Anti-PD-L1 monoclonal antibody and fusion protein with interleukin-15 (IL-15), interleukin-15 receptor 15 alpha, or interleukin-2

Abstract

The present disclosure provides proteins having antibody heavy and light variable domains that can pair to form an antigen-binding site that specifically binds to PD-L1. In certain embodiments, the proteins or antigen-binding sites form an antibody or a bifunctional antibody, such as, for example, a PD-L1 / IL-2Rβ bifunctional antibody. Also provided are pharmaceutical compositions comprising such proteins, therapeutic methods for using such proteins, and pharmaceutical compositions thereof, including for the treatment of cancer.

Description

[Technical field]

[0001] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is 300096_402WO_SEQUENCE_LISTING.txt. The text file is 220KB, was created on January 20, 2021, and has been submitted electronically via EFS-Web. [Background technology]

[0002] PD-L1 (programmed death ligand 1), also known as CD274 or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that inhibits TCR-mediated activation of IL-2 production and T cell proliferation upon binding of PD-L1 to PD-1 on T cells. Anti-PD-L1 antibodies have been used as therapeutic agents for the treatment of cancer. However, there remains a need for biotherapeutics that more effectively modulate tumor growth. Summary of the Invention

[0003] The present disclosure provides proteins having antibody heavy and light variable domains that can pair to form an antigen-binding site that specifically binds to PD-L1. The proteins or antigen-binding sites of the present disclosure may form antibodies or bifunctional antibodies, e.g., PD-L1 / IL-2Rβ bifunctional antibodies. The proteins or antigen-binding sites of the present disclosure may be used to treat or prevent cancerous or infectious conditions and disorders.

[0004] In some embodiments, disclosed herein are complementary determining region 1 (CDR1) sequences of SEQ ID NO: 3, 11, 19, 33, 52, or 63; complementary determining region 2 (CDR2) sequences of SEQ ID NO: 4, 12, 20, 34, 41, 53, or 64; complementary determining region 3 (CDR3) sequences of SEQ ID NO: 5, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, 86, or 89. and a light chain variable domain (VL) comprising: a CDR1 sequence of SEQ ID NO: 6, 14, 22, 28, 36, 47, 55, or 66; a CDR2 sequence of SEQ ID NO: 7, 15, 23, 29, 37, 42, 48, 56, 60, or 67; and a CDR3 sequence of SEQ ID NO: 8, 16, 24, 30, 38, 43, 49, 57, or 68.

[0005] In some embodiments, the antigen-binding site is present in a single chain fragment variable fragment (scFv), an antigen-binding fragment (Fab), an antibody, or similar antigen-binding protein.

[0006] In some embodiments, disclosed herein is (a) an antigen-binding site that binds to PD-L1, (i) a heavy chain variable domain (VH) comprising a complementarity determining region 1 (CDR1) of SEQ ID NO: 3, 11, 19, 33, 52, or 63; a complementarity determining region 2 (CDR2) of SEQ ID NO: 4, 12, 20, 34, 41, 53, or 64; and a complementarity determining region 3 (CDR3) of SEQ ID NO: 55, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, 86, or 89; (ii) an antigen-binding site that binds to PD-L1, comprising: a CDR1 sequence of SEQ ID NO: 6, 14, 22, 28, 36, 47, 55, or 66; a CDR2 sequence of SEQ ID NO: 7, 15, 23, 29, 37, 42, 48, 56, 60, or 67; and a light chain variable domain (VL) comprising a CDR3 sequence of SEQ ID NO: 8, 16, 24, 30, 38, 43, 49, 57, or 68; (b) A bifunctional protein comprising an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Rα) polypeptide, a wild-type interleukin-2 (IL-2) polypeptide, or an engineered IL-2 polypeptide, or a functional fragment or variant thereof.

[0007] In some aspects, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antigen binding site, protein or antibody, bifunctional protein or antibody, or a pharmaceutical composition thereof. In certain aspects, the disease is cancer.

[0008] These and other aspects of the present disclosure will become evident upon reference to the following detailed description and the accompanying drawings, in which: All references disclosed herein are incorporated by reference in their entirety as if each were individually incorporated. [Brief description of the drawings]

