Agents that bind to NKG2A and PD-L1 and uses thereof

Abstract

The present disclosure provides multispecific binding agents (e.g., multispecific antibodies such as bispecific antibodies) that bind to NKG2A and PD-L1, and uses thereof. The present disclosure provides multispecific binding agents (e.g., bispecific antibodies) that have a first binding domain that binds to NKG2A, including human NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 441,713, filed January 27, 2023, which is incorporated by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing This application contains a computer-readable Sequence Listing that has been submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The Sequence Listing XML file submitted herewith is named "14529-109-228_SEQ_LISTING.xml", was created on July 6, 2023, and is 164,926 bytes in size.

[0003] The present disclosure relates generally to binding agents, such as antibodies (including fragments thereof) that bind to both NKG2A and PD-L1 (including human NKG2A and human PD-L1), and methods of use thereof. [Background technology]

[0004] NKG2A is typically expressed on NK cells and also on T cells, especially CD8 + NKG2A is a cell surface molecule that can be expressed on T cells. Therefore, NKG2A is a potential target for relieving immune cell suppression and enhancing antitumor responses by immune cells. However, successful treatment with binding agents targeting NKG2A has not yet been achieved.

[0005] Programmed death-ligand 1 (PD-L1) is a cell surface glycoprotein ligand that specifically binds to the key immune checkpoint receptor, programmed death receptor 1 (PD-1). PD-1 is upregulated on activated T cells, B cells, and monocytes, mediating immunosuppression. While another PD-1 ligand, PD-L2, is primarily expressed on activated antigen-presenting cells (APCs), PD-L1 is widely expressed on cells of the hematopoietic lineage, such as activated T cells, B cells, monocytes, dendritic cells, and macrophages, as well as peripheral tissues, such as cardiac, skeletal, muscular, placental, pulmonary, renal, and hepatic tissues. Binding of PD-L1 to PD-1 is a negative checkpoint that can activate downstream signaling of the PD-1 receptor in T cells, thereby inhibiting T cell proliferation, cytokine production and release, and cytotoxicity. This inhibition of T cell activation and effector cytokine secretion can prevent autoimmunity and chronic infection. However, many tumor cells exploit this mechanism to protect themselves from immune attack, resulting in tumor immune evasion. Many cancers overexpress PD-L1, and its overexpression is often associated with poor prognosis. In cancer, the interaction between PD-1 and PD-L1 stimulates downstream signals that suppress T cell activation, resulting in tumor cell survival. Blockade of the interaction between PD-1 and its ligand has been proposed as an immunotherapy to enhance T cell immune responses against tumor cells, but the art remains urgently needed for agents that can inhibit or prevent the interaction between PD-1 and PD-L1.

[0006] Thus, there remains a need in the art for agents that enhance immune responses and treat diseases or disorders, such as cancer. The multispecific binding agents, compositions, and methods provided in the present disclosure fulfill this need and offer related advantages. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides multispecific binding agents (e.g., bispecific antibodies) having a first binding domain that binds to NKG2A, including human NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1). Such agents include multispecific antibodies (e.g., bispecific antibodies) that bind to NKG2A and one or more additional targets that are not NKG2A (e.g., PD-L1), such as multispecific antibodies having a first binding domain that binds to NKG2A, including human NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1). In some embodiments, such agents include multispecific antibodies (e.g., bispecific antibodies) that bind to NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each of them) and one or more additional targets other than NKG2A (e.g., PD-L1), for example, multispecific antibodies having a first binding domain that binds to NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each of them) comprising human NKG2A, and one or more additional binding domains that bind to one or more targets other than NKG2A (e.g., PD-L1), wherein the first binding domain comprises a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in any one of Tables 1 to 4, and a light chain variable (VL) region comprising the amino acid sequences of VH CDR1, VL CDR2, and VL CDR3 shown in any one of Tables 1 to 4. and a VL region comprising an amino acid sequence of any one of Tables 1 to 4 among the amino acid sequences of CDR3, or the multispecific antibody competes with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Tables 1 to 4) for binding to NKG2A, including human NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of either of them).In some embodiments, the first binding domain specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences: (1) TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139).In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), and the additional binding domain comprises a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 set forth in Table 5, and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 set forth in Table 5, or the multispecific antibody competes for binding to PD-L1 (including human PD-L1) with an antibody having a heavy chain variable region and light chain variable region set forth in Table 5.

[0008] The present disclosure also provides nucleic acids encoding the multispecific binding agents (e.g., antibodies or fragments thereof) provided herein, vectors comprising one or more of such nucleic acids, and cells comprising the nucleic acids, the vectors, or both (e.g., cells that express the multispecific binding agents).

[0009] The present disclosure also provides compositions comprising the multispecific binding agents described herein. Such compositions, in some embodiments, include multispecific antibodies (e.g., bispecific antibodies) that bind to NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) and one or more additional targets that are not NKG2A (e.g., PD-L1), e.g., multispecific antibodies having a first binding domain that binds to NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains), including human NKG2A, and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1). In some embodiments, such compositions comprise a multispecific antibody (e.g., bispecific antibody) that binds to NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each thereof) and one or more additional targets that are not NKG2A (e.g., PD-L1), for example, a multispecific antibody having a first binding domain that binds to NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each thereof) comprising human NKG2A and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1), wherein the first binding domain comprises a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 set forth in any one of Tables 1 to 4, and a light chain variable (VL) region comprising the amino acid sequences of VH CDR1, VL CDR2, and VL CDR3 set forth in any one of Tables 1 to 4. A multispecific antibody comprises a VL region comprising an amino acid sequence of any one of Tables 1 to 4 among the amino acid sequences of CDR3, or the multispecific antibody competes with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Tables 1 to 4) for binding to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or the extracellular domains of either of them).In some embodiments, the first binding domain specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences: (1) TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139).In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), and the additional binding domain comprises a heavy chain variable (VH) VH region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 set forth in Table 5, and a light chain variable (VL) VL region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 set forth in Table 5, or the multispecific antibody competes for binding to PD-L1 (e.g., human PD-L1) with an antibody having a heavy chain variable region and a light chain variable region set forth in Table 5.

[0010] The present disclosure further provides various uses of the binding agents and compositions of the present disclosure, including, for example, methods of inhibiting the interaction between HLA-E and NKG2A (e.g., NKG2A expressed on immune cells) and methods of preventing immune cell suppression or activating immune cell-mediated responses. Other aspects provided by the present disclosure include methods of treating a disease or disorder in a subject with a multispecific binding agent or composition provided by the present disclosure. Such compositions include multispecific antibodies having a first binding domain that binds to NKG2A, including human NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each of them), and one or more additional binding domains that bind to one or more targets that are not NKG2A (e.g., PD-L1), wherein the first binding domain comprises a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in any one of Tables 1 to 4, and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 shown in any one of Tables 1 to 4; or the multispecific antibodies compete for binding to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or the extracellular domains of each of them) with an antibody having the heavy chain variable region and light chain variable region described herein (e.g., Tables 1 to 4). In some embodiments, the first binding domain specifically binds to one, two, three, four, five, or all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139).In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences: (1) TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139); or (2) two, three, four, five, or all of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139).In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), and the additional binding domain comprises a heavy chain variable (VH) VH region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 set forth in Table 5, and a light chain variable (VL) VL region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 set forth in Table 5, or the multispecific antibody competes for binding to PD-L1 (e.g., human PD-L1) with an antibody having a heavy chain variable region and a light chain variable region set forth in Table 5.

[0011] In some embodiments, a binding domain that binds to NKG2A (or a complex comprising NKG2A and CD94, or their respective extracellular domains) does not bind to NKG2C. Additionally or alternatively, a binding domain that binds to NKG2A (or a first complex comprising NKG2A and CD94, or their respective extracellular domains) does not bind to a second complex comprising NKG2C and CD94. Additionally or alternatively, a binding domain that binds to NKG2A (or a first complex comprising NKG2A and CD94, or their respective extracellular domains) does not bind to a second complex comprising the extracellular domain of NKG2C and the extracellular domain of CD94.

[0012] The present disclosure further provides binding agents that bind to PD-L1 (e.g., human PD-L1), including binding agents that comprise a heavy chain variable (VH) region comprising the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 5, and a light chain variable (VL) region comprising the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth in Table 5, or binding agents that compete for PD-L1 (e.g., PD-L1) binding with an antibody having a heavy chain variable region and a light chain variable region set forth in Table 5. The present disclosure also provides multispecific binding agents (e.g., multispecific antibodies) having a first binding domain that binds to PD-L1, including human PD-L1, and one or more additional binding domains that bind to one or more targets that are not PD-L1 (e.g., NKG2A). Additionally, the present disclosure provides nucleic acids encoding the binding agents (e.g., antibodies or fragments thereof) as provided herein, vectors comprising one or more of such nucleic acids, cells expressing the nucleic acids, and compositions comprising any one or more of the above. Methods or uses of such binding agents, nucleic acids, vectors, cells, and compositions are also provided herein. [Brief explanation of the drawings]

[0013] [Figure 1-1] 1 shows exemplary results for A3 from a cell binding assay, which are further described in Examples 3 and 6. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2] Exemplary results for A3 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 3A] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 3B] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 3C] 1 shows exemplary results for A3 from a developability assay and is further described in Examples 5 and 6. [Figure 4-1] 1 shows exemplary results for A42 from a biolayer interferometry (BLI) binding assay (i.e., Octet binding assay), and is further described in Examples and 6. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] Exemplary results for A42 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 6A] Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 6B] Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 6C] Exemplary results for A42 from a developability assay are shown and further described in Examples 5 and 6. [Figure 7-1] Exemplary results for A2 from a BLI binding assay are shown and further described in Example 6. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8] Exemplary results for A2 from an HLA-E / NKG2A inhibition assay are shown and further described in Examples 4 and 6. [Figure 9A] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 9B] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 9C] 1 shows exemplary results for A2 from a developability assay and is further described in Examples 5 and 6. [Figure 10-1] 1 shows exemplary results for A11 from a BLI binding assay, further described in Example 6. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 11] 1 shows exemplary results for A11 from an HLA-E / NKG2A inhibition assay, which are further described in Examples 4 and 6. [Figure 12A] 1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 12B] 1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 12C] 1 shows exemplary results for A11 from a developability assay and is further described in Examples 5 and 6. [Figure 13A] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 13B] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 13C] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 13D] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 13E] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 13F] Exemplary results from a cell binding assay are shown and further described in Example 8. [Figure 14A] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 14B] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 14C] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 14D] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 14E] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 14F] Exemplary results from an HLA-E / NKG2A inhibition assay are shown and further described in Example 9. [Figure 15A] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 15B] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 15C] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 15D] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 15E] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 15F] Exemplary results from a PD-L1 / PD1 inhibition assay are shown and further described in Example 10. [Figure 16A] Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 16B] Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 16C] Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 16D] Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 16E]Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 16F] Exemplary results from a cytotoxicity assay are shown and further described in Example 11. [Figure 17A] Exemplary results of an in vitro functional assessment assay are shown and further described in Example 12. [Figure 17B] Statistical analysis of exemplary results of in vitro functional assessment assays is shown (NK degranulation data plotted with 95% CI differences) and is further described in Example 12. [Figure 17C-1] Statistical analysis of exemplary results of in vitro functional assessment assays is shown (NK cytotoxicity data plotted with 95% CI differences) and is further described in Example 12. [Figure 17C-2] Same as above. [Figure 17D] Statistical analysis of exemplary results of an in vitro functional assessment assay is shown (NK degranulation data, represented by the percentage of CD107a / b+ NK cells, plotted), and is further described in Example 12. [Figure 17E] Statistical analysis of exemplary results of an in vitro functional assessment assay (NK cytotoxicity data plotted as percentage of viable tumor cells) is shown and further described in Example 12. [Figure 18A-1] Exemplary results from an assay of influenza-stimulated CD8+ T cells are shown and further described in Example 12. [Figure 18A-2] Same as above. [Figure 18A-3] Same as above. [Figure 18B] Statistical analysis of exemplary results from influenza-stimulated CD8+ T cell assays is shown and further described in Example 12. [Figure 19A] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 19B] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 19C] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 19D] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 19E] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 19F] Exemplary results from SEC chromatography are shown and further described in Example 13. [Figure 20A] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 20B] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 20C] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 20D] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 20E] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 20F] Exemplary results from HIC chromatography are shown and further described in Example 13. [Figure 21A] Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 21B] Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 21C]Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 21D] Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 21E] Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 21F] Exemplary results from SMAC chromatography are shown and further described in Example 13. [Figure 22-1] 1 illustrates an exemplary bispecific antibody format used to construct the exemplary bispecific antibodies described herein. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23A-1] Exemplary results of labeling experiments with CellTrace Far Red and CellTrace Violet are shown and are further described in Example 14. Data for P12xA11(sFc) are compared to various controls at the same tested dose (2 nM). [Figure 23A-2] Same as above. [Figure 23A-3] Same as above. [Figure 23A-4] Same as above. [Figure 23B] 1 shows exemplary results of labeling experiments with CellTrace Far Red and CellTrace Violet, and is further described in Example 14. Dose-dependent co-engagement of tumor and NK cells by P12xA11(sFc) is shown. [Figure 23C-1] 1 shows exemplary results of labeling experiments with CellTrace Far Red and CellTrace Violet, and is further described in Example 14. Various target to effector (T:E) ratios used in the experiments are compared. [Figure 23C-2] Same as above. [Figure 24A-1]1 shows exemplary results demonstrating the in vitro expression of human HLA-E (hHLA-E) and human PD-L1 (hPD-L1) on engineered MC38 cells compared to unengineered MC38 cells (B-hHLA-E plus / hPD-L1 MC38 cells), and is further described in Example 15. [Figure 24A-2] Same as above. [Figure 24B-1] Exemplary results are shown showing ex vivo expression of human HLA-E (hHLA-E) and human PD-L1 (hPD-L1) on engineered MC38 cells compared to unengineered MC38 cells (B-hHLA-E plus / hPD-L1 MC38 cells), and are further described in Example 15. [Figure 24B-2] Same as above. [Figure 24B-3] Same as above. [Figure 24B-4] Same as above. [Figure 25A] Exemplary results of in vivo functional assessment are shown and are further described in Example 15. Body weight changes are plotted for all groups. [Figure 25B] 1 shows exemplary results of in vivo functional assessment, further described in Example 15. FIG. 1 shows changes in tumor volume. [Figure 25C] Exemplary results of in vivo functional assessment are shown and further described in Example 15. Treatment at 10 mg / kg body weight is noted. [Figure 26] The P12xA11 (sFc) epitope is highlighted on the surface depicting NKG2a and CD94. [Figure 27] A list of regions significantly protected from deuterium exchange is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure is based, at least in part, on novel multispecific binding agents that bind to both NKG2A and PD-L1, and their properties. Such agents include antibodies (e.g., bispecific antibodies) that bind to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and PD-L1 (e.g., human PD-L1). In certain aspects, such binding agents are useful in compositions and methods that prevent immune cell suppression or activate immune cell-mediated anti-tumor responses by inhibiting the interaction between HLA-E and NKG2A and / or PD-1 and PD-L1. In addition, the multispecific binding agents provided in this disclosure are useful for killing and / or eliminating tumor cells. The binding agents provided in this disclosure are useful in compositions and methods for treating diseases or disorders, such as cancer.

[0015] As will be appreciated, as used herein, chapter or section headings are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described.

[0016] 5.1.Definition The techniques and procedures described or referred to herein include techniques and procedures that are generally well understood and / or commonly used by those skilled in the art using conventional techniques, e.g., widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall include the plural and terms used in the plural shall include the singular. In the event that any of the explanations of terms provided conflict with any document incorporated herein by reference, the explanations of terms provided below shall control.

[0017] The term "NKG2A," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of NKG2A derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). NKG2A is also known, for example, as NK cell receptor A, NK-activating receptor NKG2A, NK-activating receptor NKG2-A / B, killer cell lectin-like receptor C1 (CD159a), CD159 antigen-like family member A, or type II integral membrane protein NKG2-A / NKG2-B. NKG2A belongs to the lectin family and forms a heterodimer with CD94 (or KLRD1), another C-type lectin expressed on NK cells. In humans, the NKG2A / CD94 complex binds to the non-classical MHC I molecule HLA-E and transmits an inhibitory signal, which activates NK cells and CD8 + It suppresses T cell activity. NKG2A is a protein encoded by the NKG2A gene (or KLRC1). The term NKG2A includes "full-length" NKG2A and any form of NKG2A, or any fragment thereof resulting from cellular processing. In some embodiments, an exemplary amino acid sequence of full-length NKG2A is shown below (see, e.g., Gene Accession Number P26715-1 in the Examples section below). In some embodiments, NKG2A includes a signal sequence. In some embodiments, NKG2A does not include a signal sequence. In some embodiments, the term NKG2A refers to a fragment of full-length NKG2A, which fragment includes the NKG2A extracellular domain. The term NKG2A also includes naturally occurring variants of NKG2A, such as SNP variants, splice variants, and allelic variants. An exemplary amino acid sequence of the extracellular domain of human NKG2A is shown below. PSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 74). An exemplary amino acid sequence of human NKG2A is shown below. RHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 141). An exemplary amino acid sequence of the extracellular domain of cynomolgus monkey NKG2A is shown below: PSTLTQKHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKEKRTWAESLLACTLKNSSLLSIDNEEEMKFLTAISPSTWTGVFRDSSQHPWVTINGLTFKHEIKDSDNAEHNCAMLHARGLKSDRCGSSKIYHCKHKL (SEQ ID NO: 77).

