Radiolabeled anti-LAG3 antibody for immunoPET imaging

Abstract

Uses of anti-LAG3 antibodies or antigen-binding fragments thereof in immunoPET imaging of tumors and treatment of patients are provided, along with compositions, formulations, and kits comprising the anti-LAG3 antibodies or antigen-binding fragments thereof.

Description

[Technical Field]

[0001] Field The present disclosure relates to LAG3 immunoPET imaging in cancer patients.

[0002] Sequence Listing An official copy of the Sequence Listing will be submitted electronically via the Patent Center contemporaneously with the present specification. The contents of the electronic Sequence Listing (11404WO01_Sequence_Listing_ST26.xml, size: 708,608 bytes, and creation date: February 16, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] background T cell costimulatory and co-inhibitory molecules (collectively termed co-signaling molecules) play a crucial role in regulating T cell activation, subset differentiation, effector function, and survival (Chen et al. 2013, Nature Rev. Immunol. 13:227-242). After T cell receptor (TCR) recognition of cognate peptide-MHC complexes on antigen-presenting cells, co-signaling receptors co-localize with the T cell receptor at the immune synapse, where they synergize with TCR signaling to promote or inhibit T cell activation and function (Flies et al. 2011, Yale J. Biol. Med. 84:409-421). Basic immune responses are controlled by the balance between costimulatory and co-inhibitory signals ("immune checkpoints") (Pardoll 2012, Nature Reviews Cancer 12:252-264). Lymphocyte activation gene-3 (LAG3) functions as one such "immune checkpoint" in mediating peripheral T cell tolerance.

[0004] LAG3 (also known as CD223) is a 503-amino acid transmembrane protein receptor expressed on activated CD4 and CD8 T cells, gamma-delta T cells, natural killer T cells, B cells, natural killer cells, plasmacytoid dendritic cells, and regulatory T cells. LAG3 is a member of the immunoglobulin (Ig) superfamily. Its primary function is to attenuate immune responses. LAG3 binding to MHC class II molecules results in the delivery of a negative signal to LAG3-expressing cells, downregulating antigen-dependent CD4 and CD8 T cell responses. LAG3 also negatively regulates the ability of T cells to proliferate, produce cytokines, and lyse target cells, a process often referred to in the prior art as T cell "exhaustion." LAG3 has also been reported to play a role in enhancing T regulatory (Treg) cell function (Pardoll 2012, Nature Reviews Cancer 12:252-264).

[0005] Immunopositron emission tomography (PET) is a diagnostic imaging tool that utilizes monoclonal antibodies labeled with positron emitters, combining the targeting properties of antibodies with the sensitivity of positron emission tomography cameras.See, for example, The Oncologist, 12: 1379 (2007); Journal of Nuclear Medicine, 52(8):1171(2011) (Non-Patent Document 4).ImmunoPET enables the visualization and quantification of antigen and antibody accumulation in vivo, and therefore can serve as an important tool for diagnosis and complementary therapy.For example, immunoPET can be useful in selecting promising patient candidates for specific therapies and monitoring the effectiveness of treatment.

[0006] As LAG3 has emerged as a target for tumor immunotherapy and infection immunotherapy, there is a need for diagnostic tools for anti-LAG3 therapy, in particular diagnostic tools that allow the detection of patient candidates suitable for said therapy. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Chen et al 2013,Nature Rev.Immunol.13:227-242 [Non-patent document 2] Flies et al 2011,Yale J.Biol.Med.84:409-421 [Non-patent document 3] Pardoll 2012, Nature Reviews Cancer 12:252-264 [Non-patent document 4] The Oncologist, 12: 1379 (2007); Journal of Nuclear Medicine, 52(8):1171(2011) Summary of the Invention

[0008] overview The present disclosure includes radiolabeled anti-LAG3 antibody conjugates for use in immunoPET imaging.

[0009] In one embodiment, the conjugate comprises an anti-LAG3 antibody or antigen-binding fragment thereof, a chelating moiety, and a positron emitter.

[0010] Provided herein are methods for synthesizing the conjugates and synthetic intermediates useful therefor.

[0011] Also provided herein is a method for imaging tissues that express LAG3, the method comprising administering to the tissue a radiolabeled anti-LAG3 antibody conjugate described herein and visualizing LAG3 expression by positron emission tomography (PET).

[0012] Also included herein is a method for imaging tissues containing cells that express LAG3, such as intratumoral lymphocytes that express LAG3, which method comprises administering to the tissue a radiolabeled anti-LAG3 antibody conjugate described herein and visualizing the expression of LAG3 by PET imaging.

[0013] Also provided herein is a method for detecting LAG3 in a tissue, the method comprising administering a radiolabeled anti-LAG3 antibody conjugate described herein to the tissue and visualizing LAG3 expression by PET imaging. In one embodiment, the tissue is present in a human subject. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer, an inflammatory disease, or an infection.

[0014] Also provided herein is a method for whole-body imaging of LAG3, the method comprising administering to a subject a radiolabeled anti-LAG3 antibody conjugate described herein and visualizing LAG3 expression by PET imaging. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder such as cancer, inflammatory disease, or infection. Whole-body imaging allows visualization of LAG3-expressing cells (e.g., T cells) and / or changes in LAG3 expression throughout the body (e.g., upregulation or downregulation).

[0015] Also included herein is a method for identifying a patient suitable for anti-tumor therapy comprising a LAG3 inhibitor, the method comprising the steps of selecting a patient having a tumor (e.g., a solid tumor), administering a radiolabeled antibody conjugate described herein, and visualizing the administered radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anti-tumor therapy comprising a LAG3 inhibitor.

[0016] Also provided herein is a method of treating a tumor, the method comprising the steps of selecting a subject having a solid tumor, determining that the solid tumor is LAG3-positive, and administering an anti-tumor therapy to the subject in need thereof. In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor. In certain embodiments, the anti-tumor therapy comprises an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor and / or an inhibitor of the PD-1 / PD-L1 signaling axis. In certain embodiments, a radiolabeled anti-LAG3 antibody conjugate described herein is administered to the subject, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) to determine whether the tumor is LAG3-positive. In certain embodiments, a radiolabeled anti-PD-1 antibody conjugate is further administered to the subject, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is PD-1 positive.

[0017] Also provided herein is a method for monitoring the effectiveness of anti-tumor therapy in a subject, the method comprising: selecting a subject having a solid tumor, the subject being treated with an anti-tumor therapy; administering a radiolabeled anti-LAG3 conjugate described herein to the subject; imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging; and determining tumor growth, wherein a change in the uptake of the conjugate or radiolabeled signal from baseline (pre-treatment baseline) indicates the effectiveness of the anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor (e.g., an anti-LAG3 antibody, e.g., REGN3767, also referred to herein as fianlimab). In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor and an inhibitor of the PD-1 / PD-L1 signaling axis. In certain embodiments, the anti-tumor therapy includes a PD-1 inhibitor (e.g., REGN2810 (also known as cemiplimab), BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Publication No. US2015-0203580), a CTLA-4 inhibitor (e.g., ipilimumab), antibodies against CD47, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Pat. No. 7,087,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 659, 660, 661, 662, 663, 664, 665, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679 ...411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, MUC16, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guerin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies that target T cells (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, cytokines such as IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, IL-2, IL-7, IL-21, and IL-15, and / or antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC).

[0018] Also included herein is a method for predicting a patient's response to anti-tumor therapy, the method comprising selecting a patient with a solid tumor and determining whether the tumor is LAG3-positive, and if the tumor is LAG3-positive, it predicts a positive response of the patient to the anti-tumor therapy. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure; and localizing the radiolabeled anti-LAG3 antibody conjugate within the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive. In some embodiments, the anti-tumor therapy is selected from the group consisting of a PD-1 inhibitor (e.g., REGN2810 (also known as cemiplimab), BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., aflibercept or U.S. Pat. No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, MUC16, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guerin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies targeting T cells (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, cytokines such as IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, IL-2, IL-7, IL-21, and IL-15, and / or antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC).

[0019] Also provided herein is a method for predicting a patient's response to anti-tumor therapy comprising a LAG3 inhibitor, the method comprising the steps of selecting a patient with a solid tumor and determining whether the tumor is LAG3-positive (e.g., whether the tumor contains LAG3-positive cells), whereby if the tumor is LAG3-positive, it indicates a positive response of the patient to anti-tumor therapy comprising a LAG3 inhibitor. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure; and localizing the radiolabeled anti-LAG3 antibody conjugate within the tumor by PET imaging, whereby the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive.

[0020] 1. A method of imaging a LAG3 positive tumor in a subject, comprising: (i) an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), administering to the subject, the antibody or antigen-binding fragment thereof has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; 89 Labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography-computed tomography (PET / CT) imaging; Also provided herein is a method for imaging a LAG3-positive tumor in a subject, comprising: In some embodiments, step (ii) of imaging the localization of the labeled antibody conjugate is performed within or near the tumor.

[0021] In some embodiments, the chelating agent is desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α''α'''-tetramethyl- ... Azacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H4 octapa, H6 phospa, H2 dedopa, H5 decapa, H2 azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N, N',N''-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cyclen), 1,4,8,11-tetraazacyclotetradecane (Cyclam), octadecane, The chelators are selected from the group consisting of dentate chelators, hexadentate chelators, phosphonate-based chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinine C and fusarinine C derivative chelators, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), and the like.

[0022] In some embodiments, the label provides about 1 mCi of radiation upon injection. In some embodiments, about 0.2 mg to about 3.0 mg of the labeled antibody conjugate is administered to a subject. In some embodiments, about 1.0 mg to about 2.0 mg of the labeled antibody conjugate is administered to a subject. In some embodiments, the antibody or antigen-binding fragment thereof may be administered to a subject in a total amount of about 10 to about 100 mg, about 20 to about 100 mg, about 20 to about 50 mg, or about 30 to about 50 mg, about 10 mg, or about 20 mg, or about 30 mg, or about 40 mg, or about 50 mg.

[0023] In some embodiments, the imaging step (ii) is performed about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days after step (i), i.e., after administering an antibody or antigen-binding fragment thereof that binds lymphocyte activation gene-3 (LAG3) to the subject. In some embodiments, the imaging step (ii) is performed about 7 days after step (i), i.e., after administering an antibody or antigen-binding fragment thereof that binds lymphocyte activation gene-3 (LAG3) to the subject.

[0024] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is selected from the group consisting of anal cancer, anaplastic thyroid carcinoma, astrocytoma, bladder cancer, bone cancer, glioblastoma multiforme, brain cancer, triple-negative breast cancer, breast cancer, cervical cancer, chondrosarcoma, clear cell carcinoma, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fibrosarcoma, gastric carcinoma, glioblastoma, head and neck cancer, hepatocellular carcinoma, jejunal cancer, renal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, metastatic cervical cancer, metastatic melanoma, myeloma, multiple myeloma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, clear cell renal carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, squamous cell carcinoma of the head and neck, stomach cancer, and the like. The cancer may be selected from the group consisting of synovial sarcoma, testicular cancer, thyroid cancer, uterine cancer, and Wilms' tumor.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO: 418 and three CDRs in the LCVR set forth in SEQ ID NO: 426. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO: 432. In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO: 418 and the LCVR set forth in SEQ ID NO: 426.

[0026] 1. A method of treating a subject, comprising: (i) administering to a subject having a tumor an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), the antibody or antigen-binding fragment thereof has three amino acid sequences in a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554. one heavy chain complementarity determining region (HCDR) and three light chain complementarity determining regions (LCDR) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; 89 Labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the imaging step of (ii) is performed 7 days after step (i), and the presence of the radiolabeled antibody conjugate in the tumor indicates the presence of LAG3-positive cells in the tumor; (iii) administering one or more doses of the anti-tumor therapy to a subject in need thereof; Also provided herein are methods of treating a subject, including:

[0027] In some embodiments, the method of treating a subject comprises: (i) administering to a subject having a tumor an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), the antibody or antigen-binding fragment thereof has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; 89 Labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the imaging step of (ii) is performed 7 days after step (i), and the presence of the radiolabeled antibody conjugate in the tumor indicates the presence of LAG3-positive cells in the tumor; (iii) if LAG3-positive cells are present in the tumor, administering one or more doses of an anti-tumor therapy to a subject in need thereof; Includes.

[0028] In some embodiments, step (iii) is performed one or more times, e.g., once, twice, three times, four times, five times, etc. In some embodiments, steps (ii) and (iii) are performed on the same day. In some embodiments, steps (i) and (ii) are repeated.

[0029] In some aspects, the method further comprises step (iv), wherein steps (i) and (ii) are repeated, with step (ii) being performed after step (iii). In some aspects, the method further comprises step (iv), wherein steps (i) and (ii) are repeated, with step (iv) being performed after step (iii). In certain embodiments, step (iii) is performed twice before step (iv).

[0030] In some embodiments, the method further comprises obtaining a tumor sample (eg, a biopsy) from the subject and determining the presence of LAG3 in the tumor sample.

[0031] In some embodiments, the method further comprises measuring the tumor response to the anti-tumor therapy. In certain embodiments, measuring the tumor response comprises a reduction in size and / or number of tumor lesions or disappearance.

[0032] In some embodiments, the label provides 1 mCi of radiation upon injection. In some embodiments, about 0.2 mg to about 3.0 mg of the labeled antibody conjugate is administered to a subject. In some embodiments, about 1.0 mg to about 2.0 mg of the labeled antibody conjugate is administered to a subject. In some embodiments, the antibody or antigen-binding fragment thereof is administered to a subject in an amount of about 2 to about 100 mg, about 20 to about 100 mg, or, for example, about 20 to about 50 mg, about 30 to about 50 mg, or about 30 mg, about 40 mg, about 50 mg, or about 100 mg.

[0033] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor may be selected from the group consisting of anal cancer, anaplastic thyroid carcinoma, astrocytoma, bladder cancer, bone cancer, glioblastoma multiforme, brain cancer, triple-negative breast cancer, breast cancer, cervical cancer, chondrosarcoma, clear cell carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, fibrosarcoma, gastric carcinoma, glioblastoma, head and neck cancer, hepatocellular carcinoma, jejunal cancer, renal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, metastatic cervical cancer, metastatic melanoma, myeloma, multiple myeloma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, clear cell renal carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, squamous cell carcinoma of the head and neck, stomach cancer, synovial sarcoma, testicular cancer, thyroid cancer, uterine cancer, and Wilms' tumor. In some embodiments, the cancer is an advanced stage cancer, i.e., a metastatic cancer. In some embodiments, the methods provided herein are useful in advanced stages of solid tumors and hematological malignancies, e.g., for treating, imaging, and / or monitoring advanced stages of solid tumors and hematological malignancies.

[0034] In some aspects, the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR of SEQ ID NO: 418 and three CDRs in the LCVR of SEQ ID NO: 426. In some aspects, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO: 432. In some aspects, the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO: 418 and the LCVR set forth in SEQ ID NO: 426. In some embodiments, the antibody or antigen-binding fragment thereof is REGN3767, (also known as fianlimab).

[0035] In some embodiments, the anti-tumor therapy is selected from the group consisting of an inhibitor of LAG3, an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody drug conjugate (ADC).

[0036] Exemplary anti-tumor therapies include anti-LAG3 antibodies, REGN2810 (also known as cemiplimab), BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, anti-CD-28 antibodies, anti-2B4 antibodies, anti-LY108 antibodies, anti-LAIR1 antibodies, anti-ICOS antibodies, anti-CD160 antibodies, anti-VISTA antibodies, aflibercept, and bevacizumab. Cetuximab, ranibizumab, sunitinib, sorafenib, pazopanib, nesbacumab, erlotinib, cetuximab, rituximab, CA9 antibody, MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin 1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin Calmette-Guerin), tumor-targeted CD3 bispecific antibodies (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, vincristine, cyclophosphamide, radiation therapy, sarilumab, dupilumab, anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC.

[0037] In some embodiments, the presence of LAG3-positive cells in a tumor identifies the subject as a candidate for anti-tumor therapy comprising an inhibitor of LAG3 or the PD-1 / PD-L1 signaling axis. In certain embodiments, the anti-tumor therapy is selected from the group consisting of an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, and an anti-PD-L1 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof selected from the group consisting of REGN2810, nivolumab, and pembrolizumab. In certain embodiments, the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a platinum-based chemotherapeutic agent. In certain embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin. In certain embodiments, the anti-tumor therapy is an anti-PD-L1 antibody or antigen-binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab. In certain embodiments, the anti-tumor therapy is an anti-PD-L1 antibody or antigen-binding fragment thereof selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 41 an anti-LAG3 antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs) in a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the group consisting of: 0, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.In certain embodiments, the anti-tumor therapy is selected from the group consisting of SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16, 20 / 22 / 24 / 28 / 30 / 32, 36 / 38 / 40 / 44 / 46 / 48, 52 / 54 / 56 / 60 / 62 / 64, 68 / 70 / 72 / 76 / 78 / 80, 84 / 86 / 88 / 92 / 94 / 96, 100 / 102 / 104 / 108 / 110 / 112, 116 / 118 / 120 / 124 / 126 / 128, 132 / 134 / 136 / 140 / 142 / 144, 148 / 150 / 152 / 156 / 158 / 160, 164 / 166 / 168 / 172 / 174 / 1 76, 180 / 182 / 184 / 188 / 190 / 192, 196 / 198 / 200 / 204 / 206 / 208, 212 / 214 / 216 / 220 / 222 / 224, 228 / 230 / 232 / 236 / 238 / 240, 244 / 246 / 248 / 252 / 254 / 256, 260 / 262 / 264 / 268 / 270 / 272, 276 / 278 / 280 / 284 / 286 / 288, 292 / 294 / 296 / 300 / 302 / 304, 308 / 310 / 312 / 316 / 318 / 320, 324 / 326 / 328 / 332 / 334 / 336, 3 40 / 342 / 344 / 348 / 350 / 352, 356 / 358 / 360 / 364 / 366 / 368, 372 / 374 / 376 / 380 / 382 / 384, 388 / 390 / 392 / 396 / 398 / 400, 404 / 406 / 408 / 412 / 414 / 416, 420 / 422 / 424 / 428 / 430 / 432, 436 / 438 / 440 / 444 / 446 / 448, 452 / 454 / 456 / 524 / 526 / 528, 460 / 462 / 464 / 524 / 526 / 528, 468 / 470 / 472 / 524 / 526 / 528, 476 / 4 An anti-LAG3 antibody or antigen-binding fragment thereof, comprising three HCDRs and three LCDRs selected from the group consisting of 78 / 480 / 524 / 526 / 528, 484 / 486 / 488 / 524 / 526 / 528, 492 / 494 / 496 / 524 / 526 / 528, 500 / 502 / 504 / 532 / 534 / 536, 508 / 510 / 512 / 532 / 534 / 536, 516 / 518 / 520 / 532 / 534 / 536, 540 / 542 / 544 / 548 / 550 / 552, and 556 / 558 / 560 / 564 / 566 / 568.In certain embodiments, the anti-tumor therapy is an anti-LAG3 antibody or antigen-binding fragment thereof comprising three HCDRs in the HCVR set forth in SEQ ID NO: 418 and three LCDRs in the LCVR set forth in SEQ ID NO: 426.

[0038] In some aspects, the anti-tumor therapy is administered in combination with a second anti-tumor therapy. In some embodiments, the second anti-tumor therapy is selected from the group consisting of an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody-drug conjugate (ADC).Exemplary second anti-tumor therapies include anti-LAG3 antibodies, REGN3767 (also known as fianlimab), REGN2810, BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, anti-CD-28 antibodies, anti-2B4 antibodies, anti-LY108 antibodies, anti-LAIR1 antibodies, anti-ICOS antibodies, anti-CD160 antibodies, anti-VISTA antibodies, and afliberce. puto, bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, nesvaccumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin Calmette-Guerin), CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, vincristine, cyclophosphamide, radiation therapy, sarilumab, dupilumab, anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC.

[0039] As used herein, a method for producing a radioactive material comprising: (i) an unlabeled anti-LAG3 antibody or antigen-binding fragment thereof; and (ii) an anti-LAG3 antibody or antigen-binding fragment thereof, which provides about 0.5 to 3.0 mCi of radioactivity. 89 and a Zr-labeled anti-LAG3 antibody conjugate, wherein the total amount of labeled and unlabeled antibody or antigen-binding fragment thereof present in the composition is about 40 mg. In some embodiments, the labeled anti-LAG3 antibody conjugate is present in the composition in an amount of about 0.2 mg to about 3 mg.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCV) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554. and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO:418 and three CDRs in the LCVR set forth in SEQ ID NO:426. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO: 432. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR set forth in SEQ ID NO: 418 and an LCVR set forth in SEQ ID NO: 426. In some specific embodiments, the antibody or antigen-binding fragment thereof is REGN3767, i.e., fianlimab.

[0041] In some embodiments, 89 The Zr-labeled anti-LAG3 antibody conjugate comprises an anti-LAG3 antibody or antigen-binding fragment thereof conjugated to desferrioxamine (DFO).

[0042] In some embodiments, 89The Zr-labeled anti-LAG3 antibody conjugate provides about 1 mCi of radioactivity. In some embodiments, the labeled anti-LAG3 antibody conjugate is present in the composition in an amount of about 1-2 mg.

[0043] In a further aspect, three heavy chain complementarity determining regions (HCDRs) in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562. linked to a portion of an anti-LAG3 antibody or its antigen-binding fragment 89 Zr radiolabel, A radiolabel that provides about 0.5 to about 3 mCi of radiation per formulation. Including, the formulation is configured for administration to a human at a total dosage of about 40 mg of the antibody or antigen-binding fragment thereof; Formulations are provided herein.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO:418 and three CDRs in the LCVR set forth in SEQ ID NO:426.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO: 432.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO:418 and the LCVR set forth in SEQ ID NO:426.

[0047] In some embodiments, the radiolabel provides about 1 mCi of radiation.

[0048] In some aspects, provided herein are methods for imaging a LAG3-positive tumor in a subject. In some embodiments, the method comprises: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), the antibody or antigen-binding fragment thereof has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; 89 Labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 40 mg and provides about 1 mCi+ / - of radiation upon injection; administering (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography-computed tomography (PET / CT) imaging, wherein the imaging is performed 7 days after step (i); In some embodiments, step (ii), imaging the localization of the labeled antibody conjugate, is performed within or near the tumor.

[0049] In some embodiments of the method, the antibody or antigen-binding fragment thereof comprises three HCDRs in the HCVR set forth in SEQ ID NO:418 and three LCDRs in the LCVR set forth in SEQ ID NO:426.