[0009] [Figure 1] ELISA binding of identified scFvs to PD-L1 is shown. [Diagram 2] AB show FACS analysis of identified scFvs binding to K562 cells engineered to express high levels of cell surface human PD-L1. [Diagram 3] Panels A-B show the competition results of PD-1 and PD-L1 for the identified scFvs in HTRF. [Figure 4] The results of a Jurkat cell NFAT reporter assay for the identified scFvs are shown. [Diagram 5] ELISA binding of affinity matured scFv to PD-L1. [Figure 6] Figure 2 shows FACS analysis of affinity matured scFv binding to K562 cells engineered to express high levels of cell surface human PD-L1. [Figure 7]1 shows the competition results of PD-1 and PD-L1 for affinity matured scFvs in HTRF. [Figure 8] NFAT reporter assay of Jurkat cells for affinity matured scFvs. [Figure 9] Antibodies, bifunctional antibodies, and alternative molecular formats are shown, including those that bind to PD-L1 to form the IL-2 cytokine, or those that bind to PD-L1 to form the IL-15 cytokine, respectively. [Figure 10] Figure 2 shows FACS analysis of anti-PD-L1 IgG antibodies binding to K562 cells engineered to express high levels of cell surface human PD-L1. [Figure 11] Jurkat cell NFAT reporter assay of anti-PD-L1 / IL-15 fusion antibodies. [Figure 12A] Figure 2 shows the results of p-STAT5 activation by anti-PD-L1 / IL-15 fusion antibodies. Figure 2 shows the activation of p-STAT5 in CD4+FoxP3- T cells. [Figure 12B] Figure 2 shows the results of p-STAT5 activation by anti-PD-L1 / IL-15 fusion antibodies. Figure 2 shows the activation of p-STAT5 in NK cells. [Figure 12C] Figure 2 shows the results of p-STAT5 activation by anti-PD-L1 / IL-15 fusion antibodies. Figure 2 shows the activation of p-STAT5 in CD8+ T cells. [Figure 12D] Figure 2 shows the results of p-STAT5 activation by anti-PD-L1 / IL-15 fusion antibodies. Figure 2 shows the activation of p-STAT5 in regulatory T cells. [Figure 13] A-B show ELISA binding of IL-2-Fc clones to IL-2 receptors. A shows ELISA binding of IL-2-Fc clones to IL-2Rα receptors. B shows ELISA binding of IL-2-Fc to IL-2Rβ receptors. [Figure 14A]Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in CD4+ T cells from donor 126. [Figure 14B] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in CD8+ T cells from donor 126. [Figure 14C] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in NK cells from donor 126. [Figure 14D] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in regulatory T cells from donor 126. [Figure 14E] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in CD4+ T cells from donor 359. [Figure 14F] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in CD8+ T cells from donor 359. [Figure 14G] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in NK cells from donor 359. [Figure 14H] Figure 1 shows p-STAT5 activation assay with IL-2-Fc clones performed on cells derived from human PBMCs of two separate donors. Figure 2 shows activation of p-STAT5 in regulatory T cells from donor 359. [Figure 15A]Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to IL-2Rα. [Figure 15B] Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to IL-2Rβ. [Figure 15C] Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 scFv Fc variants to PD-L1. [Figure 16] Figure 1 shows Jurkat cell NFAT reporter assay for the PD-1 and PD-L1 interaction blocking activity of IL-2 / anti-PD-L1 scFv Fc variants. [Figure 17A] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. Figure 2 shows p-STAT5 profiling curves for CD4+ cells from donor 359. [Figure 17B] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. Figure 2 shows p-STAT5 profiling curves for CD8+ cells from donor 359. [Figure 17C] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. Figure 2 shows p-STAT5 profiling curves for NK cells from donor 359. [Figure 17D]Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. p-STAT5 profiling curves for regulatory T cells from donor 359 are shown. [Figure 17E] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. Figure 2 shows p-STAT5 profiling curves for CD4+ cells from donor 126. [Figure 17F] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. Figure 2 shows p-STAT5 profiling curves for CD8+ cells from donor 126. [Figure 17G] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. p-STAT5 profiling curves for NK cells from donor 126 are shown. [Figure 17H] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 scFv Fc variants. p-STAT5 profiling curves for regulatory T cells from donor 126 are shown. [Figure 18A] Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to IL-2Rα. [Figure 18B] Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to IL-2Rβ. [Figure 18C]Figure 1 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to IL-2 receptor (IL-2Rα or IL-2Rβ) and PD-L1. Figure 2 shows ELISA binding of IL-2 / anti-PD-L1 Fab variants to PD-L1. [Figure 19] Jurkat cell NFAT reporter assay for PD-1 and PD-L1 interaction blocking activity of IL-2 / anti-PD-L1 Fab variants. [Figure 20] Sensorgrams of IL-2 / anti-PD-L1 Fab bifunctional variants that simultaneously bind to PD-L1 and IL-2 receptors (IL-2Rα or IL-2Rβ) are shown. [Figure 21A] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. A shows p-STAT5 profiling curves for CD4+ cells from donor 857. [Figure 21B] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 profiling curves for CD8+ cells from donor 857. [Figure 21C] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. p-STAT5 profiling curves for NK cells from donor 857 are shown. [Figure 21D] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. p-STAT5 profiling curves for regulatory T cells from donor 857 are shown. [Figure 21E]Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 profiling curves for CD4+ cells from donor 359. [Figure 21F] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 profiling curves for CD8+ cells from donor 359. [Figure 21G] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 profiling curves for NK cells from donor 359. [Fig. 21H] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells from human PBMCs of two separate donors by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 profiling curves for regulatory T cells from donor 359. [Figure 22A] Figure 1 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells in mouse splenocytes by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 activation of CD4+ T cells. [Figure 22B] Figure 1 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells in mouse splenocytes by IL-2 / anti-PD-L1 Fab variants. Figure 1 shows p-STAT5 activation of CD8+ T cells. [Figure 22C] Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells in mouse splenocytes by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 activation of NK cells. [Figure 22D]Figure 2 shows p-STAT5 activation of CD8+ T cells, NK cells, and regulatory T cells in mouse splenocytes by IL-2 / anti-PD-L1 Fab variants. Figure 2 shows p-STAT5 activation of regulatory T cells. [Diagram 23] Figure 2 shows tumor growth inhibition curves plotted from inhibition data in the humanized murine MB-231 model following 20 days of treatment with engineered anti-PD-L1 mAb (EP205 / EP206) Q3D or atezolizumab. [Figure 24A] Shows levels of human TNFα (A) and IFNγ (B) in plasma from the humanized mouse MB-231 model after 20 days of treatment with anti-PD-L1 mAb (EP205 / EP206) Q3D or atezolizumab. [Figure 24B] Shows levels of human TNFα (A) and IFNγ (B) in plasma from the humanized mouse MB-231 model after 20 days of treatment with anti-PD-L1 mAb (EP205 / EP206) Q3D or atezolizumab. [Figure 25A] Blood immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days.Profiling of CD4+FOXP3- T cells (control) is shown. [Figure 25B] Blood immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days. CD8+ T cell profiling is shown. [Figure 25C] Blood immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days.NK cell profiling is shown. [Figure 25D] Blood immune cell profiling of MC38 tumor-bearing mice after 11 days of treatment with IL-2 / anti-PD-L1 Fab variants. Regulatory T cell profiling is shown. [Figure 26A] Splenocyte immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days.Profiling of CD4+FOXP3- T cells (control) is shown. [Figure 26B] Splenocyte immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days. CD8+ T cell profiling is shown. [Figure 26C] Splenocyte immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days.NK cell profiling is shown. [Figure 26D] Splenocyte immune cell profiling of MC38 tumor-bearing mice following IL-2 / anti-PD-L1 Fab variant treatment for 11 days. Regulatory T cell profiling is shown. [Figure 27] Figure 2 shows tumor growth inhibition curves plotted from inhibition data of tumor volume over time obtained from inhibition data in B16F10-PD-L1 tumors in hPD1 transgenic mice treated with IL-2 / anti-PD-L1 Fab variants or atezolizumab for 20 days. [Figure 28] Figure 2 shows tumor growth inhibition curves plotted from tumor volume inhibition data over time obtained from MB-231 tumor NCG mouse models treated with IL-2 / anti-PD-L1 Fab variants or atezolizumab for 20 days. [Figure 29] (A-B) show the expression levels of PD-1 in T cells after treatment of human PBMCs with anti-PD-L1-Fab / IL-2 fusion protein. (A) shows PD-1 expression in CD8+ T cells after 5 days of treatment with EP290 / EP325 / EP205 or EP415 / EP325 / EP205. (B) shows PD-1 expression in regulatory T cells after 5 days of treatment with EP290 / EP325 / EP205 or EP415 / EP325 / EP205. [Diagram 30] Figures AB show binding of anti-PD-L1-Fab / IL-2 fusion proteins to IL-2Rβγ. Figure A shows expression levels of CD25, CD122, and PD-L1 in HEK Blue IL-2 cell line. Figure B shows binding of EP290 / EP325 / EP205, EP415 / EP325 / EP205 to IL-2R in the presence or absence of anti-CD25 antibody. [Diagram 31] (A-B) Tumor localization of fluorescently labeled EP415 / EP325 / EP205 in vivo. (A) Representative images of C57BL / 6N and B6N albino tumor-bearing mice 24 hours after injection of fluorescently labeled EP415 / EP325 / EP205. (B) Time-dependent enrichment of EP415 / EP325 / EP205 at the tumor site in MC-38-hPD-L1 and MC-38. [Figure 32A] Figure 1 shows tumor volume over time in humanized NCG mice and quantifies immune cells present at the tumor site. Figure 2 shows tumor growth inhibition curves of EP415 / EP325 / EP205 against anti-PD1 / PD-L1 responsive cancer cells MDA-MB-231 in humanized NCG mice. [Figure 32B] Figure 1 shows tumor volume over time in humanized NCG mice and quantifies immune cells present at the tumor site.The levels of CD4+ T cells at the tumor site are shown. [Figure 32C] Figure 1 shows tumor volume over time in humanized NCG mice and quantifies immune cells present at the tumor site. The levels of CD8+ T cells at the tumor site are shown. [Fig. 32D] Figure 1 shows tumor volume over time in humanized NCG mice and quantifies immune cells present at the tumor site. The levels of NK cells at the tumor site are shown. [Figure 32E] Figure 1 shows tumor volume over time in humanized NCG mice and quantifies immune cells present at the tumor site. Figure 2 shows levels of regulatory T cells at the tumor site. [Diagram 33] Figures 33A-B show the results of an experiment performed in a cold tumor mouse model. A shows the tumor growth inhibition curves of EP415 / EP325 / EP205 compared to vehicle control and anti-PD-L1 EP205 / EP206 against COLO205 tumors in humanized mice. B shows the corresponding body weight changes of the mice in Figure 33A. [Figure 34A] 1 shows the results of an experiment conducted in a mouse model of anti-PD1 antibody-resistant tumors. Tumor growth inhibition curves of EP415 / EP325 / EP205 against H1975 cancer cell tumors in humanized mice. [Figure 34B] 1 shows the results of an experiment performed in a mouse model of anti-PD1 antibody-resistant tumors, with the corresponding weight changes of the mice shown. [Figure 34C] Results of an experiment performed in a mouse model of anti-PD1 antibody-resistant tumors are shown, showing the number of hCD45 expressing cells per mm3 of tumor. [Fig. 34D] 1 shows the results of an experiment performed in a mouse model of anti-PD1 antibody-resistant tumors, showing the ratio of CD8+ T cells to Treg cells. [Figure 34E] Results of an experiment performed in a mouse model of anti-PD1 antibody-resistant tumors are shown, showing the number of CD8+ T cells per mm3 of tumor. [Fig. 34F] 1 shows the results of an experiment performed in a mouse model of anti-PD1 antibody-resistant tumors, depicting the ratio of NK cells to regulatory T cells. [Figure 35A] 1 shows the results of experiments performed in a cynomolgus monkey model, showing concentrations of EP415 / EP325 / EP205 detected in monkey plasma by ELISA and plotted against time. [Figure 35B] 1 shows the results of an experiment performed in a cynomolgus monkey model, showing the percentage of immune cell populations in the blood of the monkeys after administration of 0.1 mg / kg EP415 / EP325 / EP205. [Figure 35C] 1 shows the results of an experiment performed in a cynomolgus monkey model, showing the percentage of immune cell populations in the blood of the monkeys after administration of 0.5 mg / kg EP415 / EP325 / EP205. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Although anti-PD-L1 antibodies are promising immunotherapies, there remains a need to utilize biotherapeutics to more effectively modulate tumors. Immunotherapy using cytokines such as IL-2 and IL-15 has been shown to be effective in cancer treatment. Thus, antibodies targeting PD-L1 may prove to be useful immunomodulators when further targeting IL-2 and IL-15. Provided herein are novel anti-PD-L1 antibodies, scFv, and Fab polypeptides. Additionally, provided herein are bifunctional fusion proteins comprising (a) an anti-PD-L1 antibody, scFv, or Fab polypeptide, and (b) (i) IL-15, IL-15Rα, or both, or (ii) IL-2 or an engineered variant thereof.

