In some embodiments, the present disclosure provides anti-KRas antibodies that bind to and / or induce a particular conformation of KRas. In particular, provided herein are anti-KRas antibodies that open and / or stabilize the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein stabilize the SWII pocket. In some embodiments, the anti-KRas antibodies stabilize the open conformation of KRas. In some embodiments, the anti-KRas antibodies open and stabilize the SWII pocket. In some embodiments, the anti-KRas antibodies stabilize the inactive conformation of KRas. In some embodiments, the anti-KRas antibodies open and stabilize the SWII pocket to allow KRas inhibitors to bind. In some embodiments, the anti-KRas antibodies preferentially bind to the open conformation of KRas. In some embodiments, the anti-KRas antibodies bind KRas-GDP in either the open or closed conformation. In some embodiments, the anti-KRas antibodies bind KRas-GTP in either the open or closed conformation. In some embodiments, the anti-KRas antibody improves binding of a molecule to the SWII pocket. In some embodiments, the anti-KRas antibody improves binding of an inhibitor to the SWII pocket. In some embodiments, the anti-KRas antibody improves binding of a ligand to the SWII pocket. In some embodiments, the anti-KRas antibody improves binding of a covalent KRas inhibitor (e.g., a KRas inhibitor that alkylates Cys12) in the SWII pocket. In some embodiments, the anti-KRas antibody improves binding of a KRas inhibitor to the SWII pocket. G12C and enhances the covalent attachment (e.g., alkylation) of residue Cys12. In some embodiments, the anti-KRas antibody binds to KRas G12D and enhances the covalent attachment of residue Asp12. In some embodiments, the anti-KRas antibody binds to KRas G13D and enhances the covalent binding of residue Asp13.

[0142] In some embodiments, the anti-KRas antibody enhances binding of a non-covalent KRas inhibitor (e.g., a KRas inhibitor that non-covalently binds to residues 12 or 13 within the SWII pocket). G12D and enhances the non-covalent binding of residue Asp12. In some embodiments, the anti-KRas antibody binds to KRas G12D and enhances the covalent attachment of residue Asp12. In some embodiments, the anti-KRas antibody binds to KRas G12V and enhances the non-covalent binding of residue Val12. In some embodiments, the anti-KRas antibody binds to KRas G12R and enhances the non-covalent binding of residue Arg12. In some embodiments, the anti-KRas antibody binds to KRas G13D and enhances the non-covalent binding of residue Asp13. In some embodiments, the anti-KRas antibody binds to KRas G13D and enhances the covalent binding of residue Asp13. In some embodiments, the anti-KRas antibody binds to KRas Q61H and enhances the non-covalent binding of residue His61.

[0143] In some embodiments, the present disclosure provides anti-KRas antibodies that cause KRas to more frequently assume a particular conformation. In some embodiments, the anti-KRas antibodies cause KRas to occupy a particular conformation. In particular, provided herein are anti-KRas antibodies that cause KRas to more frequently contain an open SWII pocket. In some embodiments, the anti-KRas antibodies cause KRas to more frequently assume an open conformation. In some embodiments, the anti-KRas antibodies cause the KRas SWII pocket to be more frequently open and / or stabilize it. In some embodiments, the anti-KRas antibodies cause the SWII pocket to be more frequently open and stabilized so that a KRas inhibitor can bind. In such embodiments, the anti-KRas antibodies described herein can make the residue at position 12 more accessible to and / or stabilized by an inhibitor. In some embodiments, the anti-KRas antibodies increase the likelihood that a ligand will bind to the SWII pocket. In some embodiments, the anti-KRas antibodies increase the likelihood that the SWII pocket will be bound (e.g., alkylated) by a covalent KRas inhibitor. In some embodiments, the anti-KRas antibody is G12C In some embodiments, the anti-KRas antibody binds to the SWII pocket, increasing the likelihood that residue Cys12 will be bound (e.g., alkylated) by a covalent inhibitor. In some embodiments, the anti-KRas antibody increases the likelihood that the SWII pocket will be bound by a non-covalent KRas inhibitor. In some embodiments, the anti-KRas antibody binds to the KRas G12D In some embodiments, the anti-KRas antibody binds to KRas, increasing the likelihood that residue Asp12 will be bound by a non-covalent inhibitor. G12V In some embodiments, the anti-KRas antibody binds to KRas, increasing the likelihood that residue Val12 is bound by a non-covalent inhibitor. G12R and increases the likelihood that residue Arg12 will be bound by a non-covalent inhibitor. In some embodiments, the anti-KRas antibody binds to KRas G13DIn some embodiments, the anti-KRas antibody binds to KRas, increasing the likelihood that residue Asp13 will be bound by a non-covalent inhibitor. Q61H , increasing the likelihood that residue His61 will be bound by a non-covalent inhibitor.

[0144] In some embodiments, the present disclosure provides anti-KRas antibodies that affect the conformation of KRas. In some embodiments, the anti-KRas antibodies affect the structure of the KRas protein. In some embodiments, the anti-KRas antibodies change the relative frequency with which KRas occupies a particular conformation. In some embodiments, the anti-KRas antibodies change the preference of KRas for a particular conformation. In some embodiments, the anti-KRas antibodies allosterically regulate the structure of KRas. In particular, provided herein are anti-KRas antibodies that promote the opening of the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein promote the stabilization of the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein promote the stabilization of KRas. G12C In some embodiments, the anti-KRas antibodies provided herein promote stabilization of the SWII pocket in KRas. G12D In some embodiments, the anti-KRas antibodies provided herein promote stabilization of the SWII pocket in KRas. G12V In some embodiments, the anti-KRas antibodies provided herein promote stabilization of the SWII pocket in KRas. G12R In some embodiments, the anti-KRas antibodies provided herein promote stabilization of the SWII pocket in KRas. G13D In some embodiments, the anti-KRas antibodies provided herein promote stabilization of the SWII pocket in KRas. Q61HIn some embodiments, the anti-KRas antibody promotes stabilization of the SWII pocket in KRas. In some embodiments, the anti-KRas antibody promotes stabilization of the open conformation of KRas. In some embodiments, the anti-KRas antibody promotes opening and stabilization of the SWII pocket. In some embodiments, the anti-KRas antibody promotes stabilization of the inactive conformation of KRas. In some embodiments, the anti-KRas antibody promotes opening and stabilization of the SWII pocket to allow KRas inhibitors to bind. In some embodiments, the anti-KRas antibody promotes binding of a ligand to the SWII pocket. In some embodiments, the anti-KRas antibody promotes binding of an inhibitor to the SWII pocket. In some embodiments, the anti-KRas antibody promotes binding of a ligand to the SWII pocket. In some embodiments, the anti-KRas antibody promotes covalent alkylation of the SWII pocket. In some embodiments, the anti-KRas antibody promotes KRas G12C and promotes alkylation of residue Cys12. In some embodiments, the anti-KRas antibody promotes binding by a non-covalent KRas inhibitor. In some embodiments, the anti-KRas antibody promotes KRas G12D In some embodiments, the anti-KRas antibody binds to residue Asp12, and the antibody promotes binding to residue Asp12 by a non-covalent inhibitor. G12V In some embodiments, the anti-KRas antibody binds to residue Val12, and the antibody promotes binding to residue Val12 by a non-covalent inhibitor. G12R In some embodiments, the anti-KRas antibody binds to KRas, and the antibody promotes binding to residue Arg12 by a non-covalent inhibitor. G13D In some embodiments, the anti-KRas antibody binds to residue Asp13, and the antibody promotes binding to residue Asp13 by a non-covalent inhibitor. Q61H and the antibody promotes binding to residue His61 by a non-covalent inhibitor.

[0145] In some embodiments, the present disclosure provides anti-KRas antibodies that bind to and / or induce a particular conformation of KRas. In particular, provided herein are anti-KRas antibodies that interfere with the closure of the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein prevent the closure of the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein bind to and / or induce a particular conformation of KRas. G12C In some embodiments, the anti-KRas antibodies provided herein inhibit KRas and prevent closure of the SWII pocket. G12D In some embodiments, the anti-KRas antibodies provided herein inhibit KRas and prevent the closure of the SWII pocket in KRas. G12V In some embodiments, the anti-KRas antibodies provided herein inhibit KRas and prevent the closure of the SWII pocket in KRas. G12R In some embodiments, the anti-KRas antibodies provided herein inhibit KRas and prevent the closure of the SWII pocket in KRas. G13D In some embodiments, the anti-KRas antibodies provided herein inhibit KRas and prevent the closure of the SWII pocket in KRas. Q61H In some embodiments, the anti-KRas antibody prevents the SWII pocket from closing in the closed conformation. In some embodiments, the anti-KRas antibody prevents or prevents KRas from adopting a closed conformation. In some embodiments, the anti-KRas antibody prevents or prevents the closed conformation of the SWII pocket. In some embodiments, the anti-KRas antibody prevents or prevents the closure of the SWII pocket so that a KRas inhibitor can bind. In some embodiments, the anti-KRas antibody preferentially binds to an open conformation of KRas. In some embodiments, the anti-KRas antibody preferentially binds to an open conformation of the KRas SWII pocket. In some embodiments, the anti-KRas antibody reduces the likelihood that KRas is in a closed conformation. In some embodiments, the anti-KRas antibody reduces the frequency that KRas is in a closed conformation.

[0146] In some embodiments, binding of the anti-KRas antibodies disclosed herein results in the induction of a particular conformation of KRas. In particular, binding of the anti-KRas antibodies disclosed herein results in an open SWII pocket. In some embodiments, the anti-KRas antibodies provided herein inhibit KRas G12C In some embodiments, the anti-KRas antibodies provided herein inhibit KRas G12D In some embodiments, the anti-KRas antibodies provided herein inhibit KRas G12V In some embodiments, the anti-KRas antibodies provided herein inhibit KRas G12R In some embodiments, the anti-KRas antibodies provided herein inhibit KRas G13D In some embodiments, the anti-KRas antibodies provided herein inhibit KRas Q61HIn some embodiments, binding of the anti-KRas antibody results in a stably open SWII pocket. In some embodiments, binding of the anti-KRas antibody results in a SWII pocket that is more likely to be open. In some embodiments, binding of the anti-KRas antibody results in opening of the SWII pocket. In some embodiments, binding of the anti-KRas antibody results in stabilization of the SWII pocket. In some embodiments, binding of the anti-KRas antibody results in stabilization of the open conformation of KRas. In some embodiments, binding of the anti-KRas antibody results in opening and stabilization of the SWII pocket. In some embodiments, binding of the anti-KRas antibody results in stabilization of the inactive conformation of KRas. In some embodiments, binding of the anti-KRas antibody results in opening and stabilization of the SWII pocket so that a KRas inhibitor can bind. In some embodiments, binding of the anti-KRas antibody improves binding of the inhibitor to the SWII pocket. In some embodiments, binding of the anti-KRas antibody improves binding of a ligand to the SWII pocket. In some embodiments, binding of an anti-KRas antibody enhances SWII pocket covalent alkylation. In some embodiments, binding of an anti-KRas antibody to KRas G12C Conjugation to enhances alkylation of residue Cys12.

[0147] In some embodiments, the present disclosure provides anti-KRas antibodies that specifically bind to and / or specifically induce a particular conformation of KRas. In particular, provided herein are anti-KRas antibodies that specifically open the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein specifically stabilize the SWII pocket. In some embodiments, the anti-KRas antibodies provided herein specifically bind to and / or induce a particular conformation of KRas. G12D In some embodiments, the anti-KRas antibodies provided herein specifically stabilize the SWII pocket of KRas. G12V In some embodiments, the anti-KRas antibodies provided herein specifically stabilize the SWII pocket of KRas.G12R In some embodiments, the anti-KRas antibodies provided herein specifically stabilize the SWII pocket of KRas. G13D In some embodiments, the anti-KRas antibodies provided herein specifically stabilize the SWII pocket of KRas. Q61H In some embodiments, the anti-KRas antibody specifically stabilizes the SWII pocket of KRas. In some embodiments, the anti-KRas antibody specifically stabilizes the open conformation of KRas. In some embodiments, the anti-KRas antibody specifically opens and stabilizes the SWII pocket. In some embodiments, the anti-KRas antibody specifically stabilizes the inactive conformation of KRas. In some embodiments, the anti-KRas antibody specifically opens and stabilizes the SWII pocket to allow binding of KRas inhibitors. In some embodiments, the anti-KRas antibody specifically binds to the open conformation of KRas.

[0148] In some embodiments of the present disclosure, the anti-KRas antibody is a KRas alkylation conformation-specific antibody. In some embodiments, KRas alkylation conformation-specific antibodies are referred to as Class I antibodies, including, for example, antibodies 1A5, 1D6, 2C1, 1A6, 1F4, and 1B7. In some embodiments, the KRas alkylation conformation-specific antibody binds to KRas covalently linked to a KRas inhibitor (e.g., alkylated at Cys12 by the covalent inhibitor). In some embodiments, the KRas alkylation conformation-specific antibody binds in the presence of a covalently linked SWII inhibitor. In some embodiments, the KRas alkylation conformation-specific antibody binds in the presence of a covalently linked SWII ligand. In some embodiments, KRas is covalently bound to a KRas inhibitor selected from the group consisting of MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157. In another embodiment, KRas is covalently bound to a compound such as ARS1620 or GNE1952. In some embodiments, the KRas alkylation conformation-specific antibody binds to KRas in an open SWII pocket conformation. In some embodiments, the KRas alkylation conformation-specific antibody stabilizes the SWII pocket. In some embodiments, the KRas alkylation conformation-specific antibody described herein binds to KRas. G12D In some embodiments, the KRas alkylation conformation-specific antibodies described herein stabilize the SWII pocket of KRas. G12V In some embodiments, the KRas alkylation conformation-specific antibodies described herein stabilize the SWII pocket of KRas. G12R In some embodiments, the KRas alkylation conformation-specific antibodies described herein stabilize the SWII pocket of KRas. G13DIn some embodiments, the KRas alkylation conformation-specific antibodies described herein stabilize the SWII pocket of KRas. Q61H In some embodiments, the KRas alkylation conformation-specific antibodies stabilize the SWII pocket of KRas in an open conformation. In some embodiments, the KRas alkylation conformation-specific antibodies are used to detect alkylation of KRas. In some embodiments, the KRas alkylation conformation-specific antibodies described herein inhibit KRas alkylation against covalent inhibitors. G12C In some embodiments, the KRas alkylation conformation-specific antibody is used to detect binding of alkylated KRas in tumor cells in vivo. G12C- In some embodiments, KRas alkylation conformation-specific antibodies may be used to detect alkylated KRas in tumor cells in vivo. G12C- In some such embodiments, the detection may be used to detect KRas in patients treated with a KRas inhibitor (e.g., MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157). G12C It is used to monitor the alkylation of

[0149] In some embodiments, alkylated conformation-specific anti-KRas antibodies have at least 5-fold, at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater affinity for the open conformation of KRas compared to the closed conformation. In some embodiments, alkylated conformation-specific anti-KRas antibodies have a 2- to 1000-fold increased affinity for the open conformation of KRas compared to the closed conformation. In some embodiments, alkylated conformation-specific anti-KRas antibodies have a 10- to 1000-fold increased affinity for the open conformation of KRas compared to the closed conformation. In some embodiments, alkylated conformation-specific anti-KRas antibodies have a 10- to 10,000-fold increased affinity for the open conformation of KRas compared to the closed conformation. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10-100,000 fold increased affinity for the open conformation of KRas compared to the closed conformation.

[0150] In some embodiments of the present disclosure, the alkylated conformation-specific anti-KRas antibody stabilizes the open conformation of the SWII pocket of KRas described herein. In some embodiments, the anti-Kras antibody is a Class I or Class II antibody. In some embodiments, the anti-Kras antibody is 1A5. In some embodiments, the anti-Kras antibody is 1D6. In some embodiments, the antibody is 2C1. In some embodiments, the anti-Kras antibody is 1A6. In some embodiments, the antibody is 1B7. In some embodiments, the anti-Kras antibody is 1E5. In some embodiments, the anti-Kras antibody is 2H11. In some embodiments, the anti-Kras antibody is 2A3. In some embodiments, the anti-Kras antibody is 3A12. In some embodiments, the anti-Kras antibody is 4G12. In some embodiments, the anti-Kras antibody is 1F4. In some embodiments, the anti-Kras antibody is Ab1. In some embodiments, the anti-Kras antibody is Ab2. In some embodiments, the anti-Kras antibody is Ab3. In some embodiments, the anti-Kras antibody is Ab4. In some embodiments, the anti-Kras antibody is Ab5. In some embodiments, the anti-Kras antibody is Ab6. In some embodiments, the anti-Kras antibody is Ab7. In some embodiments, the anti-Kras antibody is Ab8.

