Agents binding to nkg2a and pd-l1 and uses thereof

Abstract

The present disclosure provides agents that bind to NKG2A and PD-L1 and uses thereof, in particular antibodies that bind to NKG2A and PD-L1.

Description

[0001] This application is a divisional application of Chinese national phase patent application No. 202380095902.5, filed on July 7, 2023, entitled "Pharmaceutical Agent Combined with NKG2A and PD-L1 and Its Use Thereof". Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 441,713, filed January 27, 2023, which is incorporated herein by reference in its entirety. References to sequence lists submitted electronically

[0003] This application contains a computer-readable sequence list that has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML submitted with this application is named "14529-109-228_SEQ_LISTING.xml", was created on July 6, 2023, and has a size of 164,926 bytes. Technical Field

[0004] This disclosure generally relates to binders, such as antibodies (including fragments thereof), to NKG2A and PD-L1, including human NKG2A and human PD-L1, and methods of using them. Background Technology

[0005] NKG2A is normally expressed on NK cells and can also be expressed on T cells (especially CD8 cells). + NKG2A is a cell surface molecule expressed on T cells. Therefore, it is a potential target for removing the suppression of immune cells and enhancing their anti-tumor response. However, binding agents targeting NKG2A have not yet achieved therapeutic success.

[0006] Programmed death ligand 1 (PD-L1) is a cell surface glycoprotein ligand that specifically binds to programmed death receptor 1 (PD-1), a key immune checkpoint receptor. PD-1 is upregulated on activated T cells, B cells, and monocytes and mediates immunosuppression. PD-L2 (another PD-1 ligand) is primarily expressed on activated antigen-presenting cells (APCs), while PD-L1 is widely expressed, including in hematopoietic lineage cells such as activated T cells, B cells, monocytes, dendritic cells, and macrophages, as well as in cells of peripheral tissues such as the heart, bone, muscle, placenta, lung, kidney, and liver. The binding of PD-L1 to PD-1 is a negative checkpoint that activates downstream signaling of the PD-1 receptor in T cells, thereby inhibiting proliferation, cytokine production and release, and T cell cytotoxicity. This inhibition of T cell activation and effector cytokine secretion can prevent autoimmunity and chronic infections. However, many tumor cells use this mechanism to protect themselves from immune attack, leading to tumor immune evasion. Many cancers overexpress PD-L1, and this overexpression is often associated with poor prognosis. In cancer, the PD-1 / PD-L1 interaction stimulates downstream signaling to suppress T cell activation, thereby leading to tumor cell survival. Although blocking the interaction between PD-1 and its ligand has been proposed as an immunotherapeutic approach to enhance T cell immune responses against tumor cells, there is still an urgent need in the field for agents that can inhibit or prevent the PD-1 / PD-L1 interaction.

[0007] Therefore, there remains a need in the art for agents that enhance immune responses and treat diseases or conditions such as cancer. The multispecific binders, compositions, and methods described herein meet this need and offer related advantages. Summary of the Invention

[0008] This disclosure provides multispecific binding agents (e.g., bispecific antibodies) having a first binding domain to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains), including human NKG2A; and one or more additional binding domains to one or more non-NKG2A targets (e.g., PD-L1). Such agents include multispecific antibodies (e.g., bispecific antibodies) that bind to NKG2A and one or more additional non-NKG2A targets (e.g., PD-L1), such as multispecific antibodies having a first binding domain to NKG2A (including human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional binding domains to one or more non-NKG2A targets (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 their respective extracellular domains) and one or more additional targets other than NKG2A (e.g., PD-L1), such as multispecific antibodies having a first binding domain binding to NKG2A (including human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional binding domains binding to one or more non-NKG2A targets (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 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 of Tables 1 to 4, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and light chain variable region described herein (e.g., Tables 1 to 4) for binding to NKG2A. (or a complex containing NKG2A and CD94 or their respective extracellular domains), including human NKG2A.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 containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139). In some embodiments, the first binding domain specifically binds to a conformational epitope formed of a set of amino acid residues comprising at least one amino acid residue from: (1) 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); or (2) each of 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 set of amino acid residues comprising at least one amino acid residue from: (1) one of the following amino acid sequences located on the surface of NKG2A: TWEESL (SEQ ID NO: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) each of two, three, four, five, or all of the following amino acid sequences located 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) each of two, three, four, five, or all of the following amino acid sequences located 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), and LQVNR (SEQ ID NO:139). NO:138) and AQCGSSI (SEQ ID NO:139). In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), wherein the additional binding domain comprises: a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as shown in Table 5; and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as shown in Table 5, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and light chain variable region described in Table 5 for binding to PD-L1 (including human PD-L1).

[0009] This disclosure also provides nucleic acids encoding the multispecific binders (e.g., antibodies or fragments thereof) provided herein, vectors containing one or more of such nucleic acids, and cells containing said nucleic acids, said vectors, or both (e.g., cells expressing said multispecific binders).

[0010] This disclosure also provides compositions comprising the multispecific binders described herein. In some embodiments, such compositions comprise 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 other than NKG2A (e.g., PD-L1), such as multispecific antibodies having a first binding domain to NKG2A (including human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional binding domains to one or more non-NKG2A targets (e.g., PD-L1). In some embodiments, such compositions include a multispecific antibody (e.g., a bispecific antibody) that binds to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional targets other than NKG2A (e.g., PD-L1), such as a multispecific antibody having a first binding domain binding to NKG2A (including human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional binding domains binding to one or more non-NKG2A targets (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 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 of Tables 1 to 4, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and light chain variable region described herein (e.g., Tables 1 to 4) for binding to NKG2A. (e.g., human NKG2A) (or a complex containing NKG2A and CD94 or their respective extracellular domains). 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 containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139).In some embodiments, the first binding domain specifically binds to a conformational epitope formed of a set of amino acid residues comprising at least one amino acid residue from: (1) 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); or (2) each of 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 set of amino acid residues comprising at least one amino acid residue from: (1) one of the following amino acid sequences located on the surface of NKG2A: TWEESL (SEQ ID NO: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) each of two, three, four, five, or all of the following amino acid sequences located 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) each of two, three, four, five, or all of the following amino acid sequences located 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), and LQVNR (SEQ ID NO:139). NO:138) and AQCGSSI (SEQ ID NO:139).In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), wherein the additional binding domain comprises: a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in Table 5; and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 shown in Table 5, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and the light chain variable region described in Table 5 for binding to PD-L1 (e.g., human PD-L1).

[0011] This disclosure further provides various uses of the binders and compositions disclosed herein, including, for example, methods for inhibiting the interaction between HLA-E and NKG2A (e.g., NKG2A expressed on immune cells), and methods for preventing the inhibition or activation of immune cell-mediated responses. Other aspects provided herein include methods for treating a subject's disease or condition with the multispecific binders or compositions provided herein. Such compositions include multispecific antibodies having a first binding domain to NKG2A (including human NKG2A) or a complex comprising NKG2A and CD94 or their respective extracellular domains) and one or more additional binding domains to one or more non-NKG2A targets (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 as shown in any 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 as shown in any of Tables 1 to 4, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and 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 their respective extracellular domains). 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 containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139).In some embodiments, the first binding domain specifically binds to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from: (1) 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); or (2) each of 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 set of amino acid residues comprising at least one amino acid residue from: (1) one of the following amino acid sequences located on the surface of NKG2A: TWEESL (SEQ ID NO: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) each of two, three, four, five, or all of the following amino acid sequences located 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) each of two, three, four, five, or all of the following amino acid sequences located 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), and LQVNR (SEQ ID NO:139). NO:138) and AQCGSSI (SEQ ID NO:139).In some embodiments, the additional binding domain binds to PD-L1 (e.g., human PD-L1), wherein the additional binding domain comprises: a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 shown in Table 5; and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 shown in Table 5, or wherein the multispecific antibody competes with an antibody having the heavy chain variable region and the light chain variable region described in Table 5 for binding to PD-L1 (e.g., human PD-L1).

[0012] In some embodiments, the binding domain to NKG2A (or a complex comprising NKG2A and CD94 or their respective extracellular domains) does not bind to NKG2C. Alternatively, the binding domain 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. Alternatively, the binding domain 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 domains of NKG2C and CD94.

[0013] This disclosure further provides binding agents to PD-L1 (e.g., human PD-L1), including binding agents comprising: a heavy chain variable (VH) region comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as shown in Table 5, and a light chain variable (VL) region comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as shown in Table 5; or binding agents that compete with antibodies having the heavy chain variable region and light chain variable region described in Table 5 for binding to PD-L1 (e.g., PD-L1). This disclosure also provides multispecific binding agents (e.g., multispecific antibodies) having a first binding domain to PD-L1, including human PD-L1, and one or more additional binding domains to one or more non-PD-L1 targets (e.g., NKG2A). Furthermore, this disclosure provides nucleic acids encoding such binding agents (e.g., antibodies or fragments thereof) provided herein, vectors comprising one or more of such nucleic acids, and cells expressing them, as well as compositions comprising any or more of the above. This article also provides methods or uses for such binders, nucleic acids, carriers, cells, and compositions. Attached Figure Description

[0014] Figure 1 Exemplary results for A3 obtained by cell binding assays are shown, which are further described in Examples 3 and 6.

[0015] Figure 2Exemplary results for A3 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0016] Figures 3A to 3C Exemplary results for A3 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0017] Figure 4 Exemplary results for A42 obtained by biomembrane interferometry (BLI) binding assay (i.e., Octet binding assay), which is further described in Examples and 6, are shown.

[0018] Figure 5 Exemplary results regarding A42 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0019] Figures 6A to 6C Exemplary results for A42 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0020] Figure 7 Exemplary results for A2 obtained by BLI binding assays are shown, which are further described in Example 6.

[0021] Figure 8 Exemplary results for A2 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0022] Figures 9A to 9C Exemplary results for A2 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0023] Figure 10 Exemplary results for A11 obtained by BLI-based assays are shown, which are further described in Example 6.

[0024] Figure 11 Exemplary results regarding A11 obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Examples 4 and 6.

[0025] Figures 12A to 12C Exemplary results for A11 obtained from exploitability measurements are shown, which are further described in Examples 5 and 6.

[0026] Figures 13A to 13F Exemplary results obtained from a cell binding assay are shown, which is further described in Example 8.

[0027] Figures 14A to 14F Exemplary results obtained from HLA-E / NKG2A inhibition assays are shown, which are further described in Example 9.

[0028] Figures 15A to 15F Exemplary results obtained from a PD-L1 / PD1 inhibition assay are shown, which is further described in Example 10.

[0029] Figures 16A to 16F Exemplary results obtained from a cytotoxicity assay are shown, which is further described in Example 11.

[0030] Figures 17A to 17E Provide exemplary results of in vitro functional evaluation assays and their statistical analysis. Figure 17A ()( Figure 17B Plotting NK threshing data as a result of differences at 95% CI; Figure 17C Plotting NK cell toxicity data as shown by differences at 95% CI; Figure 17D Plotting NK degranulation data as represented by the percentage of CD107a / b+ NK cells; and Figure 17E (NK cell toxicity data plotted as a percentage of live tumor cells), which is further described in Example 12.

[0031] Figures 18A to 18B Provides CD8+ T cell assays stimulated by influenza ( Figure 18A ) and its statistical analysis ( Figure 18B The exemplary results obtained are further described in Example 12.

[0032] Figures 19A to 19F Exemplary results obtained by SEC chromatography are shown, which are further described in Example 13.

[0033] Figures 20A to 20F Exemplary results obtained by HIC chromatography are shown, which are further described in Example 13.

[0034] Figures 21A to 21F Exemplary results obtained by SMAC chromatography are shown, which are further described in Example 13.

[0035] Figure 22 An exemplary bispecific antibody format is shown for constructing the exemplary bispecific antibody described herein.

[0036] Figures 23A to 23C Exemplary results of CellTrace Far Red and CellTrace Violet labeling experiments are provided, which are further described in Example 14. Figure 23ACompare P12 x A11, sFc data with various controls at the same test dose (2 nM). Figure 23B The study showed a dose-dependent co-binding of P12 x A11, sFc-induced tumors with NK cells. Figure 23C Compare the various target / effectant (T:E) ratios used in the experiment.

[0037] Figures 24A to 24B Provides display outside the body ( Figure 24A ) and isolated ( Figure 24B Exemplary results comparing the expression of human HLA-E (hHLA-E) and human PD-L1 (hPD-L1) on engineered MC38 cells (B-hHLA-E+ / hPD-L1 MC38 cells) with that on unengineered MC38 cells are further described in Example 15.

[0038] Figures 25A to 25C Exemplary results of in vivo functional evaluation are provided, which are further described in Example 15. Figure 24A Plot the weight changes in all groups, and Figure 25B It shows changes in tumor volume. Figure 25C The treatment was concentrated at a dose of 10 mg / kg body weight.

[0039] Figure 26 The surface reproduction of NKG2a and CD94 highlights the P12 x A11, sFc epitope.

[0040] Figure 27 Show a list of areas that are clearly protected from deuterium exchange. Detailed Implementation

[0041] This disclosure is based, at least in part, on novel multispecific binders 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 complexes comprising NKG2A and CD94 or their respective extracellular domains) and PD-L1 (e.g., human PD-L1). In some aspects, such binders can be used in compositions and methods that inhibit the interaction between HLA-E and NKG2A and / or between PD-1 and PD-L1, thereby preventing the suppression of immune cells or activating immune cell-mediated antitumor responses. Furthermore, the multispecific binders provided herein can be used to kill and / or remove tumor cells. The binders provided herein can be used in compositions and methods for treating diseases or conditions such as cancer.

[0042] It should be understood that the chapter or sub-chapter titles used in this document are for organizational purposes only and should not be construed as limiting and / or separating the topics described.

[0043] 5.1. Definition The techniques and procedures described or mentioned herein include those generally well understood and / or commonly used techniques and procedures as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009); Monoclonal Antibodies: Methods and Protocols (edited by Albitar, 2010); and Antibody Engineering, Volumes 1 and 2 (edited by Kontermann and Dübel, 2nd edition, 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terminology will be used, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any description of the terms set forth and any reference incorporated herein by reference, the description of the terms set forth below shall prevail.

[0044] Unless otherwise specified, the term "NKG2A" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any native NKG2A derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), canines, and rodents (e.g., mice and rats). NKG2A is also referred to, for example, as NK cell receptor A, NK receptor activating NKG2A, NK receptor activating NKG2-A / B, killer cell lectin-like receptor C1 (CD159a), CD159 antigen-like family member A, or type II NKG2-A / NKG2-B integrated membrane protein. NKG2A belongs to the lectin family and forms a heterodimer with CD94 (or KLRD1), another type C lectin expressed by NK cells. The NKG2A / CD94 complex binds to HLA-E (a non-classical MHC I molecule) in the human body and transduces inhibitory signals, thereby inhibiting NK and CD8. +T cell activity. NKG2A is a protein encoded by the NKG2A gene (or KLRC1). The term NKG2A covers “full-length” NKG2A, as well as any form of NKG2A or any fragment thereof produced in the cell. In some embodiments, an exemplary amino acid sequence of full-length NKG2A is provided below (see, for example, gene accession number P26715-1 in the Examples section below). In some embodiments, NKG2A includes a signal sequence. In some embodiments, NKG2A does not include a signal sequence. In some embodiments, the term NKG2A refers to a fragment of full-length NKG2A containing the extracellular domain of NKG2A. The term NKG2A also covers naturally occurring variants of NKG2A, such as SNP variants, splice variants, and allelic variants. An exemplary amino acid sequence of the extracellular domain of human NKG2A is provided below: PSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO:74). An exemplary amino acid sequence of human NKG2A is provided below: RHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO:141). An exemplary amino acid sequence of the extracellular domain of cynomolgus monkey (NKG2A) is provided below: PSTLTQKHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKEKRTWAESLLACTLKNSSLLSIDNEEEMKFLTAISPSTWTGVFRDSSQHPWVTINGLTFKHEIKDSDNAEHNCAMLHARGLKSDRCGSSKIYHCKHKL (SEQID NO:77).

[0045] In some embodiments, as used herein, the term NKG2A refers to the NKG2A epitope. Alternatively or additionally, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domain of NKG2A and CD94. In other embodiments, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domain of NKG2A and CD94, but not only an epitope on CD94 itself. Alternatively or additionally, as used herein, the term NKG2A refers to an epitope of a complex comprising NKG2A and CD94 or a complex comprising the extracellular domain of NKG2A and CD94, but not only an epitope on NKG2A itself. In some implementations, as used herein, the term NKG2A refers to an epitope of a complex containing NKG2A and CD94 or a complex containing the extracellular domains of NKG2A and CD94, but not only an epitope on NKG2A itself or only on CD94 itself.

[0046] In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope. Alternatively, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of an NKG2A stabilized by forming a complex with CD94. In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of a complex comprising NKG2A and CD94, and only on NKG2A itself. Alternatively, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of the extracellular domain of an NKG2A stabilized by forming a complex with the extracellular domain of CD94. In some embodiments, as used herein, the term NKG2A refers to an NKG2A epitope located on the surface of a complex comprising the extracellular domain of NKG2A and CD94, and only on the extracellular domain of NKG2A itself.

[0047] Unless otherwise specified, the term "NKG2C" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any naturally occurring NKG2C from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). NKG2C is also known as, for example, KLRC2, CD159c, NKG2-C, NKG2C, killer cell lectin-like receptor C2. NKG2C is a protein encoded by the NKG2C gene (or KLRC2). The term NKG2C encompasses “full-length” NKG2C, as well as any form of NKG2C or any fragment thereof produced by processing in cells. In some embodiments, NKG2C includes a signaling sequence. In some embodiments, NKG2C does not include a signaling sequence. In some embodiments, the term NKG2C refers to a fragment of full-length NKG2C containing the NKG2C extracellular domain. The term NKG2C also encompasses naturally occurring variants of NKG2C, such as SNP variants, splice variants, and allelic variants. The NKG2C gene is described in various databases with the following ID numbers: HGNC 6375; NCBI Entrez Gene3822; Ensembl ENSG00000205809; OMIM® 602891; and UniProtKB / Swiss-Prot P26717. Exemplary extracellular domains of human NKG2C are shown in the following examples section (see SEQ ID NO:75).

[0048] In some embodiments, as used herein, the term NKG2C refers to the NKG2C epitope. Alternatively or additionally, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domain of NKG2C and CD94. In other embodiments, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domain of NKG2C and CD94, but not only an epitope on CD94 itself. Alternatively or additionally, as used herein, the term NKG2C refers to an epitope of a complex comprising NKG2C and CD94 or a complex comprising the extracellular domain of NKG2C and CD94, but not only an epitope on NKG2C itself. In some implementations, as used herein, the term NKG2C refers to an epitope of a complex containing NKG2C and CD94 or a complex containing the extracellular domains of NKG2C and CD94, but not only an epitope on NKG2C itself or only on CD94 itself.