[0050] In some embodiments of the method, once the subject is determined to contain LAG3-positive cells in the tumor, the method further comprises administering one or more doses of an anti-tumor therapy to the subject.

[0051] In some aspects, provided herein are methods for imaging LAG3 expression throughout the body. In some embodiments, the method comprises: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), the antibody or antigen-binding fragment thereof has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; 89 Labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 40 mg and provides about 1 mCi+ / - of radiation upon injection; administering (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography-computed tomography (PET / CT) imaging, wherein the imaging is performed 7 days after step (i); Includes.

[0052] In certain embodiments, the subject has a disease or disorder, such as cancer, inflammatory disease, or infection. Whole-body imaging allows visualization of LAG3-expressing cells (e.g., T cells) and / or changes in LAG3 expression throughout the body (e.g., upregulation or downregulation).

[0053] Additional aspects provide kits comprising any of the compositions or formulations described. In some embodiments, the kits provide instructions for PET imaging 7 days after administration of the composition or formulation to a patient. [Brief explanation of the drawings]

[0054] [Figure 1] 1 shows the UV / VIS spectrum of DFO-modified anti-LAG3 antibody (mAb1-DFO). [Figure 2] HPLC-SEC of DFO-modified anti-LAG3 antibody. [Figure 3] For Study 1, radioSEC-HPLC of isotype-DFO-conjugate after 89Zr radiolabeling is shown. [Figure 4] For Study 1, radio-SEC-HPLC of anti-LAG3-DFO-conjugate after 89Zr radiolabeling is shown. [Figure 5] For Study 2, radio-SEC-HPLC of anti-LAG3-DFO-conjugate after 89Zr radiolabeling is shown. [Figure 6] For Study 1, UV280-SEC-HPLC chromatogram and radioactive iTLC trace of the isotype-DFO-conjugate after 89Zr radiolabeling are shown. [Figure 7] For Study 1, UV280-SEC-HPLC chromatogram and radioactive iTLC trace of anti-LAG3-DFO-conjugate after 89Zr radiolabeling are shown. [Figure 8] For Study 2, UV280-SEC-HPLC chromatogram and radioactive iTLC trace of anti-LAG3-DFO-conjugate after 89Zr radiolabeling are shown. [Figure 9]Representative images of 89Zr-DFO-mAb1 injected at a protein dose of 5 mg / kg (Ms01) or 0.03 mg / kg (Ms14) are provided, demonstrating specific targeting of 89Zr-DFO-mAb1 to Raji / hPBMC tumors using 0.03 mg / kg 89Zr-DFO-mAb1 and blocking with 5 mg / kg 89Zr-DFO-mAb1. Specific uptake in the spleen and lymph nodes is seen with the lower dose of 89Zr-DFO-mAb1, 0.03 mg / kg. [Figure 10] 1 provides characteristics of the melanoma samples tested in Example 7. [Figure 11] LAG3 expression in tissue samples from PBMC / Raji xenografts (obtained 27 and 15 days after tumor implantation) and melanoma clinical samples is shown. [Figure 12] 1 provides a schematic diagram of the therapeutic dosing regimen used in Example 8. [Figure 13] We provide data demonstrating that REGN2810 anti-human PD-1 Ab and mAb1 anti-human LAG3 increase LAG3+ T cells and PD-1+ T cells, respectively, in the tumor microenvironment. [Figure 14] 14A and 14B are schematic diagrams showing the protocols for parts A and B of the clinical trial detailed in Example 10. [Figure 15]Figures 15A, 15B, 15C, and 15D show the pharmacokinetics of 89Zr-DFO-REGN3767 (also referred to herein as fianlimab, a conjugated and radiolabeled anti-LAG3 antibody having the HCVR / LCVR sequence pair of SEQ ID NOs: 418 / 426, H4sH15482P, or conjugated and radiolabeled mAb1). The activity (SUV average) of 89Zr-DFO-REGN3767 in the blood pool over time is measured by PET scan (Figure 15A) and in whole blood venous samples (Figure 15B). Each line represents a different tracer protein dose level. Confidence intervals (shaded regions) are given for dose levels (20 and 40 mg) that include more than two patients. FIG. 15C shows the clearance of radiolabeled antibody in serum (mL / hr), and FIG. 15D provides the area under the curve (AUC; kBq*h / mL) associated with various tracer doses. [Figure 16] Figures 16A and 16B show the uptake of 89Zr-DFO-REGN3767 in tumor lesions. Figure 16A shows a violin plot (SUVmax) of tracer uptake in tumors per dose level. Dots represent individual tumor lesions, the thick dashed line represents the median, and the thin dotted lines indicate quartiles. Figure 16B provides the tumor-to-blood ratio per dose level over time. The geometric mean SUVmax of all lesions in a patient was divided by the mean SUV of the aorta for that patient. Dots represent the geometric mean tumor-to-blood ratio per dose level at a given time point. The gray dotted line is a reference for a ratio of 1. [Figure 17] Figures 17A, 17B, 17C, and 17D provide examples of tracer uptake in tumor lesions. A transaxial CT image is shown on the left, and a PET / CT fusion image is shown on the right. Figures 17A and 17B provide images of a patient with dMMR colon cancer and high tracer uptake. The arrow indicates the primary lesion in the colon. Figures 17C and 17D provide images of a patient with chondrosarcoma and relatively low tracer uptake. The arrow indicates a lung metastasis in the right lung. PET images are on a scale of 0 to 8 SUV. [Figure 18]Figures 18A and 18B show the uptake of Zr-DFO-REGN3767 in lymphoid tissues. Zr-DFO-REGN3767 uptake (SUV average) in the spleen (Figure 18A) and bone marrow (Figure 18B) at different imaging time points after tracer injection. Each line represents a different tracer protein dose level. [Figure 19] Correlation between response to therapy and tracer uptake in tumors is shown using a violin plot of tracer uptake in tumor lesions per RECIST response category. White dots represent geometric means with 95% confidence intervals, and dark dots represent individual tumor lesions. TME = tumor microenvironment, N = number of patients, PD = progressive disease, SD = stable disease, PR = partial response. [Figure 20] Figures 20A and 20B show the biodistribution of 89Zr-DFO-REGN3767. Figure 20A is a maximum intensity projection (scale of 0 to 8 SUV) of a 89Zr-REGN3767 positron emission tomography (PET) scan 7 days after tracer injection using a 40 mg dose. The arrow indicates the tumor lesion. Other areas of high activity include the spleen, liver, and transverse colon. Figure 20B shows tracer uptake (SUV mean) in normal tissues; the tissues evaluated are presented on the x-axis. The bars represent tracer uptake (mean + standard deviation) at days 0, 2, 4, and 7 after tracer injection. [Figure 21] Tracer uptake in tumor lesions of pMMR and dMMR tumors is shown. White dots represent geometric means with 95% confidence intervals, and dark dots represent individual tumor lesions. Abbreviations: SUV: standardized uptake value; pMMR: normal mismatch repair; dMMR: mismatch repair deficient; MSI: microsatellite instability. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0056] The term "LAG3" refers to the lymphocyte-activation gene-3 protein, also known as CD223, an immune checkpoint receptor or T-cell co-inhibitor. The amino acid sequence of full-length LAG3 is provided in GenBank under accession number NP_002277.4 and is also referred to herein as SEQ ID NO: 582. The term "LAG3" also includes protein variants of LAG3 having the amino acid sequences of SEQ ID NOs: 574, 575, or 576. The term "LAG3" includes recombinant LAG3 or fragments thereof. The term also encompasses LAG3 or fragments thereof linked to a signal sequence, such as a histidine tag, mouse or human Fc, or the signal sequence of ROR1. For example, the term includes the sequence exemplified by SEQ ID NO: 575, which includes mouse Fc (mIgG2a) linked at the C-terminus to amino acid residues 29-450 of the full-length extracellular domain of LAG3. The protein variant exemplified by SEQ ID NO: 574 comprises a histidine tag attached at the C-terminus to amino acid residues 29 to 450 of the full-length extracellular domain LAG3. Unless specified as from a non-human species, the term "LAG3" refers to human LAG3.

[0057] LAG3 is a member of the immunoglobulin (Ig) superfamily. It is a type 1 transmembrane protein with four extracellular Ig-like domains (D1 to D4) and is expressed in intratumoral lymphocytes (including activated T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and regulatory T cells). The LAG3 receptor binds to MHC class II molecules present on antigen-presenting cells (APCs).

[0058] As used herein, the term "T cell co-inhibitory factor" refers to a ligand and / or receptor that regulates immune responses through T cell activation or suppression. The term "T cell co-inhibitory factor" is also known as a T cell co-signaling molecule, and includes, but is not limited to, lymphocyte activation gene 3 protein (LAG3, also known as CD223), programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin-3 (TIM3), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT, also known as VSIG9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, also known as CD305), inducible T cell co-stimulatory molecule (ICOS, also known as CD278), B7-1 (CD80), and CD160.

[0059] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (C H 1 domain, C H 2 domain and C H Each light chain consists of a light chain variable region ("LCVR" or "V L "), and the light chain constant region (C L ) V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally occurring, or may be artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0060] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature as being capable of omitting one or two CDRs for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs have no amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0061] Based on previous studies, CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from regions of the Kabat CDRs outside the Chothia CDRs (e.g., residues H60-H65 in CDRH2 are often unnecessary). When a CDR or its residue(s) is omitted, it is typically replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0062] The anti-LAG3 monoclonal antibodies discussed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (in some cases), reduced immunogenicity, etc. Antibodies and antigen-binding fragments and those obtained in this general manner are encompassed by the present disclosure.

[0063] The present disclosure also includes anti-LAG3 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes anti-LAG3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0064] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences.

[0065] As used herein, the term "multispecific antigen-binding molecule" refers to bispecific, trispecific, or multispecific antigen-binding molecules and antigen-binding fragments thereof. Multispecific antigen-binding molecules may be specific for different epitopes of more than one target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. A multispecific antigen-binding molecule may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or noncovalently bound to each other. The term "multispecific antigen-binding molecule" includes antibodies of the present disclosure that can be linked to or coexpressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other methods) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule with a second binding specificity. According to the present disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In certain embodiments, an antibody of the present disclosure is operably linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody having a second binding specificity. Bispecific and multispecific antibodies of the present disclosure are described elsewhere herein.

[0066] The terms "specifically bind" or "specifically binds to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1x10 -8 can be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller K D indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and similar methods. As described herein, antibodies that specifically bind to LAG3 have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, multispecific antibodies that bind to one domain of LAG3 and one or more additional antigens, or bispecific antibodies that bind to two different regions of LAG3, are nevertheless considered to be "specifically binding" antibodies as used herein.

[0067] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to LAG3.

[0068] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds to LAG3 or a fragment thereof is substantially free of antibodies that specifically bind to antigens other than LAG3).

[0069] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of biomolecular interactions in real time through the detection of changes in protein concentration within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

[0070] "K D The term " ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0071] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes generally contain residues that are part of a structural epitope and directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural and / or specific charge characteristics.

[0072] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence homology in at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known algorithm of sequence homology, such as FASTA, BLAST, or GAP.

[0073] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using default gap weights, such as with the programs GAP or BESTFIT, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, non-homologous residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a scale of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence identity or sequence homology between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0074] By the phrase "therapeutically effective amount" is meant the amount that is administered to produce the desired effect. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0075] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a chronic viral infection, cancer, or an autoimmune disease.

[0076] As used herein, the phrase "administering a radiolabeled anti-LAG3 antibody conjugate described herein to a tissue" refers to administering the anti-LAG3 antibody conjugate to a subject intravenously, intramuscularly, etc., such that the radiolabeled anti-LAG3 conjugate is delivered to a tissue containing LAG3-expressing cells.

[0077] II. Radiolabeled LAG3 Antibody Immunoconjugates for ImmunoPET Imaging Provided herein are radiolabeled antigen binding proteins that bind to LAG3. Various embodiments of radiolabeled antigen binding proteins that bind to LAG3, their methods of making, and their methods of use are described in U.S. Patent No. 10,905,784, the entire contents of which are incorporated herein by reference. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to a positron emitter. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to one or more chelating moieties, where the chelating moiety is a chemical moiety capable of chelating a positron emitter.

[0078] In some embodiments, an antigen binding protein, e.g., an antibody, that binds to LAG3 is provided, wherein the antigen binding protein that binds to LAG3 has the following structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently 0 or 1 in each occurrence, and at least one of the z's is 1.

[0079] In some embodiments, the radiolabeled antigen binding protein comprises a compound of formula (I): MLA-[LM Z ] k (I) wherein A is a protein that binds to LAG3, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1.

[0080] In certain embodiments, the radiolabeled antigen binding protein comprises a compound of formula (II): A-[LM] k (II) where A is a protein that binds to LAG3, L is a chelating moiety, M is a positron emitter, and k is an integer from 1 to 30.

[0081] In some embodiments, the following structure: AL k wherein A is a protein that binds to LAG3, L is a chelating moiety, and k is an integer between 1 and 30, and wherein the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging.

[0082] Suitable binding proteins, chelating moieties, and positron emitters are provided below.

[0083] A. LAG3-binding proteins Suitable LAG3 binding proteins are those that specifically bind to LAG3, including those described in PCT / US16 / 56156, which is incorporated herein by reference in its entirety. Exemplary anti-LAG3 binding proteins of the present disclosure are the antibodies listed in Table 1 of PCT / US16 / 56156, and are also provided below.

[0084] [Table 1]

[0085] Table 1 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-LAG3 antibodies.

[0086] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that comprises an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0087] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0088] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), comprising any of the HCVR amino acid sequences listed in Table 1, paired with an LCVR amino acid sequence listed in Table 1. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pairs are SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, Selected from the group consisting of 306 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, 554 / 562. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 386 / 394 (e.g., H4sH15479P), 418 / 426 (e.g., H4sH15482P), or 538 / 546 (e.g., H4sH14813N). In certain other embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 458 / 464 (e.g., H4sH15498P2), 162 / 170 (e.g., H4H15483P), and 579 / 578 (e.g., H4H15482P).

[0089] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0090] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0091] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0092] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0093] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0094] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0095] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1, paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 392 / 400 (e.g., H4sH15479P), 424 / 432 (e.g., H4sH15482P), and 544 / 552 (e.g., H4sH14813N).

[0096] In some embodiments, the binding protein is an antibody or antigen-binding fragment that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 388-390-392-396-398-400 (e.g., H4sH15479P), 420-422-424-428-430-432 (e.g., H4sH15482P), and 540-542-544-548-550-552 (e.g., H4sH14813N).

[0097] In some embodiments, the binding protein is an antibody or antigen-binding fragment that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-LAG3 antibodies listed in Table 1. For example, in some embodiments, the binding protein is an antibody or antigen-binding fragment that comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386 / 394 (e.g., H4sH15479P), 418 / 426 (e.g., H4sH15482P), or 538 / 546 (e.g., H4sH14813N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the identified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani, et al., J. Mol. Biol. 273:927-948 (1997); and Martin, et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0098] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that competes for specific binding to LAG3 with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively. In certain embodiments, the binding protein is the REGN3767 antibody (also known as fianlimab).

[0099] Additional exemplary anti-LAG3 antibodies useful herein include LAG525 (and other LAG3 antibodies disclosed in U.S. Patent No. 20100233183), leratolimab (and other LAG3 antibodies disclosed in U.S. Patent No. 20110150892), GSK2831781 (and other LAG3 antibodies disclosed in U.S. Patent No. 20140286935), MGD013 (and other LAG3 antibodies disclosed in WO2015200119), and those disclosed in U.S. Patent No. 20160222116, U.S. Patent Application Publication No. 20170022273, U.S. Patent Application Publication No. 20170097333, U.S. Patent Application Publication No. No. 20170137517, U.S. Patent Application Publication No. 20170267759, U.S. Patent Application Publication No. 20170290914, U.S. Patent Application Publication No. 20170334995, WO 2016126858, WO 2016200782, WO 2017087589, WO 2017087901, WO 2017106129, WO 2017149143, WO 2017198741, WO 2017219995, and WO 2017220569.

[0100] Also provided herein are isolated antibodies and antigen-binding fragments thereof that inhibit LAG3 binding to MHC class II. In some embodiments, the antibodies or antigen-binding fragments thereof that inhibit LAG3 binding may bind to the same epitope on LAG3 as MHC class II, or may bind to a different epitope on LAG3 than MHC class II. In certain embodiments, the antibodies of the present disclosure that inhibit LAG3 binding to MHC class II comprise a CDR of an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and a CDR of an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0101] In alternative embodiments, the present disclosure provides antibodies and antigen-binding fragments thereof that do not inhibit LAG3 binding to MHC class II.

[0102] In some embodiments, the binding proteins are antibodies and antigen-binding fragments thereof that specifically bind to LAG3 from humans or other species. In certain embodiments, the antibodies may bind to human LAG3 and / or cynomolgus monkey LAG3.

[0103] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that cross-competes for binding to LAG3 with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0104] In one embodiment, the binding protein is an isolated antibody or antigen-binding fragment having one or more of the following characteristics: (a) inhibits binding of LAG3 to MHC class II; (b) specifically binds to human LAG3 and / or cynomolgus monkey LAG3; (c) blocks LAG3-induced impairment of T cell activation and rescues T cell signaling; and (d) suppresses tumor growth and increases survival in subjects with cancer.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to LAG3 in an agonistic manner, i.e., enhance or stimulate the binding and / or activity of LAG3, hi other embodiments, the antibody may specifically bind to LAG3 in an antagonistic manner, i.e., inhibit LAG3 from binding to its ligand.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to LAG3 in a neutral manner, i.e., bind to LAG3 but do not inhibit, enhance, or stimulate its binding or activity.

[0107] In certain embodiments, the antibody or antigen-binding fragment is bispecific, having a first binding specificity for LAG3 and a second binding specificity for a second target epitope. The second target epitope may be a different epitope on LAG3 or a different epitope on a different protein. In certain embodiments, the second target epitope may be on a different cell, including a different T cell, B cell, tumor cell, or virus-infected cell.

[0108] In certain embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to human lymphocyte activation gene 3 (LAG3) protein is provided, wherein the antibody or antigen-binding fragment thereof has (a) a binding dissociation equilibrium constant (K) of less than about 10 nM as measured by a surface plasmon resonance assay at 25° C. D ) binds to monomeric human LAG3 (using the assay format defined in Example 3 of PCT / US16 / 56156 or a substantially similar assay); (b) has a K of less than about 8 nM as measured by surface plasmon resonance assay at 37°C. D (c) a K of less than about 1.1 nM as measured by surface plasmon resonance assay at 25°C. D (d) a K of less than about 1 nM as measured by surface plasmon resonance assay at 37°C. D(e) an EC50 of less than about 8 nM as measured by flow cytometry assay; 50 (f) an EC50 of less than about 2.3 nM as measured by flow cytometry assay; 50 (g) binds to mfLAG3-expressing cells with an IC of less than about 32 nM as determined by a cell adhesion assay; 50 (h) inhibiting the binding of hLAG3 to human MHC class II with an IC of less than about 30 nM as determined by a cell adhesion assay; 50 (i) inhibits hLAG3 binding to mouse MHC class II by more than 90%, as determined by a cell adhesion assay; and (j) has an EC of less than about 9 nM, as determined by a luciferase reporter assay. 50 and (k) an EC50 of less than about 1.2 nM as determined by a fluorescent assay. 50 and binds to activated CD4+ and CD8+ T cells.

[0109] In some embodiments, the antibodies and antigen-binding fragments thereof have a dissociation half-life (t ) of greater than about 1.6 minutes at 25° C. or 37° C. as measured by surface plasmon resonance, such as using the assay format set forth in Example 3 of PCT / US16 / 56156 or a substantially similar assay. 1 / 2 In certain embodiments, the antibody or antigen-binding fragment may bind to LAG3 with a half-life (t) of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1100 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., such as using an assay format set forth in Example 3 of PCT / US16 / 56156 (e.g., mAb capture or antigen capture format), or a substantially similar assay.1 / 2 ) binds to LAG3.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 8 nM as measured by the flow cytometry assay set forth in Example 5 of PCT / US16 / 56156, or a substantially similar assay. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by a flow cytometry assay, such as using the assay format of Example 5 of PCT / US16 / 56156 or a substantially similar assay. 50 and binds to hLAG3-expressing cells.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 2.5 nM as measured by the flow cytometry assay set forth in Example 5 of PCT / US16 / 56156, or a substantially similar assay. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 2 nM, or less than about 1 nM, as measured by a flow cytometry assay, such as using the assay format of Example 5 of PCT / US16 / 56156 or a substantially similar assay. 50 and binds to mfLAG3-expressing cells.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof has an IC of less than about 32 nM as determined using a cell adhesion assay, such as that set forth in Example 7 of PCT / US16 / 56156 or a substantially similar assay. 50and blocks LAG3 binding to MHC class II (e.g., human HLA-DR2). In certain embodiments, the antibody or antigen-binding fragment thereof has an IC of less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by a cell adhesion assay, such as using the assay format set forth in Example 7 of PCT / US16 / 56156 or a substantially similar assay. 50 blocks LAG3 binding to human MHC class II.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof has an IC of less than about 30 nM as determined using a cell adhesion assay, such as that set forth in Example 7 of PCT / US16 / 56156 or a substantially similar assay. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an IC of less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by a cell adhesion assay, such as using the assay format set forth in Example 7 of PCT / US16 / 56156 or a substantially similar assay. 50 blocks mouse LAG3 binding to human MHC class II.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof blocks the binding of LAG3 to human or mouse MHC class II by greater than 90% as measured by the cell adhesion assay defined in Example 7 of PCT / US16 / 56156 or a substantially similar assay.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than 9 nM as measured by a T cell / APC luciferase reporter assay as defined in Example 8 of PCT / US16 / 56156, or a substantially similar assay. 50In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a T cell / APC luciferase reporter assay, such as using the assay format set forth in Example 8 of PCT / US16 / 56156 or a substantially similar assay. 50 and inhibits LAG3-induced T cell downregulation.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 1.2 nM as measured by the fluorescent assay set forth in Example 9 of PCT / US16 / 56156 or a substantially similar assay. 50 and binds to activated cynomolgus monkey CD4+ and CD8+ T cells. In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 1.1 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by a fluorescence assay, such as using the assay format set forth in Example 9 of PCT / US16 / 56156 or a substantially similar assay. 50 Binds to activated cynomolgus monkey CD4+ and CD8+ T cells.