[0151] In some embodiments of the present disclosure, the anti-KRas antibody is an alkylating conformation-specific KRas antibody. In some embodiments, the alkylating conformation-specific KRas antibody binds to KRas and induces a conformation of KRas in which the SWII pocket is open. In some embodiments, the alkylating conformation-specific KRas antibody initially binds to KRas with a closed SWII pocket and induces a conformational change in KRas such that the SWII pocket opens. In some embodiments, the binding of the alkylating conformation-specific KRas antibody opens the SWII pocket. In some embodiments, the alkylating conformation-specific KRas antibody promotes the opening of the SWII pocket. In some embodiments, the alkylating conformation-specific KRas antibody changes the conformation of KRas. In some embodiments, the alkylating-inducing KRas antibody is a class II antibody. In some embodiments, the KRas alkylating conformation-specific antibody is a class II antibody. In some embodiments, the anti-FAPKRas antibody is 1E5, 2H11, 2A3, 3A12, 1F4, or 4G12. In some embodiments, the anti-KRas antibody is 1E5, 2H11, 2A3, 3A12, 1F4, 4G12, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is 2H11, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-KRas antibody is Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is 1E5. In some embodiments, the anti-Kras antibody is 2H11. In some embodiments, the anti-Kras antibody is 2A3. In some embodiments, the anti-Kras antibody is 3A12. In some embodiments, the anti-Kras antibody is 4G12. In some embodiments, the anti-Kras antibody is 1F4. In some embodiments, the anti-Kras antibody is Ab1. In some embodiments, the anti-Kras antibody is Ab2. In some embodiments, the anti-Kras antibody is Ab3.In some embodiments, the anti-Kras antibody is Ab4. In some embodiments, the anti-Kras antibody is Ab5. In some embodiments, the anti-Kras antibody is Ab6. In some embodiments, the anti-Kras antibody is Ab7. In some embodiments, the anti-Kras antibody is Ab8.

[0152] In some embodiments, the alkylated conformation-specific KRas antibodies of the present disclosure comprise covalently linked KRas G12C In the absence of an inhibitor or SWII ligand, the anti-KRas antibody stabilizes the open conformation of the SWII pocket. In some embodiments, the anti-KRas antibody initially binds to KRas with an open SWII pocket and stabilizes the SWII pocket as described herein. In some embodiments, the alkylated conformation-specific KRas antibodies of the present disclosure bind to non-covalently bound KRas. G12C The anti-KRas antibody stabilizes the open conformation of the SWII pocket in the absence of an inhibitor or SWII ligand. In some embodiments, the open conformation of the SWII pocket is considered stabilized when it is more likely to be open than in a wild-type KRas protein not bound by such an antibody. In some embodiments, the open conformation of the SWII pocket is considered induced when the open conformation is present more frequently than normally present. In some embodiments, the anti-KRas antibody locks the KRas SWII pocket in an open conformation. In some embodiments, the anti-KRas antibody prevents the SWII pocket from closing. In some embodiments, the alkylated conformation-specific KRas antibody inhibits KRas. G12C and improve the non-covalent binding affinity of several G12C inhibitors for wild-type KRas.

[0153] In some embodiments, the alkylation-inducing anti-KRas antibody binds to non-alkylated KRas-GDP and alkylated KRas-GDP with approximately the same affinity. In some embodiments, the anti-KRas antibody binds to non-alkylated KRas-GDP and alkylated KRas-GDP with affinities that are within 10-fold, 5-fold, or 2-fold of each other. In some embodiments, the anti-KRas antibody binds to non-alkylated KRas-GDP and alkylated KRas-GDP with affinities that are between 10-fold and 2-fold of each other.

[0154] In some embodiments, the present disclosure provides anti-KRas antibodies or antigen-binding fragments thereof that bind to human KRas, wherein the human KRas is a KRas mutant. G12C Mutations at codon 12 of KRas are prevalent in cancers in which KRAS is most common (i.e., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC)) (Haigis, KM, Trends Cancer 2017 3:10). KRas G12C is the specific allele of KRAS targeted by a compound that covalently binds to the mutated residue Cys12 as described above.

[0155] In some embodiments, the anti-KRas antibodies of the present disclosure bind to mutant KRas proteins. In some embodiments of the present disclosure, the mutant Kras is Kras G12V In some embodiments, the Kras mutant is Kras G12R In some embodiments, the Kras mutant is Kras Q61H In some embodiments, the Kras mutant is Kras G12D In some embodiments, the Kras mutant is Kras G13D is.

[0156] In some embodiments, the isolated antibody or antigen-binding fragment comprises a KRas antibody covalently linked (e.g., alkylated) to a small molecule. G12CIn some embodiments, the anti-KRas antibody is an alkylated conformation-specific KRas antibody that binds -GDP. In some embodiments, the anti-KRas antibody is a KRas antibody covalently bound by a small molecule. G12C - an alkylated conformation-specific KRas antibody that binds GDP. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to MRTX849. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to AMG-510. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to GDC-6036. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to ARS-3248. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to LY3499446. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to JNJ-74699157. In some embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

[0157] In some embodiments, the isolated antibody or antigen-binding fragment is a KRas antibody covalently linked (e.g., alkylated) to a small molecule in a clinical sample as described herein. G12C In some embodiments, the anti-KRas antibody is an alkylated conformation-specific KRas antibody that binds -GDP. In some embodiments, the anti-KRas antibody binds KRas covalently bound by a small molecule in a tumor sample obtained from a patient as described herein. G12C In some such embodiments, the KRas antibody is an alkylated conformation-specific KRas antibody that binds to -GDP. G12C -GDP is covalently attached (e.g., alkylated) to MRTX849. In some such embodiments, KRas G12C-GDP is covalently attached (e.g., alkylated) to AMG-510. In some such embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to GDC-6036. In some such embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to ARS-3248. In some such embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to LY3499446. In some such embodiments, KRas G12C -GDP is covalently attached (e.g., alkylated) to JNJ-74699157. In some such embodiments, KRas G12C -GDP is covalently linked (e.g., alkylated) to MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157. In some embodiments, the isolated antibody or antigen-binding fragment is an alkylated conformation-specific KRas antibody that binds to KRas-GDP as described herein and is used in a biomarker assay for determining the level of target engagement as described herein.

[0158] In some embodiments, the anti-Kras antibody inhibits KRas G12C In some embodiments, the anti-KRas antibody binds to KRas -GDP. G12C -When GDP is alkylated, KRas G12C -GDP binds to KRas with higher affinity than when it is not alkylated. G12C In some embodiments, the anti-Kras antibody binds to KRas-GDP. G12D In some embodiments, the anti-Kras antibody binds to KRas-GDP. G12V In some embodiments, the anti-Kras antibody binds to KRas-GDP. G12R In some embodiments, the anti-Kras antibody binds to KRas-GDP. G13D In some embodiments, the anti-Kras antibody binds to KRas-GDP. Q61HIn some embodiments, the anti-KRas antibody binds to mutant KRas-GDP with greater affinity when the mutant KRas-GDP is bound by a covalent or non-covalent KRas inhibitor than when the mutant KRas-GDP is not bound by a covalent or non-covalent KRas inhibitor.

[0159] In some embodiments, the alkylated conformation-specific anti-KRas antibody has at least 5-fold, at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater affinity for alkylated KRas-GDP than for non-alkylated KRas-GDP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 2- to 1000-fold increased affinity for alkylated KRas-GDP over non-alkylated KRas-GDP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 1000-fold increased affinity for alkylated KRas-GDP over non-alkylated KRas-GDP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 1000-fold increased affinity for alkylated KRas-GDP over non-alkylated KRas-GDP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 10,000-fold increased affinity for alkylated KRas-GDP over non-alkylated KRas-GDP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 100,000-fold increased affinity for alkylated KRas-GDP over non-alkylated KRas-GDP.

[0160] In some embodiments, the alkylated conformation-specific anti-KRas antibody has at least 5-fold, at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater affinity for alkylated KRas-GTP than for non-alkylated KRas-GTP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 2- to 1000-fold increased affinity for alkylated KRas-GTP over non-alkylated KRas-GTP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 1000-fold increased affinity for alkylated KRas-GTP over non-alkylated KRas-GTP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 1000-fold increased affinity for alkylated KRas-GTP over non-alkylated KRas-GTP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 10,000-fold increased affinity for alkylated KRas-GTP over non-alkylated KRas-GTP. In some embodiments, the alkylated conformation-specific anti-KRas antibody has a 10- to 100,000-fold increased affinity for alkylated KRas-GTP over non-alkylated KRas-GTP.

[0161] In some embodiments, the isolated antibody or antigen-binding fragment comprises a KRas antibody covalently linked to a small molecule. G12D In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds to -GDP. G12D In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds -GTP. G12V In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds to -GDP. G12V In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds -GTP.G12R In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds to -GDP. G12R In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds -GTP. Q61H In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds to -GDP. Q61H In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds -GTP. G13D In some embodiments, the isolated antibody or antigen-binding fragment is an anti-KRas antibody that binds to -GDP. G13D -GTP-binding anti-KRas antibody.

[0162] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody 2H11, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody 2H11, as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 2H11 and / or a VH and / or VL of antibody 2H11 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody opens and stabilizes the SWII pocket of KRas.

[0163] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab1 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab1 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab1 and / or a VH and / or VL of antibody Ab1 inhibits KRas mutant KRas. G12C In some embodiments, antibody Ab1 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab1 opens and stabilizes the SWII pocket of KRas.

[0164] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab2 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab2 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab2 and / or a VH and / or VL of antibody Ab2 inhibits KRas-mutated KRas. G12C In some embodiments, antibody Ab2 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab2 opens and stabilizes the SWII pocket of KRas.

[0165] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab3 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab3 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab3 and / or a VH and / or VL of antibody Ab3 inhibits KRas mutant KRas. G12CIn some embodiments, the antibody Ab3 is an alkylation conformation-specific KRas antibody. In some embodiments, the antibody Ab3 opens and stabilizes the SWII pocket of KRas.

[0166] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab4 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab4 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab4 and / or a VH and / or VL of antibody Ab4 inhibits KRas mutant KRas. G12C In some embodiments, the antibody Ab4 is an alkylation conformation-specific KRas antibody. In some embodiments, the antibody Ab4 opens and stabilizes the SWII pocket of KRas.

[0167] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab5 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab5 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab5 and / or a VH and / or VL of antibody Ab5 inhibits KRas mutant KRas. G12C In some embodiments, antibody Ab5 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab5 opens and stabilizes the SWII pocket of KRas.

[0168] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab6 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab6 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab6 and / or a VH and / or VL of antibody Ab6 inhibits KRas mutant KRas. G12C In some embodiments, antibody Ab6 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab6 opens and stabilizes the SWII pocket of KRas.

[0169] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab7, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab7, as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab7 and / or a VH and / or VL of antibody Ab7 inhibits KRas mutant KRas. G12C In some embodiments, antibody Ab7 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab7 opens and stabilizes the SWII pocket of KRas.

[0170] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six HVRs of antibody Ab8 as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody Ab8 as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody Ab8 and / or a VH and / or VL of antibody Ab8 inhibits KRas mutant KRas. G12CIn some embodiments, antibody Ab8 is an alkylation conformation-specific KRas antibody. In some embodiments, antibody Ab8 opens and stabilizes the SWII pocket of KRas.

[0171] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 16, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 16. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 16. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0172] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 99, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 99. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 99. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 91, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0173] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 100, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 100. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 100. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 92, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0174] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 101. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 101, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 101. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 101. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 93, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0175] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 102, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 102. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 102. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 94, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0176] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 103, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 103. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 103. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 95, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0177] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 104, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 104. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 104. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 96, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0178] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 105. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 105, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 105. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 105. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 97, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0179] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 106, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 106. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 106. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0180] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 15, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 15. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 15. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0181] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 99. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 100. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 101. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 103. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 104. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 105. In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 15 and a VH comprising the amino acid sequence of SEQ ID NO: 106.

[0182] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0183] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 91, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0184] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 92, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0185] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 93, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0186] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 94, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0187] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 95, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0188] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 96, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0189] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 97, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0190] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0191] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 16; and a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0192] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 99; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0193] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a VH having the sequence set forth in SEQ ID NO: 100; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a VL having the sequence set forth in SEQ ID NO: 15.

[0194] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 101; and a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0195] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 102; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0196] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 103; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0197] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 104; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0198] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 105; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0199] In another aspect, an anti-KRas antibody is provided, comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 106; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 15.

[0200] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1A5, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 1A5, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 1A5 and / or the VH and / or VL of antibody 1A5 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0201] In some embodiments, the anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 8, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 8. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

[0202] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 7, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 7. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0203] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:7 and a VH comprising the amino acid sequence of SEQ ID NO:8.

[0204] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0205] In another aspect, an anti-KRas antibody is provided that comprises a VH CDR1, a VH CDR2, and a VH CDR3 that comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO:8; and a VL CDR1, a VL CDR2, and a VL CDR3 that comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO:7.

[0206] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1D6, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 1D6, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 1D6 and / or the VH and / or VL of antibody 1D6 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0207] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 24, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 24. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 24. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22.

[0208] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 23, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 23. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted from SEQ ID NO: 23. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19.

[0209] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:23 and a VH comprising the amino acid sequence of SEQ ID NO:24.

[0210] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19.

[0211] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 24; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 23.

[0212] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 2C1, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody 2C1, as shown in Tables 4 and 5. In certain embodiments, anti-KRas antibodies comprising one, two, three, four, five, or six CDRs of antibody 2C1 and / or a VH and / or VL of antibody 2C1 are capable of inhibiting KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0213] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 32, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 32. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 32. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30.

[0214] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 31, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 31. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 31. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0215] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:31 and a VH comprising the amino acid sequence of SEQ ID NO:32.

[0216] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0217] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 32; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 31.

[0218] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 4G12, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 4G12, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 4G12 and / or the VH and / or VL of antibody 4G12 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody opens and stabilizes the SWII pocket of KRas.

[0219] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 40, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 40. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 40. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38.

[0220] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 39, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 39. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 39. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0221] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:39 and a VH comprising the amino acid sequence of SEQ ID NO:40.

[0222] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0223] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 40; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 39.

[0224] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1A6, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 1A6, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 1A6 and / or the VH and / or VL of antibody 1A6 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0225] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 48, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 48. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 48. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46.

[0226] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 47, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 47. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0227] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:47 and a VH comprising the amino acid sequence of SEQ ID NO:48.

[0228] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0229] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 48; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 47.

[0230] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1F4, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises a VH and / or VL of antibody 1F4, as shown in Tables 4 and 5. In certain embodiments, an anti-KRas antibody comprising one, two, three, four, five, or six CDRs of 1F4 and / or a VH and / or VL of antibody 1F4 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0231] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 88, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 88. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 88. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 84, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 86.

[0232] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 87, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 87. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 87. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

[0233] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:87 and a VH comprising the amino acid sequence of SEQ ID NO:88.

[0234] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 84, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 86, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

[0235] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 88; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 87.

[0236] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1B7, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 1B7, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 1B7 and / or the VH and / or VL of antibody 1B7 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket of KRas.

[0237] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 56, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 56. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54.

[0238] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 55. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 55, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 55. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 55. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51.

[0239] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:55 and a VH comprising the amino acid sequence of SEQ ID NO:56.

[0240] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51.

[0241] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 56; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 55.

[0242] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 1E5, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 1E5, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 1E5 and / or the VH and / or VL of antibody 1E5 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody opens and stabilizes the SWII pocket of KRas.