[0049] Unless otherwise specified, the term "HLA-E" refers to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any naturally occurring HLA-E or its ortholog from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). HLA-E is also referred to, for example, as major histocompatibility complex E; class I HLA histocompatibility antigen α chain E; class I MHC antigen E; HLA-6.2; class Ib MHC antigen; HLA-E; or QA1. HLA-E is a naturally occurring protein in the human body. HLA-E HLA-E is a gene-encoded protein. It belongs to class I HLA heavy chain paralogs and is approximately 45 kDa and anchored in the membrane. The term HLA-E encompasses “full-length” HLA-E, as well as any form of HLA-E or any fragment thereof produced by cellular processing. In some embodiments, HLA-E contains a signaling sequence. In some embodiments, HLA-E does not include a signaling sequence. In some embodiments, the term HLA-E refers to a fragment of full-length HLA-E containing the extracellular domain of HLA-E. The term HLA-E also encompasses naturally occurring variants of HLA-E, such as SNP variants, splice variants, and allelic variants. The HLA-E gene is described in various databases with the following ID numbers: HGNC 4962; NCBI Entrez Gene 3133; Ensembl ENSG00000204592; OMIM®143010; and UniProtKB / Swiss-Prot P13747.

[0050] Unless otherwise specified, the term "CD94" refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any natural CD94 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), 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-non-self recognition. It forms a complex with NKG2A or NKG2C on cytotoxic and regulatory lymphocyte subsets to recognize HLA-E, a non-classical Ib major histocompatibility (MHC) molecule carrying a signal sequence derived from classical Ia MHC and non-classical Ib MHC molecules. CD94-NKG2A acts as an immunosuppressive receptor and is a key inhibitory receptor on natural killer (NK) cells that regulates their activation and effector functions. CD94-NKG2C acts as an immune-activating receptor and is an HLA-E receptor on cytotoxic lymphocyte subsets that recognizes a signal sequence-derived peptide from a non-classical class Ib MHC HLA-G molecule. The term CD94 covers “full-length” CD94, as well as any form of CD94 or any fragment thereof produced in the cell. In some embodiments, CD94 contains a signal sequence. In some embodiments, CD94 does not contain a signal sequence. In some embodiments, the term CD94 refers to a fragment of full-length CD94 containing the extracellular domain of CD94. The term CD94 also covers naturally occurring variants of CD94, such as SNP variants, splice variants, and allelic variants. The CD94 gene is described in various databases with the following ID numbers: HGNC 6378; NCBI Entrez Gene 3824; Ensembl ENSG00000134539; OMIM® 602894; and UniProtKB / Swiss-Prot Q13241. An exemplary amino acid sequence of human CD94 is provided below: SFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI (SEQ ID NO: 140).

[0051] Unless otherwise specified, the terms “programmed cell death ligand-1 (PD-L1)”, “programmed death ligand-1”, “PD-1 ligand 1”, or similar terms refer to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any native PD-L1 derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). PD-L1, also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. PD-L1 is one of two naturally occurring cell surface glycoprotein ligands of PD-1 (the other being 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. It is known in the art that PD-L1 downregulates T cell activation and cytokine secretion upon binding to PD-1. The term PD-L1 encompasses “full-length” PD-L1, as well as any form of PD-L1 or any fragment thereof produced during cellular processing. The term PD-L1 also encompasses 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 provided below (exemplary extracellular domain = underlined text): MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 100). The term PD-L1 also encompasses other related PD-L1 peptides including fragments, derivatives (e.g., substituted, deleted, truncated, and inserted variants), fusion peptides, and interspecies homologs that maintain PD-L1 activity and / or are sufficient to generate an anti-PD-L1 immune response. Those skilled in the art will understand that the PD-L1 binders (e.g., antibodies) described herein can bind to PD-L1 peptides, PD-L1 peptide fragments, PD-L1 antigens, and / or PD-L1 epitopes. An epitope may be part of a larger PD-L1 antigen, which may be part of a larger PD-L1 peptide fragment, which in turn may be part of a larger PD-L1 peptide. PD-L1 can exist in natural or denatured forms. The PD-L1 peptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. PD-L1 peptides may comprise peptides having the same amino acid sequence as corresponding PD-L1 peptides derived from nature. Orthologs of PD-L1 peptides are also well known in the art.

[0052] Unless otherwise specified, the terms “programmed cell death protein-1 (PD-1),” “programmed cell death protein-1,” “PD-1 receptor,” or similar terms refer to a polypeptide (“polypeptide” and “protein” are used interchangeably herein) or any native PD-1 derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). PD-1, also known as CD279 (differentiation cluster 279), is an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on pre-activated T cells in vivo 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: the immunoreceptor tyrosine repressive motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). The term PD-1 encompasses “full-length” PD-1, as well as any form of PD-1 or any fragment thereof produced by processing in the cell. The term PD-1 also encompasses naturally occurring variants of PD-1, such as SNP variants, splice variants, and allelic variants. Upon T cell stimulation, PD-1 is known in the art to recruit the tyrosine phosphatase SHP-2 to the ITSM motif within its cytoplasmic tail, particularly causing dephosphorylation of effector molecules involved in the CD3 T cell signaling cascade, such as CD3ζ, PKCθ, and ZAP70 (Carter et al. (2002) Eur J Immunol 32:634-43). An exemplary full-length amino acid sequence of human PD-1 is provided below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 101).

[0053] As used herein, the term "conjugate" or its grammatical equivalent refers to a molecule (e.g., an antibody) having one or more antigen-binding sites that bind to antigens. In some embodiments, the conjugates described herein are antibodies (including multispecific antibodies and antibody fragments, such as antigen-binding fragments or epitope-binding fragments) or other peptide-based molecules that bind to NKG2A (e.g., human NKG2A) and / or PD-L1 (e.g., human PD-L1), as well as conjugates of antibodies, antibody fragments, or peptide-based molecules (e.g., antibody-drug conjugates).

[0054] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein and in the broadest sense, and specifically cover, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of a heavy-chain-only antibody. Exemplary single-domain antibodies include, but are not limited to, antibodies naturally lacking a light chain, such as antibodies from camel species (e.g., llamas); single-domain antibodies derived from conventional four-chain antibodies; engineered antibodies; and single-domain backbones other than those derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. VHH can also be derived from other species besides camelids that produce naturally occurring heavy chain antibodies lacking the light chain. Antibodies also include antibody fragments (and / or polypeptides containing antibody fragments) that retain NKG2A and / or PD-L1 binding characteristics. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate-domain antibodies, single-domain antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, bifunctional antibodies, di-diabody antibodies, disulfide-linked Fv (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments composed of non-covalently coupled heavy and light chain variable regions). Generally, the variable (V) region can be any suitable arrangement of immunoglobulin heavy chain (VH) and / or light chain (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the V region can be a dimer and contain VHH-VHH, VH-VH, VH-VL, or VL-VL dimers binding NKG2A and / or PD-L1. Where necessary, VH and VL can be covalently coupled directly or via a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of the antibody. CDRs (also known as “minimum recognition units” or “hypervariates”) can be obtained by constructing polynucleotides encoding one or more CDRs of interest.Such polynucleotides are prepared, for example, by using polymerase chain reaction to synthesize variable regions using mRNA from antibody-producing cells as templates (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies”, Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (ed.), p. 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies”, Monoclonal Antibodies: Principles and Applications, Birch et al. (ed.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single-domain antibodies, macrobodies, microbodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, variable domains of neoantigen receptors (v-NAR), and double-single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies containing VH and / or VL further contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include epitope-binding fragments of any of the above. The antibodies described herein may belong to any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0055] When used with reference to binders (e.g., antibodies) as described herein, the term “monospecific” means a binder having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0056] When used as a reference binder (e.g., an antibody), the term "bispecific" means that the binder can specifically bind to at least two different epitopes, such as two binding sites, each formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or each formed by a pair of VHH domains binding to different antigens or different epitopes on the same antigen. Such bispecific binders (e.g., antibodies) may have a 1+1 format (containing one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binders (e.g., antibodies) may have a 2+1 or 1+2 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binder (e.g., an antibody) contains two antigen binding sites, each can bind to a different epitope. Such bispecific binders (e.g., antibodies) can bind to two different epitopes on the same antigen (e.g., epitopes on NKG2A or PD-L1).

[0057] The term "identity" or "percentage of identity" in the context of two or more nucleic acids or peptides refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence (with gaps introduced where necessary), are identical or have a specified percentage of identical nucleotide or amino acid residues, without taking into account any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or peptides are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured by sequence comparison algorithms or by visual inspection, they have 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% nucleotide or amino acid residue identity. In some embodiments, identity exists in regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value between these lengths of the amino acid sequence. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical throughout the full length of the compared sequence (e.g., the coding region of a target protein or antibody). In some embodiments, identity exists in regions of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value between these lengths of the nucleotide sequence. In some embodiments, identity exists in regions longer than 60-80 bases (e.g., at least about 80-1000 bases or longer), and in some embodiments, the sequences are substantially identical throughout the full length of the compared sequence (e.g., the nucleotide sequence encoding the protein of interest).

[0058] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue with a side chain having similar chemical characteristics. Families of amino acid residues with similar side chains are generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine replacing tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of peptides, soluble proteins, and / or antibodies of this disclosure do not eliminate the binding of the peptide, soluble protein, or antibody containing the said amino acid sequence to the target binding site. Methods for identifying conserved amino acid substitutions that do not eliminate binding are well known in the art.

[0059] The term "peptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may include (e.g., interspersed with) non-amino acids. The term also covers amino acid polymers that have been naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipoylation, acetylation, phosphorylation, or any other operation or modification, such as partial linkage or conjugation (directly or indirectly) to, for example, a labeled component or a drug (e.g., a toxin). The definition also includes, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that, because the peptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the peptides may exist in single-chain or single-chain dimer form.

[0060] As used herein, an "antigen" is a portion or molecule containing an epitope that a binding agent (e.g., an antibody) can bind. Therefore, an antigen can be bound by an antibody. In some embodiments, the antigen bound by the binding agent (e.g., an antibody) described herein is NKG2A (e.g., human NKG2A) or a fragment thereof, including fragments containing one or more domains of NKG2A. In some embodiments, the antigen bound by the binding agent (e.g., an antibody) described herein is PD-L1 (e.g., human PD-L1) or a fragment thereof, including fragments containing one or more domains of PD-L1.

[0061] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. For example, in the case of a peptide antigen, the epitope can be an adjacent amino acid of a peptide, such as human NKG2A or human PD-L1 ("linear" epitope), or the epitope can comprise amino acids from two or more non-adjacent regions of the peptide ("conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will understand that, in general, a linear epitope may or may not depend on secondary, tertiary, or quaternary structures. For example, in some embodiments, an antibody binds to a set of amino acids, regardless of whether it folds into a native three-dimensional protein structure. In other embodiments, the amino acid residues constituting the epitope require the antibody to exhibit a specific conformation (e.g., bending, twisting, turning, or folding) in order to recognize and bind to the epitope.

[0062] When an antibody and a reference antibody recognize the same, overlapping, or adjacent epitopes in three-dimensional space, the antibody binds to the reference antibody at an “epitope”, “substantially identical epitope”, or “identical epitope.” The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competition assay, which can be configured in many different formats, such as using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and radioactive, fluorescent, or enzyme labeling is used to measure the ability of an unlabeled antibody to block the binding of a labeled antibody.

[0063] "Epitope grouping" is a method of grouping antibodies based on the epitopes they recognize. More specifically, epitope grouping includes methods and systems for identifying the epitope recognition characteristics of different antibodies using a combination of competitive assays and computational methods, which cluster antibodies based on their epitope recognition characteristics and identify antibodies with different binding specificities.

[0064] As used herein, the terms “specific binding,” “specific recognition,” “immunospecific binding,” “selective binding,” “immunospecific recognition,” and “immunospecific” are similar terms in the context of antibodies and refer to molecules that bind to antigens (e.g., epitopes), such binding being understood by those skilled in the art. In some embodiments, “specific binding” means, for example, that the interaction of a peptide or molecule with an epitope, protein, or target molecule is more frequent, faster, longer-lasting, more affinity, or a combination thereof than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may typically bind to other peptides or polypeptides with lower affinity, which is determined by, for example, an immunoassay, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), an antibody or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with a higher affinity than any cross-reactive antigen. Typically, the specific or selective reaction will be at least twice the background signal or noise and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed., 1989). In some embodiments, as determined, for example by fluorescence activated cell sorting (FACS) analysis or RIA, the degree of binding of the antibody or antigen-binding domain to a “non-target” protein is less than about 10% of the binding of the antibody or antigen-binding domain to its specific target antigen. In some embodiments, molecules that specifically bind to an antigen bind to the antigen with a Ka at least 2 log, 2.5 log, 3 log, 4 log, or greater than the Ka at which the molecule binds to another antigen. In some embodiments, molecules that specifically bind to an antigen do not cross-react with other proteins. In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other non-NKG2A proteins. In some embodiments, "specific binding" means, for example, a peptide or molecule at a K+ level of about 0.1 mM or less, but more typically less than about 1 µM. D Binding to a protein or target. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration of at least about 0.1 µM or less, at least about 0.01 µM or less, or at least about 1 nM or less. DTarget binding. Due to sequence identity among homologous proteins in different species, specific binding may include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding may include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, an antibody or molecule that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, such as binding to a single target. Thus, in some embodiments, a peptide or molecule may specifically bind to more than one target. In some embodiments, multiple targets may be bound by the same antigen-binding site on the peptide or molecule. For example, in some cases, an antibody may contain two identical antigen-binding sites, each specifically binding to the same epitope on two or more proteins. In some alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities. Generally, but not necessarily, the term “binding” refers to “specific binding.”

[0065] "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 that reflects the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be determined by the dissociation constant (K). D ( ) indicates. Affinity can be measured by commonly used methods known in the art, including those described herein. Low-affinity antibodies typically bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind to antigens more quickly and tend to remain bound for a longer period. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. In one embodiment, “K D "or "K D The "value" can be measured via biomembrane interferometry (BLI) using, for example, the Octet QK384 system (ForteBio, Menlo Park, CA). Alternatively, K... DIt can also be performed in a radiolabeled antigen-binding assay (RIA) using, for example, the Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881), or using a surface plasmon resonance (SPR) assay via BIACORE™, using, for example, BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). “On-rate” or “rate of association” or “association rate” or “k on "and "off-rate" or "rate of dissociation" or "dissociation rate" or "k off "The same SPR or BLI techniques described above can also be used, for example, with the Octet QK384 system (ForteBio, Menlo Park, CA) or BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ) for determination."

[0066] When used in the context of binders (e.g., antibodies), the term "competition" or any grammatical variation thereof means that binders compete for the same epitope or binding site on the target, including competition between such binders as determined by assays in which the binder under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or a reference antigen-binding protein, such as a reference antibody) to a common antigen (e.g., NKG2A or PD-L1). Various types of competitive binding assays can be used to determine whether the test binder 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 may be used include solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (see, for example, Stahli et al. (1983), Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al. (1986), J. Immunol. 137:3614-3619 or Cheung et al. (1990) Virology 176:546-552); solid-phase direct labeling assay; solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see, for example, Morel et al. (1988), Molec. Immunol. 25:7-15); and direct labeling RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve using purified antigens (e.g., NKG2A, such as human NKG2A; or PD-L1, such as human PD-L1) bound to a solid surface or cell with an unlabeled test antigen-binding protein (e.g., an antibody testing NKG2A or an antibody testing PD-L1) 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 cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies bound to the same epitope as the reference antibody and / or antibodies bound to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to cause steric hindrance (e.g., similar epitopes or overlapping epitopes).Typically, when a competitive antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 20%, such as at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or a higher percentage.

[0067] As used herein, the terms "constant region" or "constant domain" are well-known antibody terms in the art and refer to antibody portions, such as the carboxyl-terminal portions of the light and / or heavy chains, that do not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The term also includes portions of immunoglobulin molecules that have a generally more conserved amino acid sequence relative to the immunoglobulin variable domain.

[0068] Antibody "effector functions" refer to biological activities attributable to the antibody's Fc region (e.g., the native Fc region or the Fc region with amino acid sequence variations), and vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0069] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine residue (residue 447, according to the EU numbering system) of the Fc region may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. An exemplary Fc region sequence is provided below (CH2 domain = bold text; CH3 domain = underlined text): CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:83).

[0070] The “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (e.g., antibody-dependent phagocytosis, i.e., ADCP); downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require combining the Fc region with a binding region or binding domain (e.g., antibody variable region or domain) and can be assessed using the various assays disclosed.

[0071] "Natural sequence Fc regions" contain the same amino acid sequence as the Fc regions found in nature and have not been artificially manipulated, modified, and / or altered (e.g., isolated, purified, selected, including other sequences such as variable region sequences or combinations with other sequences such as variable region sequences). Natural sequence human Fc regions include natural sequence human IgG1 Fc regions (non-A and A isoforms); natural sequence human IgG2 Fc regions; natural sequence human IgG3 Fc regions; and natural sequence human IgG4 Fc regions, as well as their naturally occurring variants.

[0072] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution in the native Fc region or the parental peptide Fc region, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, compared to the native Fc region or the parental peptide Fc region. The variant Fc region described herein may have at least about 80% homology with the native Fc region and / or the parental peptide Fc region, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may have loss of effector function (e.g., silenced Fc). An exemplary variant Fc region (“silent Fc”) sequence is provided below (CH2 domain = bold text, where amino acid changes are underlined; CH3 domain = underlined text): CPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL K APIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:84).

[0073] As used herein, when referring to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50–70 kDa, wherein the amino-terminal portion comprises a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion comprises one or more constant regions. "Heavy chain" can refer to any different type, such as α (alpha), δ (delta), ε (epsilon), γ (gamma), and µ (mu), based on the amino acid sequence of the constant regions, which respectively generate the IgA, IgD, IgE, IgG, and IgM antibody classes, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0074] As used herein, when referring to antibodies, the term "light chain" can refer to a polypeptide chain of approximately 25 kDa, wherein the N-terminal portion comprises a variable region of approximately 100 to approximately 110 or more amino acids, and the C-terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, two distinct types exist, referred to as κ (kappa) or λ (lambda). The amino acid sequences of light chains are well known in the art.

[0075] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody containing amino acid residues that interact with an antigen and confer specificity and affinity to the antigen (e.g., a CDR). As used herein, “antigen-binding fragment” includes “antibody fragment,” which contains portions of an antibody that include one or more CDRs, such as an antigen-binding region or variable region of an antibody.

[0076] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-individual genotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0077] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, including molecules containing one or more antigen-binding sites that bind to NKG2A antigen and / or PD-L1 antigen.

[0078] Antibodies can be any type (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) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG) or their classes (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclasses.