[0018] In some embodiments, the term NKG2A as used herein refers to the NKG2A epitope. Additionally or alternatively, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94. In further embodiments, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94, but not an epitope on CD94 itself. Additionally or alternatively, the term NKG2A as used herein refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domain of NKG2A and CD94, but not an epitope on NKG2A itself. In some embodiments, the term NKG2A, as used herein, refers to an epitope of a complex comprising NKG2A and CD94, or a complex comprising the extracellular domains of NKG2A and CD94, but not an epitope on NKG2A alone or CD94 alone.

[0019] In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope. Additionally or alternatively, as used herein, the term NKG2A refers to an NKG2A epitope that is present on the surface of NKG2A stabilized by forming a complex with CD94. In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope that is present on the surface of a complex comprising NKG2A and CD94, as well as on the NKG2A epitope alone. Additionally or alternatively, as used herein, the term NKG2A refers to an NKG2A epitope that is present on the surface of the NKG2A extracellular domain stabilized by forming a complex with the CD94 extracellular domain. In some embodiments, the term NKG2A, as used herein, refers to an NKG2A epitope that is present on the surface of a complex comprising NKG2A and the extracellular domain of CD94, as well as on the NKG2A epitope alone.

[0020] The term "NKG2C," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of NKG2C derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). NKG2C is also known, e.g., as KLRC2, CD159c, NKG2-C, NKG2C, and killer cell lectin-like receptor C2. NKG2C is a protein encoded by the NKG2C gene (or KLRC2). The term NKG2C includes "full-length" NKG2C and any form of NKG2C, or any fragment thereof resulting from cellular processing. In some embodiments, NKG2C includes a signal sequence. In some embodiments, NKG2C does not include a signal sequence. In some embodiments, the term NKG2C refers to a fragment of full-length NKG2C, which fragment comprises the NKG2C extracellular domain. The term NKG2C also includes natural variants of NKG2C, such as SNP variants, splice variants, and allelic variants. The NKG2C gene is listed in various databases under the following ID numbers: HGNC6375, NCBI Entrez Gene3822, Ensembl ENSG00000205809, OMIM® 602891, and UniProtKB / Swiss-ProtP26717. An exemplary extracellular domain of human NKG2C is shown in the Examples section below (see SEQ ID NO: 75).

[0021] In some embodiments, the term NKG2C as used herein refers to the NKG2C epitope. Additionally or alternatively, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94. In further embodiments, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94, but not an epitope on CD94 itself. Additionally or alternatively, the term NKG2C as used herein refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domain of NKG2C and CD94, but not an epitope on NKG2C itself. In some embodiments, the term NKG2C, as used herein, refers to an epitope of a complex comprising NKG2C and CD94, or a complex comprising the extracellular domains of NKG2C and CD94, but not an epitope on NKG2C alone or CD94 alone.

[0022] The term "HLA-E," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) derived from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats), or any native form of HLA-E or its ortholog. HLA-E is also known, for example, as major histocompatibility complex class I, E, HLA class I histocompatibility antigen, alpha chain E, MHC class I antigen E, HLA-6.2, MHC class Ib antigen, HLA-E, or QA1. In humans, HLA-E is a protein encoded by the HLA-E gene. HLA-E belongs to the HLA class I heavy chain paralog, is approximately 45 kDa, and is membrane-anchored. The term HLA-E includes "full-length" HLA-E and any form of HLA-E or any fragment thereof resulting from cellular processing. In some embodiments, HLA-E includes a signal sequence. In some embodiments, HLA-E does not include a signal sequence. In some embodiments, the term HLA-E refers to a fragment of full-length HLA-E, which fragment includes the HLA-E extracellular domain. The term HLA-E also includes natural variants of HLA-E, such as SNP variants, splice variants, and allelic variants. The HLA-E gene is listed in various databases under the ID numbers HGNC4962, NCBI Entrez Gene3133, Ensembl ENSG00000204592, OMIM® 143010, and UniProtKB / Swiss-Prot P13747.

[0023] The term "CD94," unless otherwise indicated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of CD94 from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). CD94 is also known, for example, as killer cell lectin-like receptor D1, killer cell lectin-like receptor subfamily D member 1, natural killer cell antigen CD94, NK cell receptor, or KP43. CD94 is a protein encoded by the KLRD1 gene. CD94 is an immune receptor involved in self-nonself discrimination. On cytotoxic and regulatory lymphocyte subsets, CD94 recognizes, in complex with NKG2A or NKG2C, classical major histocompatibility (MHC) class Ia molecules and the nonclassical MHC class Ib molecule HLA-E carrying self-peptides derived from the signal sequence of nonclassical MHC class Ib molecules. CD94-NKG2A acts as an immunosuppressive receptor and is an important inhibitory receptor for natural killer (NK) cells, regulating NK cell activation and effector function. CD94-NKG2C acts as an immunoactivating receptor on a subset of cytotoxic lymphocytes that recognizes HLA-E carried by peptides derived from the signal sequence of non-classical MHC class Ib HLA-G molecules. The term CD94 includes "full-length" CD94 and any form of CD94 or fragments thereof resulting from cellular processing. In some embodiments, CD94 includes a signal sequence. In some embodiments, CD94 does not include a signal sequence. In some embodiments, the term CD94 refers to a fragment of full-length CD94, which fragment includes the CD94 extracellular domain. The term CD94 also includes naturally occurring variants of CD94, such as SNP variants, splice variants, and allelic variants.The CD94 gene is listed in various databases under the following ID numbers: HGNC6378, NCBI Entrez Gene 3824, Ensembl ENSG00000134539, OMIM® 602894, and UniProtKB / Swiss-Prot Q13241. An exemplary amino acid sequence of human CD94 is shown below. SFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI (SEQ ID NO: 140).

[0024] The terms "programmed death-ligand-1 (PD-L1)," "programmed death-ligand-1," "PD-1 ligand 1," or similar terms, unless otherwise indicated, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of PD-L1 from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and in humans, it is a protein encoded by the CD274 gene. PD-L1 is one of two naturally occurring cell-surface glycoprotein ligands for PD-1 (the other is PD-L2). Like PD-1, PD-L1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. PD-L1 is known in the art to downregulate T cell activation and cytokine secretion upon binding to PD-1. The term PD-L1 includes "full-length" PD-L1 and any form of PD-L1 or any fragment thereof that results from cellular processing. The term PD-L1 also includes naturally occurring variants of PD-L1, such as SNP variants, splice variants, and allelic variants. An exemplary full-length amino acid sequence of human PD-L1 is shown below (exemplary extracellular domain = underlined text): MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 100). Other related PD-L1 polypeptides further encompassed by the term PD-L1 include fragments, derivatives (e.g., substitution, deletion, truncation, and insertion variants), fusion polypeptides, and interspecies homologs that retain PD-L1 activity and / or are sufficient to generate an anti-PD-L1 immune response. As will be apparent to one of skill in the art, the PD-L1-binding agents (e.g., antibodies) described herein can bind to a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 antigen, and / or a PD-L1 epitope. An epitope may be a portion of a larger PD-L1 antigen, which may be a portion of a larger PD-L1 polypeptide fragment, which in turn may be a portion of a larger PD-L1 polypeptide. PD-L1 may exist in both native and denatured states. The PD-L1 polypeptides described herein may be isolated from a variety of sources, such as human tissue species, or from another source, or may be prepared by recombinant or synthetic methods. A PD-L1 polypeptide may include a polypeptide that has the same amino acid sequence as a corresponding naturally occurring PD-L1 polypeptide. Orthologs of PD-L1 polypeptides are also known in the art.

[0025] The terms "programmed cell death-1 (PD-1)," "programmed death-1," "PD-1 receptor," or similar terms, unless otherwise indicated, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any native form of PD-L from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). PD-1, also known as CD279 (cluster of differentiation 279), is an immunoinhibitory receptor belonging to the CD28 family. In vivo, PD-1 is expressed primarily on previously activated T cells and binds to two ligands, PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. PD-1 further contains two cytoplasmic tyrosine-based signaling motifs: an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The term PD-1 includes "full-length" PD-1 and any form of PD-1 or any fragment thereof resulting from cellular processing. The term PD-1 also includes natural variants of PD-1, such as SNP variants, splice variants, and allelic variants. It is known in the art that, after T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail to dephosphorylate, among other effector molecules involved in the CD3 T cell signaling cascade, such as CD3 zeta, PKC theta, and ZAP70 (Carter et al. (2002) Eur J Immunol 32:634-43). An exemplary full-length amino acid sequence of human PD-1 is shown below. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 101).

[0026] As used herein, the term "binding agent" or grammatical equivalents refers to a molecule (e.g., an antibody) that has one or more antigen-binding sites that bind to an antigen. In some embodiments, binding agents as described herein are antibodies (including multispecific antibodies and antibody fragments, e.g., antigen-binding or epitope-binding fragments), or other peptide-based molecules, as well as conjugates of antibodies, antibody fragments, or peptide-based molecules (e.g., antibody-drug conjugates) that bind to NKG2A (such as human NKG2A) and / or PD-L1 (such as human PD-L1).

[0027] The terms "antibody," "immunoglobulin," and "Ig" are used interchangeably herein and are used in the broadest sense to specifically encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies with full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of a heavy chain-only antibody. Exemplary single domain antibodies include, but are not limited to, antibodies naturally lacking light chains, e.g., those derived from camelids (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. VHHs may also be derived from other non-Camelidae species that are capable of producing heavy chain antibodies that naturally lack light chains. Antibodies also include antibody fragments (and / or polypeptides comprising antibody fragments) that retain binding properties to NKG2A and / or PD-L1. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, dual variable domain antibodies, single variable domains, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, didiabodies, disulfide-linked Fvs (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments consisting of non-covalently linked heavy and light chain variable regions). Generally speaking, the variable (V) region domains may be immunoglobulin heavy (VH) and / or light (VL) chain variable domains in any suitable arrangement.For example, antibodies also include tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region domains may be dimeric and may comprise VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind to NKG2A and / or PD-L1. If desired, VH and VL may be covalently linked, either directly or via a linker, to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as being included in the category of "antibody fragments." Another form of antibody fragment is a peptide comprising one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also referred to as "minimal recognition units" or "hypervariable regions") can be obtained by constructing polynucleotides encoding one or more of the relevant CDRs. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).Antibody fragments may be incorporated into, for example, single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of novel antigen receptors (v-NARs), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or a VL further comprise a light chain constant region and / or a heavy chain constant region, e.g., one or more constant regions (including one or more of an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, and / or an IgG4 constant region). In some embodiments, an antibody can comprise an epitope-binding fragment of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0028] The term "monospecific," when used in reference to a binding agent (e.g., an antibody), as used herein, denotes a binding agent that has one or more binding sites that each bind to the same epitope of the same antigen.

[0029] The term "multispecific," when used in reference to a binding agent (e.g., an antibody), means that the binding agent is capable of specifically binding to at least two distinct epitopes, e.g., two binding sites formed by a pair of antibody heavy chain variable domains (VH) and light chain variable domains (VL), or by a VHH domain, bind to different antigens or different epitopes on the same antigen. Such bispecific binding agents (e.g., antibodies) may have a 1+1 format (one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binding agent (e.g., antibody) formats may be 2+1 or 1+2 formats (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope), or 2+2 formats (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., antibody) contains two antigen-binding sites, each may bind to a different epitope. Such bispecific binding agents (e.g., antibodies) may bind to two different epitopes on the same antigen (e.g., epitopes on NKG2A or PD-L1).

[0030] The term "identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of the same nucleotides or amino acid residues, when compared and aligned for maximum correspondence (introducing gaps, if necessary) and not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. Algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that, when compared and aligned for maximum correspondence, the nucleotide or amino acid residues are at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of amino acid sequence that is at least about 10 residues in length, at least about 20 residues in length, at least about 40-60 residues in length, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 residues, e.g., at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies. In some embodiments, the identity exists over a region of nucleotide sequence that is at least about 10 bases in length, at least about 20 bases in length, at least about 40-60 bases in length, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences to which they are compared, e.g., the nucleotide sequences encoding the proteins of interest.

[0031] "Conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical properties.Families of amino acid residues with similar side chains have generally been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of tyrosine with phenylalanine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not prevent the polypeptide, soluble protein, or antibody comprising that amino acid sequence from binding to a target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0032] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can contain non-amino acids (e.g., interrupted by non-amino acids). The term also includes amino acid polymers that are modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation (directly or indirectly) to a moiety, e.g., a labeling component or a drug (e.g., a toxin). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as polypeptides containing other modifications known in the art. Because the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in some embodiments, the polypeptides can exist as single chains or single-chain dimers.

[0033] As used herein, an "antigen" is a moiety or molecule that comprises an epitope to which a binding agent (e.g., an antibody) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen bound by a binding agent (e.g., an antibody) described herein is NKG2A (e.g., human NKG2A) or a fragment thereof, including fragments that comprise one or more domains of NKG2A. In some embodiments, the antigen bound by a binding agent (e.g., an antibody) described herein is PD-L1 (e.g., human PD-L1) or a fragment thereof, including fragments that comprise one or more domains of PD-L1.

[0034] As used herein, "epitope" is a term used in the art and refers to a local region of an antigen to which an antibody can bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of the polypeptide (a "linear" epitope), or it can include amino acids from two or more non-contiguous regions of the polypeptide (a "conformational" epitope, a "non-linear" epitope, or a "discontinuous" epitope), such as human NKG2A or human PD-L1. It will be apparent to those skilled in the art 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 group of amino acids regardless of whether the group is folded into the native three-dimensional protein structure. In other embodiments, antibodies require the amino acid residues that make up the epitope to adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0035] When two antibodies recognize the same epitope, overlapping epitope, or adjacent epitope in three-dimensional space, the antibody binds to an "epitope," or "essentially the same epitope" or "the same epitope" as a reference antibody. The most widely used rapid method for determining whether two antibodies bind to the same epitope, overlapping epitope, or adjacent epitope in three-dimensional space is a competitive assay, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive, fluorescent, or enzyme label.

[0036] "Epitope binning" is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning clusters antibodies based on their epitope recognition properties and includes methods and systems for distinguishing the epitope recognition properties of various antibodies using competitive assays combined with computational processes to identify antibodies with distinct binding specificities.

[0037] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," "selectively bind," "immunospecifically recognize," and "immunospecific" are synonymous in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope), as one of skill in the art would understand binding. In some embodiments, "specifically bind" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, rapidly, for a longer duration, with higher affinity, or some combination thereof, than does alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may generally bind other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, a Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), an OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds or specifically binds to an antigen when it binds to the antigen with higher affinity than any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs). Typically, a specific or selective response results in a response with at least 2x background signal or background noise, and may be more than 10x background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion of binding specificity. In some embodiments, the extent to which an antibody or antigen-binding domain binds to a "non-target" protein is less than about 10% of the extent to which the antibody or antigen-binding domain binds to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka that is at least 2, 2.5, 3, 4, or more logs higher than the Ka of the molecule when binding to another antigen.In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins. In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other proteins that are not NKG2A. In some embodiments, "specifically binds" refers, for example, to a polypeptide or molecule that binds to a protein or target with a K of about 0.1 mM or less (more usually less than about 1 μM). D In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. D Specific binding means binding to more than one target. Specific binding can include polypeptides or molecules that recognize proteins or targets in more than one species due to sequence identity between homologous proteins in different species. Similarly, specific binding can include polypeptides or molecules that recognize more than one protein or target due to homology within certain regions of the polypeptide sequences of different proteins. It is understood that in some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not require (but can include) exclusive binding, e.g., binding to one target. Thus, a polypeptide or molecule can, in some embodiments, specifically bind to more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, an antibody can, in certain cases, contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific, containing at least two antigen-binding sites with different specificities. Generally, but not necessarily, reference to "binding" means "specific binding."

[0038] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding agent such as an antibody) and its binding partner (e.g., an antigen such as NKG2A). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate quickly, whereas high affinity antibodies generally bind antigens more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. In one embodiment, "K" is used to measure affinity. D " or "K D The "K value" may be measured by biolayer interferometry (BLI), for example, using an OctetQK384 system (ForteBio, Menlo Park, CA). D may be measured in a radiolabeled antigen binding assay (RIA), such as an RIA performed with a Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881), or using a Biacore™ surface plasmon resonance (SPR) assay using, for example, a Biacore™-2000 or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ). "Association rate" or "k on " and "dissociation rate" or "k off " can also be determined using the same SPR or BLI techniques described above, for example, using an OctetQK384 system (ForteBio, Menlo Park, CA) (for SPR), or a Biacore™-2000 (for SPR) or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ) (for BLI).