[0011] The present disclosure provides antigen-binding sites that bind to human PD-L1. These antigen-binding sites can bind to various epitopes in the extracellular domain of PD-L1. Proteins and protein complexes that contain such antigen-binding sites, such as antibodies, bifunctional antibodies, antibody-drug conjugates, immunocytokines, and bispecific T cell engagers, as well as immune effector cells (e.g., T cells) that express proteins (e.g., chimeric antigen receptors (CARs)) that contain such antigen-binding sites, are useful in treating diseases such as cancer associated with PD-L1. The present disclosure also provides pharmaceutical compositions that include such proteins, protein complexes, and immune effector cells, as well as therapeutic methods for using such proteins, protein complexes, immune effector cells, and pharmaceutical compositions, including, but not limited to, cancer treatment. Various aspects of the antigen-binding sites described in this disclosure are described in the following sections; however, aspects of the antigen-binding sites described in a particular section of this disclosure are not to be construed as being limited to any particular section.

[0012] definition Before describing this disclosure in more detail, it may be helpful to an understanding of the disclosure to provide definitions of certain terms used herein. Additional definitions are set out throughout this disclosure.

[0013] Unless otherwise defined herein, scientific and technical terms shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural terms and plural terms shall include the singular terms.

[0014] In the present description, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and fractions thereof (such as 1 / 10 and 1 / 100 of an integer), if appropriate, unless otherwise indicated. Also, any numerical range described herein for any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise indicated. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have," and "comprise" are used interchangeably, and these terms and variations thereof are intended to be interpreted as non-limiting.

[0015] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which it does not occur.

[0016] In addition, it should be understood that individual constructs or groups of constructs derived from various combinations of the structures and subunits described herein are disclosed by this application to the same extent as if each construct or group of constructs was individually set forth, and thus selection of a particular structure or particular subunit is within the scope of this disclosure.

[0017] The term "consisting essentially of" is not equivalent to "comprising" and refers to a particular material or step of a claim or to something that does not materially affect the basic characteristics of the claimed subject matter. For example, a domain, region, or module of a protein (e.g., a binding domain, hinge region, or linker) or a protein (which may have one or more domains, regions, or modules) may be defined as a domain, region, module, or protein whose amino acid sequence in combination comprises at most 20% of the length of the domain, region, module, or protein (e.g., "Consists essentially of" a particular amino acid sequence if it includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal or interdomain amino acids) that contribute at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) to the total amino acid sequence of the protein and that do not substantially affect (i.e., do not reduce activity by more than 50%, e.g., 40%, 30%, 25%, 15%, 10%, 5% or 1% or less) the activity of the domain(s), region(s), module(s) or protein (e.g., target binding affinity of a binding protein).

[0018] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., a hydrogen, a carboxyl group, an amino group, and an α carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0019] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can produce several different types of changes in the sequence, including substitutions, insertions, or deletions of nucleotide(s) or amino acid(s).

[0020] "Conservative substitutions" refer to amino acid substitutions that do not significantly affect or change the binding characteristics of a particular protein. Generally, conservative amino acid substitutions are those in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be classified into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic group may include, for substitution purposes, Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys. Large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton, (1984), Proteins, W.H. Freeman and Company.

[0021] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Protein applies to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and to non-naturally occurring amino acid polymers. Variants of the proteins, peptides, and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined or reference amino acid sequence described herein.

[0022] As used herein, a "nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound containing covalently linked nucleotides that may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), including mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), including cDNA, genomic DNA, and synthetic DNA, either of which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or the non-coding (antisense) strand. A nucleic acid molecule that codes for an amino acid sequence includes all nucleotide sequences that code for the same amino acid sequence. Some versions of a nucleotide sequence may include intron(s) to the extent that the intron(s) are removed during co-transcription or via post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy in the genetic code, or by splicing.

[0023] Variants of the nucleic acid molecules of the present disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90% identical, preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a nucleic acid molecule of a defined or reference polynucleotide as described herein, or hybridize to the polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42° C. Variants of the nucleic acid molecule retain the ability to encode a binding domain thereof having a functionality as described herein, such as binding to a target molecule.

[0024] As used herein, "percent sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. A preferred method for determining sequence identity is designed to obtain the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). In addition, non-homologous sequences may be ignored for comparison purposes. Percent sequence identity referred to herein is calculated over the length of the reference sequence unless otherwise indicated. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations may be performed using BLAST programs (e.g., BLAST2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST program can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that when sequence analysis software is used for the analysis, the results of the analysis will be based on the "default settings" of the referenced program, by which is meant the set of values ​​or parameters that originally load with the software when it is first initialized.

[0025] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a nucleic acid may be part of a vector and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate) and still be isolated in that such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide.

[0026] A "functional variant" is a polypeptide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but differs in composition slightly (e.g., one base, atom, or functional group is different, added, or removed) so that the polypeptide or encoded polypeptide can perform at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of the present disclosure has "similar binding," "similar affinity," or "similar activity" if it does not show a 50% or less decrease in performance in a selected assay compared to the parent or reference polypeptide, such as, for example, an assay for measuring binding affinity (e.g., Biacore® or tetramer staining to measure association (Ka) or dissociation (KD) constants).

[0027] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that contains only a domain, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion, or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" when the functional portion or functional fragment exhibits a 50% or less decrease in performance (preferably 20% or 10% or less, or less than the log difference in affinity compared to the parent or reference) in a selected assay compared to the parent or reference polypeptide.

[0028] As used herein, the terms "engineered," "recombinant," or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic modification or that has been modified by the introduction of a foreign or heterologous nucleic acid molecule, where such modification or modification is introduced by genetic engineering (i.e., human intervention). Genetic modifications include, for example, modifications that introduce expressible nucleic acid molecules that code for functional RNA, proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of the genetic material of a cell. Additional modifications include, for example, non-coding regulatory regions where the modification alters expression of a polynucleotide, gene, or operon.

[0029] As used herein, "heterologous" or "non-endogenous" or "foreign" refers to any gene, protein, compound, nucleic acid molecule or activity that is not native to a host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to a host cell or subject that is being modified. Heterologous, non-endogenous, or foreign includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or modified such that the structure, activity, or both are different between the native gene, protein, compound, or nucleic acid molecule and the modified one. In certain embodiments, a heterologous, non-endogenous, or foreign gene, protein, or nucleic acid molecule (e.g., a receptor, ligand, etc.) may not be endogenous to a host cell or subject, but instead, a nucleic acid encoding such a gene, protein, or nucleic acid molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc., and the added nucleic acid molecule may be integrated into the genome of the host cell or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or foreign polynucleotide or gene encoding a polypeptide may be homologous to a naturally occurring polynucleotide or gene and may encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, and likewise the encoded polypeptide or activity, may be derived from the same species, a different species, or a combination thereof.

[0030] In certain embodiments, a nucleic acid molecule or a portion thereof native to a host cell will be considered heterologous to the host cell if it has been modified or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been modified by a heterologous expression control sequence or by an endogenous expression control sequence that is not normally associated with the nucleic acid molecule native to the host cell. In addition, the term "heterologous" can refer to a biological activity that is different, altered, or not endogenous to the host cell. As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as multiple separately regulated genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or as a combination thereof.

[0031] The term "endogenous" or "native" as used herein refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

[0032] The term "expression" as used herein refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The nucleic acid molecule to be expressed is usually operably linked to an expression control sequence (e.g., a promoter).

[0033] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the function of the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with each other so that the function of one does not affect the other.

[0034] As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or as a combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, the two or more heterologous nucleic acid molecules can be introduced on separate vectors as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a host chromosome at a single site or multiple sites, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of encoding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0035] The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule (or a fusion protein of the present disclosure, if the context clearly indicates). The (polynucleotide) construct may be in a vector (e.g., bacterial vector, viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that can transport another nucleic acid molecule. A vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, e.g., Geurts et al., Mol. Ther. 8:108, 2003: Mates et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those that can replicate autonomously (episomal vectors), deliver polynucleotides to a cellular genome (e.g., viral vectors), or express the nucleic acid molecule to which they are linked (expression vectors).

