NKG2A binder and its uses

Abstract

This disclosure provides NKG2A binders and their uses, and in particular provides antibodies, including monospecific and multispecific antibodies, such as bispecific antibodies.

Description

[0001] This application is a divisional application of Chinese national phase patent application No. 202480021010.5, filed on January 26, 2024, entitled "NKG2A binder and its use". Cross-references to related applications

[0002] 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 which are incorporated herein by reference in their entirety. sequence list

[0003] This application contains a computer-readable sequence list that has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence list XML file submitted with this application is named "14529-150-228_SEQ_LISTING.xml", was created on January 17, 2024, and has a size of 103,086 bytes. 1. Technical Field

[0004] This disclosure generally relates to binding agents, such as antibodies (including fragments thereof) that bind to NKG2A, including human NKG2A, and methods of using them. 2. Background Technology

[0005] NKG2A is a cell surface molecule that is normally expressed on NK cells and can also be expressed on T cells, especially CD8 cells. + NKG2A is expressed on T cells. Therefore, it is a potential target for removing the suppression of immune cells and enhancing the anti-tumor response induced by immune cells. However, treatments using conjugates targeting NKG2A have not yet been successful. Therefore, there remains a need in the art for agents that enhance immune responses and treat diseases or conditions such as cancer. The conjugates, compositions, and methods provided herein meet this need and offer relevant advantages. 3. Summary of the Invention

[0006] This disclosure provides NKG2A binders, including human NKG2A binders. Such binders include antibodies that bind to NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains), such as monospecific or multispecific (e.g., bispecific) antibodies that bind to NKG2A. In some embodiments, such binders bind to the same NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains) epitopes as antibodies comprising the CDRs described herein (e.g., Tables 1-4). In some embodiments, such binders bind to the same NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains) epitopes as antibodies comprising the heavy chain variable region and light chain variable region described herein (e.g., Tables 1-4). In some embodiments, the NKG2A binder specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A binder specifically binds to a conformational epitope formed by a group of amino acid residues, said group of amino acid residues comprising at least one amino acid residue from: (1) 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), or (2) each of two, three, four, five, or all 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 binder specifically binds to a conformational epitope formed by a group of amino acid residues, said group of amino acid residues comprising at least one amino acid residue from: (1) one of the following amino acid sequences located 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) each of two, three, four, five, or all of the following amino acid sequences located 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).

[0007] This disclosure also provides a nucleic acid encoding the NKG2A binder (e.g., an antibody or a fragment thereof, such as an antigen-binding fragment) provided herein, a vector comprising one or more of such nucleic acids, and a cell comprising the nucleic acid, the vector, or both (such as a cell expressing the binder).

[0008] This disclosure also provides compositions comprising an NKG2A binder. In some embodiments, such compositions comprise an antibody that binds to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), such as a monospecific or multispecific (e.g., bispecific) antibody that binds to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains). In some embodiments, such compositions comprise an antibody that binds to an NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) epitope substantially the same as antibodies comprising the CDRs described herein (e.g., Tables 1-4) (e.g., human NKG2A). In some embodiments, such compositions comprise an antibody that binds to an NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) epitope substantially the same as antibodies comprising the heavy chain variable region and light chain variable region described herein (e.g., Tables 1-4) (e.g., human NKG2A). In some embodiments, the NKG2A binder specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A binder specifically binds to a conformational epitope formed by a group of amino acid residues, said group of amino acid residues comprising at least one amino acid residue from: (1) 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), or (2) each of two, three, four, five, or all 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 binder specifically binds to a conformational epitope formed by a group of amino acid residues comprising at least one amino acid residue from: (1) one of the following amino acid sequences located 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) each of two, three, four, five, or all of the following amino acid sequences located 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).

[0009] 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 the extracellular domains of NKG2A and CD94. Alternatively, the binding agent does not bind to NKG2C. Alternatively, the binding agent does not bind to a second complex comprising NKG2C and CD94. Alternatively, the binding agent does not bind to a second complex comprising the extracellular domains of NKG2C and CD94.

[0010] This disclosure also provides compositions comprising a nucleic acid encoding an NKG2A binder (e.g., an antibody or a fragment thereof, such as an antigen-binding fragment) provided herein; a vector comprising one or more nucleic acids; or cells comprising the nucleic acid, the vector, or both (e.g., cells expressing the binder).

[0011] This disclosure also provides various uses of the conjugates and compositions of the present invention, including, for example, methods for inhibiting the interaction between HLA-E and NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains) (such as those expressed on immune cells), and methods for preventing the inhibition or activation of immune cell-mediated responses by immune cells. Other aspects provided herein include methods for treating a subject with a disease or condition using the NKG2A conjugates or compositions provided herein. Such compositions include antibodies that bind to NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains), such as monospecific or multispecific (e.g., bispecific) antibodies that bind to NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains) (e.g., human NKG2A). 4. Description of the attached drawings

[0012] Figure 1 Exemplary results for A3 obtained by cell binding assays are shown, which are further described in Examples 3 and 6.

[0013] Figure 2 Exemplary results for A3 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0014] Figures 3A-3C Exemplary results for A3 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0015] Figure 4Exemplary results for A42 obtained by biological layer interferometry (BLI) binding assay (i.e., Octet binding assay) are shown, which is further described in Example 6.

[0016] Figure 5 Exemplary results regarding A42 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0017] Figures 6A-6C Exemplary results for A42 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0018] Figure 7 Exemplary results for A2 obtained by BLI binding assays are shown, which are further described in Example 6.

[0019] Figure 8 Exemplary results for A2 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0020] Figures 9A-9C Exemplary results for A2 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0021] Figure 10 Exemplary results for A11 obtained by BLI-based assays are shown, which are further described in Example 6.

[0022] Figure 11 Exemplary results regarding A11 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0023] Figures 12A-12C Exemplary results for A11 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0024] Figure 13 The surface rendering of NKG2A and CD94 highlights the tabletops.

[0025] Figure 14 A list of regions that clearly avoid deuterium exchange is shown. 5. Detailed Implementation

[0026] This disclosure is based, at least in part, on novel NKG2A binders and their properties. Such binders 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), including antibodies that bind to human NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains). In some aspects, such binders can be used to inhibit the interaction between HLA-E and NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) (e.g., NKG2A or a complex comprising NKG2A expressed on immune cells), thereby preventing the inhibition or activation of immune cell-mediated antitumor responses in compositions and methods. Additionally, the NKG2A binders described herein (such as NKG2A-binding antibodies (e.g., monospecific or multispecific antibodies, including bispecific antibodies)) can be used to kill and / or remove tumor cells. The NKG2A binders described herein (such as NKG2A binding antibodies (e.g., monospecific or multispecific antibodies, including bispecific antibodies)) can be used in compositions and methods for treating diseases or conditions such as cancer.

[0027] It should be understood that the chapter or section headings used in this document are for organizational purposes only and should not be construed as limiting and / or separating the topics described.

[0028] 5.1. Definition The techniques and procedures described or referenced herein include those that are well understood and / or commonly used by those skilled in the art using conventional methods, such as those widely used in: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition 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, Volumes 1 and 2 (Kontermann and Dübel, eds., 2nd edition 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following terminology will be used, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any description of the terms set forth herein and any reference incorporated herein by reference, the description of the terms set forth herein shall prevail.

[0029] Unless otherwise indicated, the term "NKG2A" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any native NKG2A derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), canines, and rodents (e.g., mice and rats). NKG2A is also referred to as, for example, NK cell receptor A, NKG2A-activated NK receptor, NKG2-A / B-activated NK receptor, cytotoxic lectin-like receptor C1 (CD159a), CD159 antigen-like family member A, or NKG2-A / NKG2-B type II integrated membrane protein. NKG2A belongs to the lectin family and forms a heterodimer with another NK cell-expressed C-type lectin, CD94 (or KLRD1). The NKG2A / CD94 complex binds to HLA-E (a non-classical MHC I molecule) in humans and transduces inhibitory signals, thereby inhibiting NK and CD8. +T cell activity. NKG2A is a protein encoded by the NKG2A gene (or KLRC1). The term NKG2A covers “full-length” NKG2A, as well as any form of NKG2A or any fragment thereof produced during cellular processing. In some embodiments, exemplary amino acid sequences of full-length NKG2A are provided below (see, for example, 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 containing the NKG2A extracellular domain. The term NKG2A also covers 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 provided 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 provided below: PSTLTQKHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKEKRTWAESLLACTLKNSSLLSIDNEEEMKFLTAISPSTWTGVFRDSSQHPWVTINGLTFKHEIKDSDNAEHNCAMLHARGLKSDRCGSSKIYHCKHKL (SEQID NO:77).

[0030] In some embodiments, as used herein, the term NKG2A refers to the NKG2A epitope. Alternatively, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domains of NKG2A and CD94. In other embodiments, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domains of NKG2A and CD94, and not just an epitope on CD94 itself. Alternatively, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domains of NKG2A and CD94, and not just an epitope on NKG2A itself. In some implementations, as used herein, the term NKG2A refers to an epitope of a complex containing NKG2A and CD94 or a complex containing the extracellular domains of NKG2A and CD94, rather than an epitope only on NKG2A itself or only on CD94 itself.

[0031] In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope. Alternatively, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of NKG2A, which is stabilized by complexation with CD94. In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of a complex comprising NKG2A and CD94 and only on NKG2A itself. Alternatively, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of the extracellular domain of NKG2A, which is stabilized by complexation with the extracellular domain of CD94. In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of a complex comprising the extracellular domain of NKG2A and CD94 and only on the extracellular domain of NKG2A itself.

[0032] Unless otherwise indicated, the term "NKG2C" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any naturally occurring NKG2C from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), canines, and rodents (e.g., mice and rats). NKG2C is also referred to as, for example, KLRC2, CD159c, NKG2-C, NKG2C, and cytotoxic lectin-like receptor C2. NKG2C is a protein encoded by the NKG2C gene (or KLRC2). The term NKG2C encompasses “full-length” NKG2C, as well as any form of NKG2C or any fragment thereof produced through cellular processing. In some embodiments, NKG2C includes a signaling sequence. In some embodiments, NKG2C does not include a signaling sequence. In some embodiments, the term NKG2C refers to a fragment of full-length NKG2C containing the NKG2C extracellular domain. The term NKG2C also encompasses naturally occurring variants of NKG2C, such as SNP variants, splice variants, and allelic variants. The NKG2C gene is described in various databases with the following ID numbers: HGNC 6375; NCBI Entrez Gene3822; Ensembl ENSG00000205809; OMIM® 602891; and UniProtKB / Swiss-Prot P26717. Exemplary extracellular domains of human NKG2C are shown in the Examples section below (see SEQ ID NO:75).

[0033] In some embodiments, as used herein, the term NKG2C refers to an NKG2C epitope. Alternatively, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domains of NKG2C and CD94. In other embodiments, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domains of NKG2C and CD94, and not just an epitope on CD94 itself. Alternatively, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domains of NKG2C and CD94, and not just an epitope on NKG2C itself. In some implementations, as used herein, the term NKG2C refers to an epitope of a complex containing NKG2C and CD94 or a complex containing the extracellular domains of NKG2C and CD94, rather than an epitope only on NKG2C itself or only on CD94 itself.

[0034] Unless otherwise indicated, the term "HLA-E" refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any natural HLA-E or its heterologous homolog derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), canines, and rodents (e.g., mice and rats). HLA-E is also referred to as, for example, major histocompatibility complex E; class I HLA histocompatibility antigen α chain E; class I MHC antigen E; HLA-6.2; class Ib MHC antigen; HLA-E; or QA1. HLA-E is a protein derived from HLA-E in the human body. HLA-E HLA-E is a gene-encoded protein. It belongs to class I HLA heavy chain paralogs and is approximately 45 kDa and anchored in the membrane. The term HLA-E encompasses “full-length” HLA-E, as well as any form of HLA-E or any fragment thereof produced during cellular processing. In some embodiments, HLA-E contains a signaling sequence. In some embodiments, HLA-E does not include a signaling sequence. In some embodiments, the term HLA-E refers to a fragment of full-length HLA-E containing the HLA-E extracellular domain. The term HLA-E also encompasses naturally occurring variants of HLA-E, such as SNP variants, splice variants, and allelic variants. The HLA-E gene is described in various databases with the following ID numbers: HGNC 4962; NCBI Entrez Gene 3133; Ensembl ENSG00000204592; OMIM®143010; and UniProtKB / Swiss-Prot P13747.

[0035] Unless otherwise indicated, the term "CD94" refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any natural CD94 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), canines, and rodents (e.g., mice and rats). CD94 is also referred to as, for example, cytotoxic lectin-like receptor D1; cytotoxic 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- and non-self-discrimination. It forms a complex with NKG2A or NKG2C on cytotoxic and regulatory lymphocyte subsets, recognizing the non-classical Ib major histocompatibility (MHC) molecule HLA-E, which is loaded with a self-peptide containing a signal sequence derived from classical Ia MHC and non-classical Ib MHC molecules. CD94-NKG2A acts as an immunosuppressive receptor and is a key inhibitory receptor on natural killer (NK) cells that regulate their activation and effector functions. CD94-NKG2C acts as an immune-activating receptor and recognizes HLA-E on a subset of cytotoxic lymphocytes, the HLA-E being loaded with a peptide derived from a signal sequence from a non-classical class Ib MHCHLA-G molecule. The term CD94 covers “full-length” CD94, as well as any form of CD94 or any fragment thereof produced during cellular processing. In some embodiments, CD94 contains a signal sequence. In some embodiments, CD94 does not contain a signal sequence. In some embodiments, the term CD94 refers to a fragment of full-length CD94 containing the CD94 extracellular domain. The term CD94 also covers naturally occurring variants of CD94, such as SNP variants, splice variants, and allelic variants. The CD94 gene is described in various databases with the following ID numbers: HGNC 6378; 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).

[0036] As used herein, the term "binding agent" or its grammatical equivalent refers to a molecule (e.g., an antibody) having one or more antigen-binding sites that bind to an antigen. In some embodiments, the NKG2A binding agents described herein are antibodies (including antibody fragments, such as antigen-binding fragments or epitope-binding fragments) or other peptide-based molecules, as well as conjugates (e.g., antibody-drug conjugates) of antibodies, antibody fragments, or peptide-based molecules that bind to NKG2A (such as human NKG2A).

[0037] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein and in the broadest sense, and specifically cover, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple epitopes or single epitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy chains 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 a light chain, such as antibodies derived from camel species (e.g., rheas); single-domain antibodies derived from conventional four-chain antibodies; engineered antibodies; and single-domain backbones other than those derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, rheas, goats, rabbits, and cattle. VHH can also be derived from other species besides camelids that produce naturally occurring heavy chain antibodies lacking the light chain. Antibodies also include antibody fragments (and / or polypeptides containing antibody fragments) that retain the NKG2A binding signature. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual variable domain antibodies, single variable domain antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, bifunctional antibodies, di-diabody antibodies, disulfide-linked Fv (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments composed of non-covalently coupled heavy and light chain variable regions). Generally, the variable (V) region domain can be any suitable arrangement of immunoglobulin heavy chain (VH) and / or light chain (VL) variable domains. For example, antibodies also include tetrameric antibodies containing two heavy chain and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region domain can be a dimer and contain VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind NKG2A. If necessary, VH and VL can be covalently coupled 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 containing one or more complementarity-determining regions (CDRs) of the antibody. CDRs (also called “minimum recognition units” or “hypervariates”) can be obtained by constructing polynucleotides encoding one or more CDRs of interest.Such polynucleotides are prepared, for example, by using 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”, Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies”, Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated into, for example, single-domain antibodies, maximal antibodies, micro antibodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, variable domains (v-NARs) of neoantigen receptors, and double-single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies containing VH and / or VL also contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include epitope-binding fragments of any of the above. The antibodies described herein may belong to any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0038] When used with reference to binders (e.g., antibodies) as described herein, the term "monospecific" means a binder having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0039] When used as a reference binder (e.g., an antibody), the term "multispecific" means that the binder can specifically bind to at least two different epitopes, such as two binding sites formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or by a pair of VHH domains that bind to different antigens or different epitopes on the same antigen. Such bispecific binders (e.g., antibodies) may have a 1+1 form (containing one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binder forms (e.g., antibodies) may be 2+1 or 1+2 forms (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 forms (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binder (e.g., an antibody) contains two antigen binding sites, each can bind to a different epitope. Such bispecific binders (e.g., antibodies) can bind to two different epitopes on the same antigen (e.g., an epitope on NKG2A).

[0040] In the case of two or more nucleic acids or peptides, the term "identical" or "percentage of identity" refers to two or more sequences or subsequences being identical or having a specified percentage of identical nucleotide or amino acid residues when compared and aligned to maximum correspondence (with gaps introduced where necessary), regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are well known in the art for obtaining amino acid or nucleotide sequence alignments. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or peptides being substantially identical means having 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%, of nucleotide or amino acid residue identity when compared and aligned to maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. In some embodiments, identity exists in regions of length of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween in the amino acid sequence. In some embodiments, identity exists in regions longer than 60-80 residues (e.g., at least about 80-100 residues), and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (e.g., the coding region of a target protein or antibody). In some embodiments, identity exists in regions of length of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween in the nucleotide sequence. In some embodiments, identity exists in regions longer than 60-80 bases (e.g., at least about 80-1000 bases or more), and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (e.g., the nucleotide sequence encoding the protein of interest).

[0041] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a side chain having similar chemical characteristics. Families of amino acid residues with similar side chains are generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar 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, replacing tyrosine with phenylalanine is a conservative substitution. Generally, conservative substitutions in the sequences of peptides, soluble proteins, and / or antibodies of this disclosure do not eliminate the binding of the peptide, soluble protein, or antibody containing the amino acid sequence to the target binding site. Methods for identifying non-eliminating conserved substitutions of amino acids are well known in the art.

[0042] The term "peptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may include (e.g., interspersed with) non-amino acids. The term also covers amino acid polymers that have been naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linking or conjugating (directly or indirectly) to a portion of a labeling component or drug (e.g., a toxin). The definition also includes, for example, one or more analogues containing amino acids (including, for example, non-natural amino acids) and other modified peptides known in the art. It should be understood that because the peptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the peptide may exist in single-chain or single-chain dimer form.

[0043] As used herein, an "antigen" is a portion or molecule containing an epitope that a binding agent (e.g., an antibody) can bind to. Therefore, an antigen can be bound by an antibody. In some embodiments, the antigen bound by the binding agent (e.g., an antibody) described herein is NKG2A (e.g., human NKG2A) or a fragment thereof, including fragments containing one or more domains of NKG2A.

[0044] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen that an antibody can bind to. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. For example, in the case of a peptide antigen, an epitope can be an adjacent amino acid of the peptide ("linear" epitope), or an epitope can comprise amino acids from two or more non-adjacent regions of the peptide ("conformational," "non-linear," or "discontinuous" epitope), such as human NKG2A. Those skilled in the art will understand that, in general, a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a set of amino acids, regardless of whether said amino acids fold into a native three-dimensional protein structure. In other embodiments, the antibody requires the amino acid residues constituting the epitope to exhibit a specific conformation (e.g., bending, twisting, turning, or folding) in order to recognize and bind to the epitope.

[0045] When two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space, the antibody binds to a reference antibody at an “epitope” or “substantially identical epitope” or “identical epitope.” The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competition assay, which can be configured in many different ways, such as using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and radioactive, fluorescent, or enzyme labeling is used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody.