[0079] In some embodiments, the antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In other 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. Alternatively or additionally, the amino acid sequences of the L chains are different from each other. For example, the antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to an NKG2A antigen and the second H / L chain pair binds to a non-NKG2A (e.g., PD-L1) antigen. In some embodiments, the antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In other 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, the antibody comprises a first VHH and a second VHH, wherein the first VHH binds to an NKG2A antigen and the second VHH binds to a non-NKG2A (e.g., PD-L1) antigen. In some embodiments, the H chains and / or L chains comprise constant regions, such as human constant regions. In some embodiments, the L-chain constant region of such antibodies is a κ or λ light chain constant region, such as the human κ or λ light chain constant region. In some embodiments, the H-chain constant region of such antibodies comprises a γ heavy chain constant region, such as the human γ heavy chain constant region. In some embodiments, such antibodies comprise an IgG constant region, such as the human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0080] The antibody or fragment thereof may preferentially bind to NKG2A (e.g., human NKG2A) and / or PD-L1 (e.g., human PD-L1), meaning that the antibody or fragment thereof binds to NKG2A and / or PD-L1 with a higher affinity than it binds to a control protein (e.g., an unrelated control protein, such as chicken egg white lysozyme, or NKG2C), and / or binds to human NKG2A and / or PD-L1 with a higher 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 a portion thereof. “Specific binding” means that the antibody or fragment thereof binds to NKG2A and / or PD-L1 with an affinity at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than its affinity for an unrelated control protein (e.g., chicken egg white lysozyme). In some embodiments, the antibody or a fragment thereof may bind substantially only to NKG2A and / or PD-L1 (e.g., capable of distinguishing NKG2A and / or PD-L1 from other known peptides, for example, by means of measurable differences in binding affinity). In some embodiments, the NKG2A binder (e.g., antibody) may react with NKG2A sequences other than human NKG2A sequences (e.g., cynomolgus monkey NKG2A sequences, such as A42 described herein). In other embodiments, the NKG2A binder (e.g., antibody) does not react with non-human (e.g., cynomolgus monkey) NKG2A sequences, such as A2, A3, and A11 provided herein. In some embodiments, the PD-L1 binder (e.g., antibody) may react with PD-L1 sequences other than human PD-L1 sequences (e.g., cynomolgus monkey PD-L1 sequences). In other embodiments, the PD-L1 binder (e.g., antibody) does not react with non-human (e.g., cynomolgus monkey) PD-L1 sequences.

[0081] The term "variable region" or "variable domain" refers to a portion of the antibody light or heavy chain, typically located at the amino terminus of the light or heavy chain, having a length of approximately 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be called "VH". The variable region of the light chain may be called "VL". The term "variable" refers to the fact that the sequence of certain segments of the variable region varies widely among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. In fact, the V region consists of less variable (e.g., relatively invariant) segments of about 15-30 amino acids called framework regions (FRs), separated by shorter regions of about 15-30 amino acids with greater (e.g., highly variable) variability (e.g., extremely variable) variability (e.g., highly variable) variability (e.g., relatively invariant) variability (e.g., relatively constant) variability (e.g., relatively invariant ... The variable regions of both the heavy and light chains each contain four frames (FR1, FR2, FR3, and FR4) that primarily exhibit a β-sheet conformation, connected by three hypervariable regions. These hypervariable regions form loops connecting the β-sheet structure and, in some cases, portions of the β-sheet structure. The hypervariable regions in each chain are tightly held together by the frames and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as enabling antibodies to participate in antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variability is concentrated in the CDRs, while the less varied portions of the variable regions are called frame regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for the interaction between the antibody and its antigen. In a particular implementation, the variable area is a human variable area.

[0082] When used herein, the terms “hypervariant region,” “HVR,” “HV,” “complementarity-determining region,” or “CDR” refer to regions in the antibody variable region that are sequence-hypervariant and / or form structurally defined loops. Typically, an antibody contains six hypervariant regions: three located in the VH (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three located in the VL (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Various hypervariant region descriptions have been used and are covered herein. Kabat CDR is based on sequence variability and is the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the position of the structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering scheme, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places the inserted sequence at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents the balance between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software of Oxford Molecular (see, for example, Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable region is based on the analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are shown below.

[0083] A universal numbering system has been developed and is widely used: ImMunoGeneTics (IMGT) ®Information system (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are referred to based on their amino acid sequence and position within the light or heavy chain. Since the “position” of CDRs within the variable domain structure of immunoglobulins is conserved across species and exists in structures called loops, CDRs and framework residues are easily identified by using a numbering system that aligns the variable domain sequence based on structural features. This information can be used to transplant and replace CDR residues of immunoglobulins from one species into the receptor framework, which is typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol. Another numbering system (AHon) has been developed (309: 657-670 (2001). The correspondence between numbering systems, including, for example, the Kabat numbering system and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.) and is also described below. The various systems known in the art or described herein represent different approaches to depicting CDRs and are generally considered equivalent when used to define the same antibody. The exemplary system combination shown herein is Kabat and Chothia. Examples of residues from each of these hypervariable regions or CDRs are listed in the table below.

[0084] Exemplary CDRs based on various numbering systems

[0085] The high-variability region may include the following “extended high-variability regions”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL; and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms “high-variability region,” “HVR,” “HV,” “complementary determinant region,” and “CDR” are used interchangeably.

[0086] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes both DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that may be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. Cells that produce the binding molecules of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies are introduced. Unless otherwise stated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is referred to as the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of nascent RNA transcripts is referred to as the transcription direction; a sequence region on the DNA strand having the same sequence as the RNA transcript and being 5' end relative to the 5' end of the RNA transcript is referred to as the "upstream sequence"; a sequence region on the DNA strand having the same sequence as the RNA transcript and being 3' end relative to the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0087] The term "vector" refers to a substance used to carry or include a nucleic acid sequence, including, for example, those used to introduce a nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, cell-free genomes, and artificial chromosomes, which may include selective sequences or markers operable to stably integrate into the host cell chromosome. Additionally, a vector may include one or more selective marker genes and appropriate expression control sequences. Selective marker genes may include, for example, providing resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supplying critical nutrients absent in the culture medium. Expression control sequences may include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more nucleic acid molecules (e.g., antibody heavy and light chains or antibodies VH and VL) are intended to be co-expressed, the two nucleic acid molecules may be inserted into, for example, a single expression vector or separate expression vectors. For single-vector expression, the encoding nucleic acid may be operably ligated to a shared expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the nucleic acid molecule into the host cell can be confirmed using methods well-known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting of gene product expression, or other suitable analytical methods for testing the expression of an introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules are expressed in amounts sufficient to produce the desired product (e.g., the binding agent described herein), and will further understand that expression levels can be optimized to obtain sufficient expression using methods well-known in the art.

[0088] As used herein, the term “pharmaceutical acceptable” means approved by federal or state regulatory agencies or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0089] "Excipient" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freunds' adjuvant (complete or incomplete)), or medium. In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition, 1990). In one implementation, each component is "pharmaceutically acceptable" in the sense that it is compatible with other components of the pharmaceutical preparation and suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to cells or mammals exposed to it at the doses and concentrations used. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffer solution. In some embodiments, the excipient is a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid excipients, especially for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. The composition may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions (including formulations) may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Compositions, including pharmaceutical compounds, may contain preventatively or therapeutically effective amounts of, for example, isolated or purified forms of NKG2A and / or PD-L1 binders (e.g., antibodies), and suitable amounts of excipients to provide a form suitable for administration to a subject (e.g., a patient). The formulation should be suitable for the administration method.

[0090] An "effective amount" is generally an amount sufficient to achieve the following effects: reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent or delay the occurrence of symptoms and / or underlying causes, and / or improve or repair damage caused by or related to a disease, condition, or disorder. In some implementations, the effective amount is a therapeutically effective amount or a preventatively effective amount.

[0091] As used herein, the term "therapeuticly effective amount" refers to an amount of a pharmaceutical agent (e.g., an antibody or any other pharmaceutical agent described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, condition, or disorder and / or associated symptoms. A therapeutically effective amount of a pharmaceutical agent (including a therapeutic agent) may be an amount desired to: (i) slow, delay, or improve the progression or course of a given disease, condition, or disorder; (ii) reduce, delay, or improve the recurrence, development, or onset of a given disease, condition, or disorder; and / or (iii) improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than the administration of a pharmaceutical agent described herein). The "therapeuticly effective amount" of the substances / molecules / pharmaceutical agents (e.g., bispecific antibodies) disclosed herein may vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the substance / molecule / pharmaceutical agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effect of the substance / molecule / pharmaceutical agent exceeds any toxic or harmful effect. In some implementations, the term "therapeutic effective amount" refers to the amount of a multispecific binder that effectively "treats" a disease, symptom, or ailment of a subject or mammal.

[0092] The term “treatment” or any grammatical variation thereof means reducing and / or improving the severity and / or duration of a given disease, condition or disorder, and / or associated symptoms, such as (i) slowing, delaying or improving the progression or course of a given disease, condition or disorder; (ii) reducing, delaying or improving the recurrence, development or onset of a given disease, condition or disorder; and / or (iii) improving or enhancing the preventive or therapeutic effect of another therapy (e.g., a therapy other than the application of the multispecific binder described herein).

[0093] "Prophylactic effective amount" is an amount of pharmaceutical composition that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of a disease, ailment, or disorder, or reducing the likelihood of the onset (or recurrence) of a disease, ailment, or disorder or related symptoms.

[0094] A complete therapeutic or preventative effect may not occur with a single dose and may only occur after a series of doses. Therefore, an effective dose for treatment or prevention may be administered in one or more doses.

[0095] The terms “about” and “approximately” refer to a variation of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0096] As used herein, comparative terms such as decrease, reduction, increase, or any grammatical variation thereof may refer to a change relative to a reference value. In some embodiments, such a change may refer to a multiple of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 10, 20, 30, 40, 100, or more than 100 times the reference value. In some implementations, such changes may refer to approximately 1%, or approximately 2%, or approximately 3%, or approximately 4%, or approximately 5%, or approximately 6%, or approximately 7%, or approximately 8%, or approximately 9%, or approximately 10%, or approximately 20%, or approximately 30%, or approximately 40%, or approximately 50%, or approximately 60%, or approximately 70%, or approximately 80%, or approximately 90%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99% of the reference value.

[0097] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this disclosure and claims include the plural forms.

[0098] In some implementations, the terms "first," "second," "third," "fourth," and similar terms in component names are used to distinguish and identify more than one component that shares some commonality in the name. For example, "first antibody" and "second antibody" are used to distinguish two antibodies.

[0099] It should be understood that when the term "comprising" is used to describe an embodiment herein, other similar embodiments described using the terms "consisting of" and / or "substantially consisting of" are also provided. It should also be understood that when the phrase "substantially consisting of" is used to describe an embodiment herein, other similar embodiments described using the term "consisting of" are also provided.

[0100] The term "between" as used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0101] As used herein in phrases such as “A and / or B”, the term “and / or” is intended to include A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B and / or C”, the term “and / or” is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0102] The terms “optional” or “optionally” mean that the situation described below may or may not occur, and therefore this specification includes instances where the situation occurs and instances where the situation does not occur.

[0103] 5.2. Multispecific binders 5.2.1. NKG2A Binding Domain The multispecific binders provided herein comprise one or more NKG2A binding domains. In some embodiments, multispecific binders (e.g., bispecific antibodies) that bind to NKG2A are described herein. 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., containing 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 binder (e.g., a bispecific antibody) may bind to NKG2A expressed on the surface of mammalian (e.g., human) cells, including immune cells (e.g., NK cells or T cells) that express NKG2A. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein bind to NKG2A extracellular epitopes of cells exposed to, for example, immune cells. In some embodiments, a multispecific binder (e.g., a bispecific antibody) that binds to NKG2A, such as human NKG2A or a portion thereof, is described herein. In some embodiments, NKG2A is human NKG2A. In some embodiments, the multispecific binders provided herein are human NKG2A binders (e.g., antibodies that bind to human NKG2A). In some embodiments, the multispecific binders to NKG2A disclosed herein (e.g., bispecific antibodies) bind to both human and cynomolgus monkey NKG2A. In other embodiments, the multispecific binders to NKG2A disclosed herein (e.g., bispecific antibodies) bind to human NKG2A but not to cynomolgus monkey NKG2A. In some embodiments, multispecific binders (e.g., bispecific antibodies) that bind to a complex comprising NKG2A and CD94 or a complex comprising the extracellular domains of NKG2A and CD94 are described herein.

[0104] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein are available in quantities of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) D The antibody binds to NKG2A (e.g., human NKG2A). Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure, including by RIA, performed, for example, in Fab format with the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biomembrane interferometry (BLI) or surface plasmon resonance (SPR) assays using OCTET. ® Using, for example, OCTET ® Red96 system; or via BIACORE ® Using, for example, BIACORE ® TM-2000 or BIACORE ® TM-3000. The terms "on-rate," "rate of association," "association rate," or "kon" can also be used with the same biomembrane interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using, for example, OCTET. ® Red96, BIACORE ® TM-2000 or BIACORE ® TM-3000 system, BIACORE ® TM-8K or BIACORE ® Measurements were taken using the TM-8K+ system.

[0105] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein do not bind to NKG2C (or complexes comprising NKG2C and CD94 or their respective extracellular domains) (e.g., human NKG2C and / or cynomolgus monkey NKG2C). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein do not bind to human NKG2C. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein do not bind to human NKG2C or cynomolgus monkey NKG2C. In other embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein bind to NKG2A (e.g., human NKG2A) (or complexes comprising NKG2A and CD94 or their respective extracellular domains) with a higher affinity than to NKG2C (e.g., human NKG2C) (or complexes comprising NKG2A and CD94 or their respective extracellular domains). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least twice as strong as their binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least five times as strong as their binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 10 times stronger than their binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 100 times stronger than their binding affinity for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein have a binding affinity for NKG2A (e.g., human NKG2A) (or a complex comprising NKG2A and CD94 or their respective extracellular domains) that is at least 1000 times stronger than that for NKG2C (e.g., human NKG2C) (or a complex comprising NKG2C and CD94 or their respective extracellular domains).

[0106] In some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein comprise the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of any of the antibodies described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and / or VL CDR3 depicted in Tables 1 to 4. Therefore, in some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein comprise any, any two and / or all three heavy chain CDRs and / or any, any two and / or all three light chain CDRs: (a) an antibody named A2; (b) an antibody named A3; (c) an antibody named A11; and (d) an antibody named A42, as shown in Tables 1 to 4. In some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein comprise any, any two, and / or all three heavy chain CDRs and any, any two, and / or all three light chain CDRs: (a) an antibody named A2; (b) an antibody named A3; (c) an antibody named A11; and (d) an antibody named A42, as shown in Tables 1 through 4.

[0107] In some embodiments, the multispecific binder (e.g., a bispecific antibody) comprises a VH region and / or a VL region of any of the binders described herein, wherein the VH region comprises VH CDR1, VH CDR2, and / or VH CDR3, and the VL region comprises VL CDR1, VL CDR2, and / or VL CDR3 (see, for example, any of Tables 1 through 4). Therefore, in some embodiments, the multispecific binder (e.g., a bispecific antibody) described herein comprises any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from Table 1. In some embodiments, the multispecific binder (e.g., a bispecific antibody) described herein comprises any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from Table 2. In some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein comprise any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from Table 3. In some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein comprise any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from Table 4.

[0108] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise (i) VHCDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 45, or SEQ ID NO: 64; and / or (ii) VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 46, SEQ ID NO: 65, or SEQ ID NO: 73.

[0109] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and / or VL CDR1, VL CDR2, and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDRs are based on Chothia numbering. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above, such as a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated in Section 5.1 above.

[0110] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13, and 18; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 8, 14, 19, and 24; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 9, 15, and 20; and / or (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16, and 21; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, and 22; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 17, and 23.

[0111] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1, VHCDR2 containing the amino acid sequence of SEQ ID NO:2, and VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0112] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:7, VHCDR2 containing the amino acid sequence of SEQ ID NO:8, and VH CDR3 containing the amino acid sequence of SEQ ID NO:9; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:10, VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0113] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:12, VHCDR2 containing the amino acid sequence of SEQ ID NO:2, and VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0114] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:13, VHCDR2 containing the amino acid sequence of SEQ ID NO:14, and VH CDR3 containing the amino acid sequence of SEQ ID NO:15; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:16, VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL CDR3 containing the amino acid sequence of SEQ ID NO:17.

[0115] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:18, VHCDR2 containing the amino acid sequence of SEQ ID NO:19, and VH CDR3 containing the amino acid sequence of SEQ ID NO:20; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:21, VL CDR2 containing the amino acid sequence of SEQ ID NO:22, and VL CDR3 containing the amino acid sequence of SEQ ID NO:23.

[0116] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1, VHCDR2 containing the amino acid sequence of SEQ ID NO:24, and VH CDR3 containing the amino acid sequence of SEQ ID NO:3; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0117] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and / or VL CDR1, VL CDR2, and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDRs are based on Chothia numbering. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above, such as a combination of Kabat and Chothia.

[0118] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13, and 18; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 32, 35, 39, and 44; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 33, 36, and 40; and / or (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 34, 37, and 41; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 11, and 42; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 38, and 43.

[0119] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1, VHCDR2 containing the amino acid sequence of SEQ ID NO:27, and VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:29, VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0120] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:7, VHCDR2 containing the amino acid sequence of SEQ ID NO:32, and VH CDR3 containing the amino acid sequence of SEQ ID NO:33; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:34, VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0121] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:12, VHCDR2 containing the amino acid sequence of SEQ ID NO:27, and VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:29, VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0122] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 13, VHCDR2 containing the amino acid sequence of SEQ ID NO: 35, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 36; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 37, VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 38.

[0123] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:18, VHCDR2 containing the amino acid sequence of SEQ ID NO:39, and VH CDR3 containing the amino acid sequence of SEQ ID NO:40; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:41, VL CDR2 containing the amino acid sequence of SEQ ID NO:42, and VL CDR3 containing the amino acid sequence of SEQ ID NO:43.

[0124] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1, VHCDR2 containing the amino acid sequence of SEQ ID NO:44, and VH CDR3 containing the amino acid sequence of SEQ ID NO:28; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:29, VL CDR2 containing the amino acid sequence of SEQ ID NO:30, and VL CDR3 containing the amino acid sequence of SEQ ID NO:31.

[0125] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VL CDR1, VL CDR2, and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDRs are based on Chothia numbering. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above, such as a combination of Kabat and Chothia.

[0126] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:47, 51, 54, 55, and 59; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:48, 52, 56, 60, and 63; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:49, 53, 57, and 61; and (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:4, 10, 16, and 21; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:5, 11, and 22; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:50, 58, and 62.

[0127] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VHCDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0128] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:51, VHCDR2 containing the amino acid sequence of SEQ ID NO:52, and VH CDR3 containing the amino acid sequence of SEQ ID NO:53; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:10, VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0129] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:54, VHCDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0130] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:55, VHCDR2 containing the amino acid sequence of SEQ ID NO:56, and VH CDR3 containing the amino acid sequence of SEQ ID NO:57; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:16, VL CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL CDR3 containing the amino acid sequence of SEQ ID NO:58.

[0131] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:59, VHCDR2 containing the amino acid sequence of SEQ ID NO:60, and VH CDR3 containing the amino acid sequence of SEQ ID NO:61; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:21, VL CDR2 containing the amino acid sequence of SEQ ID NO:22, and VL CDR3 containing the amino acid sequence of SEQ ID NO:62.

[0132] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VHCDR2 containing the amino acid sequence of SEQ ID NO:63, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:4, VL CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0133] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and / or VL CDR1, VL CDR2, and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDRs are based on IMGT numbering. In some embodiments, the CDRs are based on Kabat numbering. In some embodiments, the CDRs are based on AbM numbering. In other embodiments, the CDRs are based on Chothia numbering. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above, such as a combination of Kabat and Chothia.