[0243] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 64, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 64. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 64. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62.

[0244] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 63, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 63. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 63. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 57; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 58; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

[0245] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:63 and a VH comprising the amino acid sequence of SEQ ID NO:64.

[0246] In another aspect, an anti-KRas antibody is provided comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 57, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

[0247] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 64; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 63.

[0248] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 2A3, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 2A3, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of antibody 2A3 and / or the VH and / or VL of antibody 2A3 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody opens and stabilizes the SWII pocket of KRas.

[0249] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 72, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 72. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 72. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70.

[0250] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 71, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 71. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 71. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 65; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 66; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 67.

[0251] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:71 and a VH comprising the amino acid sequence of SEQ ID NO:72.

[0252] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 65, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 67.

[0253] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 72; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 71.

[0254] In some embodiments, the anti-KRas antibody comprises one, two, three, four, five, or six CDRs of antibody 3A12, as shown in Tables 2 and 3. In some embodiments, the anti-KRas antibody comprises the VH and / or VL of antibody 3A12, as shown in Tables 4 and 5. In certain embodiments, the anti-KRas antibody comprising one, two, three, four, five, or six CDRs of 3A12 and / or the VH and / or VL of antibody 3A12 inhibits KRas mutant KRas. G12C In some embodiments, the anti-KRas antibody is an alkylation conformation-specific KRas antibody. In some embodiments, the anti-KRas antibody opens and stabilizes the SWII pocket of KRas.

[0255] In some embodiments, an anti-KRas antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In certain embodiments, the VH sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 80, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 80. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 80. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In certain embodiments, the VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78.

[0256] In another embodiment, an anti-KRas antibody is provided that comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79. In certain embodiments, the VL sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 79, while retaining the ability to bind to KRas as an anti-KRas antibody comprising SEQ ID NO: 79. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 79. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In certain embodiments, the VL comprises one, two or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0257] In one embodiment, the anti-Kras antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:79 and a VH comprising the amino acid sequence of SEQ ID NO:80.

[0258] In another aspect, an anti-KRas antibody is provided comprising a VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 76, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78, and a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

[0259] In another aspect, an anti-KRas antibody is provided comprising a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VH having the sequence set forth in SEQ ID NO: 80; and a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a VL having the sequence set forth in SEQ ID NO: 79.

[0260] In another aspect, an anti-KRas antibody is provided that comprises a VH of any of the embodiments provided above, and a VL of any of the embodiments provided above.

[0261] In a further aspect of the invention, the anti-KRas antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some embodiments, the anti-KRas antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the anti-KRas antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0262] In one embodiment of the present invention, the anti-KRas antibody according to the above embodiments binds to one or more amino acid epitopes of human KRas. In some embodiments, the anti-KRas antibody binds to one or more, two or more, three or more, four or more, five or more, six or more, or all of amino acids W99, K5, L6, V7, S39, D54, L54, Y71, T74, and / or G75 of human KRas, wherein human KRas comprises the amino acid sequence SEQ ID NO: 90. In some embodiments, the anti-KRas antibody binds to W99 of human KRas. In some embodiments, the anti-KRas antibody binds to residues from SW1 and SW2 of human KRas. In some embodiments, the anti-KRas antibody binds to SW2 of human KRas. In some embodiments, the anti-KRas antibody binds to the amino acid residues set forth in Tables A-D below. In some embodiments, the anti-KRas antibody binds within 3.5, 4.0, or 4.5 angstroms (Å) of a residue listed in Table A. In some embodiments, the anti-KRas antibody binds within 3.5, 4.0, or 4.5 Å of a residue listed in Table B. In some embodiments, the anti-KRas antibody binds within 3.5, 4.0, or 4.5 Å of a residue listed in Table C. In some embodiments, the anti-KRas antibody binds within 3.5, 4.0, or 4.5 Å of a residue listed in Table D. Table A: Contact residues of 2H11 JPEG0007822322000006.jpg137170 Table B: Contact residues of 2C1 JPEG0007822322000007.jpg99170Table C: Contact residues of 1E5 JPEG0007822322000008.jpg162170 Table D: Contact residues of 3A12 JPEG0007822322000009.jpg150170

[0263] In some embodiments, the contact residues of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8 are as shown in Figures 11A and 11B.

[0264] In another aspect provided herein, there is provided a fusion protein comprising a KRas protein (e.g., SEQ ID NO: 90) or a fragment thereof and a Fab, scFv, or IgG of an antibody described herein. In one such embodiment, the fusion protein comprises a KRas protein or a fragment thereof described herein, an Fab, scFv, or IgG of an antibody described herein, wherein n is at least 1 (Gly-Ser). )n and a Fab, scFv, or IgG described herein. In one such embodiment, n is an integer from 1 to 5, 1 to 8, 1 to 10, or 1 to 20. In one such embodiment, the KRas protein or fragment thereof is fused to the N-terminus of a Fab described herein. In one such embodiment, a linker is present between the KRas and the N-terminus of the Fab. In another embodiment, the KRas protein or fragment thereof is attached to the C-terminus of the Fab. In one such embodiment, a linker is present between the N-terminus of the KRas protein and the C-terminus of the Fab.

[0265] In one embodiment of the fusion proteins described herein, the Fab has the HC sequence: EVQLQESGPGLVKPPGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGSSSWYDLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 107), and LC sequence: GLNDIFEAQKIEWHEGSENLYFQSTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETSLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAINN TKSFEDIHHYREQIKRVKDSEDVPMVLVGNKSDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKGGGGSGGGGSGGGGSGGGGSSVLTQP PSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDERLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 108) Includes.

[0266] In another embodiment of the fusion protein described herein, the Fab has the HC sequence: GLNDIFEAQKIEWHEGSENLYFQSTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETSLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAINNTKS FEDIHHYREQIKRVKDSEDVPMVLVGNKSDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKGGGGSGGGGSGGGGSGGGGSEVQLQESGPGLVK PPGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGSSSWYDLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 109) and LC sequence: SVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDERLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 110) Includes.

[0267] In a further aspect of the invention, an anti-KRas antibody according to any of the above embodiments or described herein is conjugated to a heterologous moiety, agent, or label. Examples of suitable labels are the numerous labels known for use in immunoassays, including moieties that can be directly detected, such as fluorescent dye labels, chemiluminescent labels, and radioactive labels, as well as moieties such as enzymes that must react or be induced to be detected. Examples of such labels include radioisotopes. 32 P,14 C. 125 I, 3 H, and 131 I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, HRP, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and These include glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, heterocyclic oxidases such as uricase and xanthine oxidase coupled with HRP, lactoperoxidase, or microperoxidase, biotin (detectable with MUG, e.g., avidin, streptavidin, streptavidin-HRP, and streptavidin-β-galactosidase), spin labels, bacteriophage labels, and stable free radicals.

[0268] In another aspect, provided herein is an anti-KRas antibody according to any of the above embodiments, or a composition comprising one or more of the anti-KRas antibodies described herein. Also provided herein are nucleic acids encoding the anti-KRas antibodies described herein, vectors comprising the nucleic acids, and host cells comprising the vectors. In some embodiments, the host cells are isolated or purified. In some embodiments, the host cells are cell culture media.

[0269] iii. Method of generation 1. Polyclonal antibodies The antibodies of the present invention may include polyclonal antibodies. Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant are injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include human KRas or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation. The mammal can then be bled and the serum assayed for anti-KRas antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus.

[0270] 2. Monoclonal antibodies The antibodies of the invention may alternatively be monoclonal antibodies, which may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0271] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0272] The hybridoma cells thus prepared are seeded and grown in an appropriate medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (called the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0273] The fusion partner myeloma cells fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to selective media that select against the unfused parent cells. Myeloma cell lines are derived from mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 and derivatives such as X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0274] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0275] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

[0276] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, or dialysis.

[0277] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not naturally produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Rev. 130:151-188 (1992).

[0278] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing extremely large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0279] In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones of the library. Binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0280] The variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked by a short, flexible peptide, or as Fab fragments in which each is fused to a constant domain and interacts non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12:433455 (1994).

[0281] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0282] Screening of the library can be accomplished by a variety of techniques known in the art, for example, human KRas can be used to coat the wells of an adsorption plate, expressed on host cells attached to the adsorption plate, or used for cell sorting, or conjugated to biotin for capture by streptavidin-coated beads, or any other method for panning a display library.

[0283] Selection of antibodies with slow dissociation kinetics (and good binding affinity) can be facilitated by using long washes and monovalent phage display as described in Bass et al., Proteins, 8:309314 (1990) and WO 92 / 09690, and low coating density of antigen as described in Marks et al., Biotechnol., 10:779783 (1992).

[0284] Any of the anti-KRas antibodies of the present invention can be obtained by designing a suitable antigen screening procedure to select a phage clone of interest, and then constructing a full-length anti-KRas antibody clone using the Fv sequence from the phage clone of interest and an appropriate constant region (Fc) sequence as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0285] 3. Selection of Conformation-specific Anti-KRas Antibodies The methods provided herein can be used to screen for antibodies that bind to a specific conformation of human KRas. G12C -KRas with higher affinity than GTP G12C For example, this method can be used to screen for antibodies that bind to KRas-GDP. G12C -GDP, ii) alkylated KRas G12C -GDP, and iii) KRas bound to a non-hydrolyzable GTP analog. G12C and (b) contacting KRas bound to a non-hydrolyzable GTP analog. G12C It alkylates KRas with higher affinity than G12C -GDP and non-alkylated KRas G12C -selecting an antibody that binds to GDP.

[0286] For example, synthetic antibody libraries and distinct conformational forms of human KRas G12C For example, an in vitro selection strategy can be used to select GDP-bound KRas, either alkylated or unalkylated. G12C and non-alkylated KRas bound to GMPPcp (a non-hydrolyzable GTP mimetic). G12C Synthetic phage libraries can be used to identify small molecules. G12C Biotinylated KRas alkylated with the inhibitor GNE-1952 G12C Biopanning can also be performed by incubating the KRas receptor with -GDP in solution (Li Liansheng et al., WO 2017058768). Other small molecules, such as ARS-853 and ARS-1620, can covalently bind to Cys12, thereby targeting KRas receptors. G12C can be used to lock KRas in the open SWII conformation. G12C Excess non-biotinylated KRas in solution to select for its unique conformation in -GDP G12C -GDP and KRas G12C Selection may be performed in the presence of -GMPPcp. G12C -GDP can be biotinylated and recovered, allowing KRas to remain in the open conformation. G12C -Specific to GDP+GNE-1952 and KRas G12C -GDP and KRas G12C -Antibodies not specific for GMPPcp can be enriched.

[0287] Selection of conformation-specific anti-KRas antibodies can be performed, for example, using existing synthetic Fab phage display libraries (CV Lee et al., J Mol Biol 2004;340:1073-1093; WC Liang et al., J Mol Biol 2007;366:815-829). Pooled libraries can be used to screen for biotinylated KRas. G12CiBinding to -GDP+GNE1952 in solution can be repeated 3-4 times (initially ranging from 500 nM to 10 nM). This solution can be captured on NeutrAvidin beads (Promega), blocked with 5 μM biotin, washed three times for 30 seconds with PBS+0.5% BSA+0.1% Tween 20 (PBSBT), and eluted with 100 mM HCl. Eluted phage can be neutralized with 1 M Tris-HCl pH 8.0 before overnight amplification in E. coli XL1-blue (Stratagene) supplemented with M13-KO7 helper phage (New England Biolabs). Alkylated KRas G12C To enrich for specific binders, 1 μM soluble KRas G12C -GDP or KRas G12C Selection can be performed in the presence of excess -GMPPcp. After selection, individual colonies can be picked and grown overnight at 30°C in 96-well deep-well plates in 2xYT medium supplemented with carbenicillin and helper phage. Phage supernatants can be used to transfect KRas G12Ci -GDP+GNE1952, KRas G12C -GDP and KRas G12C -GMPPcp can be used in a phage ELISA to identify clones specific for conformation-specific KRas targets.

[0288] iv. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-KRas antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the antibody light chain and / or heavy chain). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and a nucleic acid encoding an amino acid sequence comprising the antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphocytic cell (e.g., a YO, NSO, or Sp20 cell). In one embodiment, a method of making an anti-KRas antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0289] For recombinant production of an anti-KRas antibody, nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0290] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) Following expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0291] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0292] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). TM(which describes the technology).

[0293] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV 1 line; human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV 1); African green monkey kidney cells (VER0-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep 02); mouse mammary tumor (MMT 060562); TRI cells, such as those described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0294] v. Assay The anti-KRas antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0295] 1. Binding Assays and Other Assays In one embodiment, the antibodies of the present invention are tested for their antigen-binding activity by known methods, such as ELISA, Western blot, etc. Binding affinity can be measured by methods common in the art. In one embodiment, the K D is the solution binding affinity of the Fab in the presence of a titration series of unlabeled antigen at a minimum concentration ( 125 I) Radiolabeled antigen binding assay (RIA) is performed using the Fab version of the antibody and the antigen molecule, as described by equilibrating the Fab with labeled antigen and then capturing the bound antigen on a plate coated with an anti-Fab antibody (Chen, et al., (1999) J. Mol. Biol. 293:865-881). To establish the conditions for this assay, microtiter plates (Dynex) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [I]-antigen is mixed with serially diluted Fabs of interest (consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., (1997), Cancer Res. 57:4593-4599). The Fabs of interest are then incubated overnight, although incubation may be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature for 1 hour. The solution is then removed, and the plate is washed eight times with PBS containing 0.1% Tween-20. Once the plate has dried, 150 μl / well of scintillant (MicroScint-20; Packard) is added, and the plate is counted for 10 minutes in a Topcount gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0296] According to another embodiment, K D is measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, NJ) with an antigen CM5 chip immobilized at 10 response units (RU) at 25°C. In some embodiments, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen can be diluted to about 3-10 μg / ml (e.g., 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injecting at a flow rate of about 3-10 μL / min (e.g., 5 μL / min) to achieve approximately 10 response units (RU) of bound protein. After injection of antigen, ethanolamine (e.g., 1 mM) can be injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab as described herein (e.g., concentrations from 0.78 nM to 500 nM) were incubated at 25°C in TWEEN 20. TM The injection can be performed in PBS containing a surfactant (PBST) (e.g., 0.05%). The injection flow rate can be approximately 10-50 μL / min (e.g., 25 μL / min). The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) can be calculated by simultaneously fitting the association and dissociation sensorgrams using, for example, a one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). D ) is k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate by the surface plasmon resonance assay is 10 6 M -1 s -1If the association rate exceeds 100 kJ / s, the association rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (e.g., excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of an anti-antigen antibody (Fab form) at a concentration of 10-50 nM (e.g., 20 nM) in PBS at a pH of about 6.8-7.5 (e.g., 7.2) at 25 °C. Measurements can be performed using a spectrophotometer (Aviv Instruments) equipped with a stopped flow or an 8000 series SLM-AMINCO with a stirred cuvette. TM It can be performed in the presence of increasing concentrations of antigen, as measured by a spectrophotometer such as ThermoSpectronic.

[0297] In another aspect, a competition assay can be used to identify additional anti-KRas antibodies that compete with any of the anti-KRas antibodies described herein for binding to human KRas. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) of KRas. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. Humana Press, Totowa, NJ.

[0298] In an exemplary competitive assay, immobilized human KRas protein is incubated in a solution containing a first labeled antibody (e.g., a first labeled anti-KRas antibody) that binds to KRas and a second unlabeled antibody (e.g., a second unlabeled anti-KRas antibody) that is being tested for its ability to compete with the first antibody for binding to KRas. The second antibody may be present in hybridoma supernatant. As a control, immobilized KRas is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to KRas, excess unbound antibody is removed and the amount of label associated with immobilized KRas is measured. If the amount of label associated with immobilized KRas is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to KRas. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Competitive assays can also be performed in the manner described above using FACS, using cells transfected with KRas and expressed on the cell surface. In addition, ELISA using KRas can also be used for competitive assays.