[0046] Epitope clustering is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope clustering includes methods and systems that use a combination of competitive assays and computational methods to distinguish the epitope recognition characteristics of different antibodies, so as to cluster antibodies based on their epitope recognition characteristics and identify antibodies with different binding specificities.

[0047] As used herein, the terms “specific binding,” “specific recognition,” “immunospecific binding,” “selective binding,” “immunospecific recognition,” and “immunospecific” are similar terms in the context of antibodies and refer to molecules that bind to antigens (e.g., epitopes), as such binding is understood by those skilled in the art. In some embodiments, “specific binding” means, for example, that the interaction of a peptide or molecule with an epitope, protein, or target molecule is more frequent, faster, longer-lasting, more affinity, or a combination thereof than with alternative substances (including related and unrelated proteins). For example, molecules that specifically bind to antigens may generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIACORE™, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), OctetQK384 systems (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, the antibody or antigen-binding domain binds to or specifically binds to the antigen when it binds with a higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction will be at least twice the background signal or noise and may exceed 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed., 1989). In some embodiments, the antibody or antigen-binding domain binds to less than about 10% of the antibody or antigen-binding domain binds to its specific target antigen, for example, as determined by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, the Ka of a molecule specifically bound to an antigen is at least 2 log, 2.5 log, 3 log, 4 log, or higher than the Ka of the molecule bound to another antigen. In some embodiments, the molecule specifically bound to an antigen does not cross-react with other proteins. In another specific embodiment, the molecule specifically bound to the antigen does not cross-react with other non-NKG2A proteins. In some embodiments, "specific binding" means, for example, a peptide or molecule at a K+ level of about 0.1 mM or less, but more typically less than about 1 µM. D Binding to a protein or target. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration 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. DTarget binding. Due to sequence identity between homologous proteins in different species, specific binding may include a peptide or molecule recognizing proteins or targets in more than one species. Similarly, due to homology in certain regions of the peptide sequences of different proteins, specific binding may include a peptide or molecule recognizing more than one protein or target. It should be understood that in some embodiments, a peptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, such as binding to a single target. Thus, in some embodiments, a peptide or molecule may specifically bind to more than one target. In some embodiments, multiple targets may be bound by the same antigen-binding site on the peptide or molecule. For example, an antibody may, in some cases, contain two identical antigen-binding sites, each of which specifically binds to the same epitopes on two or more proteins. In some alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities. Generally, but not necessarily, the term “binding” means “specific binding.”

[0048] "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 binder, such as an antibody) and its bound complex (e.g., an antigen, such as NKG2A). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its bound complex Y can generally be determined by the dissociation constant (K). D ( ) indicates. Affinity can be measured by commonly used methods known in the art, including those described herein. Low-affinity antibodies typically bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind to antigens more quickly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. In one embodiment, “K D "or "K D The "value" can be measured using biolayer interferometry (BLI) using, for example, the Octet QK384 system (ForteBio, Menlo Park, CA). Alternatively, K... DIt can also be measured in, for example, a radiolabeled antigen-binding assay (RIA) using the Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881), or using surface plasmon resonance (SPR) assays via BIACORE™, using, for example, BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ). The "on-rate of association / association rate" or "k" is used. 缔合 "and dissociation rate (off-rate / rateof dissociation / dissociation rate)" or "k 解离 "The same SPR or BLI technology described above can also be used, for example, the Octet QK384 system (ForteBio, Menlo Park, CA) or BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ) to determine the result."

[0049] When used in the context of NKG2A binders (e.g., antibodies), the term "competition" or any grammatical variation thereof means that binders compete for the same epitope or binding site on the target. This includes competition between such binders, as determined by assays in which the binder under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or a reference antigen-binding protein, such as a reference antibody) to a common antigen (e.g., NKG2A). Many types of competitive binding assays can be used to determine whether a test binder competes with a reference molecule for binding to NKG2A (e.g., human NKG2A). Examples of assays that may be used include solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see, for example, Stahli et al., (1983), Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., (1986), J. Immunol. 137:3614-3619 or Cheung et al., (1990) Virology 176:546-552); solid-phase direct labeling assay; solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see, for example, Morel et 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 using a purified antigen (e.g., NKG2A, such as human NKG2A) bound to a solid surface or cell carrying 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 labeling bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, an excess of the test antigen-binding protein is present. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to a neighboring epitope, said neighboring epitope being close enough to the reference bound epitope that the antibody exhibits steric hindrance (e.g., similar epitopes or overlapping epitopes). Typically, when an overabundance of a competing antibody is present, it will inhibit the specific binding of the reference antibody to the common antigen by at least 20%, such as at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.In some cases, the binding was inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0050] As used herein, the terms "constant region" or "constant domain" are well-known antibody terms in the art and refer to antibody portions, such as the carboxyl-terminal portions of the light and / or heavy chains, that do not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The term also includes portions of immunoglobulin molecules that have a generally more conserved amino acid sequence relative to immunoglobulin variable domains.

[0051] Antibody "effector functions" refer to those biological activities attributable to the Fc region of the antibody (e.g., the native Fc region or the Fc region with amino acid sequence variations), and vary from antibody isotype to isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0052] In this document, the term "Fc region" is used to define the C-terminal region of the 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 the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to its C-terminus. The C-terminal lysine residue (residue 447, according to the EU numbering system) of the Fc region may be removed, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are provided below (CH2 domain = bold text; CH3 domain = underlined text): CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:83).

[0053] The “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (such as antibody-dependent phagocytosis, i.e., ADCP); downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require combining the Fc region with a binding region or binding domain (e.g., antibody variable region or domain) and can be assessed using the various assays disclosed.

[0054] The “native sequence Fc region” comprises an amino acid sequence that is identical to the naturally occurring Fc region and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, including other sequences such as variable region sequences) or combined with them). Native sequence human Fc regions include the native sequence human IgG1 Fc region (non-A and A allotypes); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0055] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to 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 has at least one amino acid substitution in the native Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, compared to the native Fc region or the Fc region of the parent polypeptide. The variant Fc region described herein may have at least about 80% homology with the native Fc region and / or the Fc region of the parent polypeptide, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may lose effector function (e.g., silence Fc). An exemplary variant Fc region (“silent Fc”) sequence is provided below (CH2 domain = bold text, where amino acid changes are underlined; CH3 domain = underlined text): CPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL K APIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:84).

[0056] As used herein, when referring to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50 to 70 kDa, wherein the amino-terminal portion comprises a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion comprises one or more constant regions. Based on the amino acid sequence of the constant domains, "heavy chain" can refer to any different type, such as α (alpha), δ (delta), ε (epsilon), γ (gamma), and µ (mu), which respectively generate the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0057] As used herein, when referring to antibody use, the term "light chain" may refer to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion comprises a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, two different types exist, such as κ (kappa) or λ (lambda). The amino acid sequences of light chains are well known in the art.

[0058] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody containing amino acid residues that interact with an antigen and confer specificity and affinity to the antigen (e.g., a CDR). As used herein, “antigen-binding fragment” includes “antibody fragment,” which contains portions of an antibody that include one or more CDRs, such as an antigen-binding region or variable region of the antibody.

[0059] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-individual genotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0060] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, including molecules containing one or more antigen-binding sites that bind to the NKG2A antigen.

[0061] Antibodies can be 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 their classes (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclasses.

[0062] In some embodiments, the antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In other embodiments, the H chains have identical amino acid sequences and the L chains have identical amino acid sequences. In other embodiments, the H chains have different amino acid sequences. Or, additionally, the L chains have different amino acid sequences. For example, the antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to an NKG2A antigen and the second H / L chain pair binds to another NKG2A antigen or a non-NKG2A antigen. In some embodiments, the antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In other embodiments, the VHHs have identical amino acid sequences. In other embodiments, the VHHs have different amino acid sequences. For example, the antibody comprises a first VHH and a second VHH, wherein the first VHH binds to an NKG2A antigen and the second VHH binds to another NKG2A antigen or a non-NKG2A antigen. In some embodiments, the H and / or L chains comprise constant regions, such as human constant regions. In some embodiments, the L-chain constant region of such antibodies is a κ or λ light chain constant region, such as the human κ or λ light chain constant region. In some embodiments, the H-chain constant region of such antibodies comprises a γ heavy chain constant region, such as the human γ heavy chain constant region. In some embodiments, such antibodies comprise an IgG constant region, such as the human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0063] Antibodies or fragments thereof may preferentially bind to NKG2A (or complexes containing NKG2A and CD94 or their respective extracellular domains), such as human NKG2A, meaning that antibodies or fragments thereof bind to NKG2A with a higher affinity than they bind to control proteins (e.g., unrelated control proteins, such as chicken egg white lysozyme, or NKG2C), and / or bind to human NKG2A with a higher affinity than they bind to unrelated control proteins. For example, antibodies or fragments thereof may specifically recognize and bind to NKG2A or portions thereof. “Specific binding” means that the affinity of the antibody or fragment thereof for NKG2A is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than that for unrelated control proteins (e.g., chicken egg white lysozyme). In some embodiments, the antibody or a fragment thereof may bind substantially only to NKG2A (e.g., enabling the differentiation of NKG2A from other known peptides, for example, due to measurable differences in binding affinity). In some embodiments, the NKG2A binder (e.g., antibody) may react with NKG2A sequences other than the human NKG2A sequence (e.g., cynomolgus monkey NKG2A sequences, such as A42 as described herein). In other embodiments, the NKG2A binder (e.g., antibody) does not react with non-human (e.g., cynomolgus monkey) NKG2A sequences (such as A2, A3, and A11 as provided herein).

[0064] The term "variable region" or "variable domain" refers to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain. It is approximately 120 to 130 amino acids long in the heavy chain and approximately 100 to 110 amino acids long in the light chain, and serves to bind and define the specific antigen for each particular antibody. The variable region of the heavy chain may be called "VH". The variable region of the light chain may be called "VL". The term "variable" refers to the fact that certain segments of the variable region in an antibody vary considerably in sequence. The V region mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. In fact, the V region consists of a less variable (e.g., relatively invariant) segment called the frame region (FR), which has approximately 15-30 amino acids, separated by a shorter, more variable (e.g., highly variable) region called the "hypervariant region" or alternatively the "complementarity-determining region (CDR)". The variable regions of both the heavy and light chains each contain four frames (FR1, FR2, FR3, and FR4), which are largely β-sheet configured and connected by three hypervariable regions, forming loops that connect β-sheet structures and, in some cases, form part of a β-sheet structure. The hypervariable regions in each chain are held together tightly by the frames and, together with hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as enabling antibodies to participate in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequence variations of the variable regions are significant between different antibodies. Sequence variability is concentrated in the CDR, while the less variable portions of the variable regions are called frame regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction between the antibody and its antigen. In a particular implementation, the variable region is the human variable region.

[0065] When used in this paper, the terms “hypervariant region,” “HVR,” “HV,” “complementarity-determining region,” or “CDR” refer to regions in the antibody variable region that are highly variable in sequence and / or form structurally defined loops. Generally, an antibody contains six hypervariant regions: three located in the VH region (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three located in the VL region (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Various hypervariant region descriptions are used and covered in this paper. Kabat CDR is based on sequence variability and is the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia actually refers to the location of a structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places 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; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent the balance between the Kabat CDR and the Chothia structural loop and are used using Oxford Molecular's AbM antibody modeling software (see, for example, Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). “Contact” hypervariable regions are based on analysis of the available complex crystal structures. Residues from each of these hypervariable regions or CDRs are labeled below.

[0066] A universal numbering system has been developed and is widely used: ImMunoGeneTics (IMGT) ®Information system (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are referred to based on both their amino acid sequence and their position within the light or heavy chain. Since the “position” of CDRs within the structure of immunoglobulin variable domains is conserved across species and exists in structures called loops, CDRs and framework residues are easily identified by using a numbering system that compares variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins from one species into receptor frameworks that are typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol Another numbering system (AHon) has been developed. Correspondences between numbering systems (including, for example, the Kabat numbering and the IMGT unique numbering system) are well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.) and are also described below. Various systems known in the art or described herein represent different ways of depicting CDRs, and are generally considered equivalent when used to define the same antibody. The exemplary system shown herein combines Kabat and Chothia. Residues from each of these hypervariable regions or CDRs are illustrated in the table below.

[0067] Exemplary CDRs based on various numbering systems

[0068] The high-variability region may include the following “extended high-variability regions”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 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 “high-variability region,” “HVR,” “HV,” “complementary determinant region,” and “CDR” are used interchangeably.

[0069] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. Cells that produce the binding molecules of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells to which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left-handed end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-handed direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of a newly generated RNA transcript is referred to as the transcription direction; a sequence region on a DNA strand having the same sequence as the RNA transcript and being 5' relative to the 5' end of the RNA transcript is referred to as the “upstream sequence”; a sequence region on a DNA strand having the same sequence as the RNA transcript and being 3' relative to the 3' end of the RNA transcript is referred to as the “downstream sequence”.

[0070] The term "vector" refers to a substance used to deliver or include a nucleic acid sequence (including, for example, to introduce a nucleic acid sequence into a host cell). Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selected sequences or markers operable to stably integrate into the chromosome of a host cell. Additionally, a vector may include one or more optional marker genes and appropriate expression control sequences. Optional marker genes may be included, for example, to provide resistance to antibiotics or toxins, to supplement auxotrophic deficiencies, or to supply essential nutrients not present in the culture medium. Expression control sequences may include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, etc., well known in the art. When two or more nucleic acid molecules (e.g., antibody heavy and light chains or antibodies VH and VL) are co-expressed, both nucleic acid molecules may be inserted into, for example, a single expression vector or independent expression vectors. In the case of single-vector expression, the encoding nucleic acid is operably ligated to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm the introduction of the nucleic acid molecule into the host cell. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA; or immunoblotting for gene product expression; or other suitable analytical methods for testing the expression of an introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules are expressed in amounts sufficient to produce the desired product (e.g., the NKG2A binder as described herein), and will further understand that methods well known in the art can be used to optimize expression levels to achieve adequate expression.

[0071] As used herein, the term “pharmaceutical acceptable” means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in animals, and more particularly in humans.

[0072] "Excipient" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, humectants, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freunds' adjuvant (complete or incomplete)), or medium. In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) peptides; 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 carbohydrates, including glucose, mannose, or dextrin; 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 edition, 1990). In one implementation, each component is "pharmaceutically acceptable" in the sense that it is compatible with other components of the pharmaceutical preparation and suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.See, for example, Lippincott Williams and Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to cells or mammals exposed to it at the doses and concentrations used. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffer solution. In some embodiments, the excipient is a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid excipients, especially for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. The composition may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions (including formulations) may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Compositions (including pharmaceutical compounds) may contain a preventive or therapeutically effective amount, for example, an isolated or purified form of an NKG2A binder (e.g., an antibody), and an appropriate amount of excipients to provide a form suitable for administration to a subject (e.g., a patient). The formulation should be suitable for the administration regimen.

[0073] An "effective amount" is generally an amount sufficient to achieve the following effects: reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent or delay the occurrence of symptoms and / or underlying causes, and / or improve or repair damage caused by or related to a disease, condition, or disorder. In some implementations, the effective amount is a therapeutic effective amount or a preventive effective amount.

[0074] As used herein, the term "therapeuticly effective amount" refers to an amount of an agent (e.g., an antibody or any other agent described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, condition, or disorder and / or associated symptoms. A therapeutically effective amount of an agent (including therapeutic agents) may be an amount necessary to: (i) reduce, delay, or improve the progression or course of a given disease, condition, or disorder; (ii) reduce, delay, or improve the recurrence, development, or onset of a given disease, condition, or disorder; and / or (iii) improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than the administration of an agent described herein). The "therapeuticly effective amount" of the substances / molecules / agents disclosed herein (e.g., NKG2A antibodies) may vary based on factors such as an individual's disease state, age, sex, and weight, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effect of the substance / molecule / agent exceeds any toxic or harmful effect. In some implementations, the term "therapeutic effective amount" refers to the amount of agent that effectively "treats" a disease, symptom, or ailment of a subject or mammal.

[0075] The term “treatment” or any grammatical variation thereof means the reduction and / or improvement of the severity and / or duration of a given disease, condition or disorder, and / or associated symptoms, such as (i) reducing, delaying or improving the progression or course of a given disease, condition or disorder; (ii) reducing, delaying or improving the recurrence, development or onset of a given disease, condition or disorder; and / or (iii) improving or enhancing the preventive or therapeutic effect of another therapy (e.g., a therapy other than the administration of the agents described herein).

[0076] "Prophylactic effective amount" is the amount of a pharmaceutical composition that, when administered to a subject, will have the following expected preventive effects: for example, preventing or delaying the onset (or recurrence) of a condition, disease, or disorder, or reducing the likelihood of the onset (or recurrence) of a condition, disease, or disorder or related symptoms.

[0077] A complete therapeutic or preventative effect may not occur with a single dose and may only occur after a series of doses. Therefore, an effective dose for treatment or prevention can be administered in one or more doses.

[0078] The terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

[0079] As used herein, comparative terms (such as reduction, decrease, increase, or any grammatical variation thereof) may refer to certain changes relative to a reference. In some embodiments, such changes may refer to being about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 20, or about 30, or about 40, or about 100 times or more than the reference. In some implementations, such variations may refer to approximately 1%, or approximately 2%, or approximately 3%, or approximately 4%, or approximately 5%, or approximately 6%, or approximately 7%, or approximately 8%, or approximately 9%, or approximately 10%, or approximately 20%, or approximately 30%, or approximately 40%, or approximately 50%, or approximately 60%, or approximately 70%, or approximately 80%, or approximately 90%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

[0080] Unless the context clearly specifies otherwise, the singular forms “a / an” and “the” as used in this disclosure and claims include the plural forms.

[0081] In some implementations, the terms "first," "second," "third," "fourth," and similar terms in component names are used to distinguish and identify more than one component that shares a certain identity in its name. For example, "primary antibody" and "secondary antibody" are used to distinguish two antibodies.

[0082] It should be understood that wherever the term "comprising" is used to describe an embodiment herein, other similar embodiments described using the terms "consisting of" and / or "substantially consisting of" are also provided. It should also be understood that wherever the phrase "substantially consisting of" is used to describe an embodiment herein, other similar embodiments described using the term "consisting of" are also provided.

[0083] The term "between" as used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0084] As used in phrases such as “A and / or B”, the term “and / or” is intended herein to include both A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B and / or C”, the term “and / or” is intended to cover 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).

[0085] The terms “optional” or “optionally” mean that the situation described below may or may not occur, and therefore this specification includes both the scenario in which the scenario occurs and the scenario in which the scenario does not occur.

[0086] 5.2. NKG2A binder In some embodiments, this disclosure provides NKG2A binders that can be used herein as agents and / or therapeutic agents for enhancing immune responses. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to NKG2A (or complexes comprising NKG2A and CD94 or their respective extracellular domains). Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and conjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0087] In some embodiments, this document describes an NKG2A binder (e.g., an antibody) that binds to NKG2A, including NKG2A polypeptides, NKG2A polypeptide fragments, NKG2A peptides, or NKG2A epitopes. In some embodiments, the NKG2A binder is a human or humanized antibody (e.g., containing a human constant region) that binds to NKG2A, including NKG2A polypeptides, NKG2A polypeptide fragments, NKG2A peptides, or NKG2A epitopes. In some embodiments, the NKG2A binder (e.g., an antibody, such as a human NKG2A binder) may 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 binder (e.g., an antibody) binds to NKG2A extracellular epitopes exposed on cells such as immune cells. In some embodiments, this document describes an NKG2A binder (e.g., an antibody) that binds to NKG2A (such as human NKG2A or a portion thereof). In some embodiments, NKG2A is human NKG2A. In some embodiments, the NKG2A binder is a human NKG2A binder (e.g., an antibody that binds to human NKG2A). In some embodiments, the NKG2A binder (e.g., an antibody) binds to both human and cynomolgus monkey NKG2A. In other embodiments, the NKG2A binder (e.g., an antibody) binds to human NKG2A but not cynomolgus monkey NKG2A. In some embodiments, an NKG2A binder (e.g., an antibody) is described herein that binds to a complex comprising NKG2A and CD94 or a complex comprising the extracellular domains of NKG2A and CD94.