[0117] In one embodiment, the antibody or fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that binds to LAG3, and the antibody or fragment thereof exhibits one or more of the following characteristics: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554, or at least 90%, at least 95%, at least 98%, or at least 99% of the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554, or at least 90%, at least 95%, at least 98%, or at least 99% of (ii) comprises an HCVR having an amino acid sequence of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562, or an amino acid sequence of a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence homology;(iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544, and 560, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552, and 568, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540, and 556, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an HCDR2 domain having an amino acid sequence similar to that of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548, and 564, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 526, 534, 550, and 566, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(v) a binding-dissociation equilibrium constant ( K ) of less than about 10 nM as measured by surface plasmon resonance assay at 25°C; D ) and binds to monomeric human LAG3; (vi) a K of less than about 8 nM as measured by surface plasmon resonance assay at 37°C. D (vii) a K of less than about 1.1 nM as measured by a surface plasmon resonance assay at 25°C. D (viii) a K of less than about 1 nM as measured by surface plasmon resonance assay at 37°C. D and binds to dimeric human LAG3; (ix) an EC50 of less than about 8 nM as measured by flow cytometry assay. 50 (x) an EC50 of less than about 2.3 nM as measured by flow cytometry assay; 50 (xi) an IC of less than about 32 nM as determined by a cell adhesion assay; 50 (xii) an IC of less than about 30 nM as determined by a cell adhesion assay; 50 (xiii) blocks hLAG3 binding to MHC class II by greater than 90%, as determined by a cell adhesion assay; (xiv) has an EC of less than about 9 nM, as determined by a luciferase reporter assay. 50 (xv) an EC50 of less than about 1.2 nM as determined by a fluorescent assay; 50 and binds to activated CD4+ and CD8+ T cells; and (xvi) inhibits tumor growth and increases survival in subjects with cancer.

[0118] In one embodiment, the antibody or fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that blocks LAG3 binding to MHC class II, and the antibody or fragment thereof exhibits one or more of the following characteristics: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554, or at least 90%, at least 95%, (ii) comprising an HCVR having an amino acid sequence of at least 98%, or at least 99%, a substantially similar sequence thereof with sequence homology; (iii) comprising an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence homology;(iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544, and 560, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552, and 568, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540, and 556, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an HCDR2 domain having an amino acid sequence similar to that of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548, and 564, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 526, 534, 550, and 566, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(v) a binding-dissociation equilibrium constant ( K ) of less than about 10 nM as measured by surface plasmon resonance assay at 25°C; D ) and binds to monomeric human LAG3; (vi) a K of less than about 8 nM as measured by surface plasmon resonance assay at 37°C. D (vii) a K of less than about 1.1 nM as measured by a surface plasmon resonance assay at 25°C. D (viii) a K of less than about 1 nM as measured by surface plasmon resonance assay at 37°C. D and binds to dimeric human LAG3; (ix) an EC50 of less than about 8 nM as measured by flow cytometry assay. 50 (x) an EC50 of less than about 2.3 nM as measured by flow cytometry assay; 50 (xi) an IC of less than about 32 nM as determined by a cell adhesion assay; 50 (xii) an IC of less than about 30 nM as determined by a cell adhesion assay; 50 (xiii) blocks hLAG3 binding to MHC class II by greater than 90%, as determined by a cell adhesion assay; (xiv) has an EC of less than about 9 nM, as determined by a luciferase reporter assay. 50 (xv) an EC50 of less than about 1.2 nM as determined by a fluorescent assay; 50 and binds to activated CD4+ and CD8+ T cells; and (xvi) inhibits tumor growth and increases survival in subjects with cancer.

[0119] In certain embodiments, the antibody functions to inhibit or suppress MHC class II binding activity associated with LAG3 by binding to other regions or fragments of the full-length protein having the amino acid sequence set forth in SEQ ID NO:582.

[0120] In certain embodiments, the antibody is bispecific. A bispecific antibody can bind to one epitope in one domain and also to a second epitope in a different domain of LAG3. In certain embodiments, a bispecific antibody binds to two different epitopes within the same domain. In one embodiment, a multispecific antigen-binding molecule comprises: a first antibody binding specificity, where the first binding specificity comprises the extracellular domain of LAG3 or a fragment thereof; and a second antigen binding specificity to another epitope of LAG3.

[0121] In certain embodiments, the anti-LAG3 antibody or antigen-binding fragment thereof binds to an epitope within one or more regions of LAG3, either native as exemplified by SEQ ID NO:582 or recombinantly produced as exemplified by SEQ ID NOs:574-576, or to a fragment thereof. In some embodiments, the antibody binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 29-450 of LAG3. In some embodiments, the antibody binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 1-533 of cynomolgus monkey LAG3, as exemplified by SEQ ID NO:576.

[0122] In certain embodiments, anti-LAG3 antibodies and antigen-binding fragments thereof interact with one or more epitopes present within the extracellular region of LAG3 (SEQ ID NO: 588). The epitope(s) may consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within the extracellular region of LAG3. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) in the extracellular region of LAG3. The epitope of LAG3 that the exemplary antibody H4sH15482P interacts with is defined by the amino acid sequence LRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 589), which corresponds to amino acids 28-71 of SEQ ID NO: 588. Therefore, anti-LAG3 antibodies that interact with one or more amino acids contained within the region consisting of amino acids 28 to 71 of SEQ ID NO: 588 (i.e., the sequence "LRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY" shown in SEQ ID NO: 589) are also included.

[0123] The present disclosure includes anti-LAG3 antibodies that bind to the same epitope or a portion of an epitope as any of the specific exemplary antibodies set forth in Table 1 or an antibody having the CDR sequences of any of the exemplary antibodies set forth in Table 1. Also included are anti-LAG3 antibodies that compete with any of the specific exemplary antibodies set forth in Table 1, or that compete with an antibody having the CDR sequences of any of the exemplary antibodies set forth in Table 1, for binding to LAG3 or a LAG3 fragment. For example, the present disclosure includes anti-LAG3 antibodies that cross-compete for binding to LAG3 with one or more antibodies provided herein (e.g., H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15479P, H4H15482P, H4H15483P, H4sH15498P, H4H15498P, H4H17828P2, H4H17819P, and H4H17823P).

[0124] The antibodies and antigen-binding fragments described herein specifically bind to LAG3 and modulate the interaction of LAG3 with MHC class II. Anti-LAG3 antibodies can bind to LAG3 with high or low affinity. In certain embodiments, the antibodies are blocking antibodies that bind to LAG3 and block the interaction of LAG3 with MHC class II. In some embodiments, the blocking antibodies of the present disclosure block the binding of LAG3 to MHC class II and / or stimulate or enhance T cell activation. In some embodiments, the blocking antibodies are useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer or chronic viral infections. When administered to a subject in need thereof, the antibodies can reduce chronic infections with viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV) in the subject. These can be used to inhibit the growth of tumor cells in a subject. They can also be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating cancer or viral infections. In certain embodiments, anti-LAG3 antibodies that bind to LAG3 with low affinity are used as multispecific antigen-binding molecules, in which a first binding specificity binds to LAG3 with low affinity and a second binding specificity binds to an antigen selected from the group consisting of a different epitope of LAG3 and another T-cell co-inhibitory factor.

[0125] In some embodiments, the antibody binds to LAG3 and reverses the anergic state of exhausted T cells. In certain embodiments, the antibody binds to LAG3 and inhibits regulatory T cell activity. In some embodiments, the antibody may be useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer, viral infections, bacterial infections, fungal infections, or parasitic infections. When administered to a subject in need thereof, the antibody may reduce chronic infection with viruses such as HIV, LCMV, or HBV in the subject. They may be used to inhibit the growth of tumor cells in a subject. They may be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating cancer or viral infections.

[0126] In certain embodiments, the antibodies of the present disclosure are agonistic antibodies that bind to LAG3 and enhance the interaction of LAG3 with MHC class II. In some embodiments, activating antibodies enhance the binding of LAG3 to MHC class II and / or inhibit or suppress T cell activation. Activating antibodies of the present disclosure may be useful for inhibiting an immune response and / or treating an autoimmune disease in a subject.

[0127] Certain anti-LAG3 antibodies can bind to and neutralize the activity of LAG3, as determined by in vitro or in vivo assays. The ability of an antibody to bind to and neutralize the activity of LAG3 can be measured using any standard method known to those skilled in the art, including the binding or activity assays described herein.

[0128] Non-limiting, exemplary in vitro assays for measuring binding activity are shown in the examples provided in PCT / US16 / 56156. In Example 3, the binding affinity and kinetic constants of human anti-LAG3 antibodies were determined by surface plasmon resonance, with measurements performed on a Biacore 4000 or T200 instrument. In Example 4, a blocking assay was used to determine cross-competition between anti-LAG3 antibodies. Examples 5 and 6 describe antibody binding to cells overexpressing LAG3. In Example 7, a binding assay was used to determine the ability of anti-LAG3 antibodies to block the MHC class II-binding ability of LAG3 in vitro. In Example 8, a luciferase assay was used to determine the ability of anti-LAG3 antibodies to neutralize LAG3 in T cells. In Example 9, a fluorescence assay was used to determine the ability of anti-LAG3 antibodies to bind to activated monkey CD4+ and CD8+ T cells.

[0129] Unless otherwise specifically stated, the term "antibody," as used herein, is understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule"), as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody, or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to LAG3. An antibody fragment may include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide of a multispecific antigen-binding molecule or a fragment thereof. Antigen-binding fragments of antibodies may be obtained from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0130] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0131] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may also include a domain.

[0132] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a VH -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H or V LIt may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (e.g., via disulfide bonds) of the domains.

[0133] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0134] The anti-LAG3 antibodies and antibody fragments of the present disclosure include proteins with amino acid sequences that differ from those of the described antibodies but retain the ability to bind to LAG3. Such mutant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially the same biological activity as the described antibodies. Similarly, the antibody-encoding DNA sequences of the present disclosure encompass sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but that are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0135] Two antigen-binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered to be equivalents or pharmaceutical substitutes if the extent of absorption is comparable but the rate of absorption is not, but the antibodies can be considered bioequivalent because the difference in absorption rate is intentional, reflected in the labeling, and is not considered medically significant for the particular pharmaceutical agent being tested, for example.

[0136] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0137] In one embodiment, two antigen binding proteins are bioequivalent if there is no expected increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy, when a patient is switched between the reference product and the biological product one or more times compared to continuous therapy without such switching.

[0138] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms and to the known extent of such mechanisms for one or more conditions of use.

[0139] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluid; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0140] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody (e.g., mutations that eliminate or remove glycosylation).

[0141] According to certain embodiments of the present disclosure, the anti-LAG3 antibody comprises one or more mutations in the Fc domain that enhance or weaken antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides a method for identifying a C H 2 or C HThe present invention also includes anti-LAG3 antibodies containing mutations in the FcRn region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can result in an extended serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications may include, for example, a modification at position 250 (e.g., E or Q); a modification at positions 250 and 428 (e.g., L or F); a modification at positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or a modification at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at positions 250 and / or 428; or a modification at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L (e.g., T250Q and M428L) modifications; and 307 and / or 308 (e.g., 308F and / or 308P) modifications. In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0142] For example, the disclosure includes anti-LAG3 antibodies comprising an Fc domain, wherein the Fc domain comprises one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N428L); 34S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes an anti-LAG3 antibody comprising an Fc domain containing the S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0143] The present disclosure provides chimeric heavy chain constant (C H ) region, and H The region may contain C from more than one immunoglobulin isotype. H For example, the antibodies of the present disclosure may comprise fragments of C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with some or all of the three domains. H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the present disclosure comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge, and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein H Antibodies comprising the region, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Patent Publication No. 20140243504, the disclosure of which is hereby incorporated by reference in its entirety.) In certain embodiments, the Fc region comprises a sequence selected from the group consisting of SEQ ID NOs: 569, 570, 571, 572, and 573.

[0144] B. Positron Emitter and Chelating Moiety Suitable positron emitters include, but are not limited to, those that form stable complexes with chelating moieties and have suitable physical half-lives for the purposes of immunoPET imaging. Exemplary positron emitters include: 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Suitable positron emitters also include, but are not limited to, Y. 76 Br and 124 I, as well as prosthetic groups, e.g., 18 These include, but are not limited to, those introduced by F, including those that directly bind to LAG3 binding proteins.

[0145] The chelating moiety described herein is a chemical moiety covalently attached to a LAG3 binding protein, e.g., an anti-LAG3 antibody, that can chelate with a positron emitter, i.e., can react with a positron emitter to form a coordinate chelate complex. Suitable moieties include those that allow efficient loading of specific metals and form metal chelator complexes that are sufficiently stable for in vivo diagnostic applications, such as immunoPET imaging. Exemplary chelating moieties include those that minimize positron emitter dissociation and accumulation in bone mineral, plasma proteins, and / or bone marrow deposits to an extent suitable for diagnostic applications.

[0146] Examples of chelating moieties include positron emitters 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Examples include, but are not limited to, those that form a stable complex with Y. Exemplary chelating moieties include those described in Nature Protocols, 5(4): 739, 2010; Bioconjugate Chem., 26(12): 2579 (2015); Chem Commun (Camb), 51(12): 2301 (2015); Mol. Pharmaceutics, 12: 2142 (2015); Mol. Imaging Biol., 18: 344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007 / s00259-016-3499-x; Bioconjμgate Chem., 26(12): 2579 (2015); WO2015 / 140212A1; and U.S. Patent No. 5,639,879.

[0147] Exemplary chelating moieties include desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α''α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H4 octapa, H6 phospa, H2 dedopa, H5 decapa, H2 azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoyl) methyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cyclen), 1,4,8,11-tetraazacyclotetradecane (Cyclam), octadentate chelators, octanodentate bifunctional chelators, such as DFO* (which can be conjugated to antibodies via DFO*-pPhe-NCS) (Vugt et al., Eur J Nucl Med Mol Imaging (2017) 44:286-295), hexadentate chelators, phosphonate-based chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinine C and fusarinine C derivative chelators, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), and the like.

[0148] In some embodiments, a chelating moiety is covalently attached to a LAG3 binding protein, e.g., an antibody or antigen-binding fragment thereof, via a linker moiety that covalently attaches the chelating portion of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed from the reaction between a reactive moiety on the LAG3 binding protein, e.g., a cysteine ​​or lysine on the antibody, and a reactive moiety attached to a chelator, including, for example, a p-isothiocyanatobenyl group and the reactive moieties provided in the conjugation methods below. In addition, such linker moieties optionally contain chemical groups used to adjust the polarity, solubility properties, steric interactions, rigidity, and / or length between the chelating moiety and the LAG3 binding protein.

[0149] C. Preparation of Radiolabeled Anti-LAG3 Conjugate Radiolabeled anti-LAG3 protein conjugates can be prepared by (1) reacting a LAG3 binding protein, e.g., an antibody, with a molecule comprising a positron emitter chelator and a moiety reactive to the desired conjugation site on the LAG3 binding protein, and (2) adding the desired positron emitter.

[0150] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which can be naturally occurring or engineered, for example, present on the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, those resulting from mutation, insertion, or reduction of antibody disulfide bonds. Methods for producing cysteine-engineered antibodies include, but are not limited to, those disclosed in International Publication No. 2011 / 056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on an antibody, to achieve a desired stoichiometry, and / or to achieve a desired chelator-to-antibody ratio. Such conjugation methods are known to those skilled in the art and include, but are not limited to, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, as well as those described in J. Clin. Immunol., 36:100 (2016), the entire contents of which are incorporated herein by reference. A suitable moiety reactive with the desired conjugation site generally allows for efficient and easy conjugation of the LAG3 binding protein, e.g., an antibody, with the positron emitter chelator. Moieties reactive with lysine and cysteine ​​sites include electrophilic groups known to those skilled in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, e.g., a p-isothiocyanatobenyl group, or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0151] When the chelator is desferrioxamine (DFO), suitable reactive moieties include, but are not limited to, isothiocyanatobenzyl groups, n-hydroxysuccinimide esters, 2,3,5,6 tetrafluorophenol esters, n-succinimidyl-S-acetylthioacetate, and those described in BioMed Research International, Vol 2014, Article ID 203601, which is incorporated herein by reference in its entirety. In certain embodiments, the LAG3 binding protein is an antibody, and the molecule comprising the positron emitter chelator and a moiety reactive with the conjugation site is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO): The file is TIFF2026506001000002.tif57128.

[0152] Addition of the positron emitter is accomplished by incubating the LAG3 binding protein chelator conjugate with the positron emitter for a time sufficient to allow coordination of the positron emitter to the chelator, for example, by performing the methods described in the Examples provided herein, or substantially similar methods.

[0153] D. Exemplary Embodiments of Conjugates The present disclosure includes a radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to human LAG3 and a positron emitter. The present disclosure also includes a radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to human LAG3, a chelating moiety, and a positron emitter.

[0154] In some embodiments, the chelating moiety is 89 The chelating moiety comprises a chelator capable of complexing with Zr. In certain embodiments, the chelating moiety comprises desferrioxamine. In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine.

[0155] In some embodiments, the positron emitter is 89 In some embodiments, less than 1.0% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.9% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.8% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.7% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.6% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.5% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.4% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.3% of the anti-LAG3 antibodies are conjugated to a positron emitter, less than 0.2% of the anti-LAG3 antibodies are conjugated to a positron emitter, or less than 0.1% of the anti-LAG3 antibodies are conjugated to a positron emitter.

[0156] In some embodiments, the conjugate has a chelating moiety to antibody ratio of 1-2.

[0157] In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine and the positron emitter is 89 In another particular embodiment, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine and the positron emitter is 89 Zr, and the chelating moiety to antibody ratio of the conjugate is 1-2.

[0158] In some embodiments, provided herein is an antigen binding protein that binds to LAG3, wherein said antigen binding protein that binds to LAG3 has the following structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently 0 or 1 in each occurrence, and at least one of the z's is 1. In certain embodiments, the radiolabeled antigen binding protein is covalently linked to one or more moieties having the formula (I): MLA-[LM Z ] k (I) wherein A is a protein that binds to LAG3, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1.

[0159] In some embodiments, L is: The file is TIFF2026506001000003.tif56128.

[0160] In some embodiments, M is 89 It is Zr.

[0161] In some embodiments, k is an integer from 1 to 2. In some embodiments, k is 1.

[0162] In some embodiments, -LM is: The file is TIFF2026506001000004.tif59128.

[0163] Also, the compound of formula (III): TIFF2026506001000005.tif70128, 89 Also included in the disclosure is a method for synthesizing a radiolabeled antibody conjugate, comprising contacting A with Zr, wherein A is an antibody or antigen-binding fragment thereof that binds to LAG3. In certain embodiments, the compound of formula (III) is synthesized by contacting an antibody or antigen-binding fragment thereof that binds to LAG3 with p-SCN-Bn-DFO.

[0164] a compound of formula (III) 89 The product of the reaction between Zr is also provided herein.

[0165] Compounds of formula (III): TIFF2026506001000006.tif66128 is provided herein, wherein A is an antibody or antigen-binding fragment thereof that binds to LAG3, and k is an integer between 1 and 30. In some embodiments, k is 1 or 2.

[0166] In some embodiments, the following structure: AL k Provided herein are compositions comprising a conjugate having the formula: wherein A is a protein that binds to LAG3, L is a chelating moiety, and k is an integer between 1 and 30, wherein the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging. In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity of about 1 to about 20 mCi per 1 to 100 mg (e.g., 1 to 50 mg) of protein that binds to LAG3. In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity of 20 mCi or less, 15 mCi or less, or 10 mCi or less, e.g., about 3 to about 20 mCi, about 5 to about 20 mCi, about 1 to about 15 mCi, about 3 to about 15 mCi, about 5 to about 15 mCi, about 1 to about 10 mCi, or about 3 to about 10 mCi, per 1 to 100 mg (e.g., 1 to 50 mg) of protein that binds to LAG3.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof has a binding dissociation equilibrium constant (K) of less than about 2 nM at 37° C. as measured by a surface plasmon resonance assay. D ) and binds to monomeric human LAG3.

[0168] In some embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 1.5 nM in a surface plasmon resonance assay at 25° C. D and binds to monomeric human LAG3.

[0169] In some embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 90 pM at 37° C. as measured by a surface plasmon resonance assay.D and binds to dimeric human LAG3.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 20 pM in a surface plasmon resonance assay at 25° C. D and binds to dimeric human LAG3.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human LAG3 with a reference antibody comprising a complementarity-determining region (CDR) of an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1 and a CDR of an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences shown in Table 1. In some embodiments, the reference antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair set forth in Table 1. In some embodiments, the reference antibody is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 342 / 346, 350 / 360, 362 / 370, 374 / 382, 382 / 390, 394 / 402, 406 / 414, 416 / 420, 422 / 430, 432 / 440, 442 / 450, 458 / 466, 474 / 482, 490 / 498, 506 / 514, 522 / 530, 542 / 550, 562 / 570, 582 / 590, 594 / 602, 606 / 614, 616 / 620, 622 / 630, 632 / 640, 642 / 650, 652 / 666, 662 / 670, 674 / 682, 682 / 690, 706 / 714, 722 / 730, 742 / 750, 762 / 770, 782 / 790, 794 / 802, and HCVR / LCVR amino acid sequence pairs selected from the group consisting of 38 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof enhances binding of LAG3 to MHC class II. In some embodiments, the antibody or antigen-binding fragment thereof inhibits binding of LAG3 to MHC class II. In some embodiments, the antibody or antigen-binding fragment thereof does not increase or decrease LAG3 binding to its ligand.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554. and a CDR of an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562. In certain embodiments, the isolated antibody is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330 , 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562. In certain embodiments, the isolated antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386 / 394, 418 / 426, 538 / 546, 577 / 578, 579 / 578, and 580 / 581.

[0174] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human LAG3, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1.

[0175] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human LAG3, and the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0176] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human LAG3, and the antibody or antigen-binding fragment thereof comprises: (a) a HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and (b) a LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised in any one of the heavy chain variable region (HCVR) sequences listed in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in any one of the light chain variable region (LCVR) sequences shown in Table 1.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540, and 556; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544, and 560; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548, and 564; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 526, 534, 550, and 566; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552, and 568; Includes.

[0179] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 332, 342 / 350, 362 / 370, 382 / 390, 406 / 414, 422 / 432, 442 / 450, 462 / 470, 482 / 490, 490 / 518, 522 / 532, 542 / 550, 562 / 570, 582 / 590, 590 / 602, 606 / 614, 612 / 622, 622 / 632, 632 / 642, 642 / 650, 652 / 662, 652 / 672, 662 / 682, 674 / 682, 682 / 690, 706 / 714, 722 / 732, 742 / 752, 762 / 772, 782 / 792, 790 / 802, 806 / 814, 812 / 822, 822 / 832, 832 / 842, 842 / 8 In some embodiments, the antibody or antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 30, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

[0180] E. Scale-up Manufacturing for Production of Anti-LAG3 Antibody-Chelator Conjugate Included in the present disclosure is a scale-up manufacturing process for producing anti-LAG3 antibodies conjugated to chelators. The anti-LAG3 antibody-chelator conjugates are in a form suitable for radiolabeling.

