NKG2A BINDING AGENTS AND USES THEREOF

Abstract

The present disclosure provides NKG2A-binding agents (e.g., antibodies (including monospecific antibodies and multispecific antibodies such as bispecific antibodies)) and uses thereof. The present disclosure provides NKG2A-binding agents (including human NKG2A-binding agents). Such agents include antibodies that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), e.g., monospecific antibodies or multispecific (e.g., bispecific) antibodies that bind to NKG2A. In some embodiments, such binding agents bind to the same epitope on NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A) as antibodies comprising the CDRs described herein (e.g., Tables 1-4).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 512,432, filed July 7, 2023, and U.S. Provisional Patent Application No. 63 / 441,705, filed January 27, 2023, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a computer-readable Sequence Listing that has been submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The Sequence Listing XML file submitted herewith is named "14529-150-228_SEQ_LISTING.xml," was created on January 17, 2024, and is 103,086 bytes in size.

[0003] The present disclosure relates generally to binding agents, eg, antibodies (including fragments thereof) that bind to NKG2A (including human NKG2A), and methods of use thereof. [Background technology]

[0004] NKG2A is typically expressed on NK cells and also on T cells, especially CD8 + NKG2A is a cell surface molecule that may be expressed on T cells. Therefore, NKG2A is a potential target for removing the suppressive state of immune cells and enhancing anti-tumor responses by immune cells. However, successful treatment with binding agents targeting NKG2A has not yet been achieved. Therefore, there remains a need in the art for agents that enhance immune responses and treat diseases or disorders, such as cancer. The binding agents, compositions, and methods provided in the present disclosure fulfill this need and provide related advantages. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides NKG2A-binding agents (including human NKG2A-binding agents). Such agents include antibodies that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), e.g., monospecific or multispecific (e.g., bispecific) antibodies that bind to NKG2A. In some embodiments, such binding agents bind to the same epitope on NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A) as antibodies comprising the CDRs described herein (e.g., Tables 1-4). In some embodiments, such binding agents bind to the same epitope on a complex comprising NKG2A and CD94 or their respective extracellular domains (e.g., human NKG2A) as antibodies comprising the heavy chain variable regions and light chain variable regions described herein (e.g., Tables 1-4). In some embodiments, the NKG2A-binding agent specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A-binding agent specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from (1) one of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91); or (2) two, three, four, five, or all of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agent specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from (1) one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91); or (2) two, three, four, five, or all of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91).

[0006] The present disclosure also provides nucleic acids encoding the NKG2A-binding agents (e.g., antibodies or fragments thereof, e.g., antigen-binding fragments) provided herein, vectors comprising one or more such nucleic acids, and cells comprising the nucleic acids, the vectors, or both (e.g., cells that express the binding agents).

[0007] The present disclosure also provides compositions comprising NKG2A-binding agents. Such compositions, in some embodiments, comprise an antibody that binds to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), e.g., a monospecific or multispecific (e.g., bispecific) antibody that binds to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains). Such compositions, in some embodiments, comprise an antibody that binds to essentially the same epitope on NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A) as an antibody comprising CDRs described herein (e.g., Tables 1-4). Such compositions, in some embodiments, comprise an antibody that binds to essentially the same epitope on NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A) as an antibody comprising a heavy chain variable region and a light chain variable region described herein (e.g., Tables 1-4). In some embodiments, the NKG2A-binding agent specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A-binding agent specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from (1) one of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91); or (2) two, three, four, five, or all of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agent specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from (1) one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91); or (2) two, three, four, five, or all of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91).

[0008] In some embodiments, the binding agent binds to NKG2A. In some embodiments, the binding agent binds to a complex comprising NKG2A and CD94. In some embodiments, the binding agent binds to a complex comprising NKG2A and the extracellular domain of CD94. Additionally or alternatively, the binding agent does not bind to NKG2C. Additionally or alternatively, the binding agent does not bind to a second complex comprising NKG2C and CD94. Additionally or alternatively, the binding agent does not bind to a second complex comprising the extracellular domain of NKG2C and the extracellular domain of CD94.

[0009] The present disclosure also provides compositions comprising a nucleic acid encoding an NKG2A-binding agent (e.g., an antibody or fragment thereof, e.g., an antigen-binding fragment) provided herein, a vector comprising one or more nucleic acids, or a cell comprising the nucleic acid, the vector, or both (e.g., a cell that expresses the binding agent).

[0010] The present disclosure further provides various uses of the binding agents and compositions of the present disclosure, including, for example, methods for inhibiting the interaction of HLA-E with NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., NKG2A expressed on an immune cell or said complex), and methods for preventing immune cell suppression or activating immune cell-mediated responses. Other aspects provided by the present disclosure include methods of treating a disease or disorder in a subject with an NKG2A-binding agent or composition provided by the present disclosure. Such compositions include antibodies that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A). [Brief explanation of the drawings]

[0011] [Figure 1-1]1 shows exemplary results for A3 from a cell binding assay and is further described in Examples 3 and 6. [Figure 1-2] 1 shows exemplary results for A3 from a cell binding assay and is further described in Examples 3 and 6. [Figure 1-3] 1 shows exemplary results for A3 from a cell binding assay and is further described in Examples 3 and 6. [Figure 2] Exemplary results for A3 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 3A] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 3B] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 3C] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 4-1] 1 shows exemplary results for A42 from a biolayer interferometry (BLI) binding assay (i.e., Octet binding assay), and is further described in Examples and 6. [Figure 4-2] 1 shows exemplary results for A42 from a biolayer interferometry (BLI) binding assay (i.e., Octet binding assay), and is further described in Examples and 6. [Figure 4-3] 1 shows exemplary results for A42 from a biolayer interferometry (BLI) binding assay (i.e., Octet binding assay), and is further described in Examples and 6. [Figure 5] Exemplary results for A42 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 6A] Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 6B]Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 6C] Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 7-1] Exemplary results for A2 from a BLI binding assay are shown and further described in Example 6. [Figure 7-2] Exemplary results for A2 from a BLI binding assay are shown and further described in Example 6. [Figure 7-3] Exemplary results for A2 from a BLI binding assay are shown and further described in Example 6. [Figure 8] Exemplary results for A2 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 9A] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 9B] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 9C] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 10-1] 1 shows exemplary results for A11 from a BLI binding assay, further described in Example 6. [Figure 10-2] 1 shows exemplary results for A11 from a BLI binding assay, further described in Example 6. [Figure 10-3] 1 shows exemplary results for A11 from a BLI binding assay, further described in Example 6. [Figure 11] 1 shows exemplary results for A11 from an HLA-E / NKG2A inhibition assay, which are further described in Examples 4 and 6. [Figure 12A]1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 12B] 1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 12C] 1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 13] NKG2A and CD94 are depicted on the surface with epitopes highlighted. [Figure 14] A list of regions significantly protected from deuterium exchange is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure is based, at least in part, on novel NKG2A-binding agents and their properties. Such agents include antibodies (monospecific or multispecific antibodies, including bispecific antibodies) that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), including antibodies that bind to human NKG2A (or a complex comprising human NKG2A and human CD94 or their respective extracellular domains). In certain aspects, such binding agents are useful in compositions and methods that prevent immune cell suppression or activate immune cell-mediated anti-tumor responses by inhibiting the interaction of HLA-E with NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., a complex comprising NKG2A or NKG2A and CD94 expressed on immune cells). In addition, the NKG2A-binding agents, e.g., NKG2A-binding antibodies (e.g., monospecific or multispecific antibodies, including bispecific antibodies) described herein, are useful for killing and / or eliminating tumor cells. The NKG2A-binding agents, e.g., NKG2A-binding antibodies (e.g., monospecific antibodies or multispecific antibodies (including bispecific antibodies)) described herein are useful in compositions and methods for treating diseases or disorders, e.g., cancer.

[0013] As will be appreciated, as used herein, chapter or section headings are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described.

[0014] 5.1.Definition The techniques and procedures described or referred to herein include techniques and procedures that are generally well understood and / or commonly used by those skilled in the art using conventional techniques, e.g., widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall include the plural and terms used in the plural shall include the singular. In the event that any of the explanations of terms provided conflict with any document incorporated herein by reference, the explanations of terms provided below shall control.

[0015] The term "NKG2A," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of NKG2A derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). NKG2A is also known, for example, as NK cell receptor A, NK-activating receptor NKG2A, NK-activating receptor NKG2-A / B, killer cell lectin-like receptor C1 (CD159a), CD159 antigen-like family member A, or type II integral membrane protein NKG2-A / NKG2-B. NKG2A belongs to the lectin family and forms a heterodimer with CD94 (or KLRD1), another C-type lectin expressed on NK cells. In humans, the NKG2A / CD94 complex binds to the non-classical MHC I molecule HLA-E and transmits an inhibitory signal, which activates NK cells and CD8 + It suppresses T cell activity. NKG2A is a protein encoded by the NKG2A gene (or KLRC1). The term NKG2A includes "full-length" NKG2A and any form of NKG2A, or any fragment thereof resulting from cellular processing. In some embodiments, an exemplary amino acid sequence of full-length NKG2A is shown below (see, e.g., Gene Accession Number P26715-1 in the Examples section below). In some embodiments, NKG2A includes a signal sequence. In some embodiments, NKG2A does not include a signal sequence. In some embodiments, the term NKG2A refers to a fragment of full-length NKG2A, which fragment includes the NKG2A extracellular domain. The term NKG2A also includes naturally occurring variants of NKG2A, such as SNP variants, splice variants, and allelic variants. An exemplary amino acid sequence of the extracellular domain of human NKG2A is shown below. PSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 74). An exemplary amino acid sequence of human NKG2A is RHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 92). An exemplary amino acid sequence of the extracellular domain of cynomolgus monkey NKG2A is shown below. PSTLTQKHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKEKRTWAESLLACTLKNSSLLSIDNEEEMKFLTAISPSTWTGVFRDSSQHPWVTINGLTFKHEIKDSDNAEHNCAMLHARGLKSDRCGSSKIYHCKHKL (SEQ ID NO: 77).

[0016] In some embodiments, the term NKG2A as used herein refers to the NKG2A epitope. Additionally or alternatively, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94. In further embodiments, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94, but not an epitope on CD94 itself. Additionally or alternatively, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94, but not an epitope on NKG2A itself. In some embodiments, the term NKG2A, as used herein, refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domains of NKG2A and CD94, but not an epitope on NKG2A alone or CD94 alone.

[0017] In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope. Additionally or alternatively, as used herein, the term NKG2A refers to an NKG2A epitope that is present on the surface of NKG2A stabilized by forming a complex with CD94. In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope that is present on the surface of a complex comprising NKG2A and CD94, as well as on the NKG2A epitope alone. Additionally or alternatively, as used herein, the term NKG2A refers to an NKG2A epitope that is present on the surface of the NKG2A extracellular domain stabilized by forming a complex with the CD94 extracellular domain. In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope that is present on the surface of a complex comprising NKG2A and the extracellular domain of CD94, as well as on the NKG2A epitope alone.

[0018] The term "NKG2C," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of NKG2C derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). NKG2C is also known, e.g., as KLRC2, CD159c, NKG2-C, NKG2C, and killer cell lectin-like receptor C2. NKG2C is a protein encoded by the NKG2C gene (or KLRC2). The term NKG2C includes "full-length" NKG2C and any form of NKG2C, or any fragment thereof resulting from cellular processing. In some embodiments, NKG2C includes a signal sequence. In some embodiments, NKG2C does not include a signal sequence. In some embodiments, the term NKG2C refers to a fragment of full-length NKG2C, which fragment comprises the NKG2C extracellular domain. The term NKG2C also includes natural variants of NKG2C, such as SNP variants, splice variants, and allelic variants. The NKG2C gene is listed in various databases under the following ID numbers: HGNC6375, NCBI Entrez Gene3822, Ensembl ENSG00000205809, OMIM® 602891, and UniProtKB / Swiss-Prot P26717. An exemplary extracellular domain of human NKG2C is shown in the Examples section below (see SEQ ID NO: 75).

[0019] In some embodiments, the term NKG2C as used herein refers to the NKG2C epitope. Additionally or alternatively, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94. In further embodiments, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94, but not an epitope on CD94 itself. Additionally or alternatively, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94, but not an epitope on NKG2C itself. In some embodiments, the term NKG2C, as used herein, refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domains of NKG2C and CD94, but not an epitope on NKG2C alone or CD94 alone.

[0020] The term "HLA-E," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats), or any native form of HLA-E or its ortholog. HLA-E is also known, for example, as major histocompatibility complex class I, E, HLA class I histocompatibility antigen, alpha chain E, MHC class I antigen E, HLA-6.2, MHC class Ib antigen, HLA-E, or QA1. In humans, HLA-E is a protein encoded by the HLA-E gene. HLA-E belongs to the HLA class I heavy chain paralog, is approximately 45 kDa, and is membrane-anchored. The term HLA-E includes "full-length" HLA-E and any form of HLA-E or any fragment thereof resulting from cellular processing. In some embodiments, HLA-E includes a signal sequence. In some embodiments, HLA-E does not include a signal sequence. In some embodiments, the term HLA-E refers to a fragment of full-length HLA-E, which fragment includes the HLA-E extracellular domain. The term HLA-E also includes natural variants of HLA-E, such as SNP variants, splice variants, and allelic variants. The HLA-E gene is listed in various databases under the ID numbers HGNC4962, NCBI Entrez Gene3133, Ensembl ENSG00000204592, OMIM® 143010, and UniProtKB / Swiss-Prot P13747.

[0021] The term "CD94," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of CD94 from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). CD94 is also known, for example, as killer cell lectin-like receptor D1, killer cell lectin-like receptor subfamily D member 1, natural killer cell antigen CD94, NK cell receptor, or KP43. CD94 is a protein encoded by the KLRD1 gene. CD94 is an immune receptor involved in self-nonself discrimination. On cytotoxic and regulatory lymphocyte subsets, CD94 recognizes, in complex with NKG2A or NKG2C, classical major histocompatibility (MHC) class Ia molecules and the nonclassical MHC class Ib molecule HLA-E carrying self-peptides derived from the signal sequence of nonclassical MHC class Ib molecules. CD94-NKG2A acts as an immunosuppressive receptor and is an important inhibitory receptor for natural killer (NK) cells, regulating NK cell activation and effector function. CD94-NKG2C acts as an immunoactivating receptor on a subset of cytotoxic lymphocytes that recognizes HLA-E carried by peptides derived from the signal sequence of non-classical MHC class Ib HLA-G molecules. The term CD94 includes "full-length" CD94 and any form of CD94 or fragments thereof resulting from cellular processing. In some embodiments, CD94 includes a signal sequence. In some embodiments, CD94 does not include a signal sequence. In some embodiments, the term CD94 refers to a fragment of full-length CD94, which fragment includes the CD94 extracellular domain. The term CD94 also includes naturally occurring variants of CD94, such as SNP variants, splice variants, and allelic variants.The CD94 gene is described in various databases under the following ID numbers: HGNC6378, NCBI Entrez Gene 3824, Ensembl ENSG00000134539, OMIM® 602894, and UniProtKB / Swiss-Prot Q13241. An exemplary amino acid sequence of human CD94 is SFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI (SEQ ID NO: 93).

[0022] As used herein, the term "binding agent" or grammatical equivalents refers to a molecule (e.g., an antibody) that has one or more antigen-binding sites that bind to an antigen. In some embodiments, an NKG2A-binding agent as described herein is an antibody (including an antibody fragment, e.g., an antigen-binding fragment or an epitope-binding fragment), or other peptide-based molecule, as well as conjugates of antibodies, antibody fragments, or peptide-based molecules (e.g., antibody-drug conjugates) that bind to NKG2A, e.g., human NKG2A.