[0088] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein are available in quantities of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) D This binds to NKG2A (e.g., human NKG2A). Various methods for measuring binding affinity are known in the art, any of which can be used to achieve the purposes disclosed herein, including RIA via, for example, using the Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); determination via biolayer interferometry (BLI) or surface plasmon resonance (SPR); and determination via OCTET. ®Using, for example, OCTET ® Red96 system; or via BIACORE ® Using, for example, BIACORE ® TM-2000 or BIACORE ® TM-3000. The "on-rate of association rate" or "k-association" can also be measured using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, employing, for example, OCTET. ® Red96, BIACORE ® TM-2000, BIACORE ® TM-3000 system, BIACORE ® TM-8K or BIACORE ® The TM-8K+ system is used to determine this.

[0089] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein do not bind to NKG2C (or complexes comprising NKG2C and CD94 or their respective extracellular domains) (e.g., human NKG2C and / or cynomolgus monkey NKG2C). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein do not bind to human NKG2C. In some embodiments, the NKG2A binders (e.g., antibodies) provided herein do not bind to human NKG2C or cynomolgus monkey NKG2C. In other embodiments, the NKG2A binders (e.g., antibodies) provided herein bind to NKG2A (e.g., human NKG2A) (or complexes comprising NKG2A and CD94 or their respective extracellular domains) with a higher affinity than to NKG2C (e.g., human NKG2C) (or complexes comprising NKG2A and CD94 or their respective extracellular domains). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein exhibit a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least twice as strong as the binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein exhibit a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least five times as strong as the binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein exhibit a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 10 times stronger than that for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein exhibit a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 100 times stronger than that for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).In some embodiments, the NKG2A binders (e.g., antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 1000 times stronger than that for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).

[0090] In some embodiments, the NKG2A binders (e.g., antibodies) described herein comprise the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of any of the antibodies described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 depicted in Tables 1-4. Therefore, in some embodiments, the NKG2A binders (e.g., antibodies) described herein comprise any, two and / or all of the following three heavy chain CDRs, and / or any, two and / or all of the following three light chain CDRs: (a) an antibody designated as A2; (b) an antibody designated as A3; (c) an antibody designated as A11; and (d) an antibody designated as A42, as shown in Tables 1-4. In some embodiments, the NKG2A binder (e.g., antibody) described herein comprises any, two, and / or all of the following three heavy chain CDRs, and / or any, two, and / or all of the following three light chain CDRs: (a) an antibody designated as A2; (b) an antibody designated as A3; (c) an antibody designated as A11; and (d) an antibody designated as A42, as shown in Tables 1-4.

[0091] In some embodiments, the NKG2A binder (e.g., an antibody) comprises a VH region and / or a VL region (see, for example, any of those in Tables 1-4) of any of the binders described herein, wherein the VH region comprises VH CDR1, VH CDR2, and / or VHCDR3, and the VL region comprises VL CDR1, VL CDR2, and / or VL CDR3. Therefore, in some embodiments, the NKG2A binder (e.g., an antibody) described herein comprises any, two, and / or all of the three heavy chain CDRs from Table 1, and / or any, two, and / or all of the three light chain CDRs. In some embodiments, the NKG2A binder (e.g., an antibody) described herein comprises any, two, and / or all of the three heavy chain CDRs from Table 2, and / or any, two, and / or all of the three light chain CDRs. In some embodiments, the NKG2A binder (e.g., antibody) described herein comprises any, two, and / or all of the three heavy chain CDRs from Table 3, and / or any, two, and / or all of the three light chain CDRs. In some embodiments, the NKG2A binder (e.g., antibody) described herein comprises any, two, and / or all of the three heavy chain CDRs from Table 4, and / or one, two, and / or all of the three light chain CDRs.

[0092] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise: (i) VH CDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:25, SEQ ID NO:45, or SEQ ID NO:64, and / or (ii) VL CDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:26, SEQ ID NO:46, SEQ ID NO:65, or SEQ ID NO:73.

[0093] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:25, and / or VL CDR1, VL CDR2, and / or VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:25, and VL CDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:26. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDR is based on a Chothia number. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above (e.g., a combination of Kabat and Chothia). Various exemplary CDR numbering systems are described and illustrated in Section 5.1 of the previous article.

[0094] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13 and 18; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 8, 14, 19 and 24; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 9, 15 and 20; and / or (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 and 21; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11 and 22; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 17 and 23.

[0095] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:1, a VH CDR2 containing the amino acid sequence of SEQ ID NO:2, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:4, a VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and a VLCDR3 containing the amino acid sequence of SEQ ID NO:6.

[0096] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:7, a VH CDR2 containing the amino acid sequence of SEQ ID NO:8, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:9; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:10, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0097] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:12, a VH CDR2 containing the amino acid sequence of SEQ ID NO:2, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:4, a VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0098] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:13, a VH CDR2 containing the amino acid sequence of SEQ ID NO:14, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:15; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:16, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:17.

[0099] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:18, VH CDR2 containing the amino acid sequence of SEQ ID NO:19, and VH CDR3 containing the amino acid sequence of SEQ ID NO:20; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:21, VL CDR2 containing the amino acid sequence of SEQ ID NO:22, and VL CDR3 containing the amino acid sequence of SEQ ID NO:23.

[0100] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:1, a VH CDR2 containing the amino acid sequence of SEQ ID NO:24, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:4, a VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0101] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:45, and / or VL CDR1, VL CDR2, and / or VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:46. In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:45, and VL CDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:46. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDR is based on the Chothia number. In other embodiments, the CDR is based on the Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above (e.g., a combination of Kabat and Chothia).

[0102] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13 and 18; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 32, 35, 39 and 44; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 33, 36 and 40; and / or (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 34, 37 and 41; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 11 and 42; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 38 and 43.

[0103] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:1, a VH CDR2 containing the amino acid sequence of SEQ ID NO:27, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:29, a VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0104] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing a VH CDR1 containing the amino acid sequence of SEQ ID NO:7, a VH CDR2 containing the amino acid sequence of SEQ ID NO:32, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:33; and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO:34, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0105] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:12, VH CDR2 containing the amino acid sequence of SEQ ID NO:27, and VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:29, VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0106] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing a VH CDR1 containing the amino acid sequence of SEQ ID NO:13, a VH CDR2 containing the amino acid sequence of SEQ ID NO:35, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:36; and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO:37, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:38.

[0107] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:18, VH CDR2 containing the amino acid sequence of SEQ ID NO:39, and VH CDR3 containing the amino acid sequence of SEQ ID NO:40; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:41, VL CDR2 containing the amino acid sequence of SEQ ID NO:42, and VL CDR3 containing the amino acid sequence of SEQ ID NO:43.

[0108] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:1, a VH CDR2 containing the amino acid sequence of SEQ ID NO:44, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:29, a VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0109] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VL CDR1, VL CDR2, and / or VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 65. In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VLCDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 65. CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, CDRs are based on IMGT numbering. In some embodiments, CDRs are based on Kabat numbering. In some embodiments, CDRs are based on AbM numbering. In other embodiments, the CDR is based on the Chothia number. In other embodiments, the CDR is based on the Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above (e.g., a combination of Kabat and Chothia).

[0110] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 51, 54, 55, and 59; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 52, 56, 60, and 63; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 53, 57, and 61; and (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16, and 21; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, and 22; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 58, and 62.

[0111] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VH CDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0112] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:51, a VH CDR2 containing the amino acid sequence of SEQ ID NO:52, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:53; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:10, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0113] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:54, VH CDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0114] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:55, a VH CDR2 containing the amino acid sequence of SEQ ID NO:56, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:57; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:16, a VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:58.

[0115] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing a VH CDR1 containing the amino acid sequence of SEQ ID NO:59, a VH CDR2 containing the amino acid sequence of SEQ ID NO:60, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:61; and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO:21, a VL CDR2 containing the amino acid sequence of SEQ ID NO:22, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:62.

[0116] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing a VH CDR1 containing the amino acid sequence of SEQ ID NO:47, a VH CDR2 containing the amino acid sequence of SEQ ID NO:63, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing a VL CDR1 containing the amino acid sequence of SEQ ID NO:4, a VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0117] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VL CDR1, VL CDR2, and / or VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the NKG2A binders (e.g., antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VLCDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO: 73. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDR is based on the Chothia number. In other embodiments, the CDR is based on the Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above (e.g., a combination of Kabat and Chothia).

[0118] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 51, 54, 55, and 59; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 52, 56, 60, and 63; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 53, 57, and 61; and (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 68, 70, and 71; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 69, and 72; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 58, and 62.

[0119] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VH CDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0120] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:51, VH CDR2 containing the amino acid sequence of SEQ ID NO:52, and VH CDR3 containing the amino acid sequence of SEQ ID NO:53; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:68, VL CDR2 containing the amino acid sequence of SEQ ID NO:69, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0121] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:54, VH CDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0122] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:55, VH CDR2 containing the amino acid sequence of SEQ ID NO:56, and VH CDR3 containing the amino acid sequence of SEQ ID NO:57; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:70, VL CDR2 containing the amino acid sequence of SEQ ID NO:69, and VL CDR3 containing the amino acid sequence of SEQ ID NO:58.

[0123] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region having a VH CDR1 containing the amino acid sequence of SEQ ID NO:59, a VH CDR2 containing the amino acid sequence of SEQ ID NO:60, and a VH CDR3 containing the amino acid sequence of SEQ ID NO:61; and a VL region having a VL CDR1 containing the amino acid sequence of SEQ ID NO:71, a VL CDR2 containing the amino acid sequence of SEQ ID NO:72, and a VL CDR3 containing the amino acid sequence of SEQ ID NO:62.

[0124] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH region containing VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VH CDR2 containing the amino acid sequence of SEQ ID NO:63, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region containing VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0125] In some embodiments, the antibody further comprises one or more frame regions of SEQ ID NO: 25, 26, 45, 46, 64, 65 and / or 73. In some embodiments, the antibody or a fragment thereof further comprises frame 1 (FR1), frame 2 (FR2), frame 3 (FR3) and / or frame 4 (FR4) sequences as described in any of SEQ ID NO: 25, 26, 45, 46, 64, 65 and 73. In some embodiments, the antibody provided herein is a humanized antibody. The frame regions described herein are defined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT or Chothia, the frame region is the amino acid residues surrounding the CDR in a variable region that takes the form from the N-terminus to the C-terminus as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the N-terminal amino acid residue of CDR1 as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residue between CDR1 and CDR2 as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residue between CDR2 and CDR3 as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue of CDR3 as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0126] In some embodiments, the NKG2A binders described herein, including human NKG2A binders (e.g., antibodies, such as monospecific or bispecific antibodies), comprise a VH region or VH domain. Alternatively, in some embodiments, the NKG2A binders described herein, including human NKG2A binders (e.g., antibodies, such as monospecific or bispecific antibodies), comprise a VL region or VL domain. In some embodiments, the NKG2A binders described herein, including human NKG2A binders (e.g., antibodies, such as monospecific or bispecific antibodies), have a combination of: (i) a VH domain or VH region; and (ii) a VL domain or VL region.

[0127] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:25. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:25 and a VL containing the amino acid sequence of SEQ ID NO:26.

[0128] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:45. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VL containing the amino acid sequence of SEQ ID NO:46. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:45 and a VL containing the amino acid sequence of SEQ ID NO:46.

[0129] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:64. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VL containing the amino acid sequence of SEQ ID NO:65. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO:64 and a VL containing the amino acid sequence of SEQ ID NO:65.

[0130] In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO: 64. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the NKG2A binder (e.g., antibody) provided herein comprises a VH containing the amino acid sequence of SEQ ID NO: 64 and a VL containing the amino acid sequence of SEQ ID NO: 73.

[0131] In some embodiments, the NKG2A binders (e.g., antibodies or fragments thereof) provided herein comprise an amino acid sequence having a certain percentage of identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or higher) relative to any antibody or fragment provided herein, such as the CDR, VH, or VL in Tables 1-4, or any full-length antibody chain as disclosed herein. In some embodiments, the NKG2A binders (e.g., antibodies or fragments thereof) provided herein comprise the CDR of any antibody or fragment provided herein (e.g., in Tables 1-4). In other embodiments, the NKG2A binder provided herein comprises an amino acid sequence having a certain percentage of identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or higher) relative to any antibody or fragment thereof provided herein, such as VH or VL in Tables 1-4, or any full-length antibody chain as disclosed herein.

[0132] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST procedures of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., fraction = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set (e.g., a score of 50 and a word length of 3) to obtain amino acid sequences homologous to the protein molecules described herein. For comparison purposes, vacancy BLAST, as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997), can be used to obtain vacancy alignments. In some embodiments, the percentage of identity between two sequences is calculated by dividing the number of residues that vary between the two aligned sequences (excluding or including conserved amino acid substitutions or degenerate nucleotide substitutions) by the number of residues of either: (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 length of the vacancy-free portion of the alignment, (v) the length of the alignment excluding dangling portions, or (vi) the length of the alignment including dangling portions. As used herein with respect to sequence alignment, a dangling portion refers to either end or both ends of the alignment, where residues in one sequence are considered to be aligned with residues (e.g., vacancy) in the other sequence. Alternatively, PSI BLAST can be used for iterative searches to detect distal relationships between molecules (as above). When using the BLAST, vacancy BLAST, and PSI BLAST procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-restricted example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without allowing vacancy. When calculating the percentage of identity, typically only exact matches are counted.

[0133] In some embodiments, the binders (e.g., antibodies) provided herein contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the binders containing said sequence are still able to bind to NKG2A. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR and / or constant regions).

[0134] In some embodiments, the positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an NKG2A binder (e.g., an antibody), including a human NKG2A binder, may vary to one, two, three, four, five, or six amino acid positions, provided that the 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%). For example, in some implementations, the location of the CDR defining any of Tables 1, 2, 3, or 4 can be varied by shifting the N-terminal and / or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids relative to the current CDR location, as long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Alternatively, in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an NKG2A binder (e.g., an antibody), including a human NKG2A binder, may vary (e.g., shorter or longer) by one, two, three, four, five, or six amino acids, provided that the 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%). For example, in some embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more CDRs described by SEQ ID NO:1-24, 27-44, 47-63 or 66-72, provided that the 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 VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more CDRs described by SEQ ID NO:1-24, 27-44, 47-63 or 66-72, provided that the 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 termini of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids, compared to one or more CDRs described by SEQ ID NO:1-24, 27-44, 47-63 or 66-72, provided that the 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%). Alternatively, in some embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described by SEQ ID NO:1-24, 27-44, 47-63, or 66-72, provided that 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%). Any method known in the art may be used to determine whether binding to NKG2A (e.g., human NKG2A) is maintained, such as the binding assays and conditions described in the "Examples" section herein.

[0135] In other embodiments, the NKG2A binders (e.g., antibodies), including human NKG2A binders, presented herein also contain conserved sequence modifications. For peptides that serve as NKG2A binders (e.g., antibodies, such as human NKG2A binders), conserved sequence modifications include conserved amino acid substitutions, which include the substitution of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. Therefore, in some embodiments, predicted non-essential amino acid residues in NKG2A are substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions that do not eliminate antigen binding and their encoding nucleotides are well known in the art (see, for example, 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 conserved sequence modifications described herein modify the amino acid sequence of NKG2A binders (e.g., antibodies), including human NKG2A binders, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modification refers to the substitution of up to 1, 2, 3, 4, 5, or 6 amino acids of the CDR, such as those described in any of Tables 1-4. Therefore, for example, each such CDR may contain up to 5 conserved amino acid substitutions, such as up to (no more than) 4 conserved amino acid substitutions, such as up to (no more than) 3 conserved amino acid substitutions, such as up to (no more than) 2 conserved amino acid substitutions, or no more than 1 conserved amino acid substitution. In some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, contain one or more (including six) CDRs that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with CDRs of A3, A2, A42, or A11 (see, for example, Tables 1, 2, 3, or 4).

[0136] In some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, contain VH and VL, said VH and VL containing the same CDR as those of A3, A2, A42, or A11 (see, for example, Tables 1, 2, 3, or 4). In some embodiments, amino acid sequence modifications do not include any modifications within the SDR. In some embodiments, amino acid sequence modifications do not include any modifications within the CDR (such as CDR1, CDR2, CDR3, or any combination thereof). Alternatively or additionally, amino acid sequence modifications are located in the frame, constant region, and / or fragment crystallizable region (Fc).

[0137] In some embodiments, the antibodies or fragments provided herein comprise a VH domain having 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 with the amino acid sequence of SEQ ID NO:25, and / or a VL domain having 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 with the amino acid sequence of SEQ ID NO:26, and the binding of the antibody or fragment thereof 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%).

[0138] In some embodiments, the antibodies or fragments provided herein comprise a VH domain having 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 with the amino acid sequence of SEQ ID NO:45, and / or a VL domain having 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 with 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%).

[0139] In some embodiments, the antibodies or fragments provided herein comprise a VH domain having 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 with the amino acid sequence of SEQ ID NO:64, and / or a VL domain having 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 with the amino acid sequence of SEQ ID NO:65, and the binding of the antibody or fragment thereof 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%).

[0140] In some embodiments, the antibodies or fragments provided herein comprise a VH domain having 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 with the amino acid sequence of SEQ ID NO: 64, and / or a VL domain having 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 with the amino acid sequence of SEQ ID NO: 73, and the binding of the antibody or fragment thereof 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%).

[0141] In some embodiments, functional epitopes can be located, for example, by combining alanine screening or hydrogen / deuterium exchange mass spectrometry (HDX-MS), to identify amino acids in NKG2A proteins (or complexes containing NKG2A and CD94 or their respective extracellular domains) essential for interaction with NKG2A binders (such as antibodies) provided herein. In some embodiments, the conformation and crystal structure of NKG2A binders (such as antibodies) bound to NKG2A can be used to identify epitopes. In some embodiments, this disclosure provides an antibody that specifically binds to the same epitope as any NKG2A binder (such as an antibody or fragment thereof) provided herein.

[0142] For example, in some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VH CDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:25, and VLCDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VH containing the amino acid sequence of SEQ ID NO:25 and VL containing the amino acid sequence of SEQ ID NO:26.

[0143] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VHCDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:45, and VL CDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:46. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VH containing the amino acid sequence of SEQ ID NO:45 and VL containing the amino acid sequence of SEQ ID NO:46.

[0144] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VHCDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:64, and VL CDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:65. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VH containing the amino acid sequence of SEQ ID NO:64 and VL containing the amino acid sequence of SEQ ID NO:65.

[0145] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VHCDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:64, and VL CDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:73. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein binds to the same epitope as an anti-NKG2A antibody, which comprises VH containing the amino acid sequence of SEQ ID NO:64 and VL containing the amino acid sequence of SEQ ID NO:73.