[0036] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to a suitable control sequence capable of effecting expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences to control the termination of transcription and translation. A vector may be a plasmid, a phage particle, a virus, or simply a potential genome insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may integrate into the genome itself, or may deliver the polynucleotide contained in the vector to the genome without vector sequences. In this specification, "plasmid", "expression plasmid", "virus" and "vector" are often used interchangeably.

[0037] The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or expressed transiently (e.g., transfected mRNA).

[0038] In certain embodiments, the polynucleotide of the present disclosure may be operably linked to certain elements of a vector. For example, the polynucleotide sequences necessary to affect the expression and processing of the linked coding sequence may be operably linked. The expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (e.g., Kozak consensus sequences); sequences that increase protein stability; and possible sequences that increase protein secretion. An expression control sequence may be operably linked when it is contiguous with a gene of interest, or it may be an expression control sequence that acts in trans or at a distance to control the gene of interest.

[0039] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentivirus vector or a gamma-retrovirus vector). Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles, Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses such as adenoviruses, herpesviruses (herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, and D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0040] A "retrovirus" is a virus with an RNA genome that is reverse transcribed into DNA using reverse transcriptase, which then integrates into the host cell genome. "Gammaretrovirus" refers to a genus of the Retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0041] "Lentiviral vector" includes HIV-based lentiviral vectors for gene delivery, which can be integrating or non-integrating, have a relatively large packaging capacity, and can transduce a variety of different cell types. Lentiviral vectors are usually generated after transient transfection of three (packaging, envelope, transfer) or more plasmids into a production cell line. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Once inside, viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0042] In certain embodiments, the viral vector can be a gamma retrovirus, such as a Moloney Murine Leukemia Virus (MLV) derived vector. In other embodiments, the viral vector can be a more complex retrovirus derived vector, such as a lentivirus derived vector. HIV-1 derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells to transduce mammalian host cells with viral particles containing a transgene are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One, 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; herpes simplex virus (HSV)-derived vectors, including amplicon vectors, replication-deficient HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998), can also be used for polynucleotide delivery.

[0043] Other vectors that can be used with the compositions and methods of the present disclosure include vectors derived from baculovirus and alphavirus (Jolly, D J. 1999. Emerging Viral Vectors. pp. 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab) or plasmid vectors (e.g., Sleeping beauty and other transposon vectors).

[0044] When the viral vector genome contains multiple polynucleotides that are expressed as separate transcripts in a host cell, the viral vector may also contain additional sequences between the two (or more) transcripts that allow for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0045] The term "host" as used herein refers to a cell or microorganism that is the target of genetic manipulation with heterologous nucleic acid molecules to produce a polypeptide of interest (e.g., an antibody of the present disclosure).

[0046] A host cell may include any individual cell or cell culture that may receive incorporation of a vector or nucleic acid or express a protein. The term also includes the progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate vectors or other materials by transformation via the use of viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), page 9.51.

[0047] "Antigen" or "Ag" as used herein refers to an immunogenic molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, complement activation, antibody-dependent cellular cytotoxicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, etc. It is readily apparent that an antigen can be synthesized, recombinantly produced, or derived from a biological sample. Exemplary biological samples that may contain one or more antigens include tissue samples, fecal samples, cells, bodily fluids, or combinations thereof. An antigen can be produced by cells that have been modified or genetically engineered to express the antigen.

[0048] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitope determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific charge characteristics as well as specific three-dimensional structural characteristics. When the antigen is or includes a peptide or protein, the epitope may be composed of consecutive amino acids (e.g., linear epitopes), or may be composed of amino acids from different parts or regions of the protein that are brought into close proximity by protein folding (e.g., discontinuous or conformational epitopes), or may be composed of non-contiguous amino acids that are in close proximity regardless of protein folding.

[0049] The terms "antigen-binding site" or "antigen-binding portion" are used interchangeably herein and refer to the portion of an antibody and / or immunoglobulin molecule that is involved in binding to an antigen and / or epitope. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. "Hypervariable regions" are three highly diverse sections within the V regions of the heavy and light chains, interspersed between relatively conserved adjacent sections, the "framework regions" ("FR"). The term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each of the heavy ("H") and light ("L") chains are referred to as "complementarity determining regions" or "CDRs". An antigen-binding site can be present in an intact antibody, in an antigen-binding fragment of an antibody that retains an antigen-binding surface, or in a recombinant polypeptide such as an scFv that uses a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide. An antigen-binding site can comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0050] The term "antibody", as used herein, encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies), antibody fusion proteins, antibodies for heterodimers in engineered proteins, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0051] "Antibody fragment" refers to a polypeptide or protein other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0052] Numbering of the CDRs and framework regions may follow any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and (See Pluckthun, J. Mol. Bio. 309:657-670 (2001)). Equivalent residue positions can be annotated using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics, 15:298-300) to compare different molecules. The CDRs of an antigen-binding site can be determined according to known methods, such as Kabat, Chothia, EU, IMGT, and AHo, as described above. CDRs defined under these definitions usually include overlapping or subsets of amino acid residues when compared against each other. The heavy and light chain CDRs of an antibody can be defined using different numbering conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to Chothia, supra, and the light chain CDRs are defined according to Kabat, supra. CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0053] As used herein, PD-L1 (also known as "programmed death ligand 1" or CD274 in humans) refers to the protein with UniProt accession number Q0GN75 (human) and related isoforms and orthologues.

[0054] As used herein, the term "substitution" or "residue substitution" refers to the replacement of a native or wild-type residue with a different residue.

[0055] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., ligand, antibody, scFv) and its binding partner (e.g., receptor, antigen, epitope). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ), which can be expressed as the dissociation rate constant and the association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by methods known to those of skill in the art, including those described herein.

[0056] As used herein, "Fc domain" or "Fc region" refers to a polypeptide derived from the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes polypeptides having a native sequence Fc region, or variants thereof. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Examples of Fc regions are disclosed in U.S. Patent No. 7,317,091, U.S. Patent No. 8,735,545, U.S. Patent No. 7,371,826, U.S. Patent No. 7,670,600, and U.S. Patent No. 9,803,023, all of which are incorporated by reference in their entireties.

[0057] "Immunoglobulin" refers to a protein having the structure of an antibody. As an example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein in which two light chains and two heavy chains are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins may be assigned to one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subclasses, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of the constant domain. Immunoglobulins contain two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0058] A "Fab molecule" or "antigen-binding fragment" is an antigen-binding fragment of an antibody containing the variable and constant domains of the light chain and the variable and CH1 domains of the heavy chain.

[0059] "Single chain variable domain" or "scFv" refers to an antigen-binding portion comprising the variable regions of the heavy and light chains connected by a linker peptide.

[0060] "Bispecific antibodies," "bifunctional proteins," and "bifunctional antibodies" are used interchangeably throughout this disclosure and refer to artificial antibodies with two different antigen binding sites and / or antibody fusion proteins that contain at least one antigen binding site and at least one functional domain. Bispecific antibodies can refer to complete immunoglobulin proteins with two different antigen binding sites, or other molecules with two antigen binding moieties, such as fusion proteins containing two Fabs or two scFvs. In certain embodiments, bifunctional proteins and / or bifunctional antibodies can refer to pairs of proteins, fusion proteins, and / or heterodimeric proteins that contain one or more functional domains. Examples of functional domains include antigen binding sites, antibody fragments (e.g., Fabs, scFvs, etc.), antibody heavy and light chains, and cytokines (e.g., IL-2, IL-15). Bifunctional proteins or bifunctional antibodies can refer to antibodies that include a fusion with a non-antibody polypeptide, such as a cytokine. For example, the bifunctional protein can include an antibody heavy and light chain, where the heavy chain constant region is fused to IL-2, engineered IL-2, IL-15, engineered IL-15, IL-15 receptor, or engineered IL-15 receptor, or a functional fragment thereof. In addition, the bifunctional protein can include an antibody heavy and light chain, where the heavy chain constant region can form a heterodimer with a polypeptide or protein that does not contain an antigen binding site. For example, the bifunctional protein can include an IL-2 or modified IL-2 fusion protein that includes a heavy chain, a light chain, and an antibody Fc domain that can form a heterodimer with the Fc domain of the antibody heavy chain.