[0023] The terms "antibody," "immunoglobulin," and "Ig" are used interchangeably herein and are used in the broadest sense to specifically encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies with full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of a heavy chain-only antibody. Exemplary single domain antibodies include, but are not limited to, antibodies naturally lacking light chains, e.g., those derived from camelids (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. VHHs can also be derived from non-Camelidae species that can produce heavy-chain antibodies naturally lacking light chains. Antibodies also include antibody fragments (and / or polypeptides comprising antibody fragments) that retain NKG2A-binding properties. Non-limiting examples of antibody fragments include the antigen-binding and / or effector regions of antibodies, e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual variable domain antibodies, single variable domains, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, didiabodies, disulfide-linked Fvs (dsFv), single domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments consisting of non-covalently linked heavy and light chain variable regions). Generally speaking, the variable (V) region domains may be immunoglobulin heavy (VH) and / or light (VL) chain variable domains in any suitable arrangement, e.g., antibodies also include tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers.Thus, for example, the V region domain may be a dimer and may comprise a VHH-VHH, VH-VH, VH-VL, or VL-VL dimer that binds to NKG2A. If desired, the VH and VL may be covalently linked, either directly or via a linker, to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as being included in the category of "antibody fragments." Another form of antibody fragment is a peptide comprising one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also referred to as "minimal recognition units" or "hypervariable regions") can be obtained by constructing polynucleotides encoding one or more of the relevant CDRs. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).Antibody fragments may be incorporated into, for example, single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of novel antigen receptors (v-NARs), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or a VL further comprise a light chain constant region and / or a heavy chain constant region, e.g., one or more constant regions (including one or more of an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, and / or an IgG4 constant region). In some embodiments, an antibody can comprise an epitope-binding fragment of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0024] The term "monospecific," when used in reference to a binding agent (e.g., an antibody), as used herein, denotes a binding agent that has one or more binding sites that each bind to the same epitope of the same antigen.

[0025] The term "multispecific," when used in reference to a binding agent (e.g., an antibody), means that the binding agent is capable of specifically binding to at least two distinct epitopes, e.g., two binding sites formed by a pair of antibody heavy chain variable domains (VH) and light chain variable domains (VL), or by a VHH domain, bind to different antigens or different epitopes on the same antigen. Such bispecific binding agents (e.g., antibodies) may have a 1+1 format (one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binding agent (e.g., antibody) formats may be 2+1 or 1+2 formats (two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope), or 2+2 formats (two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., antibody) contains two antigen-binding sites, each may bind to a different epitope. Such bispecific binding agents (e.g., antibodies) may bind to two different epitopes on the same antigen (e.g., epitopes on NKG2A).

[0026] The term "identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of the same nucleotides or amino acid residues, when compared and aligned for maximum correspondence (introducing gaps, if necessary) and not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. Algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that, when compared and aligned for maximum correspondence, the nucleotide or amino acid residues are at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of amino acid sequence that is at least about 10 residues in length, at least about 20 residues in length, at least about 40-60 residues in length, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 residues, e.g., at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies. In some embodiments, the identity exists over a region of nucleotide sequence that is at least about 10 bases in length, at least about 20 bases in length, at least about 40-60 bases in length, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences to which they are compared, e.g., the nucleotide sequences encoding the proteins of interest.

[0027] "Conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical properties.Families of amino acid residues with similar side chains have generally been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of tyrosine with phenylalanine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not prevent the polypeptide, soluble protein, or antibody comprising that amino acid sequence from binding to a target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0028] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can contain non-amino acids (e.g., interrupted by non-amino acids). The term also includes amino acid polymers that are modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation (directly or indirectly) to a moiety, e.g., a labeling component or a drug (e.g., a toxin). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as polypeptides containing other modifications known in the art. Because the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in some embodiments, the polypeptides can exist as single chains or single-chain dimers.

[0029] As used herein, an "antigen" is a moiety or molecule that contains an epitope to which a binding agent (e.g., an antibody) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen to which a binding agent (e.g., an antibody) described herein binds is NKG2A (e.g., human NKG2A) or a fragment thereof, including fragments that include one or more domains of NKG2A.

[0030] As used herein, "epitope" is a term used in the art and refers to a local region of an antigen to which an antibody can bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of the polypeptide (a "linear" epitope), or it can include amino acids from two or more non-contiguous regions of the polypeptide (a "conformational" epitope, a "non-linear" epitope, or a "discontinuous" epitope), such as human NKG2A. In general, it will be apparent to those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether the group is folded into the native three-dimensional protein structure. In other embodiments, antibodies require the amino acid residues that make up the epitope to adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0031] When two antibodies recognize the same epitope, overlapping epitope, or adjacent epitope in three-dimensional space, the antibody binds to an "epitope," or "essentially the same epitope" or "the same epitope" as a reference antibody. The most widely used rapid method for determining whether two antibodies bind to the same epitope, overlapping epitope, or adjacent epitope in three-dimensional space is a competitive assay, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive, fluorescent, or enzyme label.

[0032] "Epitope binning" is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning clusters antibodies based on their epitope recognition properties and includes methods and systems for distinguishing the epitope recognition properties of various antibodies using competitive assays combined with computational processes to identify antibodies with distinct binding specificities.

[0033] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," "selectively bind," "immunospecifically recognize," and "immunospecific" are synonymous in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope), as one of skill in the art would understand binding. In some embodiments, "specifically bind" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, rapidly, for a longer duration, with higher affinity, or some combination thereof, than does alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may generally bind other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, a Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), an OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds or specifically binds to an antigen when it binds to the antigen with higher affinity than any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs). Typically, a specific or selective response results in a response with at least 2x background signal or background noise, and may be more than 10x background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion of binding specificity. In some embodiments, the extent to which an antibody or antigen-binding domain binds to a "non-target" protein is less than about 10% of the extent to which the antibody or antigen-binding domain binds to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka that is at least 2, 2.5, 3, 4, or more logs higher than the Ka of the molecule when binding to another antigen.In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins. In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other proteins that are not NKG2A. In some embodiments, "specifically binds" refers, for example, to a polypeptide or molecule that binds to a protein or target with a K of about 0.1 mM or less (more usually less than about 1 μM). D In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. D Specific binding means binding to more than one target. Specific binding can include polypeptides or molecules that recognize proteins or targets in more than one species due to sequence identity between homologous proteins in different species. Similarly, specific binding can include polypeptides or molecules that recognize more than one protein or target due to homology within certain regions of the polypeptide sequences of different proteins. It is understood that in some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not require (but can include) exclusive binding, e.g., binding to one target. Thus, a polypeptide or molecule can, in some embodiments, specifically bind to more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, an antibody can, in certain cases, contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific, containing at least two antigen-binding sites with different specificities. Generally, but not necessarily, reference to "binding" means "specific binding."

[0034] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding agent such as an antibody) and its binding partner (e.g., an antigen such as NKG2A). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate quickly, whereas high affinity antibodies generally bind antigens more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. In one embodiment, "K" is used to measure affinity. D " or "K D The "K value" may be measured by biolayer interferometry (BLI), for example, using an OctetQK384 system (ForteBio, Menlo Park, CA). D may be measured in a radiolabeled antigen binding assay (RIA), such as an RIA performed with a Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881), or using a Biacore™ surface plasmon resonance (SPR) assay using, for example, a Biacore™-2000 or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ). "Association rate" or "k on " and "dissociation rate" or "k off " can also be determined using the same SPR or BLI techniques described above, for example, using an OctetQK384 system (ForteBio, Menlo Park, CA) (for SPR), or a Biacore™-2000 (for SPR) or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ) (for BLI).

[0035] The term "compete," or any of its grammatical variations, when used in the context of an NKG2A-binding agent (e.g., an antibody), refers to binding agents that compete for the same epitope or same binding site on a target, including competition between such binding agents as determined by an assay in which the binding agent under investigation prevents or inhibits a reference molecule (e.g., a reference ligand or reference antigen-binding protein, e.g., a reference antibody) from specifically binding to a common antigen (e.g., NKG2A). Many types of competitive binding assays can be used to determine whether a test binding agent competes with a reference molecule for binding to NKG2A (e.g., human NKG2A). Examples of assays that can be used include direct solid-phase radioimmunoassay (RIA), indirect solid-phase RIA, direct solid-phase enzyme immunoassay (EIA), indirect solid-phase EIA, sandwich competition assay (see, e.g., Stahl et al., (1983) Methods in Enzymology 9:242-253), direct solid-phase biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung, et al., (1990) Virology 176:546-552), direct solid-phase labeling assay, direct solid-phase labeling sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), direct solid-phase labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and direct solid-phase labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press). al., (1988) Molec. Immunol. 25:7-15), and direct labeling RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen (e.g., NKG2A, e.g., human NKG2A) bound to a solid surface or cells bearing either an unlabeled test antigen-binding protein (e.g., a test NKG2A antibody) or a labeled reference antigen-binding protein (e.g., a reference NKG2A antibody).Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of a test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to adjacent epitopes (e.g., similar or overlapping epitopes) sufficiently close to the epitope bound by the reference antibody that the antibodies sterically hinder the binding. Typically, when a competing antibody is present in excess, it will inhibit specific binding of the reference antibody to a common antigen by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0036] As used herein, the term "constant region" or "constant domain" is a well-known antibody technical term and refers to the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but that can exhibit various effector functions, e.g., interaction with Fc receptors. The term includes portions of immunoglobulin molecules that have amino acid sequences that are generally more conserved than immunoglobulin variable domains.

[0037] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0038] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to the carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering a nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are shown below (CH2 domain = bold text, CH3 domain = underlined text): [ka]

[0039] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (such as antibody-dependent cellular phagocytosis, or ADCP), down-regulation of cell surface receptors (e.g., B cell receptors (BCRs)), and the like. Such effector functions generally require an Fc region in combination with a binding region or domain (e.g., an antibody variable region or domain), and can be assessed using various assays, such as those disclosed.

[0040] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature, and has not been manipulated, modified, and / or altered by man (e.g., has not been isolated, purified, selected (e.g., has not contained or combined with other sequences, e.g., variable region sequences)). Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0041] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region comprises at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions described herein can be at least about 80% identical to the native-sequence Fc region and / or the Fc region of the parent polypeptide, or at least about 90% identical thereto, e.g., at least about 95% identical thereto. The variant Fc regions described herein can lack effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are shown below (CH2 domain = bold text (amino acid changes underlined), CH3 domain = underlined text). [ka]

[0042] As used herein, the term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of about 50 to 70 kDa, the amino-terminal portion of which contains a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains one or more constant regions. "Heavy chain" can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of its constant domain, where alpha confers the IgA class of antibodies, delta the IgD class of antibodies, epsilon the IgE class of antibodies, gamma the IgG class of antibodies, and mu the IgM class of antibodies, including IgG subclasses such as IgG1, IgG2, IgG3, and IgG4.

[0043] As used herein, the term "light chain," when used in reference to an antibody, can refer to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of its constant domain, there are two distinct types, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0044] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and confer specificity and affinity on the binding fragment, domain, or region for that antigen (e.g., CDRs). As used herein, "antigen-binding fragment" includes antibody portions that contain one or more CDRs, e.g., "antibody fragments" that contain the antigen-binding or variable region of the antibody.

[0045] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0046] In some embodiments, antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen binding sites that bind to an NKG2A antigen.

[0047] An antibody can be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or classes (e.g., human IgG1, human IgG2, human IgG3, or IgG4) or subclasses thereof.

[0048] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. Additionally or alternatively, the amino acid sequences of the L chains are different from each other. For example, the antibody comprises a first H chain / L chain pair and a second H chain / L chain pair, where the first H chain / L chain pair binds to an NKG2A antigen and the second H chain / L chain pair binds to another NKG2A antigen or an antigen other than NKG2A. In some embodiments, the antibody is a two-chain antibody unit comprising a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHHs are identical. In other embodiments, the amino acid sequences of the VHHs are different from each other. For example, an antibody comprises a first VHH and a second VHH, where the first VHH binds to an NKG2A antigen and the second VHH binds to another NKG2A antigen or an antigen other than NKG2A. In some embodiments, the H chain and / or L chain comprise a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa light chain constant region or a lambda light chain constant region, e.g., a human kappa light chain constant region or a human lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody comprises a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., the constant region of IgG1, IgG2, IgG3, and / or IgG4).

[0049] The antibody or fragment thereof may preferentially bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), e.g., human NKG2A, meaning that the antibody or fragment thereof binds to NKG2A with greater affinity than it binds to a control protein (e.g., an unrelated control protein, e.g., hen egg white lysozyme or NKG2C) and / or binds to human NKG2A with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind to NKG2A or a portion thereof. "Specific binding" means that the antibody or fragment thereof binds to NKG2A with an affinity that is at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, or 10,000-fold greater than its affinity to an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind substantially exclusively to NKG2A (e.g., be able to distinguish NKG2A from other known polypeptides, e.g., based on a measurable difference in binding affinity). In some embodiments, the NKG2A-binding agent (e.g., antibody) may react with NKG2A sequences other than human NKG2A sequences (e.g., cynomolgus NKG2A sequences, such as A42 described herein). In other embodiments, the NKG2A-binding agent (e.g., antibody) does not react with non-human (e.g., cynomolgus) NKG2A sequences, such as A2, A3, and A11 provided in this disclosure.

[0050] The term "variable region" or "variable domain" refers to the portion of an antibody light or heavy chain generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length in the heavy chain and approximately 100-110 amino acids in the light chain, which is responsible for the binding and specificity of each particular antibody for its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that the sequences of certain segments of the variable region vary significantly among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) regions of about 15-30 amino acids, known as framework regions (FRs), separated by shorter, more variable (e.g., highly variable) regions known as "hypervariable regions" or alternatively, "complementarity-determining regions (CDRs)." The heavy and light chain variable regions each contain four framework regions (FR1, FR2, FR3, and FR4) that primarily adopt a β-sheet configuration, connected by three hypervariable regions, which form connecting loops that, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the framework regions and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant regions are not directly involved in antibody binding to antigens but are involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary greatly in sequence among different antibodies. The sequence variability is concentrated in the CDRs, while the less variable parts of the variable regions are called framework regions (FRs).The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with its antigen. In a specific embodiment, the variable regions are human variable regions.

[0051] The terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR," as used herein, refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions: three in the VH (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three in the VL (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Numerous hypervariable region descriptions are in use and are included herein. Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead, Chothia refers to the location of a structural loop (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the loop (this variation is due to the Kabat numbering scheme allowing for insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecula's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are shown below.

[0052] A universal numbering system has been developed and widely adopted (ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003))). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, CDR and framework residues can be readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from an immunoglobulin of one species into an acceptor framework, typically derived from a human antibody. An additional numbering system (AHon) was developed in Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). Correspondence between numbering systems (including, e.g., Kabat numbering and the IMGT-specific numbering system) is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also exemplified below. The various systems known in the art or described herein represent different ways of delineating CDRs and are often considered equivalent when used to define the same antibody. The exemplary system shown herein is a combination of Kabat and Chothia. Residues from each of these hypervariable regions or CDRs are illustrated in the table below. [Table A]

[0053] The hypervariable region may comprise the "hypervariable region" at positions 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and positions 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR" are used interchangeably.

[0054] "Polynucleotide" or "nucleic acid," when used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA polymerase or RNA polymerase or by a synthetic reaction. A polynucleotide may include modified nucleotides, such as methylated nucleotides and their analogs. Cells producing binding molecules of the disclosure may include parent hybridoma cells into which nucleic acid encoding the antibody has been introduced, as well as bacterial and eukaryotic host cells. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction in which a nascent RNA transcript is added from 5' to 3' is called the transcription direction; the region of the DNA strand that has the same sequence as the RNA transcript and is 5' to the 5' end of the RNA transcript is called the "upstream sequence," and the region of the DNA strand that has the same sequence as the RNA transcript and is 3' to the 3' end of the RNA transcript is called the "downstream sequence."

[0055] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence (e.g., for the purpose of introducing a nucleic acid sequence into a host cell). Examples of vectors that can be used include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can contain a selection sequence or marker that is operable for stable integration into a host cell chromosome. In addition, the vector can contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients that are not present in the culture medium. Expression control sequences can include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both the heavy and light chains of an antibody or both the VH and VL of an antibody), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or can be linked to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting for gene product expression, polymerase chain reaction (PCR) amplification or immunoblotting of mRNA, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. One skilled in the art will know that the nucleic acid molecule will be expressed in sufficient amounts to produce the desired product (e.g., an NKG2A-binding agent as described herein), and will further know that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0056] The term "pharmaceutically acceptable," as used herein, means recognized by a regulatory agency of the Federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly, in humans.

[0057] "Excipient" refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, wetting agents, lubricants, flavoring agents, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle. In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids, antioxidants (including ascorbic acid), low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides, and other sugar chains (including glucose, mannose, or dextrins), chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio.See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed to the excipient at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution. In some embodiments, the excipient is a sterile liquid, such as water or oil, including petroleum oils (animal, vegetable, or synthetic), such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions of the present disclosure can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, if desired. The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.Oral compositions (including formulations) can contain standard excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions (including pharmaceutical compounds) can contain a prophylactically or therapeutically effective amount of an NKG2A binding agent (e.g., an antibody), e.g., in isolated or purified form, together with appropriate amounts of excipients so as to provide a form for proper administration to a subject (e.g., a patient). The formulation should be compatible with the mode of administration.