[0146] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to one of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to two of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A binder (e.g., antibody) provided herein specifically binds to three of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91).In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to four of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to five of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 86), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 90), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 91). In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to all of the following NKG2A peptide fragments: NKG2A peptide fragments containing the amino acid sequence TWEESL (SEQ ID NO: 86), NKG2A peptide fragments containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 87), NKG2A peptide fragments containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 88), NKG2A peptide fragments containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 89), NKG2A peptide fragments containing the amino acid sequence LQVNR (SEQ ID NO: 90), and NKG2A peptide fragments containing the amino acid sequence AQCGSSI (SEQ ID NO: 91).

[0147] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues, said set of amino acid residues comprising 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following three 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following four 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following five 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 binder (e.g., an antibody) provided herein specifically binds to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each 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 set of amino acid residues forming the conformational epitope comprises one amino acid residue from an amino acid sequence mentioned above in this paragraph. In some embodiments, the set of amino acid residues forming the conformational epitope comprises two amino acid residues from an amino acid sequence mentioned above in this paragraph. In some embodiments, the set of amino acid residues forming the conformational epitope comprises three amino acid residues from an amino acid sequence mentioned above in this paragraph. In some embodiments, the set of amino acid residues forming the conformational epitope comprises four amino acid residues from an amino acid sequence mentioned above in this paragraph. In some embodiments, the amino acid residue set forming the conformational epitope comprises five amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the amino acid residue set forming the conformational epitope comprises more than five amino acid residues from the amino acid sequences mentioned above in this paragraph.

[0148] In some embodiments, the NKG2A binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues, said set of amino acid residues comprising at least one amino acid residue from one of the following amino acid sequences located 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following amino acid sequences located 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following three amino acid sequences located 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following four amino acid sequences located 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 binders (e.g., antibodies) provided herein specifically bind to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following five amino acid sequences located 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 binder (e.g., an antibody) provided herein specifically binds to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following amino acid sequences located 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 set of amino acid residues forming the conformational epitope comprises one amino acid residue from an amino acid sequence mentioned above in this paragraph. In some embodiments, the set of amino acid residues forming the conformational epitope comprises two amino acid residues from an amino acid sequence mentioned above in this paragraph. In some embodiments, the set of amino acid residues forming the conformational epitope comprises three amino acid residues from an amino acid sequence mentioned above in this paragraph. In some embodiments, the amino acid residue set forming the conformational epitope comprises four amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the amino acid residue set forming the conformational epitope comprises five amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the amino acid residue set forming the conformational epitope comprises more than five amino acid residues from the amino acid sequences mentioned above in this paragraph.

[0149] In some embodiments, the NKG2A binder (e.g., antibody) provided herein competitively and specifically binds to NKG2A with either the anti-NKG2A antibody or fragment thereof described herein.

[0150] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:25, and VLCDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO:25 and VL containing the amino acid sequence of SEQ ID NO:26.

[0151] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:45, and VLCDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:46. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO:45 and VL containing the amino acid sequence of SEQ ID NO:46.

[0152] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:64, and VLCDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:65. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO:64 and VL containing the amino acid sequence of SEQ ID NO:65.

[0153] In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as described by VH containing the amino acid sequence of SEQ ID NO:64, and VLCDR1, VL CDR2, and VL CDR3 as described by VL containing the amino acid sequence of SEQ ID NO:73. In some embodiments, the NKG2A binding agent (e.g., antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO:64 and VL containing the amino acid sequence of SEQ ID NO:73.

[0154] In some embodiments, the NKG2A binder binds to a first complex comprising the extracellular domains of NKG2A and CD94, but not to a second complex comprising the extracellular domains of NKG2C and CD94. In other embodiments, the NKG2A binder comprises six CDRs of an antibody designated A3. In other embodiments, the NKG2A binder comprises the six CDRs listed in a column of Table 1. In some embodiments, the NKG2A binder comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 25 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 26. In some embodiments, the NKG2A binder comprises the heavy chain variable region as illustrated in SEQ ID NO: 25 and the light chain variable region as illustrated in SEQ ID NO: 26.

[0155] In some embodiments, the NKG2A binder binds to a first complex comprising the extracellular domains of NKG2A and CD94, but not to a second complex comprising the extracellular domains of NKG2C and CD94. In other embodiments, the NKG2A binder comprises six CDRs of an antibody designated A2. In other embodiments, the NKG2A binder comprises six CDRs as listed in a column of Table 2. In some embodiments, the NKG2A binder comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 45 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 46. In some embodiments, the NKG2A binder comprises the heavy chain variable region as illustrated in SEQ ID NO: 45 and the light chain variable region as illustrated in SEQ ID NO: 46.

[0156] In some embodiments, the NKG2A binder binds to a first complex comprising the extracellular domains of NKG2A and CD94, but not to a second complex comprising the extracellular domains of NKG2C and CD94. In other embodiments, the NKG2A binder comprises six CDRs of an antibody designated A42. In other embodiments, the NKG2A binder comprises six CDRs as listed in a column of Table 3. In some embodiments, the NKG2A binder comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 64 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 65. In some embodiments, the NKG2A binder comprises the heavy chain variable region as illustrated in SEQ ID NO: 64 and the light chain variable region as illustrated in SEQ ID NO: 65.

[0157] In some embodiments, the NKG2A binder binds to a first complex comprising the extracellular domains of NKG2A and CD94, but not to a second complex comprising the extracellular domains of NKG2C and CD94. In other embodiments, the NKG2A binder comprises six CDRs of an antibody designated A11. In other embodiments, the NKG2A binder comprises six CDRs as listed in a column of Table 4. In some embodiments, the NKG2A binder comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 64 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 73. In some embodiments, the NKG2A binder comprises the heavy chain variable region as illustrated in SEQ ID NO: 64 and the light chain variable region as illustrated in SEQ ID NO: 73.

[0158] In some embodiments, the binder exhibits excellent developability based on assays known in the art, such as various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and upright monolayer adsorption chromatography (SMAC). In some embodiments, the binder exhibits excellent developability based on measurements of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0159] In some embodiments, NKG2A binders, including human NKG2A binders described herein (e.g., antibodies, such as monospecific or bispecific antibodies), comprise a heavy chain having a combination of: (i) a VH as described herein (such as in any of Tables 1-4); and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:81).

[0160] Another exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:85). In other embodiments, the carboxyl terminus (C-terminus) of VH is directly or indirectly conjugated to the amino terminus (N-terminus) of one or more heavy chain constant domains.

[0161] In some embodiments, NKG2A binders, including human NKG2A binders (e.g., antibodies, such as monospecific or bispecific antibodies), as described herein comprise a light chain having a combination of: (i) a VL domain as described herein (such as in any of Tables 1-4); and (ii) a light chain constant domain (CL). Exemplary light chains (e.g., for pairing with IgG heavy chains) comprise any of the VL sequences described herein and the following CL amino acid sequences: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:82). In other embodiments, the C-terminus of VL is directly or indirectly conjugated to the N-terminus of CL.

[0162] In some embodiments, the binders provided herein inhibit HLA-E / NKG2A (such as HLA-E / NKG2A / CD94) signaling. Such inhibition can be measured, for example, as detailed in Example 4.

[0163] In some embodiments, NKG2A binders, including human NKG2A binders described herein (e.g., antibodies, such as monospecific or bispecific antibodies), comprise: (a) a heavy chain having a combination of: (i) the VH described herein (such as in any 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) the VL described herein (such as in any of Tables 1-4), and (ii) a light chain constant domain (CL or CL1) in IgG form. Exemplary NKG2A binders (e.g., antibodies) comprise an IgG heavy chain containing any VH sequence as described herein and an amino acid sequence of SEQ ID NO: 81 or 85, and a light chain containing any VL sequence as described herein and an amino acid sequence of SEQ ID NO: 82.

[0164] In some embodiments, this document provides an NKG2A binding protein comprising any 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 containing the same VH region and two light chains containing the same VL region.

[0165] In some embodiments, the NKG2A binding protein is a monoclonal antibody, including mouse, chimeric, humanized, or human antibodies. In some embodiments, the anti-NKG2A antibody is an antibody fragment, such as scFv. In some embodiments, the NKG2A binding protein is a fusion protein comprising the anti-NKG2A antibody provided herein. In other embodiments, the NKG2A binding protein is a multispecific antibody comprising the anti-NKG2A antibody provided herein or a fragment thereof.

[0166] Other exemplary NKG2A binding molecules are described in more detail in the following sections. In some embodiments, the anti-NKG2A antibody or antigen-binding protein according to any of the above embodiments may be incorporated, alone or in combination, into any of the features described in sections 5.2.1 to 5.2.4 below.

[0167] Table 1: Antibody clone A3

[0168] Table 2: Antibody clone A2

[0169] Table 3: Antibody clone A42

[0170] Table 4: Antibody clone A11

[0171] 5.2.1. Antibody fragments Even though the term "antibody" is sometimes used in the phrase "antibody or a fragment thereof" herein, it should be understood that, as used herein, the term "antibody" also includes various antibody fragments, such as antigen-binding fragments or epitope-binding fragments. Therefore, when the term "antibody" is used alone without the subsequent "fragments thereof" or a similar term, it should be understood that the term "antibody" includes antibody fragments, such as antigen-binding fragments or epitope-binding fragments. The antibodies described herein include, but are not limited to, immunoglobulin molecules and the immunologically active portions of immunoglobulin molecules.

[0172] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to antigens. 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 comprising a light chain and a portion of a heavy chain bridged by disulfide bonds); Fab' (e.g., antibody fragments containing a single antigen-binding domain comprising a Fab and an additional heavy chain portion joined by a hinge region); F(ab')2 (e.g., two Fab' molecules joined by interchain disulfide bonds in the heavy chain hinge region; the Fab' molecules may target the same or different epitopes); bispecific Fab (e.g., Fab molecules having two antigen-binding domains, each of which may target different epitopes); single chains containing variable regions, also known as scFv (e.g., variable, antigen-binding-determining regions of a single light and heavy chain of an antibody linked together by, for example, 10-25 amino acid chains); disulfide-linked Fvs, or dsFvs (e.g., variable, antigen-binding-determining regions of a single light and heavy chain of an antibody linked together by disulfide bonds); and bispecific scFvs. (e.g., scFv or dsFv molecules having two antigen-binding domains, each of which may target different epitopes); bifunctional antibodies (e.g., dimerized scFvs 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; the two antigen-binding regions of the bifunctional antibody may target the same or different epitopes); trifunctional antibodies (e.g., trimerized scFvs formed in a manner similar to bifunctional antibodies, but with the three antigen-binding domains forming a single complex; the three antigen-binding domains may target the same or different epitopes); and tetrafunctional antibodies (e.g., tetramerized scFvs formed in a manner similar to bifunctional antibodies, but with the four antigen-binding domains forming a single complex; the four antigen-binding domains may target the same or different epitopes).

[0173] Various techniques have been developed for generating antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, 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 generated directly from recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be generated in *E. coli* (…). E. coliThese fragments are expressed and secreted by yeast or insect cells, thereby allowing for the convenient production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed 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 method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments, having extended in vivo half-lives and containing rescue receptor-binding epitope residues, are described, for example, in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In some embodiments, the antibody is a single-chain Fv fragment (scFv) (see, for example, WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458). Fv and scFv have complete combinatorial sites without constant regions; therefore, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the N-terminus or C-terminus of the scFv (see, for example, Borrebaeck, ed., ibid.). Antibody fragments can also be “linear antibodies,” as described in the references cited above. Such linear antibodies can be monospecific or multispecific, such as bispecific antibodies.

[0174] 5.2.2. Humanized Antibodies This disclosure provides humanized antibodies that bind to NKG2A, including human NKG2A. The humanized antibodies of this disclosure may contain one or more CDRs from the VH and / or VL disclosed herein, such as those shown in Tables 1-4. Various methods for humanizing non-human antibodies are known in the art. For example, the humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as “input” residues and are typically derived from an “input” variable domain. Humanized antibodies binding 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).

[0175] In some cases, humanized antibodies are constructed via CDR grafting, where the amino acid sequences of six CDRs from the parental non-human antibody (e.g., rodent) are grafted onto the 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 CDR actually contact the antigen, and referred to these residues as "specificity-determining residues" or SDRs. In SDR transplantation techniques, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36: 25-34, 2005).

[0176] Selecting the human variable domains (both light and heavy chains) used for preparing humanized antibodies is crucial for reducing antigenicity. For example, according to the so-called "best-fit" method, sequences of variable domains for non-human (e.g., rodent) antibodies are screened against an entire library of known human variable domain sequences. The human sequence 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 approach uses a specific frame derived from a specific subgroup of the light or heavy chain, sharing a common sequence among all human antibodies. Several different humanized antibodies can use the same frame (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 derived from the most abundant human subclass (V L 6 subgroups I (V) L 6I) and V H Subgroup III (V) H The shared sequence of III). In another approach, human germline genes are used at the source of the frame region.

[0177] In an alternative paradigm based on CDR comparisons (referred to as hyperhumanization), frame homology is irrelevant. The method consists of comparing non-human sequences with human germline functional gene lineages. Genes encoding typical structures identical to or closely related to mouse sequences are then selected. Subsequently, among genes sharing typical structures with non-human antibodies, those with the highest homology within the CDR are selected as frame donors. Finally, non-human CDRs are transplanted onto these frames (see, for example, Tan et al.). J. Immunol . 169: 1119-1125, 2002).

[0178] Typically, further humanization of the antibody is required while maintaining its affinity for the antigen and other beneficial biological properties. To achieve this, humanized antibodies are prepared according to a method that analyzes parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and demonstrate the possible three-dimensional conformations of selected candidate immunoglobulin sequences can be used. 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 the Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). These demonstrated examinations allow for the analysis of the possible roles of residues in the function of candidate immunoglobulin sequences, for example, analyzing residues that affect the ability of candidate immunoglobulins to bind their antigens. In this way, framework residues can be selected from receptors and input sequences and combined to achieve desired antibody characteristics, such as increased affinity for target antigens. Generally, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0179] Another approach for antibody humanization is based on an antibody humanization metric called Human String Content (HSC). This method compares the genetic profiles of mouse sequences with those of human germline genes and scores the differences as HSC. Then, by maximizing the HSC of the target sequence rather than using a measure of overall identity, the target sequence is humanized to produce a variety of different humanized variants. See, for example, Lazar et al. Mol. Immunol. 44: 1986-1998, 2007.

[0180] In addition to the methods described above, empirical methods can also be used to generate and select humanized antibodies. These methods include those based on large libraries that generate humanized variants and those that select the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as through bacterial colony screening (see, for example, 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).

[0181] In the framework library approach, a set of residue variants is introduced at specific locations within a framework, followed by selection from a library to choose the framework that best supports the transplanted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter). J. Mol. Biol . 224: 487-499, 1992), or from Baca et al. ( J. Biol. Chem (272: 10678-10684, 1997) a more limited set of target residues identified.

[0182] In frame reorganization, the entire frame is combined with non-human CDRs rather than generating a library of combinatorial variants of selected residues (see, for example, Dall'Acqua et al.). Methods 36: 43-60, 2005. Libraries can be screened for binding in a two-step selection procedure (first humanizing VL, then VH). Alternatively, a single-step framework shuffling procedure can be used. Such methods have been shown to be more efficient than two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermostability (see, for example, Damschroder et al., 2005). Mol. Immunol. 44: 3049-60, 2007).

[0183] The "humanizing" approach is based on experimental identification using the minimum specific determinant (MSD) and relies on sequential substitution of non-human fragments into a human framework library and assessment of binding. It begins with the CDR3 region of the non-human VH and VL chains and progressively substitutes other regions of the non-human antibody into the human framework, including CDR1 and CDR2 of both VH and VL. This approach typically induces epitope retention and identification of antibodies from multiple subclasses with different human V segment CDRs. Humanizing allows the isolation of antibodies with 91-96% homology to human germline gene antibodies. See, for example, Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007.

[0184] "Human engineering" methods involve altering non-human antibodies or antibody fragments, such as mouse or chimeric antibodies or fragments, by specifically changing the amino acid sequence of the antibody to produce a modified antibody that has reduced immunogenicity in humans but still retains the desired binding properties of the original non-human antibody. Typically, the techniques involve classifying amino acid residues of non-human (e.g., mouse) antibodies into "low-risk," "intermediate-risk," or "high-risk" residues. Classification is performed using an overall risk / reward calculation, which assesses the predicted benefit of performing a specific substitution (e.g., immunogenicity in humans) against the risk that the substitution would affect the folding of the resulting antibody and / or its substitution by human residues. Specific human amino acid residues to be substituted at designated locations (e.g., low-risk or intermediate-risk) in the non-human (e.g., mouse) antibody sequence can be selected by comparing the amino acid sequence from the variable region of the non-human antibody with corresponding regions of a specific or shared human antibody sequence. Based on the comparison, amino acid residues at low-risk or intermediate-risk locations in the non-human sequence can substitute for corresponding residues in the human antibody sequence. The techniques for preparing human engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994); U.S. Patents 5,766,886, 5,770,196, 5,821,123 and 5,869,619, and WO 93 / 11794.

[0185] 5.2.3. Antibody Variants This covers modifications to antibodies binding to NKG2A as described herein. For example, it may be necessary 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. Therefore, it is contemplated that variants of antibodies binding to NKG2A as described herein can be prepared and are included in this disclosure. In some embodiments, the antibody variant is an antibody with an amino acid sequence variation compared to the original antibody, for example, having substitutions, deletions, or insertions of one or more amino acids as described above. For example, the variation compared to the original antibody or peptide can be a substitution, deletion, or insertion of one or more codons encoding the antibody or peptide that causes an amino acid sequence change (e.g., conserved substitution). Sites of interest for substitutional mutagenesis include CDRs, FRs, and / or constant regions. For example, antibody variants can be prepared by introducing suitable nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or peptide. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of an antibody.

[0186] Chemical modification Other exemplary modifications include, for example, chemical modifications by covalently linking any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / capping groups, proteolytic degradation, linking to cellular ligands or other proteins, or conjugating to one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more non-classical amino acids.

[0187] In some implementations, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Adding or removing glycosylation sites from an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0188] When the antibody presented herein fuses with the Fc region, it alters the carbohydrates attached thereto. Native antibodies produced by mammalian cells typically contain branched-chain biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-bonds. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc linked to a “stem” in the bifacial oligosaccharide structure. In some embodiments, the oligosaccharides in the bound molecules provided herein may be modified to produce variants with certain improved properties.

[0189] In other embodiments, when the antibody provided herein is fused to the Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose linked (directly or indirectly) to said Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297, as measured by, for example, MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of the Fc region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have improved ADCC functionality. See, for example, U.S. Patent Publications 2003 / 0157108 and 2004 / 0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: U.S. Patent Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent Publication No. 2002 / 0164328; U.S. Patent Publication No. 2004 / 0093621; U.S. Patent Publication No. 2004 / 0132140; U.S. Patent Publication No. 2004 / 0110704; U.S. Patent Publication No. 2004 / 0110282; U.S. Patent Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 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 lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys 249:533-545 (1986); US Patent Publication No. US 2003 / 0157108; and WO 2004 / 056312) and gene knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 Gene knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. , 94(4):680-688 (2006); and WO2003 / 085107).