[0039] The term "compete," or any of its grammatical variations, when used in the context of a binding agent (e.g., an antibody), refers to binding agents that compete for the same epitope or same binding site on a target, including competition between such binding agents as determined by an assay in which the binding agent under investigation prevents or inhibits specific binding of a reference molecule (e.g., a reference ligand or reference antigen-binding protein, e.g., a reference antibody) to a common antigen (e.g., NKG2A or PD-L1). Many types of competitive binding assays can be used to determine whether a test binding agent competes with a reference molecule for binding to NKG2A (e.g., human NKG2A) or PD-L1 (e.g., human PD-L1). Examples of assays that can be used include direct solid-phase radioimmunoassay (RIA), indirect solid-phase RIA, direct solid-phase enzyme immunoassay (EIA), indirect solid-phase EIA, sandwich competition assay (see, e.g., Stahl et al., (1983) Methods in Enzymology 9:242-253), direct solid-phase biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung, et al., (1990) Virology 176:546-552), direct solid-phase labeling assay, direct solid-phase labeling sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), direct solid-phase labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and direct solid-phase labeling RIA using I-125 labeling (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press). al., (1988) Molec. Immunol. 25:7-15), and direct label RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82).Typically, such assays involve the use of purified antigen (e.g., NKG2A, e.g., human NKG2A, or PD-L1, e.g., human PD-L1) bound to a solid surface or cells bearing either an unlabeled test antigen-binding protein (e.g., a test NKG2A antibody or a test PD-L1 antibody) or a labeled reference antigen-binding protein (e.g., a reference NKG2A antibody or a reference PD-L1 antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to adjacent epitopes (e.g., similar or overlapping epitopes) sufficiently close to the epitope bound by the reference antibody so that the antibodies sterically hinder the antibody. Typically, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0040] As used herein, the term "constant region" or "constant domain" is a well-known antibody technical term and refers to the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but that can exhibit various effector functions, e.g., interaction with Fc receptors. The term includes portions of immunoglobulin molecules that have amino acid sequences that are generally more conserved than immunoglobulin variable domains.

[0041] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0042] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to the carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering a nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are shown below (CH2 domain = bold text, CH3 domain = underlined text): [ka]

[0043] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (such as antibody-dependent cellular phagocytosis, or ADCP), down-regulation of cell surface receptors (e.g., B cell receptors (BCRs)), and the like. Such effector functions generally require an Fc region in combination with a binding region or domain (e.g., an antibody variable region or domain), and can be assessed using various assays, such as those disclosed.

[0044] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature, and has not been manipulated, modified, and / or altered by man (e.g., has not been isolated, purified, selected (e.g., has not contained or combined with other sequences, e.g., variable region sequences)). Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0045] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region comprises at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions described herein can be at least about 80% identical to the native-sequence Fc region and / or the Fc region of the parent polypeptide, or at least about 90% identical thereto, e.g., at least about 95% identical thereto. The variant Fc regions described herein can lack effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are shown below (CH2 domain = bold text (amino acid changes underlined), CH3 domain = underlined text). [ka]

[0046] As used herein, the term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of about 50 to 70 kDa, the amino-terminal portion of which contains a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains one or more constant regions. "Heavy chain" can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of its constant domain, where alpha confers the IgA class of antibodies, delta the IgD class of antibodies, epsilon the IgE class of antibodies, gamma the IgG class of antibodies, and mu the IgM class of antibodies, including IgG subclasses such as IgG1, IgG2, IgG3, and IgG4.

[0047] As used herein, the term "light chain," when used in reference to an antibody, can refer to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of its constant domain, there are two distinct types, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0048] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and confer specificity and affinity on the binding fragment, domain, or region for that antigen (e.g., CDRs). As used herein, "antigen-binding fragment" includes antibody portions that contain one or more CDRs, e.g., "antibody fragments" that contain the antigen-binding region or variable region of the antibody.

[0049] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0050] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen binding sites that bind to the NKG2A antigen and / or the PD-L1 antigen.

[0051] An antibody can be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or classes (e.g., human IgG1, human IgG2, human IgG3, or IgG4) or subclasses thereof.

[0052] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. Additionally or alternatively, the amino acid sequences of the L chains are different from each other. For example, the antibody comprises a first H chain / L chain pair and a second H chain / L chain pair, where the first H chain / L chain pair binds to an NKG2A antigen and the second H chain / L chain pair binds to an antigen other than NKG2A (e.g., PD-L1). In some embodiments, the antibody is a two-chain antibody unit comprising a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHHs are identical. In other embodiments, the amino acid sequences of the VHHs are different from each other. For example, an antibody comprises a first VHH and a second VHH, where the first VHH binds to an NKG2A antigen and the second VHH binds to an antigen other than NKG2A (e.g., PD-L1). In some embodiments, the H chain and / or L chain comprise a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa light chain constant region or a lambda light chain constant region, e.g., a human kappa light chain constant region or a human lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody comprises a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., the constant region of IgG1, IgG2, IgG3, and / or IgG4).

[0053] The antibody or fragment thereof may preferentially bind to NKG2A (such as human NKG2A) and / or PD-L1 (such as human PD-L1), meaning that the antibody or fragment thereof binds to NKG2A and / or PD-L1 with greater affinity than it binds to a control protein (e.g., an unrelated control protein, e.g., hen egg white lysozyme or NKG2C), and / or binds to human NKG2A and / or human PD-L1 with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind to NKG2A and / or PD-L1, or respective portions thereof. "Specific binding" means that the antibody or fragment thereof binds to NKG2A and / or PD-L1 with an affinity that is at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, or 10,000-fold greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind substantially exclusively to NKG2A and / or PD-L1 (e.g., be able to distinguish NKG2A and / or PD-L1 from other known polypeptides, e.g., based on a measurable difference in binding affinity). In some embodiments, the NKG2A-binding agent (e.g., an antibody) may react with an NKG2A sequence other than a human NKG2A sequence (e.g., a cynomolgus monkey NKG2A sequence, such as A42 described herein). In other embodiments, the NKG2A-binding agent (e.g., antibody) does not react with non-human (e.g., cynomolgus) NKG2A sequences, such as A2, A3, and A11 provided herein. In some embodiments, the PD-L1-binding agent (e.g., antibody) may react with a PD-L1 sequence other than a human PD-L1 sequence (e.g., a cynomolgus PD-L1 sequence). In other embodiments, the PD-L1-binding agent (e.g., antibody) does not react with non-human (e.g., cynomolgus) PD-L1 sequences.

[0054] The term "variable region" or "variable domain" refers to the portion of an antibody light or heavy chain generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length in the heavy chain and approximately 100-110 amino acids in the light chain, which is responsible for the binding and specificity of each particular antibody for its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that the sequences of certain segments of the variable region vary significantly among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) regions of about 15-30 amino acids, known as framework regions (FRs), separated by shorter, more variable (e.g., highly variable) regions known as "hypervariable regions" or alternatively, "complementarity-determining regions (CDRs)." The heavy and light chain variable regions each contain four framework regions (FR1, FR2, FR3, and FR4) that primarily adopt a β-sheet configuration, connected by three hypervariable regions, which form connecting loops that, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the framework regions and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant regions are not directly involved in antibody binding to antigens but are involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary greatly in sequence among different antibodies. The sequence variability is concentrated in the CDRs, while the less variable parts of the variable regions are called framework regions (FRs).The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with its antigen. In a specific embodiment, the variable regions are human variable regions.

[0055] The terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR," as used herein, refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions: three in the VH (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three in the VL (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Numerous hypervariable region descriptions are in use and are included herein. Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead, Chothia refers to the location of a structural loop (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the loop (this variation is due to the Kabat numbering scheme allowing for insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecula's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are shown below.

[0056] A universal numbering system has been developed and widely adopted (ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003))). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, CDR and framework residues can be readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from an immunoglobulin of one species into an acceptor framework, typically derived from a human antibody. An additional numbering system (AHon) was developed in Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). Correspondence between numbering systems (including, e.g., Kabat numbering and the IMGT-specific numbering system) is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also exemplified below. The various systems known in the art or described herein represent different ways of delineating CDRs and are often considered equivalent when used to define the same antibody. The exemplary system shown herein is a combination of Kabat and Chothia. Residues from each of these hypervariable regions or CDRs are illustrated in the table below. [Table 13]

[0057] The hypervariable region may comprise the "hypervariable region" at positions 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and positions 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR" are used interchangeably.

[0058] "Polynucleotide" or "nucleic acid," when used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA polymerase or RNA polymerase or by a synthetic reaction. A polynucleotide may include modified nucleotides, such as methylated nucleotides and their analogs. Cells producing binding molecules of the disclosure may include parent hybridoma cells into which nucleic acid encoding the antibody has been introduced, as well as bacterial and eukaryotic host cells. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction in which a nascent RNA transcript is added from 5' to 3' is called the transcription direction; the region of the DNA strand that has the same sequence as the RNA transcript and is 5' to the 5' end of the RNA transcript is called the "upstream sequence," and the region of the DNA strand that has the same sequence as the RNA transcript and is 3' to the 3' end of the RNA transcript is called the "downstream sequence."

[0059] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence (e.g., for the purpose of introducing a nucleic acid sequence into a host cell). Examples of vectors that can be used include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can contain a selection sequence or marker that is operable for stable integration into a host cell chromosome. In addition, the vector can contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients that are not present in the culture medium. Expression control sequences can include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both the heavy and light chains of an antibody or both the VH and VL of an antibody), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting for gene product expression, polymerase chain reaction (PCR) amplification or immunoblotting of mRNA, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. One skilled in the art will know that the nucleic acid molecule will be expressed in sufficient amounts to produce the desired product (e.g., a binding agent as described herein), and will further know that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0060] The term "pharmaceutically acceptable," as used herein, means recognized by a regulatory agency of the Federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly, in humans.

[0061] "Excipient" refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, wetting agents, lubricants, flavoring agents, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle. In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids, antioxidants (including ascorbic acid), low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides, and other sugar chains (including glucose, mannose, or dextrins), chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio.See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed to the excipient at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution. In some embodiments, the excipient is a sterile liquid, such as water or oil, including petroleum oils (animal, vegetable, or synthetic), such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions of the present disclosure can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, if desired. The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.Oral compositions (including formulations) can contain standard excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions (including pharmaceutical compounds) can contain a prophylactically or therapeutically effective amount of an NKG2A-binding agent and / or PD-L1-binding agent (e.g., antibody), e.g., in isolated or purified form, together with appropriate amounts of excipients to provide the form for proper administration to a subject (e.g., a patient). The formulation should be compatible with the mode of administration.

[0062] An "effective amount" generally is an amount sufficient to reduce the severity and / or frequency of a symptom, eliminate a symptom and / or its underlying cause, prevent or delay the onset of a symptom and / or its underlying cause, and / or ameliorate or repair damage caused by or associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0063] The term "therapeutically effective amount," as used herein, refers to an amount of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or its associated symptoms. A therapeutically effective amount of an agent (including a therapeutic agent) can be the amount necessary to (i) reduce, delay, or ameliorate the progression or progression of a given disease, disorder, or condition; (ii) reduce, delay, or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition; and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administering an agent described herein). A "therapeutically effective amount" of a substance / molecule / agent (e.g., a bispecific antibody) of the present disclosure can vary according to factors such as the individual's condition, age, sex, and weight, and the ability of the substance / molecule / agent to elicit a desired response in an individual. A therapeutically effective amount includes an amount in which any toxic or adverse effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a multispecific binding agent effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0064] The term "treating," or any of its grammatical variations, refers to reducing and / or ameliorating the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith, for example, (i) reducing, delaying, or ameliorating the progression or onset of a given disease, disorder, or condition; (ii) reducing, delaying, or ameliorating the recurrence, development, or onset of a given disease, disorder, or condition; and / or (iii) improving or enhancing the prophylactic or therapeutic effects of another therapy (e.g., a therapy other than administering a multispecific binding agent described herein).

[0065] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., prevents or delays the onset (or recurrence) of a disease, disorder, or condition, or reduces the likelihood of the onset (or recurrence) of a disease, disorder, or condition or associated symptom(s).

[0066] The full therapeutic or prophylactic effect may not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more doses.

[0067] The terms "about" and "approximately" refer to a variation of no more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

[0068] As used herein, comparative terms, such as "reduce," "decrease," "increase," or any of their grammatical variations, can refer to a certain variation from its reference value. In some embodiments, such a variation can refer to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 100-fold higher than the reference value. In some embodiments, such a variation can refer to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of a reference value.

[0069] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0070] In some embodiments, the terms "first," "second," "third," "fourth," and similar terms in the names of components are used to distinguish and identify more than one component that shares a particular identity in those names. For example, "first antibody" and "second antibody" are used to distinguish between two antibodies.

[0071] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described using the words "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described using the term "consisting of" are also provided.

[0072] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0073] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or," when used in phrases such as "A, B and / or C," is intended to include each of the following embodiments: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0074] The term "optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes cases where the circumstance occurs and cases where the circumstance does not occur.

[0075] 5.2. Multispecific Binding Agents NKG2A-Binding Domain The multispecific binding agents provided herein comprise one or more NKG2A-binding domains. In some embodiments, described herein are multispecific binding agents (e.g., bispecific antibodies) that bind to NKG2A. As used herein, NKG2A refers to an NKG2A polypeptide, an NKG2A polypeptide fragment, an NKG2A peptide, or an NKG2A epitope. In some embodiments, the NKG2A-binding domain is derived from a human or humanized antibody (e.g., an antibody comprising a human framework region) that binds to NKG2A (including an NKG2A polypeptide, an NKG2A polypeptide fragment, an NKG2A peptide, or an NKG2A epitope). In some embodiments, the multispecific binding agent (e.g., bispecific antibody) can bind to NKG2A expressed on the surface of mammalian (e.g., human) cells, including immune cells (e.g., NK cells or T cells) that express NKG2A. In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) provided herein bind to an NKG2A extracellular epitope exposed on a cell, e.g., an immune cell. In some embodiments, described herein are multispecific binding agents (e.g., bispecific antibodies) that bind to NKG2A, e.g., human NKG2A, or a portion thereof. In some embodiments, the NKG2A is human NKG2A. In some embodiments, a multispecific binding agent provided herein is a human NKG2A binding agent (e.g., an antibody that binds to human NKG2A). In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) that binds to NKG2A as disclosed herein binds to both human NKG2A and cynomolgus NKG2A. In other embodiments, a multispecific binding agent (e.g., a bispecific antibody) that binds to NKG2A as disclosed herein binds to human NKG2A but not cynomolgus NKG2A. In some embodiments, described herein are multispecific binding agents (e.g., bispecific antibodies) that bind to a complex comprising NKG2A and CD94, or a complex comprising the extracellular domains of NKG2A and CD94.

[0076] In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) provided herein bind to NKG2A (e.g., human NKG2A) with a dissociation constant (K D ) 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 M). Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure, including, for example, by RIA using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81), by Octet®, for example, using the Octet® Red 96 system, or by Biacore®, for example, using a Biacore® TM-2000 or Biacore® TM-3000, or by surface plasmon resonance (SPR) assays. The "association rate" or "k" may be determined by the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) methods described above, for example, using Octet® Red 96, a Biacore® TM-2000, a Biacore® TM-3000 system, a Biacore® TM-8K or a Biacore® TM-8K+ system.

[0077] In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein do not bind to NKG2C (or a complex comprising NKG2C and CD94 or their respective extracellular domains) (e.g., human NKG2C and / or cynomolgus NKG2C). In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein do not bind to human NKG2C. In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein do not bind to human NKG2C or cynomolgus NKG2C. In other embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein bind to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) with higher affinity than to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of a multispecific binding agent (e.g., a bispecific antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least twice as high as its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of a multispecific binding agent (e.g., a bispecific antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least five times as high as its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of a multispecific binding agent (e.g., a bispecific antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 10 times greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).In some embodiments, the binding affinity of a multispecific binding agent (e.g., a bispecific antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 100-fold greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the binding affinity of a multispecific binding agent (e.g., a bispecific antibody) provided herein to NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) is at least 1000-fold greater than its binding affinity to NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).

[0078] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, e.g., the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as set forth in Tables 1-4. Accordingly, in some embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises any one, any two, and / or all three of the heavy chain CDRs and / or any one, any two, and / or all three of the light chain CDRs from (a) the antibody designated A2, (b) the antibody designated A3, (c) the antibody designated A11, and (d) the antibody designated A42, as set forth in Tables 1-4. In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) described herein comprise any one, any two, and / or all three of the heavy chain CDRs and any one, any two, and / or all three of the light chain CDRs from (a) the antibody designated A2, (b) the antibody designated A3, (c) the antibody designated A11, and (d) the antibody designated A42, as shown in Tables 1-4.

[0079] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) comprises the VH region (including VH CDR1, VH CDR2, and / or VH CDR3) and / or the VL region (including VL CDR1, VL CDR2, and / or VL CDR3) of any one of the binding agents described herein (see, e.g., any one of Tables 1-4). Accordingly, in some embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises any one, any two, and / or all three of the heavy chain CDRs and / or any one, any two, and / or all three of the light chain CDRs of Table 1. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises any one, any two, and / or all three of the heavy chain CDRs and / or any one, any two, and / or all three of the light chain CDRs of Table 2. In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) described herein comprise any one, any two, and / or all three of the heavy chain CDRs and / or any one, any two, and / or all three of the light chain CDRs of Table 3. In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) described herein comprise any one, any two, and / or all three of the heavy chain CDRs and / or one, two, and / or three of the light chain CDRs of Table 4.