[0058] An "effective amount" generally is an amount sufficient to reduce the severity and / or frequency of a symptom, eliminate a symptom and / or its underlying cause, prevent or delay the onset of a symptom and / or its underlying cause, and / or ameliorate or repair damage caused by or associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0059] The term "therapeutically effective amount," as used herein, refers to an amount of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or its associated symptoms. A therapeutically effective amount of an agent (including a therapeutic agent) can be the amount necessary to (i) reduce, delay, or ameliorate the progression or progression of a given disease, disorder, or condition; (ii) reduce, delay, or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition; and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administering an agent described herein). A "therapeutically effective amount" of a substance / molecule / agent (e.g., an NKG2A antibody) of the present disclosure can vary according to factors such as the individual's condition, age, sex, and weight, and the ability of the substance / molecule / agent to elicit a desired response in an individual. A therapeutically effective amount includes an amount in which any toxic or adverse effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a drug effective to "treat" a disease, disorder or condition in a subject or mammal.

[0060] The term "treating," or any of its grammatical variations, refers to reducing and / or ameliorating the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith, for example, (i) reducing, delaying, or ameliorating the progression or onset of a given disease, disorder, or condition; (ii) reducing, delaying, or ameliorating the recurrence, development, or onset of a given disease, disorder, or condition; and / or (iii) improving or enhancing the prophylactic or therapeutic effects of another therapy (e.g., a therapy other than administering an agent described herein).

[0061] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., prevents or delays the onset (or recurrence) of a disease, disorder, or condition, or reduces the likelihood of the onset (or recurrence) of a disease, disorder, or condition or associated symptom(s).

[0062] The full therapeutic or prophylactic effect may not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more doses.

[0063] The terms "about" and "approximately" refer to a variation of no more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

[0064] As used herein, comparative terms, such as "reduce," "decrease," "increase," or any of their grammatical variations, can refer to a certain variation from its reference value. In some embodiments, such a variation can refer to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 100-fold higher than the reference value. In some embodiments, such a variation can refer to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of a reference value.

[0065] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0066] In some embodiments, the terms "first," "second," "third," "fourth," and similar terms in the names of components are used to distinguish and identify more than one component that shares a particular identity in those names. For example, "first antibody" and "second antibody" are used to distinguish between two antibodies.

[0067] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described using the words "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described using the term "consisting of" are also provided.

[0068] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0069] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or," when used in phrases such as "A, B and / or C," is intended to include each of the following embodiments: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0070] The term "optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes cases where the circumstance occurs and cases where the circumstance does not occur.

[0071] NKG2A-binding agents In some embodiments, the present disclosure provides NKG2A-binding agents that can be used in the present disclosure as agents to enhance immune responses and / or therapeutic agents. Such agents include antibodies (e.g., monospecific or multispecific (including bispecific)) that bind to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, as well as variants thereof with improved or decreased affinity or other properties.

[0072] In some embodiments, described herein are NKG2A-binding agents (e.g., antibodies) that bind to NKG2A (including an NKG2A polypeptide, an NKG2A polypeptide fragment, an NKG2A peptide, or an NKG2A epitope). In some embodiments, the NKG2A-binding agent is a human or humanized antibody (e.g., an antibody comprising a human constant region) that binds to NKG2A (including an NKG2A polypeptide, an NKG2A polypeptide fragment, an NKG2A peptide, or an NKG2A epitope). In some embodiments, the NKG2A-binding agent (e.g., antibody), e.g., a human NKG2A-binding agent, can bind to NKG2A expressed on the surface of mammalian (e.g., human) cells (including immune cells (e.g., NK cells or T cells) that express NKG2A). In some embodiments, the NKG2A-binding agent (e.g., antibody) binds to an NKG2A extracellular epitope exposed on a cell, e.g., an immune cell. In some embodiments, described herein are NKG2A-binding agents (e.g., antibodies) that bind to NKG2A, e.g., human NKG2A, or a portion thereof. In some embodiments, the NKG2A is human NKG2A. In some embodiments, the NKG2A-binding agent is a human NKG2A-binding agent (e.g., an antibody that binds to human NKG2A). In some embodiments, the NKG2A-binding agent (e.g., an antibody) binds to both human NKG2A and cynomolgus NKG2A. In other embodiments, the NKG2A-binding agent (e.g., an antibody) binds to human NKG2A but not cynomolgus NKG2A. In some embodiments, described herein are NKG2A-binding agents (e.g., antibodies) that bind to a complex comprising NKG2A and CD94, or a complex comprising the extracellular domains of NKG2A and CD94.

[0073] In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein bind to NKG2A (e.g., human NKG2A) with a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 M or less, e.g., 10 -8 M~10-13M, e.g., 10 -9M ∼10 M). Various methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including, for example, by RIA using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81), by Octet®, for example, using the Octet® Red 96 system, or by Biacore®, for example, using a Biacore® TM-2000 or Biacore® TM-3000, or by surface plasmon resonance (SPR) assays. The "association rate" or "k" may be determined by the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) methods described above, for example, using Octet® Red 96, a Biacore® TM-2000, a Biacore® TM-3000 system, a Biacore® TM-8K or a Biacore® TM-8K+ system.

[0074] In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein do not bind to NKG2C (or a complex comprising NKG2C and CD94 or their respective extracellular domains) (e.g., human NKG2C and / or cynomolgus NKG2C). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein do not bind to human NKG2C. In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein do not bind to human NKG2C or cynomolgus NKG2C. In other embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein bind to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) with higher affinity than to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of an NKG2A-binding agent (e.g., an antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least twice as strong as its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of an NKG2A-binding agent (e.g., an antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least five times as strong as its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of an NKG2A-binding agent (e.g., an antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 10 times greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).In some embodiments, the binding affinity of an NKG2A-binding agent (e.g., an antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 100-fold greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of an NKG2A-binding agent (e.g., an antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 1000-fold greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).

[0075] In some embodiments, an NKG2A-binding agent (e.g., an antibody) described herein comprises the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, e.g., the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as set forth in Tables 1-4. Accordingly, in some embodiments, an NKG2A-binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three of the heavy chain CDRs and / or any one, any two, and / or all three of the light chain CDRs from (a) the antibody designated A2, (b) the antibody designated A3, (c) the antibody designated A11, and (d) the antibody designated A42, as set forth in Tables 1-4. In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein comprise any one, any two, and / or all three of the heavy chain CDRs and any one, any two, and / or all three of the light chain CDRs from (a) the antibody designated A2, (b) the antibody designated A3, (c) the antibody designated A11, and (d) the antibody designated A42, as shown in Tables 1-4.

[0076] In some embodiments, an NKG2A-binding agent (e.g., an antibody) comprises the VH region (including VH CDR1, VH CDR2, and / or VH CDR3) and / or the VL region (including VL CDR1, VL CDR2, and / or VL CDR3) of any one of the binding agents described herein (see, e.g., any one of Tables 1-4). Accordingly, in some embodiments, an NKG2A-binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 1. In some embodiments, an NKG2A-binding agent (e.g., an antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 2. In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein comprise any one, any two, and / or all three heavy chain CDRs, and / or any one, any two, and / or all three light chain CDRs, from Table 3. In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein comprise any one, any two, and / or all three heavy chain CDRs, and / or one, two, and / or three light chain CDRs, from Table 4.

[0077] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided in this disclosure comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25, SEQ ID NO:45, or SEQ ID NO:64, and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26, SEQ ID NO:46, SEQ ID NO:65, or SEQ ID NO:73.

[0078] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to the Chothia numbering system. In other embodiments, the CDRs are numbered by Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and exemplified in Section 5.1 above.

[0079] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by the present disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20, and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0080] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0081] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0082] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0083] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0084] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0085] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0086] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to the Chothia numbering system. In other embodiments, the CDRs are determined by Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0087] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by the present disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 32, 35, 39, and 44, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 33, 36, and 40; and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 34, 37, and 41, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 11, and 42, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 38, and 43.

[0088] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0089] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0090] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0091] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0092] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

[0093] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0094] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to Chothia numbering. In other embodiments, the CDRs are numbered according to Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0095] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62.

[0096] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0097] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0098] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0099] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58.

[0100] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62.

[0101] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0102] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to Chothia numbering. In other embodiments, the CDRs are numbered according to Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0103] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 68, 70, and 71, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 72, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62.

[0104] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0105] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0106] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0107] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58.

[0108] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 71, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62.

[0109] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0110] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 25, 26, 45, 46, 64, 65, and / or 73. In some embodiments, the antibody or fragment thereof further comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs: 25, 26, 45, 46, 64, 65, and 73. In some embodiments, the antibody provided herein is a humanized antibody. The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, when CDRs are determined by, for example, Kabat, IMGT, or Chothia, the framework region is the amino acid residues surrounding the CDR in the variable region in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1; when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between the amino acid residues of CDR1 and CDR2; when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between the amino acid residues of CDR2 and CDR3; and when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR4 is defined as the amino acid residues C-terminal to the amino acid residues of CDR3.

[0111] In some embodiments, an NKG2A-binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) (including the human NKG2A-binding agents described herein) comprises a VH region or VH domain. Additionally or alternatively, in some embodiments, an NKG2A-binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) (including the human NKG2A-binding agents described herein) comprises a VL region or VL domain. In some embodiments, an NKG2A-binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) (including the human NKG2A-binding agents described herein) has a combination of (i) a VH domain or VH region and (ii) a VL domain or VL region.

[0112] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0113] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0114] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0115] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0116] In certain embodiments, an NKG2A-binding agent (e.g., an antibody or fragment thereof) provided in this disclosure comprises an amino acid sequence that is a particular percent identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any antibody or fragment thereof provided in this disclosure, e.g., a CDR, VH, or VL in Tables 1-4, or any full-length antibody chain as disclosed herein. In some embodiments, an NKG2A-binding agent (e.g., an antibody or fragment thereof) provided in this disclosure comprises a CDR of any antibody or fragment thereof provided in this disclosure, e.g., in Tables 1-4. In further embodiments, the NKG2A-binding agents provided herein comprise an amino acid sequence that is a particular percentage identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any antibody or fragment thereof provided herein, e.g., a VH or VL of Tables 1-4, or any full-length antibody chain as disclosed herein.

[0117] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), which has been modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with NBLAST nucleotide program parameters set, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residues in the alignment that differ between the two sequences (excluding or including conservative amino acid substitution(s) or degenerate nucleotide substitution(s)) by the number of residues in one of the following: (i) the full length of the shorter sequence, (ii) the full length of the longer sequence, (iii) the average length of the two sequences, (iv) the total ungapped portion of the alignment, (v) the length of the alignment (excluding overhangs), or (vi) the length of the alignment (including overhangs).As used herein, an overhang, in the context of a sequence alignment, refers to a region at either or both ends of the alignment where residues from one sequence do not align with residues from the other sequence (e.g., a gap is considered). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding program (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted in calculating percent identity.

[0118] In some embodiments, binding agents (e.g., antibodies) provided herein comprise sequences that contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the binding agent retains the ability to bind to NKG2A. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDRs (e.g., in the FR and / or constant regions).

[0119] In some embodiments, one or more of the CDRs along the VH region (e.g., CDR1, CDR2, or CDR3) and / or one or more positions of the CDRs along the VL region (e.g., CDR1, CDR2, or CDR3) of an NKG2A-binding agent (e.g., antibody) described herein (including human NKG2A-binding agents) can be varied by 1 amino acid position, 2 amino acid positions, 3 amino acid positions, 4 amino acid positions, 5 amino acid positions, or 6 amino acid positions, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the positions defining a CDR of any of Tables 1, 2, 3, or 4 can be varied by shifting the N-terminal and / or C-terminal boundaries of that CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to the current CDR position, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more of the CDRs along the VH region (e.g., CDR1, CDR2, or CDR3) and / or one or more of the CDRs along the VL region (e.g., CDR1, CDR2, or CDR3) of an NKG2A-binding agent (e.g., antibody) described herein (including human NKG2A-binding agents) can vary by 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or more (e.g., can be shorter or longer), so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).For example, in some embodiments, the CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein can be 1, 2, 3, 4, 5 or more amino acids shorter than one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In other embodiments, the CDR1, CDR2 and / or CDR3 of the VH and / or VL described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In some embodiments, the amino terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%).Additionally or alternatively, in some embodiments, the carboxy terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Maintained binding to NKG2A (e.g., human NKG2A) can be confirmed using any method known in the art, such as the binding assays and binding conditions described in the "Examples" section herein.

[0120] In other embodiments, the NKG2A-binding agents (e.g., antibodies) (including human NKG2A-binding agents) provided herein that bind to NKG2A further comprise conservative sequence modifications. With respect to polypeptides that are NKG2A-binding agents (e.g., antibodies), e.g., human NKG2A-binding agents, conservative sequence modifications include conservative amino acid substitutions, including substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. Thus, in some embodiments, a predicted non-essential amino acid residue in NKG2A is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions that do not eliminate antigen binding and the nucleotides encoding those amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acid sequence of the NKG2A-binding agent (e.g., antibody), including human NKG2A-binding agents. In some embodiments, an amino acid sequence modification refers to up to 1, 2, 3, 4, 5, or 6 amino acid substitutions in a CDR, e.g., a CDR set forth in any one of Tables 1 to 4. Thus, for example, each such CDR may comprise up to 5 conservative amino acid substitutions, e.g., up to 4 (or no more than 4) conservative amino acid substitutions, e.g., up to 3 (or no more than 3) conservative amino acid substitutions, e.g., up to 2 (or no more than 2) conservative amino acid substitutions or no more than 1 conservative amino acid substitution.In some embodiments, the NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent) comprises one or more (including six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CDRs of A3, A2, A42, or A11 (see, e.g., Tables 1, 2, 3, or 4).

[0121] In some embodiments, the NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent) comprises a VH and a VL that comprise CDRs identical to the CDRs of A3, A2, A42, or A11 (see, e.g., Tables 1, 2, 3, or 4). In some embodiments, the amino acid sequence modifications do not include any modifications within the SDRs. In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (e.g., CDR1, CDR2, CDR3, or any combination thereof). Additionally or alternatively, the amino acid sequence modifications are in the framework, constant region, and / or fragment crystallizable region (Fc).

[0122] In some embodiments, the antibodies or fragments provided herein comprise a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26, and the binding of the antibody or fragment to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0123] In some embodiments, the antibodies or fragments provided herein comprise a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46, and the binding of the antibody or fragment to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0124] In some embodiments, the antibodies or fragments provided herein comprise a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65, and the binding of the antibody or fragment to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0125] In some embodiments, the antibodies or fragments provided herein comprise a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73, and the binding of the antibody or fragment to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0126] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning or hydrogen / deuterium exchange mass spectrometry (HDX-MS) to identify amino acids in the NKG2A protein (or a complex comprising NKG2A and CD94 or their respective extracellular domains) required for interaction with an NKG2A-binding agent (e.g., an antibody) provided herein. In some embodiments, the three-dimensional and crystal structures of an NKG2A-binding agent (such as an antibody) that binds to NKG2A can be used to identify its epitope. In some embodiments, the present disclosure provides antibodies that specifically bind to the same epitope as any of the NKG2A-binding agents (e.g., antibodies or fragments thereof) provided herein.

[0127] For example, in some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0128] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0129] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0130] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0131] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by the present disclosure specifically binds to one of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91). In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided by the present disclosure specifically binds to two of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to three of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to four of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to five NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 91).

[0132] In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of two of the following amino acid sequences: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of three of the following amino acid sequences: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of four of the following amino acid sequences: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of five of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the amino acid sequences TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the group of amino acid residues that form the conformational epitope includes one amino acid residue from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes two amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes three amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes four amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes five amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes more than five amino acid residues from the amino acid sequence set forth above in this paragraph.

[0133] In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of two of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following three amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following four amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following five amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 86), SIISPSSWIGV (SEQ ID NO: 87), FRNSSHHPW (SEQ ID NO: 88), IKDSDNAEL (SEQ ID NO: 89), LQVNR (SEQ ID NO: 90), and AQCGSSI (SEQ ID NO: 91). In some embodiments, the group of amino acid residues that form the conformational epitope includes one amino acid residue from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes two amino acid residues from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes three amino acid residues from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes four amino acid residues from the amino acid sequences set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes five amino acid residues from the amino acid sequences set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes more than five amino acid residues from the amino acid sequences set forth above in this paragraph.