[0134] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise: (a) a VH region comprising VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:47, 51, 54, 55, and 59; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:48, 52, 56, 60, and 63; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:49, 53, 57, and 61; and (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:66, 68, 70, and 71; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:67, 69, and 72; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:50, 58, and 62.

[0135] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VHCDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0136] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:51, VHCDR2 containing the amino acid sequence of SEQ ID NO:52, and VH CDR3 containing the amino acid sequence of SEQ ID NO:53; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:68, VL CDR2 containing the amino acid sequence of SEQ ID NO:69, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0137] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:54, VHCDR2 containing the amino acid sequence of SEQ ID NO:48, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0138] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:55, VHCDR2 containing the amino acid sequence of SEQ ID NO:56, and VH CDR3 containing the amino acid sequence of SEQ ID NO:57; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:70, VL CDR2 containing the amino acid sequence of SEQ ID NO:69, and VL CDR3 containing the amino acid sequence of SEQ ID NO:58.

[0139] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:59, VHCDR2 containing the amino acid sequence of SEQ ID NO:60, and VH CDR3 containing the amino acid sequence of SEQ ID NO:61; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:71, VL CDR2 containing the amino acid sequence of SEQ ID NO:72, and VL CDR3 containing the amino acid sequence of SEQ ID NO:62.

[0140] In some embodiments, the multispecific binder (e.g., bispecific antibody) provided herein comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:47, VHCDR2 containing the amino acid sequence of SEQ ID NO:63, and VH CDR3 containing the amino acid sequence of SEQ ID NO:49; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:66, VL CDR2 containing the amino acid sequence of SEQ ID NO:67, and VL CDR3 containing the amino acid sequence of SEQ ID NO:50.

[0141] In some embodiments, the NKG2A binding domain provided herein further comprises one or more frame regions of SEQ ID NO:25, 26, 45, 46, 64, 65, and / or 73. In some embodiments, the NKG2A binding domain further comprises frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4) sequences as shown in any of SEQ ID NO:25, 26, 45, 46, 64, 65, and 73. In some embodiments, the NKG2A binding domain provided herein is derived from a humanized antibody. The frame regions described herein are defined based on the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT, or Chothia, the frame region is the amino acid residues surrounding the CDR in the variable region, which, from the N-terminus to the C-terminus, follows the format: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as an amino acid residue at the N-terminus of an amino acid residue in CDR1, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as an amino acid residue between amino acid residues in CDR1 and CDR2, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as an amino acid residue between amino acid residues in CDR2 and CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as an amino acid residue at the C-terminus of an amino acid residue in CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0142] In some embodiments, the NKG2A binding domain described herein comprises a VH region or VH domain. Alternatively, in some embodiments, the NKG2A binding domain described herein comprises a VL region or VL domain. In some embodiments, the NKG2A binding domain described herein has a combination of: (i) a VH region or VH domain; and (ii) a VL region or VL domain.

[0143] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 25 and a VL containing the amino acid sequence of SEQ ID NO: 26.

[0144] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 45. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VL containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 45 and a VL containing the amino acid sequence of SEQ ID NO: 46.

[0145] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 64. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VL containing the amino acid sequence of SEQ ID NO: 65. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 64 and a VL containing the amino acid sequence of SEQ ID NO: 65.

[0146] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 64. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a VH containing the amino acid sequence of SEQ ID NO: 64 and a VL containing the amino acid sequence of SEQ ID NO: 73.

[0147] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a certain percentage of identity with respect to any antibody or fragment thereof provided herein, such as the CDR, VH, or VL in Tables 1 through 4, or the full-length antibody chain as disclosed herein (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or a higher percentage). In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein comprise a CDR of any antibody or fragment thereof provided herein, such as in Tables 1 through 4. In other embodiments, the bispecific antibodies provided herein comprise a certain percentage of identity relative to any antibody or fragment thereof provided herein, such as VH or VL in Tables 1 to 4, or the full-length antibody chain as disclosed herein (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or a higher percentage).

[0148] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Non-limiting examples of mathematical algorithms for comparing two sequences include the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, for example, for fraction = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set, for example, for a fraction of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy-aligned sequences for comparative purposes, Gapped BLAST, as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997), can be used. In some embodiments, the percentage of identity between two sequences is calculated by dividing the number of residues that vary between the two aligned sequences (excluding or including conserved amino acid substitutions or degenerate nucleotide substitutions) by the number of residues in any 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 length of the vacancy-free portion of the alignment, (v) the length of the alignment excluding protrusions, or (vi) the length of the alignment including protrusions. As used herein, a protrusion in sequence alignment refers to either end or both ends of an alignment in which residues of one sequence are considered to be aligned without residues (e.g., vacancy) in the other sequence. Alternatively, PSI BLAST can be used for iterative searches to detect long-distance relationships between molecules (as above). When using BLAST, BLAST with vacancies, and PSI BLAST procedures, the default parameters of the corresponding procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the Global Information Network, ncbi.nlm.nih.gov). Another non-restricted example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, allowing for the presence or absence of vacancy. When calculating the percentage of identity, typically only exact matches are counted.

[0149] In some embodiments, the multispecific binders (e.g., bispecific antibodies) provided herein contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the multispecific binders containing said sequence are still able to bind to NKG2A. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR and / or constant regions).

[0150] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the NKG2A binding domain described herein may vary by one, two, three, four, five, or six amino acid positions, provided that binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position of the CDR defining any of Tables 1, 2, 3, or 4 may be varied by shifting the N-terminal and / or C-terminal boundary of the CDR relative to the current CDR position by one, two, three, four, five, or six amino acids, provided that binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Alternatively or in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the NKG2A binding domain described herein may vary (e.g., shorten or lengthen) by one, two, three, four, five, or more amino acids, provided that the binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described in SEQ ID NO:1-24, 27-44, 47-63 or 66-72, provided that the binding to NKG2A (e.g. human NKG2A) is maintained (e.g. substantially maintained, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% maintained). In other embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described in SEQ ID NO:1-24, 27-44, 47-63 or 66-72, provided that the binding to NKG2A (e.g. human NKG2A) is maintained (e.g. substantially maintained, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% maintained).In some embodiments, the amino termini of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described in SEQ ID NO: 1-24, 27-44, 47-63 or 66-72, provided that the binding to NKG2A (e.g. human NKG2A) is maintained (e.g. substantially maintained, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% maintained). Alternatively or additionally, in some embodiments, the carboxyl termini of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids relative to one or more of the CDRs described in SEQ ID NO: 1-24, 27-44, 47-63 or 66-72, provided that binding to NKG2A (e.g., human NKG2A) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% maintained). Any method known in the art can be used to determine whether binding to NKG2A (e.g., human NKG2A) is maintained, for example, the binding assays and conditions described in the “Examples” section herein.

[0151] In other embodiments, the multispecific binder (e.g., a bispecific antibody) also includes conserved sequence modifications (e.g., in the NKG2A binding domain). Conserved sequence modifications include conserved amino acid substitutions, which involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Therefore, in some embodiments, a predicted non-essential amino acid residue in NKG2A is replaced by another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions that do not eliminate antigen binding and their encoding nucleotides are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA94:412-417 (1997)). In some embodiments, the conserved sequence modifications described herein alter the amino acid sequence of multispecific binders (e.g., bispecific antibodies), including human NKG2A binders, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modification refers to the substitution of up to 1, 2, 3, 4, 5, or 6 amino acids in the CDR, such as those described in any of Tables 1 through 4. Thus, for example, each such CDR may contain up to 5 conserved amino acid substitutions, such as up to (no more than) 4 conserved amino acid substitutions, such as up to (no more than) 3 conserved amino acid substitutions, such as up to (no more than) 2 conserved amino acid substitutions, or no more than 1 conserved amino acid substitution. In some implementations, the NKG2A binding domain contains one or more (including six) CDRs that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDRs of A3, A2, A42, or A11 (see, for example, Tables 1, 2, 3, or 4).

[0152] In some embodiments, the NKG2A binding domain contains VH and VL comprising the same CDR as A3, A2, A42, or A11 (see, for example, Tables 1, 2, 3, or 4). In some embodiments, the amino acid sequence modification does not include any modification within the SDR. In some embodiments, the amino acid sequence modification does not include any modification within the CDR (e.g., CDR1, CDR2, CDR3, or any combination thereof). Alternatively or additionally, the amino acid sequence modification is within the frame, constant region, and / or fragment crystallizable region (Fc).

[0153] In some embodiments, the multispecific binder provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 25, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 26, and the binding of the multispecific binder 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).

[0154] In some embodiments, the multispecific binder provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 45, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 46, and the binding of the multispecific binder 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).

[0155] In some embodiments, the multispecific binder provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 64, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 65, and the binding of the multispecific binder 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).

[0156] In some embodiments, the multispecific binder provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 64, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 73, and the binding of the multispecific binder 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).

[0157] In some embodiments, functional epitopes can be identified, for example, by combination alanine scanning or hydrogen / deuterium exchange mass spectrometry (HDX-MS) to locate amino acids in the NKG2A protein (or complexes comprising NKG2A and CD94 or their respective extracellular domains) required to interact with multispecific binders, their NKG2A binding domains, and / or anti-NKG2A antibodies provided herein (e.g., in the following paragraphs). In some embodiments, the conformation and crystal structure of multispecific binders to NKG2A, their NKG2A binding domains, and / or anti-NKG2A antibodies (e.g., provided in the following paragraphs) can be used to identify epitopes. In some embodiments, this disclosure provides a multispecific antibody comprising an NKG2A binding domain, its NKG2A binding domain, and / or anti-NKG2A antibody, or a fragment thereof, specifically bound to the same epitope as any of the multispecific binders disclosed herein (e.g., provided in the following paragraphs).

[0158] For example, in some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VLCDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO: 26.

[0159] In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH CDR1, VH CDR2, and VHCDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH containing the amino acid sequence of SEQ ID NO: 45 and VL containing the amino acid sequence of SEQ ID NO: 46.

[0160] In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH CDR1, VH CDR2, and VHCDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65. In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 65.

[0161] In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH CDR1, VH CDR2, and VHCDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the NKG2A binding domain provided herein binds to the same epitope as that bound by the anti-NKG2A antibody, comprising VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 73.

[0162] In some embodiments, the NKG2A binding domain provided herein specifically binds to one of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence 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 containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domain provided herein specifically binds to three of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139).In some embodiments, the NKG2A binding domain provided herein specifically binds to four of the following NKG2A polypeptide fragments: an NKG2A polypeptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A polypeptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A polypeptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A polypeptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A polypeptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A polypeptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domain provided herein specifically binds to five of the following NKG2A peptide fragments: an NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 134), an NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), an NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), an NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), an NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and an NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139). In some embodiments, the NKG2A binding domain provided herein specifically binds to all of the following NKG2A peptide fragments: NKG2A peptide fragment containing the amino acid sequence TWEESL (SEQ ID NO: 134), NKG2A peptide fragment containing the amino acid sequence SIISPSSWIGV (SEQ ID NO: 135), NKG2A peptide fragment containing the amino acid sequence FRNSSHHPW (SEQ ID NO: 136), NKG2A peptide fragment containing the amino acid sequence IKDSDNAEL (SEQ ID NO: 137), NKG2A peptide fragment containing the amino acid sequence LQVNR (SEQ ID NO: 138), and NKG2A peptide fragment containing the amino acid sequence AQCGSSI (SEQ ID NO: 139).

[0163] In some embodiments, the NKG2A binding domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues, said amino acid residues comprising 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 domain provided herein specifically binds to a conformational epitope formed by a set of amino acid residues comprising at least one amino acid residue from each of the following amino acid sequences: TWEESL (SEQ ID NO: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 comprising at least one amino acid residue from each of the following three 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 domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues comprising at least one amino acid residue from each of the following four 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 domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues comprising at least one amino acid residue from each of the following five 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 domain provided herein specifically binds to a conformational epitope formed by a group of amino acid residues, said amino acid residues comprising at least one amino acid residue from each 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 histamine residues forming the conformational epitope comprise one amino acid residue from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise two amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise three amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise four amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise five amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise more than five amino acid residues from the amino acid sequences mentioned above in this paragraph.

[0164] In some embodiments, the NKG2A binding domain provided herein specifically binds to a conformational epitope located on the surface of NKG2A, comprising 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 domain provided herein specifically binds to a conformational epitope located on the surface of NKG2A, comprising at least one amino acid residue from each 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 domain provided herein specifically binds to a conformational epitope located on the surface of NKG2A, comprising at least one amino acid residue from each of the following three 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 domain provided herein specifically binds to a conformational epitope located on the surface of NKG2A, comprising at least one amino acid residue from each of the following four 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 domain provided herein specifically binds to a conformational epitope located on the surface of NKG2A, comprising at least one amino acid residue from each of the following five 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 domain provided herein specifically binds to a conformational epitope formed by a set of amino acid residues on the surface of NKG2A, said amino acid residues comprising at least one amino acid residue from each 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 histamine residues forming the conformational epitope comprise one amino acid residue from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise two amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise three amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise four amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise five amino acid residues from the amino acid sequences mentioned above in this paragraph. In some embodiments, the histamine residues forming the conformational epitope comprise more than five amino acid residues from the amino acid sequences mentioned above in this paragraph.

[0165] In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with either the anti-NKG2A antibody or a fragment thereof described herein.

[0166] In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO: 26.

[0167] In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO: 45 and VL containing the amino acid sequence of SEQ ID NO: 46.

[0168] In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65. In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 65.

[0169] In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the multispecific binder (e.g., a bispecific antibody) provided herein competitively and specifically binds to NKG2A with an anti-NKG2A antibody comprising VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 73.

[0170] In some embodiments, the antibodies derived from the NKG2A binding arm of the present invention exhibit excellent developability based on assays known in the art, such as various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and vertical monolayer adsorption chromatography (SMAC). In some embodiments, the antibodies derived from the NKG2A binding arm of the present invention exhibit excellent developability based on measurements of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0171] 5.2.2. PD-L1 binding domain This document also provides a conjugate that binds to PD-L1. In some embodiments, the conjugate comprises one or more PD-L1 binding domains. In some embodiments, the multispecific conjugates provided herein further comprise one or more PD-L1 binding domains. In some embodiments, multispecific conjugates (e.g., bispecific antibodies) that bind to PD-L1 are described herein. 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 the conjugates of the present invention, such as multispecific conjugates, is derived from a human or humanized antibody (e.g., containing a human framework region) that binds to PD-L1, said PD-L1 comprising a PD-L1 polypeptide, a PD-L1 polypeptide fragment, a PD-L1 peptide, or a PD-L1 epitope. In some embodiments, the conjugate (e.g., an antibody, a multispecific conjugate, or a bispecific antibody) can bind to PD-L1 expressed on the surface of mammalian (e.g., human) cells, said cells including PD-L1-expressing immune cells (e.g., T cells) and / or PD-L1-expressing cancer or tumor cells. In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) bind to PD-L1 extracellular domains exposed on cells such as immune cells and / or cancer or tumor cells. In some embodiments, conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) that bind to PD-L1, such as human PD-L1 or a portion thereof, are described herein. In some embodiments, the PD-L1 is human PD-L1. In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) are human PD-L1 conjugates (e.g., antibodies that bind to human PD-L1). In some embodiments, the conjugates provided herein bind to both human PD-L1 and cynomolgus monkey PD-L1. In other embodiments, the conjugates provided herein bind to human PD-L1 but not to cynomolgus monkey PD-L1.

[0172] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) are available in quantities of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) D(e.g., human PD-L1). Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure, including those described herein, for example, in section 5.2.1 above.

[0173] In some embodiments, the binders described herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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 sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and / or VL CDR3 depicted in Table 5. Therefore, in some embodiments, the binders described herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from an antibody named P12 as shown in Table 5. In some embodiments, the binding agents described herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) comprise any one, any two, and / or all three heavy chain CDRs and any one, any two, and / or all three light chain CDRs from an antibody named P12, as shown in Table 5.

[0174] In some embodiments, the binder (e.g., an antibody, a multispecific binder, or a bispecific antibody) comprises a VH region and / or a VL region of any of the binders described herein, wherein the VH region comprises VH CDR1, VH CDR2, and / or VHCDR3, and the VL region comprises VL CDR1, VL CDR2, and / or VL CDR3 (see, for example, Table 5). Therefore, in some embodiments, the binder described herein (e.g., an antibody, a multispecific binder, or a bispecific antibody) comprises any, any two, and / or all three heavy chain CDRs and / or any, any two, and / or all three light chain CDRs from Table 5.

[0175] In some embodiments, the conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) provided herein comprise (i) VH CDR1, VH CDR2, and VHCDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98; and / or (ii) VL CDR1, VL CDR2, and VLCDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99. In some embodiments, the conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) provided herein comprise VH CDR1, VH CDR2, and / or VHCDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98, and / or VL CDR1, VL CDR2, and / or VLCDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99. The CDR sequence may be determined according to a well-known numbering system or a combination thereof. In some embodiments, the CDR is based on an IMGT number. In some embodiments, the CDR is based on a Kabat number. In some embodiments, the CDR is based on an AbM number. In other embodiments, the CDR is based on a Chothia number. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on a combination of any two or more of the numbering systems mentioned above, such as a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated in Section 5.1 above.

[0176] In some embodiments, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein comprise: (a) a VH region comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13, and 18; (2) a VHCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 86, 89, 91, 94, and 97; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 88, 93, and 96.

[0177] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0178] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0179] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0180] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0181] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0182] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise 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.

[0183] In some embodiments, the PD-L1 binding domain in the binder (e.g., antibody, multispecific binder, or bispecific antibody) provided herein further comprises one or more frame regions of SEQ ID NO:98 and 99. In some embodiments, the PD-L1 binding domain further comprises frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4) sequences as shown in any of SEQ ID NO:98 and 99. In some embodiments, the PD-L1 binding domain provided herein is derived from a humanized antibody. The frame regions described herein are determined based on the boundaries of the CDR numbering system as described in Section 5.2.1 above.

[0184] In some embodiments, the PD-L1 binding domain described herein comprises a VH region or VH domain. Alternatively, in some embodiments, the PD-L1 binding domain described herein comprises a VL region or VL domain. In some embodiments, the PD-L1 binding domain described herein has a combination of: (i) a VH region or VH domain; and (ii) a VL region or VL domain.

[0185] In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) comprise a VH containing the amino acid sequence of SEQ ID NO:98. In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) comprise a VL containing the amino acid sequence of SEQ ID NO:99. In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) comprise a VH containing the amino acid sequence of SEQ ID NO:98 and a VL containing the amino acid sequence of SEQ ID NO:99.

[0186] In some embodiments, the conjugates provided herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) comprise an amino acid sequence having a certain percentage of identity (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or higher) relative to any antibody or fragment thereof provided herein, such as the CDR, VH, or VL in Table 5. In other embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise an amino acid sequence having a certain percentage of identity (e.g., at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or higher) relative to any antibody or fragment thereof provided herein, such as VH or VL in Table 5, or the full-length antibody chain disclosed herein.

[0187] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the binders (e.g., antibodies, multispecific binders, or bispecific antibodies) containing said sequence retain their ability to bind to PD-L1. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence have been substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR, constant region, and / or Fc region).