[0181] Good manufacturing processes are followed in all aspects of production, including maintaining a sterile environment, performing aseptic procedures, keeping records of all processes, and documenting product quality, purity, strength, and identity, and any deviations therefrom.

[0182] The scaled-up manufacturing process, in some embodiments, is much faster than the research and development manufacturing process, hi some embodiments, the scaled-up manufacturing process may take less than 12 hours, or less than 10 hours, or less than 8 hours, or less than 6 hours, or less than 4 hours, or less than about 2 hours.

[0183] In some embodiments, the first step involves ultrafiltration and diafiltration (UFDF) of the anti-LAG3 antibody using a 30-50 kDa membrane to remove excipients, conjugation-interfering species, and salts that inhibit the conjugation process. Exemplary membrane polymers include polyethersulfone (PES), cellulose acetate (CA), and regenerated cellulose (RC). In this step, the antibody is buffer-exchanged into a low ionic strength, non-interfering buffer. The buffer pH can be about 4.5 to about 6, or about 5 to about 6, or about 5.3 to about 5.7, or about 5.5. Buffer systems contemplated herein include any buffer system lacking primary amines. Exemplary buffers include acetate, phosphate, or citrate buffers. The buffer provides protein stability during pre-conjugation treatment. The process volume can be further reduced to concentrate the antibody, which can then be sterile-filtered.

[0184] After UFDF prior to conjugation, the concentrated and filtered antibody can be transferred to an amine-free carbonate buffer system, which can have a pH in the range of about 8.5 to about 9.6, or about 9.0 to about 9.6, or about 9.2 to about 9.4, or about 9.4 to about 9.6, or a pH of about 9.4.

[0185] A chelator in a solvent, such as DFO, can be added to a buffer system containing the antibody to a target concentration, and additional solvent can be added to the solution to the desired ratio. The chelator can be added in molar excess to the antibody, e.g., 3.5-5:1 chelator to antibody. The total reaction volume can be up to 5 L.

[0186] The reaction temperature and reaction time are inversely proportional. For example, if the reaction temperature is high, the reaction time is shorter. If the reaction temperature is low, the reaction time is longer. Illustratively, at temperatures above about 18°C, the reaction may take less than 2 hours, and at temperatures below 18°C, the reaction may take more than 2 hours.

[0187] The conjugation reaction can be terminated by quenching it with, for example, the addition of acetic acid.

[0188] In some embodiments, conjugation of the antibody with deferoxamine is performed to produce a DFO-mAb conjugate. In some embodiments, conjugation of the antibody with p-SCN-Bn-deferoxamine is performed to produce a DFO-mAb conjugate.

[0189] Exemplary solvents for chelators include DMSO and DMA. The subsequent UFDF process utilizes membranes, which are selected based on the solvent system used in the conjugation process. For example, DMA dissolves PES membranes, so the two membranes could not be used in the same system.

[0190] Carbonate buffers are not favorable for the stability of conjugates during long-term storage. Therefore, once the antibody-chelator conjugates are formed, they can be buffer-exchanged into a buffer specifically selected for long-term storage and stability. Exemplary buffers include citrate, acetate, phosphate, arginine, and histidine buffers. An additional UFDF step can be performed to remove residual salts and provide the appropriate concentration, excipient level, and pH of the conjugated monoclonal antibody. The resulting antibody-chelator conjugate can be sterile-filtered and stored for subsequent formulation.

[0191] III. Methods Using Radiolabeled Immunoconjugates Immune checkpoint inhibitors can induce durable responses in multiple tumor types. Lymphocyte activation gene 3 (LAG3) is one of the immune checkpoints for which therapeutic antibodies are being developed. Information on the biodistribution of these antibodies remains limited. The radiolabeled LAG3 antibodies described herein (e.g., 89Zr-DFO-REGN3767, also referred to herein as a conjugated and radiolabeled anti-LAG3 antibody, H4sH15482P, or conjugated and radiolabeled mAb1, having the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 418 / 426, has been determined to be useful for PET imaging of specific tumors at specific tracer protein doses and specific imaging time points in patients with cancer.

[0192] The field of cancer immunotherapy has shown rapid development over the past decade. Monoclonal antibody-based therapies targeting programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) have solidified their place in the treatment of multiple different tumor types using checkpoint blockade (Ribas and Wolchok, Cancer Immunotherapy Science. 2018;359(6382):1350-5). Furthermore, multiple inhibitory or stimulatory receptors have been identified for which therapeutic antibodies have been developed.

[0193] One inhibitory receptor is lymphocyte-activation gene-3 (LAG3). As mentioned above, LAG3 is a transmembrane protein whose extracellular domain is primarily expressed on activated T cells, B cells, and natural killer cells and is closely related to the CD4 coreceptor, which binds to the major histocompatibility complex class II (MHC-II) on antigen-presenting cells (Lythgoe et al., Gene of the month: lymphocyte-activation gene 3 (LAG3). J Clin Pathol. 2021;74(9):543-7). However, the exact mechanism by which LAG3 exerts its inhibitory effect is not yet fully understood (Chocarro et al., Understanding LAG3 signaling. Int J Mol Sci. 2021;22(10):5282).

[0194] Relatlimab, the first anti-LAG3 antibody, was approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in combination with nivolumab for the treatment of patients with metastatic melanoma (Tawbi et al., Relatlimab and nivolumab vs nivolumab in untreated advanced melanoma. N Engl J Med. 2022;386(1):24-34). The combination of relatlimab and nivolumab resulted in increased efficacy compared with nivolumab monotherapy. Furthermore, relatlimab and nivolumab showed a more favorable safety profile than the combination of ipilimumab and nivolumab (Tawbi et al., Relatlimab and nivolumab vs nivolumab in untreated advanced melanoma. N Engl J Med. 2022;386(1):24-34).

[0195] Molecular imaging can predict response to cancer immunotherapy (Bensch et al., 89 Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer. Nat Med.2018;24(12):1852-8, Niemeijer et al., Whole body PD-1 and PD-L1 positron emission tomography in patients with non-small-cell lung cancer. Nat Commun.2018;9(1):4664, Kok et al., 89 Zr-pembrolizumab imaging as a non-invasive approach to assess clinical response to assessment of PD-1 blockade in cancer. Ann Oncol. 2022;33(1):80-8), providing information on immune cell distribution in healthy tissue.

[0196] In certain aspects, the present disclosure provides diagnostic and therapeutic methods using the radiolabeled antibody conjugates of the present disclosure.In various embodiments of diagnostic and therapeutic methods, the administration of radiolabeled anti-LAG3 antibody conjugates can include the radiolabeled conjugates administered as part of a mixture that also contains unlabeled anti-LAG3 antibodies to constitute a total dose.It is also understood that in various embodiments of diagnostic and therapeutic methods described herein, visualization of LAG3 expression can be achieved by positron emission tomography (PET) imaging, alone or in combination with other known imaging techniques, including but not limited to, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans, etc.

[0197] According to one aspect, the present disclosure provides a method for detecting LAG3 in a tissue, the method comprising administering a radiolabeled anti-LAG3 antibody conjugate provided herein to the tissue and visualizing LAG3 expression by positron emission tomography (PET). In certain embodiments, the tissue comprises a cell or cell line. In certain embodiments, the tissue is present in a subject, and the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject has a disease or disorder associated with T cell activation, for example, selected from the group consisting of cancer, infectious disease, and inflammatory disease. In one embodiment, the subject has cancer. Various non-limiting examples of cancer are described elsewhere herein. In certain embodiments, the infection is caused by, for example, Rickettsia bacteria, Bacillus, Klebsiella, meningococci and gonococci, Proteus, pneumonococci, Pseudomonas, Streptococci, Staphylococci, Serratia, Borrelia, Bacillus anthricis, Chlamydia, Clostridium, Corynebacterium diphtheriae, Legionella, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium lepromatosis, Mycobacterium leprosy ... It is a bacterial infection caused by Salmonella lepromatosis, Salmonella, Vibrio cholerae, and Yersinia pestis.In certain embodiments, the infectious disease is a viral infection caused by, for example, human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), herpesvirus (e.g., VZV, HSV-I, HAV-6, HSV-II, CMV, and Epstein-Barr virus), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV). In certain embodiments, the infection is a parasitic infection caused by, for example, Entamoeba spp., Enterobius vermicularis, Leishmania spp., Toxocara spp., Plasmodium spp., Schistosoma spp., Taenia solium, Toxoplasma gondii, and Trypanosoma cruzi. In certain embodiments, the infection is caused by, for example, Aspergillus (such as fumigatus and niger), Blastomyces dermatitidis, Candida (such as albicans, krusei, glabrata, and tropicalis), Coccidioides immitis, Cryptococcus neoformans, Genus Mucorales (such as mucor, absidia, and rhizopus), Histoplasma capsulatum, and Paracoccidioides brasiliensis. It is a fungal infection caused by Bacillus brasiliensis and Sporothrix schenkii.

[0198] According to one aspect, the present disclosure provides a method for imaging a tissue expressing LAG3, the method comprising administering a radiolabeled anti-LAG3 antibody conjugate of the present disclosure to the tissue and visualizing LAG3 expression by positron emission tomography (PET). In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained within a tumor cell culture or tumor cell line. In one embodiment, the tissue is contained within a tumor lesion in a subject. In one embodiment, the tissue is an intratumoral lymphocyte within the tissue. In one embodiment, the tissue comprises LAG3-expressing cells.

[0199] According to one aspect, the present disclosure provides a method for measuring response to therapy, wherein the response to therapy is measured by measuring inflammation. The method according to this aspect includes administering a radiolabeled antibody conjugate provided herein to a subject in need thereof and visualizing LAG3 expression by positron emission tomography (PET). In certain embodiments, the inflammation is present in a tumor in the subject. In certain embodiments, increased LAG3 expression correlates with increased inflammation in the tumor. In certain embodiments, the inflammation is present in infected tissue in the subject. In certain embodiments, decreased LAG3 expression correlates with decreased inflammation in the infected tissue. In certain embodiments, decreased LAG3 expression correlates with decreased inflammation in tissue associated with decreased tumor mass.

[0200] According to one aspect, the present disclosure provides a method for measuring response to therapy, wherein the response to therapy is measured by measuring inflammation. The method according to this aspect includes (i) administering a radiolabeled antibody conjugate provided herein to a subject in need thereof and visualizing LAG3 expression by positron emission tomography (PET), and (ii) repeating step (i) one or more times after initiation of therapy. In certain embodiments, inflammation is present in tissue in the subject. In certain embodiments, increased LAG3 expression correlates with increased inflammation in the tissue. In certain embodiments, decreased LAG3 expression correlates with decreased inflammation in the tissue. In certain embodiments, the LAG3 expression visualized in (i) is compared to the LAG3 expression visualized in (ii).

[0201] According to one aspect, the present invention provides a method for determining whether a patient is suitable for anti-tumor therapy comprising a LAG3 inhibitor, the method comprising the steps of selecting a patient having a tumor, e.g., a patient having a solid tumor, administering a radiolabeled antibody conjugate of the present invention, and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anti-tumor therapy comprising a LAG3 inhibitor.

[0202] According to one aspect, the present invention provides a method for identifying candidates for anti-tumor therapy comprising a LAG3 inhibitor and an inhibitor of the PD-1 / PD-L1 signaling axis, the method comprising selecting a patient having a tumor, e.g., a solid tumor, administering a radiolabeled antibody conjugate of the present invention, and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anti-tumor therapy comprising a LAG3 inhibitor. In some embodiments, the patient is further administered a radiolabeled anti-PD-1 conjugate, and the administered radiolabeled anti-PD-1 conjugate is localized within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the patient as suitable for anti-tumor therapy comprising an inhibitor of the PD-1 / PD-L1 signaling axis.

[0203] Also included herein is a method for predicting a patient's response to anti-tumor therapy, the method comprising selecting a patient having a tumor, e.g., a solid tumor, and determining whether the tumor is LAG3-positive, wherein if the tumor is LAG3-positive, it predicts a positive response of the patient to the anti-tumor therapy. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure; and localizing the radiolabeled anti-LAG3 antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive.

[0204] In some embodiments, the anti-tumor therapy includes a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Publication No. US2015-0203580), a CTLA-4 inhibitor (e.g., ipilimumab, ), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of another T cell co-inhibitory factor or ligand (e.g., CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Patent No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors against VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens (e.g., CA9, MUC16, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guerin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), cytotoxic agents, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC).

[0205] In some embodiments, the anti-tumor therapy is selected from cemiplimab, nivolumab, ipilimumab, pembrolizumab, and combinations thereof.

[0206] According to one aspect, the present invention provides a method for predicting a patient's response to an anti-tumor therapy comprising a LAG3 inhibitor, the method comprising: selecting a patient having a tumor, e.g., a solid tumor; and determining whether the tumor is LAG3-positive, wherein if the tumor is LAG3-positive, a positive response of the patient is predicted. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure; and localizing the radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive.

[0207] According to one aspect, the present disclosure provides a method for predicting a patient's response to an anti-tumor therapy comprising a LAG3 inhibitor in combination with an inhibitor of the PD-1 / PD-L1 signaling axis, the method comprising: selecting a patient having a tumor, e.g., a solid tumor; and determining whether the tumor is LAG3-positive and PD-1-positive, wherein a positive response of the patient is predicted if the tumor is LAG3-positive and PD-1-positive. In a specific embodiment, the tumor is determined to be LAG3-positive by administering a radiolabeled anti-LAG3 conjugate; and localizing the radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive. In certain embodiments, the tumor is determined to be PD-1 positive by further administering a radiolabeled anti-PD-1 conjugate; and localizing the radiolabeled anti-PD-1 conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate within the tumor indicates that the tumor is PD-1 positive.

[0208] According to one aspect, the present disclosure provides a method for detecting a LAG3-positive tumor in a subject. The method according to this aspect includes selecting a subject having a tumor, e.g., a solid tumor, administering to the subject a radiolabeled antibody conjugate of the present disclosure, and determining localization of the radiolabeled antibody conjugate by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive.

[0209] According to one aspect, the present disclosure provides a method for imaging a LAG3-positive tumor in a subject. In some aspects, the method includes: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), wherein at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter. 89 and (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography-computed tomography (PET / CT) imaging. In some embodiments, the imaging step (ii) is performed about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 days after step (i). In certain embodiments, the imaging step (ii) is performed 7 days after step (i). In some embodiments, imaging the localization of the labeled antibody conjugate is performed in or near the tumor.

[0210] According to one aspect, the present disclosure provides a method for whole-body imaging of LAG3, the method comprising administering to a subject a radiolabeled anti-LAG3 antibody conjugate described herein and visualizing LAG3 expression by PET imaging. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer, an inflammatory disease, or an infection. Whole-body imaging allows visualization of LAG3-expressing cells (e.g., T cells) and / or changes in LAG3 expression (e.g., upregulation or downregulation) throughout the body.

[0211] According to one aspect, the present disclosure provides a method of treating a subject, comprising: (i) administering to a subject having a tumor an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3), wherein at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter. 89 (ii) imaging the localization of the labeled antibody conjugate within the tumor by positron emission tomography (PET), wherein the imaging in (ii) is performed 7 days after step (i), and the presence of the radiolabeled antibody conjugate in the tumor indicates the presence of LAG3-positive cells within the tumor; and (iii) administering one or more doses of an anti-tumor therapy to a subject in need thereof.

[0212] The anti-tumor therapy administered in step (iii) is administered at least once, but it is contemplated herein that it may be administered several times as needed over time. For example, the anti-tumor therapy can be administered once, twice, three times, four times, or more. In some embodiments, step (iii) is performed once. In some embodiments, step (iii) is performed twice. In some embodiments, step (iii) is performed three times. In some embodiments, step (ii) and step (iii) are performed on different days. In some embodiments, step (ii) and step (iii) are performed on the same day.

[0213] It is further contemplated that the steps of administering (i) and imaging (ii) can be repeated, i.e., performed more than once. In some embodiments, the method of treatment further comprises step (iv), in which steps (i) and (ii) are repeated. In some embodiments, the method of treatment further comprises step (iv), in which steps (i) and (ii) are repeated, with the second performance of step (ii) being performed after step (iii). In some embodiments, the method of treatment further comprises step (iv), in which steps (i) and (ii) are repeated, with the second performance of steps (i) and (ii) being performed after step (iii). In other words, step (iv) can be performed after step (iii) in some embodiments, or the order of steps can be step (i), step (ii), a second time step (i), step (iii), a second time step (ii) in some embodiments. In some embodiments, step (iii) is performed at least once, at least twice, or at least three times before step (iv).

[0214] At any time during treatment, it may be useful to obtain a tumor biopsy. In some embodiments, the method further comprises obtaining a tumor sample from the subject. In certain embodiments, the presence of LAG3, PD-1, or another biomarker in the tumor sample is evaluated. In some embodiments, the method further comprises obtaining a tumor sample from the subject and determining the presence of LAG3 in the tumor sample via techniques known in the art, such as immunohistochemical analysis.

[0215] At any time during treatment, it may be useful to measure tumor response to anti-tumor therapy. Therapy efficacy can be evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST v1.1 or iRECIST), including progression-free survival (PFS), overall survival (OS), and ORR (objective response rate), which is defined as the proportion of patients who achieve the best overall response of CR (complete response) or PR (partial response). (Seymour et al., iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017;18(3):e143-e52; Eisenhauer et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). EJ Cancer 2009;45:228-247). In some embodiments, the method further comprises measuring tumor response to anti-tumor therapy. In certain embodiments, the methods include measuring tumor response by assessing the reduction or disappearance of size and / or number of tumor lesions.

[0216] In some aspects, the antibody or antigen-binding fragment thereof is administered to a subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation, e.g., 0.5 mCi + / - 20% radiation, 1.0 mCi + / - 20% radiation, 2.0 mCi + / - 20% radiation, or 3.0 mCi + / - 20% radiation. In certain embodiments, the label provides about 1.0 mCi of radiation upon injection.

[0217] Thus, a portion of an antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter. 89The remaining portion is "cold" or "unlabeled" antibody or antigen-binding fragment thereof, which is added to make up the total dose. Thus, in some embodiments, a portion of the antibody or antigen-binding fragment thereof is conjugated but not labeled with a positron emitter, and / or in some embodiments, a portion of the antibody or antigen-binding fragment thereof is not conjugated (and therefore unlabeled).

[0218] In some embodiments, a subject, i.e., a subject in need thereof, is administered a dose, i.e., an amount of about 20 mg or less, a dose of about 15 mg or less, a dose of about 10 mg or less, a dose of about 5 mg or less, e.g., a dose of about 3 mg or less, about 0.2 mg to about 3.0 mg, or about 1.0 mg to about 2.0 mg, or about 1 mg, 2 mg, 3 mg, 5 mg, or 10 mg of a radiolabeled anti-LAG3 antibody conjugate.

[0219] In some embodiments, the antibody or antigen-binding fragment thereof is administered to a subject in a total amount of about 2 mg to about 100 mg, e.g., about 10 mg to about 100 mg, about 20 to about 100 mg, about 20 to about 50 mg, about 30 mg to about 50 mg, or about 20 to about 40 mg, or about 10 mg, or about 20 mg, or about 30 mg, or about 40 mg, about 50 mg, or about 100 mg. In some embodiments, the total amount of the antibody or antigen-binding fragment thereof is about 2 mg to about 100 mg, e.g., about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 20 mg to about 50 mg, about 30 mg to about 50 mg, or about 20 mg to about 40 mg, or about 10 mg, or about 20 mg, or about 30 mg, or about 40 mg, about 50 mg, or about 100 mg. In some embodiments, the total amount of the antibody or antigen-binding fragment thereof is about 2 mg to about 100 mg, e.g., about 2 mg to about 100 mg, conjugated to a chelating moiety and a positron emitter. 89 and (b) a portion of an antibody or antigen-binding fragment thereof that is conjugated with a positron emitter but is not labeled, and / or a portion of an antibody or antigen-binding fragment thereof that is not conjugated. The labeled portion (a) is considered "hot" and the unlabeled portion (b) is considered "cold."

[0220] In some embodiments, the subject is administered an antibody or antigen-binding fragment thereof, wherein the labeled moiety is in an amount of about 0.2 mg to about 3.0 mg, and the total amount of the antibody or antigen-binding fragment administered to the subject is about 2 mg to about 100 mg, or about 20 to about 50 mg. In some aspects, the subject is administered an antibody or antigen-binding fragment thereof, wherein the labeled moiety is in an amount of about 1.0 mg to about 2.0 mg, and the total amount of the antibody or antigen-binding fragment administered to the subject is about 40 mg.

[0221] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or signs of cancer or has been diagnosed with cancer (including a tumor, e.g., a solid tumor) and is in need of treatment for that condition or diagnosis based on that symptom or diagnosis. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may have a primary tumor or a metastatic tumor and / or be diagnosed with one or more symptoms or signs, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal bloating, chest pain / tightness, enlarged spleen, and elevated levels of cancer-associated biomarkers (e.g., CA125, i.e., MUC16). This phrase includes subjects with primary or established cancer. In certain embodiments, the phrase includes a human subject having and / or in need of treatment for a tumor, such as colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer, and brain cancer. The term includes subjects having primary or metastatic tumors (advanced malignancies). In certain embodiments, the phrase "subject in need thereof" includes patients having tumors that are resistant to, unresponsive to, or not adequately controlled by prior treatment (e.g., treatment with anticancer drugs). For example, the phrase includes subjects who have been treated with one or more lines of prior treatment, such as chemotherapy (e.g., carboplatin or docetaxel). In certain embodiments, the phrase "subject in need thereof" includes patients having tumors, including solid tumors, that have subsequently recurred or metastasized despite receiving one or more lines of prior treatment. In certain embodiments, the phrase "a subject in need thereof" includes, but is not limited to, cancer, rheumatoid arthritis, arteriosclerosis, periodontal disease, hay fever, heart disease, coronary artery disease, infection, bronchitis, dermatitis, meningitis, asthma, tuberculosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, hepatitis, sinusitis, psoriasis, allergies, fibrosis, lupus, vasculitis, ankylosing spondylitis, Graves' disease, celiac disease, fibromyalgia, and transplant rejection.