[0080] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises (i) a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25, SEQ ID NO:45, or SEQ ID NO:64, and / or (ii) a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26, SEQ ID NO:46, SEQ ID NO:65, or SEQ ID NO:73.

[0081] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to the Chothia numbering system. In other embodiments, the CDRs are numbered by Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and exemplified in Section 5.1 above.

[0082] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20, and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0083] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0084] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0085] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0086] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0087] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0088] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0089] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to the Chothia numbering system. In other embodiments, the CDRs are determined by Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0090] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 32, 35, 39, and 44, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 33, 36, and 40, and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 34, 37, and 41, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 11, and 42, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 38, and 43.

[0091] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0092] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0093] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0094] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0095] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

[0096] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0097] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to Chothia numbering. In other embodiments, the CDRs are numbered according to Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0098] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62.

[0099] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0100] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0101] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0102] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58.

[0103] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62.

[0104] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0105] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to Chothia numbering. In other embodiments, the CDRs are numbered according to Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia.

[0106] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 54, 55, and 59, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 52, 56, 60, and 63, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 53, 57, and 61; and (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 68, 70, and 71, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 72, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, and 62.

[0107] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0108] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 53, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0109] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0110] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58.

[0111] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 61, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 71, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 62.

[0112] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0113] In some embodiments, the NKG2A-binding domain provided herein further comprises one or more framework regions of SEQ ID NOs: 25, 26, 45, 46, 64, 65, and / or 73. In some embodiments, the NKG2A-binding domain further comprises a framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs: 25, 26, 45, 46, 64, 65, and 73. In some embodiments, the NKG2A-binding domain provided herein is derived from a humanized antibody. The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, when CDRs are determined by, for example, Kabat, IMGT, or Chothia, the framework region is the amino acid residues surrounding the CDR in the variable region in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1; when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between the amino acid residues of CDR1 and CDR2; when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between the amino acid residues of CDR2 and CDR3; and when defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR4 is defined as the amino acid residues C-terminal to the amino acid residues of CDR3.

[0114] In some embodiments, the NKG2A-binding domains described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, the NKG2A-binding domains described herein comprise a VL region or VL domain. In some embodiments, the NKG2A-binding domains described herein have a combination of (i) a VH domain or VH region and (ii) a VL domain or VL region.

[0115] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0116] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0117] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0118] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0119] In certain embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein comprise an amino acid sequence that is a particular percent identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any of the antibodies or fragments thereof provided herein, e.g., the CDRs, VH, or VL of Tables 1-4, or to a full-length antibody chain as disclosed herein. In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein comprise the CDRs of any of the antibodies or fragments thereof provided herein, e.g., in Tables 1-4. In further embodiments, the bispecific antibodies provided herein comprise an amino acid sequence that is a particular percentage identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any of the antibodies or fragments thereof provided herein, e.g., a VH or VL of Tables 1-4, or a full-length antibody chain as disclosed herein.

[0120] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), which has been modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with NBLAST nucleotide program parameters set, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residues in the alignment that differ between the two sequences (excluding or including conservative amino acid substitution(s) or degenerate nucleotide substitution(s)) by the number of residues in one of the following: (i) the full length of the shorter sequence, (ii) the full length of the longer sequence, (iii) the average length of the two sequences, (iv) the total ungapped portion of the alignment, (v) the length of the alignment (excluding overhangs), or (vi) the length of the alignment (including overhangs).As used herein, an overhang, in the context of a sequence alignment, refers to a region at either or both ends of the alignment where residues from one sequence do not align with residues from the other sequence (e.g., a gap is considered). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding program (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted in calculating percent identity.

[0121] In some embodiments, multispecific binding agents (e.g., bispecific antibodies) provided herein comprise sequences that contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the multispecific binding agent retains the ability to bind to NKG2A. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDRs (e.g., in the FR and / or constant regions).

[0122] In some embodiments, one or more of the CDRs along the VH region (e.g., CDR1, CDR2, or CDR3) and / or one or more positions of the CDRs along the VL region (e.g., CDR1, CDR2, or CDR3) of the NKG2A binding domains described herein can be varied by 1 amino acid position, 2 amino acid positions, 3 amino acid positions, 4 amino acid positions, 5 amino acid positions, or 6 amino acid positions, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the positions defining a CDR of any of Tables 1, 2, 3, or 4 can be varied by shifting the N-terminal and / or C-terminal boundaries of that CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to the current CDR position, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more of the CDRs along the VH region (e.g., CDR1, CDR2, or CDR3) and / or one or more of the CDRs along the VL region (e.g., CDR1, CDR2, or CDR3) of the NKG2A binding domains described herein can vary by 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or more (e.g., can be shorter or longer), so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).For example, in some embodiments, the CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein can be 1, 2, 3, 4, 5 or more amino acids shorter than one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In other embodiments, the CDR1, CDR2 and / or CDR3 of the VH and / or VL described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In some embodiments, the amino terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%).Additionally or alternatively, in some embodiments, the carboxy terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1-24, 27-44, 47-63 or 66-72, so long as binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Maintained binding to NKG2A (e.g., human NKG2A) can be confirmed using any method known in the art, such as the binding assays and binding conditions described in the "Examples" section herein.

[0123] In other embodiments, the multispecific binding agent (e.g., bispecific antibody) further comprises a conservative sequence modification (e.g., in the NKG2A-binding domain). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Thus, in some embodiments, a predicted non-essential amino acid residue in NKG2A is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions that do not eliminate antigen binding and the nucleotides encoding those amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acid sequence of a multispecific binding agent (e.g., a bispecific antibody), including a human NKG2A-binding agent. In some embodiments, the amino acid sequence modifications refer to up to one, two, three, four, five, or six amino acid substitutions in a CDR, e.g., a CDR listed in any one of Tables 1-4. Thus, for example, each such CDR may include up to five conservative amino acid substitutions, e.g., up to four (or no more than four) conservative amino acid substitutions, e.g., up to three (or no more than three) conservative amino acid substitutions, e.g., up to two (or no more than two) conservative amino acid substitutions or no more than one conservative amino acid substitution. In some embodiments, the NKG2A binding domain comprises one or more (including six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of A3, A2, A42 or A11 (see, e.g., Tables 1, 2, 3 or 4).

[0124] In some embodiments, the NKG2A binding domain comprises a VH and a VL that comprise CDRs identical to the CDRs of A3, A2, A42, or A11 (see, e.g., Tables 1, 2, 3, or 4). In some embodiments, the amino acid sequence modifications do not include any modifications within the SDRs. In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (e.g., CDR1, CDR2, CDR3, or any combination thereof). Additionally or alternatively, the amino acid sequence modifications are in the framework, constant region, and / or fragment crystallizable region (Fc).

[0125] In some embodiments, a multispecific binding agent provided herein comprises a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26, and the binding of the multispecific binding agent to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0126] In some embodiments, a multispecific binding agent provided herein comprises a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46, and the binding of the multispecific binding agent to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0127] In some embodiments, a multispecific binding agent provided herein comprises a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65, and the binding of the multispecific binding agent to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0128] In some embodiments, a multispecific binding agent provided herein comprises a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73, and the binding of the multispecific binding agent to NKG2A (e.g., human NKG2A) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0129] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning or hydrogen / deuterium exchange mass spectrometry (HDX-MS) to identify amino acids in the NKG2A protein (or a complex comprising NKG2A and CD94 or their respective extracellular domains) required for interaction with a multispecific binding agent, its NKG2A-binding domain, and / or an anti-NKG2A antibody presented herein (e.g., in the paragraphs below). In some embodiments, the three-dimensional and crystal structures of a multispecific binding agent that binds to NKG2A, its NKG2A-binding domain, and / or an anti-NKG2A antibody (e.g., presented in the paragraphs below) can be used to identify the epitope. In some embodiments, the present disclosure provides a multispecific binding agent, its NKG2A-binding domain, as disclosed herein, and / or a multispecific antibody comprising an NKG2A-binding domain that specifically binds to the same epitope as any of the anti-NKG2A antibodies or fragments thereof presented herein (e.g., presented in the paragraphs below).

[0130] For example, in some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0131] In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0132] In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0133] In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, an NKG2A-binding domain provided by this disclosure binds to the same epitope as an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0134] In some embodiments, an NKG2A binding domain provided by the present disclosure specifically binds to one of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domain provided herein specifically binds to two of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domains provided herein specifically bind to three of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139).In some embodiments, the NKG2A binding domains provided herein specifically bind to four NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domains provided herein specifically bind to five NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domain provided herein specifically binds to all of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment comprising the amino acid sequence of TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment comprising the amino acid sequence of SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment comprising the amino acid sequence of FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment comprising the amino acid sequence of IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment comprising the amino acid sequence of LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment comprising the amino acid sequence of AQCGSSI (SEQ ID NO: 139).

[0135] In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of two of the following amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of three of the amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of four of the amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139).In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of five of the amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domains provided herein specifically bind to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the amino acid sequences: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the group of amino acid residues that form the conformational epitope includes one amino acid residue from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes two amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes three amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes four amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes five amino acid residues from the amino acid sequence set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes more than five amino acid residues from the amino acid sequence set forth above in this paragraph.

[0136] In some embodiments, the NKG2A-binding domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from one of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domain provided by the present disclosure specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of two of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domain provided by the present disclosure specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following three amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domain provided by the present disclosure specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following four amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139).In some embodiments, the NKG2A-binding domain provided by the present disclosure specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following five amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A-binding domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues that includes at least one amino acid residue from each of the following amino acid sequences on the surface of NKG2A: TWEESL (SEQ ID NO: 134), SIISPSSWIGV (SEQ ID NO: 135), FRNSSHHPW (SEQ ID NO: 136), IKDSDNAEL (SEQ ID NO: 137), LQVNR (SEQ ID NO: 138), and AQCGSSI (SEQ ID NO: 139). In some embodiments, the group of amino acid residues that form the conformational epitope includes one amino acid residue from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes two amino acid residues from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes three amino acid residues from an amino acid sequence described above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes four amino acid residues from the amino acid sequences set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes five amino acid residues from the amino acid sequences set forth above in this paragraph. In some embodiments, the group of amino acid residues that form the conformational epitope includes more than five amino acid residues from the amino acid sequences set forth above in this paragraph.

[0137] In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) provided herein specifically bind to NKG2A competitively with any one of the anti-NKG2A antibodies or fragments thereof described herein.

[0138] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0139] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0140] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65.

[0141] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) provided herein specifically binds to NKG2A competitively with an anti-NKG2A antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 73.

[0142] In some embodiments, antibodies derived from the NKG2A-binding arms of the present disclosure have superior developability based on assays known in the art, such as various chromatographic methods, including size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and stand-up monolayer adsorption chromatography (SMAC). In some embodiments, antibodies derived from the NKG2A-binding arms of the present disclosure have superior developability based on measurements of percentage monomer, solubility, and / or antibody aggregation or precipitability.

[0143] 5.2.2.PD-L1 Binding Domain Also provided herein are binding agents that bind to PD-L1. In some embodiments, the binding agent comprises one or more PD-L1 binding domains. In some embodiments, the multispecific binding agents provided herein further comprise one or more PD-L1 binding domains. In some embodiments, described herein are multispecific binding agents (e.g., bispecific antibodies) that bind to PD-L1. As used herein, PD-L1 refers to a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 peptide, or a PD-L1 epitope. In some embodiments, the PD-L1 binding domain in a binding agent, e.g., a multispecific binding agent, of the present disclosure is derived from a human or humanized antibody (e.g., comprising a human framework region) that binds to PD-L1 (including a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 peptide, or a PD-L1 epitope). In some embodiments, the binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) can bind to PD-L1 expressed on the surface of mammalian (e.g., human) cells, including immune cells (e.g., T cells) that express PD-L1 and / or cancer or tumor cells that express PD-L1. In some embodiments, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein bind to a PD-L1 extracellular epitope exposed on a cell, e.g., an immune cell, and / or a cancer or tumor cell. In some embodiments, described herein are binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) that bind to PD-L1, e.g., human PD-L1, or a portion thereof. In some embodiments, the PD-L1 is human PD-L1. In some embodiments, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein are human PD-L1 binding agents (e.g., antibodies that bind to human PD-L1). In some embodiments, the binding agents provided herein bind to both human PD-L1 and cynomolgus PD-L1, hi other embodiments, the binding agents provided herein bind to human PD-L1 but not cynomolgus PD-L1.

[0144] In some embodiments, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein bind to PD-L1 (e.g., human PD-L1) with a dissociation constant (K D ) 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 M). A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including those methods described herein, e.g., in Section 5.2.1 above.

[0145] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) described herein comprises the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any anti-PD-L1 antibody, such as the amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 set out in Table 5. Accordingly, in some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs, and / or any one, any two, and / or all three light chain CDRs from the antibody designated P12, as set out in Table 5. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs, and any one, any two, and / or all three light chain CDRs, from the antibody designated P12, as shown in Table 5.

[0146] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) comprises the VH region (including VH CDR1, VH CDR2, and / or VH CDR3) and / or the VL region (including VL CDR1, VL CDR2, and / or VL CDR3) of any one of the binding agents described herein (see, e.g., Table 5). Thus, in some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs, and / or any one, any two, and / or all three light chain CDRs from Table 5.

[0147] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and / or a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. CDR sequences can be determined by any well-known numbering system or combination thereof. In some embodiments, the CDRs are numbered according to the IMGT numbering system. In some embodiments, the CDRs are numbered according to the Kabat numbering system. In some embodiments, the CDRs are numbered according to the AbM numbering system. In other embodiments, the CDRs are numbered according to Chothia numbering. In other embodiments, the CDRs are numbered according to Contact numbering. In some embodiments, the CDR sequences are determined by a combination of any two or more of the above numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and exemplified in Section 5.1 above.

[0148] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided in this disclosure comprises (a) a VH region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 89, 91, 94, and 97; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 90, 92, and 95; and (b) a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 93, and 96.

[0149] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0150] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 90, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0151] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0152] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 92, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93.

[0153] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 95, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96.

[0154] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0155] In some embodiments, the PD-L1 binding domain in a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein further comprises one or more framework regions of SEQ ID NOs: 98 and 99. In some embodiments, the PD-L1 binding domain further comprises the sequence of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) as set forth in any one of SEQ ID NOs: 98 and 99. In some embodiments, the PD-L1 binding domain provided herein is derived from a humanized antibody. The framework regions described herein are determined based on the boundaries of the CDR numbering system, as described in Section 5.2.1 above.

[0156] In some embodiments, the PD-L1 binding domains described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, the PD-L1 binding domains described herein comprise a VL region or VL domain. In some embodiments, the PD-L1 binding domains described herein have a combination of (i) a VH domain or VH region and (ii) a VL domain or VL region.

[0157] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided by this disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ ID NO: 99.

[0158] In certain embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided in this disclosure comprises an amino acid sequence that is a particular percent identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any antibody or fragment thereof provided in this disclosure, e.g., a CDR, VH, or VL in Table 5, or a full-length antibody chain as disclosed herein. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided in this disclosure comprises the CDR of any antibody or fragment thereof shown herein, e.g., in Table 5. In further embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein comprises an amino acid sequence that is a particular percent identical (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any antibody or fragment thereof provided herein, e.g., a VH or VL in Table 5, or a full-length antibody chain as disclosed herein. Determining the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using methods such as those described in Section 5.2.1, above.

[0159] In some embodiments, binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, such that the binding agent (e.g., antibody, multispecific binding agent, or bispecific antibody) comprising that sequence retains the ability to bind to PD-L1. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted from the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDRs (e.g., in the FR, constant region, and / or Fc region).

[0160] In some embodiments, one or more of the CDRs along the VH region (e.g., CDR1, CDR2 or CDR3) and / or one or more positions of the CDRs along the VL region (e.g., CDR1, CDR2 or CDR3) of the PD-L1 binding domains described herein may be varied by 1 amino acid position, 2 amino acid positions, 3 amino acid positions, 4 amino acid positions, 5 amino acid positions or 6 amino acid positions, so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). For example, in some embodiments, the positions defining a CDR in Table 5 can be varied by shifting the N-terminal and / or C-terminal boundaries of that CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to the current CDR position, so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more of the CDRs along the VH region (e.g., CDR1, CDR2 or CDR3) and / or one or more of the CDRs along the VL region (e.g., CDR1, CDR2 or CDR3) of the PD-L1 binding domains described herein can vary by 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids or more (e.g., can be shorter or longer), so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).For example, in some embodiments, the CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein can be one, two, three, four, five or more amino acids shorter than one or more of the CDRs set forth by SEQ ID NOs: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In other embodiments, the CDR1, CDR2 and / or CDR3 of the VH and / or VL described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs set forth by SEQ ID NOs: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In some embodiments, the amino terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, provided that binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., is substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%).Additionally or alternatively, in some embodiments, the carboxy terminus of CDR1, CDR2 and / or CDR3 of a VH and / or VL described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs set forth by SEQ ID NOs: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, so long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). Maintained binding to PD-L1 (e.g., human PD-L1) can be confirmed using any method known in the art, such as the binding assays and binding conditions described in the "Examples" section herein.