[0299] In another embodiment, a gel shift assay can be used to identify the interaction between the anti-KRas antibody of the present invention and a target protein such as human KRas. In an exemplary gel shift assay, human KRas is pre-incubated with an anti-KRas antibody. The KRas pre-incubated with the anti-KRas antibody and the control KRas not pre-incubated are subjected to gel electrophoresis, and the secondary antibody is examined. The mobility of the pre-incubated KRas and the KRas are compared, and the difference in mobility between the pre-incubated KRas and the control KRas indicates the interaction between KRas and the anti-KRas antibody.

[0300] 2. Crystal structure In some embodiments, the crystal structure of a complex between an anti-KRas antibody of the invention and human KRas is solved, e.g., KRas and an anti-KRas antibody can be purified, crystallized in complex, and the structure determined by X-ray crystallography.

[0301] B. How to Use Anti-KRas Antibodies In certain embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for detecting the presence of KRas, KRas-GDP, and / or alkylated KRas in a biological sample. In certain embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for quantifying KRas, KRas-GDP, and / or alkylated KRas in a sample. In some embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for quantifying KRas in a sample. G12C , KRas G12C -GDP and / or alkylated KRas G12C In some embodiments, any of the anti-KRas antibodies provided herein or compositions comprising such antibodies are useful for quantifying KRas. G12V or KRas G12V In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GDP. G12R or KRas G12R In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GDP. G12D or KRas G12D In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GDP. G13D or KRas G13D In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GDP. Q61H or KRasQ61H - Useful for quantifying GDP.

[0302] In certain embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for detecting the presence of KRas, KRas-GTP, and / or alkylated KRas in a biological sample. In certain embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for quantifying KRas, KRas-GTP, and / or alkylated KRas in a sample. In some embodiments, any of the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for quantifying KRas in a sample. G12C , KRas G12C -GTP, and / or alkylated KRas G12C In some embodiments, any of the anti-KRas antibodies provided herein or compositions comprising such antibodies are useful for quantifying KRas. G12V or KRas G12V In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GTP. G12R or KRas G12R In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GTP. G12D or KRas G12D In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GTP. G13D or KRas G13D In some embodiments, any of the anti-KRas antibodies or compositions comprising such antibodies provided herein are useful for quantifying KRas-GTP. Q61H or KRas Q61H It is useful for quantifying -GTP.

[0303] In one embodiment provided herein, a KRas protein (e.g., KRasG12C Methods for measuring target engagement of one or more KRas inhibitors described herein against a tumor are provided. In one embodiment, the method comprises: (a) obtaining a sample (e.g., a tumor sample described herein) from a patient described herein; (b) contacting the sample with an anti-KRas antibody or antigen-binding fragment thereof described herein; and (c) measuring the level of KRas bound by the anti-KRas antibody. In one such embodiment, the KRas inhibitor is MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

[0304] In some embodiments provided herein, a KRas protein (e.g., KRas G12C In some such embodiments, biomarker assays are provided for measuring target engagement of one or more KRas inhibitors described herein for targeting a KRas protein. In some such embodiments, the biomarker assay measures target engagement in a clinical setting from clinical samples collected from patients treated with one or more KRas inhibitors selected from the group consisting of MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, and JNJ-74699157. In some such embodiments, the biomarker assay is used to determine dosages of the KRas inhibitors described herein for such patients.

[0305] In certain embodiments, labeled anti-KRas antibodies are provided that can be used to detect or quantify KRas, KRas-GDP, and / or alkylated KRas described herein. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties, such as enzymes or ligands, that are indirectly detected, e.g., via enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H, and131 These include, but are not limited to, fluorophores such as I, rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0306] In certain embodiments, labeled anti-KRas antibodies are provided that can be used to detect or quantify KRas, KRas-GTP, and / or alkylated KRas as described herein. Labels include, but are not limited to, those described above.

[0307] In certain embodiments, any of the anti-KRas antibodies as provided herein, or compositions comprising such antibodies, detects the KRas antibodies described herein in an immunoassay. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In some embodiments, the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for detecting the presence of KRas, alkylated KRas, and / or alkylated KRas, particularly including KRas-GDP. G12CIn some embodiments, the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for detecting the presence of KRas-GDP and / or KRas bound to a covalent KRas inhibitor described herein. G12D or KRas G13D It is useful for detecting the presence of KRas.

[0308] As described below, anti-KRas antibodies, or compositions comprising such antibodies, can be used in a variety of different assays, including, but not limited to, ELISA and immunohistochemistry.

[0309] In certain embodiments, any of the anti-KRas antibodies as provided herein, or compositions comprising such antibodies, detects the KRas antibodies described herein in an immunoassay. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In some embodiments, the anti-KRas antibodies provided herein, or compositions comprising such antibodies, are useful for detecting the presence of KRas, such as KRas-GTP, and / or alkylated KRas, particularly including KRas-GTP bound to a covalent KRas inhibitor described herein. G12D or KRas G13D It is useful for detecting the presence of KRas.

[0310] i.ELISA (enzyme-linked immunosorbent assay) In some embodiments, the anti-KRas antibody is an antibody that inhibits KRas as described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H

[0013] The present invention relates to an ELISA assay for detecting the presence and / or amount of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. Accordingly, provided herein are methods for detecting KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, including ELISA assays that utilize anti-KRas antibodies as capture reagents for KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. In the first step of the assay, a biological sample containing or suspected of containing KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas is contacted with and incubated with a capture (or coating) antibody, allowing the capture antibody to capture or bind to KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas so that it can be detected in the detection step. The detecting step involves the use of a detectable antibody that, upon contact with any of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, binds to KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, if any, are present. The detecting means is used to detect the label on the antibody and, thus, the presence or amount of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas.

[0311] In certain embodiments, the assay utilizes the following steps.

[0312] First step The first step of the assay herein involves assaying KRas as described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61HA biological sample containing or suspected of containing KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly containing KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, is contacted with and incubated with an immobilized capture (or coating) reagent that is an anti-KRas antibody. In some embodiments, these anti-KRas antibodies are monoclonal antibodies and can be derived from any species. In some embodiments, these anti-KRas antibodies are rodent antibodies, and in further embodiments, mouse or rat antibodies, and in further embodiments, mouse antibodies.

[0313] In various embodiments, the anti-KRas antibody is an anti-KRas antibody disclosed herein. The anti-KRas antibody may be any of the Class I or Class II antibodies disclosed herein. For example, in some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 12), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 91), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 92), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 93), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 94), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 95), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 96), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 97), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 98), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO: 1), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence LQDHDYPLT (SEQ ID NO: 3), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 4), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 5), and the amino acid sequence GFYVRNWFDP (SEQ ID NO: 6). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 17), a CDR-L3 comprising the amino acid sequence AASSLQS (SEQ ID NO: 18), and a CDR-L3 comprising the amino acid sequence QQYYSYPFT (SEQ ID NO: 19), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO: 20), a CDR-H2 comprising the amino acid sequence AISSSGSSTYYADSVKG (SEQ ID NO: 21), and a CDR-H3 comprising the amino acid sequence DQGGYGYPGESWFDY (SEQ ID NO: 22). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 25), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 26), and a CDR-L3 comprising the amino acid sequence QQSYSPPWT (SEQ ID NO: 27), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 29), and a CDR-H3 comprising the amino acid sequence AFYSYMDV (SEQ ID NO: 30). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 33), a CDR-L2 comprising the amino acid sequence LGSNRAS (SEQ ID NO: 34), and a CDR-L3 comprising the amino acid sequence MQALQTPLT (SEQ ID NO: 35), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 36), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 37), and a CDR-H3 comprising the amino acid sequence ERTILTGYYGFDY (SEQ ID NO: 38).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 41), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 42), and a CDR-L3 comprising the amino acid sequence GTWDSSLTGYV (SEQ ID NO: 43), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 44), a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 45), and a CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 46). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 81), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 82), and a CDR-L3 comprising the amino acid sequence AAWDDSLSGWV (SEQ ID NO: 83), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 84), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 85), and a CDR-H3 comprising the amino acid sequence SFGPYAFDV (SEQ ID NO: 86). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 49), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 50), and the amino acid sequence GTWDSSLTGWV (SEQ ID NO: 51), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 52), a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 53), and a CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 54).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 57), a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 58), and a CDR-L3 comprising the amino acid sequence NSRDSSGNHWV (SEQ ID NO: 59), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 60), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 61), and a CDR-H3 comprising the amino acid sequence TNNYGYRYFDY (SEQ ID NO: 62). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 65), a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 66), and a CDR-L3 comprising the amino acid sequence NSRDSTDNHLWV (SEQ ID NO: 67), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 68), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 69), and a CDR-H3 comprising the amino acid sequence ATSSGYYYFDY (SEQ ID NO: 70). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence GTWDNSLSVWV (SEQ ID NO: 75), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 76), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 77), and a CDR-H3 comprising the amino acid sequence GKGIVGWGFFGMDV (SEQ ID NO: 78).

[0314] Immobilization is traditionally achieved by insolubilizing the capture reagent before or after the assay procedure by adsorption to a water-insoluble matrix or surface (U.S. Pat. No. 3,720,760) or non-covalent or covalent binding (e.g., using glutaraldehyde or carbodiimide-based cross-linkers, with or without prior activation of the support with nitric acid and a reducing agent as described, for example, in U.S. Pat. No. 3,645,852 or Rotmans et al.; J. Immunol. Methods, 57:87-98 (1983)), e.g., by immunoprecipitation. In some embodiments, the capture antibody is conjugated to biotin and bound to a streptavidin-coated surface. In other embodiments, the capture antibody is conjugated to a protein tag, such as a His tag or GST, and bound to a suitable surface, e.g., a nickel- or copper-coated surface, or a glutathione-coated surface.

[0315] The solid phase used for immobilization can be any essentially water-insoluble inert support or carrier useful in immunoassays, including supports in the form of surfaces, particles, porous matrices, etc. Examples of commonly used supports include small sheets, SEPHADEX® gel, polyvinyl chloride, plastic beads, and assay plates or test tubes made from polyethylene, polypropylene, polystyrene, etc., including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for immobilizing the capture reagent. In some embodiments, the immobilized capture reagent is coated onto a microtiter plate. In some embodiments, the solid phase used is a multi-well microtiter plate, such as the NUNC MAXISORB, which can be used to analyze multiple samples at once. TMor IMMULONT TM As sold as Microtest TM or MAXISORP TM 96-well ELISA plate.

[0316] The solid phase is coated with the capture reagent, as defined above, which may be linked by non-covalent, covalent, or physical bonds as appropriate. Techniques for linking include those described in U.S. Pat. No. 4,376,110 and references therein. In the case of covalent linking, the plate or other solid phase is incubated with the capture reagent and a cross-linking agent under conditions well known in the art, e.g., for 1 hour at room temperature.

[0317] Cross-linking agents commonly used to attach capture reagents to solid-phase substrates include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters, including, for example, 3,3'-dithiobis(succinimidopropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-((p-azidophenyl)-dithio)propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.

[0318] If a 96-well plate is utilized, it can be coated with a mixture of capture reagents, typically diluted in a buffer such as 0.05M sodium carbonate, by incubating for at least about 10 hours. In some embodiments, incubation occurs at least overnight at a temperature of about 4-20°C, or about 4-8°C, and at a pH of about 8-12, about 9-10, or about 9.6. If a shorter coating time (1-2 hours) is desired, a 96-well plate with a nitrocellulose filter bottom (Millipore MULTISCREEN TM) or can be coated at 37° C. Plates may be stacked and coated well in advance of the assay itself, which can then be run manually, semi-automatically, or automatically, e.g., using robotics, on multiple samples simultaneously.

[0319] The coated plate is then typically treated with a blocking agent that nonspecifically binds to and saturates the binding sites, preventing unwanted binding of free ligand to excess sites on the wells. Examples of suitable blocking agents for this purpose include gelatin, bovine serum albumin, egg albumin, casein, and nonfat milk. Blocking is typically performed at ambient temperature for about 1 to 4 hours, or about 1.5 to 3 hours.

[0320] After coating and blocking, standards (purified KRas, KRas-GDP, and / or alkylated KRas) or the biological sample to be analyzed, appropriately diluted, are added to the immobilized phase. In certain embodiments, the dilution is about 5-15%, or about 10%, by volume. Buffers that can be used for dilution for this purpose include: (a) 0.5% BSA, 0.05% TWEEN 20; TM Detergent (P20), 0.05% PROCLIN TM (b) phosphate-buffered saline (PBS) containing 300 antibiotics, 5 mM EDTA, 0.25% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) detergent, 0.2% beta-gamma globulin, and 0.35 M NaCl; (b) 0.5% bovine serum albumin (BSA), 0.05% P20, and 0.05% PROCLIN TM 300, pH 7 containing PBS, (c) 0.5% BSA, 0.05% P20, 0.05% PROCLIN TM (d) PBS containing 0.5% BSA, 0.05% P20, and 0.05% PROCLIN. TM300, PBS containing 5 mM EDTA, 0.2% beta-gamma globulin, and 0.35 M NaCl; and (e) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN. TM 300, PBS containing 5 mM EDTA, 0.25% CHAPS, and 0.35 M NaCl. TM 300 acts as a preservative, TWEEN 20 TM acts as a detergent to eliminate non-specific binding.

[0321] The amount of capture reagent used is large enough to give a good signal compared to the standard, but large enough to capture the KRas described herein in the sample. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In certain embodiments, the amount of biological sample added is such that the immobilized capture reagent does not exceed the maximum expected levels of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. In certain embodiments, the amount of biological sample added is such that the immobilized capture reagent does not exceed the maximum expected levels of KRas described herein that would be expected in the biological sample after appropriate dilution of the sample. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H This expected level is primarily determined by the amount of KRas in the particular biological sample being analyzed, as described herein, in molar excess of the maximum molar concentration of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly KRas-GDP, KRas-GTP, and / or alkylated KRas. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61HThe present invention relies on the known correlation between the concentration levels of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly KRas, and the clinical status of the patient. Thus, for example, adult patients may have very high levels of the KRas described herein in their serum. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H and a maximum expected concentration of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, while a child will have a maximum expected concentration of KRas as described herein in the child's serum based on the dose administered. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In particular, they are likely to have lower levels of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas.

[0322] The concentration of the capture reagent is determined based on the KRas concentration described herein, taking into account the necessary dilution of the biological sample. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H The final concentration of the capture reagent can be determined by the desired concentration range of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. The final concentration of the capture reagent can also be empirically determined to maximize the sensitivity of the assay in the desired range. Generally, the molar excess is suitably determined by the concentration of KRas, as described herein, in the biological sample after any appropriate dilution of the sample. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61HIn particular, the molar concentration of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas is less than about 10-fold the maximum expected molar concentration of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas.

[0323] The incubation conditions for the sample and the immobilized capture reagent are selected to maximize the sensitivity of the assay, minimize dissociation, and maximize the activity of the KRas molecules present in the sample, as described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H The incubation time is typically about 10 hours or less. In various embodiments, the incubation time is selected to ensure that any KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, including, among others, binds to the immobilized capture reagent. Incubation is accomplished at a substantially constant temperature, such as at or about room temperature, ranging from about 0° C. to about 40° C. Incubation times are typically about 10 hours or less. In various embodiments, incubation times are determined based on the KRas concentration as described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H The incubation time is about 0.5 to 3 hours, or about 1.5 to about 3 hours, at or about room temperature to maximize binding of the capture reagent to KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H The duration of the incubation may be longer if protein inhibitors are added to prevent proteases in the biological fluid from degrading KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including:

[0324] At this stage, the pH of the incubation mixture will typically be in the range of about 4 to 9.5, or about 6 to 9, or about 7 to 8. The pH of the incubation buffer is selected to maintain significant levels of specific binding of the capture reagent to the captured KRas, KRas-GDP, KRas-GTP, or alkylated KRas. A variety of buffers can be used to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, and barbiturates. The particular buffer used is not critical to the present invention, although some buffers may be preferred over others for individual assays.

