Anti-IL2RA antibodies and uses thereof

Abstract

Anti-IL2RA (interleukin-2 receptor alpha chain) antibodies, antigen-binding fragments, and uses thereof are provided.

Description

[Technical Field]

[0001] Priority claims This application claims priority to PCT / CN2023 / 077623, filed February 22, 2023. The entire contents of the above-mentioned application are incorporated herein by reference.

[0002] The present disclosure relates to anti-IL2RA (interleukin-2 receptor alpha chain) antibodies and uses thereof. [Background technology]

[0003] Cancer is currently one of the diseases with the highest mortality rates among humans. According to statistics from the World Health Organization, the number of cancer cases and deaths worldwide reached 14 million and 8.2 million, respectively, in 2012. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million deaths.

[0004] The recent clinical and commercial success of anti-cancer antibodies has generated significant interest in antibody-based therapies, and there is a need to develop antibodies for use in a variety of antibody-based therapeutic approaches to treat cancer or autoimmune diseases. Summary of the Invention

[0005] The present disclosure relates to anti-IL2RA antibodies, antigen-binding fragments thereof, and uses thereof.

[0006] In one aspect, the disclosure provides a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence. and a light chain variable region comprising an amino acid sequence at least 80% identical to the VH CDR3 amino acid sequence, in some embodiments, the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following:

[0007] (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43, 44, and 45, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4, 5, and 6, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46, 47, and 48, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 10, 11, and 12, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 13, 14, and 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 16, 17, and 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, and 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, and 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 43, 44, and 45, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 46, 47, and 48, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, and 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (11) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (12) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; (13) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 37, 38, and 39, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively; and (14) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 40, 41, and 42, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively.

[0008] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively.

[0009] In some embodiments, the antibody or antigen-binding fragment specifically binds to human IL2RA or monkey IL2RA. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody). In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv), or the antibody is a multispecific antibody (e.g., a bispecific antibody).

[0010] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide, (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 53; (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 52; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 55; (4) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 54; (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 56; (7) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (8) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 57; (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (10) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 58; (11) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (12) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 59; (13) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (14) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein in some embodiments, the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 60; (15) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 53; (16) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 55; (17) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (18) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (19) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (20) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; or (21) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, wherein in some embodiments, the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61. Concerning nucleic acids.

[0011] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, or SEQ ID NOs: 22, 23, and 24, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively.

[0012] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, or SEQ ID NOs: 25, 26, and 27, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively.

[0013] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, or SEQ ID NOs: 28, 29, and 30, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, or SEQ ID NOs: 31, 32, and 33, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, or SEQ ID NOs: 34, 35, and 36, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, or SEQ ID NOs: 37, 38, and 39, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, or SEQ ID NOs: 40, 41, and 42, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively.

[0014] In some embodiments, the VH, when paired with the VL, specifically binds human IL2RA, or the VL, when paired with the VH, specifically binds human IL2RA or simian IL2RA. In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof. In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is a cDNA.

[0015] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure relates to a vector comprising two of the nucleic acids described herein, in some embodiments, the vectors encoding a VH region and a VL region that both bind IL2RA. In one aspect, the disclosure relates to a pair of vectors, in some embodiments, each vector comprising one of the nucleic acids described herein, and in some embodiments, encoding a VH region and a VL region that both bind IL2RA.

[0016] In one aspect, the disclosure relates to a cell comprising a vector or a pair of vectors described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein. In one aspect, the disclosure relates to a cell comprising two of the nucleic acids described herein. In some embodiments, the two nucleic acids together encode a VH region and a VL region that both bind to IL2RA.

[0017] In one aspect, the disclosure relates to a method for producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) recovering the antibody or antigen-binding fragment produced by the cell.

[0018] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to IL2RA, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence; in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:52 and the selected VL sequence is SEQ ID NO:53; (2) the selected VH sequence is SEQ ID NO:54 and the selected VL sequence is SEQ ID NO:55; (3) the selected VH sequence is SEQ ID NO:56 and the selected VL sequence is SEQ ID NO:61; (4) the selected VH sequence is SEQ ID NO:57 and the selected VL sequence is SEQ ID NO:61; (5) the selected VH sequence is SEQ ID NO:58 and the selected VL sequence is SEQ ID NO:61; (6) the selected VH sequence is SEQ ID NO:59 and the selected VL sequence is SEQ ID NO:61; and (7) the selected VH sequence is SEQ ID NO:60 and the selected VL sequence is SEQ ID NO:61.

[0019] In some embodiments, the VH comprises the sequence of SEQ ID NO: 52 and the VL comprises the sequence of SEQ ID NO: 53. In some embodiments, the VH comprises the sequence of SEQ ID NO: 54 and the VL comprises the sequence of SEQ ID NO: 55. In some embodiments, the VH comprises the sequence of SEQ ID NO: 56 and the VL comprises the sequence of SEQ ID NO: 61. In some embodiments, the VH comprises the sequence of SEQ ID NO: 57 and the VL comprises the sequence of SEQ ID NO: 61. In some embodiments, the VH comprises the sequence of SEQ ID NO: 58 and the VL comprises the sequence of SEQ ID NO: 61. In some embodiments, the VH comprises the sequence of SEQ ID NO: 59 and the VL comprises the sequence of SEQ ID NO: 61. In some embodiments, the VH comprises the sequence of SEQ ID NO: 60 and the VL comprises the sequence of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment specifically binds to human IL2RA or simian IL2RA. In some embodiments, the antibody or antigen-binding fragment is a human antibody or humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv), or the antibody is a multispecific antibody (e.g., a bispecific antibody).

[0020] In one aspect, the present disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0021] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to IL2RA, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence; in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:52 and the selected VL sequence is SEQ ID NO:53; (2) the selected VH sequence is SEQ ID NO:54 and the selected VL sequence is SEQ ID NO:55; (3) the selected VH sequence is SEQ ID NO:56 and the selected VL sequence is SEQ ID NO:61; (4) the selected VH sequence is SEQ ID NO:57 and the selected VL sequence is SEQ ID NO:61; (5) the selected VH sequence is SEQ ID NO:58 and the selected VL sequence is SEQ ID NO:61; (6) the selected VH sequence is SEQ ID NO:59 and the selected VL sequence is SEQ ID NO:61; and (7) the selected VH sequence is SEQ ID NO:60 and the selected VL sequence is SEQ ID NO:61.

[0022] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently attached to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0023] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, as described herein. In some embodiments, the subject has solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, and breast cancer. In some embodiments, the methods described herein further comprise administering to the subject a therapeutically effective amount of an anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

[0024] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or antibody drug conjugate described herein.

[0025] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or antibody drug conjugate described herein.

[0026] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

[0027] In one aspect, the present disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof described herein.

[0028] As used herein, the term "cancer" refers to cells capable of autonomous proliferation. Examples of such cells include cells with an abnormal state or condition characterized by rapid proliferation of cell growth. The term is intended to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. It also includes malignant tumors of various organ systems, e.g., head and neck, respiratory, cardiovascular, renal, reproductive, hematological, nervous system, liver, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate and / or testicular tumors, non-small cell lung cancer, small intestine cancer, glioma, and small intestine cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, such as naturally occurring cancers, cancers caused by exposing a patient to a carcinogen, cancers caused by the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancers caused by infectious diseases, e.g., viral infections. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. It also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases can arise from the myeloid, lymphoid, or erythroid lineages, or their precursor cells. Blood cancers are cancers that begin in blood-forming tissues, such as the bone marrow, or in cells of the immune system. Examples of blood cancers include leukemia, lymphoma, and multiple myeloma.

[0029] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0030] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0031] As used herein, the term "human antibody" means an antibody encoded by endogenous nucleic acid derived from a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human antibodies are recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial cells or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cows) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0032] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different species (e.g., an antibody derived from two different mammalian species, such as a human and a murine antibody). A non-limiting example of a chimeric antibody is an antibody that contains variable domain sequences (e.g., all or part of the light and / or heavy chain variable domain sequences) of a non-human (e.g., murine) antibody and the constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0033] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and contains sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from hypervariable (e.g., CDR) regions of the recipient antibody are replaced by residues from hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, a humanized antibody contains nearly all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology techniques well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0034] As used herein, the term "single-chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) that are capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0035] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by this disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0036] As used herein, the phrases "specifically binding" and "specifically binds," when referring to an antibody, mean that the antibody interacts preferably with its target molecule (e.g., IL2RA) over other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to molecules containing a particular structure, rather than all molecules in general. An antibody that specifically binds to a target molecule can also be referred to as a target-specific antibody. For example, an antibody that specifically binds to an IL2RA molecule can be referred to as an IL2RA-specific antibody or an anti-IL2RA antibody.