[0161] In other embodiments, the binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) further comprises a conservative sequence modification (e.g., in the PD-L1-binding domain). Conservative sequence modifications are described in further detail above in Section 5.2.1. In some embodiments, the conservative sequence modifications described herein modify 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the amino acid sequence of the binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody), including human PD-L1-binding agents. In some embodiments, the amino acid sequence modifications refer to up to 1, 2, 3, 4, 5, or 6 amino acid substitutions in a CDR, e.g., a CDR listed in Table 5. Thus, for example, each such CDR may comprise up to five conservative amino acid substitutions, such as up to four (or no more than four) conservative amino acid substitutions, for example up to three (or no more than three) conservative amino acid substitutions, for example up to two (or no more than two) conservative amino acid substitutions, or no more than one conservative amino acid substitution. In some embodiments, the PD-L1 binding domain comprises one or more (including six) CDRs that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of P12 (see, e.g., Table 5).

[0162] In some embodiments, the PD-L1 binding domain comprises a VH and VL that comprise CDRs identical to the CDRs of P12 (see, e.g., Table 5). In some embodiments, the amino acid sequence modifications do not include any modifications within the SDRs. In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (e.g., CDR1, CDR2, CDR3, or any combination thereof). In further embodiments, the amino acid sequence modifications are in the framework, constant region, and / or Fc region.

[0163] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein comprises a VH domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 98, and / or a VL domain that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 99, and the binding of the binding agent to PD-L1 (e.g., human PD-L1) 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%).

[0164] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning or hydrogen / deuterium exchange mass spectrometry (HDX-MS), to identify amino acids in the PD-L1 protein required for interaction with a multispecific binding agent, its PD-L1-binding domain, and / or an anti-PD-L1 antibody provided herein (e.g., in the paragraphs below). In some embodiments, the three-dimensional structure and crystal structure of a multispecific binding agent that binds to PD-L1, its PD-L1-binding domain, and / or an anti-PD-L1 antibody (e.g., as provided in the paragraphs below) may be used to identify the epitope. In some embodiments, the present disclosure provides a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) comprising a multispecific binding agent, its PD-L1-binding domain, as disclosed herein, and / or a PD-L1-binding domain that specifically binds to the same epitope as any of the anti-PD-L1 antibodies or fragments thereof provided herein (e.g., as provided in the paragraphs below).

[0165] For example, in some embodiments, a PD-L1 binding domain provided in this disclosure binds to the same epitope as an anti-PD-L1 antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, a PD-L1 binding domain provided in this disclosure binds to the same epitope as an anti-PD-L1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ ID NO: 99.

[0166] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided in this disclosure specifically binds to PD-L1 competitively with any one of the anti-PD-L1 antibodies or fragments thereof described herein.

[0167] In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein specifically binds to PD-L1 competitively with an anti-PD-L1 antibody comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) provided herein specifically binds to PD-L1 competitively with an anti-PD-L1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ ID NO: 99.

[0168] In yet another aspect, the disclosure provides a binding agent (e.g., an antibody or fragment thereof) that binds to PD-L1, wherein the binding agent comprises one or more of the CDRs described above. In some embodiments, the disclosure provides a binding agent (e.g., an antibody or fragment thereof) that binds to PD-L1, wherein the binding agent comprises a VH and / or VL described above. In some embodiments, the binding agent comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO:98, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, the VH region comprises (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 89, 91, 94, and 97; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 90, 92, and 95; and the VL region comprises (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 93, and 96.

[0169] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0170] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:89, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:90, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:88.

[0171] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0172] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 92, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93.

[0173] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 95, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96.

[0174] In some embodiments, the binding agent comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87, and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88.

[0175] In some embodiments, a binding agent provided by this disclosure comprises a VH region comprising the amino acid sequence of SEQ ID NO:98 and a VL region comprising the amino acid sequence of SEQ ID NO:99.

[0176] 5.2.3. Multispecific antibodies Provided herein are multispecific binding agents that bind to NKG2A and an antigen other than NKG2A (such as PD-L1). In some embodiments, the multispecific binding agent comprises one or more NKG2A-binding domains and one or more non-NKG2A antigen-binding domains (such as one or more PD-L1-binding domains). In certain embodiments, the multispecific binding agents of the present disclosure are multispecific antibodies that comprise one or more NKG2A-binding domains independently selected from the NKG2A-binding domains described above in Section 5.2.1, and one or more PD-L1-binding domains independently selected from the PD-L1-binding domains described above in Section 5.2.2.

[0177] In some specific embodiments, the present disclosure provides (i) a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46; (iii) a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65; or (iv) a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64. a VL comprising the amino acid sequence of SEQ ID NO: 73; and one or more PD-L1 binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98; and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99.

[0178] In some embodiments, the present disclosure provides a multispecific binding agent comprising one or more NKG2A-binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 26, respectively, and one or more PD-L1-binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99, respectively. In some embodiments, the multispecific binding agents provided herein comprise one NKG2A-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical pairs of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain having a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises one NKG2A-binding domain (e.g., a domain having two identical pairs of VH and VL regions that bind to PD-L1).

[0179] In some embodiments, the present disclosure provides a multispecific binding agent comprising one or more NKG2A-binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 46, respectively, and one or more PD-L1-binding domains comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99, respectively. In some embodiments, the multispecific binding agents provided herein comprise one NKG2A-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical pairs of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain having a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises one NKG2A-binding domain (e.g., a domain having two identical pairs of VH and VL regions that bind to PD-L1).

[0180] In some specific embodiments, the present disclosure provides a multispecific binding agent comprising one or more NKG2A-binding domains each comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65, and one or more PD-L1-binding domains each comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the multispecific binding agents provided by the present disclosure comprise one NKG2A-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical pairs of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain having a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises one NKG2A-binding domain (e.g., a domain having two identical pairs of VH and VL regions that bind to PD-L1).

[0181] In some specific embodiments, the present disclosure provides a multispecific binding agent comprising one or more NKG2A-binding domains each comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 73, and one or more PD-L1-binding domains each comprising a VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the multispecific binding agents provided herein comprise one NKG2A-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain comprising a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical pairs of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain having a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises one NKG2A-binding domain (e.g., a domain having two identical pairs of VH and VL regions that bind to PD-L1).

[0182] In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical or different pairs of VH and VL regions that bind to NKG2A) and one PD-L1-binding domain (e.g., a domain having a pair of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises one NKG2A-binding domain (e.g., a domain having a pair of VH and VL regions that bind to NKG2A) and two PD-L1-binding domains (e.g., a domain having two identical or different pairs of VH and VL regions that bind to PD-L1). In some embodiments, a multispecific binding agent provided by the present disclosure comprises two NKG2A-binding domains (e.g., a domain having two identical or different pairs of VH and VL regions that bind to NKG2A) and two PD-L1-binding domains (e.g., a domain having two identical or different pairs of VH and VL regions that bind to PD-L1). In some embodiments, the multispecific binding agent further comprises one or more heavy chain constant regions (such as CH1, CH2 and / or CH3) and / or one or more light chain constant regions (such as CL).

[0183] In some embodiments, multispecific binding agents provided herein have the antibody format shown in any of the three panels of Figure 22. In some embodiments, multispecific binding agents provided herein have the antibody format shown in any of the three panels of Figure 22, but with its PD-L1 arm swapped for its NKG2A arm. For example, in a 1x1 format, chain 1 and chain 2 are named after the binding site for NKG2A (and thus are referred to as the NKG2A arm rather than the PD-L1 arm, as featured in the "1x1" panel of Figure 22), while chain 3 and chain 4 form the binding site for PD-L1 (and thus are referred to as the PD-L1 arm rather than the NKG2A arm, as featured in the "1x1" panel of Figure 22). Additionally or alternatively, in some embodiments, multispecific binding agents provided herein have the antibody format shown in any of the three panels of Figure 22, but with the knob mutation(s) in one CH3 C-terminal to CH2 swapped with the hole mutation(s) in the other CH3 C-terminal to CH2. For example, in a 1x1 format, the knob mutation(s) can be in the CH3 C-terminal to CH2 of chain 1, and the hole mutation(s) can be in the CH3 C-terminal to CH2 of chain 3.

[0184] In some embodiments, mutations are introduced into one or more of the heavy chain constant regions (CH1, CH2 and / or CH3) and / or one or more light chain constant regions (such as CL) to achieve one or more of the following: (i) destabilize homodimers formed by the polypeptides of the multispecific antibody; (ii) stabilize the multispecific antibodies as described herein (referred to herein as heterodimers); (iii) facilitate proper assembly of the multispecific antibodies as described herein; (iv) favor heterodimerization over homodimerization of the constituent polypeptide chains; (v) improve the yield of the multispecific antibodies as described herein; and (vi) improve the purity of the multispecific antibodies as described herein. Various mutations that promote preferential heterodimerization have been developed, such as knob-in-hole (KIH or KiH) mutations (see, e.g., U.S. Pat. Nos. 5,731,168, 5,807,706, 5,821,333, and 8,216,805), disulfide-stabilizing KIH mutations (see, e.g., U.S. Pat. Nos. 7,951,917, 8,642,745, and 9,409,989), and other mutations (see, e.g., WO 2022 / 125986). Each of the patents and / or patent publications cited herein is incorporated by reference in its entirety.

[0185] Certain exemplary multispecific antibody formats are described in the Examples section below and in any of the three panels of Figure 22, however, any multispecific antibody format known in the art can be used and is contemplated as being included in this disclosure.

[0186] The multispecific binding agents (e.g., bispecific antibodies) described herein may be bispecific, trispecific, or of greater multispecificity. Such agents may comprise multispecific antibodies. In some embodiments, multispecific antibodies, e.g., bispecific antibodies, are monoclonal antibodies with binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody against NKG2A having a first binding domain for a first epitope on NKG2A and a second binding domain for a second epitope on NKG2A). In some embodiments, multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of antibodies described herein, e.g., the CDR sequences listed in Tables 1-5. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein have binding specificities for two targets, e.g., NKG2A and PD-L1. In some embodiments, the bispecific antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0187] In some embodiments, one binding specificity of a multispecific antibody provided herein is for NKG2A, a second binding specificity of a multispecific antibody provided herein is for PD-L1, and one or more additional binding specificities are for any other target(s) (e.g., antigens). In some embodiments, a multispecific antibody can comprise more than one target (e.g., antigen) binding domain, with each different binding domain being specific for a different target. In some embodiments, the additional target is an immune checkpoint regulator (e.g., a negative checkpoint regulator). In some embodiments, the additional target is expressed on an immune cell. In some embodiments, the additional target is expressed on a tumor cell or a cancer cell.

[0188] In some embodiments, a multispecific (e.g., bispecific) antibody molecule can bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) binding agent as disclosed herein can bind to one or more epitopes on a first target (e.g., NKG2A) and one or more epitopes on a second target (e.g., PD-L1).

[0189] Methods for producing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0190] 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.

[0191] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety appended, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG formats can include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and / or orthogonal Fab.

[0192] In some embodiments, BslgG comprises a heavy chain that has been engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., a κλ-body), and the use of a heterodimeric Fc region. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art, including knobs-into-holes, duobodies, azymetric, charge pairs, HA-TF, SEEDbodies, and Protein A affinity differentials.

[0193] Another bispecific antibody format is an IgG with an additional antigen-binding moiety added. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Non-limiting examples of adducted IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. In some embodiments, an exemplary antibody format is a B-Body format for monospecific or multispecific (e.g., bispecific) antibodies, as described, for example, in WO2018 / 075692, U.S. Patent Application Publication No. 2018 / 0118811, and U.S. Patent Application Publication No. 2021 / 0155692.

[0194] Bispecific (Bs) antibody (BsAb) fragments are a form of bispecific antibody molecule lacking some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, a bispecific antibody fragment comprises heavy and light chain regions linked by a peptide linker that allows efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

[0195] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or function. In some embodiments, dock-and-lock (DNL) technology can be used to generate bispecific antibody molecules with higher valency. For example, fusions of bispecific antibodies to albumin-binding proteins or human serum albumin can be constructed to extend the serum half-life of the antibody fragments. In some embodiments, chemical conjugation, e.g., of antibodies and / or antibody fragments, can be used to generate BsAb molecules. Exemplary bispecific antibody conjugates include the CovX-body format, in which a low-molecular-weight drug is site-specifically conjugated to a single reactive lysine on each Fab arm or antibody or fragment thereof. In some embodiments, the conjugation improves serum half-life.

[0196] Methods for producing multispecific (including bispecific) antibodies are known in the art. For example, multispecific (including bispecific) antibodies can be produced by separately expressing the component antibodies in different host cells followed by purification / assembly, or by expressing the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be achieved by various methods known in the art, including affinity chromatography.

[0197] In some embodiments, the multispecific binding agents (e.g., bispecific antibodies) disclosed herein can be provided in any antibody format disclosed herein or known in the art. By way of non-limiting example, in some embodiments, the multispecific binding agents (e.g., bispecific antibodies) can be provided in any antibody format disclosed herein or known in the art.

[0044] As non-limiting examples, the multispecific binding agents (e.g., bispecific antibodies) can be provided in any antibody format, including Fabs-in-tandem-lg (FIT-lg), DVD-lg, hybrid hybridomas (quadromas or tetradomas), anticalin platforms (Pieris), diabodies, single-chain diabodies, tandem single-chain Fv fragments, TandAb, trispecific Ab (Affimed), Dart (dual affinity retargeting) (Macrogenics), bispecific Xmab (Xencor), bispecific T cell engager (Bite, Amgen, 55 kDa), triplebodies, tribodies (multifunctional recombinant antibody derivatives that are Fab-scFv fusion proteins (CreativeBiolabs)), duobody platforms (Genmab), dock and The following can be selected: lock platform, knobs-into-holes (KIH) platform, humanized bispecific IgG antibody (REGN1979) (Regeneron), Mab2 bispecific antibody (F-Star), DVD-lg (dual variable domain immunoglobulin (Abbott)), kappa-lambda body, TBTI (tetravalent bispecific tandem Ig), B body, and CrossMab (Roche).

[0198] In some specific embodiments, bispecific antibodies provided by this disclosure are in a format shown in any of the three panels of Figure 22. In some embodiments, bispecific antibodies provided by this disclosure are in a 1x1 format, as shown in panel "1x1" of Figure 22. In some embodiments, bispecific antibodies provided by this disclosure are in a 1x2 format, as shown in panel "1x2" of Figure 22. In some embodiments, bispecific antibodies provided by this disclosure are in a 2x1 format, as shown in panel "2x1" of Figure 22.

[0199] In some specific embodiments, the multispecific antibodies provided herein comprise four polypeptides: a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a first CH2, and a second CH3; a second polypeptide comprising, from N-terminal to C-terminal, a first VH and a third CH3; a third polypeptide comprising, from N-terminal to C-terminal, a second VL, a CL, a second CH2, and a fourth CH3; and a fourth polypeptide comprising, from N-terminal to C-terminal, a second VH and a CH1. These four polypeptides form two binding domains. In some embodiments, the first polypeptide and the second polypeptide (e.g., the first VL and the first VH) form a binding domain that binds to PD-L1, and the third polypeptide and the fourth polypeptide (e.g., the second VL and the second VH) form a binding domain that binds to NKG2A. In other embodiments, the first polypeptide and the second polypeptide (e.g., the first VL and first VH) form a binding domain that binds to NKG2A, and the third polypeptide and the fourth polypeptide (e.g., the second VL and second VH) form a binding domain that binds to PD-L1. In some embodiments, the amino acid sequences of the first CH3, the second CH3, the third CH3, and the fourth CH3, or any subgroup thereof, are different from each other. In some embodiments, the second CH3 and the fourth CH3 undergo knobs-in-holes assembly. Additionally or alternatively, the amino acid sequences of the first CH2 and the second CH2 are identical to each other. In other embodiments, the amino acid sequences of the first CH2 and the second CH2 are different from each other.

[0200] In some specific embodiments, the multispecific antibodies provided herein comprise five polypeptides: a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a first CH2, and a second CH3; a second polypeptide comprising, from N-terminal to C-terminal, a first VH and a third CH3; a third polypeptide comprising, from N-terminal to C-terminal, a second VL, a first CL, a third VL, a second CL, a second CH2, and a fourth CH3; a fourth polypeptide comprising, from N-terminal to C-terminal, a second VH and a first CH1; and a fifth polypeptide comprising, from N-terminal to C-terminal, a third VH and a second CH1. In some embodiments, the amino acid sequences of the second VL and the third VL are identical. Additionally or alternatively, the amino acid sequences of the first CL and the second CL are identical. In some embodiments, the amino acid sequences of the second VH and the third VH are identical. Additionally or alternatively, the amino acid sequences of the first CH1 and the second CH1 are identical. In some embodiments, the amino acid sequences of the first CH3, the second CH3, the third CH3, and the fourth CH3, or any subgroup thereof, are different from each other. In some embodiments, the second CH3 and the fourth CH3 assemble knobs-in-holes. Additionally or alternatively, the amino acid sequences of the first CH2 and the second CH2 are identical to each other. In other embodiments, the amino acid sequences of the first CH2 and the second CH2 are different from each other. In some embodiments, the fourth polypeptide is the same as the fifth polypeptide. The five polypeptides form three binding domains. In some embodiments, the first and second polypeptides (e.g., their first VL and first VH) form a binding domain that binds to PD-L1, the third and fourth polypeptides (e.g., their second VL and second VH) form a first binding domain that binds to NKG2A, and the third and fifth polypeptides (e.g., their third VL and third VH) form a second binding domain that binds to NKG2A. In some embodiments, the first and second NKG2A binding domains are the same.In other embodiments, the first and second NKG2A-binding domains are different from each other. In some embodiments, the first and second polypeptides (e.g., their first VL and first VH) form a binding domain that binds to NKG2A, the third and fourth polypeptides (e.g., their second VL and second VH) form a first binding domain that binds to PD-L1, and the third and fifth polypeptides (e.g., their third VL and third VH) form a second binding domain that binds to PD-L1. In some embodiments, the two PD-L1-binding domains are the same.