[0190] Binding molecules containing the antibodies provided herein also possess a bipartite oligosaccharide, for example, in which a bipartite oligosaccharide linked to the Fc region is bipartite via GlcNAc. 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. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Publication No. 2005 / 0123546 (Umana et al.). Variants are also provided in which at least one galactose residue in the oligosaccharide is linked to the Fc region. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0191] In molecules containing the antibodies of the present invention and the Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby creating Fc region variants. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0192] In some embodiments, this application covers variants having some, but not all, effector functions, making these variants ideal candidates for applications where the in vivo half-life of the binding molecule is critical, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the binding molecule does not have FcγR binding capacity (and therefore may lack ADCC activity), but retains FcRn binding capacity. Non-limiting examples of in vitro assays for evaluating the ADCC activity of the molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, 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); US Patent No. 5,821,337 (see Bruggemann, M. et al.) J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CYTOTOX 96). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, in vivo, such as in animal models (e.g., Clynes et al.), can be used. Proc. Nat ' Acad. Sci. USA The ADCC activity of the molecule of interest can be assessed in animal models disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, 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)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB, et al.). Int ' l. Immunol. 18(12):1759-1769 (2006)).

[0193] Binding molecules with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 replacing alanine (US Patent No. 7,332,581).

[0194] Certain variants with improved or reduced FcR binding are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al.) J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0195] In some embodiments, the variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbers of residues) in the Fc region. In some embodiments, modifications are made in the Fc region that alter (i.e., improve or reduce) C1q binding and / or complement-dependent cytotoxicity (CDC), as in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. As described in 164: 4178-4184 (2000).

[0196] A binding molecule with an extended half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994) is described in US2005 / 0014934A1 (Hinton et al.). Those molecules contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at one or more of the following 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, for example, substitution of Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0197] In some embodiments, it may be necessary to generate cysteine-engineered antibodies, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiols are thereby located at accessible sites on the antibody and can be used to conjugate the antibody with other parts (such as pharmaceutical parts or linker-pharmaceutical parts) to produce immunoconjugates as further described herein.

[0198] Other known covalent modifications of antibodies are included within the scope of this disclosure. Covalent modifications include reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the antibody. Other modifications include deamidation of glutamine acyl and asparagine acyl residues to the corresponding glutamine acyl and asparagine acyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of the lysine, arginine, and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl group.

[0199] The NKG2A-bound antibody disclosed herein can also be modified to form a chimeric molecule comprising an NKG2A-bound antibody fused or conjugated to another heterologous polypeptide or amino acid sequence or small molecule compound, such as an immune activator (e.g., cytokines), an epitope tag (see, for example, Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, for example, Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi, eds., 1999)).

[0200] This document also provides fusion proteins comprising the antibody and heteropeptide of this disclosure that bind to NKG2A. In some embodiments, the heteropeptide fused to or chemically conjugated with the antibody gene can be used to target the antibody to cells expressing NKG2A on their cell surface. The gene-fused or chemically conjugated antibodies are described in more detail in the following sections.

[0201] In vitro affinity maturation In some implementations, antibody variants with improved properties (such as affinity, stability, or expression levels) compared to the parent antibody can be prepared via in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. The antibody library is displayed on the surface of an organism (e.g., bacteriophage, bacteria, yeast, or mammalian cells) or bound to its encoding mRNA or DNA (e.g., covalently or non-covalently). Affinity selection of the displayed antibody allows the isolation of the organism or complex carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods (such as phage display) typically produce antibody fragments with affinity in the low nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinity for the target antigen.

[0202] Phage display is a widely used method for antibody display and selection. Antibodies are displayed on the surface of Fd or M13 phages in the form of fusions with phage coat proteins. Selection involves exposing antibodies to an antigen to allow the phage to bind to its target; this is a process called “panning.” Phages bound to the antigen are recovered and used to infect bacteria to generate phages for further rounds of selection. For reviews, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0203] In yeast display systems (see, for example, Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can fuse to the adhesion subunit of the yeast lectin protein Aga2p, which is linked to the yeast cell wall via a disulfide bond with Aga1p. Display of the protein via Aga2p causes it to protrude away from the cell surface, thereby minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling yeast with biotin-labeled antigens and minor reagents, such as streptavidin conjugated with a fluorophore. Changes in antibody surface expression can be measured by immunofluorescence labeling of hemagglutinin or c-Myc epitope tags to side-attached single-chain antibodies (e.g., scFv). Studies have shown that expression is correlated with the stability of the displayed protein, and therefore antibodies can be selected for improved stability and affinity (see, for example, Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of eukaryotic yeast cells, thus utilizing endoplasmic reticulum chaperone proteins and quality control mechanisms. Once fully matured, antibody affinity can be conveniently “tied” while displayed on the yeast surface, eliminating the need for expression and purification of individual clones. A theoretical limitation of yeast surface display is that the functional library size is potentially smaller than that of other display methods; however, recent methods using yeast cell mating systems have yielded libraries with estimated sizes of 10-1. 14 Combinatorial diversity (see, for example, U.S. Patent Publication No. 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0204] During ribosome display, an antibody-ribosome-mRNA (ARM) complex is generated for selection in a cell-free system. A DNA library encoding a specific antibody library is fused with a spacer sequence lacking a stop codon. This spacer sequence remains linked to the peptidyl tRNA during translation and occupies a ribosomal tunnel, thus allowing the protein of interest to protrude from the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to a surface-bound ligand, allowing simultaneous separation of the antibody and its encoding mRNA via ligand affinity capture. The ribosome-bound mRNA is then reverse transcribed into cDNA, which can then undergo mutagenesis and be used for the next round of selection (see, for example, Fukuda et al., NucleicAcids Res. 34:e127 (2006)). During mRNA display, the covalent bond between the antibody and the mRNA is established using puromycin as a transfer molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55(2001)).

[0205] 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 the 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 round of selection (e.g., by non-proofreading polymerase) because no library must be transformed after any diversification step. In some embodiments, mammalian display systems can be used.

[0206] Diversity can also be introduced into the CDRs of an antibody library via targeted or random introduction. The former involves sequentially targeting all CDRs of the antibody via high or low levels of mutagenesis, or targeting isolated somatic hypermutation hotspots (see, for example, Ho et al., J. Biol. Chem. 280:607-17 (2005)), or residues suspected of affecting affinity experimentally or for structural reasons. Diversity can also be introduced by replacing naturally diverse regions via DNA shuffling or similar techniques (see, for example, Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, for example, U.S. Patent Publication No. 2004 / 0005709) to target hypervariable loops extending into frame region residues (see, for example, Bond et al., J. Mol. Biol. 348:699-709 (2005)). Other methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Other methods for generating antibody libraries and / or maturing antibody affinity are disclosed, for example, in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publications Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0207] Library screening can be achieved using various 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 attached to adsorption discs or used for cell sorting, or conjugated with biotin to capture with streptavidin-coated beads or used in any other method for panning and displaying libraries.

[0208] For reviews of in vitro affinity maturation methods, see, for example, Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and the references therein.

[0209] Antibody internalization assays can be used to determine receptor-mediated endocytosis when bound to an antibody. 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 evaluate the antibody's ability to deliver treatment to the site of interest or cells. A non-limiting exemplary assay is briefly described below. Target cells of interest are seeded at an appropriate seeding density (e.g., in a 96-well U-shaped dish), and a test antibody, such as a fluorescent compound, horseradish peroxidase (HRP) reagent, radiolabeled compound, or biotin, is labeled with a signal reporting reagent. The test antibody is then incubated with the target cells at an appropriate molar ratio. After incubation, unbound antibodies are removed by washing. Cells may be retained on ice or incubated at 37°C for a period of time to promote internalization. Cells may then be incubated for a period of time in the presence of a stop reagent to inhibit internalization. Subsequently, the cells are washed and incubated with a signal display reagent. The final signal can be studied using a plate reader or imaging instrument and analysis software. For example, flow cytometry can be used to measure the mean fluorescence intensity (MFI) of cells, and a decrease in MFI can indicate antibody internalization, antibody dissociation, or a combination of both. Cell imaging can be scanned and acquired to analyze signal intensity, size, and shape. Alternatively, cells can be lysed to release the internalized antibody. This antibody is then captured in a microtiter plate coated with a specific antigen that produces the antibody. The bound antibody in the wells is detected using alkaline phosphatase or HRP-conjugated secondary antibodies and chromogenic substrates. Based on this disclosure, those skilled in the art will readily recognize alternative detectable labels for antibodies and methods for detecting internalized labeled antibodies. Any method known in the art for determining antibody internalization may be used in this disclosure.

[0210] 5.2.4. Other binding agents containing antibodies In some embodiments, the antibody or fragment thereof provided herein is part of a larger binding agent. Non-limiting exemplary binding agents comprising the antibody or fragment thereof provided herein are described below.

[0211] This disclosure provides NKG2A binders (e.g., antibodies) having a masking portion and / or a cleavable portion, wherein one or more NKG2A binding domains of the NKG2A binder (e.g., antibody) are masked (e.g., via the masking portion) and / or activated (e.g., via the cleavable portion). Techniques for masking NKG2A binders (e.g., antibodies) are well known in the art, including SAFEbody masking techniques (see, for example, U.S. Patent Publication No. 2019 / 0241886) and Probody masking techniques (see, for example, U.S. Patent Publication No. 2015 / 0079088). Such techniques can be used to produce masked and / or activated NKG2A binders (e.g., antibodies). Such masked and / or activatable NKG2A binders (e.g., antibodies) can also be used to prepare conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable ADCs (AADCs), said conjugates comprising any of the NKG2A binders of this disclosure (e.g., antibodies, such as human NKG2A binders), including those directly or indirectly linked to another agent (such as a drug and / or an immune activator (such as a cytokine)). For example, the NKG2A binders of this disclosure (e.g., antibodies, such as human NKG2A binders) can be covalently bound to one or more agents, such as drugs and / or immune activators (such as cytokines), via synthetic linkers.

[0212] Where necessary, NKG2A binders (e.g., antibodies), including human NKG2A binders, are linked or conjugated (directly or indirectly) to a portion having effector functions such as cytotoxic activity (e.g., a chemotherapeutic fraction or radioisotope), immune recruitment, or regulatory activity. The linked or conjugated portion (directly or indirectly) includes cytotoxic drugs (e.g., toxins, such as aurostatins) or non-cytotoxic drugs, such as signal transduction modulators, kinases, or a masking portion that masks one or more binding domains of the NKG2A binder (e.g., antibody), or a cleavable portion that allows activation of the NKG2A binder by exposing one or more binding domains of the NKG2A binder (e.g., antibody) in the tumor microenvironment as a masked conjugate by cleaving the cleavable portion. The portion promoting immune recruitment may include other antigen binders, such as viral proteins that selectively bind to cells of the innate and / or adaptive immune system. Alternatively or additionally, NKG2A binders (e.g., antibodies), including human NKG2A binders, may optionally be linked or conjugated (directly or indirectly) to a portion that facilitates separation from a mixture (e.g., a tag) or to a portion having reporter gene activity (e.g., a detection tag or reporter protein). It should be understood that the characteristics of NKG2A binders (e.g., antibodies), including human NKG2A binders, described herein also extend to peptides containing NKG2A binder fragments.

[0213] In some embodiments, NKG2A binders (e.g., antibodies) that bind to human NKG2A (including NKG2A binders) as described herein may be linked or conjugated (directly or indirectly) to a peptide, thereby inducing the production of an activatable antibody. In some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, are linked or conjugated (directly or indirectly) to another agent. In some embodiments, the other agent is a drug that produces an ADC or AADC when the antibody of the ADC contains both a masking portion and a cleavable portion.

[0214] In some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, described herein are conjugated or recombined (directly or indirectly) to therapeutic agents (e.g., cytotoxic agents or cytokines), diagnostic agents, or detectable agents. Conjugated or recombined antibodies (including masked or activatable conjugates) can be used, for example, to treat or prevent diseases, conditions, or disorders, such as NKG2A-mediated diseases, conditions, or disorders. Conjugated or recombined NKG2A binders (e.g., antibodies) (including masked or activatable conjugates) can be used, for example, to monitor or predict the onset, development, progression, and / or severity of NKG2A-mediated diseases, conditions, or disorders.

[0215] Such diagnostics and detections can be achieved, for example, by coupling an NKG2A binder (e.g., an antibody) with a detectable substance including, for example, the following: enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, including but not limited to streptavidin / biotin or avidin / biotin; fluorescent materials, including but not limited to umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; luminescent materials, including but not limited to luminol; bioluminescent materials, including but not limited to luciferase, luciferin, or aequorin; chemiluminescent materials, including but not limited to acridine-based compounds or HALOTAG; and radioactive materials, including but not limited to 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), Technetium ( 99 Tc), thallium 201 Ti, gallium68 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、 142 Pr, 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 Sn; positron-emitting metals obtained using various positron emission tomography (PET) scans; and non-radioactive paramagnetic metal ions.

[0216] This document also describes NKG2A binders (e.g., antibodies) that recombinantly link or conjugate (directly or indirectly, covalently or non-covalently) to a heterologous protein or polypeptide or fragment thereof, such as polypeptides (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce fusion proteins, and their uses. Specifically, this document describes fusion proteins comprising an antigen-binding fragment (e.g., CDR1, CDR2, and / or CDR3 comprising VH and / or VL) of an NKG2A binder (e.g., an antibody), including a human NKG2A binder, and a heterologous protein, polypeptide, or peptide. In some embodiments, the heterologous protein, polypeptide, or peptide to which the NKG2A binder (e.g., an antibody) is linked can be used to target the NKG2A binder to specific cells (e.g., cells expressing NKG2A, including immune cells). Other non-restricted heterologous proteins, polypeptides, or peptides linked to NKG2A binders (such as antibodies) can serve as internalization signals or enable tumor cells to connect with immune cells.

[0217] Furthermore, NKG2A binders (e.g., antibodies), including human NKG2A binders, described herein can be linked (directly or indirectly) to a marker or “tag” sequence, such as a peptide, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is a hexahistine peptide, such as the tag provided in the pQE vector (see, for example, QIAGEN, Inc.), and others, many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistine facilitates the purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin (“HA”) tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37:767-78); and “FLAG” tags.

[0218] Methods for linking or conjugating (directly or indirectly) portions (including peptides) to antibodies are well known in the art, and any of these methods can be used to prepare the antibody-drug conjugates or fusion proteins described herein.

[0219] In some embodiments, the NKG2A binder (e.g., antibody) described herein is a fusion protein. As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of a binder (e.g., antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of an antibody). In some embodiments, the fusion protein retains the biological activity of the NKG2A binder. In some embodiments, the fusion protein comprises a VH region, a VL region, VH CDRs (one, two, or three VH CDRs), and / or VL CDRs (one, two, or three VL CDRs) of an NKG2A antibody, wherein 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 and the VL or light chain of an NKG2A antibody, which are separated by a linker (such as a cleavable linker).

[0220] Fusion proteins can be produced, for example, through techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of NKG2A binders (e.g., antibodies), including those with high affinity and low dissociation rates, as described herein (see, for example, U.S. Patent 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 implementations, NKG2A binders, including human NKG2A binders, can be altered by random mutagenesis prior to recombination via error-prone PCR, random nucleotide insertion, or other methods. The polynucleotide encoding the NKG2A binder described herein can recombine with one or more components, motifs, segments, parts, domains, fragments, etc., of one or more heterologous molecules.

[0221] The NKG2A binders (e.g., antibodies), including human NKG2A binders, described herein can also be linked to solid supports, which can be used for the immunoassay or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0222] The NKG2A binders (e.g., antibodies), including human NKG2A binders, described herein can also be linked or conjugated (directly or indirectly) to secondary antibodies to form antibody heteroconjugates.

[0223] Linkers can be “cleavable portions” that facilitate the release of conjugating or coupling agents into cells, but this document also covers non-cleavable linkers. Linkers used in conjugates (e.g., ADCs or AADCs) of this 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, such as citrulline-valine or phenylalanine-lysine), photostable linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

[0224] Antibody-agent conjugates (including those where the agent is a drug for preparing ADC or AADC) can be prepared using various bifunctional protein conjugators, such as N-(β-maleimide propoxy)succinimide ester (BMPS); N-ε-maleimide octanoyl-oxysuccinimide ester (ECMS); N-γ-maleimide octanoyl-oxysuccinimide ester (GMBS); 1,6-hexane-bis-vinyl sulfone (HBVS); succinimide-4-(-N-maleimide methyl)cyclohexane-1-carboxy-(6-amidohexanoate)). (LC-SMCC); m-maleimide benzoyl-N-hydroxysuccinimide (MBS); 4-(4-N-maleimide phenyl)butyric acid hydrazide (MPBH); 3-(bromoacetamyl)propionate succinimide (SBAP); iodoacetic acid succinimide (SIA); (4-iodoacetyl)aminobenzoic acid succinimide (SIAB); succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC); 4-(p-maleimide phenyl)butyric acid succinimide (SMPB); succinimide-6-(β-maleimide propionamido)hexanoate (SMPH); N-(ε-maleimide octanoyloxy) Sulfonyl succinimide ester (sulfon-ECMS); N-(γ-maleiminobutyryloxy)sulfonyl succinimide ester (sulfon-GMBS); N-(κ-maleiminobutadecoyloxy)sulfonyl succinimide ester (sulfon-KMUS); m-maleiminobenzoyl-N-hydroxysulfonyl succinimide ester (sulfon-MBS); (4-iodo-acetyl)aminobenzoic acid sulfonyl succinimide ester (sulfon-SIAB); 4-(N-maleimino-methyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester (sulfon-SMCC); 4-(p-maleiminophenyl)butyric acid sulfonyl succinimide ester (sulfon-SMPB); and succinimide-(4-vinyl sulfone)benzoate (SVSB).

[0225] This disclosure also covers conjugates of antibodies and agents (including drugs used to prepare ADCs or AADCs) that can be prepared using any suitable method disclosed in the art (see, for example, Bioconjugate Techniques (Hermanson, ed., 2nd edition, 2008)).

[0226] Conventional conjugation strategies for antibodies and agents (including drugs used to prepare ADCs or AADCs) have been based on random conjugation chemistry involving ε-amino groups of Lys residues or thiol groups of Cys residues, which produce heterogeneous conjugates. Recently developed techniques allow for site-specific conjugation with antibodies, resulting in homogeneous loading and avoiding alterations in antigen binding or pharmacokinetics of conjugate subsets. These techniques include the engineering of “thiomab” containing cysteine ​​substitutions at positions on both the heavy and light chains, which provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, for example, Junutula et al., 2008, J. Immunol. Meth. 332:41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another approach, selenocysteine ​​is cotranslatorily inserted into the antibody sequence by recoding the stop codon UGA from the stop codon to a selenocysteine ​​insertion, thereby allowing site-specific covalent conjugation at the nucleophilic selenool group of selenocysteine ​​in the presence of other natural amino acids (see, for example, Hofer et al., 2008, Proc. Natl. Acad. Sci. USA105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0227] In some embodiments, the NKG2A binders (e.g., antibodies), including human NKG2A binders described herein, are bound to agents such as immune activators or cytotoxic agents. In some embodiments, the NKG2A binders (e.g., antibodies), including human NKG2A binders disclosed herein, may optionally be conjugated to one or more cytotoxic agents disclosed herein or known in the art to produce an ADC or AADC. 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 inserters. 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, non-bound active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modizol A chain, α-broomitin, tung oil protein (Aleurites fordii), carnation protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, jatropha toxin (curcin), croton toxin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. In some embodiments, the cytotoxic agent is a radioactive isotope to produce a radioconjugate or radioconjugate agent. Various radioactive nuclei can be used to produce radioactive conjugates, including but not limited to... 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 212Bi. Conjugates of polypeptides or molecules with one or more small molecule toxins (such as calicheamicin, maytansinoids, trichothecene toxins and CC1065, and derivatives of these toxins with toxic activity) may also be used. Various bifunctional protein conjugates are used to prepare conjugates of peptides or molecules with cytotoxic agents. These bifunctional protein conjugates include N-succinimide-3-(2-pyridinedithiool)propionate (SPDP), iminothiones (IT), bifunctional derivatives of imide esters (such as dimethyl adipamide HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), bis-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0228] In other embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, are conjugated to pharmaceuticals such as signal transduction modulators, apoptosis-promoting agents, mitosis inhibitors, antitumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, anti-hormonal agents, corticosteroids, photosensitizers, oligonucleotides, radionuclides, radiosensitizers, topoisomerase inhibitors (such as camptothecin or analogues), and tyrosine kinase inhibitors. In some embodiments, mitosis inhibitors are dolasatin, auristatin, maytansine, and plant alkaloids. In some embodiments, pharmaceuticals are dolasatin, auristatin, maytansine, and plant alkaloids. Examples of auristatin are monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE). Examples of maytansine-like antibiotics include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of: actinomycine, anthracycline, kazimidic acid, and duocarmycin. An example of anthracycline is pyrrolobenzodiazepine (PBD). An example of anthracycline is PNU-anthracycline, such as PNU-159682 or its derivatives.