[0061] "Modulating an immune response" may include one or more of increasing T effector cell responses (e.g., cytotoxicity against tumor cells and virally infected cells), increasing B cell activation, restoring lymphocyte activation and proliferation, increasing expression of IL-2 receptors, increasing T cell responsiveness, increasing natural killer cell activity or lymphokine-activated killer (LAK) cell activity, decreasing regulatory T cell responses against other T cells, and the like.

[0062] "Treatment," "treating," or "ameliorating" refers to the medical management of a subject's (e.g., patient's) condition, disease, or disorder, which may be curative, result in the alleviation of one or more symptoms, result in a reduction in tumor size and / or severity, inhibit measurable growth of a tumor or the onset of symptoms, or any combination thereof.

[0063] An "effective amount" or "therapeutically effective amount" may refer to the amount of a therapeutic agent that provides a desired physiological change, such as an immunomodulatory and / or anti-cancer effect. The desired physiological change may be, for example, a reduction in disease symptoms, or a decrease in disease severity, or a reduction in disease progression. In the context of cancer, the desired physiological change may include, for example, tumor regression, a reduction in the rate of tumor progression, a reduction in the level of a cancer biomarker, a reduction in symptoms associated with cancer, prevention or delay of metastasis, or clinical remission.

[0064] As used herein, the term "inhibit" refers to a decrease in a particular activity (e.g., immune suppression or tumor growth). Unless otherwise specified, an activity can be considered inhibited if it is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% when measured by methods disclosed herein or known in the art.

[0065] "Cancer antigen" refers to a molecule that is preferentially expressed by cancer cells. Examples of cancer antigens include CD19, CD20, ROR1, fibroblast activation protein alpha, and carcinoembryonic antigen (CEA).

[0066] "Tumor microenvironment inhibitors" refer to agents that promote tumor growth and suppress one or more conditions or cell types present in the local environment surrounding the tumor. For example, bevacizumab can suppress the tumor microenvironment by reducing angiogenesis in the tumor microenvironment.

[0067] The techniques of recombinant DNA, molecular cloning, and gene expression used in this disclosure are known in the art and described in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, New York, 2001, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, 1999.

[0068] Further definitions are provided throughout the detailed description.

[0069] Antigen-binding site that binds to PD-L1 In one aspect, the application provides an antigen-binding site that binds to PD-L1 (e.g., human PD-L1). Exemplary sequences that can be used within or as antigen-binding sites, such as CDR, VH, VL, and scFv sequences, are listed in Table 1. The amino acid positions and CDR sequences are identified according to the IMGT numbering scheme. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0070] In certain embodiments, the antigen-binding site that binds to PD-L1 has a complementarity determining region 1 (CDR1) sequence of SEQ ID NO: 11, 3, 19, 33, 52, or 63; a complementarity determining region 2 (CDR2) sequence of SEQ ID NO: 12, 4, 20, 34, 41, 53, or 64; and a complementarity determining region 3 (CDR2) sequence of SEQ ID NO: 89, 5, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, or 86. and a light chain variable domain (VL) comprising a CDR1 sequence of SEQ ID NO: 47, 6, 14, 22, 28, 36, 55, or 66; a CDR2 sequence of SEQ ID NO: 48, 7, 15, 23, 29, 37, 42, 56, 60, or 67; and a CDR3 sequence of SEQ ID NO: 49, 8, 16, 24, 30, 38, 43, 57, or 68.

[0071] In certain embodiments, the antigen-binding site that binds to PD-L1 comprises an antibody heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL of the same antibody disclosed in Table 1. In certain embodiments, the antigen binding site comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 of the VH and VL sequences of the antibodies disclosed in Table 1, as determined under the IMGT system, the EU system, the Kabat system (Kabat et al., (1991), Sequences of Proteins of Immunological Interest, NIH Publication, No. 91-3242, Bethesda), the Chothia system (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196:901-917), MacCallum (see MacCallum, R M. et al., (1996), J. Mol. Biol. 262:732-745), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 of an antibody disclosed in Table 1.

[0072] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP164-F04. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:2. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 1 and 2, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

[0073] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP170-E06. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 9 and 10, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively.

[0074] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP161-G08. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 18. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 17 and 18, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively.

[0075] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP161-F08. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:25, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:26. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 25 and 26, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 27, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 27, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively.

[0076] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP161-F04. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 31, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 32. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 31 and 32, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively.

[0077] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP280-E04. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 39, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 40. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 39 and 40, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 3, 41, and 5, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 42, and 43, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 3, 41, and 5, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 42, and 43, respectively.

[0078] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP171-H02. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 44, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 45. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 44 and 45, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 46, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 46, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0079] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP173-H11. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:50, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:51. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 50 and 51, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 52, 53, and 54, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 55, 56, and 57, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 52, 53, and 54, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 55, 56, and 57, respectively.

[0080] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP172-F10. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:58, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:59. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 58 and 59, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 46, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 60, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 46, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 60, and 49, respectively.

[0081] In certain embodiments, the antigen-binding site described in the present application is derived from 2018EP280-E01. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:61, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:62. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 61 and 62, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63, 64, and 65, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 66, 67, and 68, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63, 64, and 65, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 66, 67, and 68, respectively.

[0082] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-E02. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:69, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:70. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 69 and 70, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 71, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 71, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0083] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-E10. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 72, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 73. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 72 and 73, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 74, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 74, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0084] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-C05. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 75, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 76. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 75 and 76, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 77, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 77, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0085] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-F02. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 78, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 79. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 78 and 79, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 80, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 80, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0086] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-B01. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 81, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 82. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 81 and 82, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 83, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 83, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0087] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-H10. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 84, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 84 and 85, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 86, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 86, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0088] In certain embodiments, the antigen-binding site described in the present application is derived from 2019EP69-F03. For example, in certain embodiments, the antigen-binding site described in the present application comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 87, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 88. In certain embodiments, the antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined under IMGT, EU, Kabat, Chothia, MacCallum, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 87 and 88, respectively. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 89, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 89, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0089] In certain embodiments, an antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 87, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 88, wherein the VH comprises a CDR3 comprising an amino acid sequence variant of SEQ ID NO: 89 having at least one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 89. In some embodiments, the VH CDR3 comprises or consists of SEQ ID NO: 71, 74, 77, 80, 83, or 86. In certain embodiments, the VH further comprises a CDR1 and a CDR2 comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively. In certain embodiments, the VL comprises a CDR1, a CDR2, and a CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising a CDR1 having the sequence of SEQ ID NO: 11, a CDR2 having the sequence of SEQ ID NO: 12, and a CDR3 comprising a variant of SEQ ID NO: 89 having at least 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 89; and (b) a VL comprising a CDR1, a CDR2, and a CDR3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively.

[0090] In each of the foregoing embodiments, it is contemplated herein that the VH and / or VL sequences that bind PD-L1 may contain amino acid changes (e.g., substitutions, deletions or additions of at least 1, 2, 3, 4, 5, or 10 amino acids) in the framework regions of the VH and / or VL without affecting the ability to bind PD-L1. For example, it is contemplated herein that the VH and VL sequences that bind PD-L1 (e.g., in an scFv) may contain heterodimerization mutations of cysteines that promote the formation of disulfide bridges between the VH and VL of the scFv.

[0091] In some embodiments, the antigen-binding sites disclosed herein bind to human PD-L1 with a KD of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by surface plasmon resonance (SPR).

[0092] In some embodiments, the antigen-binding site disclosed herein binds to human PD-L1 with an EC50 of less than about 160 nM, less than about 10 nM, less than about 1.5 nM, less than about 1.2 nM, less than about 1.0 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, or less than about 0.4 nM as measured by enzyme-linked immunosorbent assay (ELISA).