[0325] Optional Second Step In the optional second step of the assay, the biological sample is separated from the immobilized capture reagent (e.g., by washing) to remove uncaptured KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. The solution used for washing is typically a buffer ("wash buffer") with a pH determined using the buffer and considerations described above for the incubation step, ranging from about 6 to 9. Washing may be performed three or more times. The temperature for washing is typically refrigerated to room temperature, and a constant temperature is maintained throughout the assay, typically between about 0 and 40°C, or between about 4 and 30°C. For example, the wash buffer can be placed in a reservoir on ice at 4°C before washing, and a plate washer can be used for this step. Additionally, if there is concern that the captured KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas may dissociate to some extent in subsequent steps, a cross-linking agent or other suitable agent may be added at this stage to allow the currently bound KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas to become covalently bound to the capture reagent.

[0326] The third step In the next step, the immobilized capture reagent containing either bound KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas is contacted with a detectable antibody at a temperature of about 20-40°C or about 36-38°C. The exact temperature and time for contacting the two depend primarily on the detection method used. For example, when 4-methylumbelliferyl-β-galactoside (MUG), streptavidin-HRP, or streptavidin-β-galactosidase is used as the detection method, contacting can be performed overnight (e.g., about 15-17 hours or more) to maximize signal amplification. The detectable antibody can be either polyclonal or monoclonal, but is preferably monoclonal to reduce background noise. In some embodiments, the same anti-KRas antibody is used for both coating and detection in the assay. In other embodiments, different anti-KRas antibodies can be used for coating and detection, selected to minimize background noise.

[0327] In some embodiments, the detectable antibody is an antibody from a non-human species that binds to a human antibody. In some embodiments, the detectable antibody is an anti-huIgG Fc antibody. In some embodiments, the detectable antibody is a mouse anti-huIgG Fcγ antibody. In some embodiments, the detectable antibody is directly detectable. In certain embodiments, the detectable antibody is biotinylated. In such cases, the detection means for the biotinylated label can be avidin or streptavidin-HRP, and the readout of the detection means can be fluorometric or colorimetric. In some embodiments, the antibody is conjugated to HRP and the detection means is colorimetric.

[0328] A molar excess of a detectable antibody relative to the maximum expected concentration of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas (as described above) is added to the washed plate. This antibody (directly or indirectly detectable) is typically a monoclonal antibody, although any antibody can be used. The affinity of the detection antibody must be high enough to detect small amounts of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, but not so high that it pulls KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas from the capture reagent.

[0329] Fourth step In the final step of the assay, the level of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas from the sample that is bound to the capture reagent, if any, is measured using a detectable antibody detection means. If the biological sample is from a clinical patient, the measuring step involves comparing the response resulting from the above three steps to a standard curve to determine the level of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas relative to a known amount.

[0330] The antibody attached to the immobilized capture reagent is either directly labeled or indirectly detected by washing away excess first antibody and then adding a molar excess of a second, labeled antibody directed against the IgG of the animal species of the first antibody. In the latter indirect assay, labeled antiserum against the first antibody is added to the sample to generate labeled antibodies in situ.

[0331] The label used for the first antibody or the second antibody may be a KRas antibody as described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H Any detectable function that does not interfere with the binding of free KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas to the anti-KRas antibody, particularly including:

[0332] Examples of suitable labels are the numerous labels known for use in immunoassays, including moieties that can be detected directly, such as fluorescent dye labels, chemiluminescent labels, radioactive labels, and moieties such as enzymes that must react or be induced in order to be detected. Examples of such labels include radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131 I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, HRP, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and These include glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, heterocyclic oxidases such as uricase and xanthine oxidase coupled with HRP, lactoperoxidase, or microperoxidase, biotin (detectable with MUG, e.g., avidin, streptavidin, streptavidin-HRP, and streptavidin-β-galactosidase), spin labels, bacteriophage labels, and stable free radicals.

[0333] Conventional methods for covalently binding these labels to proteins or polypeptides are available. For example, antibodies can be tagged with the above-mentioned fluorescent, chemiluminescent, and enzyme labels using coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, and bis-diazotized benzidine. See, for example, U.S. Patent No. 3,940,475 (fluorescence analysis) and U.S. Patent No. 3,645,090 (enzymes); Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Methods, 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982).

[0334] Conjugation of such labels, including enzymes, to antibodies is a standard procedure for those skilled in the art of immunoassay technology. See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166. Alternatively, commercially available, appropriately labeled antibodies can be used.

[0335] After the final labeled antibody is added, the amount of bound antibody is determined by washing away excess unbound labeled antibody, then measuring the amount of bound label using a detection method appropriate for the label and correlating the measured amount with the amount of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas in the biological sample. For example, in the case of an enzyme, the amount measured by color development will be a direct measure of the amount of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas present. Specifically, when HRP is the label, the substrate TMD may be used, and color may be detected using a read wavelength of 450 nm and a reference wavelength of 620 nm or 630 nm.

[0336] In one example, after washing the enzyme-labeled second antibody against the first unlabeled antibody from the immobilized phase, color or chemiluminescence is developed and measured by incubating the immobilized capture reagent with a substrate for the enzyme, and the concentrations of KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas are then calculated by comparing the color or chemiluminescence generated by standard KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas run in parallel.

[0337] ii. Immunohistochemistry (IHC) In some embodiments, the anti-KRas antibodies of the present disclosure detect, by immunohistochemistry (IHC), the KRas antibodies described herein. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61HThese methods are used to detect KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas, particularly including KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas. Thus, in some embodiments, provided herein are methods for detecting KRas, such as KRas-GDP, KRas-GTP, and / or alkylated KRas, in tissue samples using immunohistochemistry. Immunohistochemistry (IHC) involves the localization of a target (e.g., an antigen such as KRas, KRas-GDP, KRas-GTP, and / or alkylated KRas) and / or a subset of target-presenting cells in tissue sections by using directly labeled (direct IHC) or indirectly labeled (indirect IHC) binding domains (e.g., anti-KRas antibodies) that react with the target through target-binding-domain interactions. These interactions are then visualized by the referenced labels.

[0338] In some embodiments of the method of the present invention, it is envisaged that said immunohistochemistry is characterized by the following steps: (a) providing a tissue sample containing a subset of cells and / or a means (e.g., a slide) containing the subset of cells bound by a binding domain (e.g., an anti-KRas antibody); (b) optionally, fixing the tissue sample; (c) optionally, dehydrating the tissue sample; (d) optionally, paraffinizing the tissue sample; (e) directly or indirectly detecting the binding domain (e.g., an anti-KRas antibody), thereby detecting a subset of cells.

[0339] "Fixing" or "fixation" refers to a fixation procedure suitable for preparing a target / cell subset / tissue sample containing said cell subset for a subsequent IHC procedure. In particular, "fixation" is performed to ensure preservation of tissue structure and cellular morphology. Suitable fixation conditions are well known and are disclosed herein. Alternatively, preservation of tissue / subset by deep freezing (e.g., in liquid nitrogen) is also envisaged.

[0340] All the above pretreatment steps / measures fall within the scope of the term "fixation", i.e. fixation specifically includes fixation with fixative agents such as formaldehyde, paraformaldehyde; and / or deep freezing of tissue samples / cell subsets, and / or optionally also embedding of tissue / cell subsets in paraffin or a similar agent. It should be understood that the gist of the present invention lies in the surprising discovery that it is advantageous to bind a binding domain (e.g., a primary antibody specific for the target) to a target before subjecting tissues / subsets etc. presenting said target to a fixation procedure, since the fixation procedure may affect the quantity and / or quality of the target, thereby undesirably altering the results.

[0341] Tissues / subsets can also be paraffinized (usually after fixation).

[0342] The means and methods for implementing different IHC protocols are well known to those skilled in the art and are, are / can be adapted to the particular tissue / cell subset / target of interest without further explanation.

[0343] iii. Surface plasmon resonance In one embodiment, the antibodies or antigen-binding fragments thereof described herein can facilitate the identification and / or development of new chemical entities to generate the drug candidates described herein. In one embodiment, the SWII pocket of KRas is opened and / or stabilized as described herein. In one embodiment, opening and / or stabilizing the SWII pocket of KRas can increase the probability of higher affinity compound binding, thereby increasing the rate at which weakly binding compounds are detected. In embodiments, the antibodies or antigen-binding fragments thereof described herein may not increase the affinity of compound binding, but instead stabilize the conformation of KRas.

[0344] Surface plasmon resonance (SPR), or various forms of surface interferometry, generates an optical evanescent field at the sensing surface that is sensitive to the accumulation of biomolecules on the sensing surface by monitoring the associated change in the average refractive index near the sensing surface. The evanescent field decays exponentially with distance from the surface, defining the depth of refractive index sensitivity of the surface. Typically, the penetration depth of this field is on the order of 200–300 nM, providing a three-dimensional probe volume that can be fully exploited by using coupled hydrogels to support bimolecular complex formation; hydrogel-coated sensor chips are widely available.

[0345] Hydrogels can be generated by chemically grafting polysaccharide chains (e.g., linear, although some branching is permitted) onto a flat surface, forming a hydrogel that extends 10-200 nm from the sensing surface. In one embodiment, these chains are derivatized to contain reactive groups that allow target molecules to be attached to the hydrogel. In one embodiment, the target can be bound to a concentration of 20-50 mg / ml within the hydrogel, although concentrations up to 5-fold above and below this limit are acceptable. The resulting response is proportional to the molecular volume of the molecule being bound, the number of target molecules present, and the refractive index contrast between the molecule and the surrounding buffer.

[0346] In one aspect, provided herein is a biosensing surface for measuring the binding of a KRas inhibitor compound to a KRas mutant described herein. In one embodiment, the surface is pre-bound to an antibody or antigen-binding fragment thereof described herein. In one embodiment, provided herein is a biosensing surface for measuring the binding of a compound to a KRas mutant described herein, wherein: the biosensing surface comprises a hydrogel in which a KRas protein and an antibody or antigen-binding fragment thereof described herein are co-localized; the KRas protein and the antibody or antigen-binding fragment thereof have sufficient freedom within the hydrogen to engage with each other to form an affinity complex; wherein the local concentrations of the KRas protein and the antibody or antigen-binding fragment thereof exceed the dissociation affinity constant by at least 10-fold, and the local concentrations promote the formation of an affinity complex; the percentage of unbound KRas protein and antibody or antigen-binding fragment thereof is less than about 50%; A KRas inhibitor compound is injected over the biosensing surface for at least 5 seconds; Binding of the KRas inhibitor compound to the antibody or fragment thereof is measured on at least one sensing channel.

[0347] In one embodiment of the surface, the percentage of unbound KRas protein and antibody or antigen-binding fragment thereof is less than about 40%, 30%, 25%, 20%, or 10%.

[0348] In some embodiments, the hydrogel has a thickness of about 10 nm to 500 nm, 10 nm to 300 nm, 10 to 250 nm, or about 10 to 200 nm. In some embodiments, the hydrogel comprises streptavidin.

[0349] In one embodiment, the KRas protein is biotinylated. In some embodiments, the KRas protein is KRas G12C In some embodiments, the KRas protein is KRas G12D In some embodiments, the Kras protein is Kras G12V In some embodiments, the KRas protein is KRas G12C In some embodiments, the KRas protein is KRas G12R In some embodiments, the Kras protein is Kras G13D In some embodiments, the Kras protein is Kras Q61HIn one embodiment, the KRas protein is at a concentration of about 50-1000 nM, 50-750 nM, 50-500 nM, 100-1000 nM, 100-750 nM, 100-500 nM, or about 100-250 nM in the buffer prior to application to the biosensing surface. In some embodiments, the concentration of the KRas protein in the hydrogel is 0.5-2 mM, 0.5-1.5 mM, 0.5-1 mM, 0.75-2 mM, 0.75-1.5 mM, 0.75-1 mM, 0.9-2 mM, or 0.9-1.5 mM.

[0350] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab described herein. In one embodiment, the antibody or antigen-binding fragment thereof is injected at a concentration of about 50, 100, 150, 200, 250, 500, or 1000 nM. In one embodiment, the antibody or antigen-binding fragment thereof is injected at a concentration of about 150-200 nM. In one embodiment, the antibody or antigen-binding fragment thereof is a Fab of 2H11.

[0351] In some embodiments, the biosensing surface is attached to a BIACORE sensor chip. In some embodiments, measurements are performed on at least two channels. In one such embodiment, at least one channel is a standard (e.g., blank) sensing channel.

[0352] In one embodiment, the KRas inhibitor compound is present at a concentration of about 0.025 μM to 500 μM, 0.025 μM to 250 μM, 0.025 μM to 100 μM, 0.025 μM to 50 μM, 0.025 μM to 25 μM, 0.025 μM to 10 μM, 0.03 μM to 500 μM, 0.03 μM to 250 μM, 0.0 In some embodiments, the KRas inhibitor compound is infused at a concentration of 3 μM to 100 μM, 0.03 μM to 50 μM, 0.03 μM to 25 μM, 0.03 μM to 10 μM, 0.05 μM to 500 μM, 0.05 μM to 250 μM, 0.05 μM to 100 μM, 0.05 μM to 50 μM, 0.05 μM to 25 μM, or 0.05 μM to 10 μM. In some embodiments, the KRas inhibitor compound is infused at the concentrations indicated above at a rate of about 10, 25, 50, 100, 150, or about 250 μL / min. In some embodiments, the KRas inhibitor compound is infused at the rates and concentrations described herein for about 5, 7, 8, 9, 10, 15, 20, or about 25 seconds. In one embodiment, the KRas inhibitor compound is injected over the hydrogel at a rate of about 100 μL / min at a concentration of about 0.04-10 μM for about 10 seconds. In some embodiments, the KRas inhibitor compound is provided as a series of different concentrations (e.g., a series of 2, 3, 4, 5, 6, 7, 8, or 9 different concentrations). In some embodiments, each different concentration is injected over the hydrogel as described herein.

[0353] In another aspect, provided herein is a method of screening compounds for anti-KRas inhibitor activity, the method comprising measuring binding of the compound to a described KRas mutant protein, wherein the KRas mutant protein binds to an antibody or antigen-binding fragment thereof described herein, and wherein binding is measured using a biosensing surface described herein.

[0354] In one embodiment, KRas and an antibody or antigen-binding fragment thereof described herein are conjugated simultaneously within the hydrogel. In another embodiment, KRas is added prior to conjugation with an antibody or antigen-binding fragment thereof described herein. In yet another embodiment, the antibody or antigen-binding fragment thereof is added prior to conjugation with KRas.

[0355] Further provided herein is a method for measuring binding of a KRas mutant protein to an antibody or antigen-binding fragment thereof described herein, the method comprising: contacting a biosensing surface described herein with a KRas protein described herein to form a KRas-bound biosensing surface; contacting a biosensing surface bound to KRas with an antibody or antigen-binding fragment thereof described herein, wherein the anti-KRas antibody is in molar excess relative to the KRas protein; and Detecting the binding and affinity of antibodies or antigen-binding fragments thereof to KRas proteins using surface plasmon resonance Includes.

[0356] In one such embodiment, the biosensing surface is coated with avidin, hi another such embodiment, the KRas protein is biotinylated.

[0357] Further provided herein is a method of measuring binding of a KRas mutant protein to a KRas antibody described herein, the method comprising: contacting a biosensing surface described herein with an antibody or antigen-binding fragment thereof described herein to form a biosensing surface bound to an anti-KRas antibody; contacting a biosensing surface conjugated to an anti-KRas antibody with a KRas protein described herein, wherein the anti-KRas antibody is in molar excess relative to the KRas protein; and Detecting the binding and affinity of antibodies or antigen-binding fragments thereof to KRas proteins using surface plasmon resonance Includes.

[0358] In one such embodiment, the biosensing surface is coated with avidin. In another such embodiment, the antibody or antigen-binding fragment thereof is biotinylated.