[0134] In some embodiments, the NKG2A-binding agents (e.g., antibodies) provided in this disclosure specifically bind to NKG2A competitively with any one of the anti-NKG2A antibodies or fragments thereof described herein.

[0135] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0136] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0137] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0138] In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, an NKG2A-binding agent (e.g., an antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0139] In some embodiments, the NKG2A-binding agent binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the NKG2A-binding agent comprises six CDRs of the antibody A3. In further embodiments, the NKG2A-binding agent comprises six CDRs as listed in one of the columns of Table 1. In some embodiments, the NKG2A-binding agent comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:25 and three CDRs of a light chain variable region as set forth in SEQ ID NO:26. In some embodiments, the NKG2A-binding agent comprises a heavy chain variable region as set forth in SEQ ID NO:25 and a light chain variable region as set forth in SEQ ID NO:26.

[0140] In some embodiments, the NKG2A-binding agent binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the NKG2A-binding agent comprises six CDRs of antibody A2. In further embodiments, the NKG2A-binding agent comprises six CDRs as listed in one of the columns of Table 2. In some embodiments, the NKG2A-binding agent comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:45 and three CDRs of a light chain variable region as set forth in SEQ ID NO:46. In some embodiments, the NKG2A-binding agent comprises a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:46.

[0141] In some embodiments, the NKG2A-binding agent binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the NKG2A-binding agent comprises six CDRs of the antibody A42. In further embodiments, the NKG2A-binding agent comprises six CDRs as listed in one of the columns of Table 3. In some embodiments, the NKG2A-binding agent comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:64 and three CDRs of a light chain variable region as set forth in SEQ ID NO:65. In some embodiments, the NKG2A-binding agent comprises a heavy chain variable region as set forth in SEQ ID NO:64 and a light chain variable region as set forth in SEQ ID NO:65.

[0142] In some embodiments, the NKG2A-binding agent binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In a further embodiment, the NKG2A-binding agent comprises six CDRs of the antibody A11. In a further embodiment, the NKG2A-binding agent comprises six CDRs as listed in one of the columns of Table 4. In some embodiments, the NKG2A-binding agent comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:64 and three CDRs of a light chain variable region as set forth in SEQ ID NO:73. In some embodiments, the NKG2A-binding agent comprises a heavy chain variable region as set forth in SEQ ID NO:64 and a light chain variable region as set forth in SEQ ID NO:73.

[0143] In some embodiments, the binding antibodies have good developability based on assays known in the art, such as various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and stand-up monolayer adsorption chromatography (SMAC). In some embodiments, the binding agents have good developability based on measurements of percentage monomer, solubility, and / or antibody aggregation or precipitability.

[0144] In some embodiments, the NKG2A-binding agents (e.g., antibodies, such as monospecific or bispecific antibodies) described herein, including human NKG2A-binding agents, comprise a heavy chain having a combination of (i) a VH described herein, e.g., in any one of Tables 1-4, and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). Exemplary IgG heavy chains include any VH sequence as described herein, It contains the amino acid sequences of CH1, hinge, CH2 and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81). Another exemplary IgG heavy chain comprises any VH sequence as described herein: It contains the amino acid sequence of CH1, hinge, CH2 and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 85). In a further embodiment, the carboxyl terminus (C-terminus) of the VH is conjugated directly or indirectly to the amino terminus (N-terminus) of one or more heavy chain constant domains thereof.

[0145] In some embodiments, the NKG2A-binding agents (e.g., antibodies such as monospecific or bispecific antibodies) described herein, including human NKG2A-binding agents, comprise a light chain having a combination of (i) a VL described herein, e.g., in any one of Tables 1-4, and (ii) a light chain constant domain (CL). An exemplary light chain (e.g., a light chain paired with an IgG heavy chain) comprises any of the VL sequences described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82) In a further embodiment, the C-terminus of the VL is conjugated directly or indirectly to the N-terminus of the CL.

[0146] In some embodiments, the binding agents provided herein inhibit HLA-E / NKG2A (e.g., HLA-E / NKG2A / CD94) signaling. Such inhibition can be measured, for example, as detailed in Example 4.

[0147] In some embodiments, the NKG2A-binding agents (e.g., antibodies such as monospecific or bispecific antibodies) described herein, including human NKG2A-binding agents, comprise (a) a heavy chain having a combination of (i) a VH described herein, e.g., in any one of Tables 1-4, and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3), and (b) a light chain having a combination of (i) a VL described herein, e.g., in any one of Tables 1-4, and (ii) a light chain constant domain (CL or CL1) in an IgG format. Exemplary NKG2A-binding agents (e.g., antibodies) comprise an IgG heavy chain comprising any VH sequence as described herein and the amino acid sequence of SEQ ID NO: 81 or 85, and a light chain comprising any VL sequence as described herein and the amino acid sequence of SEQ ID NO: 82.

[0148] In some embodiments, the present disclosure provides an NKG2A binding protein comprising any one of the anti-NKG2A antibodies described herein. In some embodiments, the NKG2A binding protein is an antibody comprising two heavy chains and two light chains. In some embodiments, the NKG2A binding protein is an antibody comprising two heavy chains comprising the same VH region and two light chains comprising the same VL region.

[0149] In some embodiments, the NKG2A binding protein is a monoclonal antibody, including a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the anti-NKG2A antibody is an antibody fragment, e.g., an scFv. In some embodiments, the NKG2A binding protein is a fusion protein comprising an anti-NKG2A antibody provided herein. In other embodiments, the NKG2A binding protein is a multispecific antibody comprising an anti-NKG2A antibody or a fragment thereof provided herein.

[0150] Other exemplary NKG2A binding molecules are described in further detail in the sections below. In some embodiments, an anti-NKG2A antibody or antigen binding protein according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 5.2.1-5.2.4, below. [Table 1] [Table 2] [Table 3] [Table 4]

[0151] 5.2.1. Antibody fragments Although the term "antibody" is sometimes used herein in the phrase "antibody or fragment thereof," as used herein, it should be understood that the term "antibody" also includes various antibody fragments, e.g., antigen-binding or epitope-binding fragments. Thus, when the term "antibody" is used alone, without being followed by "fragment thereof" or a similar term, it should be understood that the term "antibody" includes antibody fragments, e.g., antigen-binding or epitope-binding fragments. Antibodies provided in the present disclosure include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

[0152] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to antigen. Antibody fragments include, but are not limited to, those described in Section 5.1 above. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and a portion of a light chain and a heavy chain cross-linked by a disulfide bond), Fab' (e.g., antibody fragments containing a single antigen-binding domain containing a Fab and an additional portion of a heavy chain up to the hinge region), F(ab')2 (e.g., two Fab' molecules linked by an interchain disulfide bond at the hinge region of the heavy chain, where the Fab' molecules may be directed against the same epitope or different epitopes), bispecific Fa (ab') ... b (e.g., a Fab molecule having two antigen-binding domains, each directed against a different epitope); single chain Fvs (also known as scFvs) comprising variable regions (e.g., in which the variable antigen-binding determining regions of a single light chain and a single heavy chain of an antibody are linked together by a chain of, e.g., 10-25 amino acids); disulfide-linked Fvs, or dsFvs (e.g., in which the variable antigen-binding determining regions of a single light chain and a single heavy chain of an antibody are linked together by a disulfide bond); bispecific scFvs (e.g., an scFv or dsFv molecule having two antigen-binding domains, each of which may be directed against a different epitope), a diabody (e.g., a dimerized scFV formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv and the VL domain of the first scFv is assembled with the VH domain of the second scFv, wherein the two antigen-binding regions of the diabody may be directed against the same epitope or different epitopes), a triabody These include scFvs (e.g., trimerized scFvs formed in a manner similar to diabodies, but in which the three antigen-binding domains are assembled in a single complex, and in which the three antigen-binding domains may be directed against the same or different epitopes), and tetrabodies (e.g., tetramerized scFvs formed in a manner similar to diabodies, but in which the four antigen-binding domains are assembled in a single complex, and in which the four antigen-binding domains may be directed against the same or different epitopes).

[0153] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained via proteolysis of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17 and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be expressed and secreted from E. coli, yeast, or insect cells, allowing the facile production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture media. Fab and F(ab')2 fragments with extended in vivo half-lives, which contain salvage receptor-binding epitope residues, are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, e.g., WO 93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458). Fvs and scFvs have combined intact fragments that lack constant regions, which may be suitable for reduced nonspecific binding during in vivo use. ScFv fusion proteins can be constructed to fuse effector proteins to either the amino or carboxy terminus of the scFv (see, e.g., Borrebaeck, ed., supra). An antibody fragment may be a "linear antibody," e.g., as described in the references cited above. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

[0154] Humanized Antibodies The present disclosure provides humanized antibodies that bind to NKG2A (including human NKG2A). The humanized antibodies of the present disclosure may comprise one or more CDRs from the VH and / or VL disclosed herein, e.g., those CDRs set forth in Tables 1-4. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanized antibodies that bind to NKG2A can be generated using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8):397-402, 2000; and U.S. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120, and 4,816,567).

[0155] In some cases, the humanized antibody is constructed by CDR grafting, in which the six amino acid sequences of the CDRs of the VH and VL of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J.9:133-139, 1995) determined that only about one-third of the residues in the CDRs actually contact the antigen, and designated these residues as "specificity-determining residues" or "SDRs." In the SDR grafting method, only the residues of the SDRs are grafted onto a human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34, 2005).

[0156] The selection of human variable domains (both light and heavy chains) used to generate humanized antibodies can be important for reducing antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623). In some cases, the framework is selected from the most abundant human subclass, V L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0157] In an alternative paradigm based on CDR comparison (termed Superhumanization), framework homology is not important. The method involves comparing nonhuman sequences with the functional human germline gene repertoire. Among these genes, genes encoding a canonical structure identical to or closely related to the mouse sequence are then selected. Next, among genes that share their canonical structure with the nonhuman antibody, genes with the highest CDR homology are selected as framework donors. Finally, nonhuman CDRs are grafted onto these frameworks (see, e.g., Tan et al., J. Immunol. 169:1119-1125, 2002).

[0158] Furthermore, it is generally desirable to humanize antibodies while maintaining affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display predicted three-dimensional conformations of selected candidate immunoglobulin sequences. These programs include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-2713, 1997). Inspection of these representations permits analysis of the potential role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, for example, improved affinity for the target antigen(s). In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0159] Another method for humanizing antibodies is based on a metric called Human String Content (HSC). In this method, the mouse sequence is compared to the human germline gene repertoire, and the differences are scored as HSC. The target sequence is then humanized by maximizing the HSC, rather than using an exhaustive identity measure, to generate multiple diverse humanized variants. See, for example, Lazar et al., Mol. Immunol. 44:1986-1998, 2007.

[0160] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants may be isolated from phage display libraries, ribosome display libraries, and yeast display libraries, as well as by screening bacterial colonies (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-1116, 2005; Dufner et al., Trends Biotechnol. 24:523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21:163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17:847-60, 2004).

[0161] In the framework library approach, a collection of residue variants is introduced into a given position in the framework, and the library is then selected to select the framework that best corresponds to the grafted CDR. The substituted residues may include some or all of the "Vernier" residues identified as likely contributors to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272:10678-10684, 1997).

[0162] In framework shuffling, instead of generating a combinatorial library of selected residue variants, the entire framework is combined with a non-human CDR (see, e.g., Dall'Acqua et al., Methods 36:43-60, 2005). The library may be screened for binding in a two-step selection process, first humanizing the VL and then the VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and increased thermostability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0163] The "humaneering" method is based on experimentally identifying essential minimal specificity determinants (MSDs) and then sequentially replacing non-human fragments with a library of human frameworks and assessing binding. Starting with the CDR3 regions of the non-human VH and VL chains, other regions of the non-human antibody are progressively replaced with the human framework (including CDR1 and CDR2 of both VH and VL). This approach typically preserves and identifies epitopes of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies. See, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007.

[0164] The method of "human engineering" involves altering a non-human antibody or non-human antibody fragment, e.g., a murine antibody, chimeric antibody, murine antibody fragment, or chimeric antibody fragment, by making predetermined changes to the amino acid sequence of the antibody to generate a modified antibody with reduced immunogenicity in humans, but which nonetheless retains the desired binding properties of the original non-human antibody. Generally, this technique involves classifying amino acid residues in a non-human (e.g., murine) antibody as "low risk," "moderate risk," or "high risk" residues. The classification is performed using a comprehensive risk / reward calculation that assesses the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody and / or the risk of substituting a human residue. Specific human amino acid residues to be substituted at predetermined positions (e.g., low-risk or moderate-risk positions) in a non-human (e.g., murine) antibody sequence can be selected by aligning an amino acid sequence from the variable region of the non-human antibody with the corresponding region of a predetermined human antibody sequence or a consensus human antibody sequence. Following the alignment, amino acid residues at low-risk or moderate-risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence. Techniques for generating human-engineered proteins are described in further detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.

[0165] 5.2.3. Antibody Variants Modifications of the antibodies that bind to NKG2A described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. That is, variants of the antibodies that bind to NKG2A described herein can be prepared, and these variants are contemplated as being included in the present disclosure. In some embodiments, antibody variants are antibodies that have variations in amino acid sequence compared to the original antibody, e.g., substitutions, deletions, or insertions of one or more amino acids as described above. For example, variations can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide that result in an altered amino acid sequence compared to the original antibody or polypeptide (e.g., conservative substitutions). Relevant sites for substitutional mutagenesis include CDRs, FRs, and / or constant regions. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. It is apparent to one skilled in the art that amino acid changes can alter post-translational processing of antibodies.

[0166] chemical modification Other exemplary modifications include chemical modification, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives may include antibodies chemically modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, routine chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-classical amino acids.

[0167] In some embodiments, antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites from an antibody may conveniently be accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0168] When an antibody provided by the present disclosure is fused to an Fc region, the carbohydrate moiety attached to the antibody may be modified. Naturally occurring antibodies produced by mammalian cells typically contain a biantennary branched carbohydrate moiety, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). This carbohydrate moiety may include various carbohydrate moieties, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary carbohydrate structure. In some embodiments, carbohydrate modifications in the binding molecules provided by the present disclosure may be performed to generate variants with improved specific properties.

[0169] In other embodiments, when antibodies provided herein are fused to an Fc region, antibody variants provided herein may have a glycan structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-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 glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Fc region residue EU numbering), although Asn297 may be located approximately ±3 amino acids upstream or downstream from position 297, i.e., at positions 294 to 300, depending on minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication Nos. 2003 / 0157108 and WO2004 / 056312), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006) and WO2003 / 085107).

[0170] The antibody-containing binding molecules provided herein further comprise bisected glycans, e.g., glycans in which GlcNAc is attached to a branch of a biantennary glycan attached to the Fc region. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue on the glycan attached to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0171] In the antibodies and Fc region-containing molecules of the present disclosure, one or more amino acid modifications may be introduced into the Fc region to generate Fc region variants, which may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0172] In some embodiments, the present application contemplates variants that retain some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the binding molecule is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the binding molecule lacks FcγR binding (i.e., is likely to lack ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96I® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, for example, the C1q-binding ELISA and C3c-binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0173] Binding molecules with reduced effector function include molecules with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0174] Certain variants have been described that have improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0175] In some embodiments, the variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334 (EU numbering). In some embodiments, modifications are made in the Fc region that alter (e.g., either improve or decrease) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0176] Binding molecules with increased half-life and improved binding to fetal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These molecules comprise an Fc region with one or more substitutions in that region that improve binding to FcRn. Such Fc variants include variants having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0177] In some embodiments, it may be desirable to create a cysteine ​​engineered antibody, in which one or more of the antibody's residues are substituted with a cysteine ​​residue. In some embodiments, the substituted residues are located at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein.

[0178] Other known covalent modifications of antibodies are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of an antibody with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl residues to the corresponding glutamyl residues and asparaginyl residues to the corresponding aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of either C-terminal carboxyl group.

[0179] The antibodies of the present disclosure that bind NKG2A may be modified to form chimeric molecules comprising the antibody that binds NKG2A fused or conjugated to another heterologous polypeptide, amino acid sequence, or small molecule compound, such as an immunostimulatory agent (e.g., a cytokine), an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)), or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0180] Also provided herein are fusion proteins comprising an antibody of the present disclosure that binds to NKG2A and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated is useful for directing the antibody to cells expressing NKG2A on their surface. Genetically fused or chemically conjugated antibodies are described in further detail in the sections below.