[0093] In certain embodiments, the antigen-binding sites disclosed herein bind to human PD-L1 or an extracellular domain thereof with a KD value (affinity or better) of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or 0.5 nM or less. In certain embodiments, the antigen-binding sites of the application bind to human PD-L1 or an extracellular domain thereof with a KD value (affinity or better) of 5 nM or less. In certain embodiments, the antigen-binding site of the application binds to human PD-L1 or an extracellular domain thereof with a KD value (affinity or greater) of about or less than 2.0 nM, 2.1 nM, 2.2 nM, 2.3 nM, 2.4 nM, 2.5 nM, 2.6 nM, 2.7 nM, 2.8 nM, 2.9 nM, 3.0 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, 4.0 nM, 4.1 nM, 4.2 nM, 4.3 nM, 4.4 nM, 4.5 nM, 4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM or 5.0 nM. In certain embodiments, the antigen-binding site of the present application binds to human PD-L1 or an extracellular domain thereof with a KD value in the range of about 1.0-3.5 nM, 1.0-4.0 nM, 1.0-4.5 nM, 1.0-5.0 nM, 1.5-3.5 nM, 1.5-4.0 nM, 1.5-4.5 nM, 1.5-5.0 nM, 2.0-3.5 nM, 2.0-4.0 nM, 2.0-4.5 nM, 2.0-5.0 nM, 2.5-3.5 nM, 2.5-4.0 nM, 2.5-4.5 nM, 2.5-5.0 nM, 3.0-3.5 nM, 3.0-4.0 nM, 3.0-4.5 nM, or 3.0-5.0 nM. These KD values ​​are measured using standard binding assays, such as SPR or ELISA.

[0094] In certain embodiments, the antigen-binding sites disclosed herein bind to cells expressing human PD-L1 with an EC50 of less than about 40 nM, less than about 10 nM, less than about 8 nM, less than about 6 nM, less than about 4 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.3 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by fluorescence activated cell sorting (FACS).

[0095] In certain embodiments, the antigen binding sites disclosed herein bind to human PD-L1 displayed on the surface of a cell membrane (plasma membrane of a cell) with an EC50 value of 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or 0.5 nM or less (affinity or greater). In certain embodiments, the antigen binding sites disclosed herein bind to human PD-L1 displayed on the surface of a membrane (plasma membrane of a cell) with an EC50 value of 1 nM or less (affinity or greater). These EC50 values ​​can be measured in binding assays using cells that recombinantly or endogenously express PD-L1, such as the assays disclosed in the Example(s) below. In certain embodiments, the antibodies bind to PD-L1 from bodily fluids, tissues, and / or cells of a subject.

[0096] In certain embodiments, the antigen-binding sites disclosed herein compete with PD-1 for binding to PD-L1 or inhibit binding of PD-L1 to PD-1. In certain embodiments, the antigen-binding sites disclosed herein are present as single chain fragment variable fragments (scFv). In certain embodiments, the antigen-binding sites disclosed herein are present as antigen-binding fragments (Fab).

[0097] Proteins that have antigen-binding sites that bind to PD-L1 In certain embodiments, the present disclosure provides proteins comprising an antigen-binding site disclosed herein that binds to human PD-L1. In certain embodiments, the proteins of the disclosure comprise one or more antibody heavy chain constant regions. In certain embodiments, the antibody heavy chain constant region is a human IgG heavy chain constant region. In certain embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region. In certain embodiments, the antibody heavy chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:90.

[0098] In certain embodiments, the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V, numbered according to the EU numbering system, compared to human IgG. In some embodiments, the human IgG is an IgG1. In some embodiments, the human IgG comprises a sequence of SEQ ID NO: 90, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 90. In some embodiments, the heavy chain constant region comprises a LALAPG mutation. LALAPG mutations refer to the L234A, L235A, and P329G changes in the CH2-CH3 region of a human IgG heavy chain constant region, e.g., IgG1 (see, e.g., Schlothauer et al., Protein Engineering, Design & Selection. 29(10):457-466, 2016; Lo et al., J. Biol. Chem. 292(9):3900-3908, 2017, incorporated herein by reference). In some embodiments, the antibody heavy chain constant region comprises a knob mutation or a hole mutation. Knob-into-hole mutations are modifications to the IgG constant domain that allow heterodimerization of Fc domains that contain knob and hole mutations, respectively. Knob or hole mutations allow preferential heterodimer formation in vitro with low levels of homodimer contamination. Knob-into-hole mutations are disclosed in Merchant et al. Nat. Biotechnol. 16:677-681, 1998; and Wei et al., Oncotarget. 8(31):51037-51049, 2017, which are incorporated herein by reference. Examples of knob mutations include the S354C, T366W, and K409A mutations in the IgG heavy chain constant region.Examples of hole mutations include Y349C, T366S, L368A, F405K and Y407V mutations in the IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region comprises a LALAPG mutation and a hole or knob mutation. For example, in some embodiments, the antibody heavy chain constant region can comprise L234A, L235A, P329G, S354C, T366W and K409A mutations. In some embodiments, the antibody heavy chain constant region can comprise L234A, L235A, P329G, Y349C, T366S, L368A, F405K and Y407V mutations. In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 91 or 92. In certain embodiments, the protein comprises a first antibody heavy chain constant region comprising one or more mutations selected from S354C, T366W and K409A numbered according to the EU numbering system relative to SEQ ID NO: 90, and a second antibody heavy chain constant region comprising one or more mutations selected from Y349C, T366S, L368A, F405K and Y407V relative to SEQ ID NO: 90. In certain embodiments, the protein comprises a first antibody heavy chain constant region comprising one or more mutations selected from L234A, L235A, P329G, S354C, T366W and K409A numbered according to the EU numbering system relative to SEQ ID NO: 90, and a second antibody heavy chain constant region comprising one or more mutations selected from L234A, L235A, P329G, Y349C, T366S, L368A, F405K and Y407V relative to SEQ ID NO: 90. [Table 2]

[0099] In some embodiments, the protein that comprises an antigen-binding site that binds to PD-L1 is an antibody. In some embodiments, the antibody comprises: (a) a heavy chain (HC) that comprises, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 148 and a light chain (LC) that comprises, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 149; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 151; (c) a HC comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 152; (d) a HC comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 153; (e) a HC comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 154; %, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 156; (e) a HC comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 156 and a LC comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 157;or (f) a HC comprising, or consisting of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 159, and a LC comprising, or consisting of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 160;

[0100] In some embodiments, the antibody comprises (a) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 148 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 149; (b) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 150 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 151; (c) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 152 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 153; (d) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 154 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 155; (e) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 156 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 157; or (f) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 159 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 160.

[0101] The HC and LC sequences of exemplary proteins with a PD-L1 antigen-binding site are listed below in Table 3. Amino acid positions are identified according to the IMGT numbering scheme. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0102] In some embodiments, the antigen-binding sites disclosed herein can be present as antibodies or antigen-binding fragments thereof. The antibody can be a monoclonal antibody, a chimeric antibody, a diabody, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment, an Fv, a bispecific antibody, a bifunctional antibody, a bispecific Fab2, a bispecific (mab)2, a humanized antibody, an artificially generated human antibody, a bispecific T cell engager, a bispecific NK cell engager, a single chain antibody (e.g., a single chain Fv fragment or scFv), a triomab, a knob-into-hole (KiH) IgG with a common light chain, a crossmab, an ortho Fab IgG, a DVD-Ig, a bifunctional-IgG, an IgG-scFv, a sdFv2-Fc, a bi-nanobody, a dual affinity retargeting antibody (DART), a DART-Fc, a scFv-HSA-scFv (HSA=human serum albumin), or a dock-and-lock (DNL)-Fab3.

[0103] In certain embodiments, the antigen binding sites disclosed herein are linked to an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an antibody constant region, e.g., an antibody constant region selected from an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE heavy chain constant region; in particular, an IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant region. In another embodiment, the antigen binding sites disclosed herein can be linked to a light chain constant region selected from, e.g., a kappa or lambda (e.g., human) light chain constant region. The constant region can be altered, e.g., mutated or engineered, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In some embodiments, the antibody has effector function and can fix complement. In some embodiments, the antibody does not recruit effector cells or fix complement. In some embodiments, the antibody has reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant form that does not support binding to Fc receptors, e.g., it has a mutated or deleted Fc receptor binding region.

[0104] In certain embodiments, the antigen binding site is linked to an IgG constant region comprising hinge, CH2 and CH3 domains, with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human antibody constant region, e.g., a human IgG1 constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, an antibody Fc domain or portion thereof sufficient to bind CD16 comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a wild-type human IgG1 Fc comprising the sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90)

[0105] In some embodiments, one or more mutations may be incorporated into the constant region relative to the human IgG1 constant region, for example at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0106] In certain embodiments, the antigen binding site is linked to a portion of the antibody Fc domain sufficient to bind to CD16. Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is primarily focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 in the CH2 domain, and carbohydrate residues N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, for example by using a phage display library or a yeast surface display cDNA library, or mutations can be designed based on the known three-dimensional structure of the interaction.