[0188] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the PD-L1 binding domain described herein may vary by one, two, three, four, five, or six amino acid positions, provided that 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%). For example, in some embodiments, the position of the CDRs defining Table 5 may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDRs relative to the current CDR position by one, two, three, four, five, or six amino acids, provided that 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%). Alternatively or in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the PD-L1 binding domain described herein may vary (e.g., shorten or lengthen) by one, two, three, four, five, or more amino acids, provided that the 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). For example, in some embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NO: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, provided that the 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). In other embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NO: 1, 4-5, 7, 10-13, 16, 18, 21, 22 and 86-97, provided that the 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).In some embodiments, the amino termini of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NO: 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. substantially maintained, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). Alternatively or additionally, in some embodiments, the carboxyl termini of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NO: 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., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% maintained). Any method known in the art can be used to determine whether binding to PD-L1 (e.g., human PD-L1) is maintained, for example, the binding assays and conditions described in the “Examples” section herein.

[0189] In other embodiments, the binder (e.g., antibody, multispecific binder, or bispecific antibody) further comprises a conserved sequence modification (e.g., in the PD-L1 binding domain). Conserved sequence modifications are described in more detail in section 5.2.1 above. In some embodiments, the conserved sequence modifications described herein alter the amino acid sequence of the binder (e.g., antibody, multispecific binder, or bispecific antibody), including human PD-L1 binders, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modification refers to the substitution of up to 1, 2, 3, 4, 5, or 6 amino acids in the CDR, such as those described in Table 5. Thus, for example, each such CDR may contain up to 5 conserved amino acid substitutions, such as up to (no more than) 4 conserved amino acid substitutions, such as up to (no more than) 3 conserved amino acid substitutions, such as up to (no more than) 2 conserved amino acid substitutions, or no more than 1 conserved amino acid substitution. In some implementations, the PD-L1 binding domain contains one or more (including six) CDRs that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDRs of P12 (see, for example, Table 5).

[0190] In some embodiments, the PD-L1 binding domain contains VH and VL comprising the same CDR as P12 (see, for example, Table 5). In some embodiments, the amino acid sequence modification does not include any modification within the SDR. In some embodiments, the amino acid sequence modification does not include any modification within the CDR (e.g., CDR1, CDR2, CDR3, or any combination thereof). In other embodiments, the amino acid sequence modification is in the frame, constant region, and / or Fc region.

[0191] In some embodiments, the binders provided herein (e.g., antibodies, multispecific binders, or bispecific antibodies) comprise a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 98, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 99, and the binding of the binder 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).

[0192] In some embodiments, functional epitopes can be located, for example, by combined alanine scanning or hydrogen / deuterium exchange mass spectrometry (HDX-MS) to identify amino acids in the PD-L1 protein required to interact with the multispecific binders, their PD-L1 binding domains, and / or anti-PD-L1 antibodies provided herein (e.g., in the following paragraphs). In some embodiments, the conformation and crystal structure of the multispecific binders to PD-L1, their PD-L1 binding domains, and / or anti-PD-L1 antibodies (e.g., provided in the following paragraphs) can be used to identify epitopes. In some embodiments, this disclosure provides a binder (e.g., an antibody, a multispecific binder, or a bispecific antibody) comprising a PD-L1 binding domain, its PD-L1 binding domain, and / or anti-PD-L1 antibody, or a fragment thereof, specifically bound to the same epitope as any of the multispecific binders disclosed herein (e.g., provided in the following paragraphs).

[0193] For example, in some embodiments, the PD-L1 binding domain provided herein binds to the same epitope as that bound by an anti-PD-L1 antibody comprising VHCDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:99. In some embodiments, the PD-L1 binding domain provided herein binds to the same epitope as that bound by an anti-PD-L1 antibody comprising VH containing the amino acid sequence of SEQ ID NO:98 and VL containing the amino acid sequence of SEQ ID NO:99.

[0194] In some embodiments, the binding agent provided herein (e.g., antibody, multispecific binding agent, or bispecific antibody) competitively and specifically binds to PD-L1 with any of the anti-PD-L1 antibodies or fragments thereof described herein.

[0195] In some embodiments, the binding agents provided herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) competitively and specifically bind to PD-L1 with an anti-PD-L1 antibody comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:99. In some embodiments, the binding agents provided herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) competitively and specifically bind to PD-L1 with an anti-PD-L1 antibody comprising VH containing the amino acid sequence of SEQ ID NO:98 and VL containing the amino acid sequence of SEQ ID NO:99.

[0196] In another aspect, this document provides a PD-L1 binding agent (e.g., an antibody or fragment thereof) comprising one or more of the CDRs described above. In some embodiments, this document provides a PD-L1 binding agent (e.g., an antibody or fragment thereof) comprising the VH and / or VL described above. In some embodiments, the binding agent comprises VH CDR1, VH CDR2, and VH CDR3 as shown in VH comprising the amino acid sequence of SEQ ID NO:98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, the VH region comprises: (1) VHCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 7, 12, 13 and 18; (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 86, 89, 91, 94 and 97; and (3) VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 87, 90, 92 and 95; and the VL region comprises: (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 10, 16 and 21; (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11 and 22; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 88, 93 and 96.

[0197] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 86, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 87; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 88.

[0198] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 7, VH CDR2 containing the amino acid sequence of SEQ ID NO: 89, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 90; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 88.

[0199] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, VH CDR2 containing the amino acid sequence of SEQ ID NO: 86, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 87; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 88.

[0200] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 13, VH CDR2 containing the amino acid sequence of SEQ ID NO: 91, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 92; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence of SEQ ID NO: 11, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 93.

[0201] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 18, VH CDR2 containing the amino acid sequence of SEQ ID NO: 94, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 95; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 21, VL CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 96.

[0202] In some embodiments, the binder comprises: a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 97, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 87; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 88.

[0203] In some embodiments, the binder provided herein includes 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.

[0204] 5.2.3. Multispecific antibodies This document provides a multispecific binder that binds to NKG2A and non-NKG2A antigens (e.g., PD-L1). In some embodiments, the multispecific binder comprises one or more NKG2A binding domains and one or more non-NKG2A antigen binding domains (e.g., one or more PD-L1 binding domains). In some embodiments, the multispecific binder of the present invention is a multispecific antibody comprising one or more NKG2A binding domains, each independently selected from the NKG2A binding domains described in section 5.2.1 above, and one or more PD-L1 binding domains, each independently selected from the PD-L1 binding domains described in section 5.2.2 above.

[0205] In some specific embodiments, this document provides a multispecific binding agent comprising one or more NKG2A binding domains, each independently selected from NKG2A binding domains comprising: (i) VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26; (ii) VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46; (iii) VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65. CDR1, VL CDR2, and VL CDR3; or (iv) VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64 and VLCDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73; and one or more PD-L1 binding domains containing VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99.

[0206] In some embodiments, this document provides a multispecific binder comprising: one or more NKG2A binding domains, each comprising VHCDR1, VH CDR2, and VH CDR3 as shown in VH of the amino acid sequence comprising SEQ ID NO: 25 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL of the amino acid sequence comprising SEQ ID NO: 26; and one or more PD-L1 binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH of the amino acid sequence comprising SEQ ID NO: 98 and VL CDR1, VL CDR2, and VL CDR3 as shown in VL of the amino acid sequence comprising SEQ ID NO: 99. In some embodiments, the multispecific binder provided herein comprises an NKG2A binding domain (e.g., having a pair of VH and VL regions binding to NKG2A) and a PD-L1 binding domain (e.g., having a pair of VH and VL regions binding to PD-L1). In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two identical pairs of VH and VL regions bound to NKG2A) and one PD-L1 binding domain (e.g., having a pair of VH and VL regions bound to PD-L1).

[0207] In some embodiments, this document provides a multispecific binder comprising: one or more NKG2A binding domains, each comprising VHCDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46; and one or more PD-L1 binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99. In some embodiments, the multispecific binder provided herein comprises an NKG2A binding domain (e.g., having a pair of VH and VL regions binding to NKG2A) and a PD-L1 binding domain (e.g., having a pair of VH and VL regions binding to PD-L1). In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two identical pairs of VH and VL regions bound to NKG2A) and one PD-L1 binding domain (e.g., having a pair of VH and VL regions bound to PD-L1).

[0208] In some specific embodiments, this document provides a multispecific binder comprising: one or more NKG2A binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VLCDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 65; and one or more PD-L1 binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99. In some embodiments, the multispecific binder provided herein comprises an NKG2A binding domain (e.g., having a pair of VH and VL regions binding to NKG2A) and a PD-L1 binding domain (e.g., having a pair of VH and VL regions binding to PD-L1). In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two identical pairs of VH and VL regions bound to NKG2A) and one PD-L1 binding domain (e.g., having a pair of VH and VL regions bound to PD-L1).

[0209] In some specific embodiments, this document provides a multispecific binder comprising: one or more NKG2A binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64, and VLCDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73; and one or more PD-L1 binding domains, each comprising VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 98, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 99. In some embodiments, the multispecific binder provided herein comprises an NKG2A binding domain (e.g., having a pair of VH and VL regions binding to NKG2A) and a PD-L1 binding domain (e.g., having a pair of VH and VL regions binding to PD-L1). In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two identical pairs of VH and VL regions bound to NKG2A) and one PD-L1 binding domain (e.g., having a pair of VH and VL regions bound to PD-L1).

[0210] In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two pairs of identical or different VH and VL regions bound to NKG2A) and one PD-L1 binding domain (e.g., having a pair of VH and VL regions bound to PD-L1). In some embodiments, the multispecific binder provided herein comprises one NKG2A binding domain (e.g., having a pair of VH and VL regions bound to NKG2A) and two PD-L1 binding domains (e.g., having two pairs of identical or different VH and VL regions bound to PD-L1). In some embodiments, the multispecific binder provided herein comprises two NKG2A binding domains (e.g., having two pairs of identical or different VH and VL regions bound to NKG2A) and two PD-L1 binding domains (e.g., having two pairs of identical or different VH and VL regions bound to PD-L1). In some embodiments, the multispecific binder further comprises one or more heavy chain constant regions (e.g., CH1, CH2, and / or CH3) and / or one or more light chain constant regions (e.g., CL).

[0211] In some embodiments, the multispecific binders provided herein have Figure 22 The antibody format is shown in any of the three figures. In some embodiments, the multispecific binding agents provided herein have... Figure 22 The antibody format shown in any of the three figures is alternating between its PD-L1 arm and its NKG2A arm. For example, in the 1x1 format, the labeled chain 1 and chain 2 form the NKG2A binding site (hence the name NKG2A arm, instead of the PD-L1 arm). Figure 22 The PD-L1 arms are marked in the "1x1" diagram, while chains 3 and 4 form the PD-L1 binding sites (hence the name PD-L1 arms, replacing...). Figure 22 (NKG2A arm marked in the "1x1" diagram). Alternatively or in some embodiments, the multispecific binder provided herein has... Figure 22 The antibody format shown in any of the three figures is an example of an antibody format where a knockb mutation in one CH3 at the C-terminus of CH2 is exchanged for a hole mutation in another CH3 at the C-terminus of CH2. For example, in the 1x1 format, the knockb mutation could be in CH3 at the C-terminus of CH2 on chain 1, while the hole mutation could be in CH3 at the C-terminus of CH2 on chain 3.

[0212] In some embodiments, mutations are introduced into one or more heavy chain constant regions (e.g., CH1, CH2, and / or CH3) and / or one or more light chain constant regions (e.g., CL) to achieve one or more of the following: (i) destabilizing homodimers formed from polypeptides of multispecific antibodies; (ii) stabilizing multispecific antibodies as described herein (also referred to herein as heterodimers); (iii) promoting proper assembly of multispecific antibodies as described herein; (iv) favoring heterodimerization relative to homodimerization of component polypeptide chains; (v) increasing the yield of multispecific antibodies as described herein; and (vi) increasing the purity of multispecific antibodies as described herein. Several mutations driving preferential heterodimerization have been developed, such as the mortar (KIH or KiH) mutation (see, for example, U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, and 8,216,805), disulfide bond-stabilized KIH mutations (see, for example, U.S. Patent Nos. 7,951,917, 8,642,745, and 9,409,989), and others (see, for example, WO 2022 / 125986). Each patent and / or patent disclosure cited herein is incorporated herein by reference in its entirety.

[0213] Although the following embodiments are partially neutral Figure 22A particular exemplary multispecific antibody format has been described in any of the three figures, but upon careful consideration, any multispecific antibody format known in the art may be used and included in this disclosure.

[0214] The multispecific binding agents (e.g., bispecific antibodies) described herein may be bispecific, trispecific, or more multispecific. Such agents may include multispecific antibodies. In some embodiments, multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody against NKG2A having a first binding domain against a first epitope of NKG2A and a second binding domain against a second epitope of NKG2A). In some embodiments, multispecific (e.g., bispecific) antibodies may be constructed based on the sequence of the antibodies described herein, such as the CDR sequences listed in Tables 1 through 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 specificity to two targets, such as NKG2A and PD-L1. In some embodiments, bispecific antibodies are mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies.

[0215] In some embodiments, one of the binding specificities of the multispecific antibody provided herein is against NKG2A, a second binding specificity of the multispecific antibody provided herein is against PD-L1, and one or more additional binding specificities are against any other target (e.g., antigen). In some embodiments, the multispecific antibody may contain more than one target (e.g., antigen) binding domain, wherein different binding domains are specific to different targets. 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 immune cells. In some embodiments, the additional target is expressed on tumor or cancer cells.

[0216] In some embodiments, multispecific (e.g., bispecific) antibody molecules can bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen). In some embodiments, the multispecific (e.g., bispecific) binders 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).

[0217] Methods for preparing multispecific antibodies are known in the art, for example, through the co-expression of two immunoglobulin heavy-light chain pairs, wherein the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For further details on the generation of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0218] 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.

[0219] 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 attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As a non-limiting example, BsIgG formats may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, mortis-common LC, mortis-common assembly, charge pairs, Fab-arm exchange, SEED bodies, triomab, LUZ-Y, Fcab, κλ bodies, and / or orthogonal Fab.

[0220] In some implementations, BsIgG includes a heavy chain engineered for heterodimerization. For example, the heavy chain may be engineered to use a "mortar and pestle" strategy, a SEED platform, a common heavy chain (e.g., in κλ body form), and heterodimerization using a heterodimeric Fc region. Strategies known in the art to avoid homodimer heavy chain pairing in BsIgG include mortar and pestle, duobody, azymetric, charge pair, HA-TF, SEED body, and differential protein A affinity.

[0221] Another bispecific antibody format is IgG with an additional antigen-binding portion attached. For example, monospecific IgG can be engineered to be bispecific by attaching an additional antigen-binding unit to the monospecific IgG (e.g., at the N-terminus or C-terminus of the heavy or light chain). Exemplary additional antigen-binding units include a single-domain antibody (e.g., a variable heavy or light chain), an engineered protein backbone, and a paired antibody variable domain (e.g., a single-chain variable fragment or variable fragment). Non-limiting examples of attached 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 (quadruple). See Spiess et al., Mol. Immunol. 67(2015):95-106. In some embodiments, exemplary antibody formats are B-body formats of monospecific or multispecific (e.g., bispecific antibodies) as described, for example, in WO 2018 / 075692, U.S. Patent Publication No. 2018 / 0118811, and U.S. Patent Publication No. 2021 / 0155692.

[0222] Bispecific (Bs) antibody (BsAb) fragments are bispecific antibody molecule formats that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment includes heavy and light chain regions linked by a peptide linker, which allows for efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTE, bifunctional antibodies, DART, TandAb, scDiabody, scDiabody-CH3, bifunctional antibody-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, bifunctional antibody-Fc, tandem scFv-Fc, and intrabody.

[0223] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, a bispecific fusion protein may be linked to other proteins to add additional specificity and / or functionality. In some embodiments, a dock-and-lock (DNL) approach may be used to generate bispecific antibody molecules with higher valences. For example, a bispecific antibody fusion with an albumin-binding protein or human serum albumin may be constructed to extend the serum half-life of the antibody fragment. In some embodiments, chemical conjugations, such as chemical conjugations of antibodies and / or antibody fragments, may be used to generate BsAb molecules. Exemplary bispecific antibody conjugations include a CovX body format in which a low molecular weight drug site is specifically conjugated to a single reactive lysine residue in each Fab arm or antibody or fragment thereof. In some embodiments, the conjugation improves serum half-life.

[0224] Methods for generating multispecific antibodies (including bispecific antibodies) are known in the art. For example, multispecific antibodies (including bispecific antibodies) can be generated by separately expressing component antibodies in different host cells and subsequently purifying / assembling them, or by expressing component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by various methods known in the art, including affinity chromatography.

[0225] In some embodiments, the multispecific binders (e.g., bispecific antibodies) disclosed herein may be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, in some embodiments, the multispecific binder (e.g., bispecific antibody) may be selected from Fab tandem-lg (FIT-lg); DVD-lg; heterozygous hybridoma (tetrasomatic hybridoma or tetrasomatic hybridoma); anti-carrier protein platform (Pieris); bifunctional antibody; single-chain bifunctional antibody; tandem single-chain Fv fragment; TandAb, trispecific Ab (Affimed); Darts dual affinity retargeting (Macrogenics); bispecific Xmab (Xencor); Bispecific T-cell conjugating molecules (Bites; Amgen; 55kDa); Trifunctional antibodies; Trifunctional antibody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs); Bispecific antibody platform (Genmab); Docking-locking platform; Mortar and pestle (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-lg = dual variable domain immunoglobulin (Abbott); κ-λ body; TBTI = tetravalent bispecific tandem Ig; B-Body; and CrossMab (Roche).

[0226] In some specific implementations, the bispecific antibodies provided herein exhibit Figure 22 The format depicted in any of the three figures. In some embodiments, the bispecific antibody provided herein presents as shown in... Figure 22 The 1x1 format is depicted in the "1x1" diagram. In some embodiments, the bispecific antibody provided herein is presented as shown in... Figure 22 The 1x2 format is depicted in the "1x2" diagram. In some embodiments, the bispecific antibody provided herein presents as shown in... Figure 22 The 2x1 format depicted in the “2x1” diagram.

[0227] In some specific embodiments, the multispecific antibody provided herein comprises four polypeptides: a first polypeptide comprising, from its N-terminus to its C-terminus: a first VL, a first CH3, a first CH2, and a second CH3; a second polypeptide comprising, from its N-terminus to its C-terminus: a first VH and a third CH3; a third polypeptide comprising, from its N-terminus to its C-terminus: a second VL, a C1, a second CH2, and a fourth CH3; and a fourth polypeptide comprising, from its N-terminus to its C-terminus: a second VH and a CH1. These four polypeptides form two binding domains. In some embodiments, the first polypeptide forms a PD-L1 binding domain with the second polypeptide (e.g., its first VL and first VH), and the third polypeptide forms a NKG2A binding domain with the fourth polypeptide (e.g., its second VL and second VH). In other embodiments, the first polypeptide forms a NKG2A binding domain with the second polypeptide (e.g., its first VL and first VH), and the third polypeptide forms a PD-L1 binding domain with the fourth polypeptide (e.g., its second VL and second VH). In some embodiments, the amino acid sequences of the first CH3, second CH3, third CH3, and fourth CH3, or any subgroup thereof, are different from each other. In some embodiments, the second CH3 and fourth CH3 provide a pestle-and-mortar assembly. Alternatively or concurrently, the amino acid sequences of the first CH2 and second CH2 are identical to each other. In other embodiments, the amino acid sequences of the first CH2 and second CH2 are different from each other.