[0037] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0038] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0039] 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 this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials well known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0040] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0041] [Figure 1] Figures AB show the blocking effect of the binding between IL2RA ligands and IL2RA in the presence of anti-IL2RA antibodies 11C3, 11C12, 11D2, 13F9, and 14C2 (Figure 1A); 5D9 and 7B5 (Figure 1B). A daclizumab analog was used as a control. [Figure 2]Panels A and B show the blocking effect of murine IL2 protein binding to IL2 receptor cells in the presence of anti-IL2RA antibodies 11C3, 13F9, 11D2, 14C2, and 11C12 (Figure 2A), 5D9, and 7B5 (Figure 2B) at concentrations of 100 μg / mL, 33.3 μg / mL, or 11.1 μg / mL. A daclizumab analog was used as a control. [Figure 3] Figures 3A-B show the blocking effect of STAT5 phosphorylation in human CD3+ T cells incubated with anti-IL2RA antibodies 11C12, 11D2, 11C3, 13F9, 14C2 (Figure 3A), 7B5, and 5D9 (Figure 3B), and human IL2 (hIL2). Daclizumab analogs were used as pSTAT5-blocking antibody controls. 7G7B6 analog-SI was used as a non-pSTAT5-blocking antibody control. [Figure 4] Figure 1 shows the time course of tumor volume in B-hIL2RA mice injected with MC38 cancer cells and treated with PBS (G1), 3 mg / kg 7B5 (G2), 10 mg / kg 7B5 (G3), 3 mg / kg 5D9 (G4), 10 mg / kg 5D9 (G5), 3 mg / kg 7G7B6 analog (G6), or 10 mg / kg 7G7B6 analog (G7). [Figure 5] This shows the time course of tumor volume in B-hIL2RA mice injected with MC38 cancer cells and treated with PBS (G1), 10 mg / kg 7B5 (G2), 10 mg / kg 5D9 (G3), 10 mg / kg 7G7B6 analog-SI (G4), or 10 mg / kg 7G7B6 analog (G5). [Figure 6] Figure 1 shows the time course of tumor volume in B-hIL2RA mice injected with MC38 cancer cells and treated with PBS (G1), 10 mg / kg of 7G7B6 analog-SI (G2), 10 mg / kg of 11C3 (G3), 10 mg / kg of 13F9 (G4), 10 mg / kg of 11D2 (G5), 10 mg / kg of 11C12 (G6), or 10 mg / kg of 14C2 (G7). [Figure 7] The Kabat CDR sequences of anti-IL2RA antibodies are listed. [Figure 8] The CDR sequences of the anti-IL2RA antibody Chothia are listed below. [Figure 9] The sequences of the heavy chain variable region and light chain variable region of anti-IL2RA antibodies are listed below. [Figure 10] The amino acid sequences discussed in this disclosure are listed below. DETAILED DESCRIPTION OF THE INVENTION

[0042] Interleukin-2 (IL-2) is a cytokine that can maintain the proliferative capacity of T lymphocytes. IL-2 is the major growth factor in activated T lymphocytes, and it has been shown to be capable of driving clonal expansion and maturation of effector cells. IL-2 stimulation can also induce proliferation of natural killer (NK) cells. IL-2 mediates its biological effects through the IL-2 receptor (IL-2R) complex. IL-2R is composed of three distinct subunits: the α chain, the β chain, and the γ chain. IL-2R binds to activated CD4 - , CD8 - , CD4 + 8 + , CD4 + , and CD8 + The importance of IL2RA in T cell proliferation is demonstrated by its high affinity for IL-2. IL-2R-mediated signaling pathways include Jak3-dependent activation of Jak1, which has been shown to be an important growth signal in fibroblasts. Increased expression of sIL2RA and IL2RA protein in tumor cells has been detected in various cancers, including lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, and breast cancer. Furthermore, increased expression of IL2RA mRNA and protein has been identified in many solid tumor types, including ovarian, lung, head and neck, and breast tumors. Therefore, anti-IL2RA antibodies could potentially be used for cancer therapy.

[0043] The present invention provides examples of antibodies and antigen-binding fragments thereof that bind to IL2RA (interleukin-2 receptor α chain).

[0044] IL2RA IL2RA (interleukin-2 receptor α chain, or CD25) is a type I membrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes. IL2RA has been used as a marker to identify CD4+FoxP3+ regulatory T cells in mice, but in humans, a large proportion of resting memory T cells has been found to constitutively express IL2RA. IL2RA is expressed in most B-cell tumors, some acute nonlymphocytic leukemias, neuroblastomas, mastocytosis, and tumor-infiltrating lymphocytes. It functions as a receptor for HTLV-1 and, as a result, is expressed on tumor cells in adult T-cell lymphoma / leukemia. Its soluble form, called sIL-2R, can be elevated in these diseases and is sometimes used to track disease progression.

[0045] The interleukin-2 (IL-2) receptor is formed by the α (IL-2RA, CD25), β (IL-2RB, CD122), and γ common (IL-2RG, CD132) subunits and plays an important role in maintaining the immune system. The interleukin-2 (IL2) receptor α (IL2RA) and β (IL2RB) chains, together with the common γ chain (IL2RG), constitute the high-affinity IL2 receptor. The homodimeric α chain (IL2RA) generates the low-affinity receptor, while the homodimeric β chain (IL2RB) generates the intermediate-affinity receptor. The high-affinity receptor for IL-2 incorporates all three chains (α, β, and γ) and is present on activated T cells, activated B cells, and Treg cells. The intermediate-affinity receptor is composed of only the γ and β chains and is expressed on resting T and B cells as well as NK cells. The low-affinity receptor is composed of the α chain and is expressed on dendritic cells.

[0046] Among IL-2 receptors, IL2RA is a unique subunit that binds only IL-2, whereas CD132 binds common γc family cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21), and the CD122 subunit binds IL-15. IL2RA is constitutively expressed at high levels by regulatory T cells (Tregs), enabling them to be the first responders to IL-2 during immune responses. IL2RA promotes FOXP3 transcription by amplifying IL-2 signaling in a STAT5-dependent manner. Interestingly, single nucleotide polymorphism (SNP) studies in the IL2RA gene have been associated with several forms of autoimmunity, demonstrating that IL-2 signaling via IL2RA is a key axis in regulating tolerance. IL2RA is also important for the proliferation of effector T cells in response to IL-2 immediately after antigen stimulation.

[0047] Formation of the high-affinity tetrameric IL-2 / IL-2R complex leads to signaling via the tyrosine kinases Jak1 and Jak3 associated with IL-2Rβ and γc, respectively. Three tyrosine residues within the cytoplasmic tail of IL-2Rβ are phosphorylated to promote recruitment of the adaptor Shc (human Y338, mouse Y341), leading to activation of MAPK and PI-3K kinase pathways and primarily the Stat5 transcription factor (human Y392 and Y510, mouse Y398 and Y505), resulting in Stat5-dependent gene regulation. The tetrameric IL-2-IL-2R complex is rapidly internalized, and IL-2, IL-2Rβ, and γc are rapidly degraded, whereas IL-2Rα is recycled back to the cell surface. Therefore, functional activities requiring sustained IL-2R signaling require a continuous source of IL-2 to associate with IL-2Rα and form additional IL-2-IL-2R signaling complexes.

[0048] IL2RA is a protein encoded by the IL2RA gene in humans. Soluble IL2RA, normally a transmembrane protein, has been isolated and found to be generated by extracellular proteolysis. Alternatively spliced ​​IL2RA mRNAs have been isolated, but the significance of each is currently unknown. Mutations in this gene are associated with interleukin-2 receptor alpha deficiency.