[0107] In certain embodiments, mutations that can be incorporated into CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into Ck of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0108] In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, such as a human IgG1 antibody. In some embodiments, mutations are introduced into the antibody constant domain to allow heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from a human IgG1 constant domain, the antibody constant domain can comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to amino acids 234-332 of a human IgG1 antibody, and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0109] In some other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an antibody constant region from another mammal, such as rabbit, dog, cat, mouse, or horse.

[0110] The proteins described herein can be produced using recombinant DNA techniques known in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding a first immunoglobulin light chain can be cloned into a third expression vector; a fourth nucleic acid sequence encoding a second immunoglobulin light chain can be cloned into a fourth expression vector; the first, second, third and fourth expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0111] To achieve the highest protein yield, different ratios of the first, second, third, and fourth expression vectors can be tested to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, and microscopy.

[0112] The clones can be cultured under conditions suitable for scale-up in a bioreactor to maintain expression of proteins comprising the antigen-binding sites disclosed herein. Proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0113] Thus, in some embodiments, the present disclosure provides one or more isolated nucleic acids comprising a sequence encoding the variable region of an immunoglobulin heavy chain and / or an immunoglobulin light chain of any one of the antibodies disclosed herein. The present disclosure provides one or more expression vectors expressing the variable region of an immunoglobulin heavy chain and / or an immunoglobulin light chain of any one of the antibodies disclosed herein. Similarly, the present application provides host cells comprising one or more of the foregoing expression vectors and / or isolated nucleic acids.

[0114] Competitive assays for determining whether an antibody binds to the same epitope as the disclosed antibodies or competes for binding with the disclosed antibodies are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0115] Typically, a competitive assay involves the use of an antigen (e.g., human PD-L1 protein or fragment thereof) bound to a solid surface or expressed on a cell surface, a test PD-L1-binding antibody, and a reference antibody. The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1×, 5×, 10×, 20×, or 100×). Antibodies identified by competitive assays (e.g., competing antibodies) include antibodies that bind to the same epitope as the reference antibody, or a similar (e.g., overlapping) epitope, and antibodies that bind to adjacent epitopes close enough to sterically interfere with the epitope bound by the reference antibody.

[0116] Competitive assays can be performed in both directions to ensure that the presence of label does not interfere with or otherwise inhibit binding, e.g., in one orientation, the reference antibody is labeled and the test antibody is unlabeled, and in the second orientation, the test antibody is labeled and the reference antibody is unlabeled.

[0117] A test antibody competes with a reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1×, 5×, 10×, 20× or 100×) inhibits binding of the other antibody, e.g., by at least 50%, 75%, 90%, 95% or 99%, as measured in a competitive binding assay.

[0118] Two antibodies may be determined to bind the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies may be determined to bind overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0119] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties, including affinity and / or specificity, to improve biophysical properties, including aggregation, stability, precipitation and / or non-specific interactions, and / or to reduce immunogenicity. Diversity can be introduced into the immunoglobulin heavy chains and / or immunoglobulin light chains, for example, by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-directed mutagenesis.

[0120] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified to human germline sequences (e.g., by a process called germlining) to optimize the antibody.

[0121] Generally, an optimized antibody has at least the same, or substantially the same, affinity for its antigen as the non-optimized (or parent) antibody from which it was derived. For example, in certain embodiments, an optimized antibody has a higher affinity for an antigen when compared to the parent antibody.

[0122] When antibody is used as therapeutic agent, antibody can be conjugated to effector agent such as small molecule toxin or radionuclide using standard in vitro conjugation chemistry.When effector agent is polypeptide, antibody can be chemically conjugated to effector or can be linked to effector as fusion protein.Construction of fusion protein is within the ordinary skill of the art.

[0123] Standard in vitro conjugation chemistry can be used to conjugate the antibody to effector moiety such as small molecule toxin or radionuclide.If the effector moiety is a polypeptide, the antibody can be chemically conjugated to the effector or can be linked to the effector as a fusion protein.The construction of fusion protein is within the ordinary skill of the art.

[0124] In some embodiments, the protein is an antibody that inhibits tumor growth in vivo.

[0125] In certain embodiments, the protein is an antibody that induces secretion of IFNγ and TNFα in vivo at levels comparable to or even enhanced relative to atezolizumab.

[0126] Bifunctional Proteins In certain embodiments, disclosed herein are bifunctional proteins, e.g., bifunctional antibodies. The bifunctional protein may comprise an antigen binding site that binds to PD-L1, an IgG Fc domain, and / or a functional domain. In some embodiments, the bifunctional protein is a single protein sequence. In other embodiments, the bifunctional protein is a heterodimer formed by at least two protein sequences. The antigen binding site that binds to PD-L1 may be any of the anti-PD-L1 antigen binding sites, antibody, and / or scFv sequences disclosed herein. In some embodiments, the antigen binding site that binds to PD-L1 may comprise a Fab formed by the heavy and light chains of an antibody. In some embodiments, the antigen binding site that binds to PD-L1 may comprise an scFv. The IgG Fc domain may comprise a wild-type antibody constant region or a modified antibody constant region. Examples of modified antibody constant regions are provided herein, including, for example, knob or hole mutations and LALAPG mutations. The functional domain may comprise a cytokine, a cytokine receptor, or a functional fragment thereof. Examples of functional domains include IL-2 polypeptides, such as the IL-2 polypeptides and engineered IL-2 polypeptides disclosed in Table 4, IL-15, and IL-15Rα.Examples of bifunctional proteins are shown in Figure 9 and Table 7, including IL-2-Fc / anti-PD-L1-scFv-Fc heterodimer; IL-2-anti-PD-L1-scFv-fusion protein lacking the Fc domain; IL-2-Fc / anti-PD-L1-Fab-Fc heterodimer; anti-PD-L1-Fab-Fc-IL-2-fusion / anti-PD-L1-Fab-Fc heterodimer; anti-PD-L1-Fab-Fc-IL-2-fusion / anti-PD-L1-Fab-Fc heterodimer; anti-PD-L1-Fab b-Fc-IL-2-fusion / anti-PD-L1-Fab-Fc-IL-2-fusion dimer or heterodimer;anti-PD-L1-scFv-Fc-IL-2-fusion / anti-PD-L1-scFv-Fc-IL-2-fusion dimer or heterodimer;anti-PD-L1-scFv-Fc-IL-2-fusion / anti-PD-L1-scFv-Fc heterodimer;anti-PD-L1-scFv-Fc-IL-2-fusion / anti-PD-L1-Fab-Fc-IL-2-fusion heterodimer; anti-PD-L1-scFv-Fc / anti-PD-L1-Fab-Fc-IL-2-fusion heterodimer; anti-PD-L1-scFv-F c-IL-2-fusion / anti-PD-L1-Fab-Fc heterodimer; anti-PD-L1-Fab-Fc-IL-15-fusion / anti-PD-L1-Fab-Fc-IL-15Rα-fusion heterodimer; anti-P anti-PD-L1-scFv-Fc-IL-15-fusion / anti-PD-L1-scFv-Fc-IL-15Rα-fusion heterodimers; anti-PD-L1-Fab-Fc-IL-15-fusion / anti-PD-L1-scFv-Fc-IL-15Rα-fusion heterodimers; and anti-PD-L1-scFv-Fc-IL-15-fusion / anti-PD-L1-Fab-Fc-IL-15Rα-fusion heterodimers.