[0228] In some specific embodiments, the multispecific antibody provided herein comprises five polypeptides: a first polypeptide comprising, from N-terminus to C-terminus: a first VL, a first CH3, a first CH2, and a second CH3; a second polypeptide comprising, from N-terminus to C-terminus: a first VH and a third CH3; a third polypeptide comprising, from N-terminus to C-terminus: 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-terminus to C-terminus: a second VH and a first CH1; and a fifth polypeptide comprising, from N-terminus to C-terminus: a third VH and a second CH1. In some embodiments, the second VL and the third VL have the same amino acid sequence. Alternatively or additionally, the first CL and the second CL have the same amino acid sequence. In some embodiments, the second VH and the third VH have the same amino acid sequence. Alternatively or additionally, the first CH1 and the second CH1 have the same amino acid sequence. 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 provide a pestle-and-mortar assembly. Alternatively, the amino acid sequences of the first CH2 and the second CH2 are identical. 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 identical to the fifth polypeptide. The five polypeptides form three binding domains. In some embodiments, the first polypeptide forms a binding domain to PD-L1 with the second polypeptide (e.g., its first VL and first VH), the third polypeptide forms a first binding domain to NKG2A with the fourth polypeptide (e.g., its second VL and second VH), and the third polypeptide forms a second binding domain to NKG2A with the fifth polypeptide (e.g., its third VL and third VH). In some embodiments, the first NKG2A binding domain and the second NKG2A binding domain are identical. In other embodiments, the first NKG2A binding domain and the second NKG2A binding domain are different from each other. In some embodiments, the first polypeptide forms a binding domain to NKG2A with the second polypeptide (e.g., its first VL and first VH), the third polypeptide forms a first binding domain to PD-L1 with the fourth polypeptide (e.g., its second VL and second VH), and the third polypeptide forms a second binding domain to PD-L1 with the fifth polypeptide (e.g., its third VL and third VH). In some embodiments, the two PD-L1 binding domains are identical.

[0229] In other, more specific embodiments, the multispecific antibody provided herein comprises five polypeptides: a first polypeptide comprising, from N-terminus to C-terminus: first VL, first CH3, second VL, second CH3, first CH2, and third CH3; a second polypeptide comprising, from N-terminus to C-terminus: first VH and fourth CH3; a third polypeptide comprising, from N-terminus to C-terminus: second VH and fifth CH3; a fourth polypeptide comprising, from N-terminus to C-terminus: third VL, first CH1, second CH2, and sixth CH3; and a fifth polypeptide comprising, from N-terminus to C-terminus: third VH and CH1. In some embodiments, the first VL and second VL have the same amino acid sequence. Alternatively or additionally, the first CH3 and second CH3 have the same amino acid sequence. In some embodiments, the first VH and second VH have the same amino acid sequence. Alternatively or additionally, the fourth CH3 and fifth CH3 have the same amino acid sequence. In some embodiments, the second polypeptide is the same as the third polypeptide. In some embodiments, the amino acid sequences of the first CH3, second CH3, third CH3, fourth CH3, fifth CH3, and sixth CH3, or any subgroup thereof, are different from each other. In some embodiments, the third CH3 and the sixth CH3 provide a mortar assembly. Alternatively or additionally, the amino acid sequences of the first CH2 and the second CH2 are identical. 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 forms a first binding domain to PD-L1 with the second polypeptide (e.g., its first VL and first VH), the first polypeptide forms a second binding domain to PD-L1 with the third polypeptide (e.g., its second VL and second VH), and the fourth polypeptide forms a binding domain to NKG2A with the fifth polypeptide (e.g., its third VL and third VH). In some embodiments, the two PD-L1 binding domains are identical. In other embodiments, the first polypeptide forms a first binding domain to NKG2A with a second polypeptide (e.g., its first VL and first VH), the first polypeptide forms a second binding domain to NKG2A with a third polypeptide (e.g., its second VL and second VH), and the fourth polypeptide forms a binding domain to PD-L1 with a fifth polypeptide (e.g., its third VL and third VH). In some embodiments, the two NKG2A binding domains are identical. In some embodiments, the first NKG2A binding domain and the second NKG2A binding domain are different from each other.

[0230] In some embodiments, one or more of the CH3 sequences contain GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (aa 116 to aa 222 of SEQ ID NO:102; SEQ ID NO:83).

[0231] In some embodiments, allotropic mutations D356E and L358M are generated in the CH3 sequence as disclosed herein. In some embodiments, one or more allotropic mutations (e.g., either or both of D356E and L358M) are in one or more of the CH3 sequence immediately adjacent to the C-terminus of the CH2 sequence, for example... Figure 22 Any one or both of the unmarked CH3 in each of the three figures shown. Alternatively, as described in more detail in Stickler et al. (Genes Immun. 2011 Apr; 12(3):213-221), any one or more of the CH3 sequence are engineered to reduce the risk of antibody immunogenicity by replacing a specific amino acid of one allotype with a specific amino acid of another allotype and are referred to herein as allotype mutations.

[0232] Alternatively or concurrently, the CH3 sequence is engineered to include a club-and-mortar mutation. In some embodiments, the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence (e.g., when forming a binding agent containing a polypeptide, one CH3 in one polypeptide dimers with another CH3 in a different polypeptide) (e.g., Figure 22 In any of the three figures shown, of the two CH3 groups (without * markings) in each construct, one CH3 contains a club-shaped mutation (e.g., T366W), while the other CH3 contains a mortar-shaped mutation (e.g., any one, any two, or all three of T366S, L368A, and Y407V). In other embodiments, one CH3 adjacent to the C-terminus of VH or VL in a VH / VL pair (e.g., VH and VL forming a binding domain in a binder containing VH and VL) contains a club-shaped mutation (e.g., T366W), while the other CH3 (adjacent to the C-terminus of VL or VH in the same VH / VL pair) contains a mortar-shaped mutation (e.g., any one, any two, or all three of T366S, L368A, and Y407V).

[0233] Those skilled in the art will understand that a domain pair, such as CH3 / CH3, VH / VL, VL / VH, CH2 / CH2, CH1 / CL, or CL / CH1, refers to another antibody domain (e.g., VH, VL, CH1, CH2, CH3, or CH) in a different polypeptide chain that dimerizes with an antibody domain (e.g., VL, VH, CL, CH2, CH3, or CH) in a polypeptide chain when forming a binding agent containing a polypeptide.

[0234] In some embodiments, in a CH3 / CH3 pair (e.g., when forming a binding agent containing a polypeptide, one CH3 in one polypeptide dimers with another CH3 in a different polypeptide), one CH3 contains the Y349C mutation, while the other CH3 contains the S354C mutation. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in a VH / VL pair contains the S354C mutation, while the other CH3 (immediately adjacent to the C-terminus of the VL or VH in the same VH / VL pair) contains the Y349C mutation. Alternatively or additionally, the CH3 sequence is engineered to allow it to form disulfide bonds in the antibody, for example, to stabilize the mortar mutation as described above.

[0235] Alternatively, any or more of the CH3 sequence may be engineered to include one or more other mutations, provided that said one or more mutations do not significantly reduce the antibody's affinity and / or stability, nor significantly increase the risk of immunogenicity. In some embodiments, any or more of the CH3 sequence may be engineered to include mutations as described in WO 2022 / 125986.

[0236] In some embodiments, in the CH3 / CH3 pair, one CH3 contains the S354C mutation, while the other CH3 contains the Y349C mutation. Alternatively or additionally, in the CH3 / CH3 pair, the E357 residue 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, the E357 residue of CH3 is substituted with W. Alternatively or additionally, in some embodiments, CH (e.g., CH3) contains the K370R mutation and dimers with the CH3 containing the E357 mutation in the binding agent. In some embodiments, one CH3 in the CH3 / CH3 pair contains the K370R mutation. Alternatively or additionally, the other CH3 in the same CH3 / CH3 pair contains the E357W mutation. In some embodiments, one CH3 in the CH3 / CH3 pair contains the K370R mutation, while the other CH3 in the same CH3 / CH3 pair contains the E357W mutation. In some embodiments, in the CH3 / CH3 pair, one CH3 contains S354C and E357W, while the other CH3 contains Y349C and K370R. In some embodiments, each CH3 in the CH3 / CH3 pair is immediately adjacent to the C-terminus of the CH2 sequence. In other embodiments, one CH3 in the CH3 / CH3 pair is immediately adjacent to the C-terminus of the VH or VL in the VH / VL pair, while the other CH3 in the CH3 / CH3 pair is immediately adjacent to the C-terminus of the VL or VH in the same VH / VL pair. In some embodiments, one CH3 immediately adjacent to the C-terminus of the VH or VL in the VH / VL pair contains the E357W mutation; while the other CH3 (immediately adjacent to the C-terminus of the VL or VH in the same VH / VL pair) contains the K370R mutation. In other embodiments, in the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence, one CH3 contains both S354C and E357W, while the other CH3 contains both Y349C and K370R.

[0237] Alternatively or additionally, one or more amino acid residues in CH3 are exchanged with one or more corresponding amino acid residues in CH1. In some embodiments, as used herein, the first amino acid residue in the first peptide corresponding to the second amino acid residue in the 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 are available to those skilled in the art, such as BLAST and / or ClustalOmega disclosed herein. In some embodiments, CH3 is immediately adjacent to the C-terminus of VH or VL. Alternatively or additionally, the one or more amino acid residues are in the N-terminal segment of CH1, for example, selected from the first to the tenth amino acid of CH1 (inclusive of each range or integer therebetween, such as the first to the fifth, the first to the third, the first, or the third). In some embodiments, CH3 contains the first amino acid residue exchanged with the first amino acid residue of CH1, also referred herein as the N-terminal amino acid residue exchanged with CH1. In some embodiments, the first amino acid residue G of CH3 is substituted with the first amino acid residue A of CH1. For ease of reference, such substitution is also referred to herein as G341A. In some embodiments, the CH3 immediately adjacent to the C-terminus of VH or VL contains G341A. Not wishing to be bound by theory, the CH3 immediately adjacent to the C-terminus of VH or VL and engineered to contain the N-terminal fragment of CH1 can improve the assembly and / or purity of the binder as disclosed herein. In some embodiments, in each CH3 / CH3 pair immediately adjacent to the C-terminus of VH or VL, one CH3 contains the S354C and E357W mutations, while the other CH3 contains the Y349C and K370R mutations. In some embodiments, in each CH3 / CH3 pair immediately adjacent to the C-terminus of VH or VL, one CH3 contains the G341A, S354C, and E357W mutations, while the other CH3 contains the Y349C and K370R mutation. In some embodiments, in each CH3 / CH3 pair immediately adjacent to the C-terminus of a CH3 cell in the VH or VL region, one CH3 cell contains the G341A, S354C, and E357W mutations, while the other CH3 cell contains the G341A, Y349C, and K370R mutations.

[0238] In some embodiments, the multispecific antibody described herein comprises one or more CH3 mutations as disclosed in WO 2022 / 125986, which is incorporated herein by reference in its entirety. In some embodiments, the multispecific antibody described herein comprises one or more CH3 domains as disclosed in WO 2022 / 125986.

[0239] Therefore, any one or more of the CH3 sequence contains any of the following: 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); OR GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108; T366W).

[0240] In some embodiments, the binders disclosed herein comprise a CH3 sequence as disclosed herein (e.g., any of SEQ ID NO: 70-74), but said CH3 lacks the C-terminal amino acid residue lysine (K). In other embodiments, CH3 is located at the C-terminus of any one or more polypeptides of the binder. In some embodiments, the C-terminal lysine is cleaved in one or more polypeptides of the binder, such as any one, any two, or any three of the following: a first polypeptide of the binder disclosed herein, a second polypeptide of the binder disclosed herein, and a third polypeptide of the binder disclosed herein.

[0241] In some embodiments, one CH3 adjacent to the C-terminus of VH or VL in the VH / VL pair contains GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:103; Y349C and K370R), while another CH3 (adjacent to the C-terminus of VL or VH in the same VH / VL pair) contains AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:106; N-terminal amino acid residues exchanged with CH1, S354C and E357W).

[0242] In some implementations, the CH3 / CH3 pair immediately adjacent to the C-terminus of the CH2 sequence (e.g.) Figure 22Of the two CH3s (without *) in each of the three figures shown, one CH3 contains GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:107; D356E, L358M, T366S, L368A, and Y407V), while the other CH3 contains GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:108; T366W).

[0243] Those skilled in the art will understand, and unless otherwise stated, that EU numbers are referred to herein when describing antibodies or mutations in fragments such as Fc or CH3. Further details can be found at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs, which are hereby incorporated in their entirety by reference and identify residues according to their position in the endogenous constant region sequence, regardless of the physical location of said residues within the range of antibody constructs described herein. For example, in CH3 composed of aa 116 to aa 222 of SEQ ID NO: 83, the first aa of CH3 (i.e., the 116th aa of SEQ ID NO: 83) is numbered 341 and referred to herein as G341; the 9th aa of CH3 is referred to herein as Y349; the 14th aa of CH3 is referred to herein as S354; the 16th aa of CH3 is referred to herein as D356; the 17th aa of CH3 is referred to herein as E357; the 18th aa of CH3 is referred to herein as L358; the 26th aa of CH3 is referred to herein as T366; the 28th aa of CH3 is referred to herein as L368; the 30th aa of CH3 is referred to herein as K370; and the 67th bit of CH3 is referred to herein as Y407. Therefore, mutations in aa residues can be added after the EU number to indicate the mutation, such as S354C, E357W, Y349C, K370R, D356E, L358M, T366W, T366S, L368A, and Y407V.

[0244] In some embodiments, the binder, as disclosed herein, comprises a variant Fc region, such as the silenced Fc disclosed herein. In some embodiments, the binder lacks one or more effector functions, such as ADCC, ADCP, or CDC. In some embodiments, the variant Fc region comprises CH2 as disclosed herein, such as CH2 containing a mutation that reduces effector function as disclosed herein.

[0245] In some embodiments, one or more of the CH2 sequence contains APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (aa 6 to aa 115, L234, L235, and P329 of SEQ ID NO: 109; SEQ ID NO: 83). Alternatively or additionally, one or more of the CH2 sequence lacks mutations that reduce the effector function of the binder. In other embodiments, one or more of the CH2 sequence contains mutations that reduce (including significantly reduce and eliminate) the effector function of multispecific binders (e.g., ADCC, ADCP, or CDC). In other embodiments, one or more of the CH2 sequence contains any one, any two, or all three of the following mutations: L234A, L235A, and P329K (according to EU numbers). In some embodiments, one or more of the CH2 sequence contains a mutation in any one, any two, or all three of the following amino acid residues: L234, L235, and P329 (according to EU numbering). In some embodiments, one or more of the CH2 sequence contains APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAK (aa 6 to aa 115, L234A, L235A, and P329K of SEQ ID NO: 110; SEQ ID NO: 84). In some embodiments, the multispecific antibody as described herein comprises one or more CH2 domains as disclosed in WO 2022 / 125986.

[0246] In some embodiments, one or more light chain constant domains (CLs) comprise RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 111) or RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82). In some embodiments, the multispecific antibody as described herein comprises one or more CL mutations as disclosed in WO 2022 / 125986. In some embodiments, the multispecific antibody as described herein comprises one or more CL domains as disclosed in WO 2022 / 125986.

[0247] In some embodiments, one or more CH1 sequences comprise ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:112; aa1 to aa103 of SEQ ID NO:85), ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO:113) or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDRKVEPKSC (SEQ ID NO:114). In some embodiments, the multispecific antibody described herein comprises one or more CH1 mutations as disclosed in WO 2022 / 125986. In some embodiments, the multispecific antibody described herein comprises one or more CH1 domains as disclosed in WO 2022 / 125986.

[0248] In some embodiments, the binders disclosed herein also include a hinge domain. For example, the hinge domain is immediately adjacent to the N-terminus of the CH2 domain, such as between CH3 and CH2 or between the light chain constant domain (CL) and CH2. Alternatively or concurrently, 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 other embodiments, the hinge domain comprises DKTHTCPPCP (SEQ ID NO: 115). In some embodiments, the multispecific antibodies described herein include one or more domain binding sites as disclosed in WO 2022 / 125986.

[0249] In some embodiments, the binders disclosed herein also include a linker. For example, the linker may be present between two variable regions within a single polypeptide (e.g., in a binder having a 2x1 or 1x2 format). In one example, the binders disclosed herein comprise a polypeptide optionally comprising, from the N-terminus to the C-terminus: VL-CL-linker-VL-CL-optional hinge-CH2-CH3. In another example, the binders disclosed herein comprise a polypeptide optionally comprising, from the N-terminus to the C-terminus: VL-CH3-linker-VL-CH3-optional hinge-CH2-CH3. In some embodiments, the linker comprises (SSSG). n (SEQ ID NO:116) or (SSG) n , where n is any positive integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. Alternatively or concurrently, the linker is about 1 amino acid long to about 30 amino acids long. In some embodiments, the linker comprises TASSGGSSSG (SEQ ID NO: 117).

[0250] In some implementations, the multispecific binders described herein have constructs as disclosed in WO 2022 / 125986.

[0251] In some embodiments, the multispecific binders disclosed herein include a first polypeptide comprising SEQ ID NO: 118. In some embodiments, the multispecific binders disclosed herein include a first polypeptide comprising SEQ ID NO: 119. In some embodiments, the multispecific binders disclosed herein include a first polypeptide comprising SEQ ID NO: 127. In some embodiments, the multispecific binders disclosed herein include a first polypeptide comprising SEQ ID NO: 128.

[0252] In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:121. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:123. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:124. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:125. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:129. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:130. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:131. In some embodiments, the multispecific binder disclosed herein includes a third polypeptide comprising SEQ ID NO:132.

[0253] In some embodiments, the multispecific binders disclosed herein include a second polypeptide comprising SEQ ID NO:120. In other embodiments, the multispecific binders disclosed herein comprise two second polypeptides, each comprising SEQ ID NO:120.

[0254] In some embodiments, the multispecific binders disclosed herein include a fourth polypeptide comprising SEQ ID NO:122 or SEQ ID NO:126. In other embodiments, the multispecific binders disclosed herein comprise two fourth polypeptides, each comprising SEQ ID NO:122 or SEQ ID NO:126.

[0255] In some embodiments, the multispecific binder 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.

[0256] In some embodiments, the multispecific binder 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 comprising SEQ ID NO:122.

[0257] In some embodiments, the multispecific binder disclosed herein comprises: a first polypeptide comprising SEQ ID NO:119; one or more (e.g., two) second polypeptides, each of which comprises SEQ ID NO:120; a third polypeptide comprising SEQ ID NO:121; and a fourth polypeptide comprising SEQ ID NO:122.

[0258] In some embodiments, the multispecific binder 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.

[0259] In some embodiments, the multispecific binder 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 comprising SEQ ID NO:126.

[0260] In some embodiments, the multispecific binder disclosed herein comprises: a first polypeptide comprising SEQ ID NO:119; one or more (e.g., two) second polypeptides, each of which comprises SEQ ID NO:120; a third polypeptide comprising SEQ ID NO:125; and a fourth polypeptide comprising SEQ ID NO:126.