[0181] In vitro affinity maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Like natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Antibody libraries are displayed on the surface of organisms (e.g., phage, bacterial, yeast, or mammalian cells) or in association with their encoding mRNA or DNA (e.g., by covalent or noncovalent attachment). Affinity selection of the displayed antibodies allows the isolation of organisms or complexes carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinities in the narrow nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinities for target antigens.

[0182] Phage display is a widely used method for the display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions to bacteriophage coat proteins. Selection involves exposing the phage-displayed antibodies to antigen to bind to their target, a process called "panning." Antigen-bound phage are removed and used to infect bacteria to generate phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002) and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0183] In the yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall through a disulfide bond to Aga1p. Display of the protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules in the yeast cell wall. The library is screened using magnetic separation and flow cytometry to select for antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent conjugated to a fluorophore, such as streptavidin. Differences in antibody surface expression can be measured through immunofluorescence labeling of either hemagglutinin or c-Myc epitope tags adjacent to the single-chain antibody (e.g., scFv). Expression has been shown to correlate with the stability of the displayed protein, so antibodies can be selected for improved stability and affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control machinery. Once maturation is complete, antibody affinity can be conveniently "fine-tuned" while displayed on the yeast surface, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is that the size of the functional library can potentially be smaller than that of other display methods, although recent approaches have used the mating system of yeast cells to generate libraries of up to 10 14 (See, e.g., U.S. Patent Application Publication No. 2003 / 0186374 and Blaise et al., Gene 342:211-18 (2004)).

[0184] Ribosome display generates antibody-ribosome-mRNA (ARM) complexes during cell-free selection. DNA libraries encoding specific antibodies are genetically engineered to spacer sequences lacking a stop codon. When translated, these remain attached to peptidyl-tRNAs, occupying the ribosomal tunnel and allowing the proteins to protrude from the ribosome and fold. The resulting complexes of mRNA, ribosomes, and proteins can be bound to surface-bound ligands, allowing for simultaneous isolation of the antibody and its encoding mRNA through affinity capture by the ligand. The ribosome-bound mRNA is then reverse-transcribed back to cDNA, which can then be mutagenized and used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish a covalent link between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0185] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each selection round, for example, by non-proofreading polymerases, since no library should be transformed after any diversification step. In some embodiments, mammalian display systems may be used.

[0186] Diversity can be introduced into the CDRs of an antibody library in a targeted manner or through random introduction. The former approach involves sequentially targeting all CDRs of an antibody through high-level or low-level mutagenesis, or targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected of affecting affinity for experimental or structural reasons. Diversity can also be introduced by substituting naturally diverse regions through DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops spanning framework region residues, utilize loop deletions and insertions in the CDRs (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), or use hybridization-based diversification (see, e.g., U.S. Patent Application Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Additional methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0187] Screening of libraries can be accomplished by a variety of techniques known in the art, for example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells affixed to adsorption plates, used in cell sorting, conjugated to biotin for capture by streptavidin-coated beads, or used in any other method for panning display libraries.

[0188] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005), Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and references therein.

[0189] Antibody internalization assays may be used to determine receptor-mediated endocytosis upon antibody binding. In some embodiments, the efficacy of certain antibody-based therapeutics depends on the antibody internalization process. In some embodiments, antibody internalization assays examine the rate and extent of antibody internalization to assess the ability of the antibody to deliver a therapeutic agent to a site or cell of interest. Non-limiting exemplary assays are described below. Target cells of interest are seeded at an appropriate seeding density (e.g., in a 96-well U-bottom plate), and a test antibody is labeled with a signal-reporting reagent, such as a fluorescent compound, horseradish peroxidase (HRP) reagent, a radiolabeled compound, or biotin. The test antibody and target cells are then incubated at an appropriate molar ratio. After incubation, unbound antibody is removed by washing. The cells can be left on ice or incubated at 37°C for a period of time to facilitate internalization. The cells can then be incubated in the presence of a stop reagent for a period of time to inhibit internalization. The cells are then washed and incubated with a signal-emitting reagent. The final signal can be examined using a plate reader or imaging device and analysis software. For example, a flow cytometer can be used to measure the mean fluorescence intensity (MFI) of the cells, and a decrease in MFI can indicate antibody internalization, antibody dissociation, or a combination of both. Cell images can be scanned and acquired to analyze signal intensity, size, and shape. Alternatively, the cells can be lysed to release the internalized antibody. This antibody is then captured on a microtiter well plate coated with the specific antigen to which the antibody was raised. Bound antibody in the wells is detected using a secondary antibody conjugated with alkaline phosphatase or HRP and a chromogenic substrate. Alternative detectable antibody labels and disclosures will be apparent to those skilled in the art. Any method known in the art for determining antibody internalization can be used in the present disclosure.

[0190] 5.2.4. Other Binding Agents Comprising Antibodies of the Disclosure In some embodiments, the antibodies or fragments thereof provided herein are part of a larger binding agent. Non-limiting exemplary binding agents comprising the antibodies or fragments provided herein are described below.

[0191] The present disclosure provides NKG2A-binding agents (e.g., antibodies) having masking and / or cleavable moieties, such that one or more of the NKG2A-binding domains of the NKG2A-binding agent (e.g., antibody) are masked (e.g., via a masking moiety) and / or activatable (e.g., via a cleavable moiety). Techniques for masking NKG2A-binding agents (e.g., antibodies) are well known in the art, including the masking technology SAFEbody (see, e.g., U.S. Patent Application Publication No. 2019 / 0241886) and the masking technology Probody (see, e.g., U.S. Patent Application Publication No. 2015 / 0079088). Such techniques can be used to generate masked and / or activatable NKG2A-binding agents (e.g., antibodies). Such masked and / or activatable NKG2A-binding agents (e.g., antibodies) are also useful for preparing conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable ADCs (AADCs) comprising any one of the NKG2A-binding agents (e.g., antibodies), e.g., human NKG2A-binding agents, of the present disclosure, including binding agents linked directly or indirectly to another agent, e.g., a drug and / or an immunoactivator (e.g., a cytokine). For example, the NKG2A-binding agents (e.g., antibodies), e.g., human NKG2A-binding agents, of the present disclosure, can be covalently linked to one or more agents, e.g., drugs and / or immunoactivators (e.g., cytokines), by a synthetic linker.

[0192] If desired, the NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent) is linked or conjugated (directly or indirectly) to a moiety that has an effector function, e.g., cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope), immune mobilization activity, or immune modulatory activity. Linked or conjugated (directly or indirectly) moieties include drugs that are cytotoxic (e.g., a toxin such as auriristatin), drugs that are not cytotoxic, e.g., signal transduction regulators such as kinases, masking moieties that mask one or more of the binding domains of the NKG2A-binding agent (e.g., an antibody), or cleavable moieties that allow the NKG2A-binding agent to be activated in the tumor microenvironment in the form of a masked conjugate by cleaving the cleavable moiety to expose one or more of the binding domains of the NKG2A-binding agent (e.g., an antibody). Moieties that promote immune mobilization can include other antigen-binding agents, e.g., viral proteins that selectively bind to cells of the innate and / or adaptive immune system. Alternatively or additionally, the NKG2A-binding agents (e.g., antibodies) (including human NKG2A-binding agents) are optionally linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety that has reporter activity (e.g., a detectable label or reporter protein). It will be apparent that the characteristics of NKG2A-binding agents (e.g., antibodies) (including human NKG2A-binding agents) described herein also extend to polypeptides, including NKG2A-binding agent fragments.

[0193] In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) that bind to NKG2A can be linked or conjugated (directly or indirectly) to a polypeptide, thereby generating an activatable antibody. In some embodiments, the NKG2A-binding agents (e.g., antibodies) (including human NKG2A-binding agents) are linked or conjugated (directly or indirectly) to an additional agent. In some embodiments, the additional agent is a drug, resulting in an ADC, or an AADC when the antibody of the ADC comprises a masking moiety and a cleavable moiety.

[0194] In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent or cytokine), or a diagnostic or detectable agent. Conjugated or recombinantly linked antibodies, including masked or activatable conjugates, can be useful, for example, to treat or prevent a disease, disorder, or condition, e.g., an NKG2A-mediated disease, disorder, or condition. Conjugated or recombinantly linked NKG2A-binding agents (e.g., antibodies), including masked or activatable conjugates, can be useful, for example, to monitor or prognose the onset, development, progression, and / or severity of an NKG2A-mediated disease, disorder, or condition.

[0195] Such diagnosis and detection can be accomplished, for example, by coupling the NKG2A binding agent (e.g., an antibody) to a detectable substance, such as, for example, an enzyme (including but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase), a prosthetic group (including but not limited to, streptavidin / biotin or avidin / biotin), a fluorescent substance (including but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin), a luminescent substance (including but not limited to, luminol), a bioluminescent substance (including but not limited to, luciferase, luciferin, or aequorin), a chemiluminescent substance (including but not limited to, acridinium-based compounds or HALOTAG), a radioactive substance (iodine ( 131 I, 125 I, 123 I and 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In and 111 In), Technonetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga and 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn or 117 ion, including but not limited to Sn), positron-emitting metals using various positron emission tomography techniques, as well as non-radioactive paramagnetic metal ions.

[0196] Also described herein are NKG2A-binding agents (e.g., antibodies) recombinantly linked or conjugated (covalently or non-covalently conjugated (directly or indirectly)) to a heterologous protein or polypeptide, or fragment thereof, e.g., a polypeptide (e.g., consisting of about 10 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, or about 100 amino acids), to generate fusion proteins, and uses thereof. In particular, described herein are fusion proteins comprising an antigen-binding fragment (e.g., a fragment comprising CDR1, CDR2, and / or CDR3 of VH and / or VL) of an NKG2A-binding agent (e.g., antibody) described herein (including human NKG2A-binding agents) and a heterologous protein, polypeptide, or peptide. In some embodiments, heterologous proteins, polypeptides, or peptides to which an NKG2A-binding agent (e.g., an antibody) is linked are useful for directing the NKG2A-binding agent to specific cells, e.g., NKG2A-expressing cells (including immune cells). Other non-limiting heterologous proteins, polypeptides, or peptides to which an NKG2A-binding agent (e.g., an antibody) is linked can be useful as internalization signals or to engage tumor cells with immune cells.

[0197] Additionally, the NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) can be linked (directly or indirectly) to a marker or "tag" sequence, e.g., a peptide, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is a hexahistidine peptide, such as the tag provided in pQE vectors (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine allows for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag (which corresponds to an epitope derived from the influenza hemagglutinin protein) (Wilson et al., 1984, Cell 37:767-78) and the "FLAG" tag.

[0198] Methods for linking or conjugating (directly or indirectly) moieties (including polypeptides) to antibodies are well known in the art, any one of which can be used to generate the antibody-drug conjugates or fusion proteins described herein.

[0199] In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein are fusion proteins. The term "fusion protein," as used herein, refers to a polypeptide comprising the amino acid sequence of a binding agent (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not normally a part of the antibody). In certain embodiments, the fusion protein retains the biological activity of the NKG2A-binding agent. In certain embodiments, the fusion protein comprises a VH region, a VL region, a VH CDR (one, two, or three VH CDRs), and / or a VL CDR (one, two, or three VL CDRs) of an NKG2A antibody, and the fusion protein binds to an NKG2A epitope, an NKG2A fragment, and / or an NKG2A polypeptide. In some embodiments, the fusion protein comprises the VH or heavy chain of an NKG2A antibody, the VL or light chain of an NKG2A antibody, separated by a linker, e.g., a cleavable linker.

[0200] Fusion proteins may be generated, for example, through the techniques of gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be used to modify the activity of NKG2A-binding agents (e.g., antibodies) as described herein, including human NKG2A-binding agents (e.g., antibodies), including NKG2A-binding agents with higher affinity and slower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some embodiments, NKG2A-binding agents (including human NKG2A-binding agents) may be modified prior to recombination by random mutagenesis via error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding the NKG2A-binding agents described herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0201] The NKG2A-binding agents (e.g., antibodies) described herein, including human NKG2A-binding agents, may be attached to a solid support, which is useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0202] The NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0203] The linker may be a "cleavable moiety" that facilitates release of the linked or conjugated agent in the cell, although non-cleavable linkers are also contemplated in the present disclosure. Linkers for use in the conjugates (e.g., ADCs or AADCs) of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent transporter-mediated multidrug resistance.

[0204] Conjugates of antibodies and drugs, including those for the preparation of ADCs or AADCs, can be prepared using a variety of bifunctional protein coupling agents, such as N-(β-maleimidopropyloxy)succinimide ester (BMPS), N-ε-maleimidocaproyl-oxysuccinimide ester (ECMS), N-γ-maleimidocaproyl-oxysuccinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4(-N-maleimidomethyl)succinimide ester (SBS), and 1,6-hexane-bis-vinylsulfone (HBVS). m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl-4-(N-maleimidomethyl)cyclohexane -1-carboxylate (SMCC), succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-ECMS), N-(γ-maleimidobutyloxy)sulfosuccinimide ester (sulfo-GMBS), N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), It may be made using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), and succinimidyl-(4-vinylsulfone)benzoate (SVSB).

[0205] The present disclosure further conjugates of antibodies and drugs (including when the drug is a drug for the preparation of an ADC or AADC), which may be prepared using any suitable method as disclosed in the art (see, e.g., Bioconjugate Technique (Hermanson ed., 2d ed. 2008)).

[0206] Traditional conjugation strategies for antibodies and drugs (including when the drug is a drug for the preparation of an ADC or AADC) are based on random conjugation chemistry involving the ε-amino group of a Lys residue or the thiol group of a Cys residue, resulting in heterogeneous conjugates. Recently developed techniques enable site-specific conjugation to antibodies, resulting in uniform loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These techniques include engineering "thiomabs" containing cysteine ​​substitutions at positions in the heavy and light chains, which introduce reactive thiol groups without inhibiting immunoglobulin folding and assembly or altering antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332:41-52 and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another method, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from STOP to a selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56 and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0207] In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) are conjugated to an agent, e.g., an immunostimulatory agent or a cytotoxic agent. In some embodiments, the NKG2A-binding agents (e.g., antibodies) disclosed herein (including human NKG2A-binding agents) can be optionally conjugated to one or more cytotoxic agents disclosed herein or known in the art to generate ADCs or AADCs. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including, but not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the cytotoxic agent is a radioconjugate or a radioisotope for generating a radioconjugated drug. A variety of radionuclides are available for generating radioconjugated drugs. 90 Y, 125 I, 131 I, 123 I, 111 In, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re and 212Conjugates of a polypeptide or molecule with one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a trichothecene, and CC1065, and derivatives of these toxins that retain toxin activity, can also be used. Conjugation of polypeptides or molecules with cytotoxic agents is carried out using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0208] In other embodiments, the NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) are conjugated to a drug, e.g., a signal transduction modulator, a proapoptotic agent, a mitotic inhibitor, an antitumor antibiotic, an immunomodulator, a nucleic acid for gene therapy, an alkylating agent, an angiogenesis inhibitor, an antimetabolite, a boron-containing agent, a chemoprotectant, a hormonal agent, an antihormonal agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor such as camptothecin or an analog thereof, and a tyrosine kinase inhibitor. In some embodiments, the mitotic inhibitor is a dolastatin, an auristatin, a maytansinoid, or a plant alkaloid. In some embodiments, the drug is a dolastatin, an auristatin, a maytansinoid, or a plant alkaloid. Exemplary auristatins are monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). Examples of maytansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracycline, calicheamicin, and duocarmycin. An example of an actinomycin is pyrrolobenzodiazepine (PBD). An example of an anthracycline is a PNU-anthracycline, such as PNU-159682 or a derivative.

[0209] The NKG2A-binding agents (e.g., antibodies) described herein (including human NKG2A-binding agents) can be monospecific, bispecific, trispecific, or of greater multispecificity. Such agents may comprise monospecific or multispecific antibodies. Multispecific antibodies, e.g., bispecific antibodies, are monoclonal antibodies with binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., bispecific antibodies against NKG2A having a first binding domain for a first epitope on NKG2A and a second binding domain for a second epitope on NKG2A). In some embodiments, monospecific and multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of antibodies described herein, e.g., the CDR sequences listed in Tables 1-4. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are murine, chimeric, human, or humanized antibodies.

[0210] In some embodiments, one of the binding specificities of the multispecific antibody is for NKG2A and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody can comprise more than one target (e.g., antigen) binding domain, each different binding domain being specific for a different target (e.g., a first binding domain that binds to NKG2A and a second binding domain that binds to another target (e.g., antigen)). In some embodiments, the second target is an immune checkpoint regulator (e.g., a negative checkpoint regulator). In some embodiments, the second target is expressed on an immune cell. In some embodiments, the second target is expressed on a tumor cell or a cancer cell.