[0222] In certain embodiments, the methods of the present disclosure are used in subjects with solid tumors. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (not cancerous) or malignant (cancer). In some embodiments, tumors are metastatic. For the purposes of this disclosure, the term "solid tumor" refers to a malignant solid tumor. This term includes different types of solid tumors designated by the cell type that forms them, i.e., sarcomas and carcinomas.

[0223] In certain embodiments, the term solid tumor includes, but is not limited to, anal cancer, anaplastic thyroid carcinoma, astrocytoma, bladder cancer, bone cancer, glioblastoma multiforme, brain cancer, triple-negative breast cancer, breast cancer, cervical cancer, chondrosarcoma, clear cell carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, fibrosarcoma, gastric carcinoma, glioblastoma, head and neck cancer, hepatocellular carcinoma, jejunal cancer, renal cancer, liver cancer, lung cancer, melanoma, mesothelioma, metastatic cervical cancer, metastatic melanoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, clear cell renal carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, squamous cell carcinoma of the head and neck, stomach cancer, synovial sarcoma, testicular cancer, thyroid cancer, uterine cancer, and Wilms' tumor. Solid tumors include advanced and metastatic cancers.

[0224] In some embodiments, the methods disclosed herein can be used in subjects with cancer, such as hematological cancers (e.g., myeloma, lymphoma (e.g., B-cell lymphoma), leukemia), brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, and melanoma. In some aspects, the cancer is advanced or metastatic, e.g., metastatic melanoma.

[0225] In some embodiments, the tumor is characterized by mismatch repair deficiency (dMMR) and microsatellite instability. dMMR tumors may be more responsive to checkpoint inhibitors. Thus, in some embodiments of the method for imaging, diagnosing, or treating tumors, determining that the tumor is LAG3 positive based on the detection of a radiolabeled anti-LAG3 antibody conjugate correlates with determining that the subject contains a dMMR tumor or a tumor characterized by microsatellite instability. Thus, in some embodiments, determining that the tumor is LAG3 positive identifies that the subject contains a dMMR tumor or a tumor characterized by microsatellite instability.

[0226] According to one aspect, the present disclosure provides a method for treating a tumor in a subject. The method according to this aspect includes selecting a subject with a tumor, for example, a solid tumor, determining that the tumor is LAG3-positive, and administering one or more doses of a LAG3 inhibitor. In certain embodiments, the tumor is determined to be LAG3-positive by administering a radiolabeled anti-LAG3 antibody conjugate of the present disclosure to the subject; and visualizing the radiolabeled antibody conjugate in the tumor by PET imaging, where the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive.

[0227] In further aspects, the method of treatment comprises administering an anti-tumor therapy. In some aspects, the method of treatment comprises administering one or more doses of an anti-tumor therapy. In some embodiments, the anti-tumor therapy comprises an inhibitor of LAG3, an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist (e.g., aflibercept or U.S. Patent Application Publication No. 7,087,097,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, MUC16, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guerin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADCs), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or any palliative care for treating cancer.

[0228] In some embodiments, the anti-tumor therapy is an anti-LAG3 antibody, REGN3767 (also known as fianlimab), REGN2810 (also known as cemiplimab), BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, an anti-CD-28 antibody, an anti-2B4 antibody, an anti-LY108 antibody, an anti-LAIR1 antibody, an anti-ICOS antibody, an anti-CD160 antibody, or an anti-VISTA antibody. , aflibercept, bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, nesbacumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin Calmette-Guerin), CD20xCD3 bispecific antibody, PSMAxCD3 bispecific antibody, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, vincristine, cyclophosphamide, radiation therapy, sarilumab, dupilumab, anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC;

[0229] In certain aspects, the anti-tumor therapy is administered in combination with a second anti-tumor therapy. In some embodiments, the second anti-tumor therapy is selected from the group consisting of an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody-drug conjugate (ADC).

[0230] In some embodiments, the anti-tumor therapy is an anti-LAG3 antibody, REGN3767 (fianlimab), REGN2810, BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, anti-CD-28 antibody, anti-2B4 antibody, anti-LY108 antibody, anti-LAIR1 antibody, anti-ICOS antibody, anti-CD160 antibody, anti-VISTA antibody, aflibercept , bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, nesvaccumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin Calmette-Guerin), CD20xCD3 bispecific antibody, PSMAxCD3 bispecific antibody, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, vincristine, cyclophosphamide, radiation therapy, sarilumab, dupilumab, anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC.

[0231] In certain embodiments, LAG3 inhibitors are used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance anti-tumor responses. Examples of cancer vaccines that can be used in combination with LAG3 inhibitors include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancers (including glioblastoma multiforme), or ALVAC-CEA (for CEA-positive cancers).

[0232] In certain embodiments, LAG3 inhibitors may be used in combination with radiation therapy in methods aimed at inducing long-lasting anti-tumor responses and / or improving survival rates in cancer patients. In some embodiments, LAG3 inhibitors (e.g., anti-LAG3 antibodies) may be administered to cancer patients before, simultaneously with, or after radiation therapy. For example, radiation therapy may be administered in one or more doses to tumor lesions, followed by administration of one or more doses of an anti-LAG3 antibody. In some embodiments, radiation therapy is administered locally to tumor lesions to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or destroy tumor cells (ablative radiation), followed by systemic administration of an anti-LAG3 antibody. For example, a patient with a brain tumor (e.g., glioblastoma multiforme) may be administered intracranial radiation therapy in combination with systemic administration of an anti-LAG3 antibody. In certain embodiments, the anti-LAG3 antibody may be administered in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or with a VEGF antagonist (eg, aflibercept).

[0233] In certain embodiments, LAG3 inhibitors may be administered in combination with one or more antiviral drugs to treat viral infections, such as those caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids.

[0234] In certain embodiments, LAG3 inhibitors may be administered in combination with one or more antibacterial agents to treat bacterial infections caused by, for example, Rickettsia, Bacillus, Klebsiella, Neisseria meningitidis, Gonococcus, Proteus, Streptococcus pneumoniae, Pseudomonas, Streptococcus, Staphylococcus, Serratia, Borrelia, Bacillus anthracis, Chlamydia, Clostridium, Corynebacterium diphtheriae, Legionella, Mycobacterium leprae, Mycobacterium lepromatosis, Salmonella, Vibrio cholerae, and Yersinia pestis. Examples of antibacterial agents include, but are not limited to, penicillin, tetracycline, cephalosporin, quinolone, lincomycin, macrolides, ketolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0235] In certain embodiments, LAG3 inhibitors can be administered in combination with one or more antifungal agents to treat fungal infections caused by, for example, Aspergillus (e.g., fumigatus, niger), Blastomyces dermatitidis, Candida (e.g., albicans, krusei, glabrata, tropicalis), Coccidioides immitis, Cryptococcus neoformans, Mucorales (e.g., Mucor, Abscissionia, Rhizopus), Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii. Examples of antifungal agents include, but are not limited to, amphotericin B, fluconazole, vorixonazole, posaconazole, itraconazole, voriconazole, anidulafungin, caspofungin, micafungin, and flucytosine.

[0236] In certain embodiments, a LAG3 inhibitor may be administered in combination with one or more antiparasitic agents to treat parasitic infections caused by, for example, Entamoeba species, pinworms, Leishmania species, Toxocara species, Plasmodium species, Schistosoma species, Taenia solium, Toxoplasma gondii, and Trypanosoma cruzi. Examples of antiparasitic agents include, but are not limited to, praziquantel, oxamniquine, metronidazole, tinidazole, nitazoxanide, dehydroemetine or chloroquine, dioxanide furoate, iodoquinone, chloroquine, paromomycin, pyrantel pamoate, albendazole, nifurtimox, and benznidazole.

[0237] The additional therapeutically active agent(s) / ingredient(s) may be administered prior to, simultaneously with, or following administration of the LAG3 inhibitor. For purposes of this disclosure, such administration regimens are considered administration of the LAG3 inhibitor "in combination" with the second therapeutically active ingredient.

[0238] In some embodiments, the method of treatment comprises selecting a subject with a bacterial, viral, fungal, or parasitic infection, determining that the affected tissue in the subject is LAG3-positive, and administering one or more doses of a therapeutic agent appropriate for the infection. In certain embodiments, the affected tissue is determined to be LAG3-positive by administering a radiolabeled anti-LAG3 conjugate of the present disclosure to the subject; and visualizing the radiolabeled antibody conjugate in the subject by PET imaging, where the presence of the radiolabeled antibody conjugate in the tissue indicates that the tissue is LAG3-positive. In certain embodiments, the administering and visualizing steps are performed one or more times to monitor the effectiveness of the therapeutic agent in treating the infection.

[0239] In some aspects, the presence of LAG3-positive cells in a tumor identifies the subject as a candidate for anti-tumor therapy comprising an inhibitor of LAG3 or the PD-1 / PD-L1 signaling axis. Thus, the anti-tumor therapy may be selected from the group consisting of an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, and an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof, e.g., REGN2810 (also known as cemiplimab), nivolumab, or pembrolizumab. In some embodiments, the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a platinum-based chemotherapeutic agent. Exemplary platinum-based chemotherapeutic agent options include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin. In some embodiments, the anti-tumor therapy is an anti-PD-L1 antibody or antigen-binding fragment thereof, e.g., atezolizumab, avelumab, or durvalumab. In some embodiments, the anti-tumor therapy is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 38 three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the group consisting of 6 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562;Sequence numbers: 4 / 6 / 8 / 12 / 14 / 16, 20 / 22 / 24 / 28 / 30 / 32, 36 / 38 / 40 / 44 / 46 / 48, 52 / 54 / 56 / 60 / 62 / 64, 68 / 70 / 72 / 76 / 78 / 80, 84 / 86 / 88 / 92 / 94 / 96, 100 / 102 / 104 / 108 / 110 / 112, 116 / 118 / 120 / 124 / 126 / 128, 132 / 134 / 136 / 140 / 142 / 144, 148 / 150 / 152 / 156 / 158 / 160, 164 / 166 / 168 / 172 / 174 / 176, 180 / 1 82 / 184 / 188 / 190 / 192, 196 / 198 / 200 / 204 / 206 / 208, 212 / 214 / 216 / 220 / 222 / 224, 228 / 230 / 232 / 236 / 238 / 240, 244 / 246 / 248 / 252 / 254 / 256, 260 / 262 / 264 / 268 / 270 / 272, 276 / 278 / 280 / 284 / 286 / 288, 292 / 294 / 296 / 300 / 302 / 304, 308 / 310 / 312 / 316 / 318 / 320, 324 / 326 / 328 / 332 / 334 / 336, 340 / 342 / 344 / 348 / 350 / 352, 356 / 358 / 360 / 364 / 366 / 368, 372 / 374 / 376 / 380 / 382 / 384, 388 / 390 / 392 / 396 / 398 / 400, 404 / 406 / 408 / 412 / 414 / 416, 420 / 422 / 424 / 428 / 430 / 432, 436 / 438 / 440 / 444 / 446 / 448, 452 / 454 / 456 / 524 / 526 / 528, 460 / 462 / 464 / 524 / 526 / 528, 468 / 470 / 472 / 524 / 52 a set of three HCDRs and three LCDRs selected from the group consisting of 6 / 528, 476 / 478 / 480 / 524 / 526 / 528, 484 / 486 / 488 / 524 / 526 / 528, 492 / 494 / 496 / 524 / 526 / 528, 500 / 502 / 504 / 532 / 534 / 536, 508 / 510 / 512 / 532 / 534 / 536, 516 / 518 / 520 / 532 / 534 / 536, 540 / 542 / 544 / 548 / 550 / 552, and 556 / 558 / 560 / 564 / 566 / 568;or an anti-LAG3 antibody or antigen-binding fragment thereof, comprising three HCDRs in the HCVR set forth in SEQ ID NO: 418 and three LCDRs in the LCVR set forth in SEQ ID NO: 426;

[0240] In some aspects, a method of treatment includes selecting a subject having a tumor, e.g., a solid tumor, determining that the tumor is LAG3-positive and PD-1-positive, and administering one or more doses of a LAG3 inhibitor and / or one or more doses of an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the tumor is determined to be LAG3-positive by administering a radiolabeled anti-LAG3 conjugate of the present disclosure to the subject; and visualizing the radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is LAG3-positive. In certain embodiments, a tumor is determined to be PD-1 positive by administering to a subject a radiolabeled anti-PD-1 conjugate of the present disclosure; and visualizing the radiolabeled anti-PD-1 conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled anti-PD-1 conjugate in the tumor indicates that the tumor is PD-1 positive.

[0241] Exemplary anti-PD-1 antibodies include REGN2810 (also known as cemiplimab), BGB-A317, nivolumab, pidilizumab, and pembrolizumab.

[0242] Exemplary anti-PD-L1 antibodies include atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, as well as those disclosed in Patent Publication No. 2015-0203580 and those disclosed therein.

[0243] The inhibitor of the PD-1 / PD-L1 signaling axis may be administered prior to, simultaneously with, or following administration of the LAG3 inhibitor. For purposes of this disclosure, such administration regimens will be considered administration of the LAG3 inhibitor "in combination" with the inhibitor of the PD-1 / PD-L1 signaling axis.

[0244] As used herein, the terms "treat," "treating," and the like mean alleviating symptoms, eliminating the cause of symptoms either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell mass or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, preventing or inhibiting metastasis, inhibiting metastatic tumor growth, and / or prolonging the survival of a subject.

[0245] According to one aspect, the present disclosure provides a method for monitoring the effectiveness of anti-tumor therapy in a subject, the method comprising: selecting a subject having a tumor, e.g., a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering a radiolabeled anti-LAG3 conjugate of the present disclosure to the subject; imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging; and determining tumor growth, wherein a change in the radiolabeled signal from baseline indicates the effectiveness of the anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor. In certain embodiments, the anti-tumor therapy further comprises an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).

[0246] In certain embodiments, the present disclosure provides a method for assessing changes in the inflammatory state of a tumor, the method comprising: selecting a subject having a tumor, e.g., a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering a radiolabeled anti-LAG3 conjugate provided herein to the subject; and imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging, wherein an increase in the radiolabeled signal from baseline indicates increased inflammation and the effectiveness of the anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises a LAG3 inhibitor and / or an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the anti-tumor therapy is selected from the group consisting of PD-1 inhibitors (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504), CTLA-4 inhibitors (e.g., ipilimumab), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of another T-cell co-inhibitory factor or ligand (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Pat. No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, MUC16, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD20xCD3 bispecific antibodies or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC)

[0247] As used herein, the term "baseline" with respect to LAG3 expression in tumors refers to the numerical value of the radiolabeled conjugate uptake for a subject before or at the time of administration of a dose of antitumor therapy. The radiolabeled conjugate uptake is determined using methods known in the art (see, for example, Oosting et al. 2015, J. Nucl. Med. 56: 63-69). In certain embodiments, the antitumor therapy includes a LAG3 inhibitor.

[0248] In some embodiments, serial iPET scans and tumor biopsies are performed before and after treatment with standard of care immunotherapy. Such immunotherapy may be selected from cemiplimab, nivolumab, ipilimumab, pembrolizumab, and combinations thereof.

[0249] To determine whether the anti-tumor therapy is effective, uptake of the radiolabeled conjugate is quantified at baseline and at one or more time points after administration of the LAG3 inhibitor. For example, uptake of the radiolabeled antibody conjugate (e.g., a radiolabeled anti-LAG3 antibody conjugate) is measured at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 22, 25, 29, 36, 43, 50, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 Uptake may be measured at days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more. The difference between the uptake value at a particular time point after treatment initiation and the baseline uptake value is used to ascertain whether the anti-tumor therapy is effective (tumor regression or progression).

[0250] In certain embodiments, the radiolabeled antibody conjugate is administered intravenously or subcutaneously to a subject. In certain embodiments, the radiolabeled antibody conjugate is administered intratumorally. In some embodiments, the dosage of the radiolabeled antibody conjugate is administered in a volume of about 1 mL to about 15 mL, or about 5 mL to about 12 mL, or about 5 mL, about 7 mL, about 10 mL, about 12 mL, or about 15 mL.

[0251] After administration, the radiolabeled antibody conjugate is localized in tumor.The localized radiolabeled antibody conjugate is imaged by PET imaging, and the tumor uptake of the radiolabeled antibody conjugate is measured by methods known in the art.In certain embodiments, imaging is carried out 1, 2, 3, 4, 5, 6 or 7 days after the administration of the radiolabeled conjugate.In certain embodiments, imaging is carried out on the same day as the administration of the radiolabeled antibody conjugate.

[0252] In certain embodiments, the anti-LAG3 antibody is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554. and CDRs of an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562.

[0253] In certain embodiments, the LAG3 inhibitor comprises an antibody or antigen-binding fragment thereof that specifically binds to LAG3. Exemplary anti-LAG3 antibodies are listed in Table 1 of Huo JL, Wang YT, Fu WJ, Lu N, and Liu ZS (2022). LAG3, a promising immune checkpoint in cancer immunotherapy, ranges from basic research to clinical applications. See Front. Immunol, 13:956090. In some other embodiments, the LAG3 inhibitor comprises an antibody or antigen-binding fragment thereof that specifically binds to LAG3. In one embodiment, the anti-LAG3 antibody comprises the HCVR of SEQ ID NO: 418 and the LCVR of SEQ ID NO: 426. In one embodiment, the anti-LAG3 antibody is REGN3767 (fianlimab).

[0254] IV. Compositions, Formulations, and Kits As used herein, a method for producing a radioactive material comprising: (i) an unlabeled anti-LAG3 antibody or antigen-binding fragment thereof; and (ii) an anti-LAG3 antibody or antigen-binding fragment thereof, which provides about 0.5 to 3.0 mCi of radioactivity. 89 and a Zr-labeled anti-LAG3 antibody conjugate, wherein the labeled anti-LAG3 antibody conjugate is present in the composition in an amount of about 0.2 mg to about 3 mg, and the total amount of the antibody or antigen-binding fragment thereof is present in the composition in an amount of about 2 mg to about 100 mg, e.g., about 10 to about 100 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, or about 100 mg. In certain embodiments, the composition 89 The Zr-labeled anti-LAG3 antibody conjugate provides a radioactivity of about 1 mCi. In certain embodiments, the labeled anti-LAG3 antibody conjugate is present in the composition in an amount of about 1 mg to about 2 mg.

[0255] In some embodiments, 89 The Zr-labeled anti-LAG3 antibody conjugate comprises an anti-LAG3 antibody or antigen-binding fragment thereof conjugated to desferrioxamine (DFO).

[0256] Also provided herein are formulations configured for administration to humans, comprising the compositions provided herein. Thus, provided herein are formulations configured for administration to humans at a total dosage of about 40 mg of an antibody or antigen-binding fragment thereof. The formulations include an anti-LAG3 antibody or antigen-binding fragment thereof, and a portion of the anti-LAG3 antibody or antigen-binding fragment thereof linked to the anti-LAG3 antibody or antigen-binding fragment thereof. 89 In certain embodiments, the radiolabel provides about 0.5 to about 3 mCi of radiation per formulation. In certain embodiments, the formulation 89 The Zr-labeled anti-LAG3 antibody provides approximately 1 mCi of radiation. 89 The portion of the total anti-LAG3 antibody or antigen-binding fragment thereof with which the Zr radiolabel is associated in the formulation is in an amount of about 1 mg to about 2 mg.

[0257] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: 89 Pharmaceutical compositions are provided that include a Zr-labeled anti-LAG3 antibody conjugate. The pharmaceutical composition is formulated with one or more pharmaceutically acceptable vehicles, carriers, diluents, and / or excipients. Various pharmaceutically acceptable carriers, diluents, and excipients are well known in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. In some embodiments, the pharmaceutically acceptable carrier or diluent is a buffer. Exemplary buffers include citrate, acetate, phosphate, arginine, and histidine buffers. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration. The pharmaceutical composition can be in a volume suitable for intravenous administration, for example, about 1 mL to about 15 mL, or about 2 mL, about 5 mL, about 7 mL, about 8 mL, about 10 mL, about 12 mL, or about 15 mL.

[0258] In some embodiments, the antibody or antigen-binding fragment thereof present in the composition or formulation has a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554. and three light chain complementarity determining regions (LCDRs) in the light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562. In certain embodiments, the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO: 418 and three CDRs in the LCVR set forth in SEQ ID NO: 426, an HCDR1 of SEQ ID NO: 420, an HCDR2 of SEQ ID NO: 422, an HCDR3 of SEQ ID NO: 424, an LCDR1 of SEQ ID NO: 428, an LCDR2 of SEQ ID NO: 430, and an LCDR3 of SEQ ID NO: 432, and / or comprises the HCVR set forth in SEQ ID NO: 418 and the LCVR set forth in SEQ ID NO: 426.

[0259] Also provided herein are kits containing the formulations and compositions described throughout this disclosure. In embodiments of the invention, the kits include (i) an unlabeled anti-LAG3 antibody or antigen-binding fragment thereof, and (ii) an anti-LAG3 antibody or antigen-binding fragment thereof that provides about 0.5 to 3.0 mCi of radioactivity in a container or injection device (e.g., an IV line or syringe). 89and a Zr-labeled anti-LAG3 antibody conjugate. In some embodiments, the labeled anti-LAG3 antibody conjugate is present in the container or injection device in an amount of about 0.2 mg to about 3 mg, and the total amount of the antibody or antigen-binding fragment thereof present in the container or injection device is about 40 mg. The kit can include a package insert containing information regarding the pharmaceutical composition and dosage form in the kit. Typically, such information will assist patients and physicians in effectively and safely using the enclosed pharmaceutical composition. For example, any of the following information regarding the combination of the present invention can be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdose, appropriate dosage and administration, method of delivery, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0260] In some embodiments, the kit includes instructions for PET imaging of the subject after administration of a dose of a radiolabeled composition described herein. In some embodiments, the instructions specify PET imaging about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days after administration of the composition. In some embodiments, the instructions specify PET imaging about 7 days after administration of the composition.

[0261] The compositions, formulations, and kits are useful according to any of the methods described herein, and are particularly useful for imaging LAG3-positive tumors and / or treating tumor-bearing subjects. [Example]

[0262] V. Working Examples Certain embodiments of the present disclosure are illustrated by the following non-limiting examples.