[0261] In some embodiments, the multispecific binder 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.

[0262] In some embodiments, the multispecific binder 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 comprising SEQ ID NO:122.

[0263] In some embodiments, the multispecific binder disclosed herein comprises: a first polypeptide comprising SEQ ID NO:128; one or more (e.g., two) second polypeptides, each of which comprises SEQ ID NO:120; a third polypeptide comprising SEQ ID NO:129; and a fourth polypeptide comprising SEQ ID NO:122.

[0264] In some embodiments, the multispecific binder 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.

[0265] In some embodiments, the multispecific binder 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 comprising SEQ ID NO:126.

[0266] In some embodiments, the multispecific binder disclosed herein comprises: a first polypeptide comprising SEQ ID NO:128; one or more (e.g., two) second polypeptides, each of which comprises SEQ ID NO:120; a third polypeptide comprising SEQ ID NO:132; and a fourth polypeptide comprising SEQ ID NO:126.

[0267] In some embodiments, any one, any two, any three, or all four polypeptides (e.g., any polypeptide containing any of the amino acid sequences in SEQ ID NO: 118-132) of the multispecific binders disclosed herein are modified by one or more insertions, one or more deletions, or one or more substitutions in their amino acid sequences. In other embodiments, these insertions, deletions, or substitutions are not within the CDR. In some embodiments, the modified polypeptide is at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the modified polypeptide.

[0268] In some aspects, this document 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). Alternatively 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). Alternatively 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). Alternatively or alternatively, the fourth polypeptide chain comprises an amino acid sequence as disclosed herein.

[0269] In some embodiments, the multispecific binder disclosed herein comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain as disclosed herein. In other embodiments, the C-terminal lysine of the first polypeptide chain of the multispecific binder is removed (e.g., cleaved). Alternatively or additionally, the C-terminal lysine of the second polypeptide chain of the multispecific binder is removed (e.g., cleaved). Alternatively or additionally, the C-terminal lysine of the third polypeptide chain of the multispecific binder is removed (e.g., cleaved). Alternatively or additionally, the C-terminal lysine of the first polypeptide chain and the C-terminal lysine of the second polypeptide chain of the multispecific binder are removed (e.g., cleaved). Alternatively or additionally, the C-terminal lysine of both the first and third polypeptide chains of the multispecific binder are removed (e.g., cleaved). Alternatively or additionally, the C-terminal lysine of both the second and third polypeptide chains of the multispecific binder are removed (e.g., cleaved). Alternatively or additionally, each C-terminal lysine of the first, second, and third polypeptide chains is removed (e.g., cleaved) from the multispecific binder. As used herein, a multispecific binder that removes any one, two, or all three of the C-terminal lysine amino acid residues is referred to as a C-terminal variant of the multispecific binder. Therefore, this document also provides a composition comprising a multispecific binder and one or more of its C-terminal variants, or a composition comprising one or more C-terminal variants of said multispecific binder. Those skilled in the art will understand that the compositions, methods, uses, or any other embodiments related to multispecific binders disclosed herein are also extended to compositions, methods, uses, or embodiments comprising: (i) C-terminal variants of a multispecific binder; or (ii) compositions comprising any one or more of the following: a multispecific binder and / or its C-terminal variants.

[0270] In some embodiments, the multispecific antibodies provided herein also include 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) at the N-terminus, followed by three constant domains (CH), namely CH1, CH2, and CH3. The hinge region is a segment of the heavy chain between the Fab and Fc portions. CH1, hinge, CH2, and CH3 can each be derived from any naturally occurring or artificial species and may contain variations. An exemplary IgG heavy chain contains the following amino acid sequences for CH1, hinge, CH2, and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:81).

[0271] Another exemplary IgG heavy chain contains the following amino acid sequence for CH1, hinge, CH2, and CH3: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:85).

[0272] In some embodiments, the multispecific antibodies provided herein also include one or more light chain constant domains (CLs). An exemplary light chain includes the following CL amino acid sequence: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:82).

[0273] In some embodiments, the binders provided herein inhibit HLA-E / NKG2A (e.g., HLA-E / NKG2A / CD94) signaling. Alternatively or additionally, the binders provided herein inhibit PD-1 / PD-L1 checkpoint signaling. In some embodiments, the binders provided herein promote NK cell-mediated cytotoxicity. In some embodiments, the binders provided herein promote NK cell degranulation. In some embodiments, the binders provided herein activate CD8+ T cells.

[0274] Other exemplary binding molecules are described in more detail in the following sections. In some embodiments, the multispecific binder according to any of the above embodiments may be incorporated, alone or in combination, into any of the features described in sections 5.2.4 to 5.2.7 below.

[0275] Table 1: Antibody clone A3

[0276] Table 2: Antibody clone A2

[0277] Table 3: Antibody clone A42

[0278] Table 4: Antibody clone A11

[0279] Table 5: Antibody clone P12

[0280] 5.2.4. Antibody Fragments Even though the term "antibody" is sometimes used herein in the phrase "antibody or a fragment thereof," it should be understood that, as used herein, the term "antibody" also includes various antibody fragments, such as antigen-binding fragments or epitope-binding fragments. Therefore, when the term "antibody" is used alone without the subsequent phrase "fraction thereof" or a similar term, it should be understood that the term "antibody" includes antibody fragments, such as antigen-binding fragments or epitope-binding fragments. The antibodies described herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. In some embodiments, the multispecific binders described herein comprise one or more antibody fragments.

[0281] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to antigens. Antibody fragments include, but are not limited to, those described in Section 5.1 above. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and comprising a light chain and a portion of a heavy chain bridged by disulfide bonds); Fab' (e.g., antibody fragments containing a single antigen-binding domain comprising a Fab and an additional heavy chain portion joined via a hinge region); F(ab')2 (e.g., two Fab' molecules joined by interchain disulfide bonds in the heavy chain hinge region; the Fab' molecules may target the same or different epitopes); bispecific Fab (e.g., Fab molecules having two antigen-binding domains, each of which may target different epitopes); single chains containing variable regions, also known as scFv (e.g., variable, antigen-binding-determining regions of a single light and heavy chain of an antibody linked together by a series, e.g., 10-25 amino acids); disulfide-linked Fvs or dsFvs (e.g., variable, antigen-binding-determining regions of a single light and heavy chain of an antibody linked together by disulfide bonds); and bispecific scFvs. (e.g., scFv or dsFv molecules having two antigen-binding domains, each of which may target different epitopes); bifunctional antibodies (e.g., dimerized scFvs formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv and the VL domain of the first scFv is assembled with the VH domain of the second scFv; the two antigen-binding domains of the bifunctional antibody may target the same or different epitopes); trifunctional antibodies (e.g., trimerized scFvs formed in a manner similar to bifunctional antibodies, but the three antigen-binding domains produce a single complex; the three antigen-binding domains may target the same or different epitopes); and tetrafunctional antibodies (e.g., tetramerized scFvs formed in a manner similar to bifunctional antibodies, but the four antigen-binding domains produce a single complex; the four antigen-binding domains may target the same or different epitopes).

[0282] Several techniques have been developed for generating antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be generated directly from recombinant host cells. For example, Fab, Fv, and ScFv antibody fragments can all be generated in *E. coli* (…). E. coli These fragments are expressed and secreted by yeast or insect cells, thereby allowing for the convenient production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, the Fab'-SH fragment can be directly recovered from *E. coli* and chemically conjugated to form the F(ab')2 fragment (Carter et al., 1992, Bio / Technology 10:163-67). According to another method, the F(ab')2 fragment can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments, having extended in vivo half-lives and containing rescue receptor-binding epitope residues, are described, for example, in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In some embodiments, the antibody is a single-chain Fv fragment (scFv) (see, for example, WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458). Fv and scFv have complete combinatorial sites without constant regions; therefore, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of the scFv (see, for example, Borrebaeck, ed., ibid.). Antibody fragments can also be “linear antibodies,” as described in the references cited above. Such linear antibodies can be monospecific or multispecific, such as bispecific antibodies.

[0283] 5.2.5. Humanized Antibodies This disclosure provides humanized antibodies that bind to NKG2A and PD-L1, including human NKG2A and human PD-L1. The humanized antibodies of this disclosure may contain one or more CDRs from the VH and / or VL disclosed herein, such as those shown in Tables 1 through 5. Various methods for humanizing non-human antibodies are known in the art. For example, one or more amino acid residues from a non-human source may be introduced into the humanized antibody. These non-human amino acid residues are generally referred to as “input” residues and are typically derived from an “input” variable domain. Humanized antibodies binding to NKG2A can be produced 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).

[0284] In some cases, humanized antibodies are constructed via CDR grafting, where the amino acid sequences of the six CDRs of the parental non-human antibody (e.g., rodent) are grafted onto the human antibody framework. For example, Padlan et al. ( FASEB J According to [reference needed], 9:133-139, 1995, only about one-third of the residues in the CDR actually contact the antigen, and these residues are referred to as "specificity-determining residues" or SDRs. In SDR transplantation techniques, only SDR residues are transplanted onto the human antibody framework (see, for example, Kashmiri et al., 9:133-139). Methods 36: 25-34, 2005).

[0285] The selection of human light and heavy chain variable domains for the preparation of humanized antibodies is crucial for reducing antigenicity. For example, according to the so-called "best-fit" method, variable domain sequences for non-human (e.g., rodent) antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993)). J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol.196:901). Another approach uses a specific framework derived from a common sequence of all human antibodies having a specific subgroup with a light or heavy chain. Several different humanized antibodies can use the same framework (Carter et al. (1992)). Proc. Natl. Acad. Sci. USA , 89:4285; Presta et al. (1993) J. Immunol. (151:2623). In some cases, the framework is derived from the common sequence of the most abundant human subclasses, namely V. L Subgroup 6 I (V) L 6I) and V H Subgroup III (V) H III). In another approach, human germline genes are used at the source of the frame region.

[0286] In an alternative paradigm based on CDR comparisons (called superhumanization), frame homology is irrelevant. The method consists of: comparing non-human sequences with functional human germline gene lineages; selecting genes encoding canonical structures identical to or closely related to mouse sequences; next, selecting genes with the highest homology within CDRs from among genes sharing canonical structures with non-human antibodies as frame donors; and finally, transplanting non-human CDRs onto these frames (see, for example, Tan et al.). J. Immunol . 169: 1119-1125, 2002).

[0287] Antibodies typically require humanization to retain their affinity for antigens and other beneficial biological properties. To achieve this, according to one method, humanized antibodies are prepared by analyzing parental sequences and various conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well-known to those skilled in the art. Computer programs can be used to illustrate and demonstrate the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These computer programs include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol(234: 779-815, 1993) and the Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Examining these displays allows for the analysis of the potential roles of residues in the function of candidate immunoglobulin sequences, such as analyzing residues that affect the ability of candidate immunoglobulins to bind their antigens. In this way, framework residues can be selected and combined from the acceptor and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Generally, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0288] Another approach to antibody humanization is based on an antibody humanization metric called Human String Content (HSC). This method compares mouse sequences with human germline gene lineages and scores the differences using HSC. The target sequence is then humanized by maximizing its HSC, rather than using a measure of overall identity, to produce a variety of different humanized variants. See, for example, Lazar et al. Mol. Immunol. 44: 1986-1998, 2007.

[0289] In addition to the methods described above, empirical methods can also be used to generate and select humanized antibodies. These methods include those based on the generation of large libraries of humanized variants and the selection of the best clones, using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as through bacterial colony screening (see, for example, Hoogenboom). Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

[0290] In the frame library approach, a set of residue variants is introduced at specific locations within a frame, and the library is then selected to choose the frame that best supports the transplanted CDR. The residues to be substituted may include some or all of the "Vernier" residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter). J. Mol. Biol. 224:487-499, 1992), or from Baca et al. ( J. Biol. Chem. (272: 10678-10684, 1997) identified a more limited set of target residues.

[0291] In framework shuffling, the entire framework is combined with non-human CDRs rather than generating a combinatorial library of selected residue variants (see, for example, Dall'Acqua et al.). Methods 36: 43-60, 2005). A two-step selection procedure can be used to screen libraries for binding, wherein the VL is first humanized, followed by the VH. Alternatively, a single-step framework shuffling procedure can be used. This procedure has been shown to be more efficient than the two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermostability (see, for example, Damschroder et al., 2005). Mol. Immunol. 44: 3049-60, 2007).

[0292] The "humanizing" approach is based on experimental identification using the essential minimum specific determinant (MSD) and on the sequential substitution of non-human fragments into human framework libraries and the evaluation of binding. This method begins with the CDR3 region of the non-human VH and VL chains and progressively substitutes other regions of the non-human antibody into the human framework, including the CDR1 and CDR2 of both VH and VL. This approach typically results in epitope retention and identification of antibodies from multiple subclasses with different human V region CDRs. Humanizing allows the isolation of antibodies with 91-96% homology to human germline antibodies. See, for example, Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007.

[0293] "Human engineering" methods involve altering non-human antibodies or antibody fragments, such as mouse or chimeric antibodies or fragments, by making specific changes to the amino acid sequence of an antibody to produce a modified antibody that has reduced immunogenicity in humans but still retains the desired binding properties of the original non-human antibody. Typically, this technique involves classifying amino acid residues of non-human (e.g., mouse) antibodies into "low-risk," "intermediate-risk," or "high-risk" residues. This classification is performed using an overall risk / reward calculation that evaluates the predicted benefit of performing a specific substitution (e.g., immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody and / or its substitution by human residues. Specific human amino acid residues intended to be substituted at a given position (e.g., low-risk or intermediate-risk) in the non-human (e.g., mouse) antibody sequence can be selected by comparing the amino acid sequence of a variable region from a non-human antibody with corresponding regions of a specific or common human antibody sequence. Based on the comparison, amino acid residues at low-risk or intermediate-risk positions in the non-human sequence can be substituted with corresponding residues in the human antibody sequence. Techniques for the preparation of human engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994); U.S. Patents 5,766,886, 5,770,196, 5,821,123 and 5,869,619; and WO 93 / 11794.

[0294] 5.2.6. Antibody Variants This covers modifications to the multispecific antibodies described herein that bind to NKG2A and PD-L1. For example, it may be necessary to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, variants of the antibodies described herein may be prepared and included in this disclosure after careful consideration. In some embodiments, the antibody variant is an antibody with an amino acid sequence variation relative to the original antibody, such as having substitutions, deletions, or insertions of one or more amino acids as described above. For example, the variation may be a substitution, deletion, or insertion (e.g., conserved substitution) of one or more codons encoding the antibody or peptide that causes a change in the amino acid sequence compared to the original antibody or peptide. Sites of interest induced by substitutional mutations include CDRs, FRs, and / or constant regions. For example, antibody variants may be prepared by introducing appropriate nucleotide changes into encoding DNA and / or by synthesizing the desired antibody or peptide. Those skilled in the art will understand that amino acid changes can alter the post-translational processing of antibodies (e.g., multispecific antibodies).

[0295] Chemical modification Other exemplary modifications include, for example, chemical modifications by covalently linking any type of molecule to a multispecific antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / capping groups, proteolytic cleavage, linking to cellular ligands or other proteins, or conjugating to one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, and the metabolic synthesis of tunicamycin. Additionally, antibodies may contain one or more non-canonical amino acids.

[0296] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites in the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0297] When the antibodies (e.g., bispecific antibodies) presented herein are fused to the Fc region, the linked carbohydrates can be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, usually linked to Asn297 in the CH2 domain of the Fc region via N-bonds. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc linked to the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the binding molecules provided herein may be modified to produce variants with certain improved properties.

[0298] In other embodiments, when the antibody provided herein is fused to the Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose linked (directly or indirectly) to said Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. As described, for example, in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose at Asn297 within the glycan chain relative to the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publications 2003 / 0157108 and 2004 / 0093621. Examples of disclosures relating to antibody variants that are “defucosylated” or “fucose-deficient” include: U.S. Patent Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent Publication No. 2002 / 0164328; U.S. Patent Publication No. 2004 / 0093621; U.S. Patent Publication No. 2004 / 0132140; U.S. Patent Publication No. 2004 / 0110704; U.S. Patent Publication No. 2004 / 0110282; U.S. Patent Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., ). Arch. Biochem. Biophys 249:533-545 (1986); US Patent Publication No. 2003 / 0157108; and WO 2004 / 056312) and gene knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 Gene knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng .87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. , 94(4):680-688 (2006); and WO2003 / 085107).

[0299] Binding molecules containing the antibodies provided herein further possess a sharded oligosaccharide, for example, wherein the biantennary oligosaccharide linked to the Fc region is sharded via GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Publication No. 2005 / 0123546 (Umana et al.). Variants are also provided wherein at least one galactose residue of the oligosaccharide is linked to the Fc region. Such variants may have improved CDC function. Such variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0300] In molecules containing the antibodies of the present invention and the Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby producing Fc region variants. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0301] In some embodiments, this application covers variants having some, but not all, of the effector functions, making these variants ideal candidates for applications where the in vivo half-life of the binding molecule is critical, and certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the binding molecule does not have FcγR binding capacity (and therefore may not have ADCC activity), but retains FcRn binding capacity. Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., P. roc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al. Proc. Nat' Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al.) J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used for this type of analysis include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, in vivo assays can be performed, for example in animal models such as Clynes et al. Proc. Nat'l Acad. Sci. USA The ADCC activity of the molecule of interest was assessed in animal models disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al.). J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol . 18(12):1759-1769 (2006)).

[0302] Binding molecules with reduced effector function include those with substitutes for one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted with alanine (US Patent No. 7,332,581).

[0303] Certain variations that improve or reduce the effect of combining with FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al.) J. Biol. Chem.9(2): 6591-6604 (2001). In some embodiments, the variant comprises amino acid substitutions that improve ADCC, such as substituted Fc regions at positions 298, 333, and / or 334 (EU numbers of residues) in the Fc region. In some embodiments, modifications are made in the Fc region that alter (i.e., improve or reduce) C1q binding and / or complement-dependent cytotoxicity (CDC), as in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. As described in ,164: 4178-4184(2000).

[0304] It has a prolonged half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for transferring maternal IgG to the fetus (Guyer et al.). J. Immunol. 117:587 (1976) and Kim et al., J. Immunol The binding molecules described in 24:249 (1994) are described in US2005 / 0014934A1 (Hinton et al.). Those molecules contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, the substitution of Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan and Winter. Nature , 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0305] In some embodiments, it may be necessary to generate cysteine-engineered antibodies, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In some embodiments, the substituted residues are present at accessible sites on the antibody. By replacing said residues with cysteine, reactive thiols are thereby located at accessible sites on the antibody and can be used to conjugate the antibody with other parts (e.g., pharmaceutical parts or linker-pharmaceutical parts) to produce immunoconjugates as further described herein.

[0306] Other known covalent modifications of antibodies are included within the scope of this disclosure. Covalent modifications include reacting target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the antibody. Other modifications include deamidation of glutamine acyl and asparagine acyl residues to the corresponding glutamine acyl and asparagine acyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of the lysine, arginine, and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl group.