[0211] In some embodiments, multispecific (eg, bispecific) antibody molecules are capable of binding to more than one (eg, two or more) epitopes on the same target (eg, antigen).

[0212] Methods for producing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0213] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10:1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0214] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety appended, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG formats can include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and / or orthogonal Fab.

[0215] In some embodiments, BslgG comprises a heavy chain that has been engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., a κλ-body), and the use of a heterodimeric Fc region. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art, including knobs-into-holes, duobodies, azymetric, charge pairs, HA-TF, SEEDbodies, and Protein A affinity differentials.

[0216] Another bispecific antibody format is an IgG with an additional antigen-binding moiety added. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Non-limiting examples of adducted IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. In some embodiments, an exemplary antibody format is a B-Body format for monospecific or multispecific (e.g., bispecific) antibodies, as described, for example, in WO 2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0217] Bispecific (Bs) antibody (BsAb) fragments are a form of bispecific antibody molecule lacking some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, a bispecific antibody fragment comprises heavy and light chain regions linked by a peptide linker that allows efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

[0218] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or function. In some embodiments, dock-and-lock (DNL) technology can be used to generate bispecific antibody molecules with higher valency. For example, fusions of bispecific antibodies to albumin-binding proteins or human serum albumin can be constructed to extend the serum half-life of the antibody fragments. In embodiments, chemical conjugation, e.g., of antibodies and / or antibody fragments, can be used to generate BsAb molecules. Exemplary bispecific antibody conjugates include the CovX-body format, in which a low-molecular-weight drug is site-specifically conjugated to a single reactive lysine on each Fab arm or antibody or fragment thereof. In some embodiments, the conjugation improves serum half-life.

[0219] Methods for producing multispecific (including bispecific) antibodies are known in the art. For example, multispecific (including bispecific) antibodies can be produced by separately expressing the component antibodies in different host cells followed by purification / assembly, or by expressing the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be achieved by various methods known in the art, including affinity chromatography.

[0220] In some embodiments, the NKG2A-binding agents (e.g., antibodies) disclosed herein (including human NKG2A-binding agents) can be provided in any antibody format disclosed herein or known in the art. By way of non-limiting example, in some embodiments, the NKG2A-binding agent (e.g., antibody) (including human NKG2A-binding agents) is selected from the group consisting of Fabs-in-tandem-lg (FIT-lg), DVD-lg, hybrid hybridomas (quadromas or tetradomas), anticalin platforms (Pieris), diabodies, single-chain diabodies, tandem single-chain Fv fragments, TandAb, trispecific Ab (Affimed), Dart (dual affinity retargeting) (Macrogenics), bispecific Xmab (Xencor), bispecific T cell engager (Bite, Amgen, 55 kDa), triplebodies, tribodies (multifunctional recombinant antibody derivatives that are Fab-scFv fusion proteins (CreativeBiolabs)), duobody platforms (Genmab), dock and The following can be selected: lock platform, knobs-into-holes (KIH) platform, humanized bispecific IgG antibody (REGN1979) (Regeneron), Mab2 bispecific antibody (F-Star), DVD-lg (dual variable domain immunoglobulin (Abbott), kappa-lambda body, TBTI (tetravalent bispecific tandem Ig), and CrossMab (Roche).

[0221] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an NKG2A-binding domain and one or more additional binding domains that bind to one or more targets that are not NKG2A. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an NKG2A-binding domain comprising a VH and / or VL amino acid sequence as disclosed herein, e.g., an amino acid sequence of Table 1, Table 2, Table 3, or Table 4.

[0222] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to NKG2A, and that comprise a VH CDR and a VL CDR described herein, e.g., a VH CDR and a VL CDR as shown in Table 1, Table 2, Table 3, or Table 4.

[0223] In some embodiments, the NKG2A-binding agent is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the first binding domain comprises six CDRs of antibody A3. In some embodiments, the first binding domain comprises six CDRs as listed in one of the columns of Table 1. In some embodiments, the first binding domain comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:25 and three CDRs of a light chain variable region as set forth in SEQ ID NO:26. In some embodiments, the first binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:25 and a light chain variable region as set forth in SEQ ID NO:26. In some embodiments, the second binding domain binds to a different epitope in the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first complex or the second complex.

[0224] In some embodiments, the NKG2A-binding agent is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the first binding domain comprises six CDRs of antibody A2. In some embodiments, the first binding domain comprises six CDRs as listed in one of the columns of Table 2. In some embodiments, the first binding domain comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:45 and three CDRs of a light chain variable region as set forth in SEQ ID NO:46. In some embodiments, the first binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:46. In some embodiments, the second binding domain binds to a different epitope in the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first complex or the second complex.

[0225] In some embodiments, the NKG2A-binding agent is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the first binding domain comprises six CDRs of antibody A42. In some embodiments, the first binding domain comprises six CDRs as listed in one of the columns of Table 3. In some embodiments, the first binding domain comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:64 and three CDRs of a light chain variable region as set forth in SEQ ID NO:65. In some embodiments, the first binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:64 and a light chain variable region as set forth in SEQ ID NO:65. In some embodiments, the second binding domain binds to a different epitope in the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first complex or the second complex.

[0226] In some embodiments, the NKG2A-binding agent is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and the extracellular domain of CD94. In further embodiments, the first binding domain comprises six CDRs of antibody AA11. In some embodiments, the first binding domain comprises six CDRs as listed in one of the columns of Table 4. In some embodiments, the first binding domain comprises three CDRs of a heavy chain variable region as set forth in SEQ ID NO:64 and three CDRs of a light chain variable region as set forth in SEQ ID NO:73. In some embodiments, the first binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:64 and a light chain variable region as set forth in SEQ ID NO:73. In some embodiments, the second binding domain binds to a different epitope in the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first complex or the second complex.

[0227] In another aspect, the antibodies or antigen-binding fragments thereof provided herein can be part of an engineered cell surface receptor, such as a chimeric antigen receptor (CAR). Typically, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0228] In some embodiments, the present disclosure provides a CAR comprising an extracellular domain comprising one or more antibodies or fragments thereof provided herein. In some embodiments, the extracellular domain of a CAR provided herein comprises the VH CDRs and VL CDRs disclosed herein, e.g., the CDRs as set forth in Table 1, Table 2, Table 3, or Table 4.

[0229] The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain suitable for use in the CAR described herein can be derived from a natural protein. Alternatively, the domain can be a synthetic and non-natural protein segment that is thermodynamically stable in a cell membrane, such as a hydrophobic protein segment. The transmembrane domain is classified based on the three-dimensional structure of the transmembrane domain. For example, the transmembrane domain can form an α-helix, a complex with more than one α-helix, a β-barrel, or any other stable structure that can penetrate the phospholipid bilayer of a cell.

[0230] The CARs of the present disclosure comprise an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to the specialized function of a cell. T cell effector functions can be, for example, cytolytic activity or helper activity (including cytokine secretion). Thus, the term "cytoplasmic signaling domain" refers to the portion of a protein that transmits an effector function signal to instruct the cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be utilized, although in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transmit the effector function signal.

[0231] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain that consists essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function.

[0232] In addition to antigen-specific signal stimulation, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of the cells. In some embodiments, the CAR comprises at least one costimulatory signaling domain. The term "costimulatory signaling domain," as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function.

[0233] The CAR of the present disclosure may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. The hinge domain is an amino acid segment generally located between two domains of a protein, and may allow the protein to be flexible and one or both of these domains to move relative to each other. Any amino acid sequence that allows such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0234] A CAR of the present disclosure may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that directs a polypeptide to a desired site within a cell.

[0235] Other engineered transmembrane receptors, including antibodies or fragments provided in this disclosure, are also included in this disclosure.

[0236] 5.3. Nucleic Acids, Vectors and Cells Additionally, provided are nucleic acids encoding an NKG2A-binding agent (e.g., an antibody or antibody fragment) or fusion polypeptide as disclosed herein, nucleic acids complementary to the nucleic acids, vectors comprising a nucleic acid as disclosed herein, and cells comprising any one or more of an NKG2A-binding agent as disclosed herein, a nucleic acid as disclosed herein, or a vector as disclosed herein. In some embodiments, the cells express the NKG2A-binding agent. In some embodiments, the cells replicate the nucleic acid or vector. In some embodiments, provided are materials for producing NKG2A-binding agents, e.g., human NKG2A-binding agents, and fragments thereof. For example, an isolated cell may produce an NKG2A-binding agent (e.g., an antibody or antibody fragment). In this regard, the cell (e.g., an isolated cell) may produce an antibody or fragment thereof comprising a VH and VL as disclosed herein. In some embodiments, a polynucleotide described herein may comprise one or more nucleic acid sequences encoding an NKG2A-binding agent (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotide is an isolated polynucleotide and / or a recombinant polynucleotide. In various aspects, the isolated polynucleotide comprises a nucleotide sequence encoding a VH and / or a VL, wherein the VH and VL comprise complementarity determining regions (CDRs) identical to those disclosed herein.

[0237] As used herein, the term "complementary" refers to specific binding between polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they generate a predetermined or detectable level of signal in the hybridization assay. Polynucleotide moieties are complementary to each other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., less than about 3 bases) of mismatch, insertion, or deletion sequences may be present. The term "stringent assay conditions" refers to conditions that are compatible with forming nucleic acid binding pairs, e.g., probe and target mRNA binding pairs, that are sufficiently complementary to provide the desired level of specificity in the assay, but are generally incompatible with forming binding pairs between binding members that are insufficiently complementary to provide the desired specificity. The term "stringent assay conditions" generally refers to the combined hybridization and washing conditions.

[0238] In some embodiments, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expression of the one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying the polynucleotides in the host cell to produce useful quantities of the polynucleotides and for expressing the binding agent, e.g., an antibody or antibody fragment, using recombinant techniques.

[0239] In some embodiments, the one or more vectors are expression vectors in which one or more polynucleotides are operably linked to one or more polynucleotides comprising expression control sequences. Autonomously replicating recombinant expression constructs, such as plasmids and viral DNA vectors, incorporating one or more polynucleotides encoding antibody sequences that bind to NKG2A are specifically contemplated. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct will be utilized. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, while operator sequences are generally selected for their ability to regulate gene expression. The expression construct may also include sequences encoding one or more selectable markers that allow host cells containing the construct to be identified. The expression construct may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. In some embodiments, the expression construct may also include sequences necessary for replication in a host cell.

[0240] Exemplary expression control sequences include promoter / enhancer sequences, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985), the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993), the Tie promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995), the simian virus 40 promoter, DRA (Downregulated in adenoma; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007), MCT1 (monocarboxylate transporter 1; Cuff et al., J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007), and the cytomegalovirus promoter (Cuff et al., J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007). For expression in mammalian cells, the promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In another variation, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include an appropriate polyadenylation sequence (e.g., SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0241] If desired, the one or more polynucleotides may also optionally contain a nucleotide sequence encoding a secretory signal peptide fused in-frame to the polypeptide sequence. The secretory signal peptide directs secretion of the antibody polypeptide by cells expressing the one or more polynucleotides and is cleaved by the cell from the secreted polypeptide. The one or more polynucleotides may further optionally contain a sequence whose only intended function is to facilitate large-scale production of the vector. For gene therapy, polynucleotides can be produced and administered using procedures described in the literature for various transgenes. See, e.g., Isner et al., Circulation, 91:2687-2692, 1995 and Isner et al., Human Gene Therapy, 7:989-1011, 1996.

[0242] In some embodiments, the polynucleotide may further comprise additional sequences that facilitate uptake by a host cell and expression of the antibody or fragment thereof (and / or any other peptide). Some embodiments utilize a "naked" transgene (e.g., a transgene without a viral, liposomal, or other vector to facilitate transfection) encoding an antibody or fragment thereof described herein.

[0243] The polynucleotides of the present disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA can be double-stranded or single-stranded (where a single strand can be both the coding strand and the non-coding (antisense) strand). In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0244] The present disclosure further relates to variants of the polynucleotides described herein, which variants encode, for example, fragments, analogs, and / or derivatives of the binding molecules of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a binding molecule of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, provided that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with alternative nucleotides, or up to 5% of the total number of nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can be made at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and can be interspersed among nucleotides in the reference sequence either individually or in one or more contiguous groups within the reference sequence.

[0245] The polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not change the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing codons in human mRNA to codons preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0246] In some embodiments, polynucleotide variants are generated to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0247] Any suitable vector may be used to introduce one or more polynucleotides encoding the antibodies or fragments thereof into the host. Exemplary vectors that have been described include replication-deficient retroviral vectors, including lentiviral vectors (Kim et al., J. Virol., 72(1):811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46), parvoviral vectors, such as adeno-associated viral (AAV) vectors (U.S. Pat. Nos. 5,474,9351, 5,139,941, 5,622,856, 5,658,776, 5,773,289, 5,789,390, 5,834,441, 5,863,541, 5,851,521, 5,252,479; Gnatenko et al., J. Virol., 72(1):811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46), parvoviral vectors, such as adeno-associated viral (AAV) vectors (U.S. Pat. Nos. 5,474,9351, 5,139,941, 5,622,856, 5,658,776, 5,773,289, 5,789,390, 5,834,441, 5,863,541, 5,851,521, 5,252,479, Gnatenko et al., J. Virol., 72(1):811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46), and other vectors. al., J. Invest. Med., 45:87-98, 1997), adenovirus (AV) vectors (U.S. Patent Nos. 5,792,453, 5,824,544, 5,707,618, 5,693,509, 5,670,488, 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992; Stratford Perricaudet et al., J. Clin. Invest., 90:626-630, 1992; and Rosenfeld et al., J. Clin. Invest., 90:626-630, 1992). al., Cell, 68:143-155, 1992), adenovirus-adeno-associated virus chimeras (U.S. Pat. No. 5,856,152), or vaccinia or herpes virus vectors (U.S. Pat. Nos. 5,879,934, 5,849,571, 5,830,727, 5,661,033, 5,328,688), Lipofectin-mediated gene transfer (BRL), liposomal vectors (U.S. Pat. No. 5,631,237), and combinations thereof.Any of these expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). Optionally, the viral vector is made replication-deficient, for example, by deleting or disrupting certain genes required for viral replication.

[0248] Other non-viral delivery mechanisms that have been contemplated include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979; Felgner, Sci Am., 276(6):102-6, 1997; Felgner, Hum Gene Ther., 7(15):1791-3, 1996), sonication of cells (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene bombardment with high-velocity microprojectiles (Yang et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), and other methods have been proposed. USA, 87:9568-9572, 1990) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993).

[0249] The vector (or antibody or fragment thereof, or nucleic acid as disclosed herein) may be encapsulated in a liposome. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275(5301):810-814, (1997). Also contemplated are various commercial approaches involving "lipofection" technology. In some embodiments, liposomes may be complexed with hemagglutinating virus (HVJ), which has been shown to facilitate fusion with cell membranes and promote cell entry of liposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989). In some embodiments, liposomes are complexed or utilized in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In some embodiments, liposomes are complexed or utilized in conjunction with both HVJ and HMG-1. Such expression constructs have been successfully used for in vitro and in vivo transfer and expression of nucleic acids. In some embodiments, an NKG2A-binding agent (e.g., an antibody), including a human NKG2A-binding agent, is included in the liposome to target the liposome to cells (e.g., immune cells) that express NKG2A on their surface.