[0263] Example 1: Generation of human antibodies against LAG3 Human antibodies against LAG3 were generated using a fragment of LAG3 spanning approximately amino acids 29-450 of GenBank accession NP_002277.4 (SEQ ID NO: 582) genetically fused to a mouse Fc region. The immunogen, along with an adjuvant to stimulate the immune response, was administered directly to VELOCIMMUNE® mice (genetically engineered mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions) according to the method described in U.S. Patent No. 8,502,018 B2, or to humanized universal light chain (ULC) VelocImmune® mice according to the method described in WO 2013022782. Antibody immune responses were monitored by LAG3-specific immunoassays. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing LAG3-specific antibodies. Using this technique and the immunogens described above, several anti-LAG3 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) with human variable regions and mouse constant regions were obtained. Fully human versions of antibodies can be generated by replacing the mouse constant regions with human constant regions. Examples of antibodies generated in this manner from VELOCIMMUNE® mice are designated H1M14985N, H1M14987N, H2M14811N, H2M14885N, H2M14926N, H2M14927N, H2M14931N, H2M18336N, H2M18337N, and H4H14813N.

[0264] Anti-LAG3 antibodies were isolated directly from antigen-positive B cells obtained from one of the immunized mice, without fusion with myeloma cells, according to the method described in U.S. Patent No. 7,582,298 (incorporated herein by reference in its entirety). Using this method, several anti-LAG3 antibodies (i.e., antibodies with human variable domains and human constant domains) were obtained. Exemplary antibodies generated by this method are designated as follows: H4H15477P, H4H15483P, H4H15484P, H4H15491P, H4H17823P, H4H17826P2, H4H17828P2, H4sH15460P, H4sH15462P, H4sH15463P, H4sH15464P, H4sH15466P, H4sH15467P, H4sH15470P , H4sH15475P, H4sH15479P, H4sH15480P, H4sH15482P, H4sH15488P, H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, H4sH15567P2, and H4H17819P.

[0265] Exemplary antibodies H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, and H4sH15567P2 were generated from B cells from ULC VELOCIMMUNE® mice.

[0266] The biological properties of exemplary antibodies generated according to the methods of this example are detailed in the Examples below.

[0267] Example 2: Binding of anti-LAG3 antibody H4sH15482P to p-SCN-Bn-DFO To modify the parent anti-LAG3 antibody, H4sH15482P (HCVR / LCVR sequence pair: SEQ ID NO: 418 / 426; hereafter abbreviated as mAb1) and an isotype control antibody suitable for radiolabeled immunoPET studies, the chelator p-SCN-bn-deferoxamine (DFO; Macrocylics, product code B-705) was conjugated to these antibodies.

[0268] For modification, mAb1 was first buffer exchanged from histidine buffer into PBS, pH 7.2, by dialysis overnight at 4°C (Slide A-Lyzer Dialysis Cassette G2 10k MWCO; ThermoScientific), and then buffer exchanged again into a buffer consisting of 50 mM carbonate buffer, 150 mM NaCl, pH 9.0 (conjugation buffer) using a PD-10 column (GE Healthcare, catalog number 17-0851-01). To determine the concentration after buffer exchange, samples were analyzed by MacVector sequence-based extinction coefficient of 223400 M -1 cm -1 The chromatographic data were measured on a Nanodrop 2000 UV / VIS spectrometer (Thermo Scientific) using a pH of 1.0 and a molecular weight of 145,709 g / mol (see Table 2). In a 15 mL polypropylene tube, 1485.24 μL of mAb1 (70 mg) was added to 5374.8 μL of conjugation buffer. A 139 μL solution of DFO in DMSO was added in quarter portions to the mAb1 solution, gently mixing by pipetting up and down each time. The final solution was 10 mg / mL mAb1 in conjugation buffer, 2% DMSO, and a 3-fold molar excess of DFO. This solution was incubated in a 37°C water bath without further agitation.

[0269] After allowing to stand at 37°C for 30 minutes, the solution was immediately passed through a PD-10 desalting column (GE Healthcare, product number 17-0851-01). The column was pre-equilibrated with a pH 5.4 buffer containing 250 mM NaAcO (preparation buffer). The volume of the solution was reduced by approximately 50% using a 10K MWCO concentrator (Amicon Ultra-15 Centrifugal Filter Unit, EMD Millipore, catalog number UFC901024). The final solution was sterile filtered using a syringe filter (Acrodisc 13 mm syringe filter, Pall Corporation, catalog number 4602). The concentration and DFO-to-antibody ratio (DAR) were then measured by UV / VIS spectroscopy. See Figure 1. For absorbance measurements, DFO-conjugated antibodies were measured against formulation buffer at 252 nm (A252), 280 nm (A280), and 600 nm (A600). For calculations, the following equation was used: The background was corrected for each absorbance using TIFF2026506001000007.tif9128.

[0270] Antibody conjugates were subjected to aggregation testing using SEC chromatography. 25 μg of sample was injected onto a Superdex 200 column (GE Healthcare, part number: 17-5175-01) using a PBS mobile phase (0.75 mL / min) and monitored at 280 nm. See Figure 2. Antibody integrity was assessed by SDS-PAGE 4-20% Tris / Gly precast gel (Novex) loaded with 2 μg of sample. Antibody concentration, conjugate concentration, and DAR were calculated using the following equation: Calculation of antibody concentration: TIFF2026506001000008.tif14128 Calculation of conjugate concentration: TIFF2026506001000009.tif13149DAR calculation: Calculations were performed using TIFF2026506001000010.tif14128.

[0271] [Table 2]

[0272] [Table 3]

[0273] Example 3: Monoclonal antibody conjugated with DFO 89 Zr chelation For use in ImmunoPET in vivo testing, anti-LAG3 antibody conjugated with DFO, mAb1, and an isotype control antibody conjugated with DFO were 89 Radiolabeled with Zr.

[0274] The DFO-conjugated antibodies were initially brought to 1.25 mg / mL in 1 M HEPES, pH 7.2. The composition of the DFO-Ab conjugate solution for each test is listed in Table 4. Separately, 89 Zr solutions were prepared using the corresponding compositions for each test shown in Table 5. 89 Zr-oxalate solution was obtained from 3D Imaging, Inc. The final radioactivity of the solution was first confirmed using a Capintec CRC-25R dose calibrator (Capintec #520), then immediately combined with the DFO-Ab conjugate solution, mixed gently (by pipetting up and down), and then incubated at room temperature for 45 minutes.

[0275] After incubation, the mixture was transferred to a desalting column (either a PD-10 (GE Healthcare, part number: 17-0851-01) for Test 1 or a NAP-5 (GE Healthcare, part number: 17-0853-02) for Test 2) pre-equilibrated with 250 mM sodium acetate at pH 5.4 for gravity desalting. For Test 1, the reaction mixture was loaded onto the PD-10 column. After the contents of the reaction entered the column bed, the flow-through was discarded. The product was eluted with 250 mM sodium acetate at pH 5.4 (formulation buffer) and the eluate was collected according to the manufacturer's instructions. For Test 2, the mixture was transferred to a NAP-5 column and the flow-through was discarded. The product was eluted with 250 mM sodium acetate at pH 5.4 (formulation buffer) and the eluate was collected according to the manufacturer's instructions. The Ab concentration was then measured by UV / VIS spectroscopy and calculated using the appropriate extinction coefficient and absorbance value at 280 nm using the following equation: Concentration (mg / mL) = Absorbance at 280 nm ÷ Extinction coefficient at 280 nm (listed in Table 6) was calculated by

[0276] The final mass, measured in grams, was recorded in Table 7. Radioactivity was then measured using a dose calibrator, and the results are shown in Table 7. The final sample (5 μg) was analyzed using SEC-HPLC (Agilent 1260 with Lablogic Radio-TLC / HPLC Detector, SCAN-RAM) with UV 280 and radioisotope detectors in series. A Superdex 200 Increase column was used with a PBS mobile phase at a flow rate of 0.75 mL / min. The radioactive tracer was detected in the total protein peak (approximately 10-16 minutes) and in the unlabeled peak. 89 By comparing the integration value of the Zr peak (approximately 25 min), the radiochemical purity (100% - unlabeled) was confirmed. 89 The percentage of monomer purity was determined by UV280 tracing by comparing the incorporation of monomer (approximately 16 min) with the peak of high molecular weight (HMW) species (10 min to approximately 15 min).

[0277] The specific radioactivity and percent protein recovery of each radiolabeled conjugate were determined using the following formulas: a. Mass of conjugate in mg = concentration in mg / mL × mass of solution in grams b. Specific activity in mCi / mg = activity of vial in mCi ÷ mass of conjugate in mg c. Protein recovery = starting conjugate mass (mg) ÷ mass of conjugate in mg

[0278] Finally, the appearance was noted and recorded in Table 7. The results are summarized in Table 7 below. The Radio-SEC-HPLC chromatograms shown in Figures 3-5 confirm a radiochemical purity of at least 98%. The UV280-HPLC SEC chromatograms shown in Figures 6-8 confirm a highly pure monomeric product (>90%).

[0279] [Table 4]

[0280] [Table 5]

[0281] [Table 6]

[0282] [Table 7]

[0283] Example 4: Immunoreactivity The immunoreactivity (IR) of radiolabeled anti-LAG3 antibodies and isotype control antibodies was measured as follows: In these assays, 20 ng of each 89 Zr-labeled antibody was diluted to 15 × 10 in a final volume of 1 mL. 6 MC38-cOVA / eGFP-mLAG3 - / -hLAG3 Tg The samples were incubated for 45 minutes (37°C, 5% CO2) with continuous mixing and then washed twice with medium to remove unbound antibody. The radioactivity of the test cell pellets was then measured using the same 20 ng of 89 Two reference standards containing Zr-labeled antibodies were counted in an automated gamma counter (2470Wizard2, Perkin Elmer). The mean of the standards was used as a measure of total activity to determine the percentage immunoreactivity for the samples.

[0284] As shown in Table 8, 89 The Zr-labeled anti-LAG3 antibody retained immunoreactivity after conjugation and radiolabeling with an IR of 86%.

[0285] [Table 8]

[0286] Example 5: Selective localization of radiolabeled anti-LAG3 antibodies to LAG3-positive tumors in mice Tumor implantation and treatment group assignment: For in vivo imaging studies, we used LAG3-positive tumor lines. First, we used the mouse colon cancer cell line MC38-cOVA / eGFP-mLAG3. - / - hLAG3 Tg The MC38-cOVA / eGFP-mLAG3 cells were used. Here, cells overexpressing human LAG3 and full-length chicken ovalbumin fused with eGFP were introduced by lentiviral transduction (pLVX EF1a and pLKO SSFV, respectively). - / - hLAG3 Tg For tumor allografts, 1 x 10 6 Cells were implanted subcutaneously into the left flank of male NCr nude mice (Taconic, Hudson, NY). Tumors were 100–150 mm 3 Once the average volume reached 100 mg / kg (approximately 7 days after implantation), mice were randomized into 5 groups and treated with either the test antibody or 89 A Zr-radiolabeled control antibody was administered.

[0287] 89 Administration and biodistribution of Zr-DFO-mAb1 For initial studies in nude mice bearing MC38 / ova / LAG3 tumors, mice were administered 50±1 μCi of 50 μCi at a protein dose of approximately 0.6 mg / kg. 89 Zr-labeled antibodies were administered. For biodistribution studies, mice were euthanized 6 days after administration and blood was collected by cardiac puncture. Tumor and normal tissues were excised, placed in counting tubes, and weighed. Then, the mice were incubated in CPM. 89 Zr counting data were collected by measuring samples on an automatic gamma counter (Wizard 2470, Perkin Elmer). All tissues were also weighed, and the percent injected dose per gram (%ID / g) of each sample was calculated using standards prepared from the injected material.

[0288] Summary of results and conclusions In this example, NCr mice bearing MC38 / ova / hLAG3 tumors were treated with a final dose of 50 μCi / mouse. 89 Mice received either Zr-conjugated anti-LAG3 mAb1 or unconjugated antibody. The mice were then allowed to rest for 6 days for blood, tumor, and tissue collection. The %ID / g for each sample was calculated for all samples. The mean %ID / g for each antibody is shown in Table 9. The significantly higher uptake in the MC38 / ova / hLAG3 tumor was evident compared with other normal tissues, with 43.1% tumor uptake significantly higher than the next highest uptake of 6.6%ID / g observed in the thymus. The specificity of anti-LAG3 mAb1 uptake in the tumor is evident, as the 7.8% tumor uptake observed with unconjugated antibody was significantly lower.

[0289] [Table 9]

[0290] Example 6: Selective localization of radiolabeled anti-LAG3 antibodies to Raji / PBMC tumors in mice This example describes the in vivo imaging and ex vivo biodistribution of a zirconium-89 labeled DFO-anti-LAG3 antibody conjugate in NSG mice co-transplanted with Raji cells and human PBMCs.

[0291] The exemplary antibody used in this example was mAb1, which contains the HCVR / LCVR of SEQ ID NOs: 418 / 426.

[0292] Tumor implantation and treatment group assignment: To demonstrate the specificity of the radiolabeled antibody for LAG3 targeting, 2 x 10 6 5 x 10 Raji cells and 5 x 10 5 Human PBMCs (Lot 0151029, ReachBio Research Labs) were co-implanted into the right flank of female NSG mice (8-10 weeks old, Jackson Labs). 14 days after tumor implantation, mice were randomized into four groups and received various protein doses. 89 Zr-DFO-mAb1 was injected intravenously.

[0293] 89 Zr-DFO-mAb1 administration and PET / CT imaging: Mice bearing Raji / hPBMC tumors were treated with 5, 0.3, 0.1, or 0.03 mg / kg of IFN-γ on day 14 after tumor implantation. 89 Mice receiving doses of 0.1 and 0.03 mg / kg were injected with approximately 30 or 9 μCi of radiolabeled Zr-DFO-mAb1, respectively. 89 Mice receiving 5 mg / kg or 0.3 mg / kg of protein received radiolabeled Zr-DFO-mAb1. 89 Approximately 30 μCi of Zr-DFO-mAb1 was administered, supplemented with additional mAb1 (L5) not conjugated to DFO to adjust the final total protein amount administered.

[0294] PET imaging of antibody localization 89Evaluation was performed 6 days after administration of Zr-DFO-mAb1. PET / CT images were acquired using a Sofie Biosciences G8 PET / CT (Sofie Biosciences and Perkin Elmer). The instrument was 89 The detector was pre-calibrated for Zr detection. The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. Mice were anesthetized using isoflurane and maintained under a continuous flow of isoflurane during imaging. Static 10-minute images were collected using G8 acquisition software and then reconstructed using pre-configured settings. Image data were corrected for attenuation and other parameters. CT images were collected after PET acquisition and then co-registered with the PET images. Images were processed using VivoQuant post-processing software (Invicro Imaging Services).

[0295] 89 Biodistribution of Zr-DFO-mAb1 For biodistribution studies, mice were treated with 100 mg / kg of 10 ... 89 The animals were euthanized (6 days after Zr-DFO-mAb1 administration) and blood was collected via cardiac puncture. Raji / hPBMC tumor and normal tissues were excised, placed in counting tubes, and weighed. Then, the animals were euthanized in CPM. 89 Zr counting data were collected by measuring the samples on an automatic gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (%ID / g) of each sample was calculated using standards prepared from the injected material.

[0296] Summary of results and conclusions This study investigated the role of LAG3 expressed on human lymphocytes in subcutaneous Raji / hPBMC tumors grown in NSG mice. 89 Figure 1 shows antigen-specific targeting of Zr-DFO-mAb1. 89 Blocking doses of Zr-DFO-mAb1 are 0.3, 0.1, and 0.03 mg / kg in Raji / hPBMC tumors.89 Compared to the lower dose of Zr-DFO-mAb1, the blood uptake rate (%ID / g) increased and the tumor uptake rate (%ID / g) decreased (Table 10). Furthermore, as the protein dose decreased, the mean tumor-to-blood ratio increased, demonstrating specificity for LAG3 in vivo (Table 10). In addition to targeting LAG3 expressed in Raji / hPBMC tumors, the 0.3, 0.1, and 0.03 mg / kg doses 89 Lower doses of Zr-DFO-mAb1 showed targeting to the spleen and axillary lymph nodes of tumor-bearing mice. 89 Representative PET images (Figure 9) taken 6 days after administration of Zr-DFO-mAb1 show significantly increased tumor, spleen, and axillary lymph node activity at 0.03 mg / kg compared to 5 mg / kg. 89 1 shows higher targeting of Zr-DFO-mAb1.

[0297] [Table 10]

[0298] Example 7: LC-PRM-MS quantification of LAG3 in Raji / PBMC xenografts and clinical samples Frozen tissue samples (Raji / PBMC tumor, mouse spleen, and melanoma tissue; see Figure 10 for source and characteristics of melanoma tissue) were lysed in lysis buffer (8 M urea in 50 mM NH4HCO3 with 1% RapiGest). Tissues were cut into small pieces and homogenized in 1 mL of lysis buffer in a tight Dounce homogenizer. Lysates were incubated on ice for 30 minutes with sonication for 30 seconds every 10 minutes to obtain complete protein extracts. Lysates were centrifuged at 14,000 g for 10 minutes. Protein concentrations were measured by BCA assay. Each sample was diluted to 1 mg / mL, centrifuged at 14,000 g for 10 minutes, and stored in multiple aliquots at -80°C.

[0299] Untransplanted NSG mouse spleen lysate was used as a surrogate matrix to generate a standard curve for LAG3 quantification. LAG3.Fc was added to every 100 μg of mouse spleen lysate at final concentrations of 0.39–50 ng / mg per mg of protein (1:2 serial dilutions). Standards, xenografts, and clinical melanoma lysates were precipitated overnight in 900 μL of cold acetone and then denatured in 90 μL of 8 M urea / TCEP buffer at 37°C for 1 hour. Heavy chain-labeled human LAG3 peptide (FVWSSLDTPSQR 13 C6 15 N4) was added to all samples as an internal standard. Standard and test samples were alkylated with IAA for 30 min at room temperature, then digested with lys-C (1:100 w / w) for 4 h and further digested with trypsin (1:20 w / w) overnight at 37 °C. Samples were quenched with 10% FA to reach a final volume of 100 μL.

[0300] Each treated sample (2 μL) was injected onto a pre-equilibrated nano C18 trap column and separated using a simple nano C18 separation column. The flow rate was 250 nL / min (mobile phase A: water:formic acid / 100:0.1 [V:V] and mobile phase B: acetonitrile:formic acid / 100:0.1 [V:V]). Retention times and peak areas were determined using Skyline software. A calibration curve was generated using the LAG3.Fc reference standard (unlabeled LAG3 peptide FVWSSLDTPSQR, generated by trypsin digestion of hLAG3) versus the internal standard (stable isotope-labeled LAG3 peptide). 12 C6 14 The peak area ratios of the LAG3 and N4 peaks were generated by plotting the peak area ratios of the LAG3 and N4 peaks. The concentration of LAG3 in each sample was calculated using linear regression. The lowest concentration of the LAG3 reference standard (0.39 ng / mg protein) was within the dynamic range of the assay and was defined as the lower limit of quantitation of the assay.

[0301] Summary of results and conclusions LAG3 quantification was performed on tissue samples from four Raji / PBMC xenografts from 27 days after tumor implantation, five xenografts from 15 days after tumor implantation, and 10 melanoma clinical samples. Tissue weight, protein amount, extraction yield, and LAG3 expression are listed in Table 11. Tumor density was estimated to be 1 g / mL, and Bmax was calculated using the following equation: Calculations based on TIFF2026506001000020.tif12167.

[0302] Five of ten melanoma tissue samples were detected as LAG3 positive, with an average expression level of 2.52±1.87 nM, which is similar to the Raji / PBMC model at day 27 (3.79±1.93 nM) and day 15 (6.06±4.04 nM). See Table 11 and Figure 11.

[0303] [Table 11]

[0304] Example 8: Therapy with REGN2810 (anti-human PD-1 Ab) and mAb1 (anti-human LAG3 Ab) upregulates human LAG3 and PD-1 expression on T cells in the tumor microenvironment This study demonstrated that treatment with REGN2810 and mAb1 inhibited Regeneron's proprietary PD-1 hu / hu / LAG3 hu / huUsing double-humanized immunocompetent mice, we evaluated the regulation of human LAG3 and PD-1 expression levels on T cells in the tumor microenvironment. The tumor cell line used in this experiment was the murine colon carcinoma cell line MC38 (obtained from the National Cancer Institute at the Frederick, MD, Laboratory of Tumor Immunology and Biology), which was engineered in-house to express full-length chicken ovalbumin fused to eGFP and is therefore designated MC38-cOVA / eGFP. Human LAG3 expression levels were assessed ex vivo in both enzymatically dissociated CD4 and CD8 T cells from tumors extracted from tumor-bearing double-humanized mice. All surface staining was performed using commercially available fluorescent dyes directly conjugated to antibodies (anti-human LAG3 antibody: eBioscience, clone 3DS223H; anti-human PD-1 antibody: BioLegend, clone EH12.2H7) according to standard protocols. Briefly, tumor cells were washed once with PBS, once with ice-cold staining buffer, and stained with anti-human PD-1 or anti-human LAG3 antibodies directly conjugated with commercially available fluorescent dyes in staining buffer for 30 minutes on ice in the dark, followed by a second wash with 2 ml of PBS. Fixable dye eFluor506 was also included according to the manufacturer's protocol (eBioscience). Samples were acquired on a BD FACSCanto II™ IVD10 with DIVA v8. Data were further analyzed with FlowJo v10.0.6 or later.

[0305] Summary of results and conclusions Table 12 and Figure 12 provide a schematic overview of the treatment dosing regimen in the preclinical tumor setting. 6 MC38-cOVA / eGFP cells were transfected with PD-1 hu / hu / LAG3 hu / hu The tumors were subcutaneously implanted into doubly humanized immunocompetent mice. Approximately 11 days after tumor implantation, the mice were treated with a 100 mm 3 Treatment was initiated on days 0 and 4, as indicated. Tumor samples were collected 3 days after the second dose on day 7.

[0306] [Table 12]

[0307] As shown in Table 13, the combination of anti-human PD-1 (REGN2810) and anti-human LAG3 (mAb1) significantly inhibited tumor growth in the MC38-cOVA / eGFP syngeneic tumor model in double-humanized mice. Tumor-bearing mice (approximately 100 mm 3 Tumors (with tumor size of 10 ... 2 In the control group, tumor size ranged from 300 to 869 mm. 3 The range is 548 mm, with a median 3 The REGN2810 treatment group showed a reduction in tumor size (121-721 mm). 3 , median 466mm 3 ), but the difference did not reach statistical significance. The mAb1-treated group showed no difference from either of the isotype control groups (203–721 mm 3 , median is 592 mm 3 ), and the combination therapy significantly delayed tumor growth (113–621 mm 3 , median is 289 mm 3 , p<0.01).