[0201] In other more specific embodiments, the multispecific antibodies provided herein comprise five polypeptides: a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a second VL, a second CH3, a first CH2, and a third CH3; a second polypeptide comprising, from N-terminal to C-terminal, a first VH and a fourth CH3; a third polypeptide comprising, from N-terminal to C-terminal, a second VH and a fifth CH3; a fourth polypeptide comprising, from N-terminal to C-terminal, a third VL, a first CL, a second CH2, and a sixth CH3; and a fifth polypeptide comprising, from N-terminal to C-terminal, a third VH and a CH1. In some embodiments, the amino acid sequences of the first VL and the second VL are identical. Additionally or alternatively, the amino acid sequences of the first CH3 and the second CH3 are identical. In some embodiments, the amino acid sequences of the first VH and the second VH are the same. Additionally or alternatively, the amino acid sequences of the fourth CH3 and the fifth CH3 are the same. In some embodiments, the second polypeptide and the third polypeptide are the same. In some embodiments, the amino acid sequences of the first CH3, the second CH3, the third CH3, the fourth CH3, the fifth CH3, and the sixth CH3, or any subgroup thereof, are different from each other. In some embodiments, the second CH3 and the fourth CH3 assemble knobs-in-holes. Additionally or alternatively, the amino acid sequences of the first CH2 and the second CH2 are identical to each other. In other embodiments, the amino acid sequences of the first CH2 and the second CH2 are different from each other. The five polypeptides form three binding domains. In some embodiments, the first polypeptide and the second polypeptide (e.g., their first VL and first VH) form a first binding domain that binds to PD-L1, the first polypeptide and the third polypeptide (e.g., their second VL and second VH) form a second binding domain that binds to PD-L1, and the fourth polypeptide and the fifth polypeptide (e.g., their third VL and third VH) form a binding domain that binds to NKG2A. In some embodiments, the two PD-L1 binding domains are the same.In other embodiments, the first polypeptide and the second polypeptide (e.g., their first VL and first VH) form a first binding domain that binds to NKG2A, the first polypeptide and the third polypeptide (e.g., their second VL and second VH) form a second binding domain that binds to NKG2A, and the fourth polypeptide and the fifth polypeptide (e.g., their third VL and third VH) form a binding domain that binds to PD-L1. In some embodiments, the two NKG2A binding domains are the same. In some embodiments, the first and second NKG2A binding domains are different from one another.

[0202] In some embodiments, any one or more of the CH3 sequences comprises GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 102, aa 116 to aa 222 of SEQ ID NO: 83).

[0203] In some embodiments, the following isoallotypic mutations are made in a CH3 sequence as disclosed herein: D356E and L358M. In some embodiments, one or more isoallotypic mutations (e.g., one or both of D356E and L358M) are in one or more of the CH3 sequences immediately adjacent to the C-terminus of the CH2 sequence, e.g., one or both of the CH3 sequences not marked with an * in each construct illustrated in any of the three panels of Figure 22. Additionally or alternatively, one or more of the CH3 sequences are engineered to reduce the risk of immunogenicity of the antibody by replacing certain amino acids of one allotype with amino acids of another allotype, referred to as isoallotypic mutations, as described in more detail in Stickler et al. (Genes Immun. 2011 Apr;12(3):213-221).

[0204] Additionally or alternatively, the CH3 sequence is engineered to contain a knobs-into-holes mutation. In some embodiments, in a CH3 / CH3 pair (e.g., in which one CH3 in one polypeptide dimerizes with another CH3 in a different polypeptide when forming a binder comprising the polypeptides), in the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence (e.g., the two CH3s not marked with an * in each construct illustrated in any of the three panels of FIG. 22), one CH3 contains a knob mutation (e.g., T366W) and the other CH3 contains a hole mutation (e.g., any one, any two, or all three of T366S, L368A, and Y407V). In other embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair (e.g., the VH and VL form a binding domain in a binder comprising the VH and VL) comprises a knob mutation (e.g., T366W), and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises a hole mutation (e.g., any one, any two, or all three of T366S, L368A, and Y407V).

[0205] As will be understood by those skilled in the art, a pair of domains, e.g., a CH3 / CH3 pair, a VH / VL pair, a VL / VH pair, a CH2 / CH2 pair, a CH1 / CL pair, or a CL / CH1 pair, refers to the dimerization of one antibody domain (e.g., VH, VL, CH1, CH2, CH3, or CL) in one polypeptide chain with another antibody domain (e.g., VL, VH, CL, CH2, CH3, or CH, respectively) in a different polypeptide chain when forming a binding agent comprising the polypeptide.

[0206] In some embodiments, in a CH3 / CH3 pair (e.g., in which one CH3 from one polypeptide dimerizes with the other CH3 from a different polypeptide when forming a binder comprising the polypeptides), one CH3 comprises a Y349C mutation and the other CH3 comprises a S354C mutation. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair comprises a S354C mutation, and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises a Y349C mutation. Additionally or alternatively, the CH3 sequence has been engineered to allow disulfide bond formation in the antibody, e.g., to stabilize knobs-into-holes mutations as described above.

[0207] Additionally or alternatively, any one or more of the CH3 sequences have been engineered to include other mutation(s), provided that the mutation(s) do not significantly reduce the affinity and / or stability of the antibody or significantly increase the immunogenicity risk of the antibody, hi some embodiments, any one or more of the CH3 sequences have been engineered to include mutations as described in WO2022 / 125986.

[0208] In some embodiments, in a CH3 / CH3 pair, one CH3 comprises a S354C mutation and the other CH3 comprises a Y349C mutation. Additionally or alternatively, in a CH3 / CH3 pair, E357 of one CH3 is substituted with a hydrophobic or aromatic amino acid. In various embodiments, the hydrophobic amino acid residue is selected from the group consisting of isoleucine (I), leucine (L), methionine (M), proline (P), and valine (V). In various embodiments, the aromatic amino acid is selected from the group consisting of histidine (H), tryptophan (W), phenylalanine (F), and tyrosine (Y). In some embodiments, E357 of the CH3 is substituted with W. Additionally or alternatively, in some embodiments, the CH (e.g., CH3) in the binder comprises a CH3 comprising a K370R mutation dimerized to the E357 mutation. In some embodiments, one CH3 of a CH3 / CH3 pair comprises a K370R mutation. Additionally or alternatively, the other CH3 of the same CH3 / CH3 pair comprises an E357W mutation. In some embodiments, one CH3 of a CH3 / CH3 pair comprises a K370R mutation, and the other CH3 of the same CH3 / CH3 pair comprises an E357W mutation. In some embodiments, one CH3 of a CH3 / CH3 pair comprises S354C and E357W, and the other CH3 comprises Y349C and K370R. In some embodiments, each CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the CH2 sequence. In other embodiments, one CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the VH or VL of a VH / VL pair, and the other CH3 of a CH3 / CH3 pair is immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL of a VH / VL pair contains an E357W mutation, and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) contains a K370R mutation.In another embodiment, in the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence, one CH3 contains both S354C and E357W, and the other CH3 contains both Y349C and K370R.

[0209] Additionally or alternatively, one or more amino acid residues in CH3 are replaced with one or more corresponding amino acid residues in CH1. In some embodiments, as used herein, a first amino acid residue in a first peptide that corresponds to a second amino acid residue in a second peptide refers to the first amino acid residue aligned with the second amino acid residue in a sequence alignment between the first and second peptides. Alignment methods, such as BLAST as disclosed herein and / or Clustal Omega, are available to those skilled in the art. In some embodiments, CH3 is immediately adjacent to the C-terminus of VH or VL. Additionally or alternatively, one or more amino acid residues are in the N-terminal fragment of CH1, e.g., selected from the first through tenth amino acids of CH1 (including each range or integer therebetween, e.g., the first through fifth amino acids, the first through third amino acids, the first amino acid, or the third amino acid). In some embodiments, the CH3 comprises a first amino acid residue exchanged with the first amino acid residue of the CH1, also referred to as the N-terminal amino acid residue exchanged with the CH1. In some embodiments, the first amino acid residue of the CH3 (which is G) is replaced with the first amino acid residue of the CH1 (which is A). Such a substitution is also referred to herein for ease of reference as G341A. In some embodiments, the CH3 immediately adjacent to the C-terminus of the VH or VL comprises G341A. Without wishing to be bound by theory, a CH3 immediately adjacent to the C-terminus of the VH or VL and engineered to comprise an N-terminal fragment of the CH1 can improve the assembly and / or purity of binders as disclosed herein. In some embodiments, in a CH3 / CH3 pair (where each CH3 is immediately adjacent to the C-terminus of the VH or VL), one CH3 comprises the following mutations: S354C and E357W, and the other CH3 comprises the following mutations: Y349C and K370R.In some embodiments, in a CH3 / CH3 pair (wherein each CH3 is immediately adjacent to the C-terminus of a VH or VL), one CH3 comprises the following mutations: G341A, S354C, and E357W, and the other CH3 comprises the following mutations: Y349C and K370R. In some embodiments, in a CH3 / CH3 pair (wherein each CH3 is immediately adjacent to the C-terminus of a VH or VL), one CH3 comprises the following mutations: G341A, S354C, and E357W, and the other CH3 comprises the following mutations: G341A, Y349C, and K370R. In some embodiments, in a CH3 / CH3 pair (wherein each CH3 is immediately adjacent to the C-terminus of a VH or VL), one CH3 comprises the following mutations: S354C and E357W, and the other CH3 comprises the following mutations: G341A, Y349C, and K370R.

[0210] In some embodiments, a multispecific antibody as described herein comprises one or more CH3 mutations as disclosed in WO2022 / 125986 (herein incorporated by reference in its entirety). In some embodiments, a multispecific antibody as described herein comprises one or more CH3 domains as disclosed in WO2022 / 125986.

[0211] Therefore, any one or more of the CH3 sequences may be: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 102, aa 116 to aa 222 of SEQ ID NO: 83), GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103, Y349C and K370R), AQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104, N-terminal amino acid residues exchanged with CH1, Y349C and K370R), GQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 105, S354C and E357W), AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106, N-terminal amino acid residues exchanged with CH1, S354C and E357W), GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 107, D356E, L358M, T366S, L368A and Y407V), or GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108, T366W) Contains one of the following:

[0212] In some embodiments, a binding agent as disclosed herein comprises a CH3 sequence as disclosed herein (e.g., any one of SEQ ID NOs: 70-74), but the CH3 lacks a C-terminal lysine (K) amino acid residue. In further embodiments, the CH3 is at the C-terminus of any one or more of the binding agent polypeptides. In some embodiments, the C-terminal lysine is truncated in one or more of the binding agent polypeptides, e.g., any one, any two, or any three of a first polypeptide of a binding agent as disclosed herein, a second polypeptide of a binding agent as disclosed herein, and a third polypeptide of a binding agent as disclosed herein.

[0213] In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair comprises GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103, Y349C and K370R), and the other CH3 (immediately adjacent to the C-terminus of the VL or VH of the same VH / VL pair) comprises AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106, N-terminal amino acid residues exchanged with CH1, S354C and E357W).

[0214] In some embodiments, in the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence (e.g., the two CH3s not marked with an * in each construct illustrated in any of the three panels of Figure 22), one CH3 comprises GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 107, D356E, L358M, T366S, L368A, and Y407V), and the other CH3 comprises GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108, T366W).

[0215] As will be appreciated by those of skill in the art, and unless otherwise specified, when describing mutations in antibodies or fragments thereof, e.g., Fc or CH3, reference is made herein to EU numbering, see further details at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs (herein incorporated by reference in its entirety), and residues are identified according to their position in the endogenous constant region sequence, regardless of the physical location of the residue within the chain of the antibody constructs described herein. For example, in a CH3 consisting of aa 116 to aa 222 of SEQ ID NO: 83, the first aa of the CH3 (i.e., aa 116 of SEQ ID NO: 83 is numbered 341 and is referred to herein as G341), the 9th aa of the CH3 is referred to herein as Y349, the 14th aa of the CH3 is referred to herein as S354, the 16th aa of the CH3 is referred to herein as D356, the 17th aa of the CH3 is referred to herein as E357, and the 18th aa of the CH3 is referred to herein as S354. is referred to herein as L358, the 26th aa of its CH3 is referred to herein as T366, the 28th aa of its CH3 is referred to herein as L368, the 30th aa of its CH3 is referred to herein as K370, and the 67th aa of its CH3 is referred to herein as Y407. Thus, the mutated aa residues can be added after the EU numbering to identify the mutation, such as S354C, E357W, Y349C, K370R, D356E, L358M, T366W, T366S, L368A, and Y407V.

[0216] In some embodiments, the binding agent as disclosed herein comprises a variant Fc region, e.g., a silent Fc region as disclosed herein. In some embodiments, the binding agent lacks one or more effector functions, e.g., ADCC, ADCP, or CDC. In some embodiments, the variant Fc region comprises a CH2 as disclosed herein, e.g., a CH2 comprising a mutation that reduces effector function, as disclosed herein.

[0217] In some embodiments, any one or more of the CH2 sequences comprises APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 109, aa 6 to aa 115, L234, L235, and P329 of SEQ ID NO: 83). Additionally or alternatively, any one or more of the CH2 sequences lack a mutation that reduces an effector function of the binding agent. In other embodiments, any one or more of the CH2 sequences comprises a mutation that reduces (including significantly reduces and eliminates) an effector function (e.g., ADCC, ADCP, or CDC) of the multispecific binding agent. In further embodiments, any one or more of the CH2 sequences comprise any one, any two, or all three of the following mutations: L234A, L235A, and P329K (according to EU numbering). In some embodiments, any one or more of the CH2 sequences comprise mutations at any one, any two, or all three of the following amino acid residues: L234, L235, and P329 (according to EU numbering). In some embodiments, any one or more of the CH2 sequences comprise APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAK (SEQ ID NO:110, aa 6 to aa 115 of SEQ ID NO:84, L234A, L235A, and P329K). In some embodiments, a multispecific antibody as described herein comprises one or more CH2 domains as disclosed in WO2022 / 125986.

[0218] In some embodiments, one or more light chain constant domains (CL) comprise RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 111) or RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82). In some embodiments, a multispecific antibody as described herein comprises one or more CL mutations as disclosed in WO2022 / 125986. In some embodiments, a multispecific antibody as described herein comprises one or more CL domains as disclosed in WO2022 / 125986.

[0219] In some embodiments, one or more CH1 sequences comprise ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 112, aa 1 to aa 103 of SEQ ID NO: 85), ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 113), or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDRKVEPKSC (SEQ ID NO: 114). In some embodiments, a multispecific antibody as described herein comprises one or more CH1 mutations as disclosed in WO2022 / 125986. In some embodiments, a multispecific antibody as described herein comprises one or more CH1 domains as disclosed in WO2022 / 125986.

[0220] In some embodiments, a binding agent as disclosed herein further comprises a hinge domain. For example, the hinge domain is immediately adjacent to the N-terminus of the CH2 domain, e.g., between CH3 and CH2 or between the light chain constant domain (CL) and CH2. Additionally or alternatively, the hinge domain is immediately adjacent to the C-terminus of the CL and the N-terminus of the light chain variable domain (VL). In a further embodiment, the hinge domain comprises DKTHTCPPCP (SEQ ID NO: 115). In some embodiments, a multispecific antibody as described herein comprises one or more domain junctions as disclosed in WO2022 / 125986.

[0221] In some embodiments, a binding agent as disclosed herein further comprises a linker. For example, a linker can be provided between two variable regions in a single polypeptide (e.g., in a binding agent having a 2x1 format or a 1x2 format). In one example, a binding agent as disclosed herein comprises a polypeptide comprising, optionally from its N-terminus to its C-terminus, VL-CL-linker-VL-CL-optional hinge-CH2-CH3. In another example, a binding agent as disclosed herein comprises a polypeptide comprising, optionally from its N-terminus to its C-terminus, VL-CH3-linker-VL-CH3-optional hinge-CH2-CH3. In some embodiments, the linker is (SSSG) n (SEQ ID NO: 116) or (SSG) n where n is any positive integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. Additionally or alternatively, the linker is from about 1 amino acid to about 30 amino acids in length. In some embodiments, the linker comprises TASSGGSSSG (SEQ ID NO: 117).

[0222] In some embodiments, a multispecific binding agent as described herein has a construct as disclosed in WO2022 / 125986.