[0049] Detailed descriptions of IL2RA and its functions are found in, for example, Goudy, Kevin, et al., "Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity," Clinical Immunology 146.3 (2013): 248-261; Kuhn, Deborah J., and Q. Ping Dou. "The role of interleukin-2 receptor alpha in cancer," Front Biosci 10 (2005): 1462-1474; Malek, Thomas R., and Iris Castro. "Interleukin-2 receptor signaling: at the interface between tolerance and immunity," Immunity 33.2 (2010): 153-165; and Jiang et al., "Role of IL-2 in cancer immunotherapy," Oncoimmunology 5.6(2016):e1163462; both of which are incorporated by reference in their entireties.

[0050] The present disclosure provides anti-IL2RA antibodies, antigen-binding fragments thereof, and methods of using these anti-IL2RA antibodies and antigen-binding fragments to inhibit tumor growth and treat various diseases, including, for example, cancer.

[0051] Anti-IL2RA antibodies and antigen-binding fragments The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to IL2RA (e.g., human IL2RA). The antibodies and antigen-binding fragments described herein are capable of binding to IL2RA. In some embodiments, these antibodies do not block the binding of human IL2RA to an IL2RA ligand (e.g., human IL2). In some embodiments, these antibodies do not block the binding of IL2 protein to IL2 receptor cells. In some embodiments, these antibodies do not significantly inhibit human IL2-induced STAT5 phosphorylation. In some embodiments, these antibodies can enhance immune responses because they do not block the IL2 / IL2RA signaling pathway. In some embodiments, these antibodies can initiate complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, these antibodies bind to cells expressing IL2RA.

[0052] The present invention provides, for example, anti-IL2RA antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2, and modified antibodies thereof (including, for example, chimeric antibodies, humanized antibodies, and human antibodies).

[0053] CDR sequences for 5D9 and 5D9-derived antibodies (e.g., humanized antibodies) include the heavy chain variable domain CDRs set forth in SEQ ID NOS: 1, 2, and 3, and the light chain variable domain CDRs set forth in SEQ ID NOS: 43, 44, and 45, as defined by the Kabat definition. CDRs can also be defined by the Chothia system. Under the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 22, 23, and 24, and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 43, 44, and 45.

[0054] CDR sequences for 7B5 and 7B5-derived antibodies include, as defined by the Kabat definition, heavy chain variable domain CDRs SEQ ID NOS: 4, 5, 6, and light chain variable domain CDRs SEQ ID NOS: 46, 47, 48. Under the Chothia definition, heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 25, 26, 27, and light chain variable domain CDRs are set forth in SEQ ID NOS: 46, 47, 48.

[0055] CDR sequences for 11C3 and 11C3-derived antibodies include, as defined by the Kabat definition, heavy chain variable domain CDRs SEQ ID NOs: 7, 8, 9 and light chain variable domain CDRs SEQ ID NOs: 49, 50, 51. Under the Chothia definition, heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 28, 29, 30 and light chain variable domain CDRs are set forth in SEQ ID NOs: 49, 50, 51.

[0056] CDR sequences for 11C12 and antibodies derived from 11C12 include, as defined by the Kabat definition, heavy chain variable domain CDRs SEQ ID NOS: 10, 11, 12 and light chain variable domain CDRs SEQ ID NOS: 49, 50, 51. Under the Chothia definition, heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 31, 32, 33 and light chain variable domain CDRs are set forth in SEQ ID NOS: 49, 50, 51.

[0057] CDR sequences for 11D2 and 11D2-derived antibodies include, as defined by the Kabat definition, heavy chain variable domain CDRs SEQ ID NOs: 13, 14, 15 and light chain variable domain CDRs SEQ ID NOs: 49, 50, 51. Under the Chothia definition, heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 34, 35, 36 and light chain variable domain CDRs are set forth in SEQ ID NOs: 49, 50, 51.

[0058] CDR sequences for 13F9 and 13F9-derived antibodies, as defined by the Kabat definition, include the heavy chain variable domain CDRs SEQ ID NOs: 16, 17, 18 and the light chain variable domain CDRs SEQ ID NOs: 49, 50, 51. Under the Chothia definition, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 37, 38, 39 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 49, 50, 51.

[0059] CDR sequences for 14C2 and 14C2-derived antibodies include, as defined by the Kabat definition, heavy chain variable domain CDRs SEQ ID NOS: 19, 20, 21 and light chain variable domain CDRs SEQ ID NOS: 49, 50, 51. Under the Chothia definition, heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 40, 41, 42 and light chain variable domain CDRs are set forth in SEQ ID NOS: 49, 50, 51.

[0060] The amino acid sequence for the heavy chain variable region of the 5D9 antibody is shown in SEQ ID NO: 52. The amino acid sequence for the light chain variable region of the 5D9 antibody is shown in SEQ ID NO: 53.

[0061] The amino acid sequence for the heavy chain variable region of the 7B5 antibody is shown in SEQ ID NO: 54. The amino acid sequence for the light chain variable region of the 7B5 antibody is shown in SEQ ID NO: 55.

[0062] The amino acid sequence for the heavy chain variable region of the 11C3 antibody is shown in SEQ ID NO: 56. The amino acid sequence for the light chain variable region of the 11C3 antibody is shown in SEQ ID NO: 61.

[0063] The amino acid sequence for the heavy chain variable region of the 11C12 antibody is shown in SEQ ID NO: 57. The amino acid sequence for the light chain variable region of the 11C12 antibody is shown in SEQ ID NO: 61.

[0064] The amino acid sequence for the heavy chain variable region of the 11D2 antibody is shown in SEQ ID NO: 58. The amino acid sequence for the light chain variable region of the 11D2 antibody is shown in SEQ ID NO: 61.

[0065] The amino acid sequence for the heavy chain variable region of the 13F9 antibody is shown in SEQ ID NO: 59. The amino acid sequence for the light chain variable region of the 13F9 antibody is shown in SEQ ID NO: 61.

[0066] The amino acid sequence for the heavy chain variable region of the 14C2 antibody is shown in SEQ ID NO: 60. The amino acid sequence for the light chain variable region of the 14C2 antibody is shown in SEQ ID NO: 61.

[0067] Also provided are amino acid sequences for the heavy and light chain variable regions of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52, 54, 56, 57, 58, 59, or 60. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53, 55, or 61. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, both of which bind to IL2RA.

[0068] In some embodiments, the variable regions are fully human, e.g., derived from human heavy chain immunoglobulin locus sequences (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin locus sequences (e.g., a combination of human IGKV and IGKJ genes).

[0069] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1 to 3, SEQ ID NOs: 4 to 6, SEQ ID NOs: 7 to 9, SEQ ID NOs: 10 to 12, SEQ ID NOs: 13 to 15, SEQ ID NOs: 16 to 18, SEQ ID NOs: 19 to 21, SEQ ID NOs: 22 to 24, SEQ ID NOs: 25 to 27, SEQ ID NOs: 28 to 30, SEQ ID NOs: 31 to 33, SEQ ID NOs: 34 to 36, SEQ ID NOs: 37 to 39, and SEQ ID NOs: 40 to 42, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 43 to 45, SEQ ID NOs: 46 to 48, and SEQ ID NOs: 49 to 51.

[0070] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody can have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, and 3 amino acid sequences and selected VL CDR1, 2, and 3 amino acid sequences are shown in Figure 7 (CDRs according to the Kabat definition) and Figure 8 (CDRs according to the Chothia definition).

[0071] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0072] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 6 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0073] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0074] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0075] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0076] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0077] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0078] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 24 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0079] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 27 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0080] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0081] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 33 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0082] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0083] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 39 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0084] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 42 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0085] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 45 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0086] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 48 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0087] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 51 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0088] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, CDRs are determined according to the Kabat definition scheme. In some embodiments, CDRs are determined according to the Chothia definition scheme. In some embodiments, CDRs are determined according to a combination of the Kabat and Chothia definition schemes. In some embodiments, CDRs are determined according to the IMGT definition. In some embodiments, CDRs are determined according to the contact definition.

[0089] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to IL2RA. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising, or consisting of, an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 53. In some embodiments, the selected VH sequence is SEQ ID NO: 54 and the selected VL sequence is SEQ ID NO: 55. In some embodiments, the selected VH sequence is SEQ ID NO: 56 and the selected VL sequence is SEQ ID NO: 61. In some embodiments, the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO: 61. In some embodiments, the selected VH sequence is SEQ ID NO: 58 and the selected VL sequence is SEQ ID NO: 61. In some embodiments, the selected VH sequence is SEQ ID NO: 59 and the selected VL sequence is SEQ ID NO: 61. In some embodiments, the selected VH sequence is SEQ ID NO: 60 and the selected VL sequence is SEQ ID NO: 61.