[0308] [Table 13]

[0309] The REGN2810 anti-human PD-1 Ab and mAb1 anti-human LAG3 Ab increased LAG3 and PD-1 T cells, respectively, in the tumor microenvironment, as seen in Figure 13. Tumors from individual mice were dissociated using a GentalMAC (Miltenyi Biotech) according to the manufacturer's protocol. Samples were stained with a panel of Abs and analyzed by flow cytometry. The presented data were pre-gated for FSC / SSC, viability, singlets, and CD45+CD3+ cells, and then further gated for CD4 or CD8 T cells. Human LAG3 and human PD-1 expression was assessed among the different groups. To exclude potential Ab cross-competition, the REGN2810- and combination-treated groups were excluded from human PD-1 analysis. Similarly, the mAb1- and combination-treated groups were also excluded from human LAG3 analysis. After two therapeutic doses, REGN2810 significantly increased the frequency of human LAG3+ CD4 T cells in the tumor microenvironment by approximately 24% (p=0.0006), and appeared to have a direct regulatory role on LAG3 expression on CD8 T cells at the tested dosing regimen. Interestingly, mAb1 also increased the frequency of human PD-1+ CD4 (p=0.0026) and CD8 T cells (p=0.0249) in the tumor microenvironment by approximately 28%, respectively. See Figure 13.

[0310] The results of the tests carried out here are 89 This clearly demonstrates that Zr-labeled anti-LAG3 antibodies can significantly and specifically localize to tumors. One can envision a scenario in which anti-LAG3 antibodies could be used to select patients with LAG3-positive tumors for subsequent treatment with LAG3 inhibitors, either alone or in combination with other anti-cancer therapeutics, including inhibitors of the PD-1 / PD-L1 signaling axis.

[0311] Example 9: Scaled-up manufacturing process for producing DFO-anti-LAG3 antibody conjugates This example details a scaled-up manufacturing process for preparing an anti-LAG3 antibody (mAb, H4sH15482P described herein) suitable for radiolabeling by conjugating p-SCN-bn-deferoxamine (DFO) to the antibody: (1) a pre-mAb ultrafiltration and diafiltration (UFDF) process removes excipients that inhibit the conjugation process; (2) pre-conjugation UFDF is followed by conjugation of the mAb with p-SCN-bn-deferoxamine to produce a DFO-mAb conjugate; and (3) post-conjugation UFDF to remove residual salts provides the appropriate concentration, excipient levels, and pH for the conjugated monoclonal antibody. The resulting DFO-mAb conjugate is then provided in a buffered state with improved stability for subsequent formulation.

[0312] (1) Ultrafiltration and diafiltration (UFDF) before conjugation 100 g of mAb was placed on a Sius Prostream (TangenX Technology Corporation) membrane (membrane capacity ≤ 500 g / m 2 The antibody was buffer exchanged into 5 mM acetate buffer with pH 5.50 using HCl (HCl ...

[0313] (2) Conjugation The concentrated and filtered antibody (20 g) was transferred to a conjugation vessel containing an amine-free carbonate buffer system (56 mM carbonate, 167 mM sodium chloride, pH 9.40) with a slight level of residual acetate. DFO (25 mM p-SCN-Bn-deferoxamine) was dissolved in DMSO and added to the conjugation vessel with additional DMSO such that DMSO was present at a final volume of 5%. DFO was added in molar excess at a ratio of 4.5:1 DFO to mAb. The total reaction volume was equal to 2.0 L. The buffer system was mixed throughout the addition of reaction components and throughout the reaction time.

[0314] The reaction temperature was controlled over a specific time period by using an equation relating temperature to reaction time. In this case, the reaction temperature was held at 20±2° C. for 180 minutes. The reaction was quenched by adding 2 M acetic acid (23 mL / L) to obtain a solution with a pH of 6.

[0315] (3) UFDF after conjugation After the conjugation step, the quenched DFO-mAb conjugation solution was buffer-exchanged into histidine buffer (10 mM histidine, pH 5.50, with 0.0005% (w / v) ultra-purified polysorbate 80 added as a shear protectant) to remove residual process salts, DMSO, and unreacted DFO. After diafiltration, the solution was concentrated and then formulated. The histidine buffer was selected for long-term storage of the protein at -80°C. The same Sius Prostream membrane mentioned in step (1) was used in the final UFDF step. The resulting concentrated DFO-mAb conjugate solution was sterile-filtered using the Sartopore 2 filter described above.

[0316] UV-DAR (target of 1.5) and protein concentration determination were performed as described in Example 2.

[0317] [Table 14]

[0318] Example 10: LAG3 iPET imaging in cancer patients prior to immune checkpoint inhibitor therapy Purpose: Zirconium-89 labeled LAG3 antibody ( 89 A study using Zr-DFO-REGN3767, herein a conjugated and radiolabeled anti-LAG3 antibody having the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 418 / 426, H4sH15482P, or a conjugated and radiolabeled mAb1; REGN3767 is also known as fianlimab, was conducted in patients with locally advanced or metastatic solid tumors. The objective was to evaluate the efficacy of a radiolabeled anti-LAG3 antibody ( 89 The objective of this study was to evaluate the safety of Zr-DFO-REGN3767 and to determine the optimal tracer protein dose and imaging time point to gain insight into its systemic distribution.

[0319] Patients and methods Methods: Patients with metastatic solid tumors received 37MBq (1mCi) 89Patients received intravenous Zr-DFO-REGN3767, followed by PET / CT scans on days 0, 2, 4, and 7. When medically feasible, tumor biopsies were performed after the imaging procedures. Patients then received the programmed cell death protein 1 (PD-1) antibody cemiplimab alone or in combination with standard-of-care platinum-containing chemotherapy. Therapy efficacy was evaluated according to RECIST 1.1 and iRECIST (Seymour et al., iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017;18(3):e143-e52; Eisenhauer et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). EJ Cancer 2009;45:228-247). Using the Accurate tool (Boellaard R., Quantitative oncology molecular analysis suite: ACCURATE. J Nucl Med. 2018; 59:1753), PET scans were analyzed by placing regions of interest (VOIs), measuring tracer uptake, and calculating the mean normal tissue standardized uptake value (SUV). 平均 ) and the maximum standardized uptake value (SUV) of the tumor lesion. 最大 ) Tumor biopsies were immunohistochemically stained for LAG3 expression.

[0320] Patient population Patients with a histologically confirmed diagnosis of locally advanced or metastatic solid tumors that could benefit from PD-1 antibody therapy, with or without platinum-based chemotherapy, were included. Other inclusion criteria were age ≥18 years, Eastern Cooperative Oncology Group performance status 0–1, life expectancy ≥12 weeks, and measurable disease (i.e., the presence of at least one measurable lesion) according to RECIST v1.1 (Eisenhauer et al., New response evaluation criteria in solid tumors: Revised RECIST Guidelines (version 1.1). EJ Cancer 2009;45:228–247). The ECOG performance status scale describes a patient's level of function in terms of their ability to care for themselves, perform daily activities, and physical abilities (e.g., walking, working). Patients with an ECOG status of 0–1 are fully active and able to maintain all predisease performance without limitations, or are ambulatory with limitations in physically strenuous activities and are able to perform light or sedentary tasks, such as light housework and office work.

[0321] All patients provided written informed consent.

[0322] Study design The data provided herein were obtained from the open-label, non-randomized imaging clinical trial NCT04706715.

[0323] This study consisted of two parts: In Part A, the optimal tracer protein dose and imaging time point were evaluated in 16 patients. Then, in Part B, 22 patients were imaged using the optimal imaging conditions determined in Part A before starting treatment and again after starting the second treatment cycle. 89undergo Zr-DFO-REGN3767 PET / CT scan. See Figure 14A - Part A, which shows the study design for the dose escalation phase of the study, and Figure 14B - Part B, which shows the study design for the dose expansion phase of the study.

[0324] Patients received 37 MBq (1 mCi, 10 mL, 1–2 mg of labeled Ab). 89 They received the prescribed dose of Zr-DFO-REGN3767. Additional unlabeled REGN3767 (fianlimab) was added to achieve a total tracer protein dose of 2, 5, 10, 20, or 40 mg. PET / CT scans were performed on days 0, 2, 4, and 7 after tracer injection. The mean standardized uptake value (SUV) in the blood pool on day 4 was 平均 ) but other with well-known dynamics 89 The total tracer protein dose was considered sufficient if it was equivalent over time to the Zr-monoclonal antibody (Bensch et al., Comparative biodistribution analysis across four different 89 Zr-monoclonal antibody tracers—The first step towards imaging warehouse. Theranostics. 2018;8(16):4295-304). Tumor biopsies were obtained immediately after the PET / CT scan on day 7, whenever medically feasible, before initiating intravenous cemiplimab treatment (350 mg every 3 weeks) with or without platinum-based chemotherapy. Tumor response assessments were performed every 9 weeks after treatment initiation according to RECIST v1.1 and iRECIST (Seymour et al., iRECIST: Guideline for response criteria for use in study test immunotherapeutics. Lancet Oncol. 2017;18(3):e143-e52).

[0325] Evaluation items Part A: The main objective is to89 To determine the Zr-DFO-REGN3767 dose and optimal PET imaging time point, based on available clinical data, in patients with histologically or cytologically documented locally advanced or metastatic solid tumors who may benefit from treatment with cemiplimab + / - platinum-based chemotherapy. 89 By measuring SUV on Zr-DFO-REGN3767 PET scans, 89 To evaluate the PK of Zr-DFO-REGN3767, and 89 The objective of this study was to evaluate the safety of Zr-DFO-REGN3767.

[0326] Part B: The primary objective is to 89 To assess the heterogeneity of Zr-DFO-REGN3767 antibody tumor uptake, to correlate tumor tracer uptake with tumor and immune cell LAG3 expression as assessed by biopsy, to correlate tumor tracer uptake with response to cemiplimab with or without platinum-based chemotherapy, and to assess changes in tumor and normal organ uptake after two cycles of cemiplimab with or without chemotherapy.

[0327] Patients enrolled in Part B will undergo a PET scan at baseline and another PET scan after starting the second treatment cycle. 89 Zr-DFO-REGN3767 tracer uptake was quantified and expressed as standardized uptake values ​​(SUVs) in defined regions of interest (VOIs) in the PET scans. Results from both PET scans were compared to assess changes in imaging tracer uptake over time.

[0328] Additional Purposes Additional objectives include correlating normal organ tracer uptake with potential immune-related adverse events, immune infiltration as determined by immunohistochemistry (IHC) and other molecular biomarkers. 89These include assessing correlations of Zr-DFO-REGN3767 uptake, assessing immunogenicity by baseline and on-treatment ADA formation, and evaluating the PK of REGN3767.

[0329] 89 Zr-DFO-REGN3767 PET The REGN3767 antibody was conjugated with p-SCN-Bn-deferoxamine (DFO) and then purified according to the guidelines of the Japanese Ministry of Health, Labour and Welfare. 89 Radiolabeled with Zr-oxalate. Protein dose was adjusted to the intended target by adding unconjugated REGN3767. 89 Zr-DFO-REGN3767 injection was administered in doses of 2 to 40 mg, equivalent to approximately 37 MBq. 89 The product contained Zr-DFO-REGN3767 with a radiochemical purity of >95%. The product was sterile, endotoxin-free, and had a pH of 5.0-6.0. 89 The immunoreactivity of Zr-DFO-REGN3767 was ≥60%.

[0330] PET scans were acquired in whole-body mode (foot-to-vertex trajectory) and combined with low-dose CT scans for attenuation correction and anatomical referencing. PET scans were performed using a Biograph Vision Quadra PET / CT camera with a 106 cm long axial field of view (Siemens Healthineers, Knoxville, Tennessee, USA) (Prenosil et al., Performance characteristics of the Biograph Vision Quadra PET / CT system with a long axial field of view using the NEMA NU 2-2018 Standard. J Nucl Med. 2022;63(3):476-84). PET acquisitions were performed in two bed positions: from head to upper thigh and from upper thigh to foot. The scan duration per bed position varied depending on the number of days after tracer injection and bed position to obtain sufficient counting statistics. For the skull vertex to upper thigh, acquisitions were performed with a scan duration of 15 minutes on day 0, 20 minutes on days 2 and 4, and 40 minutes on day 7. For the second bed position covering the legs and feet, acquisitions were performed with a scan duration of 5 minutes on day 0, 7 minutes on days 2 and 4, and 11 minutes on day 7. PET data were acquired using a maximum number of ring differences (MRD) of 322 crystal rings, although at the time of this study, image reconstruction could be performed with an MRD of only 85 crystal rings. All PET images were acquired at a multicenter 89Reconstruction was performed using an algorithm from a Zr-monoclonal antibody PET scan study (van Sluis et al., EARL compliance and imaging optimization on the Biograph Vision Quadra PET / CT using phantom and clinical data. Eur J Nucl Med Mol Imaging. 2022;49(13):4652-4660). The Accurate tool was used for region-of-interest (VOI)-based background and lesion analysis. Tumor lesions were identified on a contrast-enhanced CT scan performed before the start of therapy. 89 Spherical VOIs were placed on tumor lesions on Zr-DFO-REGN3767 PET / CT images using the Accurate tool (Boellaard, Quantitative oncology molecular analysis suite: ACCURATE. J Nucl Med. 2018;59:1753). To account for partial volume effects, PET analysis was performed on the longest tumor lesion >1 cm and on malignant lymph nodes >1 cm in short axis (Gallivanone et al., A partial volume effect correction tailored for 18 F-FDG-PET oncological studies. Biomed Res Int. 2013;2013:780458). Tumor lesions with little or no tracer uptake in the vicinity of background tissues with high activity were excluded from PET analysis to avoid inaccurate measurements. Biodistribution was assessed by placing spherical VOIs of a certain size for each organ. Tracer uptake was corrected for body weight and injected dose and expressed as standardized uptake values ​​(SUV). For tumor lesions, uptake was SUV 最高 uptake in normal organs is reported as SUV 平均 was reported as.

[0331] Blood and tissue analysis Blood samples for pharmacokinetic analysis were collected 30 min, 2 days, 4 days, and 7 days after tracer injection. Radionuclide concentrations in blood were measured in serum and whole blood. 89 It was evaluated by Zr radioactivity measurement.

[0332] 89 The in vivo stability of Zr-DFO-REGN3767 was assessed in whole blood and serum samples using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (Giesen et al., Probody therapeutic design of 89 Zr-CX-072 promotes accumulation in PD-L1-expressing tumors compared to normal mouse lymphoid tissue. Clin Cancer Res. 2020;26(15):3999-4009). The gel was exposed to a supersensitive phosphor plate (PerkinElmer) at -20°C for 3 days, and then autoradiography was performed to visualize intact lymphoid tissue. 89 Zr-DFO-REGN3767 and radioactive degradation products were detected. Exposures were captured using a Cyclone phosphor imager. Images were analyzed using ImageJ (version 1.53k) (data not shown).

[0333] Tumor specimens were formalin-fixed and paraffin-embedded (FFPE). Four-micrometer tumor tissue sections were stained with hematoxylin and eosin and then stained for negative control antibodies, LAG3, CD3, CD8, CD4, MMR proteins, and PD-L1.

[0334] All tissue blocks were analyzed for radioactivity by autoradiography. 89Zr-DFO-REGN3767 was used as a standard to correlate autoradiographic intensity to the percentage of injected dose per pixel. Standards (0.1%, 0.05%, and 0.01%) were prepared on silica gel on TLC Al foil (Sigma-Aldrich). Along with the standard on the TLC foil, whole FFPE tumor tissue blocks were exposed to ultrasensitive or multisensitive phosphor storage plates (PerkinElmer) for 7 days. Exposures were captured using a Cyclone phosphor imager (data not shown).

[0335] statistical analysis Data were analyzed using GraphPad Prism (version 8.4.2).

[0336] result Sixteen patients were enrolled between January 2022 and August 2022. Patient characteristics at enrollment are shown in Table 15. 89 No tracer-related adverse events were observed after Zr-DFO-REGN3767 administration.

[0337] [Table 15]

[0338] 89 Zr-DFO-REGN3767 Pharmacokinetics A protein dose-dependent tracer half-life was determined (Figures 15A and 15B). At the lowest evaluated doses (2 and 5 mg), blood activity rapidly decreased over time, with little activity remaining on days 4 and 7. Increasing the total protein dose from 10 mg to 40 mg prolonged the tracer half-life in blood. At a 40 mg tracer dose, blood pool activity was high enough to allow this fully antibody-based PET tracer to accumulate in tumor lesions. Tracer excretion was mediated by both the liver and kidney, as exemplified by high activity in bile, feces, and urine.

[0339] Additional pharmacokinetic properties of the radiolabeled antibody were evaluated in serum samples. Clearance (mL / hr) is provided in Figure 15C, and area under the curve (AUC; kBq*h / mL) is shown in Figure 15D.

[0340] 89 Zr-DFO-REGN3767 tumor uptake A total of 66 tumor lesions were identified in 16 patients. Tracer uptake in tumor lesions varied for different protein dose levels, with a trend observed between total protein dose and tracer uptake (Figure 16A). Tracer uptake in tumor lesions also varied between patients (see Figure 17). Some patients showed clear visible tracer uptake in tumor lesions, while others showed moderate to low uptake. Heterogeneity was observed between tumor lesions in some patients.

[0341] The uptake in tumor lesions increased from day 0 to day 7, especially at doses of 20 and 40 mg. The tumor-to-blood ratio showed the highest contrast on day 7 after tracer injection (Figure 16B). Therefore, LAG3 PET imaging on day 7 was 89 The use of Zr-DFO-REGN3767 was deemed optimal. The geometric mean of tracer uptake in tumor lesions using the 40 mg dose was 6.2 (SD: 1.7).

[0342] 89 Zr-DFO-REGN3767 normal tissue biodistribution Biodistribution analysis revealed the highest tracer uptake in the spleen (mean: 11.2, SD: 1.6). Spleen activity increased from day 0 to day 7. Furthermore, uptake decreased with increasing tracer protein dose (Figure 18A). Bone marrow also showed minimal tracer uptake over time (mean: 2.2, SD: 0.7) (Figure 18B). To avoid partial volume effects when quantifying tracer uptake, uptake in tonsils was not quantified due to their small size. Visual tracer uptake was present in tonsils in 9 of 16 patients, and at least two patients without tracer uptake in their tonsils had previously undergone tonsillectomy. Tracer uptake in normal lymph nodes was low and barely noticeable from the background.

[0343] Other tissues in which high activity was observed were the liver (mean: 4.9, SD: 0.9) and kidney (mean: 3.0, SD: 1.2). In contrast to lymphoid tissue, the activity seen here decreased from day 0 to day 7. High activity was also observed in bile, urine, and feces.

[0344] Moderate uptake was present in the ascending colon (mean: 3.2, SD: 2.0) and small intestine (mean: 2.0, SD: 0.5). Tracer uptake was also detected in the reproductive tract: in the testes in male patients (n = 5), in the uterus in women without reproductive malignancies (n = 3), and in the ovaries of one patient.

[0345] The lowest tracer uptake was observed in mammary glandular tissue, lung, muscle, brain, cortical bone, peritoneal cavity, and subcutaneous tissue (see Table 16 and Figure 20). Tracer uptake decreased over time in most of these tissues, except for mammary glandular tissue, where it increased.

[0346] [Table 16]

[0347] In some patients, the site of inflammation is 89 Zr-DFO-REGN3767 showed uptake during PET imaging. Examples included infected sebaceous cysts, post-obstruction pulmonary infiltrates, and pulmonary infiltrates thought to be due to viral infection. Two patients in this cohort experienced immune-related adverse events (Table 17). However, at baseline, no increased tracer uptake was observed in the involved tissues.

[0348] [Table 17]

[0349] tissue analysis LAG3 density and tumor uptake (SUV) in the test samples 最高 ) and evaluated the relationship between LAG3 density in normal tissues and PET biodistribution results (SUV 平均 ) is evaluated in the same way.

[0350] For the 20 and 40 mg dose levels, tracer uptake in tumor lesions at day 7 was higher in the five patients with mismatch repair deficient (dMMR) than in the three patients with mismatch repair competent (pMMR) tumors (Figure 21).

[0351] 89 Zr-DFO-REGN3767 uptake and treatment outcomes Eleven patients received cemiplimab monotherapy (350 mg every 3 weeks), three patients were treated with cemiplimab (350 mg every 3 weeks) in combination with carboplatin (every 3 weeks for the first 6 cycles), and one patient was treated with cemiplimab (350 mg every 3 weeks) in combination with carboplatin and paclitaxel (every 3 weeks for the first 6 cycles). One patient was unable to start therapy after imaging due to clinical deterioration related to brain metastases (see Table 17). At the time of data cutoff, all patients without evident disease progression underwent at least one response-evaluating CT scan. The best overall tumor response was partial response in four patients, stable disease in three patients, and progressive disease in nine patients. As an exploratory analysis, including patients in the 20 mg and 40 mg dose cohorts, the clinical significance of the tumor lesion and / or tumor microenvironment (TME) was assessed. 89 An evaluation was performed (FIG. 19) to determine whether a relationship could be found between Zr-DFO-REGN3767 uptake and response to therapy. The higher the tracer uptake in the tumor lesion / TME, the better the response to therapy (P 傾向 =0.0064).

[0352] Summary of results 89 No Zr-DFO-REGN3767-related toxicity was observed in 16 patients. The tumor-to-blood ratio at all dose levels increased from day 0 to day 7. Therefore, PET imaging 7 days after tracer injection was considered optimal to achieve the highest contrast. A tracer protein dose of 40 mg resulted in the most favorable hemodynamics for tumor PET imaging. Tumor tracer uptake varied between patients (day 7, 40 mg, geometric mean SUV 最高 was 6.2, SD: 1.7). 89Zr-DFO-REGN3767 demonstrated specific LAG3 targeting in tumor and normal tissues. Mismatch repair-deficient (dMMR) tumors showed higher tracer uptake than mismatch repair-competent (pMMR) tumors. In normal tissues, high tracer uptake was observed in the spleen (mean: 11.2, SD: 1.6) and bone marrow (mean: 2.2, SD: 0.7) at 40 mg. Areas of inflammation also showed tracer uptake. For the 20 and 40 mg dose levels, higher tumor tracer uptake was associated with response to therapy.

[0353] Essay 89 This Phase I clinical trial of Zr-DFO-REGN3767 PET imaging demonstrated that LAG3 PET imaging is safe and feasible in patients with advanced solid tumors. Optimal imaging results were obtained using a 40 mg total protein dose and PET imaging 7 days after tracer injection. This study is the first in humans. 89 The systemic distribution of Zr-DFO-REGN3767 was shown. 89 Normal tissue biodistribution of Zr-DFO-REGN3767 revealed tracer accumulation in the spleen and bone marrow, where T cells are known to reside. Furthermore, sites of inflammation showed increased tracer uptake. Tumor tracer uptake varied between and sometimes even within patients and was found to correlate with response to therapy.