[0307] The antibodies of this disclosure that bind to NKG2A and PD-L1 can also be modified to form chimeric molecules, which comprise antibodies fused to or conjugated to another heterologous polypeptide or amino acid sequence or small molecule compound, such as an immune activator (e.g., cytokine), an epitope tag (see, for example, Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)), or the Fc region of an IgG molecule (see, for example, Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi, eds., 1999)).

[0308] This document also provides fusion proteins comprising the multispecific antibodies and heterologous peptides of this disclosure that bind to NKG2A and PD-L1. In some embodiments, the heterologous peptides genetically fused with or chemically conjugated to the antibody can be used to target the antibody to cells having NKG2A and / or PD-L1 expressed on their cell surface. The genetically fused or chemically conjugated antibodies are described in more detail in the following sections.

[0309] In vitro affinity maturation In some implementations, antibody variants with improved properties (e.g., affinity, stability, or expression levels) compared to the parent antibody can be prepared via in vitro affinity maturation. Similar to the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. The antibody library is displayed on the surface of an organism (e.g., bacteriophage, bacteria, yeast, or mammalian cells) or associated with its encoding mRNA or DNA (e.g., covalently or non-covalently). Affinity selection of the displayed antibody allows the isolation of the organism or complex carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods (e.g., phage display) typically produce antibody fragments with affinity in the low nanomolar concentration range. Affinity-matured antibodies can have nanomolar or even picomolar affinity for the target antigen.

[0310] Phage display is a widely used method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 phages in the form of fusions with phage coat proteins. Selection involves exposing the antibody to an antigen to allow the phage to display and bind to its target; this method is called “panning.” Phages bound to the antigen are recovered and bacteria are infected with said phages to produce phages for further rounds of selection. For reviews, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0311] In yeast display systems (see, for example, Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesion subunit of the yeast lectin protein Aga2p, which is linked to the yeast cell wall via a disulfide bond with Aga1p. Protein display via Aga2p causes the protein to protrude away from the cell surface, thereby minimizing potential interactions with other molecules on the yeast cell wall. Libraries are screened using magnetic separation and flow cytometry to select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling yeast with biotinylated antigen and a second reagent (e.g., streptavidin conjugated with a fluorophore). Changes in antibody surface expression can be measured via immunofluorescence labeling of hemagglutinin or c-Myc epitope tags to side-attached single-chain antibodies (e.g., scFv). It has been shown that expression is correlated with the stability of the displayed protein, and therefore antibodies can be selected for improved stability and affinity (see, for example, Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of eukaryotic yeast cells, thus utilizing endoplasmic reticulum chaperone proteins and quality control mechanisms. Once fully matured, antibody affinity can be conveniently “tied” simultaneously with display on the yeast surface, thereby eliminating the need for expression and purification of each clone. The theoretical limitation of yeast surface display is that the functional library size is potentially smaller than that of other display methods; however, recent methods using yeast cell-co ... 14 Combinatorial diversity (see, for example, U.S. Patent Publication 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0312] During ribosome display, an antibody-ribosome-mRNA (ARM) complex is generated for selection in a cell-free system. A DNA library encoding a specific antibody library is genetically fused with a spacer sequence lacking a stop codon. This spacer sequence remains linked to the peptidyl tRNA during translation and occupies a ribosomal tunnel, thereby allowing the protein of interest to protrude from and fold from the ribosome. The resulting complex of mRNA, ribosome, and protein can bind to a surface-bound ligand, allowing simultaneous separation of the antibody and its encoding mRNA via ligand affinity capture. The ribosome-bound mRNA is then reverse transcribed into cDNA, which can then undergo mutation induction and be used for the next round of selection (see, for example, Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). During mRNA display, the covalent bond between the antibody and the mRNA is generated using puromycin as a translinker molecule (Wilson et al., Proc.Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0313] Because these methods are performed entirely in vitro, they offer two main advantages over other selection techniques. First, library diversity 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 round of selection (e.g., by uncorrected polymerase) because no library must be transformed after any diversification step. In some implementations, mammalian display systems can be used.

[0314] Diversity can also be introduced into the CDRs of an antibody library either through targeted approaches or random introduction. Methods for introducing diversity through targeted approaches include sequentially targeting all CDRs of the antibody via high- or low-level mutagenesis, or targeting isolated somatic hypermutation hotspots (see, for example, Ho et al., J. Biol. Chem. 280:607-17 (2005)), or targeting residues suspected of affecting affinity, either experimentally or for structural reasons. Diversity can also be introduced by replacing naturally different regions using DNA shuffling or similar techniques (see, for example, Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patents 5,565,332 and 6,989,250). Alternative techniques employ loop deletions and insertions in the CDR or utilize hybridization-based diversification (see, for example, U.S. Patent Publication No. 2004 / 0005709) to target hypervariable loops extending into the frame region residues (see, for example, Bond et al., J. Mol. Biol. 348:699-709 (2005)). Other methods for generating diversification in the CDR are disclosed, for example, in U.S. Patent No. 7,985,840. Other methods that can be used to generate antibody libraries and / or mature antibody affinity are disclosed, for example, in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publications Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0315] Library screening can be achieved using a variety of techniques known in the art. For example, antibodies can be immobilized on solid carriers, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells attached to an adsorption plate or used for cell sorting, or conjugated with biotin to capture with streptavidin-coated beads or used in any other method for panning and displaying libraries.

[0316] For reviews of in vitro affinity maturation methods, see, for example, Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and the references therein.

[0317] Antibody internalization assays can be used to determine receptor-mediated endocytosis when bound to an antibody. In some embodiments, the efficacy of certain antibody-based therapeutics depends on the antibody internalization process. In some embodiments, antibody internalization assays examine the rate and extent of antibody internalization to evaluate the antibody's ability to deliver treatment to a site of interest or cells. A non-limiting exemplary assay is simply described below. Target cells of interest are seeded at an appropriate seeding density (e.g., in a 96-well U-shaped plate), and the 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 is then incubated with the target cells at an appropriate molar ratio. After incubation, unbound antibodies are removed by washing. Cells may be retained on ice or incubated at 37°C for a period of time to promote internalization. Cells may then be incubated for a period of time in the presence of a termination reagent to inhibit internalization. Subsequently, the cells are washed and incubated with a signal display reagent. The final signal can be studied using a plate reader or imaging instrument and analysis software. For example, flow cytometry can be used to measure the mean fluorescence intensity (MFI) of cells, and a decrease in MFI can indicate antibody internalization, antibody dissociation, or a combination of both. Cell images can be scanned and acquired to analyze signal intensity, size, and shape. Alternatively, cells can be lysed to release the internalized antibody. This antibody is then captured in a microtiter plate coated with a specific antigen that induces antibody production. The bound antibody in the wells is detected using alkaline phosphatase or HRP-conjugated secondary antibody and a chromogenic substrate. Alternative antibody detection labels and methods for detecting internalized labeled antibodies will be apparent to those skilled in the art. Any method known in the art for determining antibody internalization can be used in this disclosure.

[0318] 5.2.7. Other binding agents containing multispecific antibodies 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 will be described below.

[0319] This disclosure provides conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) having a masking moiety and / or a cleavable moiety, wherein one or more of the NKG2A binding domain and / or PD-L1 binding domain of the conjugate (e.g., via the masking moiety) are masked (e.g., via the masking moiety) and / or activated (e.g., via the cleavable moiety). Techniques for masking antibodies are well known in the art, including SAFEbody masking techniques (see, for example, U.S. Patent Publication No. 2019 / 0241886) and Probody masking techniques (see, for example, U.S. Patent Publication No. 2015 / 0079088). Such techniques can be used to produce masked and / or activated conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies). Such masked and / or activatable conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) can also be used to prepare conjugates comprising any of the conjugates disclosed herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies), including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable ADCs (AADCs), said conjugates including those directly or indirectly linked to another pharmaceutical agent such as a drug and / or an immune activator (e.g., cytokines). For example, the conjugates of this disclosure (e.g., antibodies, multispecific conjugates, or bispecific antibodies) can be covalently bound to one or more pharmaceutical agents such as drugs and / or immune activators via synthetic linkers.

[0320] Where necessary, the conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) provided herein are linked or conjugated (directly or indirectly) to a portion having effector functions such as cytotoxic activity (e.g., a chemotherapeutic fraction or a radioisotope), immune recruitment, or regulatory activity. The linked or conjugated portions (directly or indirectly) include cytotoxic drugs (e.g., toxins such as aurostatins) or non-cytotoxic drugs, such as signal transduction modulators such as kinases or masking fractions that mask one or more binding domains of the conjugates provided herein, or cleavable fractions that allow activation of the conjugate by exposing one or more binding domains in the tumor microenvironment as a masked conjugate. Fractions that promote immune recruitment may include other antigen-binding agents, such as viral proteins that selectively bind to cells of the innate and / or adaptive immune system. Alternatively or additionally, the binding agents (e.g., antibodies, multispecific binding agents, or bispecific antibodies) provided herein optionally link or conjugate (directly or indirectly) to a portion that facilitates separation from a mixture (e.g., a tag) or a portion having reporter gene activity (e.g., a detection marker or reporter protein). It should be understood that the characteristics of the binding agents described herein also extend to polypeptides containing binding agent fragments.

[0321] In some embodiments, the multispecific binders (e.g., bispecific antibodies) described herein may be linked or conjugated (directly or indirectly) to a polypeptide, thereby inducing the generation of an activatable antibody. In some embodiments, the multispecific binders provided herein are linked or conjugated (directly or indirectly) to an additional agent. In some embodiments, the additional agent is a drug that generates an ADC or AADC when the antibody of the ADC comprises a masking portion and a cleavable portion.

[0322] In some embodiments, the conjugates described herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent or cytokine) or a diagnostic or diagnostic agent. Conjugated or recombinantly linked antibodies, including masked or activated conjugates, may be useful, for example, for treating or preventing diseases, symptoms, or disorders. Conjugated or recombinantly linked multispecific conjugates (e.g., bispecific antibodies), including masked or activated conjugates, may be useful, for example, for monitoring or predicting the onset, development, progression, and / or severity of diseases, symptoms, or disorders.

[0323] Such diagnostics and detections can be achieved, for example, by conjugating a binder (e.g., an antibody, a multispecific binder, or a bispecific antibody) to a detectable substance including, for example, the following: enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, including but not limited to streptavidin / biotin or avidin / biotin; fluorescent materials, including but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, including but not limited to luminol; bioluminescent materials, including but not limited to luciferase, luciferin, or aequorin; chemiluminescent materials, including but not limited to acridine-based compounds or HALOTAG; and radioactive materials, including but not limited to iodine (…). 131 I, 125 I, 123 I and 121 I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 115 In、 113 In、 112 In and 111 In), Technetium ( 99 Tc), thallium 201 Ti, gallium 68 Ga and 67 Ga), Palladium (103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F) 153 Sm、 177 Lu、 159 Gd, 149 Pm, 140 La、 175 Yb、 166 Ho、 90 Y、 47 Sc、 186 Re、 188 Re、 142 Pr、 105 Rh、 97 Ru、 68 Ge 57 Co、 65 Zn, 85 Sr、 32 P, 153 Gd, 169 Yb、 51 Cr 54 Mn, 75 Se、 113 Sn or 117 Sn; positron-emitting metals obtained using various positron emission tomography methods; and non-radioactive paramagnetic metal ions.

[0324] This document also describes multispecific binders (e.g., bispecific antibodies) that recombinantly link or conjugate (directly or indirectly covalently or non-covalently) to heterologous proteins or peptides, such as peptides (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce fusion proteins, and their uses. In some embodiments, the heterologous protein, peptide, or peptide linked to the binder (e.g., antibody, multispecific binder, or bispecific antibody) can be used to target the binder to specific cells (e.g., cells expressing NKG2A, such as immune cells; and / or cells expressing PD-L1, including immune cells, and / or cancer or tumor cells). Other non-restricted heterologous proteins, peptides, or peptides linked to the binder (e.g., antibody, multispecific binder, or bispecific antibody) can be used as internalization signals or to bind tumor cells to immune cells.

[0325] Furthermore, the binding agents described herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) can be linked (directly or indirectly) to a label or “tag” sequence, such as a peptide, to facilitate purification. In some embodiments, the label or tag amino acid sequence is a hexahistine peptide, such as the tags provided in pQE vectors (see, for example, QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistine facilitates the purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin (“HA”) tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37:767-78), and “FLAG” tags.

[0326] Methods for linking or conjugating (directly or indirectly) portions (including peptides) to antibodies are well known in the art, and any of these methods can be used to prepare the antibody-drug conjugates or fusion proteins described herein.

[0327] In some embodiments, the binding agents described herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) are fusion proteins. As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of a binding agent (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of an antibody). In some embodiments, the fusion protein retains the biological activity of the binding agent.

[0328] Fusion proteins can be produced, for example, via gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”). DNA shuffling can be used to alter the activity of binders (e.g., antibodies, multispecific binders, or bispecific antibodies) with high affinity and low dissociation rates as described herein (see, for example, U.S. Patents 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some implementations, the binding agent provided herein can be altered prior to recombination by undergoing error-prone PCR, random nucleotide insertion, or other methods of random mutation induction. The polynucleotide encoding the binding agent described herein can recombine with one or more components, motifs, segments, parts, domains, fragments, etc., of one or more heterologous molecules.

[0329] The conjugates described herein (e.g., antibodies, multispecific conjugates, or bispecific antibodies) can also be linked to solid carriers, thereby enabling their use in the immunoassay or purification of target antigens. Such solid carriers include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0330] The binding agents described herein (e.g., antibodies, multispecific binding agents, or bispecific antibodies) may also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0331] Linkers can be “cleavable portions” that facilitate the release of conjugated or ligated drugs into cells, but non-cleavable linkers are also covered herein. Linkers used in conjugates (e.g., ADCs or AADCs) of this disclosure include, but are not limited to, acid-instable linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photostable linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

[0332] Antibody-pharmaceutical conjugates, including those in which the pharmaceutical agent is a drug for preparing ADCs or AADCs, can be prepared using a variety of bifunctional protein conjugates, such as N-(β-cis-butenylpropoxy)succinimide ester (BMPS); N-ε-cis-butenylpropoxy succinimide ester (ECMS); N-γ-cis-butenylpropoxy succinimide ester (GMBS); 1,6-hexane-bis-vinyl sulfone (HBVS); succinimide-4(-N-cis-butenylpropoxy) ester. (Iminemethyl)cyclohexane-1-carboxy-(6-amidohexanoate) (LC-SMCC); m-cis-butenyldiimide benzoyl-N-hydroxysuccinimide (MBS); 4-(4-N-cis-butenyldiimidephenyl)butyric acid hydrazide (MPBH); succinimide-3-(bromoacetamyl)propionate (SBAP); succinimide-iodoacetate (SIA); succinimide-4-(N-cis-butenyldiimide)aminobenzoate (SIAB); Methylcyclohexane-1-carboxylate (SMCC); succinimide-4-(p-cis-butenylimide-phenyl)butyrate (SMPB); succinimide-6-(β-cis-butenylimide-propamido)hexanoate (SMPH); N-(ε-cis-butenylimide-octanoyloxy)sulfosuccinimide (sulfon-ECMS); N-(γ-cis-butenylimide-butanoyloxy)sulfosuccinimide (sulfon-GMBS); N-(κ-cis-butenylimide-undecanyloxy)sulfosuccinimide (Sulfo-KMUS); m-cis-butenyl benzoyl-N-hydroxysulfosuccinimide (Sulfo-MBS); sulfosuccinimide (4-iodo-acetyl)aminobenzoate (Sulfo-SIAB); sulfosuccinimide 4-(N-cis-butenyl-methyl)cyclohexane-1-carboxylate (Sulfo-SMCC); sulfosuccinimide 4-(p-cis-butenyl-phenyl)butyrate (Sulfo-SMPB); and succinimide-(4-vinyl sulfone)benzoate (SVSB).

[0333] This disclosure further covers conjugates of antibodies and pharmaceutical agents, including those where the pharmaceutical agent is a drug for preparing an ADC or AADC, which can be prepared using any suitable method disclosed in the art (see, for example, Bioconjugate Techniques (Hermanson, ed., 2nd edition, 2008)).

[0334] Conventional conjugation strategies for antibodies and pharmaceuticals (including those for the preparation of ADCs or AADCs) are based on random conjugation chemistry involving ε-amino groups of Lys residues or thiol groups of Cys residues, resulting in heteroconjugates. Newly developed techniques allow for site-specific conjugation with antibodies, producing homogeneous loading and avoiding antigen-binding or pharmacokinetic changes in conjugate subsets. These techniques include the engineering of “thiomab” which involves cysteine ​​substitutions at sites on both the heavy and light chains that provide reactive thiol groups without disrupting immunoglobulin folding and assembly or altering antigen-binding positions (see, for example, Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another approach, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from the termination codon into a selenocysteine ​​insertion sequence, thereby allowing site-specific covalent conjugation at the nucleophilic selenool group of selenocysteine ​​in the presence of other natural amino acids (see, for example, Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry48(50):12047-57).

[0335] In some embodiments, the conjugates (antibodies, multispecific conjugates, or bispecific antibodies) described herein are conjugated to agents such as immune activators (e.g., cytokines) or cytotoxic agents. In some embodiments, the conjugates (e.g., antibodies, multispecific conjugates, or bispecific antibodies) described herein may optionally be conjugated to one or more cytotoxic agents disclosed herein or known in the art to produce an ADC or AADC. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including but not limited to methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other inserters. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants, or animals, including but not limited to diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, sarcin, tung oil protein (Aleuritesfordii), carnation protein, pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, jatropha toxin, croton toxin, soapwort (Sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the cytotoxic agent is a radioactive isotope used to produce a radioconjugate or radioconjugate agent. Many radionuclides can be used to generate radioconjugates, including but not limited to... 90 Y、 125 I, 131 I, 123 I, 111 In、 131 In、 105 Rh、 153 Sm、 67 Cu、 67 Ga、 166 Ho、 177 Lu、 186 Re、 188 Re and 212Bi. Conjugates of polypeptides or molecules and one or more small molecule toxins may also be used, such as calicheamicin, maytansinoids, trichothene, and CC1065, as well as derivatives of these toxins with toxic activity. A variety of bifunctional protein conjugates are used to prepare conjugates of peptides or molecules and cytotoxic agents, such as N-succinimide-3-(2-pyridinedithiol)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imide esters (e.g., dimethyl adipamide ester HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)hexamet...

Claims

1. A multispecific antibody or a fragment thereof, said multispecific antibody or fragment comprising a first binding domain to NKG2A and a second binding domain to PD-L1, wherein said first binding domain comprises: (i) VH CDR1, VH CDR2 and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 64 and VL CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 73; (ii) VH CDR1, VH CDR2 and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25 and VL CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26; (iii) VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 45, and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 46; or (iv) VH CDR1, VH CDR2 and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:64 and VL CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:65; Optionally, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are determined according to the Kabat numbering scheme, the Chothia numbering scheme, the AbM numbering scheme, the Contact numbering scheme, the IMGT numbering scheme, or a combination thereof.

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

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

50.

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

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

6.

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

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

31.

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

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

50.

10. The multispecific antibody or fragment thereof as claimed in claim 1, wherein the first binding domain comprises VH comprising the amino acid sequence of SEQ ID NO: 64 and VL comprising the amino acid sequence of SEQ ID NO: 73.

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