[0090] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison). The length of the reference sequence aligned for comparison is at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, sequence comparisons and determination of percent identity between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0091] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs shown in Figure 7 or Figure 8, or has the sequence shown in Figure 9. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to IL2RA.

[0092] Anti-IL2RA antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multimeric, multispecific (e.g., bispecific), human, chimeric (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0093] Antibody fragments are suitable for use in the provided methods, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to IL2RA retain the ability to bind to IL2RA. Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight association, which can be essentially covalent, for example, in an scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs, or a subset thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for a given antigen) can have the ability to recognize and bind to antigen, although usually with lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0094] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein," Journal of Virology 70.3 (1996):1863-1872, the entire contents of which are incorporated herein by reference. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, the entire contents of which are incorporated herein by reference.

[0095] Antibodies and antigen-binding fragments The present disclosure provides various antibodies and antigen-binding fragments thereof derived from the anti-IL2RA antibodies described herein. Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains: light chains and heavy chains. Non-limiting examples of antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. The antibody can comprise two identical copies of the light chain and two identical copies of the heavy chain, each containing one variable domain (or variable region, V H ), and heavy chains containing multiple constant domains (or constant regions) linked together via disulfide bonds within their constant domains to form the "stem" of the antibody. L Each light chain, containing a constant domain (or constant region) and one light chain, binds to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0096] Hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0097] Methods for identifying CDR regions of antibodies by analyzing their amino acid sequences are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422~439; Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated by reference herein in its entirety.

[0098] CDRs are important for recognizing the epitope of an antigen. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0099] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences between IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in Immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated by reference in its entirety herein.

[0100] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is, or is homologous to, an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0101] Also provided are antibody fragments suitable for use in the methods described herein. Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments covalently linked, generally near their carboxy termini, by hinge cysteines between them. Other chemical linkages of antibody fragments are well known in the art.

[0102] Diabodies are small antibody fragments that contain two antigen-binding sites, a VH connected to a VL (VH and VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be paired with the complementary domains of another chain and create two antigen-binding sites.

[0103] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0104] The antibodies and antibody fragments of the disclosure can be modified in the Fc region to confer desired effector functions or serum half-lives.

[0105] Antibody multimerization can be achieved by natural antibody aggregation or by chemical or recombinant conjugation techniques well known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing antibody homodimers and other higher order antibody multimers.

[0106] Alternatively, antibody homodimers can be formed by chemical coupling techniques well known in the art. For example, heterobifunctional cross-linkers, including but not limited to SMCC (4-(maleimidomethyl)cyclohexane-1-carboxylate succinimidyl) and SATA (N-S-acetylthioacetate N-succinimidyl), can be used to form antibody multimers. An exemplary procedure for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is the use of autophilic T15 peptide, as described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0107] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers. This method is described, for example, in WO 96 / 27011, incorporated by reference in its entirety.

[0108] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, incorporated herein by reference in its entirety.

[0109] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumins, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or prolong the biological activity of the antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture medium or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0110] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinplastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

[0111] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, for increased efficacy. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0112] In some embodiments, an scFv has one heavy chain variable domain and one light chain variable domain, hi some embodiments, an scFv has two heavy chain variable domains and two light chain variable domains.

[0113] In some embodiments, the sequences of the antibodies or antigen-binding fragments thereof described herein (e.g., CDR or VH / VL sequences) can be used to generate bispecific antibodies that target IL2RA and an additional antigen (e.g., OX40, CD3, 4-1BB, CD314, CD47, PD-1, CTLA4, CD40, or PDL1).

[0114] Antibody characteristics In some embodiments, the antibodies or antigen-binding fragments thereof described herein are unable to block the binding between IL2RA and an IL2RA ligand (e.g., IL2). Thus, the antibodies do not inhibit the IL2RA signaling pathway upon binding to IL2RA. In some embodiments, the antibodies are capable of upregulating an immune response. In some embodiments, the antibodies are capable of reducing tumor volume in an animal.

[0115] In some embodiments, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 Specifically binds to IL2RA (e.g., human IL2RA or monkey IL2RA) with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0116] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0117] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10-10 Super M, 1×10 -11 Over M or 1 x 10 -12 It's over M.

[0118] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human IL2RA (SEQ ID NO: 64) and / or monkey IL2RA (e.g., SEQ ID NO: 65). In some embodiments, the antibody does not bind to human IL2RA and / or monkey IL2RA.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not inhibit IL2-induced STAT5 phosphorylation, or inhibit STAT5 phosphorylation by less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%.

[0120] In some embodiments, the antibodies or antigen-binding fragments thereof described herein exhibit a tumor growth inhibition (TGI) percentage of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TV %). In some embodiments, the antibody has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after initiation of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after initiation of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula:

number

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are IL2RA antagonists. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are IL2RA agonists.

[0122] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are non-toxic, and in some embodiments, no significant difference in body weight can be observed between treatment and control groups, for example, at 0.3 mg / kg, 1 mg / kg, 10 mg / kg, or 25 mg / kg.

[0123] In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) to kill tumor cells.

[0124] In some embodiments, the antibody or antigen-binding fragment comprises a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0125] In some embodiments, the antibody or antigen-binding fragment is capable of inducing complement-dependent cytotoxicity (CDC).

[0126] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4, hi some embodiments, the antibody is a human IgG1 antibody.

[0127] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0128] In some embodiments, the Fc has SI mutations (S239D and I332E mutations in EU numbering).

[0129] Method for producing anti-IL2RA antibody An isolated fragment (e.g., the extracellular region) of human IL2RA can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. Animals can be injected with the antigenic peptide or protein two or more times (e.g., two, three, or four times).

[0130] The full-length polypeptide or protein can be used, or an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of IL2RA and encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein. As mentioned above, the full-length sequence of human IL2RA is well known in the art (SEQ ID NO: 64). In some embodiments, an Fc-tagged human IL2RA protein (an Fc-fusion protein containing all or a portion of the human IL2RA extracellular domain, e.g., positions 22 to 213 of SEQ ID NO: 64) is used as an immunogen.

[0131] An immunogen is typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of human IL2RA). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0132] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with an IL2RA polypeptide or an antigenic peptide thereof (e.g., a portion of IL2RA, such as the extracellular domain) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized IL2RA polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein G or protein A chromatography, to obtain an IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titer is at its highest, antibody-producing cells are obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Hybridoma production techniques are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY)). For example, hybridoma cells producing monoclonal antibodies are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest using a standard ELISA assay.

[0133] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that makes up the antigen-binding site or domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments have increased affinity for a target protein, e.g., IL2RA. Any combination of deletion, insertion, and / or a combination can be achieved in an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered by introducing amino acid changes into the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites, or by introducing new post-translational modifications into the antibody or antigen-binding fragment.

[0134] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0135] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0136] Humanized antibodies typically have a human framework (FR) into which non-human CDRs have been grafted. Thus, a humanized antibody has one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable domain. Humanization can essentially be performed, for example, by substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. These methods are described, for example, in Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse," Nature 321.6069 (1986):522; Riechmann et al. "Reshaping human antibodies for therapy," Nature 332.6162 (1988):323; and Dall'Acqua et al. "Antibody humanization by framework shuffling," Methods 36.1 (2005):43-60, each of which is incorporated by reference herein in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which significantly less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically murine antibodies in which some CDR residues and some FR residues have been substituted by residues from analogous sites in human antibodies.

[0137] The selection of human VH and VL domains used in the production of humanized antibodies is crucial to reducing immunogenicity. According to the so-called "best-fit" method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human FR for the humanized antibody (Sims et al., "A humanized CD18 antibody can block function without cell destruction," The Journal of Immunology 151.4 (1993):2296-2308; Chothia, et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of molecular biology 196.4 (1987):901-917).

[0138] Furthermore, it is important to humanize antibodies while retaining high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that can illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0139] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-IL2RA antibody contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with the sequence present in the light or heavy chain of the original antibody.