[0359] iv. Methods for detecting KRas-GDP in samples Provided herein are methods for detecting KRas-GDP in a sample. In some embodiments, an anti-KRas antibody of the present disclosure binds to human KRas, wherein the antibody binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP). Accordingly, in some embodiments, an anti-KRas antibody is used to detect KRas-GDP in a sample. In some embodiments, KRas-GDP is detected using various techniques known in the art, as described above. In some embodiments, KRas-GDP is detected using ELISA. In some embodiments, KRas-GDP is detected using immunohistochemistry, as provided herein. In some embodiments, KRas-GDP is detected using surface plasmon resonance (SPR). In some embodiments, KRas-GDP is detected using a BIOACORE SPR instrument. In some embodiments, KRas-GDP is detected using flow cytometry. In some embodiments, KRas-GDP is detected using fluorescence-activated cell sorting (FACS). In some embodiments, KRas-GDP is detected using immunoprecipitation. In some embodiments, KRas-GDP is detected using affinity electrophoresis, such as electrophoretic mobility shift assay. In some embodiments, KRas-GDP is detected using fluorescence polarization / anisotropy. In some embodiments, KRas-GDP is detected using affinity purification coupled with mass spectrometry. In some embodiments, KRas-GDP is detected using biolayer interferometry. In some embodiments, KRas-GDP is detected using microscale thermophoresis (MST). In some embodiments, KRas-GDP is detected using a labeled KRas antibody.

[0360] Provided herein is a method for detecting KRas-GDP in a sample in which KRas is a KRas mutant. In some embodiments, the KRas mutant is an oncogenic Kras. In some embodiments, the KRas mutant is a KRas G12C In some embodiments, the KRas mutant is KRas G12R In some embodiments, the Kras mutant is Kras G12V In some embodiments, the Kras mutant is Kras Q61H In some embodiments, the Kras mutant is Kras G12D In some embodiments, the Kras mutant is Kras G13D is.

[0361] In some embodiments, any of the anti-KRas antibodies described herein is used to detect KRas-GDP in a sample. In some embodiments, the anti-KRas antibody is a Class I antibody. In some embodiments, the anti-KRas antibody is a Class II antibody. In some embodiments, the anti-KRas antibody is an alkylation conformation-specific antibody. In some embodiments, the anti-KRas antibody is an alkylation-inducing antibody. In some embodiments, the anti-KRas antibody opens the SWII pocket. In some embodiments, the anti-KRas antibody stabilizes the SWII pocket. In some embodiments, the anti-Kras antibody is 1E5. In some embodiments, the anti-Kras antibody is 2H11. In some embodiments, the anti-Kras antibody is 2A3. In some embodiments, the anti-Kras antibody is 3A12. In some embodiments, the anti-Kras antibody is 4G12. In some embodiments, the anti-Kras antibody is 1A5. In some embodiments, the anti-Kras antibody is 1D6. In some embodiments, the anti-Kras antibody is 2C1. In some embodiments, the anti-Kras antibody is 1A6. In some embodiments, the anti-Kras antibody is 1B7. In some embodiments, the anti-Kras antibody is 1F4. For example, in some embodiments, the anti-Kras antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO:9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO:10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO:11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO:12), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO:13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO:14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 91), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 92), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 93), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 94), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 95), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 96), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 97), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10), and a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence GSSIWSSN (SEQ ID NO: 98), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13), and a CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO: 1), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence LQDHDYPLT (SEQ ID NO: 3), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 4), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 5), and the amino acid sequence GFYVRNWFDP (SEQ ID NO: 6). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 17), a CDR-L3 comprising the amino acid sequence AASSLQS (SEQ ID NO: 18), and a CDR-L3 comprising the amino acid sequence QQYYSYPFT (SEQ ID NO: 19), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO: 20), a CDR-H2 comprising the amino acid sequence AISSSGSSTYYADSVKG (SEQ ID NO: 21), and a CDR-H3 comprising the amino acid sequence DQGGYGYPGESWFDY (SEQ ID NO: 22).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 25), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 26), and a CDR-L3 comprising the amino acid sequence QQSYSPPWT (SEQ ID NO: 27), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 29), and a CDR-H3 comprising the amino acid sequence AFYSYMDV (SEQ ID NO: 30). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 33), a CDR-L2 comprising the amino acid sequence LGSNRAS (SEQ ID NO: 34), and a CDR-L3 comprising the amino acid sequence MQALQTPLT (SEQ ID NO: 35), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 36), a CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 37), and a CDR-H3 comprising the amino acid sequence ERTILTGYYGFDY (SEQ ID NO: 38). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 41), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 42), and a CDR-L3 comprising the amino acid sequence GTWDSSLTGYV (SEQ ID NO: 43), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 44), a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 45), and a CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 46).In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 81), a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 82), and a CDR-L3 comprising the amino acid sequence AAWDDSLSGWV (SEQ ID NO: 83), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 84), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 85), and a CDR-H3 comprising the amino acid sequence SFGPYAFDV (SEQ ID NO: 86). In some embodiments, the anti-KRas antibody comprises a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 49), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 50), and a light chain variable region comprising the amino acid sequence GTWDSSLTGWV (SEQ ID NO: 51), a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 52), and a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 53). and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 57), a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 58), and a CDR-L3 comprising the amino acid sequence NSRDSSGNHWV (SEQ ID NO: 59), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 60), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 61), and a CDR-H3 comprising the amino acid sequence TNNYGYRYFDY (SEQ ID NO: 62). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 65), a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 66), and a CDR-L3 comprising the amino acid sequence NSRDSTDNHLWV (SEQ ID NO: 67), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 68), a CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 69), and a CDR-H3 comprising the amino acid sequence ATSSGYYYFDY (SEQ ID NO: 70). In some embodiments, the anti-KRas antibody comprises a light chain variable region comprising a CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence GTWDNSLSVWV (SEQ ID NO: 75), and a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 76), a CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 77), and a CDR-H3 comprising the amino acid sequence GKGIVGWGFFGMDV (SEQ ID NO: 78).

[0362] In some embodiments, the methods provided herein can be used to quantify (or determine the amount of) KRas-GDP in a sample. In some embodiments, the methods provided herein can be used to measure the abundance of KRas-GDP in a sample. In some embodiments, the methods provided herein can be used to measure the abundance of KRas-GDP bound to a KRas inhibitor described herein in a sample. In some embodiments, the amount of KRas-GDP is determined relative to a standard. For example, in some embodiments, quantitative Western blot can be used to quantify the abundance of KRas-GDP. In some embodiments, KRas-GDP is quantified using ELISA. In some embodiments, KRas-GDP is quantified using immunohistochemistry. In some embodiments, KRas-GDP is quantified using flow cytometry. In some embodiments, KRas-GDP is quantified using fluorescence-activated cell sorting (FACS). In some embodiments, KRas-GDP is quantified after immunoprecipitation. In some embodiments, KRas-GDP is quantified using affinity electrophoresis, such as an electrophoretic mobility shift assay. In some embodiments, KRas-GDP is quantified using affinity purification coupled with mass spectrometry. In some embodiments, KRas-GDP is quantified after purification. In some embodiments, KRas-GDP is quantified after purification by high performance liquid chromatography (HPLC). In some embodiments, KRas-GDP is quantified after purification by size exclusion chromatography.

[0363] In some embodiments, the method includes detecting KRas-GDP in a sample. In some embodiments, KRas-GDP is detected in a biological sample. In some embodiments, the biological sample is a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum, and plasma, ascites, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, urine, and other bodily constituents that may contain KRas-GDP. In various embodiments, the sample is a bodily sample from any animal. In various embodiments, the sample is a human sample. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject, for example, when detecting KRas-GDP in a clinical sample. In some embodiments, the biological sample is from a clinical patient or a patient with an oncogenic KRas mutation. In some embodiments, the biological sample is from a clinical patient or a patient with an oncogenic KRas mutation who has been administered a KRas inhibitor described herein. In some embodiments, the biological sample is a clinical patient or KRas G12C In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12D In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12V In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12R In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G13D In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. Q61H The biological sample is derived from a patient with an oncogenic mutation. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient. In certain embodiments, the biological sample is urine. In certain embodiments, the biological sample is urine from a clinical patient.

[0364] In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient. In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient. G12C In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12D In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12V In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12R In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient having an oncogenic mutation. G13D In some embodiments, KRas-GDP is detected in a sample derived from a cancer patient having an oncogenic mutation. Q61H It is detected in samples from cancer patients who carry oncogenic mutations.

[0365] In some embodiments, the methods provided herein can be used to quantify (or determine the amount of) KRas-GTP in a sample. In some embodiments, the methods provided herein can be used to measure the abundance of KRas-GTP in a sample. In some embodiments, the methods provided herein can be used to measure the abundance of KRas-GTP bound to a KRas inhibitor described herein in a sample. In some embodiments, the amount of KRas-GTP is determined relative to a standard. For example, in some embodiments, quantitative Western blot can be used to quantify the abundance of KRas-GTP. In some embodiments, KRas-GTP is quantified using ELISA. In some embodiments, KRas-GTP is quantified using immunohistochemistry. In some embodiments, KRas-GTP is quantified using flow cytometry. In some embodiments, KRas-GTP is quantified using fluorescence-activated cell sorting (FACS). In some embodiments, KRas-GTP is quantified after immunoprecipitation. In some embodiments, KRas-GTP is quantified using affinity electrophoresis, such as electrophoretic mobility shift assay. In some embodiments, KRas-GTP is quantified using affinity purification coupled with mass spectrometry. In some embodiments, KRas-GTP is quantified after purification. In some embodiments, KRas-GTP is quantified after purification by high performance liquid chromatography (HPLC). In some embodiments, KRas-GTP is quantified after purification by size exclusion chromatography.

[0366] In some embodiments, the method includes detecting KRas-GTP in a sample. In some embodiments, KRas-GTP is detected in a biological sample. In some embodiments, the biological sample is a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum, and plasma, ascites, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, urine, and other bodily constituents that may contain KRas-GTP. In various embodiments, the sample is a bodily sample from any animal. In various embodiments, the sample is a human sample. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject, for example, when detecting KRas-GTP in a clinical sample. In some embodiments, the biological sample is from a clinical patient or a patient with an oncogenic KRas mutation. In some embodiments, the biological sample is from a clinical patient or a patient with an oncogenic KRas mutation who has been administered a KRas inhibitor described herein. In some embodiments, the biological sample is a clinical patient or KRas G12C In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12D In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12V In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G12R In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. G13D In some embodiments, the biological sample is derived from a patient with an oncogenic mutation. Q61H The biological sample is derived from a patient with an oncogenic mutation. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient. In certain embodiments, the biological sample is urine. In certain embodiments, the biological sample is urine from a clinical patient.

[0367] In some embodiments, KRas-GTP is detected in a sample from a cancer patient. In some embodiments, KRas-GTP is detected in a sample from a cancer patient. G12C In some embodiments, KRas-GTP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12D In some embodiments, KRas-GTP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12V In some embodiments, KRas-GTP is detected in a sample derived from a cancer patient having an oncogenic mutation. G12R In some embodiments, KRas-GTP is detected in a sample derived from a cancer patient having an oncogenic mutation. G13D In some embodiments, KRas-GTP is detected in a sample derived from a cancer patient having an oncogenic mutation. Q61H It is detected in samples from cancer patients who carry oncogenic mutations.

[0368] In some embodiments, the biological sample is a clinical patient or KRas G12C In some embodiments, the biological sample is derived from a patient treated with a covalent inhibitor (e.g., a compound that alkylates Cys12). G12C Derived from patients treated with covalent inhibitors (e.g., compounds that alkylate Cys12), KRas G12C In some embodiments, the biological sample is a clinical patient or a KRas G12D In some embodiments, the biological sample is derived from a patient treated with a covalent inhibitor (e.g., an inhibitor that covalently binds to Asp12). G12D Derived from patients treated with covalent inhibitors, the inhibitors KRas G12D In some embodiments, the biological sample is a sample from a clinical patient or a KRas G13DIn some embodiments, the biological sample is derived from a patient treated with a covalent inhibitor (e.g., an inhibitor that covalently binds to Asp13). G13D Derived from patients treated with covalent inhibitors, the inhibitors KRas G13D The level of covalent binding to is determined as described herein.

[0369] In some embodiments, the biological sample is a clinical patient or KRas G12D In some embodiments, the biological sample is derived from a patient treated with a non-covalent inhibitor of KRas. G12V In some embodiments, the biological sample is derived from a patient treated with a non-covalent inhibitor of KRas. G12R In some embodiments, the biological sample is derived from a patient treated with a non-covalent inhibitor of KRas. G13D In some embodiments, the biological sample is derived from a patient treated with a non-covalent inhibitor of KRas. Q61H In some embodiments, the biological sample is derived from a patient treated with a non-covalent inhibitor of KRas. G12C In some embodiments, the biological sample is derived from a clinical patient or a patient treated with a KRas SWII ligand. In some embodiments, the biological sample is derived from a clinical patient or a patient treated with an anti-KRas antibody.

[0370] In some embodiments, KRas-GDP is detected as part of a method for monitoring a patient's treatment for cancer. In some such embodiments, the method for monitoring a patient's treatment for cancer is carried out using a biomarker assay described herein. ... G12C In some embodiments, the patient is diagnosed with a KRas-mediated cancer. G12C In some embodiments, the patient has received a specific covalent inhibitor of KRas.G12C The specific covalent inhibitor is ARS-1952. G12C The specific covalent inhibitor is ARS-853. G12C The specific covalent inhibitor is MRTX849. G12C The specific covalent inhibitor is AMG-510. G12C The specific covalent inhibitor is GDC-6036. G12C The specific covalent inhibitor is ARS-3248. G12C A specific covalent inhibitor is LY3499446. In some embodiments, KRas G12C The specific covalent inhibitor is JNJ-74699157. In some embodiments, the patient has KRas G12D In some embodiments, the patient has received a specific covalent inhibitor of KRas G12V In some embodiments, the patient has received a specific covalent inhibitor of KRas G12R In some embodiments, the patient has received a specific covalent inhibitor of KRas G13D In some embodiments, the patient has received a specific covalent inhibitor of KRas Q61H Previous administration of specific covalent inhibitors.

[0371] In some embodiments, KRas-GDP or KRas-GTP is used to treat KRas in a patient, as described herein. G12D In some embodiments, the patient is diagnosed with a KRas-mediated cancer. G12D In some embodiments, the patient has received a specific covalent inhibitor of KRas G12D In some embodiments, KRas-GDP or KRas-GTP is administered to inhibit KRas in the patient, as described herein. G13DIn some embodiments, the patient is diagnosed with a KRas-mediated cancer. G13D In some embodiments, the patient has received a specific covalent inhibitor of KRas G13D In some embodiments, KRas-GDP or KRas-GTP is administered to inhibit KRas in the patient, as described herein. G12V In some embodiments, the patient is diagnosed with a KRas-mediated cancer. G12V In some embodiments, KRas-GDP or KRas-GTP is administered to inhibit KRas in the patient, as described herein. G12R In some embodiments, the patient is diagnosed with a KRas-mediated cancer. G12R In some embodiments, KRas-GDP or KRas-GTP is administered to inhibit KRas in the patient, as described herein. Q61H In some embodiments, the patient is diagnosed with a KRas-mediated cancer. Q61H Previous administration of specific non-covalent inhibitors.

[0372] v. Method for detecting KRas-GDP and KRas-GTP in a sample Further provided herein are methods for detecting KRas-GDP and KRas-GTP in a sample. In some embodiments, the relative amounts of KRas-GDP and KRas-GTP in the sample are determined. In some embodiments, the abundance of KRas-GDP and KRas-GTP in the sample is determined. In some embodiments, the ratio of KRas-GDP to KRas-GTP in the sample is determined. In some embodiments, an anti-KRas antibody of the present disclosure is used in combination with an anti-KRas antibody that binds to KRas-GTP with higher affinity than KRas-GDP. In some embodiments, the anti-KRas antibody that binds to KRas-GDP is labeled with a first label, and the anti-KRas antibody that preferentially binds to KRas-GTP is labeled with a second label. In some embodiments, the first and second labels are detected. In some embodiments, detection and quantification of signals from both the first and second labels allows for separate quantification of both KRas-GDP and KRas-GTP levels in a sample. In some embodiments, the anti-KRas antibody that binds KRas-GDP and the anti-KRas antibody that preferentially binds KRas-GTP are not necessarily labeled with different labels, hi some embodiments, the amounts of KRas-GDP and KRas-GTP are determined relative to a standard.