[0229] NKG2A binding agents (e.g., antibodies), including human NKG2A binding agents, described herein may have monospecific, bispecific, trispecific, or higher multispecificity. Such agents may include monospecific or multispecific antibodies. Multispecific antibodies (such as bispecific antibodies) are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody against NKG2A having a first binding domain against a first epitope of NKG2A and a second binding domain against a second epitope of NKG2A). In some embodiments, monospecific and multispecific (e.g., bispecific) antibodies may be constructed based on the sequences of the 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 antibody is a mouse, chimeric, human, or humanized antibody.

[0230] In some embodiments, a multispecific antibody may bind to NKG2A in one specific target and to any other target (e.g., an antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may comprise more than one target (e.g., an antigen) binding domain, wherein different binding domains are specific to different targets (e.g., a first binding domain binds to NKG2A and a second binding domain binds to another target (e.g., an antigen)). In some embodiments, the second target is an immune checkpoint modulator (e.g., a negative checkpoint modulator). In some embodiments, the second target is expressed on immune cells. In some embodiments, the second target is expressed on tumor cells or cancer cells.

[0231] In some implementations, multispecific (e.g., bispecific) antibody molecules can bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen).

[0232] Methods for preparing multispecific antibodies are known in the art, such as the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For further details on the generation of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0233] 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.

[0234] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with an additional antigen-binding portion attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG forms may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes co-LC, knobs-in-hole assemblies, charge pairs, Fab-arm exchanges, SEED bodies, triomab, LUZ-Y, Fcab, κλ bodies, and / or orthogonal Fab.

[0235] In some implementations, BsIgG includes heavy chains engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a "mortar and pestle" strategy, a SEED platform, a common heavy chain (e.g., in κλ body form), and the use of heterodimeric Fc regions. Strategies known in the art to avoid homodimeric heavy chain pairing in BsIgG include mortar and pestle, duobody, azymetric, charge pairs, HA-TF, SEED bodies, and differential protein A affinity.

[0236] Another form of bispecific antibody is IgG attached to an additional antigen-binding portion. For example, monospecific IgG can be engineered to have bispecificity by attaching an additional antigen-binding unit to a monospecific IgG (e.g., at the N-terminus or C-terminus of the heavy or light chain). Exemplary additional antigen-binding units include a single-domain antibody (e.g., a variable heavy or light chain), an engineered protein backbone, and a paired antibody variable domain (e.g., a single-chain variable fragment or variable segment). Non-limiting examples of attached IgG forms 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 (quadruple). See Spiess et al., Mol. Immunol. 67(2015):95-106. In some embodiments, exemplary antibody forms are, for example, monospecific or multispecific B-body forms (e.g., bispecific antibodies) as described in WO 2018 / 075692 and U.S. Patent Publication No. 2018 / 0118811.

[0237] Bispecific (Bs) antibody (BsAb) fragments are forms of bispecific antibody molecules that lack some or all of the constant antibody domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment includes heavy and light chain regions linked by a peptide linker that allows for efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTE, bifunctional antibodies, DART, TandAb, sc bifunctional antibodies, sc bifunctional antibodies-CH3, bifunctional antibodies-CH3, triple bodies, miniantibodies, TriBi miniantibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc bifunctional antibodies-Fc, bifunctional antibodies-Fc, tandem scFv-Fc, and intrabody.

[0238] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins may be linked to other proteins to add additional specificity and / or functionality. In some embodiments, a dock-and-lock (DNL) approach may be used to generate bispecific antibody molecules with higher valences. For example, bispecific antibody fusions with albumin-binding proteins or human serum albumin can extend the serum half-life of the antibody fragment. In embodiments, chemical conjugation (e.g., chemical conjugation of antibodies and / or antibody fragments) may be used to generate BsAb molecules. Exemplary bispecific antibody conjugations include CovX forms in which low molecular weight drug sites are specifically conjugated to a single reactive lysine residue in each Fab arm or antibody or fragment thereof. In some embodiments, conjugation improves serum half-life.

[0239] Methods for generating multispecific antibodies (including bispecific antibodies) are known in the art. For example, multispecific antibodies (including bispecific antibodies) can be generated by expressing component antibodies separately in different host cells and then purifying / assembling them, or by expressing component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by various methods known in the art, including affinity chromatography.

[0240] In some embodiments, the NKG2A binders (e.g., antibodies), including human NKG2A binders disclosed herein, may be available in any antibody form disclosed herein or known in the art. As a non-limiting example, in some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, may be selected from Fabs-in-tandem-lg (FIT-lg); DVD-lg; hybridomas (tetrasomatic or tetrasomatic hybridomas); anti-carrier protein platforms (Pieris); bifunctional antibodies; single-chain bifunctional antibodies; tandem single-chain Fv fragments; TandAb, trispecific Ab (Affimed); Darts dual-parental retargeting (Macrogenics); bispecific Xmab (Xencor); bispecific T-cell adaptors (Bites; Amgen; 55kDa); trisomatic antibodies; trifunctional antibodies = Fab-scFv fusion protein multifunctional recombinant antibody derivatives (CreativeBiolabs); Duobody platforms (Genmab); dock and lock platforms; kihododendron (KIH) platforms; humanized bispecific IgG antibodies (REGN1979). (Regeneron); Mab2 bispecific antibody (F-Star); DVD-lg = dual variable domain immunoglobulin (Abbott); κ-λ body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0241] 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 containing VH and / or VL amino acid sequences as disclosed herein, such as those in Tables 1, 2, 3, or 4.

[0242] In some embodiments, this document describes a multispecific (e.g., bispecific) antibody comprising a binding domain that binds to NKG2A, said binding domain comprising the VH and VL CDRs disclosed herein, such as those set forth in Tables 1, 2, 3, or 4.

[0243] In some embodiments, the NKG2A binder is a bispecific antibody comprising a first binding domain and a second binding domain. In other embodiments, the first binding domain binds to a first complex comprising extracellular domains of NKG2A and CD94, but not to a second complex comprising extracellular domains of NKG2C and CD94. In other embodiments, the first binding domain comprises six CDRs of an antibody designated A3. In some embodiments, the first binding domain comprises six CDRs as listed in a column of Table 1. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 25 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 26. In some embodiments, the first binding domain comprises the heavy chain variable region as illustrated in SEQ ID NO: 25 and the light chain variable region as illustrated in SEQ ID NO: 26. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to either the first or second complex.

[0244] In some embodiments, the NKG2A binder is a bispecific antibody comprising a first binding domain and a second binding domain. In other embodiments, the first binding domain binds to a first complex comprising extracellular domains of NKG2A and CD94, but not to a second complex comprising extracellular domains of NKG2C and CD94. In other embodiments, the first binding domain comprises six CDRs of an antibody designated A2. In some embodiments, the first binding domain comprises six CDRs as listed in a column of Table 2. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 45 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 46. In some embodiments, the first binding domain comprises the heavy chain variable region as illustrated in SEQ ID NO: 45 and the light chain variable region as illustrated in SEQ ID NO: 46. In some embodiments, the second binding domain binds to different epitopes of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to either the first or second complex.

[0245] In some embodiments, the NKG2A binder is a bispecific antibody comprising a first binding domain and a second binding domain. In other embodiments, the first binding domain binds to a first complex comprising extracellular domains of NKG2A and CD94, but not to a second complex comprising extracellular domains of NKG2C and CD94. In other embodiments, the first binding domain comprises six CDRs of an antibody designated A42. In some embodiments, the first binding domain comprises six CDRs as listed in a column of Table 3. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 64 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 65. In some embodiments, the first binding domain comprises the heavy chain variable region as illustrated in SEQ ID NO: 64 and the light chain variable region as illustrated in SEQ ID NO: 65. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to either the first or second complex.

[0246] In some embodiments, the NKG2A binder is a bispecific antibody comprising a first binding domain and a second binding domain. In other embodiments, the first binding domain binds to a first complex comprising extracellular domains of NKG2A and CD94, but not to a second complex comprising extracellular domains of NKG2C and CD94. In other embodiments, the first binding domain comprises six CDRs of an antibody designated AA11. In some embodiments, the first binding domain comprises six CDRs as listed in a column of Table 4. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region as illustrated in SEQ ID NO: 64 and three CDRs of the light chain variable region as illustrated in SEQ ID NO: 73. In some embodiments, the first binding domain comprises the heavy chain variable region as illustrated in SEQ ID NO: 64 and the light chain variable region as illustrated in SEQ ID NO: 73. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to either the first or second complex.

[0247] On the other hand, the antibodies or antigen-binding fragments provided in this article can be part of engineered cell surface receptors (such as chimeric antigen receptors (CARs)). Typically, CARs contain extracellular domains, transmembrane domains, and intracellular signal transduction domains.

[0248] In some embodiments, this document provides a CAR comprising an extracellular domain including one or more antibodies or fragments thereof provided herein. In some embodiments, the extracellular domain of the CAR provided herein comprises the VH and VL CDRs disclosed herein, such as those set forth in Tables 1, 2, 3, or 4.

[0249] The CAR disclosed herein comprises a transmembrane domain that can be directly or indirectly fused with an extracellular antigen-binding domain. The transmembrane domain may be of natural or synthetic origin. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable in the cell membrane, preferably the eukaryotic cell membrane. Transmembrane domains compatible with the CAR described herein may be obtained from naturally occurring proteins. Alternatively, it may be a synthetic, non-naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment in the cell membrane. Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain may form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of spanning the phospholipid bilayer of the cell.

[0250] The CAR disclosed herein comprises an intracellular signal transduction domain. This intracellular signal transduction domain is responsible for the activation of at least one normal effector function of immune effector cells expressing the CAR. The term "effector function" refers to a specific function of a cell. Effector functions of T cells can be, for example, cytolytic activity or helper activities, including cytokine secretion. Therefore, the term "cytoplasmic signal transduction domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specific function. While the entire cytoplasmic signal transduction domain can generally be used, in many cases, the entire strand is not necessary. Regarding the use of truncated portions of cytoplasmic signal transduction domains, such truncated portions can be used in place of the complete strand, provided that they transduce effector function signals. Therefore, the term cytoplasmic signal transduction domain means including any truncated portion of the cytoplasmic signal transduction domain sufficient to transduce effector function signals.

[0251] In some embodiments, the intracellular signaling domain comprises the major intracellular signaling domain of immune effector cells. In some embodiments, the CAR comprises an intracellular signaling domain that is substantially composed of the major intracellular signaling domain of immune effector cells. "Major intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function.

[0252] In addition to stimulating antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR includes at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as effector functions.

[0253] The CAR disclosed herein may comprise a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. A hinge domain is an amino acid segment that is typically present between two domains of a protein and allows for the flexibility of the protein and the movement of one or both of said domains relative to each other. Any amino acid sequence that provides such flexibility of the effector molecule and the movement of the extracellular antigen-binding domain relative to the transmembrane domain may be used.

[0254] The CAR disclosed herein may contain a signal peptide (also called a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that enables the polypeptide to target a desired site in the cell.

[0255] Other engineered transmembrane receptors that include the antibodies or fragments provided herein are also included in this disclosure.

[0256] 5.3. Nucleic Acids, Vectors, and Cells Additionally, a nucleic acid encoding an NKG2A binder (e.g., an antibody or antibody fragment) or a fusion polypeptide as disclosed herein is provided, along with a complementary nucleic acid; a vector comprising the nucleic acid as disclosed herein; and a cell comprising one or more of the following: an NKG2A binder as disclosed herein, a nucleic acid as disclosed herein, or a vector as disclosed herein. In some embodiments, the cell expresses an NKG2A binder. In some embodiments, the cell replicates the nucleic acid or the vector. In some embodiments, materials are provided for generating an NKG2A binder (e.g., a human NKG2A binder) and fragments thereof. For example, isolated cells may generate an NKG2A binder (e.g., an antibody or antibody fragment). In this regard, cells (e.g., isolated cells) may generate antibodies comprising VH and VL as disclosed herein, or fragments thereof. In some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding an NKG2A binder (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In each respect, the isolated polynucleotide contains nucleotide sequences encoding VH and / or VL, wherein VH and VL contain the same complementarity-determining regions (CDRs) as disclosed herein.

[0257] As used herein, the term "complementary" refers to the specific binding between polynucleotides based on the sequence of the polynucleotide. 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, for example, if they produce a given or detectable level of signal in the hybridization assay. The portions of a polynucleotide are complementary to each other when they follow conventional base pairing rules (e.g., A pairs with T (or U) and G pairs with C), but small regions (e.g., fewer than about 3 bases) of mismatched, inserted, or deleted sequences may exist. The term "stringent assay conditions" refers to conditions that are compatible with generating binding pairs of nucleic acids (e.g., probes and target mRNAs) with sufficient complementarity to provide the desired level of specificity in the assay, while generally incompatible with forming binding pairs between binding members with insufficient complementarity to provide the desired specificity. The term "stringent assay conditions" generally refers to a combination of hybridization and washing conditions.

[0258] In some implementations, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expression in a suitable host cell. Using recombinant techniques, such vectors can be used, for example, to amplify polynucleotides in a host cell to produce a suitable amount, and can be used to express binding agents, such as antibodies or antibody fragments.

[0259] In some embodiments, one or more vectors are expression vectors in which one or more polynucleotides are operatively linked to one or more polynucleotides containing expression control sequences. This is particularly covered by autonomously replicating recombinant expression constructs, such as plasmids and viral DNA vectors incorporating one or more polynucleotides encoding an antibody sequence binding NKG2A. The expression control DNA sequence includes a promoter, an enhancer, and an operator, and is typically selected based on the expression system in which the expression construct will be used. Promoter and enhancer sequences are typically selected based on their ability to enhance gene expression, while operator sequences are typically selected based on their ability to regulate gene expression. The expression construct may also include sequences encoding one or more optional markers that allow for the identification of host cells carrying the construct. The expression construct may also include sequences that facilitate and preferably promote homologous recombination in host cells. In some embodiments, the expression construct may also include sequences necessary for replication in host cells.

[0260] Exemplary expression control sequences include promoter / enhancer sequences, such as cytomegalovirus promoter / enhancer sequences (Lehner et al.). J. Clin. Microbiol ., 29: 2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther ., 4: 151, 1993); Tie promoter (Korhonen et al., Blood , 86(5): 1828-1835, 1995); simian virus 40 promoter; DRA (downregulated in adenomas; Allefai et al., Am. J. Physiol. Gastrointest. Liver Physiol ., 293: G923-G934, 2007); MCT1 (monocarboxylic acid transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liver Physiol ., G977-G979. 2005); and Math1 (mouse atonymous homolog 1; Shroyer et al., Gastroenterology (132: 2477-2478, 2007), for expression in mammalian cells, wherein the promoter is operatively 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 a suitable polyadenylated sequence (e.g., SV40 or the human growth hormone gene polyadenylated sequence) operatively linked downstream (e.g., 3') of the polypeptide coding sequence.

[0261] If necessary, the one or more polynucleotides may also optionally include a nucleotide sequence encoding a secretion signal peptide, which is fused to the polypeptide sequence within the framework. The secretion signal peptide guides the secretion of the antibody polypeptide by cells expressing the one or more polynucleotides and is cleaved from the secreted polypeptide by the cells. The one or more polynucleotides may further optionally include sequences whose intended function is to facilitate the large-scale production of the vector. Polynucleotides can be manufactured and administered for gene therapy using procedures described in the literature for various transgenes. See, for example, Isner et al. Circulation , 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy , 7: 989-1011, 1996.

[0262] In some embodiments, the polynucleotide may also contain additional sequences to facilitate uptake by the host cell and expression of the antibody or a fragment thereof (and / or any other peptide). In some embodiments, a “naked” transgene (e.g., a transgene without a virus, liposome, or other vector that promotes transfection) encoding the antibody or a fragment thereof described herein is employed.

[0263] The polynucleotides disclosed herein may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be coding or non-coding (antense) strands. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0264] This disclosure also relates to variants of the polynucleotides described herein, wherein said variants encode fragments, analogs, and / or derivatives of, for example, the binding molecules of this disclosure. In some embodiments, this disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to the polynucleotide encoding the binding molecule of this disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% identical to a reference nucleotide sequence" is intended to mean that, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence, the nucleotide sequence of the polynucleotide is identical to the reference sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted by another nucleotide, or up to 5% of all the nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' end positions of the reference nucleotide sequence or at any position between those end positions, said positions being individually distributed in the nucleotides of the reference sequence or in one or more consecutive groups within the reference sequence.

[0265] The polynucleotide variant may contain variations in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variant contains variations that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In some embodiments, the polynucleotide variant contains silent substitutions that do not cause changes in the amino acid sequence of the polypeptide (due to degeneracy of the genetic code). Polynucleotide variants can be generated for various reasons, such as optimizing codon expression in a specific host (i.e., changing codons in human mRNA to such...). Escherichia coli (Those preferred by the bacterial host). In some implementations, the polynucleotide variant contains at least one silent mutation in the non-coding or coding region of the sequence.

[0266] In some embodiments, polynucleotide variants are generated to modulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to enhance the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to reduce the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant enhances the expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant reduces the expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0267] Any suitable vector can be used to introduce one or more polynucleotides encoding antibodies or fragments thereof into the host. Exemplary vectors described include replication-defective retroviral vectors, including but not limited to lentiviral vectors (Kim et al., J. Virol. , 72(1): 811-816, 1998; Kingsman and Johnson, Scrip Magazine (October 1998, pp. 43-46); parvovirus vectors, such as adeno-associated virus (AAV) vectors (US Patent Nos. 5,474,935l; 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. Invest. Med ., 45: 87-98, 1997); adenovirus (AV) vector (US 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; StratfordPerricaudet et al., J. Clin. Invest ., 90: 626-630, 1992; and Rosenfeld et al., Cell ,68: 143-155, 1992); adenovirus-adeno-associated virus chimeras (US Patent No. 5,856,152) or vaccinia virus or herpesvirus vectors (US Patent Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688); Lipofectin-mediated gene transfer (BRL); liposome vectors (US Patent No. 5,631,237); and combinations thereof. Any of these expression vectors can be prepared using standard recombinant DNA techniques as described below: for example, Sambrook et al., Molecular Cloning, a Laboratory Manual2nd 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, viral vectors may exhibit replication defects by, for example, deleting or interrupting selected genes required for viral replication.