[0250] The cells can comprise one or more polynucleotides or one or more vectors, e.g., the cells have been transformed or transfected with one or more polynucleotides encoding an NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent), or one or more vectors comprising the one or more polynucleotides. In some embodiments, the cells express an NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent) that comprises one or more (including six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CDRs of A2, A3, A11, and / or A42 (see, e.g., Tables 1, 2, 3, and / or 4). In some embodiments, the cell expresses an NKG2A-binding agent (e.g., an antibody) (including a human NKG2A-binding agent) comprising a VH and VL that comprise CDRs identical to the CDRs of A2, A3, A11, and / or A42 (see, e.g., Tables 1, 2, 3, and / or 4). The cell can be a prokaryotic cell, such as an Escherichia coli cell (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or a eukaryotic cell, such as an animal cell (e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell, or a hybridoma cell), yeast (e.g., Saccharomyces cerevisiae), an insect cell, or a plant cell (e.g., a tobacco cell, a corn cell, a soybean cell, or a rice cell). The use of mammalian host cells can result in translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on the recombinant expression product. Similarly, polypeptides (e.g., NKG2A-binding agents (e.g., antibodies), including human NKG2A-binding agents) can be glycosylated, non-glycosylated, and / or covalently modified to include one or more water soluble polymer attachments, e.g., polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0251] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are also useful for amplifying polynucleotides and expressing polypeptides encoded by the polynucleotides. In this regard, a process for producing an NKG2A-binding agent (e.g., an antibody) may include culturing host cells and isolating the NKG2A-binding agent. Naked DNA expression constructs can be introduced into cells using particle bombardment, which relies on the ability to accelerate DNA-coated microprojectiles to high velocities, allowing them to penetrate cell membranes and enter cells without killing them (Klein et al., Nature, 327:70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on high-voltage firing to generate electrical current and thus provide motive force (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). The microprojectiles used consist of biologically inert materials, such as tungsten or gold beads. The host cell may be isolated and / or purified. The host cell may be a cell transformed in vivo to transiently or permanently express the polypeptide in vivo. The host cell may also be an isolated cell transformed ex vivo and introduced after transformation to produce the polypeptide in vivo, for example, for therapeutic purposes. The definition of host cell explicitly excludes transgenic humans.

[0252] 5.4. Fabrication method Antibodies that bind to NKG2A may be obtained by any suitable method, including (but not limited to) immunization with a population of whole cells and antibodies containing NKG2A, recombinant methods, or by screening a library of antibodies or antibody fragments using an extracellular domain epitope of NKG2A. Monoclonal antibodies may be produced using a variety of known techniques (see, e.g., Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for producing monoclonal antibodies involves immunizing an animal with human NKG2A antigen and producing hybridomas from spleen cells harvested from the animal, which are capable of producing monoclonal antibodies or antibody fragments that bind to NKG2A.

[0253] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques such as those described in Antibody Phage Display: Methods and Protocols, P.M.O. Brien and R. Aitken, eds., Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen adsorb to the antigen and are thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0254] The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments (in which VH and VL are covalently linked via a short, flexible peptide) or as Fab fragments (in which VH and VL are each fused to a constant domain and interact non-covalently), as described, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

[0255] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding clones, as described in Winter et al., supra. Libraries derived from immunized sources yield high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned without any immunization, resulting in a single source of human antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve in vitro rearrangement, as described, for example, in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0256] Screening of the library can be accomplished by a variety of techniques known in the art. For example, NKG2A (e.g., a polypeptide, fragment, or epitope of NKG2A) can be used to coat the wells of an adsorption plate, expressed on host cells attached to the adsorption plate, used in cell sorting, conjugated to biotin for capture on streptavidin-coated beads, or used in any other method for panning the display library. Selection of antibodies with slow dissociation rates (e.g., good binding affinity) can be facilitated by extensive washing and the use of monovalent phage display (such as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690), and low coating density of antigen (such as described in Marks et al., Biotechnol., 10:779-783 (1992)).

[0257] NKG2A-binding agents (e.g., antibodies) can be obtained by designing an appropriate antigen screening procedure to select relevant phage clones, and then constructing full-length NKG2A-binding agent (e.g., antibody) clones using the VH and / or VL sequences (e.g., Fv sequences) derived from the relevant phage clones, or various CDR sequences derived from the VH and VL sequences, and appropriate constant region (e.g., Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0258] Similarly, human antibodies that bind to NKG2A may be produced by any of a number of techniques, including, but not limited to, transformation of human peripheral blood cells (e.g., cells containing B lymphocytes) with Epstein-Barr virus (EBV), in vitro immunization of human B cells, fusion of splenocytes from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V-region phage libraries, or other procedures known in the art and based on the present disclosure. Methods for obtaining human antibodies from transgenic animals are further described, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8:455-58, 1997; Jakobovits et al., Ann. NY Acad. Sci., 764:525-35, 1995; Green et al., Nature Genet., 7:13-21, 1994; Lonberg et al., Nature, 368:856-859, 1994; Taylor et al., Int. Immun. 6:579-591, 1994; and U.S. Pat. No. 5,877,397.

[0259] For example, human antibodies that bind to NKG2A may be obtained from transgenic animals engineered to produce a specific human antibody in response to antigen challenge. For example, WO 98 / 24893 discloses transgenic animals with human Ig loci, which do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. Non-primate transgenic mammalian hosts capable of mounting an immune response to an immunogen have also been described, in which the antibodies have primate constant and / or variable regions and the endogenous immunoglobulin-encoding loci have been replaced or inactivated. WO 96 / 30498 discloses the use of the Cre / Lox system to modify immunoglobulin loci in mammals, e.g., to replace all or part of the constant or variable regions to form modified antibody molecules. WO 94 / 02602 discloses non-human mammalian hosts in which endogenous Ig loci have been inactivated and which have functional human Ig loci. U.S. Patent No. 5,939,598 discloses a method for producing transgenic mice lacking endogenous heavy chains and expressing an exogenous immunoglobulin locus containing one or more heterologous constant regions. Transgenic animals, such as those described herein, can be used to mount an immune response against a predetermined antigenic molecule, antibody-producing cells can be removed from the animal, and the cells can be used to generate hybridomas secreting human-derived monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art and have been used in immunizing transgenic mice, for example, as described in WO 96 / 33735. The monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.

[0260] In some embodiments, the NKG2A-binding agents described herein comprise a non-antibody protein scaffold. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, phynomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz-type inhibitors. Methods for producing such non-antibody protein scaffolds are well known in the art, any one of which can be used to produce an NKG2A-binding agent comprising a non-antibody protein scaffold (see, e.g., Simeon and Chen, Protein Cell, 9(1):3-14 (2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1):293-320 (2017)).

[0261] Various methods for producing antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). In addition, numerous publications describe techniques suitable for manipulating DNA, creating expression vectors, and transforming and culturing appropriate cells to prepare antibodies (see, for example, Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992, and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0262] NKG2A-binding agents (e.g., antibodies), including human NKG2A-binding agents, are produced using any suitable method, e.g., isolated from immunized animals, recombinantly or synthetically produced, or genetically engineered, including those described above. Antibody fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. For example, papain digestion of whole antibodies yields a 5S fragment designated F(ab')2, or pepsin digestion yields two monovalent Fab fragments and an Fc fragment. F(ab)2 can be further cleaved using a thiol-based reducing agent to generate a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described, for example, in Edelman et al., Methods in Enzymology, 1:422 Academic Press (1967), Nisonoff et al., Arch. Biochem. Biophys., 89:230-244, 1960, Porter, Biochem. J., 73:119-127, 1959, U.S. Patent No. 4,331,647, and Current Protocols in Immunology by Andrews, S. M. and Titus, J. A. (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5.

[0263] NKG2A-binding agents (e.g., antibodies), including human NKG2A-binding agents, can be genetically engineered. For example, NKG2A-binding agents (e.g., antibodies), including human NKG2A-binding agents, can include variable region domains, e.g., generated by recombinant DNA engineering. In this regard, the variable regions are optionally modified by insertions, deletions, or changes in the amino acid sequence of the antibody to generate the corresponding antibody, including those described above. Polynucleotides encoding the relevant CDRs are prepared, for example, by using the polymerase chain reaction to synthesize the variable regions using mRNA from antibody-producing cells as a template (see, for example, Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137 (Wiley Liss, Inc. 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106-110, 1991). Current antibody engineering methods allow for the construction of engineered variable region domains containing at least one CDR and, optionally, one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody. Such techniques are used, for example, to humanize an antibody or improve its affinity for a binding target. Exemplary methods for generating humanized antibodies are also described in the sections above.

[0264] Pharmaceutical Compositions In one aspect, the present disclosure further provides a composition, e.g., a pharmaceutical composition, comprising at least one binding agent provided herein (e.g., an antibody or antigen-binding fragment thereof provided herein), a nucleic acid provided herein, a vector provided herein, or a cell provided herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an NKG2A-binding agent provided herein (e.g., an antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid provided herein (e.g., a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a vector provided herein (e.g., a vector comprising a nucleic acid as disclosed herein and expressing an NKG2A-binding agent as disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a cell provided herein (e.g., a cell that comprises a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein and / or that expresses an antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient.

[0265] In some embodiments, pharmaceutical compositions provided herein are prepared for storage by mixing a binding agent, nucleic acid, vector, or cell provided herein, having the desired purity, with any physiologically acceptable excipient (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)), in aqueous solution or in lyophilized or other dried form.

[0266] The binding agents, nucleic acids, vectors, or cells of the disclosure may be formulated in any form suitable for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), as sustained release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0267] The binding agents, nucleic acids, vectors, or cells provided herein can also be encapsulated in microcapsules prepared, for example, by coacervation or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed, for example, in Remington, supra.

[0268] A variety of compositions and delivery systems are known and can be used with the binding agents, nucleic acids, vectors, or cells described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antigen-binding fragment thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of nucleic acids as part of retroviral or other vectors, etc. In another embodiment, the composition can be provided as a controlled- or sustained-release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74).In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof as described herein), or compositions provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al. (See, e.g., U.S. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326, WO 99 / 15154, and WO 99 / 20253.) Examples of polymers for use in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in the sustained release formulation are inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0269] In yet another embodiment, a controlled-release or sustained-release system can be placed in close proximity to a specific target tissue, such as the nasal passages or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, in Langer, 1990, Science 249:1527-33. Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more antibodies or antigen-binding fragments thereof as described herein (see, e.g., U.S. Pat. No. 4,526,938, WO 91 / 05548 and WO 96 / 20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89, Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97, Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 and Lam et al., 1997, Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:759-60).

[0270] 5.6.How to use In another aspect, the present disclosure provides methods of using the binding agents or compositions provided herein. In a further aspect, the present disclosure provides a binding agent or composition as disclosed herein for use as a pharmaceutical. In a further aspect, the present disclosure provides a binding agent or composition as disclosed herein for use in treating a disease or condition, such as a disease or condition disclosed herein. In one aspect, the present disclosure provides a binding agent or composition as disclosed herein for use in a method as disclosed herein.

[0271] In some embodiments, the binding agent binds to NKG2A. Additionally or alternatively, the binding agent binds to a complex comprising NKG2A and CD94. In further embodiments, the binding agent binds to a complex comprising NKG2A and the extracellular domain of CD94. In various embodiments, the NKG2A is human NKG2A. In further embodiments, the NKG2A also refers to cynomolgus NKG2A. In other embodiments, the binding agent does not bind to cynomolgus NKG2A or a complex comprising cynomolgus NKG2A. Additionally or alternatively, the binding agent does not bind to a complex comprising NKG2C and CD94 or a complex comprising the extracellular domain of NKG2C and CD94. In some embodiments, the binding agent binds to a first complex comprising NKG2A and the extracellular domain of CD94 but does not bind to a second complex comprising NKG2C and CD94.

[0272] In some embodiments, the present disclosure provides a method of inhibiting the interaction between NKG2A (e.g., expressed on and / or in a first cell) and HLA-E (e.g., expressed on and / or in a second cell), the method comprising contacting NKG2A (e.g., a first cell expressing NKG2A) with a binding agent (e.g., an antibody or fragment thereof) provided herein. In some embodiments, the present disclosure provides a use of a binding agent provided herein for inhibiting the interaction between NKG2A (e.g., expressed on and / or in a first cell) and HLA-E (e.g., expressed on and / or in a second cell), the use comprising contacting NKG2A (e.g., a first cell expressing NKG2A) with a binding agent (e.g., an antibody or fragment thereof) provided herein.

[0273] In a further aspect, the present disclosure provides a method for inhibiting the interaction of a complex comprising NKG2A and CD94, or their respective extracellular domains (e.g., expressed on and / or in a first cell), with HLA-E (e.g., expressed on and / or in a second cell), the method comprising contacting the complex comprising NKG2A and CD94 (e.g., a first cell expressing NKG2A and CD94) with a binding agent (e.g., an antibody or fragment thereof) provided herein. In some embodiments, the present disclosure provides a use of a binding agent provided herein to inhibit the interaction of a complex comprising NKG2A and CD94, or their respective extracellular domains (e.g., expressed on and / or within a first cell), with HLA-E (e.g., expressed on and / or within a second cell), comprising contacting a complex comprising NKG2A and CD94 (e.g., a first cell expressing NKG2A and CD94) with a binding agent provided herein (e.g., an antibody or fragment thereof).

[0274] In some embodiments, the methods or uses as disclosed herein do not inhibit the interaction of NKG2C (or a complex comprising NKG2C and CD94, or their respective extracellular domains) with HLA-E. In other embodiments, the methods or uses as disclosed herein inhibit the interaction of NKG2C (or a complex comprising NKG2C and CD94, or their respective extracellular domains) with HLA-E, but at a rate that is significantly lower than that of a benchmark anti-NKG2A antibody (e.g., at least about 10% lower, at least about 20% lower, at least about 30% lower, at least about 40% lower, at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, at least about 90% lower, at least about 95% lower, or at least about 99% lower).

[0275] As used herein, the term "inhibit" means to decrease or reduce. For example, in some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by 10% to 99%. In other embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by 100% (i.e., completely eliminate the interaction as measured by an assay). In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 10%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 20%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 30%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 40%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 50%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 60%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 70%.In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 80%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by at least 90%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by about 10% to 90%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by about 20% to 80%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by about 30% to 70%. In some embodiments, the binding agents provided herein inhibit the interaction of NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) with HLA-E by about 40% to 60%. In some embodiments, the NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) and HLA-E are expressed on different cells. In some embodiments, the NKG2A cell is expressed on a first cell, e.g., an immune cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, e.g., a CD8+ T cell.

[0276] Immune cells are cells of the immune system and can be cells of the lymphoid lineage. Non-limiting examples of lymphoid lineage cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and lymphocytes (B cells and T cells). T cells are a type of lymphocyte and can be characterized by expressing a T cell receptor (TCR). T cells play a central role in adaptive immune responses. T cell subtypes have various important functions in controlling and shaping immune responses. For example, cytotoxic T cells (also called cytotoxic T lymphocytes and killer T cells) are T lymphocytes that kill specific cells, such as cancer cells, cells infected with intracellular pathogens (such as viruses or bacteria), or cells that are otherwise damaged. Most cytotoxic T cells express a T cell receptor (TCR) that can recognize a specific antigen. CD8+ T cells are a subpopulation of MHC class I-restricted T cells that mediate adaptive immunity and are important for killing cancer- or virus-infected cells. NK cells are a type of cytotoxic lymphocyte essential to the innate immune system and belong to the innate lymphoid cell (ILC) family. In some embodiments, NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3-). NK cells have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, resulting in a more rapid immune response.

[0277] In some embodiments, the HLA-E is expressed on a second cell, eg, a cancer cell.

[0278] In some embodiments, the methods as disclosed herein are in vitro or ex vivo methods. In other embodiments, the methods as disclosed herein are in vivo methods. In some embodiments, the uses as disclosed herein are in vitro or ex vivo uses. In other embodiments, the uses as disclosed herein are in vivo uses. In some embodiments, the in vivo methods or in vivo uses as disclosed herein comprise administering a binding agent as disclosed herein to a subject having cells that express HLA-E, e.g., tumor cells that express HLA-E.

[0279] In other embodiments, the present disclosure provides a method of preventing or inhibiting immune cell suppression, e.g., suppression mediated by the interaction of NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) expressed on the immune cell with HLA-E (e.g., expressed on a cancer cell). In still other embodiments, the present disclosure provides a method of activating an immune cell-mediated response, e.g., an anti-tumor response. In some embodiments, the immune cell suppression is tumor / cancer-associated immune cell suppression, e.g., immune cell suppression in the tumor microenvironment. In some embodiments, the method comprises contacting the immune cell with a binding agent (e.g., an antibody or fragment thereof) provided herein. In some embodiments, the present disclosure provides a use of a binding agent provided herein to prevent immune cell suppression or to activate an immune cell-mediated response. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, e.g., a CD8 + In some embodiments, the immune cells are T cells. In some embodiments, the immune cells express NKG2A.

[0280] In some embodiments, the NKG2A-binding agents (e.g., antibodies) described herein are useful in compositions and methods for treating a disease or disorder. Accordingly, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a binding agent or pharmaceutical composition provided herein. In other embodiments, the present disclosure provides a use of a binding agent or pharmaceutical composition provided herein for treating a disease or disorder in a subject. In other embodiments, the present disclosure provides a binding agent or pharmaceutical composition provided herein for use in the manufacture of a medicament for treating a disease or disorder.

[0281] In some embodiments, treatments provided by the present disclosure include alleviating one or more symptoms associated with a disease or disorder (e.g., a cancer or tumor, or an autoimmune or inflammatory disease or disorder).