[0373] In some embodiments, KRas-GDP and KRas-GTP are detected by various means known in the art, as described above. In some embodiments, KRas-GDP and KRas-GTP are detected using ELISA. In some embodiments, KRas-GDP and KRas-GDP are detected using immunohistochemistry, as provided herein. In some embodiments, KRas-GDP and KRas-GDP are detected using surface plasmon resonance (SPR). In some embodiments, KRas-GDP and KRas-GTP are detected using a BIOACORE SPR instrument. In some such embodiments, KRas-GTP and / or KRas-GDP are detected using a biosensing surface provided herein. In some embodiments, KRas-GDP and KRas-GTP are detected using flow cytometry. In some embodiments, KRas-GDP and KRas-GTP are detected using fluorescence-activated cell sorting (FACS). In some embodiments, KRas-GDP and KRas-GTP are detected using immunoprecipitation. In some embodiments, KRas-GDP and KRas-GTP are detected using affinity electrophoresis, such as electrophoretic mobility shift assays. In some embodiments, KRas-GDP and KRas-GTP are detected using fluorescence polarization / anisotropy. In some embodiments, KRas-GDP and KRas-GTP are detected using affinity purification coupled with mass spectrometry. In some embodiments, KRas-GDP and KRas-GTP are detected using biolayer interferometry. In some embodiments, KRas-GDP and KRas-GTP are detected using microscale thermophoresis (MST).

[0374] As noted above, in some embodiments, any of the anti-KRas antibodies described herein are used to detect KRas-GDP in a sample. In some embodiments, any of the anti-KRas antibodies described herein are used to detect KRas-GTP in a sample. In some embodiments, the anti-KRas antibody is a Class I antibody. In some embodiments, the anti-KRas antibody is a Class II antibody. In some embodiments, the anti-Kras antibody is 1E5. In some embodiments, the anti-Kras antibody is 2H11. In some embodiments, the anti-Kras antibody is 2A3. In some embodiments, the anti-Kras antibody is 3A12. In some embodiments, the anti-Kras antibody is 4G12. In some embodiments, the anti-Kras antibody is 1A5. In some embodiments, the anti-Kras antibody is 1D6. In some embodiments, the anti-Kras antibody is 2C1. In some embodiments, the anti-Kras antibody is 1A6. In some embodiments, the anti-Kras antibody is 1B7. In some embodiments, the anti-Kras antibody is 1F4. In some embodiments, the anti-Kras antibody is 1E5, 2H11, 2A3, 3A12, 1F4, 4G12, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is 2H11, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is Ab1. In some embodiments, the anti-Kras antibody is Ab2. In some embodiments, the anti-Kras antibody is Ab3. In some embodiments, the anti-Kras antibody is Ab4. In some embodiments, the anti-Kras antibody is Ab5. In some embodiments, the anti-Kras antibody is Ab6. In some embodiments, the anti-Kras antibody is Ab7. In some embodiments, the anti-Kras antibody is Ab8.

[0375] In some embodiments, the anti-KRas antibody that binds to KRas-GTP is a commercially available antibody. In some embodiments, the anti-KRas antibody that binds to KRas-GTP is iDab6 (Tanaka, T. et al., EMBO J2007;26:3250-3259). In some embodiments, the anti-KRas antibody that binds to KRas-GTP is anti-Ras antibody EP1125Y (Abcam, ab52939). In some embodiments, the anti-KRas antibody that binds to KRas-GTP is KRas-2B-specific rabbit polyclonal antibody (Proteintech, catalog number 16155-1-AP). In some embodiments, the anti-KRas antibody that binds to KRas-GTP is Ras10 (Millipore, catalog number 05-516). In some embodiments, the anti-KRas antibody that binds to KRas-GTP is 3B10-2F2 (Sigma-Aldrich, catalog number WH0003845M1). In some embodiments, the anti-KRas antibody that binds to KRas-GTP is 234-4.2 (Millipore, catalog number OP24).

[0376] vi. How to obtain KRas inhibitors Further provided herein are methods for obtaining KRas inhibitors. In some embodiments, the anti-KRas antibodies of the present disclosure stabilize and / or open the KRas SWII pocket. In some embodiments, the anti-KRas antibodies of the present disclosure inhibit KRas G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In some embodiments, the anti-KRas antibodies of the present disclosure can be used to induce an open conformation of the SWII pocket of KRas. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRasQ61H In some embodiments, the anti-KRas antibodies of the present disclosure lock the KRas pocket in an open conformation. In some embodiments, this allows for screening of molecules that specifically target the open SWII pocket. In some embodiments, this allows for screening of molecules that specifically target the open SWII pocket. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H In some embodiments, the anti-KRas antibody stabilizes the open conformation of the SWII pocket, allowing for the identification of small molecules that covalently bind to the SWII pocket. In some embodiments, the anti-KRas antibody stabilizes the open conformation of the SWII pocket, allowing for the identification of small molecules that covalently bind to the SWII pocket. G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H This allows for the identification of small molecules that covalently bind to the SWII pocket of KRas. For example, in some embodiments, KRas can be bound by an anti-KRas antibody of the present disclosure that induces an open conformation of the SWII pocket, and the anti-KRas antibody-bound KRas can be used to obtain KRas inhibitors.

[0377] Thus, provided herein are methods for obtaining KRas inhibitors, comprising contacting an anti-KRas antibody with KRas, screening a library of compounds, and identifying compounds that bind to KRas. In some embodiments, compounds that bind to KRas inhibit KRas. In some embodiments, Kras inhibitors inhibit mutant KRas. In some embodiments, Kras inhibitors inhibit oncogenic Kras. In some embodiments, Kras inhibitors inhibit KRas. G12CIn some embodiments, the KRas inhibitor inhibits KRas G12R In some embodiments, the Kras inhibitor inhibits KRas G12V In some embodiments, the Kras inhibitor inhibits KRas Q61H In some embodiments, the Kras inhibitor inhibits KRas G12D In some embodiments, the Kras inhibitor inhibits KRas G13D inhibits.

[0378] In some embodiments, any of the anti-KRas antibodies disclosed herein can be used in the methods for obtaining KRas inhibitors of KRas mutants described herein. In some embodiments, the anti-Kras antibody is a Class I or Class II antibody provided herein. In some embodiments, the anti-Kras antibody is 1E5. In some embodiments, the anti-Kras antibody is 2H11. In some embodiments, the anti-Kras antibody is 2A3. In some embodiments, the anti-Kras antibody is 3A12. In some embodiments, the anti-Kras antibody is 4G12. In some embodiments, the anti-Kras antibody is 1A5. In some embodiments, the anti-Kras antibody is 1D6. In some embodiments, the anti-Kras antibody is 2C1. In some embodiments, the anti-Kras antibody is 1A6. In some embodiments, the anti-Kras antibody is 1B7. In some embodiments, the anti-Kras antibody is 1F4. In some embodiments, the anti-KRas antibody is 1E5, 2H11, 2A3, 3A12, 1F4, 4G12, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is 2H11, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-KRas antibody is Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8. In some embodiments, the anti-Kras antibody is Ab1. In some embodiments, the anti-Kras antibody is Ab2. In some embodiments, the anti-Kras antibody is Ab3. In some embodiments, the anti-Kras antibody is Ab4. In some embodiments, the anti-Kras antibody is Ab5. In some embodiments, the anti-Kras antibody is Ab6. In some embodiments, the anti-Kras antibody is Ab7. In some embodiments, the anti-Kras antibody is Ab8.

[0379] In some embodiments, high-throughput screening (HTS) is performed to identify KRas inhibitors. In some embodiments, a chemical library is screened to identify KRas inhibitors. In some embodiments, a library of natural products or naturally occurring compounds is screened to identify KRas inhibitors. In some embodiments, a peptide library is screened to identify KRas inhibitors. In some embodiments, a peptidomimetic library is screened to identify KRas inhibitors. In some embodiments, a library of antibodies or antigen-binding fragments is screened to identify KRas inhibitors. In some embodiments, a library of small molecules is screened. In some embodiments, a library of covalent inhibitors is screened. In some embodiments, a library of non-covalent inhibitors is screened.

[0380] In some embodiments, the KRas inhibitor is obtained by identifying a KRas inhibitor in a screening test. In some embodiments, the KRas inhibitor is identified by its KRas binding. In some embodiments, the KRas binding is detected through one of a variety of techniques known in the art for detecting protein-small molecule interactions (see, e.g., McFedries, A., et al. Chem. Biol. 2013 20:5). In some embodiments, the KRas binding is detected using differential scanning fluorimetry (DSF). In some embodiments, the KRas binding is detected using a thermostability shift assay. In some embodiments, the KRas binding is detected using affinity capture coupled to stable isotope labeling of amino acids in cell culture (SILAC).

[0381] In some embodiments, a KRas inhibitor is identified by its alteration in mutant KRas (e.g., a mutant KRas described herein) activity in an assay of KRas activity. In some embodiments, the assay of KRas activity is designed based on the biology of KRas. In some embodiments, a KRas inhibitor is identified by its alteration in the nucleotide binding affinity of KRas. In some embodiments, a KRas inhibitor is identified by its alteration in the ability of KRas to activate RAF kinase. In some embodiments, a KRas inhibitor is identified by its blocking of KRas RAF kinase activation. In some embodiments, a KRas inhibitor is identified by its blocking of KRas binding to RAF kinase. In some embodiments, a KRas inhibitor is identified by its blocking of a reporter indicative of KRas activation, such as a GLUT1 transcriptional reporter.

[0382] In some embodiments, the KRas inhibitor covalently binds to a mutant KRas described herein. In some embodiments, the KRas inhibitor is a covalent inhibitor (e.g., an inhibitor that alkylates KRas). In some embodiments, the KRas inhibitor covalently binds to a residue in the SWII pocket. In some embodiments, the KRas inhibitor binds to and alkylates the SWII pocket. In some embodiments, the KRas inhibitor alkylates a residue that is exposed on the surface of KRas when the SWII pocket is open. In some embodiments, the KRas inhibitor covalently modifies a residue in the SWII pocket. In some embodiments, the KRas inhibitor binds to a cysteine ​​residue in the SWII pocket of a mutant KRas described herein. In some embodiments, the KRas inhibitor alkylates a cysteine ​​residue in the SWII pocket of a mutant KRas described herein. In some embodiments, the KRas inhibitor binds to a cysteine ​​residue in the KRas SWII pocket. In some embodiments, the KRas inhibitor alkylates a cysteine ​​residue in the KRas SWII pocket. In some embodiments, the KRas inhibitor allosterically inhibits KRas. In some embodiments, the KRas inhibitor prevents mutant KRas from entering an active, GTP-bound state. In some embodiments, the KRas inhibitor locks mutant KRas in an inactive, GDP-bound state. In some embodiments, the KRas inhibitor alters the nucleotide binding affinity of mutant KRas. In some embodiments, the KRas inhibitor causes mutant KRas to preferentially bind GDP over GTP. In some embodiments, the KRas inhibitor transitions mutant KRas to an inactive form of KRas. In some embodiments, the KRas inhibitor blocks GEF-catalyzed nucleotide exchange. In some embodiments, the KRas inhibitor blocks signaling downstream of mutant KRas. In some embodiments, the KRas inhibitor is an alkylating agent. In some embodiments, the Kras inhibitor inhibits KRas. G12Cand binds to residue Cys12. In some embodiments, the mutant KRas described herein inhibits KRas G12D , KRas G12R , KRas G12V , KRas G13C , or KRas Q61H is.

[0383] In some embodiments, KRas inhibitors are identified, where such inhibitors bind to the mutant KRas described herein. In some embodiments, molecular probes for KRas are identified. In some embodiments, the KRas inhibitor is a small molecule, such as an organic or inorganic compound. In some embodiments, the small molecule is a naturally occurring small molecule or a synthetic small molecule. In some embodiments, the KRas inhibitor is a protein. In some embodiments, the KRas inhibitor is a peptide. In some embodiments, the KRas inhibitor is an antibody or antibody fragment. In some embodiments, the KRas inhibitor is a nucleic acid. In some embodiments, the KRas inhibitor obtained by the methods of the present disclosure can be used as a drug to treat cancer associated with a KRas mutation. In some embodiments, the KRas inhibitor binds to a KRas G12C It is used as a drug to treat mediated cancers.

[0384] vii. Methods for detecting KRas-GDP alkylation in biological samples Provided herein are KRas alkylation conformation-specific antibodies that specifically bind to the alkylated form of KRas. Thus, in some embodiments of the present disclosure, the anti-KRas antibody is used to detect the alkylation of KRas-GDP in a biological sample. In some embodiments, the anti-KRas antibody is used to detect the alkylation of KRas by binding of a covalent inhibitor in a biological sample. G12Cの In some embodiments, the detection is performed using a biomarker assay described herein to measure target engagement. In some embodiments, the class I antibody is used to detect KRas. G12CIn some embodiments, the class I antibody is used to detect alkylation of KRas. G12D In some embodiments, the class I antibody is used to detect covalent binding of KRas. G12D In some embodiments, the class I antibody is used to detect non-covalent binding of KRas. G12V In some embodiments, the class I antibody is used to detect non-covalent binding of KRas. G12R In some embodiments, the class I antibody is used to detect non-covalent binding of KRas. G13D In some embodiments, the class I antibody is used to detect non-covalent binding of KRas. Q61H In some embodiments, 1A5, 1D6, 2C1, 1A6, 1F4, or 1B7 are used to detect alkylated KRas or KRas non-covalently bound to a non-covalent KRas inhibitor described herein. In some embodiments, 1E5, 2H11, 2A3, 3A12, 1F4, 4G12, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8 are used to detect alkylated KRas or KRas non-covalently bound to a non-covalent KRas inhibitor described herein. In some embodiments, 2H11, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8 are used to detect alkylated KRas or KRas non-covalently bound to a non-covalent KRas inhibitor described herein. In some embodiments, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, or Ab8 are used to detect alkylated KRas or KRas non-covalently bound to a KRas non-covalent inhibitor described herein.

[0385] In some embodiments, KRas G12C KRas in subjects treated with specific covalent inhibitors G12C A method for detecting covalent binding (e.g., alkylation) of KRas is provided, the method comprising: (a) detecting a KRas G12Cand (b) detecting the antibody or antigen-binding fragment thereof bound to alkylated KRas. G12C The specific covalent inhibitor is ARS-1952. G12C The specific covalent inhibitor is ARS-853. G12C Specific covalent inhibitors are ARS-1620, MRTX849. In some embodiments, KRas G12C The specific covalent inhibitor is AMG-510. G12C The specific covalent inhibitor is GDC-6036. G12C The specific covalent inhibitor is ARS-3248. G12C A specific covalent inhibitor is LY3499446. In some embodiments, KRas G12C The specific covalent inhibitor is JNJ-74699157. In some embodiments, KRas G12C A specific covalent inhibitor is LY3537982.

[0386] In some embodiments, KRas G12D KRas in subjects treated with specific covalent inhibitors G12D A method for detecting covalent binding of a covalent KRas inhibitor to a KRas inhibitor is provided, the method comprising: (a) detecting a covalent binding of a KRas inhibitor to a KRas inhibitor; G12D (b) administering any of the anti-KRas antibodies disclosed herein to a subject after treatment with a specific covalent inhibitor; and (b) detecting the antibody or antigen-binding fragment thereof bound to KRas. G12D KRas in subjects treated with specific covalent inhibitors G12D A method for detecting non-covalent binding of a non-covalent KRas inhibitor to a KRas inhibitor is provided, the method comprising: (a) detecting a non-covalent binding of a KRas inhibitor to a KRas inhibitor; G12D(b) administering any of the anti-KRas antibodies disclosed herein to a subject after treatment with a specific non-covalent inhibitor; and (b) detecting the antibody or antigen-binding fragment thereof bound to KRas.

[0387] In some embodiments, KRas-GDP alkylation is detected by various means known in the art, as described above. In some embodiments, KRas-GDP alkylation is detected using ELISA. In some embodiments, KRas-GDP alkylation is detected using immunohistochemistry, as provided herein. In some embodiments, KRas-GDP alkylation is detected using surface plasmon resonance (SPR). In some embodiments, KRas-GDP alkylation is detected using a BIOACORE SPR instrument. In some embodiments, KRas-GDP alkylation is detected using flow cytometry. In some embodiments, KRas-GDP alkylation is detected using fluorescence-activated cell sorting (FACS). In some embodiments, KRas-GDP alkylation is detected using immunoprecipitation. In some embodiments, KRas-GDP alkylation is detected using affinity electrophoresis, such as electrophoretic mobility shift assays. In some embodiments, KRas-GDP alkylation is detected using fluorescence polarization / anisotropy. In some embodiments, the alkylation of KRas-GDP is detected using affinity purification coupled with mass spectrometry. In some embodiments, the alkylation of KRas-GDP is detected using biolayer interferometry. In some embodiments, the alkylation of KRas-GDP is detected using microscale thermophoresis (MST).