[0140] In some embodiments, antibodies are generated using mice (e.g., RenMab mice) with a humanized heavy chain immunoglobulin locus and a humanized κ chain immunoglobulin locus. The heavy chain immunoglobulin locus is a region on a chromosome containing genes for the heavy chain of an antibody. The locus can include, for example, a human IGHV (variable) gene, a human IGHD (diversity) gene, a human IGHJ (joining) gene, and a mouse heavy chain constant domain gene. The κ chain immunoglobulin locus is a region on a chromosome containing genes encoding the light chain (κ chain) of an antibody. Examples of the κ chain immunoglobulin locus include a human IGKV (variable) gene, a human IGKJ (joining) gene, and a mouse light chain constant domain gene. A detailed description of the RenMab mouse can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety. Antibodies generated by the mice have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to a human IgG constant region (eg, IgG1, IgG2, IgG3, and IgG4).

[0141] In some embodiments, mice with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenLite TMThe heavy chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (joining) gene, and the mouse heavy chain constant domain gene. The kappa chain immunoglobulin locus is a region on a chromosome that contains genes encoding the common light chain. Examples of kappa chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (joining) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mice can be found in PCT / CN2021 / 097652, which is incorporated by reference herein in its entirety.

[0142] Identity or homology to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to sequences present in the human antibody or fragment, humanized antibody or fragment, or chimeric anti-IL2RA antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity.

[0143] Further modifications can be made to the anti-IL2RA antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have some increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-lives can also be prepared using heterobifunctional cross-linkers, e.g., as described by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993):2560-2565). Alternatively, antibodies having dual Fc regions can be engineered.

[0144] In some embodiments, covalent modifications can be made to anti-IL2RA antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0145] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibody compositions may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 (position 314 in the EU numbering of Fc region residues or Kabat numbering) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody can be further modified to replace asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0146] In some embodiments, to promote production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further modified by substituting proline for serine at position 228 (EU numbering) of IgG4 (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation," Journal of Biological Chemistry 290.9 (2015):5462-5469, which is incorporated by reference in its entirety.

[0147] Recombinant vector The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors comprising the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0148] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, at, near, or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in a host cell into which the expression vector is introduced.

[0149] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0150] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may employ a replication-defective virus, in which case viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321;Flexner et al.,1989,Ann.NYAcad Sci.569:86-103;Flexner et al. al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; 91 / 02805;Berkner-Biotechniques,6:616-627,1988;Rosenfeld et al. Suitable systems are disclosed in Kolls et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be enhanced by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0151] For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral long terminal repeats, to name a few. Other suitable promoters are known to those of skill in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) positioned approximately at the end of the polypeptide to be translated.

[0152] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0153] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0154] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0155] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.

[0156] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0157] Transcription of DNA encoding the antibodies of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that serve to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0158] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0159] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in host cells during purification or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification are, inter alia, well-known and routine techniques in the art.

[0160] Treatment method The antibodies or antigen-binding fragments thereof of the present disclosure can be used for a variety of therapeutic purposes.

[0161] In one aspect, the present disclosure provides methods of treating cancer in a subject, reducing the rate of growth of tumor volume in a subject over time, reducing the risk of developing metastases, or reducing the risk of developing further metastases in a subject. In some embodiments, treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0162] In one aspect, the invention relates to a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed with, cancer), for example, breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, intrauterine cancer, nervous system cancer, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, skin cancer, or hematological malignancies. In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastroesophageal junction cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, glioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0163] In some embodiments, the anti-IL2RA antibody is designed to treat melanoma (e.g., advanced melanoma), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), B-cell non-Hodgkin's lymphoma, bladder cancer, and / or prostate cancer (e.g., metastatic hormone-refractory prostate cancer). In some embodiments, the anti-IL2RA antibody is designed to treat hepatocellular carcinoma, ovarian cancer, colon cancer, or cervical cancer. In some embodiments, the anti-IL2RA antibody is designed to treat advanced breast cancer, advanced ovarian cancer, and / or advanced refractory solid tumors. In some embodiments, the anti-IL2RA antibody is designed to treat metastatic solid tumors, NSCLC, melanoma, non-Hodgkin's lymphoma, colorectal cancer, and multiple myeloma. In some embodiments, the anti-IL2RA antibody is designed to treat melanoma, pancreatic cancer, mesothelioma, hematological malignancies (e.g., non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia), or solid tumors (e.g., advanced solid tumors). In some embodiments, the anti-IL2RA antibody is designed to treat cancer (e.g., nasopharyngeal carcinoma, bladder cancer, cervical cancer, renal cancer, or ovarian cancer).

[0164] In some embodiments, the IL2RA antibody is designed to treat acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), solid tumors, colorectal cancer, ovarian cancer, prostate cancer, melanoma, lung cancer, breast cancer, gastric cancer, esophageal squamous cell carcinoma (ESCC), leukemia, lymphoma, multiple myeloma, sarcoma, and / or head and neck cancer.

[0165] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified by a variety of methods known in the art.

[0166] In some aspects, the present disclosure relates to a method of treating an autoimmune disease or inflammation, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, or a composition comprising an antibody drug conjugate described herein.

[0167] In one aspect, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with an aberrant or unwanted immune response, such as autoimmune diseases, including alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behçet's disease, pernicious anemia, bullous pemphigoid, nodular polyarthritis, cardiomyopathy, polychondritis, celiac sprue dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary tract infections, and primary cholangitis. These conditions include, but are not limited to, hemolytic cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemia sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjogren's syndrome, Guillain-Barré, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic alveolar fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type 1), vasculitis, lichen planus, and vitiligo. Anti-IL2RA antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing disorders associated with an aberrant or unwanted immune response associated with cell, tissue, or organ transplantation, e.g., kidney, liver, and heart transplants, e.g., graft-versus-host disease (GVHD), or prevent allograft rejection. In some embodiments, the subject suffers from Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the antibody or antigen-binding fragment can be used to treat inflammation. In some embodiments, the anti-IL2RA antibody is designed to treat other diseases or disorders, such as microbial infections and allergic disorders.

[0168] In some embodiments, the antibodies or antigen-binding fragments described herein can specifically target Treg cells without blocking the interaction between IL2 and IL2RA, thereby eliminating Treg cells and reducing immunosuppression, while maintaining the IL2 / IL2RA interaction effectively stimulates T cells by activating the IL2 / IL2RA signaling pathway.

[0169] In some aspects, the present disclosure relates to a method of inhibiting an immune response in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, or a composition comprising an antibody drug conjugate described herein. In some embodiments, the subject suffers from an autoimmune disease.

[0170] As used herein, "effective amount" means an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus, dosing can be determined on an individual basis.

[0171] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to palliate, arrest, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or to palliate, arrest, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the patient's medical history, as well as the type (and / or dosage) of antibody used.

[0172] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and other agents administered to the mammal.

[0173] A typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg (mg per kg of patient body weight). In some embodiments, the dose can be less than 100 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be less than 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0174] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0175] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is overlap in the period of biological activity of the one or more additional therapeutic agents with the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0176] In some embodiments, a subject can be administered at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to monitor at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0177] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0178] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0179] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 The present invention may include one or more therapeutic agents selected from the group consisting of 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0180] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-6 antagonist (e.g., IL-6 receptor), an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0181] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0182] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, an anti-4-1BB antibody, or an anti-CD40 antibody.

[0183] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition, in any combination. Pharmaceutical compositions can be formulated in any manner known in the art.

[0184] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol, methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. The composition preparation can be formulated and enclosed in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0185] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound(s) for ease of administration and uniformity of dosage).

[0186] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0187] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0188] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats a disease (e.g., kills cancer cells) in a subject (e.g., a human subject identified as having cancer) or a subject identified as at risk for developing the disease (e.g., a subject who previously developed cancer but has now been cured), or reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and the presence of other diseases).

[0189] Exemplary doses include amounts (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, about 1 μg / kg to about 50 mg / kg, about 0.3 mg / kg to about 25 mg / kg, about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg). While these doses cover a wide range, those skilled in the art will understand that the efficacy and effective amounts of therapeutic agents comprising antibodies and antigen-binding fragments thereof can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the attending health care professional or veterinary professional (for therapeutic uses) or researcher (if still working in the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in the body.