[0354] In addition to systemic distribution, this study also showed that cemiplimab therapy resulted in higher LAG3 iPET signal in some tumors. For example, in patients with neuroendocrine bladder cancer, metastatic tumors showed higher LAG3 iPET signal at baseline (day 1 of the first cemiplimab treatment and 89 After two cycles of cemiplimab treatment (7 days after Zr-DFO-REGN3767 administration) compared with 89 The LAG3 iPET signal increased at day 7 after administration of Zr-DFO-REGN3767. Similarly, higher LAG3 iPET signals were observed at baseline (day 1 of the first cemiplimab treatment and 89was seen in patients with metastatic jejunal cancer treated with two cycles of cemiplimab compared with 7 days after administration of Zr-DFO-REGN3767 ( 89 (7 days after administration of Zr-DFO-REGN3767). iPET signals were also detected in areas of inflammation (data not shown).

[0355] Previous clinical PET imaging studies using radiotracers targeting PD-1 and PD-L1 have demonstrated that tracer uptake in tumor lesions holds predictive value for treatment with anti-PD-1 or anti-PD-L1, respectively. Interestingly, this study demonstrated that higher LAG3 tracer uptake in tumors appears to correlate with a better therapeutic response to PD-1 antibody treatment. This may be due to coexpression of LAG3 and PD-1 on tumor-infiltrating lymphocytes.

[0356] The 40 mg protein dose resulted in the most favorable hemodynamics on day 4 and was sufficiently active to allow the tracer to diffuse and accumulate in the tumor until day 7 after tracer injection (Marcucci et al., Approaches to improve tumor accumulation and interactions between monoclonal antibodies and immune cells. MAbs. 2013;5(1):34-46). Higher tracer protein doses favor clearance mechanisms and allow this tracer to partially saturate the spleen, a highly perfused sink organ. The tumor-to-blood ratio increased from day 0 to day 7 for all tracer dose levels. Thus, the highest imaging contrast was achieved 7 days after tracer injection.

[0357] 89The biodistribution of Zr-DFO-REGN3767 suggests specific LAG3 targeting, with high uptake in lymphoid tissues such as the spleen. Tracer uptake increased in these tissues from day 0 to day 7, indicating specific tracer accumulation over time. Furthermore, partial saturation was observed with increasing total protein dose. Sites of inflammation were also affected. 89The tracer was visualized with Zr-DFO-REGN3767. The liver and kidneys also showed high tracer uptake. However, in contrast to lymphatic tissue, tracer uptake decreased over time, suggesting nonspecific tracer accumulation in those organs. Moderate tracer uptake was observed in the gastrointestinal tract. This may be due to immune cells in the small intestine and colon, although fecal excretion may influence these measurements. A surprising finding was the tracer uptake seen in the testes, ovaries, and uterus. Some reports indicate that T cells and other immune cells are involved in maintaining immune tolerance and play a role in supporting ovarian follicle growth and fallopian tube formation (Yeaman et al., CD8+ T cells in human uterine endometrial lymphoid aggregates: evidence for accumulation of cells by trafficking. Immunology. 2001;102(4):434-40; Zhao et al., Testicular defense systems: immune privilege and innate immunity. Cell Mol Immunol. 2014;11(5):428-37; Gong et al., T lymphocytes and testicular immunity: A new insight into immune regulation in testes. Int J Mol Sci. 2020;22(1); Winship et al., Checkpoint inhibitor immunotherapy decreases oocyte number and quality in mice. Nat Cancer. 2022;3(8):1-13). Therefore, activity observed in these areas may reflect specific tracer accumulation.

[0358] In summary, this test 89We demonstrated that LAG3 PET imaging using Zr-DFO-REGN3767 is safe and feasible in patients with advanced solid tumors. 89 Zr-DFO-REGN3767 specifically accumulates in tumor lesions and lymphatic tissues. Optimal imaging results were achieved using a 40 mg tracer dose and PET imaging 7 days after tracer injection.

[0359] The above-described embodiments and examples are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope and encompassed by the appended claims. All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety.

[0360] [Table 18] TIFF2026506001000029.tif230170TIFF2026506001000030.tif225170TIFF2026506001000031.tif221170TIFF2026506001000032.tif225170TIFF2026506001000033.tif226170TIFF2026506001000034.tif221170TIFF2026506001000035.tif221170TIFF2026506001000036.tif225170TIFF2026506001000037.tif230170TIFF2026506001000038.tif225170TIFF2026506001000039.tif225170TIFF2026506001000040.tif221170TIFF2026506001000041.tif226170TIFF2026506001000042.tif226170TIFF2026506001000043.tif226170TIFF2026506001000044.tif226170TIFF2026506001000045.tif221170TIFF2026506001000046.tif225170TIFF2026506001000047.tif221170TIFF2026506001000048.tif225170TIFF2026506001000049.tif230170TIFF2026506001000050.tif225170TIFF2026506001000051.tif221170TIFF2026506001000052.tif221170TIFF2026506001000053.tif225170TIFF2026506001000054.tif221170TIFF2026506001000055.tif221170TIFF2026506001000056.tif225170TIFF2026506001000057.tif221170TIFF2026506001000058.tif225170TIFF2026506001000059.tif221170TIFF2026506001000060.tif225170TIFF2026506001000061.tif225170TIFF2026506001000062.tif230170TIFF2026506001000063.tif221170TIFF2026506001000064.tif226170TIFF2026506001000065.tif226170TIFF2026506001000066.tif229170TIFF2026506001000067.tif228170TIFF2026506001000068.tif224170TIFF2026506001000069.tif224170TIFF2026506001000070.tif130170.

Claims

1. 1. A method of imaging a LAG3 positive tumor in a subject, comprising: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3); the antibody or antigen-binding fragment thereof, in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; ８９ labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography computed tomography (PET / CT) imaging; 1. A method for imaging a LAG3-positive tumor in a subject, comprising:

2. The chelating agent is selected from the group consisting of desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α''α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H ４ Oktapa, H. ６ Hospa, H ２ Dedopa, H ５ Dekapa, H ２ Azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cycl 10. The method of claim 1, wherein the chelator is selected from the group consisting of 1,4,8,11-tetraazacyclotetradecane (Cyclam), octadentate chelators, hexadentate chelators, phosphonate-based chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinine C and fusarinine C derivative chelators, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), and the like.

3. The method of claim 2 , wherein the chelating agent is DFO.

4. 10. The method of claim 1, wherein the label provides about 1 mCi of radiation upon injection.

5. 10. The method of any one of the preceding claims, wherein the subject is administered about 0.2 mg to about 3.0 mg of the labeled antibody conjugate.

6. 10. The method of any one of the preceding claims, wherein the subject is administered about 1.0 mg to about 2.0 mg of the labeled antibody conjugate.

7. 10. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered to the subject in a total amount of about 20 to 100 mg.

8. 10. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered to the subject in a total amount of about 30 to 50 mg.

9. 10. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 40 mg.

10. 10. The method of any one of the preceding claims, wherein the imaging step of (ii) is performed about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days after step (i).

11. 11. The method of claim 10, wherein the imaging step (ii) is performed 7 days after step (i).

12. The method of any one of claims 1 to 11, wherein the tumor is a solid tumor.

13. The tumor is selected from the group consisting of anal cancer, anaplastic thyroid carcinoma, astrocytoma, bladder cancer, bone cancer, glioblastoma multiforme, brain cancer, triple-negative breast cancer, breast cancer, cervical cancer, chondrosarcoma, clear cell carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, fibrosarcoma, gastric carcinoma, glioblastoma, head and neck cancer, hepatocellular carcinoma, jejunal cancer, renal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, metastatic cervical cancer, metastatic melanoma, myeloma, multiple myeloma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, clear cell renal carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, squamous cell carcinoma of the head and neck, and stomach cancer.

12. The method of any one of claims 1 to 11, wherein the cancer is selected from the group consisting of: thyroid cancer, synovial sarcoma, testicular cancer, thyroid cancer, uterine cancer, and Wilms' tumor.

14. The method of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO: 418 and three CDRs in the LCVR set forth in SEQ ID NO:

426.

15. The method of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO:

432.

16. The method of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO:418 and the LCVR set forth in SEQ ID NO:

426.

17. 1. A method of treating a subject, comprising: (i) administering to a subject having a tumor an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3); the antibody or antigen-binding fragment thereof, in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; ８９ labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the imaging step of (ii) is performed 7 days after step (i), and the presence of the radiolabeled antibody conjugate in the tumor indicates the presence of LAG3-positive cells within the tumor; (iii) administering one or more doses of the anti-tumor therapy to a subject in need thereof; A method of treating a subject, comprising:

18. The chelating agent is selected from the group consisting of desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α''α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H ４ Oktapa, H. ６ Hospa, H ２ Dedopa, H ５ Dekapa, H ２ Azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cycl 18. The method of claim 17, wherein the chelator is selected from the group consisting of 1,4,8,11-tetraazacyclotetradecane (Cyclam), octadentate chelators, hexadentate chelators, phosphonate-based chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinine C and fusarinine C derivative chelators, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), and the like.

19. 19. The method of claim 18, wherein the chelating agent is DFO.

20. 18. The method of claim 17, wherein the label provides 1 mCi of radiation upon injection.

21. 21. The method of any one of claims 17 to 20, wherein the subject is administered about 0.2 mg to about 3.0 mg of the labeled antibody conjugate.

22. 22. The method of any one of claims 17 to 21, wherein the subject is administered about 1.0 mg to about 2.0 mg of the labeled antibody conjugate.

23. 23. The method of any one of claims 17 to 22, wherein the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 20 to about 100 mg.

24. The method of any one of claims 17 to 23, wherein the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 30 to about 50 mg.

25. The method of any one of claims 17 to 24, wherein the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 40 mg.

26. The method of any one of claims 17 to 25, wherein the tumor is a solid tumor.

27. The tumor is selected from the group consisting of anal cancer, anaplastic thyroid carcinoma, astrocytoma, bladder cancer, bone cancer, glioblastoma multiforme, brain cancer, triple-negative breast cancer, breast cancer, cervical cancer, chondrosarcoma, clear cell carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, fibrosarcoma, gastric carcinoma, glioblastoma, head and neck cancer, hepatocellular carcinoma, jejunal cancer, renal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, metastatic cervical cancer, metastatic melanoma, myeloma, multiple myeloma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, clear cell renal carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, squamous cell carcinoma of the head and neck, and stomach cancer.

26. The method of any one of claims 17 to 25, wherein the cancer is selected from the group consisting of: uterine cancer, synovial sarcoma, testicular cancer, thyroid cancer, uterine cancer, and Wilms' tumor.

28. 28. The method of any one of claims 17 to 27, wherein the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR of SEQ ID NO: 418 and three CDRs in the LCVR of SEQ ID NO:

426.

29. The method of any one of claims 17 to 28, wherein the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO:

432.

30. The method of any one of claims 17 to 29, wherein the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO:418 and the LCVR set forth in SEQ ID NO:

426.

31. 18. The method of claim 17, wherein the anti-tumor therapy is selected from the group consisting of an inhibitor of LAG3, an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody drug conjugate (ADC).

32. The anti-tumor therapy is selected from the group consisting of anti-LAG3 antibody, REGN2810, BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, anti-CD-28 antibody, anti-2B4 antibody, anti-LY108 antibody, anti-LAIR1 antibody, anti-ICOS antibody, anti-CD160 antibody, anti-VISTA antibody, aflibercept, bevacizumab, and ranibizumab. Mab, sunitinib, sorafenib, pazopanib, nesbacumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin 18. The method of claim 17, wherein the therapeutic agent is selected from the group consisting of: anti-CD19-DM4 ADC, anti-DS6 ...

33. 28. The method of claims 17-27, wherein the presence of LAG3-positive cells in the tumor identifies the subject as a candidate for anti-tumor therapy comprising an inhibitor of LAG3 or the PD-1 / PD-L1 signaling axis.

34. 34. The method of claim 33, wherein the anti-tumor therapy is selected from the group consisting of an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, and an anti-PD-L1 antibody or antigen-binding fragment thereof.

35. 35. The method of claim 34, wherein the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof.

36. 35. The method of claim 34, wherein the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof selected from the group consisting of REGN2810, nivolumab, and pembrolizumab.

37. 35. The method of claim 34, wherein the anti-tumor therapy is an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a platinum-based chemotherapeutic agent.

38. 38. The method of claim 37, wherein the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin.

39. 35. The method of claim 34, wherein the anti-tumor therapy is an anti-PD-L1 antibody or antigen-binding fragment thereof selected from the group consisting of atezolizumab, avelumab, and durvalumab.

40. The anti-tumor therapy is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 428 / 430, 432 / 440, 436 / 442, 446 / 450, 452 / 460, 460 / 470, 472 / 480, 482 / 490, 492 / 500, 502 / 510, 512 / 520, 514 / 530, 526 / 540, 532 / 550, 542 / 552, 546 / 554, 556 / 560, 562 / 570, 572 / 580, 574 / 580, 582 / 590, 592 / 600, 602 / 610, 614 / 620, 614 / 630, 614 / 640, 614 / 650, 614 / 660, 614 / 670, 6 35. The method of claim 34, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs) in a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the group consisting of 34 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

41. The anti-tumor therapy is selected from the group consisting of SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16, 20 / 22 / 24 / 28 / 30 / 32, 36 / 38 / 40 / 44 / 46 / 48, 52 / 54 / 56 / 60 / 62 / 64, 68 / 70 / 72 / 76 / 78 / 80, 84 / 86 / 88 / 92 / 94 / 96, 100 / 102 / 104 / 108 / 110 / 112, 116 / 118 / 120 / 124 / 126 / 128, 132 / 134 / 136 / 140 / 142 / 144, 148 / 150 / 152 / 156 / 158 / 160, 164 / 166 / 168 / 172 / 174 / 176, 180 / 18 2 / 184 / 188 / 190 / 192, 196 / 198 / 200 / 204 / 206 / 208, 212 / 214 / 216 / 220 / 222 / 224, 228 / 230 / 232 / 236 / 238 / 240, 244 / 246 / 248 / 252 / 254 / 256, 260 / 262 / 264 / 268 / 270 / 272, 276 / 278 / 280 / 284 / 286 / 288, 292 / 294 / 296 / 300 / 302 / 304, 308 / 310 / 312 / 316 / 318 / 320, 324 / 326 / 328 / 332 / 334 / 336, 340 / 342 / 344 / 348 / 350 / 352, 356 / 358 / 360 / 364 / 366 / 368, 372 / 374 / 376 / 380 / 382 / 384, 388 / 390 / 392 / 396 / 398 / 400, 404 / 406 / 408 / 412 / 414 / 416, 420 / 422 / 424 / 4 28 / 430 / 432, 436 / 438 / 440 / 444 / 446 / 448, 452 / 454 / 456 / 524 / 526 / 528, 460 / 462 / 464 / 524 / 526 / 528, 468 / 470 / 472 / 524 / 526 / 528, 476 / 478 / 480 / 524 / 35. The method of claim 34, wherein the antibody or antigen-binding fragment thereof is an anti-LAG3 antibody or antigen-binding fragment thereof, comprising three HCDRs and three LCDRs selected from the group consisting of 526 / 528, 484 / 486 / 488 / 524 / 526 / 528, 492 / 494 / 496 / 524 / 526 / 528, 500 / 502 / 504 / 532 / 534 / 536, 508 / 510 / 512 / 532 / 534 / 536, 516 / 518 / 520 / 532 / 534 / 536, 540 / 542 / 544 / 548 / 550 / 552, and 556 / 558 / 560 / 564 / 566 / 568.

42. 35. The method of claim 34, wherein the anti-tumor therapy is an anti-LAG3 antibody or antigen-binding fragment thereof comprising three HCDRs in the HCVR set forth in SEQ ID NO: 418 and three LCDRs in the LCVR set forth in SEQ ID NO:

426.

43. 35. The method of claim 17 or claim 34, wherein the anti-tumor therapy is administered in combination with a second anti-tumor therapy.

44. 44. The method of claim 43, wherein the second anti-tumor therapy is selected from the group consisting of an inhibitor of the PD-1 / PD-L1 signaling axis, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitory factor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody drug conjugate (ADC).

45. The second anti-tumor therapy is selected from the group consisting of an anti-LAG3 antibody, REGN2810, BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, an anti-CD-28 antibody, an anti-2B4 antibody, an anti-LY108 antibody, an anti-LAIR1 antibody, an anti-ICOS antibody, an anti-CD160 antibody, an anti-VISTA antibody, aflibercept, bevacizumab, and ranilizumab. Telomere, sunitinib, sorafenib, pazopanib, nesbacumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-MUC16 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin 44. The method of claim 43, wherein the therapeutic agent is selected from the group consisting of: anti-CD19-DM4 ADC, anti-DS6 ...

46. 18. The method of claim 17, wherein step (iii) is performed more than once.

47. 18. The method of claim 17, wherein steps (ii) and (iii) are performed on the same day.

48. 18. The method of claim 17, wherein steps (i) and (ii) are repeated.

49. 18. The method of claim 17, further comprising (iv) repeating steps (i) and (ii), wherein step (ii) is performed after step (iii).

50. 18. The method of claim 17, further comprising (iv) repeating steps (i) and (ii), wherein step (iv) is performed after step (iii).

51. 51. The method of claim 50, wherein step (iii) is performed twice before step (iv).

52. 22. The method of claims 17-21, further comprising obtaining a tumor sample from the subject and determining the presence of LAG3 in the tumor sample.

53. The method of claims 17 to 52, further comprising measuring tumor response to said anti-tumor therapy.

54. 54. The method of claim 53, wherein measuring the tumor response comprises a reduction or disappearance of the size and / or number of tumor lesions.

55. (i) an unlabeled anti-LAG3 antibody or antigen-binding fragment thereof; and (ii) an anti-LAG3 antibody or antigen-binding fragment thereof that provides about 0.5 to 3.0 mCi of radioactivity. ８９ a Zr-labeled anti-LAG3 antibody conjugate, the total amount of labeled and unlabeled antibody or antigen-binding fragment thereof present in the composition is about 40 mg; the antibody or antigen-binding fragment thereof, in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; composition.

56. 56. The composition of claim 55, wherein the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO: 418 and three CDRs in the LCVR set forth in SEQ ID NO:

426.

57. The composition of any one of claims 55 or 56, wherein the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO:

432.

58. 58. The composition of any one of claims 55 to 57, wherein the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO:418 and the LCVR set forth in SEQ ID NO:

426.

59. The aforementioned ８９ The composition of any one of claims 55 to 58, wherein the Zr-labeled anti-LAG3 antibody conjugate comprises the anti-LAG3 antibody or antigen-binding fragment thereof conjugated to desferrioxamine (DFO).

60. The aforementioned ８９ 60. The composition of any one of claims 55 to 59, wherein the Zr-labeled anti-LAG3 antibody conjugate provides a radioactivity of about 1 mCi.

61. 61. The composition of any one of claims 55-60, wherein the labeled anti-LAG3 antibody conjugate is present in the composition in an amount of about 1-2 mg.

62. 1. A formulation comprising: three heavy chain complementarity determining regions (HCDRs) in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; and Linked to a portion of an anti-LAG3 antibody or antigen-binding fragment thereof ８９ Zr radiolabel Including, the radiolabel provides about 0.5 to about 3 mCi of radiation per formulation; the formulation is adapted for administration to a human at a total dosage of about 40 mg of the antibody or antigen-binding fragment thereof; formulation.

63. 63. The formulation of claim 62, wherein the antibody or antigen-binding fragment thereof comprises three CDRs in the HCVR set forth in SEQ ID NO: 418 and three CDRs in the LCVR set forth in SEQ ID NO:

426.

64. The formulation of any one of claims 62 or 63, wherein the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising SEQ ID NO: 420, an HCDR2 comprising SEQ ID NO: 422, and an HCDR3 comprising SEQ ID NO: 424; an LCDR1 comprising SEQ ID NO: 428, an LCDR2 comprising SEQ ID NO: 430, and an LCDR3 comprising SEQ ID NO:

432.

65. 65. The formulation of any one of claims 62-64, wherein the antibody or antigen-binding fragment thereof comprises the HCVR set forth in SEQ ID NO:418 and the LCVR set forth in SEQ ID NO:

426.

66. 66. The formulation of any one of claims 62 to 65, wherein the radiolabel provides about 1 mCi of radiation.

67. A kit comprising the composition of any one of claims 55 to 61.

68. 68. The kit of claim 67, providing instructions for PET imaging 7 days after administration of the composition to a patient.

69. 1. A method of imaging a LAG3 positive tumor in a subject, comprising: (i) administering to the subject an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3); the antibody or antigen-binding fragment thereof, in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; ８９ labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount of about 40 mg and provides about 1 mCi of radiation upon injection; administering (ii) imaging the localization of the labeled antibody conjugate by positron emission tomography (PET) imaging or positron emission tomography computed tomography (PET / CT) imaging, wherein the imaging is performed 7 days after step (i); 1. A method for imaging a LAG3-positive tumor in a subject, comprising:

70. 70. The method of claim 69, wherein the antibody or antigen-binding fragment thereof comprises three HCDRs in the HCVR set forth in SEQ ID NO: 418 and three LCDRs in the LCVR set forth in SEQ ID NO:

426.

71. 70. The method of claim 69, wherein the subject is administered one or more doses of an anti-tumor therapy upon determination that the subject contains LAG3-positive cells within the tumor.

72. 1. A method of treating a subject, comprising: (i) administering to a subject having a tumor an antibody or antigen-binding fragment thereof that binds to lymphocyte activation gene-3 (LAG3); the antibody or antigen-binding fragment thereof, in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and 554; three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562; at least a portion of the antibody or antigen-binding fragment thereof is conjugated to a chelating moiety and a positron emitter; ８９ labeled with Zr, the antibody or antigen-binding fragment thereof is administered to the subject in an amount that provides 0.5 to 3.0 mCi + / - 20% radiation; administering (ii) imaging the localization of the labeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the imaging step of (ii) is performed 7 days after step (i), and the presence of the radiolabeled antibody conjugate in the tumor indicates the presence of LAG3-positive cells within the tumor; (iii) if LAG3-positive cells are present in the tumor, administering one or more doses of an anti-tumor therapy to the subject in need thereof; A method of treating a subject, comprising:

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