[0223] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide that comprises SEQ ID NO: 118. In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide that comprises SEQ ID NO: 119. In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide that comprises SEQ ID NO: 127. In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide that comprises SEQ ID NO: 128.

[0224] In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 121. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 123. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 124. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 125. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 129. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 130. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 131. In some embodiments, a multispecific binding agent as disclosed herein comprises a third polypeptide comprising SEQ ID NO: 132.

[0225] In some embodiments, a multispecific binding agent as disclosed herein comprises a second polypeptide that comprises SEQ ID NO: 120. In further embodiments, a multispecific binding agent as disclosed herein comprises two second polypeptides that each comprise SEQ ID NO: 120.

[0226] In some embodiments, a multispecific binding agent as disclosed herein comprises a fourth polypeptide comprising SEQ ID NO: 122 or SEQ ID NO: 126. In further embodiments, a multispecific binding agent as disclosed herein comprises two fourth polypeptides comprising SEQ ID NO: 122 or SEQ ID NO: 126, respectively.

[0227] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO:118, a second polypeptide comprising SEQ ID NO:120, a third polypeptide comprising SEQ ID NO:121, and a fourth polypeptide comprising SEQ ID NO:122.

[0228] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 118, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 123, and one or more (e.g., two) fourth polypeptides (each of the fourth polypeptides comprises SEQ ID NO: 122).

[0229] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 119, one or more (e.g., two) second polypeptides (each of the second polypeptides comprises SEQ ID NO: 120), a third polypeptide comprising SEQ ID NO: 121, and a fourth polypeptide comprising SEQ ID NO: 122.

[0230] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO:118, a second polypeptide comprising SEQ ID NO:120, a third polypeptide comprising SEQ ID NO:125, and a fourth polypeptide comprising SEQ ID NO:126.

[0231] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 118, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 124, and one or more (e.g., two) fourth polypeptides (each of the fourth polypeptides comprises SEQ ID NO: 126).

[0232] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 119, one or more (e.g., two) second polypeptides (each of the second polypeptides comprises SEQ ID NO: 120), a third polypeptide comprising SEQ ID NO: 125, and a fourth polypeptide comprising SEQ ID NO: 126.

[0233] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 127, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 129, and a fourth polypeptide comprising SEQ ID NO: 122.

[0234] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 127, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 130, and one or more (e.g., two) fourth polypeptides (each of the fourth polypeptides comprises SEQ ID NO: 122).

[0235] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 128, one or more (e.g., two) second polypeptides (each of the second polypeptides comprises SEQ ID NO: 120), a third polypeptide comprising SEQ ID NO: 129, and a fourth polypeptide comprising SEQ ID NO: 122.

[0236] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 127, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 132, and a fourth polypeptide comprising SEQ ID NO: 126.

[0237] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 127, a second polypeptide comprising SEQ ID NO: 120, a third polypeptide comprising SEQ ID NO: 131, and one or more (e.g., two) fourth polypeptides (each of the fourth polypeptides comprises SEQ ID NO: 126).

[0238] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide comprising SEQ ID NO: 128, one or more (e.g., two) second polypeptides (each of the second polypeptides comprises SEQ ID NO: 120), a third polypeptide comprising SEQ ID NO: 132, and a fourth polypeptide comprising SEQ ID NO: 126.

[0239] In some embodiments, any one, any two, any three, or all four of the polypeptides of a multispecific binding agent as disclosed herein (e.g., any polypeptide comprising any of SEQ ID NOs: 118-132) are modified by one or more insertions, one or more deletions, or one or more substitutions in their amino acid sequence. In further embodiments, these insertions, deletions, or substitutions are not within the CDRs. In some embodiments, the modified polypeptide is 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% identical to the polypeptide from which it is modified.

[0240] In some aspects, the present disclosure provides a multispecific binding agent comprising four polypeptide chains as disclosed herein: a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. In some embodiments, the first polypeptide comprises (i) an amino acid sequence as disclosed herein, or (ii) an amino acid sequence as disclosed herein, but lacking a C-terminal lysine (K). Additionally or alternatively, the second polypeptide comprises (i) an amino acid sequence as disclosed herein, or (ii) an amino acid sequence as disclosed herein, but lacking a C-terminal lysine (K). Additionally or alternatively, the third polypeptide chain comprises (i) an amino acid sequence as disclosed herein, or (ii) an amino acid sequence as disclosed herein, but lacking a C-terminal lysine (K). Additionally or alternatively, the fourth polypeptide chain comprises an amino acid sequence as disclosed herein.

[0241] In some embodiments, a multispecific binding agent as disclosed herein comprises a first polypeptide chain as disclosed herein, a second polypeptide chain as disclosed herein, a third polypeptide chain as disclosed herein, and a fourth polypeptide chain as disclosed herein. In further embodiments, the C-terminal lysine of the first polypeptide chain of the multispecific binding agent has been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of the second polypeptide chain of the multispecific binding agent has been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of the third polypeptide chain of the multispecific binding agent has been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of the first polypeptide chain and the C-terminal lysine of the second polypeptide chain of the multispecific binding agent have been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of the first polypeptide chain and the C-terminal lysine of the third polypeptide chain of the multispecific binding agent have been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of the second polypeptide chain and the C-terminal lysine of the third polypeptide chain of the multispecific binding agent have been removed (e.g., truncated). Additionally or alternatively, the C-terminal lysine of each of the first, second, and third polypeptide chains has been removed (e.g., truncated) from the multispecific binding agent. As used herein, a multispecific binding agent from which any one, any two, or all three C-terminal lysine amino acid residues have been removed is referred to as a C-terminal variant of the multispecific binding agent. Accordingly, the present disclosure also provides compositions comprising the multispecific binding agent and one or more of its C-terminal variants, or compositions comprising one or more C-terminal variants of the multispecific binding agent. As will be appreciated by those of skill in the art, compositions, methods, uses, or any other embodiments related to the multispecific binding agent as disclosed herein also extend to compositions, methods, uses, or embodiments of (i) a C-terminal variant of the multispecific binding agent, and (ii) a composition comprising the multispecific binding agent and / or any one or more of its C-terminal variants.

[0242] In some embodiments, the multispecific antibodies provided herein further comprise one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). As described above, a typical heavy chain has a variable domain (VH) followed by three constant domains (CH), namely CH1, CH2, and CH3, at the N-terminus. The hinge region is the region of the heavy chain between the Fab portion and the Fc portion. Each of CH1, hinge, CH2, and CH3 can be derived from any natural or artificial species and may include variations. An exemplary IgG heavy chain is: It comprises the amino acid sequences of CH1, hinge, CH2 and CH3 such as ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81).

[0243] Another exemplary IgG heavy chain is It comprises the amino acid sequences of CH1, hinge, CH2 and CH3 such as ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 85).

[0244] In some embodiments, the multispecific antibodies provided herein further comprise one or more light chain constant domains (CL). Exemplary light chains include: It contains the CL amino acid sequence of RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82).

[0245] In some embodiments, the binding agents provided herein inhibit HLA-E / NKG2A (e.g., HLA-E / NKG2A / CD94) signaling. Additionally or alternatively, the binding agents provided herein inhibit PD-1 / PD-L1 checkpoint signaling. In some embodiments, the binding agents provided herein promote NK cell-mediated cytotoxicity. In some embodiments, the binding agents provided herein promote NK cell degranulation. In some embodiments, the binding agents provided herein activate CD8+ T cells.

[0246] Other exemplary binding molecules are described in more detail in the following sections: In some embodiments, a multispecific binding agent according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 5.2.4-5.2.7, below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0247] 5.2.4. Antibody fragments Although the term "antibody" is sometimes used herein in the phrase "antibody or fragment thereof," as used herein, it should be understood that the term "antibody" also includes various antibody fragments, e.g., antigen-binding or epitope-binding fragments. Thus, when the term "antibody" is used alone without being followed by "fragment thereof" or a similar term, it should be understood that the term "antibody" includes antibody fragments, e.g., antigen-binding or epitope-binding fragments. Antibodies provided by the present disclosure include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. In some embodiments, multispecific binding agents provided by the present disclosure comprise one or more antibody fragments.

[0248] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to antigen. Antibody fragments include, but are not limited to, those described in Section 5.1 above. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and a portion of a light chain and a heavy chain cross-linked by a disulfide bond), Fab' (e.g., antibody fragments containing a single antigen-binding domain containing a Fab and an additional portion of a heavy chain up to the hinge region), F(ab')2 (e.g., two Fab' molecules linked by an interchain disulfide bond at the hinge region of the heavy chain, where the Fab' molecules may be directed against the same epitope or different epitopes), bispecific Fa (ab') ... b (e.g., a Fab molecule having two antigen-binding domains, each directed against a different epitope); single chain Fvs (also known as scFvs) comprising variable regions (e.g., in which the variable antigen-binding determining regions of a single light chain and a single heavy chain of an antibody are linked together by a chain of, e.g., 10-25 amino acids); disulfide-linked Fvs, or dsFvs (e.g., in which the variable antigen-binding determining regions of a single light chain and a single heavy chain of an antibody are linked together by a disulfide bond); bispecific scFvs (e.g., an scFv or dsFv molecule having two antigen-binding domains, each of which may be directed against a different epitope), a diabody (e.g., a dimerized scFV formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv and the VL domain of the first scFv is assembled with the VH domain of the second scFv, wherein the two antigen-binding regions of the diabody may be directed against the same epitope or different epitopes), a triabody These include scFvs (e.g., trimerized scFvs formed in a manner similar to diabodies, but in which the three antigen-binding domains are assembled in a single complex, and in which the three antigen-binding domains may be directed against the same or different epitopes), and tetrabodies (e.g., tetramerized scFvs formed in a manner similar to diabodies, but in which the four antigen-binding domains are assembled in a single complex, and in which the four antigen-binding domains may be directed against the same or different epitopes).

[0249] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained via proteolysis of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17 and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be expressed and secreted from E. coli, yeast, or insect cells, allowing the facile production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture media. Fab and F(ab')2 fragments with extended in vivo half-lives, which contain salvage receptor-binding epitope residues, are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, e.g., WO 93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458). Fvs and scFvs have combined intact fragments that lack constant regions, which may be suitable for reduced nonspecific binding during in vivo use. ScFv fusion proteins can be constructed to fuse effector proteins to either the amino or carboxy terminus of the scFv (see, e.g., Borrebaeck, ed., supra). An antibody fragment may be a "linear antibody," e.g., as described in the references cited above. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

[0250] Humanized Antibodies The present disclosure provides humanized antibodies that bind to NKG2A and PD-L1 (including human NKG2A and human PD-L1). The humanized antibodies of the present disclosure may comprise one or more CDRs from the VH and / or VL disclosed herein, e.g., those CDRs set forth in Tables 1-5. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanized antibodies that bind to NKG2A can be generated using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8):397-402, 2000; and U.S. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120, and 4,816,567).

[0251] In some cases, the humanized antibody is constructed by CDR grafting, in which the six amino acid sequences of the CDRs of the VH and VL of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J.9:133-139, 1995) determined that only about one-third of the residues in the CDRs actually contact the antigen, and designated these residues as "specificity-determining residues" or "SDRs." In the SDR grafting method, only the residues of the SDRs are grafted onto a human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34, 2005).

[0252] The selection of human variable domains (both light and heavy chains) used to generate humanized antibodies can be important for reducing antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623). In some cases, the framework is selected from the most abundant human subclass, V L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0253] In an alternative paradigm based on CDR comparison (termed Superhumanization), framework homology is not important. The method involves comparing nonhuman sequences with the functional human germline gene repertoire. Among these genes, genes encoding a canonical structure identical to or closely related to the mouse sequence are then selected. Next, among genes that share their canonical structure with the nonhuman antibody, genes with the highest CDR homology are selected as framework donors. Finally, nonhuman CDRs are grafted onto these frameworks (see, e.g., Tan et al., J. Immunol. 169:1119-1125, 2002).

[0254] Furthermore, it is generally desirable to humanize antibodies while maintaining affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display predicted three-dimensional conformations of selected candidate immunoglobulin sequences. These programs include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-2713, 1997). Inspection of these representations permits analysis of the potential role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, for example, improved affinity for the target antigen(s). In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0255] Another method for humanizing antibodies is based on a metric called Human String Content (HSC). In this method, the mouse sequence is compared to the human germline gene repertoire, and the differences are scored as HSC. The target sequence is then humanized by maximizing the HSC, rather than using an exhaustive identity measure, to generate multiple diverse humanized variants. See, for example, Lazar et al., Mol. Immunol. 44:1986-1998, 2007.

[0256] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include methods based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening methods. Antibody variants may be isolated from phage display libraries, ribosome display libraries, and yeast display libraries, as well as by screening bacterial colonies (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-1116, 2005; Dufner et al., Trends Biotechnol. 24:523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21:163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17:847-60, 2004).

[0257] In the framework library approach, a collection of residue variants is introduced into a given position in the framework, and the library is then selected to select the framework that best corresponds to the grafted CDR. The substituted residues may include some or all of the "Vernier" residues identified as likely contributors to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272:10678-10684, 1997).

[0258] In framework shuffling, instead of generating a combinatorial library of selected residue variants, the entire framework is combined with a non-human CDR (see, e.g., Dall'Acqua et al., Methods 36:43-60, 2005). The library may be screened for binding in a two-step selection process, first humanizing the VL and then the VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and increased thermostability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0259] The "humaneering" method is based on experimentally identifying essential minimal specificity determinants (MSDs) and then sequentially replacing non-human fragments with a library of human frameworks and assessing binding. Starting with the CDR3 regions of the non-human VH and VL chains, other regions of the non-human antibody are progressively replaced with the human framework (including CDR1 and CDR2 of both VH and VL). This approach typically preserves and identifies epitopes of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies. See, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007.

[0260] The method of "human engineering" involves altering a non-human antibody or non-human antibody fragment, e.g., a murine antibody, chimeric antibody, murine antibody fragment, or chimeric antibody fragment, by making predetermined changes to the amino acid sequence of the antibody to generate a modified antibody with reduced immunogenicity in humans, but which nonetheless retains the desired binding properties of the original non-human antibody. Generally, this technique involves classifying amino acid residues in a non-human (e.g., murine) antibody as "low risk," "moderate risk," or "high risk" residues. The classification is performed using a comprehensive risk / reward calculation that assesses the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody and / or the risk of substituting a human residue. Specific human amino acid residues to be substituted at predetermined positions (e.g., low-risk or moderate-risk positions) in a non-human (e.g., murine) antibody sequence can be selected by aligning an amino acid sequence from the variable region of the non-human antibody with the corresponding region of a predetermined human antibody sequence or a consensus human antibody sequence. Following the alignment, amino acid residues at low-risk or moderate-risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence. Techniques for generating human-engineered proteins are described in further detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.

[0261] 5.2.6. Antibody Variants Modifications of the multispecific antibodies that bind to NKG2A and PD-L1 described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, variants of the antibodies described herein can be prepared, and these variants are contemplated as being included in the present disclosure. In some embodiments, antibody variants are antibodies that have variations in amino acid sequence compared to the original antibody, e.g., substitutions, deletions, or insertions of one or more amino acids as described above. For example, variations can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide that result in an altered amino acid sequence compared to the original antibody or polypeptide (e.g., conservative substitutions). Relevant sites for substitutional mutagenesis include CDRs, FRs, and / or constant regions. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. It is apparent to one skilled in the art that amino acid changes can alter post-translational processing of antibodies (eg, multispecific antibodies).

[0262] chemical modification Other exemplary modifications include chemical modification, for example, by covalently attaching any type of molecule to the multispecific antibody. Antibody derivatives may include antibodies chemically modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, routine chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-classical amino acids.

[0263] In some embodiments, antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites from an antibody may conveniently be accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0264] When an antibody (e.g., a bispecific antibody) provided herein is fused to an Fc region, the carbohydrate moiety attached to the antibody may be modified. Naturally occurring antibodies produced by mammalian cells typically contain a branched, biantennary carbohydrate moiety, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). This carbohydrate moiety may include various carbohydrate moieties, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary carbohydrate structure. In some embodiments, carbohydrate modifications in the binding molecules provided herein may be performed to generate variants with improved specific properties.

[0265] In other embodiments, when antibodies provided herein are fused to an Fc region, antibody variants provided herein may have a glycan structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Fc region residue EU numbering), although Asn297 may be located approximately ±3 amino acids upstream or downstream from position 297, i.e., at positions 294 to 300, depending on minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 and WO2004 / 056312), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006) and WO2003 / 085107).

[0266] The antibody-containing binding molecules provided herein further comprise bisected glycans, e.g., glycans in which GlcNAc is attached to a branch of a biantennary glycan attached to the Fc region. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue on the glycan attached to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0267] In the antibodies and Fc region-containing molecules of the present disclosure, one or more amino acid modifications may be introduced into the Fc region to generate Fc region variants, which may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0268] In some embodiments, the present application contemplates variants that retain some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the binding molecule is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the binding molecule lacks FcγR binding (i.e., is likely to lack ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, for example, the C1q-binding ELISA and C3c-binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0269] Binding molecules with reduced effector function include molecules with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0270] Certain variants have been described that have improved or diminished binding to FcRs (see, e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0271] In some embodiments, the variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334 (EU numbering). In some embodiments, modifications are made in the Fc region that alter (e.g., either improve or decrease) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0272] Binding molecules with increased half-life and improved binding to fetal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These molecules comprise an Fc region with one or more substitutions in that region that improve binding to FcRn. Such Fc variants include variants having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0273] In some embodiments, it may be desirable to create a cysteine ​​engineered antibody, in which one or more of the antibody's residues are substituted with a cysteine ​​residue. In some embodiments, the substituted residues are located at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein.