[0190] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods for producing antibodies or antigen-binding fragments thereof for the various uses described herein. [Example]

[0191] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0192] Example 1. Generation of anti-IL2RA antibodies To generate antibodies against human IL2RA, RenMice (i.e., recombinant mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions, e.g., RenMabTM Mouse, RenLite TM Mice were immunized with Fc-tagged human IL2RA protein (ACROBiosystems Inc., catalog number: ILA-H5251) or a plasmid encoding the IL2RA protein. Antibody immune responses were monitored by antigen-specific immunoassay.

[0193] A total of three immunizations were performed. Immunizations were performed at two-week intervals. One week after the final immunization, retroorbital blood was collected, and antibody titers in the serum were determined by fluorescence-activated cell sorting (FACS). Two weeks later, mice with high titers were selected for impulse immunization. Human IL2RA protein or CHO-S cells expressing human IL2RA protein were used for impulse immunization via intraperitoneal injection and tail vein injection, respectively.

[0194] When a desired immune response was achieved, antigen-specific immune cells were isolated from the immunized mice to further obtain anti-IL2RA antibodies, or to obtain light and heavy chain variable region sequences of anti-IL2RA antibodies. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened and identified using single-cell technology (e.g., using the Beacon® Optofluidic System, Berkeley Lights Inc.), and antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were cloned into vectors containing sequences encoding human IgG constant regions for antibody expression. The binding affinity of the expressed antibodies to IL2RA was verified using FACS.

[0195] Exemplary antibodies obtained included 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2. The heavy and light chain variable regions of 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 are shown in Figure 9. Figure 7 shows the CDR sequences according to the Kabat definition. Figure 8 shows the CDR sequences according to the Chothia definition.

[0196] Various IgG1, IgG2, and IgG4 antibodies were produced. Regarding the name of the antibody, when the antibody VH / VL are linked to various isotypes, the isotype is added to the name. For example, when the VH and VL of 5D9 are linked to an IgG1 constant region, the antibody is named 5D9-IgG1 (or 5D9). Examples of other isotypes include 5D9-IgG2 and 5D9-IgG4. The constant region of the antibody can contain several mutations. For example, when SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of 5D9-IgG1, the resulting antibody is named 5D9-IgG1-SI (or 5D9-SI).

[0197] Example 2. Binding affinity of anti-IL2RA antibodies Affinity of anti-IL2RA antibodies to His-tagged human IL2RA protein (hIL2RA-his, ACROBiosystems Inc., Cat. No. ILA-H52H9) and His-tagged recombinant monkey (cynomolgus monkey, or Macaca fascicularis) IL2RA protein (cynoIL2RA-his, Sino Biological, Inc., Cat. No. 90265-C08H) was measured using a Biacore™ sensor chip equipped with pre-immobilized Protein A. TM (Biacore, INC, Piscataway NJ) was used to validate the surface plasmon resonance (SPR) 8K biosensor.

[0198] Purified anti-IL2RA antibody was diluted to 2 μg / mL and then subjected to Biacore centrifugation at 10 μL / min for approximately 50 seconds. TM The 8K biosensor was injected to achieve the desired protein density (e.g., approximately 150 response units (RU)). His-tagged IL2RA protein at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, or 0 nM was then injected at 30 μL / min for 180 seconds. Dissociation was monitored for 400 seconds. After the last injection of each titration, the chip was regenerated with glycine (pH 2.0, 30 μL / min for 30 seconds).

[0199] Biacore TM The k and k were obtained simultaneously by fitting the entire data set to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0200] The same method was performed for each test antibody, adjusting parameters (e.g., antibody concentration) appropriately, as would be understood by one of skill in the art. The results for the test antibodies are summarized in the table below. ISO is an antibody of the same IgG1 subtype but targeting an unrelated antigen.

[0201] [Table 1]

[0202] RG6292 is a human IgG1 monoclonal antibody targeting IL2RA that is in early clinical development at Roche for the treatment of patients with advanced and / or metastatic solid tumors; its VH and VL sequences are set forth in SEQ ID NO: 62 and SEQ ID NO: 63, respectively.

[0203] The results showed that the anti-IL2RA antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 exhibited good binding affinity to human IL2RA and monkey IL2RA.

[0204] Example 3. Binding activity of anti-IL2RA antibodies The binding activity of the anti-IL2RA antibodies (5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2) to CHO-S-hIL2RA cells (CHO-S cells expressing human IL2RA (hIL2RA, SEQ ID NO: 64)) or CHO-S-fas IL2RA cells (CHO-S cells expressing monkey (Macaca fascicularis) IL2RA (fasIL2RA, SEQ ID NO: 65)) was examined by flow cytometry.

[0205] CHO-S-hIL2RA cells or CHO-S-fasIL2RA cells were plated in a 96-well plate at 2 × 10 5 Cells were seeded at a density of 1000 cells / well. Serially diluted purified anti-IL2RA antibody (maximum concentration: 9 μg / mL, 2-fold dilutions, 11 gradient) was added to each well and incubated at 4°C for 30 minutes. After washing once with PBS, the cells were incubated with the secondary antibody Alexa Fluor® 647 anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) at 4°C for 15 minutes, followed by flow cytometry analysis.

[0206] The cells were harvested and the mean fluorescence intensity (MFI) was measured. A fitting curve was generated using Log (antibody concentration (μg / mL)) as the X-axis and mean MFI as the Y-axis. EC50 values ​​were determined, and the test results are shown in the table below.

[0207] [Table 2]

[0208] The results showed that the seven antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 all exhibited good binding activity to human IL2RA and monkey IL2RA.

[0209] Example 4. Blocking Assay Flow cytometric assay for blocking of IL2RA ligand binding to IL2RA Blockade of binding of human IL2RA ligand (biotinylated human IL-2 protein, His, Avitag, ACROBiosystems Inc., catalog number: IL2-H82E4) to human IL2RA by anti-IL2RA antibodies was tested by flow cytometry using human CHO-S-hIL2RA cells.

[0210] CHO-S-hIL2RA cells were plated in 96-well plates (cell density 2 × 10 5 Cells were seeded onto 96-well plates (11 x 1000 cells / well) and incubated overnight at 37°C. Serially diluted test antibodies (11 2-fold dilutions with a top concentration of 20 μg / mL) were incubated with the cells for 15 minutes at 4°C, after which human IL2RA ligand was added to each well. The working concentration of human IL2RA ligand was 2.5 μg / mL. The 96-well plate was incubated for 20 minutes at 4°C. After washing once with PBS, the cells were incubated with the secondary antibody APC streptavidin (BioLegend, catalog number: 405207) for 15 minutes at 4°C before flow cytometry analysis.

[0211] Mean fluorescence intensity (MFI) was measured. A fitting curve was obtained using Log(antibody concentration (μg / mL)) or antibody concentration (μg / mL) as the X-axis and Log(mean MFI) or MFI as the Y-axis. The results are shown in Figures 1A-1B.

[0212] Daclizumab is a humanized monoclonal antibody that binds to the IL-2 receptor (α subunit, CD25) on activated T cells, inhibits IL-2-mediated lymphocyte activation, and prevents the activation of inflammatory cytokine responses common to graft rejection, autoimmune diseases, and inflammatory diseases. Its VH and VL sequences are shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively.

[0213] Increasing concentrations of antibodies (5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2) did not result in a corresponding decrease in MFI, indicating that anti-IL2RA antibodies did not block the binding of human IL2RA to IL2RA ligands, whereas daclizumab analogs did block the binding of human IL2RA to IL2RA ligands.

[0214] IL2 receptor cell blocking assay This experiment was carried out to test whether the anti-IL2RA antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 could block the binding of mouse IL2 protein (mIL2) to IL2 receptor cells.

[0215] 20 μL of diluted mouse IL2 protein (mIL2, ACRO, catalog number: IL2-M52H3) (100 ng / mL) was added to a 96-well plate. Then, 20 μL of diluted antibody solution was added to each well. The working concentrations of the diluted antibody were 100 μg / mL, 33.3 μg / mL, or 11.1 μg / mL. Then, 160 μL of IL2 receptor cells (HEK293-Blue-IL2 cells, Invivogen, catalog number: hkb-il2) were seeded into the 96-well plate (cell density 5 × 10 4 The 96-well plate was incubated at 37°C and 5% CO2 for 20-24 hours. After incubation, the plate was removed and 180 μL of QUANTI-Blue TM A solution (QUANTI Blue reagent:QUANTI Blue buffer:sterilized water = 1:1:98) and 20 μL of cell supernatant were added to each well. The plate was incubated at 37 °C for 1 hour, and then placed in a microplate reader to detect the OD value at 630 nm. The results are shown in Figures 2A-2B.