[0282] Thus, in some embodiments, the present disclosure provides a method of alleviating one or more symptoms associated with cancer or a tumor in a subject, the method comprising administering to the subject an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein. In some embodiments, the present disclosure provides an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, for use in alleviating one or more symptoms associated with cancer or a tumor in a subject. In some embodiments, the present disclosure provides the use of an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for alleviating one or more symptoms associated with cancer or a tumor in a subject.

[0283] In some embodiments, the treatment provided by the present disclosure includes reducing tumor size in a subject with a tumor. Accordingly, in some embodiments, described herein are methods of reducing tumor size in a subject with a tumor, comprising administering to the subject an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein. In some embodiments, the present disclosure provides an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, for use in reducing tumor size in a subject with a tumor. In some embodiments, the present disclosure provides the use of an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for reducing tumor size in a subject with a tumor.

[0284] In some embodiments, described herein are methods of enhancing tumor cell elimination in a subject with a tumor, comprising administering to the subject an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein. In some embodiments, the present disclosure provides an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, for use in enhancing tumor cell elimination in a subject with a tumor. In some embodiments, the present disclosure provides the use of an NKG2A-binding agent (e.g., an antibody) described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for enhancing tumor cell elimination in a subject with a tumor.

[0285] "Enhancing" tumor cell elimination includes, but is not required to, a 100% enhancement of elimination. Any enhancement of the rate of elimination is contemplated. Similarly, "modulating" tumor growth refers to reducing tumor size, slowing tumor growth, or inhibiting the expansion of an existing tumor. Complete tumor disappearance is included, but is not required; either a reduction in tumor size or a slowdown in tumor growth constitutes a beneficial biological effect in a subject. In this regard, tumor cell elimination may be enhanced, for example, by at least about 5%, at least about 10%, or at least about 20% compared to the level of elimination observed in the absence of the disclosed method (e.g., a biologically matched control subject or control specimen not exposed to the agent of the method). The effect is detected, for example, by a reduction in tumor size, a reduction or maintenance of tumor marker levels, or a reduction or maintenance of tumor cell population. In some embodiments, tumor cell elimination is enhanced by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more (about 100%) compared to, for example, the rate of tumor cell elimination in the absence of the NKG2A binding agent (e.g., antibody) or pharmaceutical composition of the disclosed methods.

[0286] Also provided are methods for modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject, e.g., comprising administering to the subject a composition comprising an NKG2A-binding agent (e.g., an antibody) as disclosed herein, or another composition as disclosed herein, in an amount effective to modulate tumor growth in the subject.

[0287] In some embodiments, the disease or disorder is a cancer or tumor. In further embodiments, the cancer or tumor expresses HLA-E. In some embodiments, the subject is a human subject.

[0288] As used herein, "tumor" refers to any growth or proliferation of neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, colon cancer, kidney cancer, lung cancer, squamous cell carcinoma, myeloid leukemia, hemangioma, melanoma, astrocytoma, and glioblastoma, and other cell proliferative disease states; cancer of the heart, i.e., sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; cancer of the lung, i.e., bronchogenic carcinoma (squamous cell, small undifferentiated cell, large undifferentiated cell, adenocarcinoma), alveolar epithelial (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma; gastrointestinal cancer, i.e., cancer of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma); cancer of the stomach (carcinoma, lymphoma, leiomyosarcoma); cancer of the pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, Cancer of the small intestine (adenocarcinoma, lymphoma, carcinoid tumor, VIP-secreting tumor), cancer of the small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), cancer of the large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), cancer of the genitourinary tract, i.e., cancer of the kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia, renal cell carcinoma), cancer of the bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), cancer of the prostate (adenocarcinoma, sarcoma, small cell carcinoma of the prostate), cancer of the testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma), cancer of the liver, i.e., hepatoma (hepatocellular carcinoma)carcinoma), bile duct carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, bone cancers, i.e., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), malignant giant cell tumor, chordoma, osteochondroma (osteochondroid exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor, cancers of the nervous system, i.e., skull cancers (osteoma, hemangioma, granuloma) tumors, xanthomas, osteitis deformans), cancers of the meninges (meningiomas, meningeal sarcomas, gliomatosis), cancers of the brain (astrocytoma, medulloblastoma, glioma, ependymoma, embryonal tumors (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas), gynecological cancers, i.e., cancer of the uterus (endometrial cancer), cancer of the cervix (cervical carcinoma, pre-neoplastic cervical dysplasia), cancer of the ovaries (ovarian carcinoma), serous Cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa cell-theca cell tumor, Sertoli cell-Leydig cell tumor, dysgerminoma, malignant teratoma), cancer of the vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), cancer of the vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), cancer of the fallopian tube (carcinoma), cancer of the blood, i.e., myeloid leukemia (acute and chronic), acute lymphoblastic leukemia These include, but are not limited to, myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma), cancers of the skin, i.e., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis, cancer of the adrenal gland, i.e., neuroblastoma, and cancer of the thyroid gland (including medullary thyroid carcinoma).

[0289] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor or cancer is not a solid tumor. In further embodiments, the cancer is a leukemic cancer. In some embodiments, the tumor or cancer is a recurrent tumor or cancer. In some embodiments, the tumor or cancer is a metastatic tumor or cancer. In some embodiments, the tumor or cancer is a primary tumor or cancer. In some embodiments, the tumor or cancer goes into remission but may recur. In some embodiments, the tumor or cancer is unresectable. Additionally or alternatively, the tumor or cancer is resistant to chemotherapy or other anti-cancer therapy. In further embodiments, the cancer or tumor expresses HLA-E.

[0290] In addition, NKG2A binding agents (e.g., antibodies) may be used to alleviate or reduce side effects associated with cancer, such as bone deterioration, spinal collapse, and paralysis. In one embodiment, a subject exhibits or is at risk of exhibiting bone metastases, and an NKG2A binding agent (e.g., antibody) is administered in an amount that reduces surrounding bone deterioration. Thus, in some embodiments, the NKG2A binding agent prevents bone deterioration due to bone metastases, with or without reducing tumor cell proliferation. In some embodiments, the NKG2A binding agent (e.g., antibody) both prevents bone deterioration due to bone metastases and reduces tumor cell proliferation. Generally, the effect on tumor cell proliferation (e.g., inhibition of proliferation or lack of an effect on proliferation) depends on the microenvironment of the particular metastasis. For example, the spread of metastases in a microenvironment with a significant amount of type 1 collagen may be inhibited. In contrast, the spread of metastases in a microenvironment without a significant amount of type 1 collagen may not be inhibited, but bone deterioration observed near the metastasis may be reduced or prevented.

[0291] In other embodiments, the present disclosure provides a method of alleviating one or more symptoms associated with an autoimmune or inflammatory disease or disorder in a subject, the method comprising administering to the subject an NKG2A-binding agent (e.g., an antibody) described herein or a pharmaceutical composition described herein. In some embodiments, the present disclosure provides an NKG2A-binding agent (e.g., an antibody) described herein or a pharmaceutical composition described herein, which is a multispecific binding agent or pharmaceutical composition for use in alleviating one or more symptoms associated with an autoimmune or inflammatory disease or disorder in a subject. In some embodiments, the present disclosure provides the use of an NKG2A-binding agent (e.g., an antibody) described herein or a pharmaceutical composition described herein in the manufacture of a medicament for alleviating one or more symptoms associated with an autoimmune or inflammatory disease or disorder in a subject. Autoimmune disease refers to a disease in which the body's immune system attacks healthy cells. In further embodiments, autoimmune disease refers to a disease in which the body's adaptive immune system attacks healthy cells. Inflammatory diseases refer to conditions associated with abnormal inflammation, for example, when the inflammatory response is misdirected, typically by the immune system attacking healthy tissue (resulting in inflammation). In some embodiments, inflammatory diseases are triggered by infection with a pathogen, such as a virus or bacteria. Additionally or alternatively, inflammatory diseases refer to conditions associated with an abnormal innate immune system that binds healthy cells.

[0292] Also provided are methods of treating a disease or disorder (e.g., cancer, or an autoimmune or inflammatory disease or disorder) by administering an NKG2A-binding agent (e.g., an antibody), e.g., a human NKG2A-binding agent, or a pharmaceutical composition as disclosed herein, to a subject in need of treatment, alone or in combination with another agent.

[0293] Subjects of the methods described herein can administer one or more additional therapeutic agents in combination with the NKG2A-binding agents (e.g., antibodies) or fragments thereof described herein, or the pharmaceutical compositions described herein. The additional agents can be agents that target tumor cells or cancer cells. The additional agents can also be agents that target immune cells (e.g., NK cells or T cells). In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic efficacy of the additional agent by about 10% to 90% or about 2 to 100 times. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic efficacy of the additional agent by at least 10%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic efficacy of the additional agent by at least 20%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic efficacy of the additional agent by at least 30%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic efficacy of the additional agent by at least 40%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 50%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 60%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 70%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 80%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 90%. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 2-fold. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 5-fold. In some embodiments, the NKG2A-binding agents or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 10-fold.In some embodiments, an NKG2A-binding agent or pharmaceutical composition provided by this disclosure enhances the therapeutic effect of an additional agent by at least 20-fold. In some embodiments, an NKG2A-binding agent or pharmaceutical composition provided by this disclosure enhances the therapeutic effect of an additional agent by more than 50-fold.

[0294] The particular dosing regimen for an NKG2A-binding agent (e.g., antibody) or pharmaceutical composition as disclosed herein for a particular subject will depend, in part, on the agent used, the dosage of the agent, the route of administration, and the cause and extent of any side effects. The amount of agent (e.g., antibody) administered to a subject (e.g., a mammal such as a human) should be sufficient to produce the desired response over a reasonable time frame. Thus, in some embodiments, the amount of NKG2A-binding agent (e.g., antibody) or pharmaceutical composition described herein that is administered to a subject is an effective amount.

[0295] Suitable routes of administering an NKG2A-binding agent (e.g., antibody), e.g., a human NKG2A-binding agent (e.g., antibody), or composition described herein are well known in the art, e.g., intravenous injection (such as intravenous infusion), intratumoral injection, or injection adjacent to a tumor or cancer. More than one route can be used to administer an agent (e.g., an antibody), although certain routes can result in a more rapid and effective response than other routes.

[0296] Gene Therapy and Cell Therapy In some embodiments, compositions for use in accordance with the present disclosure comprise one or more nucleic acids encoding, or complementary thereto, an NKG2A-binding agent (e.g., an antibody or fragment thereof) provided herein. In specific embodiments, the nucleic acid is administered to a subject by gene therapy for use in a method provided herein, e.g., to prevent, manage, treat, and / or ameliorate a disease or disorder (e.g., cancer, e.g., an HLA-E-expressing cancer). Such therapy includes therapy performed by administering to a subject an expressed or expressible nucleic acid. In embodiments, the nucleic acid produces an antibody encoded by the nucleic acid, and the antibody mediates a prophylactic or therapeutic effect.

[0297] Any of the methods for recombinant gene expression (or gene therapy) available in the art can be used.

[0298] For general reviews of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Recombinant DNA technology methods that are generally known in the art and that can be used are described in Ausubel et al. (eds.), Current ...

Claims

1. An antibody or fragment thereof that binds to NKG2A, (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 46; (iii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65; or (iv) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 73; The antibody or fragment thereof, comprising any one or more of (i) to (iv):

2. (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23; and and a VL region comprising: The antibody or fragment thereof of claim 1, comprising:

3. (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; The antibody or fragment thereof according to claim 1, comprising any one or more of (i) to (vi):

4. (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 32, 35, 39, and 44; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 33, 36, and 40; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 34, 37, and 41; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 11, and 42; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 38, and 43; and and a VL region comprising: The antibody or fragment thereof of claim 1, comprising:

5. (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 43; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31; The antibody or fragment thereof according to claim 1, comprising any one or more of (i) to (vi):

6. (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62; and and a VL region comprising: The antibody or fragment thereof of claim 1, comprising:

7. (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; The antibody or fragment thereof according to claim 1, comprising any one or more of (i) to (vi):

8. (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 68, 70, and 71; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 72; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62; and and a VL region comprising: The antibody or fragment thereof of claim 1, comprising:

9. (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 71, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50; The antibody or fragment thereof according to claim 1, comprising any one or more of (i) to (vi):

10. The antibody or fragment thereof according to any one of claims 1 to 9, further comprising the sequence of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4).

11. The antibody or fragment thereof according to any one of claims 1 to 10, further comprising a human framework sequence as set forth in any one of SEQ ID NOs: 25, 26, 45, 46, 64, 65 and 73, optionally further comprising the sequence of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4).

12. (i) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) VH comprising the amino acid sequence of SEQ ID NO: 45 and VL comprising the amino acid sequence of SEQ ID NO: 46; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65; or (iv) An antibody or fragment thereof according to any one of claims 1 to 11, comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO:

73.

13. The antibody or fragment thereof according to any one of claims 1 to 12, wherein the antibody is a monoclonal antibody.

14. The antibody or fragment thereof according to any one of claims 1 to 13, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

15. Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ 15. The antibody or fragment thereof according to any one of claims 1 to 14, which is any one of a single-chain antibody molecule, a dual variable region antibody, a single variable region antibody, a linear antibody, a multispecific antibody formed from a V region or an antibody fragment.

16. The antibody or fragment thereof of any one of claims 1 to 15, conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent.

17. The antibody or fragment thereof of claim 16, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug.

18. The antibody or fragment thereof according to any one of claims 1 to 17, wherein the antibody is a multispecific antibody.

19. 19. The antibody or fragment of claim 18, wherein the multispecific antibody is a bispecific antibody.

20. A binding agent that binds to essentially the same epitope as the antibody or fragment thereof of any one of claims 1 to 19.

21. 21. The binding agent of claim 20, which is an antibody or a fragment thereof.

22. 22. The binding agent of claim 21, wherein the antibody is a multispecific antibody.

23. A binding agent that competes with the antibody or fragment thereof of any one of claims 1 to 19 for binding to human NKG2A.

24. 24. The binding agent of claim 23, which is an antibody or a fragment thereof.

25. 25. The binding agent of claim 24, wherein the antibody is a multispecific antibody.

26. A polynucleotide encoding the antibody or fragment thereof according to any one of claims 1 to 15 and 18 to 19, or the binding agent according to any one of claims 21 to 22 and 24 to 25.

27. 27. One or more vectors comprising one or more of the polynucleotides or complementary polynucleotides of claim 26.

28. A cell comprising any one or more of the antibody or fragment thereof of any one of claims 1 to 19, the binding agent of any one of claims 20 to 25, the polynucleotide of claim 26, or one or more vectors of claim 27.

29. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the antibody or fragment thereof of any one of claims 1 to 19, the binding agent of any one of claims 20 to 25, the polynucleotide of claim 26, the one or more vectors of claim 27, or the cell of claim 28.

30. A method for inhibiting the interaction between HLA-E and NKG2A, the method comprising contacting the NKG2A with an antibody or fragment thereof according to any one of claims 1 to 19, a binding agent according to any one of claims 20 to 25, or a pharmaceutical composition according to claim 29.

31. 31. The method of claim 30, wherein the NKG2A is expressed on an immune cell.

32. 32. The method of claim 31 , wherein the immune cell is a NK cell or a T cell.

33. The T cell is CD8 ＋ 33. The method of claim 32, wherein the cell is a T cell.

34. The method according to any one of claims 30 to 33, wherein the HLA-E is expressed on cancer cells.

35. 30. A method of preventing immune cell suppression or activating immune cell-mediated responses, the method comprising contacting said immune cells with the antibody or fragment thereof of any one of claims 1 to 19, the binding agent of any one of claims 20 to 25, or the pharmaceutical composition of claim 29.

36. 36. The method of claim 35, wherein the immune cell is a NK cell or a T cell.

37. The T cell is CD8 ＋ 37. The method of claim 36, wherein the cell is a T cell.

38. The method of any one of claims 35 to 37, wherein the immune cells express NKG2A.

39. The method of any one of claims 35 to 38, wherein the immune cell-mediated response is an anti-tumor response.

40. 30. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an antibody or fragment thereof of any one of claims 1 to 19, a binding agent of any one of claims 20 to 25, or a pharmaceutical composition of claim 29.

41. 41. The method of claim 40, wherein the disease or disorder is cancer.

42. 42. The method of claim 41, wherein the cancer expresses HLA-E.

43. 41. The method of claim 40, wherein the disease or disorder is an autoimmune or inflammatory disease.

44. The method of any one of claims 40 to 43, wherein the subject is a human subject.

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