[0274] Other known covalent modifications of antibodies are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of an antibody with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl residues to the corresponding glutamyl residues and asparaginyl residues to the corresponding aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of either C-terminal carboxyl group.

[0275] The antibodies of the present disclosure that bind NKG2A and PD-L1 may also be modified to form chimeric molecules comprising the antibody fused or conjugated to another, heterologous polypeptide, amino acid sequence, or small molecule compound, such as an immunostimulatory agent (e.g., a cytokine), an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)), or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0276] Also provided in the present disclosure are fusion proteins comprising a multispecific antibody of the present disclosure that binds NKG2A and PD-L1 and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated is useful for targeting the antibody to cells expressing NKG2A and / or PD-L1 on their surface. Genetically fused or chemically conjugated antibodies are described in further detail in the sections below.

[0277] In vitro affinity maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Like natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Antibody libraries are displayed on the surface of organisms (e.g., phage, bacterial, yeast, or mammalian cells) or in association with their encoding mRNA or DNA (e.g., by covalent or noncovalent attachment). Affinity selection of the displayed antibodies allows the isolation of organisms or complexes carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinities in the narrow nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinities for target antigens.

[0278] Phage display is a widely used method for the display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions to bacteriophage coat proteins. Selection involves exposing the phage-displayed antibodies to antigen to bind to their target, a process called "panning." Antigen-bound phage are recovered and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002) and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0279] In the yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall through a disulfide bond to Aga1p. Display of the protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules in the yeast cell wall. The library is screened using magnetic separation and flow cytometry to select for antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent conjugated to a fluorophore, such as streptavidin. Differences in antibody surface expression can be measured through immunofluorescence labeling of either hemagglutinin or c-Myc epitope tags adjacent to the single-chain antibody (e.g., scFv). Expression has been shown to correlate with the stability of the displayed protein, so antibodies can be selected for improved stability and affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control machinery. Once maturation is complete, antibody affinity can be conveniently "fine-tuned" while displayed on the yeast surface, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is that the size of the functional library can potentially be smaller than that of other display methods, although recent approaches have used the mating system of yeast cells to generate libraries of up to 10 14 (See, e.g., U.S. Patent Application Publication No. 2003 / 0186374 and Blaise et al., Gene 342:211-18 (2004)).

[0280] Ribosome display generates antibody-ribosome-mRNA (ARM) complexes during cell-free selection. DNA libraries encoding specific antibodies are genetically engineered to spacer sequences lacking a stop codon. When translated, these remain attached to peptidyl-tRNAs, occupying the ribosomal tunnel and allowing the proteins to protrude from the ribosome and fold. The resulting complexes of mRNA, ribosomes, and proteins can be bound to surface-bound ligands, allowing for simultaneous isolation of the antibody and its encoding mRNA through affinity capture by the ligand. The ribosome-bound mRNA is then reverse-transcribed back to cDNA, which can then be mutagenized and used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish a covalent link between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0281] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each selection round, for example, by non-proofreading polymerases, since no library should be transformed after any diversification step. In some embodiments, mammalian display systems may be used.

[0282] Diversity can be introduced into the CDRs of an antibody library in a targeted manner or through random introduction. The former approach involves sequentially targeting all CDRs of an antibody through high-level or low-level mutagenesis, or targeting isolated hotspots of somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected of affecting affinity for experimental or structural reasons. Diversity can also be introduced by substituting naturally diverse regions through DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops spanning framework region residues, utilize loop deletions and insertions in the CDRs (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), or use hybridization-based diversification (see, e.g., U.S. Patent Application Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Additional methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0283] Screening of libraries can be accomplished by a variety of techniques known in the art, for example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells affixed to adsorption plates, used in cell sorting, conjugated to biotin for capture by streptavidin-coated beads, or used in any other method for panning display libraries.

[0284] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005), Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and references therein.

[0285] Antibody internalization assays may be used to determine receptor-mediated endocytosis upon antibody binding. In some embodiments, the efficacy of certain antibody-based therapeutics depends on the antibody internalization process. In some embodiments, antibody internalization assays examine the rate and extent of antibody internalization to assess the ability of the antibody to deliver a therapeutic agent to a site or cell of interest. Non-limiting exemplary assays are described below. Target cells of interest are seeded at an appropriate seeding density (e.g., in a 96-well U-bottom plate), and a test antibody is labeled with a signal-reporting reagent, such as a fluorescent compound, horseradish peroxidase (HRP) reagent, a radiolabeled compound, or biotin. The test antibody and target cells are then incubated at an appropriate molar ratio. After incubation, unbound antibody is removed by washing. The cells can be left on ice or incubated at 37°C for a period of time to facilitate internalization. The cells can then be incubated in the presence of a stop reagent for a period of time to inhibit internalization. The cells are then washed and incubated with a signal-emitting reagent. The final signal can be examined using a plate reader or imaging device and analysis software. For example, a flow cytometer can be used to measure the mean fluorescence intensity (MFI) of the cells, and a decrease in MFI can indicate antibody internalization, antibody dissociation, or a combination of both. Cell images can be scanned and acquired to analyze signal intensity, size, and shape. Alternatively, the cells can be lysed to release the internalized antibody. This antibody is then captured on a microtiter well plate coated with the specific antigen to which the antibody was raised. Bound antibody in the wells is detected using a secondary antibody conjugated with alkaline phosphatase or HRP and a chromogenic substrate. Alternative detectable antibody labels and disclosures will be apparent to those skilled in the art. Any method known in the art for determining antibody internalization can be used in the present disclosure.

[0286] 5.2.7. Other Binding Agents Including Multispecific Antibodies of the Disclosure In some embodiments, the multispecific antibodies or fragments thereof provided herein are part of a larger binding agent. Non-limiting exemplary binding agents comprising the antibodies or fragments provided herein are described below.

[0287] The present disclosure provides binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) having masking and / or cleavable moieties such that one or more of the NKG2A-binding domain and / or PD-L1-binding domain of the binding agent (e.g., a multispecific antibody) are masked (e.g., via a masking moiety) and / or activatable (e.g., via a cleavable moiety). Techniques for masking antibodies are well known in the art, including the masking technology SAFEbody (see, e.g., U.S. Patent Application Publication No. 2019 / 0241886) and the masking technology Probody (see, e.g., U.S. Patent Application Publication No. 2015 / 0079088). Such techniques can be used to generate binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) that are masked and / or activatable. Such masked and / or activatable binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) are also useful for preparing conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable ADCs (AADCs) comprising any one of the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) of the present disclosure, including binding agents linked directly or indirectly to another agent, e.g., a drug and / or an immunoactivator (e.g., a cytokine). For example, a binding agent (e.g., an antibody, multispecific binding agent, or bispecific antibody) of the present disclosure may be covalently attached to one or more agents, e.g., a drug and / or an immunoactivator, by a synthetic linker.

[0288] If desired, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) prov...

Claims

1. A multispecific antibody or fragment thereof comprising a first binding domain that binds to NKG2A and a second binding domain that binds to PD-L1, wherein the first binding domain is: (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 46; (iii) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 65; or (iv) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 64, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 73; The multispecific antibody or a fragment thereof, comprising any one or more of (i) to (iv):

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

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

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

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

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

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

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

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

10. 10. The multispecific antibody or fragment thereof according to any one of claims 1 to 9, wherein the first binding domain optionally further comprises the sequence of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4) as set forth in any one of SEQ ID NOs: 25, 26, 45, 46, 64, 65 and 73.

11. The multispecific antibody or fragment thereof according to any one of claims 1 to 10, wherein the first binding domain further comprises a human framework sequence.

12. the first binding domain comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26; (ii) VH comprising the amino acid sequence of SEQ ID NO: 45 and VL comprising the amino acid sequence of SEQ ID NO: 46; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65; or (iv) VH comprising the amino acid sequence of SEQ ID NO: 64 and VL comprising the amino acid sequence of SEQ ID NO: 73; The multispecific antibody or fragment thereof according to any one of claims 1 to 11, comprising:

13. the second binding domain comprises a VH CDR1, a VH CDR2 and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, a VL CDR2 and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 99; the first binding domain binds to a complex comprising NKG2A and CD94, or their respective extracellular domains; the first binding domain does not bind to NKG2C or a complex comprising NKG2C and CD94 or the extracellular domains of each of them; the first binding domain also binds to cynomolgus monkey NKG2A, and / or the first binding domain does not bind to cynomolgus monkey NKG2A; A multispecific antibody or a fragment thereof according to any one of claims 1 to 12.

14. A multispecific antibody or fragment thereof comprising a first binding domain that binds to NKG2A and a second binding domain that binds to PD-L1, wherein the second binding domain comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:

99.

15. The second binding domain comprises: (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 89, 91, 94, and 97; (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 90, 92, and 95; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 93, and 96; and and a VL region comprising:

15. The multispecific antibody or fragment thereof according to claim 13 or 14, comprising:

16. the second binding domain comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 90; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 92; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 95; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; The multispecific antibody or fragment thereof according to claim 13 or 14, comprising any one or more of (i) to (vi):

17. 15. A multispecific antibody or fragment thereof according to claim 13 or 14, wherein the second binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO: 98 and a VL region comprising the amino acid sequence of SEQ ID NO:

99.

18. the multispecific antibody or fragment thereof (i) a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a first CH2, and a second CH3; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first VH and a third CH3; (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a second VL, a CL, a second CH2, and a fourth CH3; (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a second VH and a CH1; and Including, the first polypeptide and the second polypeptide form the second binding domain that binds to PD-L1, and the third polypeptide and the fourth polypeptide form the first binding domain that binds to NKG2A; Optionally, (1) the first polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 118, the second polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 121, and the fourth polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 122; or (2) the first polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 118, the second polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 123, and the fourth polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 122; or (3) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 119, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 121, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 122; or (4) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 118, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 125, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 126; or (5) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 118, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 124, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 126; or (6) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 119, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 125, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 126; or (7) The first polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 127, the second polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 129, and the fourth polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 122; or (8) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 127, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 130, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 122; or (9) The first polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 128, the second polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 129, and the fourth polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 122; or (10) The first polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 127, the second polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 132, and the fourth polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 126; or (11) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 127, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 120, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 126; or (12) The first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 128, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 120, the third polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 132, and the fourth polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:

126. A multispecific antibody or a fragment thereof according to any one of claims 1 to 17.

19. The multispecific antibody or fragment thereof according to any one of claims 1 to 18, further comprising a third binding domain.

20. 20. The multispecific antibody or fragment thereof of claim 19, wherein the third binding domain binds to NKG2A.

21. 20. The multispecific antibody or fragment thereof of claim 19, wherein the third binding domain is the same as the first binding domain.

22. the multispecific antibody or fragment thereof (i) a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a first CH2, and a second CH3; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first VH and a third CH3; (iii) a third polypeptide comprising, from N-terminal to C-terminal, a second VL, a first CL, a third VL, a second CL, a second CH2, and a fourth CH3, wherein the second VL and the third VL are the same, and the first CL and the second CL are the same; (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a second VH and a first CH1; (v) a fifth polypeptide comprising, in an N-terminal to C-terminal direction, a third VH and a second CH1; Including, the second VH and the third VH are the same, the first CH1 and the second CH1 are the same, the first polypeptide and the second polypeptide form the second binding domain that binds to PD-L1, the third polypeptide and the fourth polypeptide form the first binding domain that binds to NKG2A, and the third polypeptide and the fifth polypeptide form the third binding domain that binds to NKG2A. A multispecific antibody or fragment thereof according to claim 20.

23. The multispecific antibody or fragment thereof of claim 19, wherein the third binding domain binds to PD-L1.

24. 24. The multispecific antibody or fragment thereof of claim 23, wherein the third binding domain is the same as the second binding domain.

25. the multispecific antibody or fragment thereof (i) a first polypeptide comprising, from N-terminal to C-terminal, a first VL, a first CH3, a second VL, a second CH3, a first CH2, and a third CH3, wherein the first VL and the second VL are the same, and the first CH3 and the second CH3 are the same; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first VH and a fourth CH3; (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a second VH and a fifth CH3; (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third VL, a first CL, a second CH2, and a sixth CH3; (v) a fifth polypeptide comprising, in an N-terminal to C-terminal direction, a third VH and a CH1; Including, the first VH and the second VH are the same, the fourth CH3 and the fifth CH3 are the same, the first polypeptide and the second polypeptide form the second binding domain that binds to PD-L1, the first polypeptide and the third polypeptide form the third binding domain that binds to PD-L1, and the fourth polypeptide and the fifth polypeptide form the first binding domain that binds to NKG2A. A multispecific antibody or fragment thereof according to claim 23.

26. A polynucleotide encoding the multispecific antibody or fragment thereof according to any one of claims 1 to 25.

27. 27. One or more vectors comprising one or more polynucleotides according to claim 26, or complementary polynucleotides thereto.

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

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

30. A method for inhibiting the interaction between HLA-E and NKG2A (or a complex comprising NKG2A and CD94 or the extracellular domains of each of them), and / or inhibiting the interaction between PD-1 and PD-L1, the method comprising contacting the NKG2A (or a complex comprising NKG2A and CD94) and / or PD-L1 with the multispecific antibody or fragment thereof according to any one of claims 1 to 25, or the pharmaceutical composition according to claim 29.

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

32. The immune cells are NK cells or T cells, and optionally, the T cells are CD8 ＋ 32. The method of claim 31, wherein the cell is a T cell.

33. The method according to any one of claims 30 to 32, wherein the HLA-E is expressed on cancer cells.

34. A method for inhibiting the interaction between PD-1 and PD-L1, the method comprising contacting the PD-L1 with the multispecific antibody or fragment thereof described in any one of claims 1 to 25, or the pharmaceutical composition described in claim 29.

35. The method of any one of claims 30 to 34, wherein the PD-L1 is present on an immune cell or a cancer cell.

36. The PD-1 is expressed on immune cells, optionally on NK cells or T cells, and optionally on CD8 ＋ The method of any one of claims 30 to 35, wherein the antibody is expressed on a T cell.

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

38. 38. The method of claim 37, wherein the immune cell is a NK cell or a T cell.

39. The T cell is CD8 ＋ 39. The method of claim 38, wherein the cell is a T cell.

40. The method of any one of claims 37 to 39, wherein the immune cells express NKG2A and / or PD-1.

41. The method of any one of claims 37 to 40, wherein the immune cell-mediated response is an anti-tumor response, and optionally the tumor cells express HLA-E and / or PD-L1.

42. 30. A method of treating a disease or disorder in a subject, the method comprising administering to the subject a multispecific antibody or fragment thereof according to any one of claims 1 to 25, or a pharmaceutical composition according to claim 29.

43. 43. The method of claim 42, wherein the disease or disorder is cancer, and optionally, the cancer expresses HLA-E and / or PD-L1.

44. 43. The method of claim 42, wherein the disease or disorder is an autoimmune or inflammatory disease.

45. The method of any one of claims 42 to 44, wherein the subject is a human subject.

46. An antibody or fragment thereof that binds to PD-L1, wherein the antibody or fragment thereof comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:98, and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:

99.

47. (a) (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 89, 91, 94, and 97; (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 90, 92, and 95; and and a VH region comprising: (b) (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 88, 93, and 96; and and a VL region comprising:

47. The antibody or fragment thereof of claim 46, comprising:

48. (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 90; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (iii) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (iv) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 92; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (v) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 95; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (vi) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 88; The antibody or fragment thereof according to claim 46 or 47, comprising any one or more of (i) to (vi):

49. The antibody or fragment thereof according to any one of claims 46 to 48, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 98 and the VL region comprises the amino acid sequence of SEQ ID NO:

99.

50. A polynucleotide encoding the antibody or fragment thereof according to any one of claims 46 to 49.

51. 51. One or more vectors comprising one or more polynucleotides of claim 50, or complementary polynucleotides thereto.

52. A cell comprising any one or more of the antibody or fragment thereof of any one of claims 46 to 49, the polynucleotide of claim 50, or the one or more vectors of claim 51.

53. 52. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the antibody or fragment thereof of any one of claims 46 to 49, the polynucleotide of claim 50, the one or more vectors of claim 51, or the cell of claim 52.

54. A method for inhibiting the interaction between PD-1 and PD-L1, the method comprising contacting the PD-L1 with the antibody or fragment thereof described in any one of claims 46 to 49, or the pharmaceutical composition described in claim 53.

55. 52. A method for preventing the suppression of immune cells or activating immune cell-mediated responses, the method comprising contacting said immune cells with the antibody or fragment thereof of any one of claims 46 to 49, or the pharmaceutical composition of claim 53.

56. 52. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an antibody or fragment thereof according to any one of claims 46 to 49, or a pharmaceutical composition according to claim 53.

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