[0216] The results showed that the anti-IL2RA antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 did not block the binding of mouse IL2 protein to IL2 receptor cells, whereas the daclizumab analogs blocked the binding of mouse IL2 protein to IL2 receptor cells.

[0217] STAT5 phosphorylation blocking activity detection Human CD3 + T cells were incubated at 37°C for 2 hours and then seeded into a 96-well plate (100 μL / well). Antibodies were diluted and added to the wells and incubated with the cells for 30 minutes at 37°C and 5% CO2 (the working concentration of the diluted antibodies was 10 μg / mL). Human IL2 protein (hIL2, ACROBiosystems Inc., catalog number: IL2-H4113) (5 U / mL) was then added to each well, and the 96-well plate was incubated for 15 minutes at 37°C and 5% CO2. Cells were then harvested, and single-cell suspensions were prepared from the samples. Cells were labeled with PE anti-STAT5 phosphorylated (Tyr694) antibody (BioLegend, catalog number: 936904) followed by flow cytometry analysis.

[0218] 7G7B6 is a murine IgG2 monoclonal antibody targeting human IL2RA in preclinical development for the treatment of leukemia, and its VH and VL sequences are shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

[0219] The results are shown in Figures 3A-3B. Similar to the 7G7B6 analog-SI, the anti-IL2RA antibodies 5D9, 7B5, 11C3, 11C12, 11D2, 13F9, and 14C2 had little effect on human IL2-induced STAT5 phosphorylation, indicating that they are non-blocking pSTAT5 antibodies. The daclizumab analog significantly inhibited STAT5 phosphorylation and is therefore a blocking pSTAT5 antibody.

[0220] Example 5. Antitumor activity in the MC38 xenograft model Antitumor activity of anti-IL2RA antibodies 7B5 and 5D9 Anti-IL2RA antibodies were tested for their effect on tumor growth in vivo in a colon cancer model. 5 MC38 cells (mouse colon cancer cells) were subcutaneously injected into each B-hIL2RA mouse (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Catalog No.: 110066). Tumors in the mice were approximately 100 mm 3 When tumors reached a volume of 1000 mg / kg, mice were randomly assigned to different groups based on tumor size. Mice were then injected intraperitoneally (ip) with phosphate-buffered saline (PBS) or anti-IL2RA antibody. Details are shown in the table below.

[0221] [Table 3]

[0222] The lengths of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5 × (long axis) × (short axis). 2 It was calculated as:

[0223] The tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100%. T i is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. V i is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0224] Values ​​are expressed as mean ± SEM. T-test was performed for statistical analysis. P<0.05 was the threshold for indicating significant differences.

[0225] The weights of all mice in the different groups increased. On the day of group assignment (day 0), the mean weight of each group ranged from 21.2 g to 21.5 g. At the end of the experiment (day 21), the mean weight of each group ranged from 24.6 g to 25.6 g, and the mean weight change of each group ranged from 115.4% to 119.8%. The results showed that these anti-IL2RA antibodies were well tolerated and had no apparent toxicity to mice.

[0226] The following table summarizes the results for this experiment, including tumor volumes on the day of grouping (day 0), 11 days after grouping (day 11), and 21 days after grouping (day 21); tumor growth inhibition values ​​(TGI); and statistical differences (P values) in tumor volume and body weight between treatment and control groups.

[0227] [Table 4]

[0228] The tumor volumes of different groups of mice treated with antibody or PBS are shown in Figure 4. Compared with the positive control 7G7B6 analog (G6-G7), the anti-IL2RA antibodies (G2-G5) showed superior tumor-inhibitory effects at the same dose level and exhibited dose-dependence.

[0229] In another experiment, approximately 5 × 10 5 MC38 cells were subcutaneously injected into B-hIL2RA mice to determine the antitumor activity of 7B5 and 5D9. 3 Once tumor volume reached 1000 mg / kg, mice were randomized into control and different treatment groups based on tumor size. Grouping and treatment details are shown in the table below.

[0230] [Table 5]

[0231] The weights of mice in different groups all increased, indicating that 7B5 and 5D9 were well tolerated and had no obvious toxicity to mice. The following table summarizes the results of this experiment, including tumor volumes on the day of grouping (day 0), 10 days after grouping (day 10), and at the end of the experiment (day 21); TGI (%); and statistical differences (P values) in tumor volume and body weight between the treatment group and the control group.

[0232] [Table 6]

[0233] The tumor volumes of different groups of mice treated with antibody or PBS are shown in Figure 5, and the anti-IL2RA antibodies 7B5 and 5D9 showed superior tumor-inhibitory effects compared to 7G7B6 analog-SI and 7G7B6 analog at the dose level of 10 mg / kg.

[0234] Antitumor activity of anti-IL2RA antibodies 11C3, 11C12, 11D2, 13F9, and 14C2 Approximately 5×10 5 MC38 cells (mouse colon cancer cells) were subcutaneously injected into B-hIL2RA mice to determine the antitumor activity of 11C3, 11C12, 11D2, 13F9, and 14C2. Tumors in the mice were approximately 80-90 mm. 3 When tumors reached a volume of 1000 mg / kg, mice were randomly assigned to different groups based on tumor size. Mice were then injected intraperitoneally (ip) with PBS or anti-IL2RA antibody. Details are shown in the table below.

[0235] [Table 7]

[0236] The weights of all mice in the different groups increased. On the day of group assignment (day 0), the mean weight of each group ranged from 20.7 g to 21.7 g. At the end of the experiment (day 22), the mean weight of each group ranged from 23.8 g to 24.9 g, and the mean weight change for each group ranged from 113.5% to 117.8%. The results showed that these anti-IL2RA antibodies were well tolerated and had no apparent toxicity to mice.

[0237] The following table summarizes the results for this experiment, including tumor volumes on the day of grouping (day 0), 12 days after grouping (day 12), and 22 days after grouping (day 22); tumor growth inhibition values ​​(TGI); and statistical differences (P values) in tumor volume and body weight between treatment and control groups.

[0238] [Table 8]

[0239] The tumor volumes of different groups of mice treated with antibody or PBS are shown in Figure 6. The anti-IL2RA antibodies (G3-G7) showed superior tumor-inhibitory effects at the 10 mg / kg dose level compared to the control group PBS (G1) and the positive control 7G7B6 analog-SI (G2).

[0240] Example 6. Antitumor activity in a GL261 xenograft model Approximately 1×10 6 Mouse glioma GL261 cells were subcutaneously injected into each B-hIL2RA mouse. Tumors in the mice were approximately 80–90 mm 3 When tumors reached a volume of 1000 mg / kg, mice were randomly assigned to different groups based on tumor size. Mice were then injected intraperitoneally (ip) with PBS or anti-IL2RA antibody. Details are shown in the table below.

[0241] [Table 9]

[0242] The weights of all mice in the different groups increased. On the day of group assignment (day 0), the mean weight of each group ranged from 20.5 g to 20.9 g. At the end of the experiment (day 14), the mean weight of each group ranged from 23.3 g to 25.0 g, and the mean weight change of each group ranged from 113.5% to 119.4%. The results showed that the tested antibodies were well tolerated and had no apparent toxicity to mice.

[0243] The following table summarizes the results for this experiment, including tumor volumes on the day of grouping (day 0), 7 days after grouping (day 7), and 14 days after grouping (day 14); tumor growth inhibition values ​​(TGI); and statistical differences (P values) in tumor volume and body weight between treatment and control groups.

[0244] [Table 10]

[0245] The treatment groups (G2-G4) showed superior tumor-inhibiting effects compared to the PBS-treated control group (G1). Furthermore, 7B5 showed superior tumor-inhibiting effects compared to the positive control 7G7B6 analog, while the tumor-inhibiting effect of 5D9 was essentially equivalent to that of the positive control 7G7B6 analog.

[0246] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to the interleukin-2 receptor alpha chain (IL2RA), a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR3; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR1, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR2, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR3; The amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are one of the following: (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 43, 44, and 45, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 10, 11, and 12, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 49, 50, and 51, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, and 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 43, 44, and 45, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, and 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (11) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (12) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, and 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; (13) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, and 51, respectively; and (14) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 40, 41, and 42, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 49, 50, and 51, respectively.