[0268] Other non-viral delivery mechanisms covered 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-glucan (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), direct microinjection (Harland and Weintraub, 81: 7161-7165, 1984), J. Cell Biol. , 101: 1094-1099, 1985), DNA-loaded liposomes (Nicolau and Sene, 101: 1094-1099, 1985), 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), Cellular acoustic treatment (Fechheimer et al., Proc. Natl. Acad. Sci. USA , 84: 8463-8467, 1987), gene bombardment using high-speed micro-projectiles (Yang et al., 84: 8463-8467, 1987), Proc. Natl. Acad. Sci 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).

[0269] Vectors (or antibodies or fragments thereof, or nucleic acids as discussed herein) can be embedded in liposomes. See, for example, Ghosh and Bachhawat. Liver diseases, targeted diagnosis and therapy using specific receptors and ligands Wu G, Wu C (eds.), New York: Marcel Dekker, pp. 87-104 (1991); Radler et al. Science , 275(5301): 810-814, 1997). It also covers various commercial methods involving "liposome transfection" technology. In some embodiments, liposomes can be complexed with hemagglutinating viruses (HVJ). This has been shown to promote fusion with the cell membrane and facilitate cell entry of liposome-encapsulated DNA (Kaneda et al., 275(5301): 810-814, 1997). Science , 243: 375-378, 1989). In some implementations, liposomes are used in combination with or in combination with nuclear non-histone chromosomal protein (HMG-1) (Kato et al., 243: 375-378, 1989). J. Biol. Chem (Reference: 266: 3361-3364, 1991). In some embodiments, liposomes are used in combination with or in combination with both HVJ and HMG-1. Such expression constructs have been successfully used for the in vitro and in vivo transfer and expression of nucleic acids. In some embodiments, NKG2A binders (e.g., antibodies), including human NKG2A binders, are included in the liposomes to target the liposomes to cells (such as immune cells) that express NKG2A on their cell surface.

[0270] Cells may contain one or more polynucleotides or one or more vectors. For example, cells may be transformed or transfected with one or more polynucleotides encoding NKG2A binders (e.g., antibodies), including human NKG2A binders, or with one or more vectors containing said polynucleotides. In some embodiments, cells express NKG2A binders (e.g., antibodies), including human NKG2A binders, and contain one or more (including six) CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDRs of A2, A3, A11, and / or A42 (see, for example, Tables 1, 2, 3, and / or 4). In some embodiments, cells express NKG2A binders (e.g., antibodies), including human NKG2A binders, and contain VH and VL, said VH and said VL containing the same CDRs as those of A2, A3, A11, and / or A42 (see, for example, Tables 1, 2, 3, and / or 4). The cells can be prokaryotic cells, such as Escherichia coli (E. coli). Escherichia coli ) cells (see, for example, Pluckthun et al., Methods Enzymol ., 178: 497-515, 1989); or eukaryotic cells, such as animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells); yeast (e.g., Saccharomyces cerevisiae ( Saccharomyces cerevisiae (e.g., insect cells or plant cells, such as tobacco, corn, soybean, or rice cells). Using mammalian host cells can provide translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be necessary to confer optimal biological activity to the recombinant expression product. Similarly, peptides (e.g., NKG2A binders, such as antibodies, including human NKG2A binders) can be glycosylated or deglycosylated and / or covalently modified to include one or more water-soluble polymer linkers, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0271] Methods for introducing DNA or RNA into host cells are well-known and include transformation, transfection, electroporation, nuclear injection, or fusion with vectors such as liposomes, micelles, ghost cells, and protoplasts. Such host cells can be used to amplify polynucleotides and also to express polypeptides encoded by polynucleotides. In this regard, methods for generating NKG2A binders (e.g., antibodies) may include culturing host cells and isolating NKG2A binders. Transferring naked DNA expression constructs into cells can be achieved using particle bombardment, which depends on the ability to accelerate DNA-coated microspheres to high speeds, allowing them to penetrate the cell membrane and enter the cell without killing the cell (Klein et al.). Nature(Yang et al., 327: 70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high-voltage discharge to generate an electric current, which in turn provides the driving force (Yang et al., 327: 70-73, 1987). Proc. Natl. Acad. Sci USA (87: 9568-9572, 1990). The microspheres used are composed of biologically inert materials such as tungsten or gold beads. Host cells can be isolated and / or purified. Host cells can also be cells transformed in vivo to induce transient or permanent expression of peptides in vivo. Host cells can also be isolated cells transformed in vitro and introduced after transformation, for example, to produce peptides in vivo for therapeutic purposes. The definition of host cells explicitly excludes transgenic humans.

[0272] 5.4. Preparation method Antibodies binding to NKG2A can be obtained by any suitable method, such as (but not limited to) immunization with whole cells containing NKG2A and collection of antibodies, recombinant techniques, or screening libraries of antibodies or antibody fragments using NKG2A extracellular domain epitopes. Monoclonal antibodies can be generated using a variety of known techniques (see, for example, 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 ”, DNA Cloning 2: Expression Systems , 2nd edition, Glover et al. (eds.), page 93 (Oxford University Press 1995). An exemplary technique for generating monoclonal antibodies includes immunizing an animal with the human NKG2A antigen and generating a hybridoma from spleen cells taken from the animal. The hybridoma can produce a monoclonal antibody or antibody fragment that binds to NKG2A.

[0273] In another embodiment, monoclonal antibodies or antibody fragments can be isolated from an antibody phage library generated using the techniques described below: for example, Antibody Phage Display: Methods and Protocols, PM O'Brien and R. Aitken (eds.), Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against desired antigens. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed onto the antigen and thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched through additional antigen adsorption / elution cycles.

[0274] Variable domains can be functionally displayed on phages in the form of single-chain Fv (scFv) fragments (where VH and VL are covalently linked by a short, flexible peptide) or Fab fragments (where they are each fused to a constant domain and interact nonvalently), as exemplified by Winter et al. Ann. Rev. Immunol As described in ., 12: 433-455 (1994).

[0275] Lineages of the VH and VL genes can be cloned individually via polymerase chain reaction (PCR) and randomly recombined in a phage library, from which antigen-binding clones can then be searched, as described above by Winter et al. Libraries derived from immune sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, native lineages can be cloned to provide a single source of human antibodies against a broad range of non-autoantigens and autoantigens without any immunization, as described by Griffiths et al. EMBO J As described in , 12: 725-734 (1993). Finally, the native library can also be prepared synthetically by cloning the unrearranged V gene segment from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and achieve in vitro rearrangement, as exemplified by Hoogenboom and Winter, . J. Mol. Biol As described in ., 227:381-388 (1992).

[0276] Library screening can be achieved using various techniques known in the art. For example, NKG2A (e.g., NKG2A peptides, fragments, or epitopes) can be used to coat the pores of an adsorption disc, expressed on host cells attached to the adsorption disc, used for cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning and displaying libraries. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be achieved using prolonged washing and monovalent phage display (e.g., Bass et al.). Proteins(as described in , 8: 309-314 (1990) and WO 92 / 09690) and the low coating density of the antigen (as described by Marks et al., , 8: 309-314 (1990) and WO 92 / 09690), and the low coating density of the antigen (as described by Marks et al., , 8: 309-314 (1990) and WO Biotechnol As described in ., 10: 779-783 (1992) to facilitate.

[0277] NKG2A binders (e.g., antibodies) can be obtained by designing a suitable antigen screening procedure to select the phage clone of interest, followed by the use of VH and / or VL sequences (e.g., Fv sequences) from the phage clone of interest, or various CDR sequences from VH and VL sequences, along with a suitable constant region (e.g., Fc) sequence (described in Kabat et al.). Sequences of Proteins of Immunological Interest (5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3) to construct full-length NKG2A binding agent (e.g., antibody) clones.

[0278] Similarly, human antibodies conjugated to NKG2A can be generated by any of a variety of techniques, including but not limited to Epstein-Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization with human B cells, fusion of spleen cells from transgenic mice immunized with an inserted human immunoglobulin gene, isolation from a phage library of human immunoglobulin V region, or other procedures known in the art and based on the disclosure herein. Methods for obtaining human antibodies from transgenic animals are further described in the following literature: e.g., Bruggemann et al., Curr. Opin. Biotechnol ., 8: 455 58, 1997; Jakobovits et al., Ann. N. Y. 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. Patent No. 5,877,397.

[0279] For example, human antibodies binding to NKG2A can be obtained from transgenic animals engineered to produce specific human antibodies in response to antigen challenge. For instance, WO 98 / 24893 discloses transgenic animals possessing human Ig loci, wherein, due to the inactivation of endogenous heavy and light chain loci, the animals do not produce functional endogenous immunoglobulins. Transgenic non-primate mammalian hosts capable of generating an immune response to immunogens are also described, wherein the antibodies have primate constant regions and / or variable regions, and wherein the endogenous immunoglobulin encoding the locus is substituted or inactivated. WO 96 / 30498 discloses the use of the Cre / Lox system to modify immunoglobulin loci in mammals, such as by replacing all or part of the constant or variable regions, thereby forming modified antibody molecules. WO 94 / 02602 discloses non-human mammalian hosts possessing inactivated endogenous Ig loci and functional human Ig loci. U.S. Patent No. 5,939,598 discloses a method for preparing transgenic mice, wherein the mice lack endogenous heavy chains and express exogenous immunoglobulin loci containing one or more xenogeneic constant regions. Using transgenic animals, such as those described herein, an immune response to selected antigen molecules can be generated, and antibody-producing cells can be extracted from the animals and used to generate hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, etc., are known in the art and are used to immunize, for example, the transgenic mice described in WO 96 / 33735. The ability of monoclonal antibodies to inhibit or neutralize the biological activity or physiological effects of the corresponding proteins can be tested.

[0280] In some embodiments, the NKG2A binders described herein comprise a non-antibody protein backbone. Non-limiting examples of such non-antibody protein backbones include fibronectin backbones, anticarrier proteins, adnectin, affinity molecules, DARPin, fynomer, affitin, affilin, avimer, cysteine-rich knotting peptides, or engineered Kunitz-type inhibitors. Methods for generating such non-antibody protein backbones are well known in the art, and any of these can be used to generate NKG2A binders comprising a non-antibody protein backbone (see, for example, 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)).

[0281] Various methods for generating antibodies from polynucleotides are generally well-known. For example, the following literature describes basic molecular biology procedures: Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd edition, Cold Spring Harbor Laboratory, New York, 2001). In addition, many publications describe techniques applicable to the preparation of antibodies by manipulating DNA, generating expression vectors, and transforming and culturing appropriate cells (see, for example, Mountain and Adair, Biotechnology and Genetic Engineering Reviews Chapter 1, Tombs (ed.), *Intercept*, Andover, UK, 1992; and Current Protocols in Molecular Biology , edited by Ausubel, Wiley Interscience, New York, 1999).

[0282] NKG2A binders (e.g., antibodies), including human NKG2A binders, can be prepared using any suitable method, such as isolation from immunized animals, recombinant or synthetic production, or genetic engineering (including as described above). Antibody fragments derived from antibodies are obtained through, for example, proteolysis of the antibody. For instance, papain or pepsin digestion of a whole antibody produces a 5S fragment called F(ab')2 or two monovalent Fab fragments and one Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described in the following literature: e.g., 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 Andrews, SM and Titus, JA Current Protocols in Immunology (Coligan et al., eds.), John Wiley & Sons, New York (2003), pp. 2.8.1-2.8.10 and 2.10A.1-2.10A.5.

[0283] NKG2A binders, including human NKG2A binders (e.g., antibodies), can be genetically engineered. For example, NKG2A binders, including human NKG2A binders (e.g., antibodies), contain variable region domains, for example, generated by recombinant DNA engineering techniques. In this regard, the variable region can optionally be modified by inserting, deleting, or altering the amino acid sequence of the antibody to generate the antibody of interest, as described above. For example, the variable region can be synthesized using polymerase chain reaction (PCR) to prepare a polynucleotide encoding the CDR of interest by using mRNA from antibody-producing cells as a template (see, for example, Courtenay Luck, “Genetic Manipulation of Monoclonal Antibodies”). Monoclonal Antibodies: Production, Engineering and Clinical Application Ritter et al. (eds.), p. 166 (Cambridge University Press, 1995); Ward et al., “Genetic Manipulation and Expression of Antibodies”. Monoclonal Antibodies: Principles and Applications Birch et al. (eds.), p. 137 (Wiley Liss, Inc. 1995); and Larrick et al. Methods: A Companion to Methods in Enzymology (2: 106-110, 1991). Current antibody manipulation techniques allow the construction of engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a primary antibody and the remainder from a secondary antibody. Such techniques are used, for example, to humanize antibodies or improve their affinity for binding targets. Exemplary methods for generating humanized antibodies are also described in the preceding sections.

[0284] 5.5. Pharmaceutical Compositions On one hand, this disclosure also provides a composition, such as a pharmaceutical composition, comprising at least one of the following: a conjugate provided herein (e.g., an antibody or antigen-binding fragment thereof disclosed herein), a nucleic acid provided herein, a carrier provided herein, or a cell provided herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an NKG2A conjugate provided herein (e.g., an antibody or antigen-binding fragment thereof disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid provided herein (such as a nucleic acid encoding an antibody or antigen-binding fragment thereof disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a carrier provided herein (such as a carrier comprising a nucleic acid as disclosed herein and expressing an NKG2A conjugate as disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a cell provided herein (such as a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof disclosed herein and / or expressing an antibody or antigen-binding fragment thereof disclosed herein) and a pharmaceutically acceptable excipient.

[0285] In some embodiments, the pharmaceutical compositions provided herein are prepared for storage by mixing the binders, nucleic acids, carriers or cells provided herein with the desired purity with optional physiologically acceptable excipients (see, for example, Remington, Remington's Pharmaceutical Sciences (18th edition, 1980)) in an aqueous solution or lyophilized or other dried form.

[0286] The binders, nucleic acids, vectors, or cells disclosed herein can be formulated in any suitable form for delivery to target cells / tissues, such as in microcapsule or macroemulsion form (Remington, Same as above (Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), in sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57) or in liposome form (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0287] The binders, nucleic acids, carriers, or cells described herein can also be embedded in microcapsules, such as microcapsules prepared by coagulation techniques or interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or embedded in macroemulsions. Such techniques are disclosed, for example, by Remington, ibid.

[0288] Various compositions and delivery systems are known and can be used with conjugates, nucleic acids, vectors, or cells as described herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or their antigen-binding fragments, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), constructing nucleic acids as part of retroviruses or other vectors, etc. In another embodiment, the composition may be provided in the form of a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled release or sustained release (see, for example, Langer, ibid.; 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 may be used to achieve controlled or sustained release of the preventative or therapeutic agents (e.g., antibodies or their antigen-binding fragments as described herein) or compositions provided herein (see, for example, 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., 1989, J. Neurosurg.). 71:105-12; US Patent 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 used 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), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolic acid) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of filterable impurities, stable in storage, sterile, and biodegradable.

[0289] In another embodiment, a controlled-release or sustained-release system can be placed close to a specific target tissue (e.g., the nasal passage or lungs), thus requiring only a portion of the systemic dose (see, for example, 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, for example, U.S. Patent Nos. 4,526,938, WO91 / 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).

[0290] 5.6. Instructions for Use On the other hand, this document provides methods for using the binders or compositions provided herein. On the other hand, this document provides binders or compositions as disclosed herein for use as pharmaceutical agents. On the other hand, this document provides binders or compositions as disclosed herein for treating diseases or ailments, such as those disclosed herein. On the other hand, this document provides binders or compositions as disclosed herein for use in methods as disclosed herein.

[0291] In some embodiments, the binder binds to NKG2A. Alternatively, the binder binds to a complex comprising NKG2A and CD94. In other embodiments, the binder binds to a complex comprising the extracellular domains of NKG2A and CD94. In various embodiments, NKG2A is human NKG2A. In other embodiments, NKG2A also refers to cynomolgus monkey NKG2A. In other embodiments, the binder does not bind to cynomolgus monkey NKG2A or a complex comprising cynomolgus monkey NKG2A. Alternatively, the binder does not bind to a complex comprising NKG2C and CD94, or a complex comprising the extracellular domains of NKG2C and CD94. In some embodiments, the binder binds to a first complex comprising NKG2A and CD94, but not to a second complex comprising NKG2C and CD94.

[0292] In some embodiments, this document provides a method 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), said 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, this document provides the use of the 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), said use comprising contacting NKG2A (e.g., a first cell expressing NKG2A) with a binding agent (e.g., an antibody or fragment thereof) provided herein.

[0293] On the other hand, this document provides a method for inhibiting the interaction between a complex containing NKG2A and CD94 or their respective extracellular domains (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 the complex containing NKG2A and CD94 (e.g., a first cell expressing NKG2A and CD94) with a binding agent (e.g., an antibody or a fragment thereof) provided herein. In some embodiments, this document provides the use of the binding agent provided herein for inhibiting the interaction between a complex containing NKG2A and CD94 or their respective extracellular domains (e.g., expressed on and / or in a first cell) and HLA-E (e.g., expressed on and / or in a second cell), said use comprising contacting the complex containing NKG2A and CD94 (e.g., a first cell expressing NKG2A and CD94) with a binding agent (e.g., an antibody or a fragment thereof) provided herein.

[0294] In some embodiments, the methods or uses disclosed herein do not inhibit the interaction between NKG2C (or a complex comprising NKG2C and CD94 or their respective extracellular domains) and HLA-E. In other embodiments, the methods or uses disclosed herein inhibit the interaction between NKG2C (or a complex comprising NKG2C and CD94 or their respective extracellular domains) and HLA-E, but said inhibition is significantly less than the inhibition of a benchmark anti-NKG2A antibody (e.g., at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%).

[0295] As used herein, the term "inhibit" means reduction or decrease. For example, in some embodiments, the binders provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by 10%-99%. In other embodiments, the binders provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by 100% (i.e., complete elimination of the interaction, as measured by assay). In some embodiments, the binders provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 10%. In some embodiments, the binders provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 20%. In some embodiments, the binding agent provided herein inhibits the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 30%. In some embodiments, the binding agent provided herein inhibits the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 40%. In some embodiments, the binding agent provided herein inhibits the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 50%. In some embodiments, the binding agent provided herein inhibits the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 60%. In some embodiments, the binding agent provided herein inhibits the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 70%. In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 80%. In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by at least 90%. In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by about 10% to 90%.In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by approximately 20%–80%. In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by approximately 30%–70%. In some embodiments, the binding agents provided herein inhibit the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E by approximately 40%–60%. In some embodiments, NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and HLA-E are expressed on different cells. In some embodiments, NKG2A is expressed on a first cell (such as 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 implementations, the T cells are cytotoxic T cells, such as CD8+ T cells.

[0296] Immune cells are cells in the immune system and can be lymphocytes. Non-restricted examples of lymphocytes 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 the T cell receptor (TCR). T cells play a crucial 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 certain cells, such as cancer cells, cells infected with intracellular pathogens (such as viruses or bacteria), or cells damaged in other ways. Most cytotoxic T cells express the T cell receptor (TCR) that recognizes specific antigens. CD8+ T cells are a subset of class I MHC-restricted T cells and are mediators of adaptive immunity, essential for killing cancerous or virus-infected cells. NK cells are a type of cytotoxic lymphocyte that is crucial for the innate immune system and belong to the innate lymphoid cell (ILC) family. In some implementations, NK cells can be identified by the presence or absence of CD56 and CD3 (CD56+, CD3-). NK cells can recognize and kill stress cells in the absence of antibodies and MHC, thereby achieving a faster immune response.

[0297] In some implementations, HLA-E is expressed on second cells (such as cancer cells).