[0388] In some embodiments, alkylation of KRas-GDP is detected in a biological sample. In some embodiments, the biological sample is a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum, and plasma, ascites, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, urine, and other constituents of the body that may contain alkylated KRas. In various embodiments, the sample is a bodily sample from any animal. In various embodiments, the sample is a sample from a human.

[0389] In some embodiments, the sample is derived from a mammal. In some embodiments, the sample is derived from a human subject, for example, when detecting the alkylation status of KRas-GDP in a clinical sample. In some embodiments, the biological sample is derived from a clinical patient or a patient with an oncogenic KRas mutation. In some embodiments, the biological sample is derived from a clinical patient or a patient with KRas G12C The biological sample is derived from a patient with an oncogenic mutation. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient. In certain embodiments, the biological sample is urine. In certain embodiments, the biological sample is urine from a clinical patient.

[0390] viii. Methods for detecting KRas-GDP alkylation in vivo In some embodiments of the present ...

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 9); (ii) a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 10); and (iii) a CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 12); (ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13); and (iii) CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14) 1. An isolated antibody or antigen-binding fragment thereof comprising:

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

16.

3. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP); (a) a light chain variable region comprising: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11; and (b) a heavy chain variable region comprising: (i) a CDR-H1 comprising one of the amino acid sequences selected from the group consisting of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14 1. An isolated antibody or antigen-binding fragment thereof comprising:

4. 4. The isolated antibody or antigen-binding fragment thereof of claim 3, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region comprises one of the amino acid sequences selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO:

106.

5. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO: 1); (ii) a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 2); and (iii) CDR-L3 comprising the amino acid sequence LQDHDYPLT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 4); (ii) a CDR-H2 comprising the amino acid sequence YISSSSSSTIYYADSVKG (SEQ ID NO: 5); and (iii) CDR-H3 comprising the amino acid sequence GFYVRNWFDP (SEQ ID NO: 6) 1. An isolated antibody or antigen-binding fragment thereof comprising:

6. 6. The isolated antibody or antigen-binding fragment thereof of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

7. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 17); (ii) a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 18); and (iii) a CDR-L3 comprising the amino acid sequence QQYYSYPFT (SEQ ID NO: 19); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO: 20); (ii) a CDR-H2 comprising the amino acid sequence AISSSGSSTYYADSVKG (SEQ ID NO: 21); and (iii) CDR-H3 comprising the amino acid sequence DQGGYGYPGESWFDY (SEQ ID NO: 22) 1. An isolated antibody or antigen-binding fragment thereof comprising:

8. 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

24.

9. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to KRas bound to GDP (KRas-GDP) with higher affinity than to KRas bound to GTP (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO: 25); (ii) a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 26); and (iii) CDR-L3 comprising the amino acid sequence QQSYSPPWT (SEQ ID NO: 27); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 28); (ii) a CDR-H2 comprising the amino acid sequence SISSSSSSYIYYADSVKG (SEQ ID NO: 29); and (iii) CDR-H3 comprising the amino acid sequence AFYSYMDV (SEQ ID NO: 30) 1. An isolated antibody or antigen-binding fragment thereof comprising:

10. 10. The isolated antibody or antigen-binding fragment thereof of claim 9, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

32.

11. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 33); (ii) a CDR-L2 comprising the amino acid sequence LGSNRAS (SEQ ID NO: 34); and (iii) a CDR-L3 comprising the amino acid sequence MQALQTPLT (SEQ ID NO: 35); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO: 36); (ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 37); and (iii) CDR-H3 comprising the amino acid sequence ERTILTGYYGFDY (SEQ ID NO: 38) 1. An isolated antibody or antigen-binding fragment thereof comprising:

12. 12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

40.

13. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 41); (ii) a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 42); and (iii) a CDR-L3 comprising the amino acid sequence GTWDSSLTGYV (SEQ ID NO: 43); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 44); (ii) a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 45); and (iii) CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 46) 1. An isolated antibody or antigen-binding fragment thereof comprising:

14. 14. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 47 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

48.

15. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 81); (ii) a CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO: 82); and (iii) a CDR-L3 comprising the amino acid sequence AAWDDSLSGWV (SEQ ID NO: 83); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 84); (ii) a CDR-H2 comprising the amino acid sequence YISSSSSSTIYYADSVKG (SEQ ID NO: 85); and (iii) CDR-H3 comprising the amino acid sequence SFGPYAFDV (SEQ ID NO: 86) 1. An isolated antibody or antigen-binding fragment thereof comprising:

16. 16. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

88.

17. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 49); (ii) a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 50); and (iii) a CDR-L3 comprising the amino acid sequence GTWDSSLTGWV (SEQ ID NO: 51); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO: 52); (ii) a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 53); and (iii) CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 54) 1. An isolated antibody or antigen-binding fragment thereof comprising:

18. 18. The isolated antibody or antigen-binding fragment thereof of claim 17, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

56.

19. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO:57); (ii) a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 58); and (iii) a CDR-L3 comprising the amino acid sequence NSRDSSGNHWV (SEQ ID NO: 59); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 60); (ii) a CDR-H2 comprising the amino acid sequence SISSSSSSYIYYADSVKG (SEQ ID NO: 61); and (iii) CDR-H3 comprising the amino acid sequence TNNYGYRYFDY (SEQ ID NO: 62) 1. An isolated antibody or antigen-binding fragment thereof comprising:

20. 20. The isolated antibody or antigen-binding fragment thereof of claim 19, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 63 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

64.

21. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 65); (ii) a CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 66); and (iii) a CDR-L3 comprising the amino acid sequence NSRDSTDNHLWV (SEQ ID NO: 67); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 68); (ii) a CDR-H2 comprising the amino acid sequence SISSSSSSYIYYADSVKG (SEQ ID NO: 69); and (iii) CDR-H3 comprising the amino acid sequence ATSSGYYYFDY (SEQ ID NO: 70) 1. An isolated antibody or antigen-binding fragment thereof comprising:

22. 22. The isolated antibody or antigen-binding fragment thereof of claim 21, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 71 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

72.

23. An isolated antibody or antigen-binding fragment thereof that binds to human KRas, wherein the antibody specifically binds to GDP-bound KRas (KRas-GDP) with higher affinity than to GTP-bound KRas (KRas-GTP); (a) a light chain variable region comprising: (i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 73); (ii) a CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO: 74); and (iii) a CDR-L3 comprising the amino acid sequence GTWDNSLSVWV (SEQ ID NO: 75); and (b) a heavy chain variable region comprising: (i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 76); (ii) a CDR-H2 comprising the amino acid sequence YISSSSSSTIYYADSVKG (SEQ ID NO: 77); and (iii) CDR-H3 comprising the amino acid sequence GKGIVGWGFFGMDV (SEQ ID NO: 78) 1. An isolated antibody or antigen-binding fragment thereof comprising:

24. 24. The isolated antibody or antigen-binding fragment thereof of claim 23, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

80.

25. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 24, which is a KRas alkylation conformation-specific antibody.

26. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 24, which opens and stabilizes the SWII pocket.

27. ​​An isolated antibody or its antigen-binding fragment described in any one of claims 1 to 24, wherein the human KRas is a KRas mutant selected from the group consisting of KRas G12C, KRas G12V, KRas G12R, KRas Q61H, KRas G12D, and KRas G13D.

28. The isolated antibody or antigen-binding fragment thereof described in claim 27, wherein the human KRas is a KRas mutant selected from the group consisting of KRas G12C, KRas G12V, KRas G12D, and KRas G13D.

29. The isolated antibody or antigen-binding fragment thereof of claim 28, wherein the KRas mutant is KRas G12C.

30. The isolated antibody or antigen-binding fragment thereof of claim 29, wherein KRas G12C -GDP is alkylated with a KRas G12C specific covalent inhibitor.

31. The isolated antibody or antigen-binding fragment thereof of claim 30, which is an alkylated conformation-specific KRas antibody that binds to KRas G12C-GDP alkylated with MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

32. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 31, which stabilizes the SWII pocket of a KRas mutant protein.

33. 33. One or more isolated nucleic acids encoding the light chain variable domain and the heavy chain variable domain of the antibody or antigen-binding fragment of any one of claims 1 to 32.

34. 34. A vector comprising one or more nucleic acids of claim 33.

35. A host cell comprising the vector of claim 34.

36. 33. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 32, conjugated to a detectable label.

37. 36. A method for producing an antibody or fragment thereof that binds to KRas-GDP, comprising culturing the host cell of claim 35 under conditions suitable for expression of a vector encoding the antibody, and recovering the antibody.

38. 36. A method for producing an antibody or fragment thereof that binds to KRas-GtP, comprising culturing the host cell of claim 35 under conditions suitable for expression of a vector encoding the antibody, and recovering the antibody.

39. A method for detecting KRas-GDP in a biological sample, comprising contacting the biological sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32.

40. 40. The method of claim 39, further comprising contacting the biological sample with an antibody that binds to KRas-GTP, wherein the amount of KRas-GDP and the amount of KRas-GTP are determined.

41. A method for detecting KRas-GTP in a biological sample, comprising contacting the biological sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32.

42. 40. The method of claim 39, further comprising contacting the biological sample with an antibody that binds to KRas-GDP, wherein the amount of KRas-GTP and the amount of KRas-GDP are determined.

43. A kit comprising a KRas antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 conjugated to a detectable label and instructions for detecting the antibody or antigen-binding fragment thereof.

44. A method for obtaining an inhibitor of a KRas mutant, comprising contacting an antibody or antigen-binding fragment thereof described in any one of claims 1 to 32 with a KRas mutant, screening the compounds, and identifying a compound that binds to the KRas mutant bound to the antibody or antigen-binding fragment thereof.

45. 45. The method of claim 44, wherein the compound comprises a molecule that covalently modifies KRas in the SWII pocket.

46. 46. ​​The method of claim 45, wherein the compound comprises a covalent inhibitor that alkylates at least one residue within the SWII pocket.

47. 45. The method of claim 44, wherein the compound comprises a molecule that non-covalently modifies KRas in the SWII pocket.

48. KRas mutants are Ｇ１２Ｃ , KRas Ｇ１２Ｖ , KRas Ｇ１２Ｄ , KRas Ｇ１３Ｄ , KRas Ｇ１２Ｒ , or KRas Ｑ６１Ｈ The method according to any one of claims 44 to 47, wherein

49. A method for detecting alkylation of KRas, comprising contacting a biological sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32, and detecting the antibody or antigen-binding fragment thereof bound to alkylated KRas.

50. Detection is KRas Ｇ１２Ｃ 50. The method of claim 49, comprising detecting:

51. 51. The method of claim 49 or 50, wherein the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

52. A method for detecting alkylation of KRas in a mammal, comprising administering to the mammal an antibody or antigen-binding fragment thereof described in any one of claims 1 to 32, and detecting the antibody or antigen-binding fragment thereof bound to alkylated KRas.

53. 1. An in vitro method for detecting alkylation of KRas in a patient treated with a KRas inhibitor, comprising: (b) contacting in vitro a sample obtained from a patient with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32; (c) measuring the amount of KRas bound by the antibody or antigen-binding fragment thereof. A method comprising:

54. 54. The method of claim 53, wherein the KRas inhibitor is MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

55. 55. The method of claim 53 or 54, wherein the amount of KRas bound by the antibody or antigen-binding fragment thereof determines the dosage of the KRas inhibitor administered to the patient.

56. Detection is KRas Ｇ１２Ｃ 56. The method of any one of claims 52 to 55, comprising detecting:

57. The method of any one of claims 52 to 56, wherein the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

58. 57. The method of any one of claims 52 to 56, wherein the mammal is a human.

59. A method for treating KRas comprising administering to a subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32. Ｇ１２Ｃ A medicament for treating a cancer mediated by HIV, comprising: The antibody or antigen-binding fragment thereof is administered to a patient having such cancer.

60. KRas Ｇ１２Ｃ 60. The method of claim 59, wherein the mediated cancer is NSCLC, colon cancer, or pancreatic cancer.

61. A crystallization chaperone comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32.

62. 33. A method for crystallizing KRas, wherein KRas is optionally bound to a KRas inhibitor, comprising contacting KRas with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 32, and solving the crystal structure of the complex.

63. KRas is KRas Ｇ１２Ｃ , KRas Ｇ１２Ｄ , KRas Ｇ１２Ｖ , KRas Ｇ１２Ｒ , KRas Ｇ１３Ｄ , or KRas Ｑ６１Ｈ 63. The method of claim 62, wherein:

64. 1. A biosensing surface for measuring the binding of a compound to KRas, comprising: (i) a biosensing surface comprising a hydrogel in which a KRas protein and an antibody or antigen-binding fragment thereof according to any one of claims 1 to 32 are co-localized; (ii) KRas and the antibody or antigen-binding fragment thereof have sufficient freedom within the hydrogen to engage with each other to form an affinity complex; (iii) the local concentrations of KRas and the antibody or antigen-binding fragment thereof exceed the dissociation affinity constant by at least 10-fold, and the local concentrations promote the formation of an affinity complex; (iv) the percentage of unbound KRas protein and anti-KRas antibody is less than 50%; (v) a KRas inhibitor compound is injected over the biosensing surface for at least 5 seconds; and (vi) binding of a KRas inhibitor compound to an anti-KRas antibody is measured on at least one sensing channel; Biosensing surfaces.

65. 65. The biosensing surface of claim 64, wherein the hydrogel is about 10 nm to 500 nm, 10 nm to 300 nm, 10 to 250 nm, or about 10 to 200 nm thick.

66. 66. The biosensing surface of claim 64 or 65, wherein KRas is biotinylated.

67. 67. The biosensing surface of any one of claims 64 to 66, attached to a BIACORE sensor chip.

68. 68. A method of screening compounds for anti-KRas inhibitor activity, comprising measuring binding of the compound to KRas, wherein KRas is bound to an anti-KRas antibody, and wherein binding is measured using a biosensing surface according to any one of claims 64 to 67.

69. A method for measuring binding of a KRas mutant protein to an anti-KRas antibody described herein, comprising: (i) contacting the biosensing surface of any one of claims 64 to 67 with KRas to form a biosensing surface bound to KRas; (ii) contacting a biosensing surface bound to KRas with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof is in molar excess compared to the KRas protein; and (iii) detecting the binding and affinity of antibodies or antigen-binding fragments thereof to KRas using surface plasmon resonance; A method comprising:

70. A method for measuring binding of a KRas mutant protein to an anti-KRas antibody described herein, comprising: (i) contacting the biosensing surface of any one of claims 64 to 67 with the antibody or antigen-binding fragment thereof of any one of claims 1 to 32 to form a biosensing surface bound to an anti-KRas antibody; (ii) contacting a biosensing surface coupled to an anti-KRas antibody with KRas, wherein the antibody or antigen-binding fragment thereof is in molar excess relative to the KRas protein; and (iii) detecting the binding and affinity of antibodies or antigen-binding fragments thereof to KRas using surface plasmon resonance; A method comprising:

71. A kit for detecting alkylation of KRas in a patient treated with a KRas inhibitor, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 32, a sample is obtained from the patient; the sample is contacted with the antibody or antigen-binding fragment thereof; The amount of KRas bound by the antibody or antigen-binding fragment thereof is measured.

72. The kit described in claim 71, wherein the KRas inhibitor is MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

73. A kit described in claim 71 or 72, wherein the amount of KRas bound by the antibody or its antigen-binding fragment determines the dosage of the KRas inhibitor to be administered to the patient.

74. A kit according to any one of claims 71 to 73, wherein the detection comprises detection of KRas G12C.

75. A kit described in any one of claims 71 to 74, wherein the antibody or antigen-binding fragment thereof is a KRas alkylation conformation-specific antibody.

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