2. According to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively. The antibody or antigen-binding fragment thereof described in claim 1.

3. According to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; The antibody or antigen-binding fragment thereof described in claim 1.

4. According to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively; The antibody or antigen-binding fragment thereof described in claim 1.

5. According to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively; The antibody or antigen-binding fragment thereof described in claim 1.

6. According to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. The antibody or antigen-binding fragment thereof described in claim 1.

7. According to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. The antibody or antigen-binding fragment thereof described in claim 1.

8. According to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively. The antibody or antigen-binding fragment thereof described in claim 1.

9. the antibody or antigen-binding fragment specifically binds to human IL2RA or simian IL2RA; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody); The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody); The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A nucleic acid comprising a polynucleotide encoding a polypeptide, (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 53; (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 52; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 55; (4) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 54; (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (6) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 56; (7) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (8) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 57; (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (10) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 58; (11) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (12) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 59; (13) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (14) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, wherein the VL binds to IL2RA when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 60; (15) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 53; (16) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 55; (17) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (18) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (19) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; (20) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61; or (21) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, wherein the VH binds to IL2RA when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:

61. Nucleic acid.

13. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, or SEQ ID NOs: 22, 23, and 24, respectively; The nucleic acid of claim 12.

14. The nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively; The nucleic acid of claim 12.

15. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, or SEQ ID NOs: 25, 26, and 27, respectively; The nucleic acid of claim 12.

16. The nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; The nucleic acid of claim 12.

17. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, or SEQ ID NOs: 28, 29, and 30, respectively; The nucleic acid of claim 12.

18. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, or SEQ ID NOs: 31, 32, and 33, respectively; The nucleic acid of claim 12.

19. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, or SEQ ID NOs: 34, 35, and 36, respectively; The nucleic acid of claim 12.

20. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, or SEQ ID NOs: 37, 38, and 39, respectively; The nucleic acid of claim 12.

21. the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, or SEQ ID NOs: 40, 41, and 42, respectively; The nucleic acid of claim 12.

22. The nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively; The nucleic acid of claim 12.

23. the VH, when paired with the VL, specifically binds to human IL2RA, or the VL, when paired with the VH, specifically binds to human IL2RA or simian IL2RA; The nucleic acid according to any one of claims 12 to 22.

24. the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof; The nucleic acid according to any one of claims 12 to 23.

25. the nucleic acid encodes a single-chain variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR), The nucleic acid according to any one of claims 12 to 24.

26. The nucleic acid according to any one of claims 12 to 25, wherein the nucleic acid is a cDNA.

27. A vector comprising one or more of the nucleic acids according to any one of claims 12 to 26.

28. A vector comprising two of the nucleic acids according to any one of claims 12 to 26, wherein the vector encodes the VH region and the VL region that both bind to IL2RA.

29. A pair of vectors, each vector comprising one of the nucleic acids of any one of claims 12 to 26, and together encoding the VH region and the VL region that bind to IL2RA.

30. A cell comprising a vector according to claim 27 or 28, or a pair of vectors according to claim 29.

31. 31. The cell of claim 30, wherein the cell is a CHO cell.

32. A cell comprising one or more nucleic acids according to any one of claims 12 to 26.

33. A cell comprising two of the nucleic acids according to any one of claims 12 to 26.

34. The cell of claim 33 , wherein the two nucleic acids together encode the VH region and the VL region that both bind to IL2RA.

35. 1. A method for producing an antibody or antigen-binding fragment thereof, said method comprising: (a) culturing the cell of any one of claims 30 to 34 under conditions sufficient to produce the antibody or antigen-binding fragment thereof; (b) recovering the antibody or antigen-binding fragment produced by the cell. method.

36. An antibody or antigen-binding fragment thereof that binds to IL2RA, 1. An antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence; and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 53; (2) the selected VH sequence is SEQ ID NO:54 and the selected VL sequence is SEQ ID NO:55; (3) the selected VH sequence is SEQ ID NO:56 and the selected VL sequence is SEQ ID NO:61; (4) the selected VH sequence is SEQ ID NO:57 and the selected VL sequence is SEQ ID NO:61; (5) the selected VH sequence is SEQ ID NO:58 and the selected VL sequence is SEQ ID NO:61; (6) the selected VH sequence is SEQ ID NO:59 and the selected VL sequence is SEQ ID NO:61; (7) The selected VH sequence is SEQ ID NO: 60 and the selected VL sequence is SEQ ID NO:

61.

37. The VH comprises the sequence of SEQ ID NO: 52 and the VL comprises the sequence of SEQ ID NO: 53; 37. The antibody or antigen-binding fragment thereof of claim 36.

38. The VH comprises the sequence of SEQ ID NO: 54 and the VL comprises the sequence of SEQ ID NO: 55; 37. The antibody or antigen-binding fragment thereof of claim 36.

39. The VH comprises the sequence of SEQ ID NO: 56 and the VL comprises the sequence of SEQ ID NO: 61; 37. The antibody or antigen-binding fragment thereof of claim 36.

40. The VH comprises the sequence of SEQ ID NO: 57 and the VL comprises the sequence of SEQ ID NO: 61; 37. The antibody or antigen-binding fragment thereof of claim 36.

41. The VH comprises the sequence of SEQ ID NO: 58 and the VL comprises the sequence of SEQ ID NO: 61; 37. The antibody or antigen-binding fragment thereof of claim 36.

42. The VH comprises the sequence of SEQ ID NO: 59 and the VL comprises the sequence of SEQ ID NO: 61; 37. The antibody or antigen-binding fragment thereof of claim 36.

43. The VH comprises the sequence of SEQ ID NO: 60, and the VL comprises the sequence of SEQ ID NO:

61.

37. The antibody or antigen-binding fragment thereof of claim 36.

44. the antibody or antigen-binding fragment specifically binds to human IL2RA or canine IL2RA; An antibody or antigen-binding fragment thereof according to any one of claims 36 to 43.

45. The antibody or antigen-binding fragment is a human antibody or a humanized antibody or an antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof according to any one of claims 36 to 44.

46. the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody); An antibody or antigen-binding fragment thereof according to any one of claims 36 to 45.

47. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 36 to 46.

48. An antibody or antigen-binding fragment thereof that binds to IL2RA, a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and an antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, a VL CDR2, and a VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 52 and the selected VL sequence is SEQ ID NO: 53; (2) the selected VH sequence is SEQ ID NO:54 and the selected VL sequence is SEQ ID NO:55; (3) the selected VH sequence is SEQ ID NO:56 and the selected VL sequence is SEQ ID NO:61; (4) the selected VH sequence is SEQ ID NO:57 and the selected VL sequence is SEQ ID NO:61; (5) the selected VH sequence is SEQ ID NO:58 and the selected VL sequence is SEQ ID NO:61; (6) the selected VH sequence is SEQ ID NO:59 and the selected VL sequence is SEQ ID NO:61; and (7) The selected VH sequence is SEQ ID NO: 60 and the selected VL sequence is SEQ ID NO:

61.

49. 49. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 36 to 48 covalently attached to a therapeutic agent.

50. 50. The antibody drug conjugate of claim 49, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

51. A method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 36 to 48, or the antibody-drug conjugate of claim 49 or 50. method.

52. The subject has solid tumors, brain tumors, lung cancer, melanoma, prostate cancer, esophageal squamous cell carcinoma, leukemia, lymphoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cancer, and breast cancer.

52. The method of claim 51.

53. further comprising administering to the subject a therapeutically effective amount of an anti-OX40 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-PDL2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, an anti-4-1BB antibody, and / or an anti-CD40 antibody.

53. The method of claim 51 or 52.

54. 1. A method for reducing tumor growth rate, said method comprising: contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 36 to 48, or the antibody-drug conjugate of claim 49 or 50; method.

55. 1. A method for killing tumor cells, said method comprising: contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 36 to 48, or the antibody-drug conjugate of claim 49 or 50; method.

56. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 36 to 48, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

57. 51. An antibody drug conjugate according to claim 49 or 50, comprising: a pharmaceutically acceptable carrier. Pharmaceutical compositions.

58. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 36 to 48. Chimeric antigen receptor (CAR).

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