[0298] In some embodiments, the methods disclosed herein are in vitro or ex vivo methods. In other embodiments, the methods disclosed herein are in vivo methods. In some embodiments, the uses disclosed herein are in vitro or ex vivo uses. In other embodiments, the uses disclosed herein are in vivo uses. In some embodiments, the in vivo methods or in vivo uses disclosed herein involve administering the binder disclosed herein to a subject having cells expressing HLA-E, such as tumor cells expressing HLA-E.

[0299] In other embodiments, this document provides a method for preventing or inhibiting the suppression of immune cells (e.g., inhibition mediated by the interaction between NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) expressed on immune cells and HLA-E (e.g., expressed on cancer cells). In other embodiments, this document provides a method for activating immune cell-mediated responses (e.g., anti-tumor responses). In some embodiments, the immune cell suppression is tumor / cancer-associated immune cell suppression, such as in the tumor microenvironment. In some embodiments, the method includes contacting immune cells with a binder (e.g., an antibody or a fragment thereof) provided herein. In some embodiments, this document provides the use of the binder provided herein for preventing the suppression of immune cells or activating immune cell-mediated responses. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are cytotoxic T cells, such as CD8+. + T cells. In some implementations, immune cells express NKG2A.

[0300] In some embodiments, the NKG2A binders (e.g., antibodies) described herein can be used in compositions and methods for treating diseases or conditions. Therefore, in some embodiments, this document provides a method for treating a subject with a disease or condition, the method comprising administering to the subject the binder or pharmaceutical composition provided herein. In other embodiments, this document provides the use of the binder or pharmaceutical composition provided herein for treating a subject with a disease or condition. In other embodiments, this document provides the binder or pharmaceutical composition provided herein for manufacturing an agent for treating a disease or condition.

[0301] In some implementations, the treatments provided herein include relieving one or more symptoms associated with a disease or condition (e.g., cancer or tumor, or an autoimmune or inflammatory disease or condition).

[0302] Therefore, in some embodiments, this document provides a method for relieving one or more symptoms associated with cancer or tumor in a subject, the method comprising administering to the subject an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, this document provides an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for relieving one or more symptoms associated with cancer or tumor in a subject. In some embodiments, this document provides the use of an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for manufacturing an agent for relieving one or more symptoms associated with cancer or tumor in a subject.

[0303] In some embodiments, the treatments provided herein include reducing the size of a tumor in a subject with a tumor. Therefore, in some embodiments, a method for reducing the size of a tumor in a subject with a tumor is described herein, the method comprising administering to the subject an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein is provided for reducing the size of a tumor in a subject with a tumor. In some embodiments, the use of an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein is provided for manufacturing an agent for reducing the size of a tumor in a subject with a tumor.

[0304] In some embodiments, this document describes a method for enhancing tumor cell removal in a subject with a tumor, the method comprising administering to the subject an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, this document provides an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for enhancing tumor cell removal in a subject with a tumor. In some embodiments, this document provides use of an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for manufacturing an agent for enhancing tumor cell removal in a subject with a tumor.

[0305] "Enhanced" tumor cell removal includes, but does not require, a 100% enhancement of removal. This encompasses any enhancement of the removal rate. Similarly, "modulation" of tumor growth refers to reducing tumor size, slowing tumor growth, or inhibiting the increase of existing tumor size. This includes, but does not require, complete elimination of the tumor; any reduction in tumor size or slowing of tumor growth constitutes a beneficial biological effect on the subject. In this regard, tumor cell removal may be enhanced, for example, by at least about 5%, at least about 10%, or at least about 20%, compared to the removal levels observed in the absence of the method (e.g., in biologically matched control subjects or samples not exposed to the agents of the method). This effect is detected by, for example, a reduction in tumor size, a reduction or maintenance of tumor marker levels, or a reduction or maintenance of the tumor cell population. In some embodiments, tumor cell removal is enhanced by, for example, 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 tumor cell removal in the absence of the NKG2A binder (e.g., antibody) or pharmaceutical composition of the method.

[0306] A method for modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject is also provided. For example, the method includes administering to the subject a composition comprising an NKG2A binder (e.g., an antibody) or another composition as disclosed herein, in an amount that effectively modulates tumor growth in the subject.

[0307] In some embodiments, the disease or condition is cancer or a tumor. In other embodiments, the cancer or tumor expresses HLA-E. In some embodiments, the subject is a human subject.

[0308] As used herein, “tumor” refers to any proliferative cell growth or proliferation (whether malignant or benign), and to all precancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to: breast cancer, colon cancer, kidney cancer, lung cancer, squamous cell myeloid leukemia, hemangioma, melanoma, astrocytoma, and glioblastoma, as well as other proliferative cell disease states, including but not limited to: heart: sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma; lung: bronchial carcinoma (squamous cell bronchial carcinoma, undifferentiated small cell bronchial carcinoma, undifferentiated large cell bronchial carcinoma, etc.). Bronchiectasis, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid, vasoactive intestinal peptide tumor (vipoma)), small intestine (adenocarcinoma, lymphoma, carcinoid, Kaposi's sarcoma). Sarcoma, leiomyomas, hemangiomas, lipomas, neurofibromas, fibromas; colon (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyomas); genitourinary tract: kidneys (adenocarcinoma, Wilms' tumor, lymphoma, leukemia, renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma, small cell prostate carcinoma), testes (seminomas, teratomas, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma); liver: liver tumors (hepatocellular carcinoma), bile duct carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteoblastic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma. Sarcoma), malignant lymphoma (reticular cell sarcoma), malignant giant cell tumor chordoma, osteochondroma (osteochondroma), benign chondroma, chondroblastoma, chondromycinous fibroma, osteoid osteoma and giant cell tumor; nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, spinal sarcoma, glioma), cerebrum (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma);Gynecology: Uterus (endometrial cancer), Cervix (cervical cancer, precancerous cervical dysplasia), Ovary (ovarian cancer) serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), Vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), Vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), Fallopian tube (cancer); Hematology: Hematology (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodyplasia syndrome), Hodgkin's disease Diseases including: non-Hodgkin's lymphoma (malignant lymphoma); skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus of dysplasia, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal glands: neuroblastoma; and thyroid cancer, including medullary thyroid carcinoma.

[0309] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor or cancer is not a solid tumor. In other embodiments, the cancer is leukemia 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 has reached remission but may relapse. In some embodiments, the tumor or cancer is unresectable. Or, additionally, the tumor or cancer is resistant to chemotherapy or other anticancer therapies. In other embodiments, the cancer or tumor expresses HLA-E.

[0310] Additionally, NKG2A binders (e.g., antibodies) can be used to alleviate or reduce cancer-related side effects such as bone degeneration, vertebral collapse, and paralysis. In one scenario, the subject has bone metastases or is at risk of developing bone metastases and is given an NKG2A binder (e.g., antibody) in an amount that reduces surrounding bone degeneration. Thus, in some respects, NKG2A binder prevention is attributed to bone degeneration of bone metastases, where tumor cell proliferation is reduced or not reduced. In other respects, NKG2A binder prevention is attributed to bone degeneration of bone metastases and reduces tumor cell proliferation. Generally, the effect on tumor cell proliferation (e.g., inhibition of proliferation or no effect on proliferation) depends on the specific metastatic microenvironment. For example, metastatic proliferation in microenvironments with abundant type I collagen may be inhibited. In contrast, metastatic proliferation in microenvironments lacking abundant type I collagen may not be inhibited, but bone degeneration near the metastasis may be reduced or prevented.

[0311] In other embodiments, this document provides a method for alleviating one or more symptoms associated with an autoimmune or inflammatory disease or condition in a subject, the method comprising administering to the subject an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, this document provides an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for alleviating one or more symptoms associated with an autoimmune or inflammatory disease or condition in a subject. In some embodiments, this document provides the use of an NKG2A conjugate (e.g., an antibody) or a pharmaceutical composition described herein for manufacturing an agent for alleviating one or more symptoms associated with an autoimmune or inflammatory disease or condition in a subject. An autoimmune disease is a disease in which the body's immune system attacks healthy cells. In other embodiments, an autoimmune disease is a disease in which the body's adaptive immune system attacks healthy cells. An inflammatory disease is a condition associated with abnormal inflammation, such as when the inflammatory response is misdirected, which is typically attributed to the immune system attacking healthy tissue (causing inflammation). In some embodiments, an inflammatory disease is triggered by infection with a pathogen (such as a virus or bacteria). Or, additionally, an inflammatory disease is a condition associated with an abnormal innate immune system that attacks healthy cells.

[0312] A method for treating a disease or condition (e.g., cancer or an autoimmune or inflammatory disease or condition) is also provided, the method being carried out by administering, alone or in combination with another agent, an NKG2A binder (e.g., an antibody, such as a human NKG2A binder) or a pharmaceutical composition as disclosed herein, to a subject in need.

[0313] One or more additional therapeutic agents may be administered to a subject to the methods described herein in combination with an NKG2A conjugate (e.g., an antibody) or a fragment thereof, or a pharmaceutical composition described herein. The additional agent may be an agent targeting tumors or cancer cells. The additional agent may also be an agent targeting immune cells (e.g., NK cells or T cells). In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by about 10% to 90% or about 2 to 100 times. In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 10%. In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 20%. In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 30%. In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 40%. In some embodiments, the NKG2A conjugate or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 50%. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 60%. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 70%. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 80%. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 90%. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 2 times. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 5 times. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 10 times. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by at least 20 times. In some embodiments, the NKG2A binder or pharmaceutical composition provided herein increases the therapeutic effect of the additional agent by more than 50 times.

[0314] For a specific subject, the specific administration regimen of the NKG2A binder (e.g., antibody) or pharmaceutical composition disclosed herein will depend in part on the agent used, the amount of agent administered, the route of administration, and the cause and extent of any side effects. The amount of agent (e.g., antibody) administered to the subject (e.g., a mammal, such as a human) should be sufficient to achieve the desired response within a reasonable timeframe. Therefore, in some embodiments, the amount of the NKG2A binder (e.g., antibody) or pharmaceutical composition described herein administered to the subject is an effective amount.

[0315] Suitable routes for administering the NKG2A binders (e.g., antibodies) (such as human NKG2A binders (e.g., antibodies)) or compositions described herein are well known in the art, such as intravenous injection (e.g., intravenous infusion), intratumoral injection, or injection into a tumor or cancer adjacent to the tumor. Although more than one route may be used to administer the agent (e.g., antibody), a particular route may provide a more direct and effective response than another.

[0316] Gene and cell therapy In some embodiments, the compositions used according to this disclosure comprise one or more nucleic acids or complementary nucleic acids encoding the NKG2A binder (e.g., an antibody or a fragment thereof) provided herein. In a particular embodiment, the nucleic acid is administered to a subject for use in the methods provided herein, such as to prevent, manage, treat, and / or improve a disease or condition (e.g., cancer, such as HLA-E-expressing cancer) by means of gene therapy. Such therapies encompass those performed by administering an expressed or expressible nucleic acid to a subject. In one embodiment, the nucleic acid produces an antibody it encodes, and the antibody mediates a preventative or therapeutic effect.

[0317] Any method available in the art for recombinant gene expression (or gene therapy) can be used.

[0318] For a general review of gene therapy approaches, see Goldspiel et al., 1993, ClinicalPharmacy 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. Methods of recombinant DNA technology commonly known in the art that can be used are described in the following literature: Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0319] In one particular embodiment, the composition comprises a nucleic acid encoding an antibody or fusion protein provided herein, said nucleic acid being part of an expression vector expressing said antibody or fusion protein or its heavy or light chain in a suitable host. Specifically, such nucleic acid has a promoter operatively linked to a coding region, such as a heterologous promoter, said promoter being inducible or constitutive and optionally tissue-specific and / or tumor / cancer-specific. In another particular embodiment, a nucleic acid molecule is used in which the antibody sequence and any other desired sequence are side-mounted to regions that promote homologous recombination at desired sites in the genome, thereby providing intrachromosomal expression of the nucleic acid encoding said antibody or fusion protein (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0320] Nucleic acids can be delivered directly to the subject, in which case the subject is directly exposed to the nucleic acid or a carrier carrying the nucleic acid, or indirectly to the subject, in which case cells are first transformed with nucleic acid in vitro and then transplanted into the subject's body. These two methods are respectively referred to as in vivo or in vitro gene therapy.

[0321] In one particular embodiment, a nucleic acid sequence is administered directly in vivo, wherein the sequence is expressed to produce an encoded product. This can be achieved by any of a variety of methods known in the art, such as by constructing them as part of a suitable nucleic acid expression vector and administering the vector to make the sequence intracellular, for example by infection with a defective or attenuated retrovirus or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by using microparticle bombardment (e.g., gene gun; Biolistic, DuPont), or by coating with lipids or cell surface receptors or transfection agents, encapsulating in liposomes, microparticles, or microcapsules, or by administering them after linking them to peptides known to enter the cell nucleus, or by administering them after linking them to ligands undergoing receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target specific cell types expressing the receptor), etc. In another embodiment, a nucleic acid-ligand complex may be formed, wherein the ligand contains a fusion viral peptide to disrupt the endosome, thereby allowing the nucleic acid to avoid lysosomal degradation. In another embodiment, the nucleic acid may be targeted in vivo to achieve cell-specific uptake and expression by targeting a specific receptor (see, for example, WO 92 / 06180; WO 92 / 22635; WO 92 / 20316; WO 93 / 14188; WO 93 / 20221). Alternatively, the nucleic acid may be introduced into the cell and incorporated into the host cell DNA for expression via homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature 342:435-438).

[0322] In one particular implementation, a viral vector containing a nucleic acid sequence encoding an antibody is used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequence encoding an antibody to be used in gene therapy can be cloned into one or more vectors, which facilitates gene delivery to the subject. Further details about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the MDR1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0323] Adenoviruses are another viral vector that can be used for the recombinant generation of antibodies. Adenoviruses are particularly attractive vectors for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, causing mild disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, provide a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10, demonstrate the use of adenovirus vectors to transfer genes into the respiratory epithelium of rhesus monkeys. Other instances of adenovirus use in gene therapy can be found in the following literature: Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication WO94 / 12649; and Wang et al., 1995, Gene Therapy 2:775-783. In one particular implementation, an adenovirus vector is used.

[0324] Adeno-associated virus (AAV) can also be used (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; and U.S. Patent No. 5,436,146). In one particular embodiment, an AAV vector is used to express the anti-NKG2A antibody as provided herein. In some embodiments, the AAV contains nucleic acid encoding the VH domain. In other embodiments, the AAV contains nucleic acid encoding the VL domain. In some embodiments, the AAV contains nucleic acids encoding both the VH and VL domains. In some embodiments of the methods provided herein, a subject is administered an AAV containing nucleic acid encoding the VH domain and an AAV containing nucleic acid encoding the VL domain. In other embodiments, a subject is administered an AAV containing nucleic acids encoding both the VH and VL domains. In some embodiments, the VH and VL domains are overexpressed.

[0325] In some embodiments, oncolytic viruses can be used for the recombinant generation of the antibodies provided herein. Oncolytic viruses preferentially infect and kill cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious viral particles or virions to help destroy the remaining tumor. In one particular embodiment, the oncolytic virus is a virus that causes tumor regression when injected into a tumor. In another particular embodiment, the oncolytic virus is a virus that selectively replicates and kills cancer cells in cancer cells and spreads within the tumor. In yet another particular embodiment, the oncolytic virus is a virus that selectively replicates and kills cancer cells in cancer cells and spreads within the tumor without causing any significant damage to normal tissue. In some embodiments, in vitro or ex vivo assays known to those skilled in the art are used to determine the selectivity of the virus replicating in cancer cells relative to non-cancerous cells (e.g., healthy cells). In one embodiment, the virus selectively replicates in cancer cells if, after incubation with the virus, there is a statistically significant increase in the number of viral particles detected in cancer cells in an in vitro or ex vivo assay relative to the number of viral particles detected in non-cancerous cells (e.g., healthy cells) in the same assay. In another embodiment, the virus selectively kills cancer cells if the amount of cancer cells killed in an in vitro or ex vivo assay is statistically significant relative to the amount of non-cancerous cells (e.g., healthy cells) killed in the same assay. In one embodiment, the oncolytic virus naturally replicates preferentially in cancer cells and is nonpathogenic in humans. Oncolytic viruses may be nonpathogenic in humans due to increased sensitivity to innate antiviral signals or dependence on oncogenic signaling pathways. In some embodiments, the oncolytic virus is a parvovirus (e.g., autonomous parvovirus), myxomavirus, avian paramyxovirus (e.g., Newcastle disease virus), reovirus, or Seneca valley virus. In one embodiment, the oncolytic virus is wild-type parvovirus H1 (ParvOryx). In another embodiment, the oncolytic virus is vesicular stomatitis virus. In another embodiment, the oncolytic virus is avian paramyxovirus. In some implementations, the oncolytic virus is a genetically engineered influenza virus, measles virus, poliovirus, vaccinia virus, poxvirus, microRNA virus, alpha virus, retrovirus, rod-shaped virus, reovirus, adenovirus, herpes simplex virus, or vesicular stomatitis virus. In some implementations, such viruses are attenuated.

[0326] Another approach to gene and cell therapy involves transferring genes into cells in tissue c...

Claims

1. An antibody or a fragment thereof that binds to NKG2A, wherein the antibody or fragment thereof comprises: (i) VH CDR1, VH CDR2 and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:64, and VL CDR1, VL CDR2 and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:73; (ii) VH CDR1, VH CDR2 and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:25, and VL CDR1, VL CDR2 and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:26; (iii) VH CDR1, VH CDR2, and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:45, and VL CDR1, VL CDR2, and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:46; or (iv) VH CDR1, VH CDR2 and VH CDR3 as described in VH containing the amino acid sequence of SEQ ID NO:64, and VL CDR1, VL CDR2 and VL CDR3 as described in VL containing the amino acid sequence of SEQ ID NO:65; Optionally, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are determined according to the Kabat numbering scheme, the Chothia numbering scheme, the AbM numbering scheme, the Contact numbering scheme, the IMGT numbering scheme, or a combination thereof.

2. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof comprises... (a) The VH region, which contains: (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 51, 54, 55 and 59; (2) VH CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 48, 52, 56, 60 and 63; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 53, 57 and 61; and (b) The VL region, which includes: (1) VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 68, 70 and 71; (2) VLCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 67 and 72 or amino acid sequence VGS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 58 and 62.

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

50.

4. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof comprises... (a) The VH region, which contains: (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13 and 18; (2) VH CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 8, 14, 19 and 24; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 9, 15 and 20; and (b) The VL region, which includes: (1) VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 and 21; (2) VLCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 22 or amino acid sequences SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 17 and 23.

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

6.

6. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof comprises... (a) The VH region, which contains: (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13 and 18; (2) VH CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 27, 32, 35, 39 and 44; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 33, 36 and 40; and (b) The VL region, which includes: (1) VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 34, 37 and 41; (2) VLCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30 and 42 or amino acid sequences SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 38 and 43.

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

31.

8. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof comprises... (a) The VH region, which contains: (1) VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 51, 54, 55 and 59; (2) VH CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 48, 52, 56, 60 and 63; and (3) VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 53, 57 and 61; and (b) The VL region, which includes: (1) VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 and 21; (2) VLCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 22 or amino acid sequences SAS; and (3) VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, 58 and 62.

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

50.

10. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof further comprises a frame 1 (FR1), a frame 2 (FR2), a frame 3 (FR3), and / or a frame 4 (FR4) sequence.

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