[0127] In certain embodiments, the bifunctional protein comprises: (i) a heavy chain variable domain (VH) comprising: a CDR1 comprising the sequence of SEQ ID NO: 11, 3, 19, 33, 52, or 63; a CDR2 comprising the sequence of SEQ ID NO: 12, 4, 20, 34, 41, 53, or 64; and a CDR3 comprising the sequence of SEQ ID NO: 89, 5, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, or 86; and and (ii) an antigen-binding site that binds to PD-L1 comprising a light chain variable domain (VL) comprising: a CDR1 comprising the sequence of SEQ ID NO: 47, 6, 14, 22, 28, 36, 55, or 66; a CDR2 comprising the sequence of SEQ ID NO: 48, 7, 15, 23, 29, 37, 42, 56, 60, or 67; and a CDR3 comprising the sequence of SEQ ID NO: 49, 8, 16, 24, 30, 38, 43, 57, or 68. In certain embodiments, the antigen-binding site that binds to PD-L1 comprises at least one of (a)-(q), where (a) the VH comprises, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, and the VL comprises, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2; (b) the VH comprises, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9. (c) the VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17 and the VL comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18;(d) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26; (e) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31. 32 and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32; (f) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40; (g) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44 and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45. , 97%, 98%, 99% or 100% identical to SEQ ID NO:50; (h) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:50 and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:51;(i) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:58, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:59; (j) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61. 69, (k) the VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:69, and the VL comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:69; and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:70; (l) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:72 and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:73. , 97%, 98%, 99% or 100% identical to SEQ ID NO: 75; (m) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75 and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 76;(n) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 79; (o) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82; (p) and / or the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 84, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 85; and / or or (q) the VH comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 87, and the VL comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 88;

[0128] In some embodiments, the bifunctional protein comprises an antibody heavy chain constant region that is a human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the antibody heavy chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 90. In some embodiments, the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A, and Y407V, numbered according to the EU numbering system, compared to SEQ ID NO: 90. In some embodiments, the antibody heavy chain constant region comprises one or more mutations selected from S354C, T366W, and K409A, compared to SEQ ID NO: 90. In some embodiments, the antibody heavy chain constant region comprises one or more mutations selected from Y349C, T366S, L368A, F405K, and Y407V, numbered according to the EU numbering system, relative to SEQ ID NO: 90. In some embodiments, the bifunctional protein is a heterodimer and comprises a first and a second antibody heavy chain constant region, wherein the first antibody heavy chain constant region comprises one or more mutations selected from S354C, T366W, and K409A, numbered according to the EU numbering system, relative to SEQ ID NO: 90; and the second antibody heavy chain constant region comprises one or more mutations selected from Y349C, T366S, L368A, F405K, and Y407V, relative to SEQ ID NO: 90. In some embodiments, the antibody heavy chain constant region comprises L234A, L235A, and P329G mutations. In some embodiments, one of the first and second antibody heavy chain constant regions comprises the amino acid sequence of SEQ ID NO:91 and the second antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO:92.

[0129] In certain embodiments, the bifunctional protein comprises a functional domain, the functional domain being IL-15 or IL-15Rα, or a functional fragment thereof. IL-15 is a member of the cytokines that contain four α-helical bundles. IL-15 normally forms a complex with IL-15 receptor alpha expressed on APCs before binding to functional IL-15 receptor beta and gamma units on T cells and NK cells. IL-15 may be presented in trans to responsive cells expressing IL-2Rβ (IL15RB or CD122) and IL-2Rγ (CD132) by cells expressing the cytokine itself bound to a membrane form of the receptor alpha chain. Without wishing to be limited by theory, it is believed that the IL-15 receptor alpha sushi domain must form a complex with IL-15 before it can properly bind to the receptors beta and gamma. It has been reported that IL-15 and IL-15Rα complexes, and IL-15 / IL-15Rα sushi domain fusion proteins, are highly potent in stimulating CD8 T cells and NK cells.

[0130] In certain embodiments, a bifunctional protein disclosed herein comprises (a) a first subunit comprising an antigen binding site that binds to PD-L1, a first antibody heavy chain constant region, and an IL-15 polypeptide or a functional fragment or variant thereof; and (b) a second subunit comprising an antigen binding site that binds to PD-L1, a second antibody heavy chain constant region, and an IL-15Rα polypeptide or a functional fragment or variant thereof. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:93, or a functional fragment or variant thereof. In some embodiments, the IL-15 polypeptide comprises amino acids 50-162 of SEQ ID NO:93, or a functional fragment or variant thereof. In some embodiments, the IL-15Rα polypeptide comprises SEQ ID NO:94, or a functional fragment or variant thereof. In some embodiments, the IL-15Rα polypeptide comprises amino acids 31-97 of SEQ ID NO:94, or a functional fragment or variant thereof.

[0131] In some embodiments, the bifunctional protein disclosed herein is a heterodimer comprising an anti-PD-L1-Fab-Fc-IL-15 fusion protein and an anti-PD-L1-Fab-Fc-IL-15Rα fusion protein. For example, in some embodiments, the bifunctional protein comprises a first subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO: 186 (EP203); a second subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO: 187 (EP204); and a third subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO: 181 (EP205). In some embodiments, the first subunit comprises or consists of the amino acid sequence of SEQ ID NO: 186, the second subunit comprises or consists of the amino acid sequence of SEQ ID NO: 187; and the third subunit comprises or consists of the amino acid sequence of SEQ ID NO: 181.

[0132] In some embodiments, the bifunctional protein disclosed herein is a heterodimer comprising an anti-PD-L1-scFv-Fc-IL-15-fusion protein and an anti-PD-L1-scFv-Fc-IL-15Rα-fusion protein. For example, in some embodiments, the bifunctional protein comprises a first subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO:188 (EP207) and a second subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO:189 (EP208). In some embodiments, the bifunctional protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO:188 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO:189.

[0133] In certain embodiments, the bifunctional protein disclosed herein comprises a functional domain, where the functional domain is IL-2, e.g., wild-type IL-2 or engineered IL-2 or a functional fragment or variant thereof. The term "interleukin 2" or "IL-2" as used herein refers to IL-2 from any vertebrate source, including mammals such as humans or mice, unless otherwise indicated. The term encompasses precursor IL-2, or unprocessed IL-2, as well as any form of IL-2 resulting from cellular processing. The term also encompasses naturally occurring variants of IL-2, e.g., splice variants or allelic variants. The amino acid sequence of an example of mature human IL-2 is set forth in SEQ ID NO: 191. "Wild-type" or "naturally occurring" when used with respect to IL-2 is intended to mean the mature IL-2 molecule (e.g., SEQ ID NO: 191). The term "engineered IL-2" or "engineered IL-2 polypeptide" as used herein encompasses IL-2 having at least one residue that differs from native or wild-type IL-2, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is conjugated or fused to another molecule, such as another polypeptide. The various forms of engineered IL-2 are characterized as having at least one amino acid substitution that affects the interaction of IL-2 with IL-2Rβ and / or IL-2Rα. Identification of the various engineered forms of IL-2 described herein is made with reference to the sequence shown, for example, in SEQ ID NO: 192. In some embodiments, IL-2 is modified to include a T3A substitution. The T3A substitution may be made to the wild-type sequence. Additionally, the T3A substitution may be made to the engineered IL-2 sequences disclosed herein. Examples of wild-type and engineered IL-2 polypeptides are described in PCT / US2020 / 046244, which is incorporated herein by reference in its entirety.

[0134] IL-2 regulates lymphocyte proliferation and activation. IL-2 mediates its actions by binding to the IL-2 receptor (IL-2R), which contains up to three separate subunits. Association of all three subunits, the interleukin-2 receptor alpha chain (IL-2Rα, or CD25), the interleukin-2 receptor beta chain (IL-2Rβ, or CD122), and the interleukin-2 receptor gamma chain (IL-2Rγ, or CD132), results in the trimeric IL-2Rαβγ, the high affinity receptor for IL-2. Association of the IL-2Rβ and IL-2Rγ subunits results in the dimeric receptor IL-2Rβγ, termed the intermediate affinity IL-2R. The IL-2Rα subunit forms the monomeric low affinity IL-2 receptor. Expression of IL-2Rα is involved in the proliferation of immunosuppressive regulatory T cells (Tregs); whereas dimeric IL-2Rβγ promotes the proliferation of cytolytic CD8 + It can result in the proliferation and killing of T cells and NK cells.

[0135] In some embodiments, the bifunctional protein comprises an antigen binding site that binds to PD-L1 and comprises wild-type IL-2 (e.g., SEQ ID NO: 191). In some embodiments, the bifunctional protein comprises an antigen binding site that binds to PD-L1 and comprises engineered IL-2 (e.g., SEQ ID NO: 192). In some embodiments, the bifunctional protein comprises an antigen binding site that binds to PD-L1 and comprises engineered IL-2, wherein the engineered IL-2 polypeptide is (a) IL-2 receptor alpha (IL-2Rα) binding region 1, which comprises one or more mutations compared to wild-type IL-2 at one or more positions selected from the following: a mutation at position K35 selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C; a mutation at position R38 selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, or R38N; A mutation at position F42 selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H; A mutation at position Y45 selected from Y45S, Y45P, Y45A, Y45V, Y45C, Y45T and Y45F, and / or (b) an IL-2 receptor beta (IL-2Rβ) binding domain 2 motif comprising: X1-X2-X3-D-X4-X-5-X6-N-X7-X8-X9-X10-X11-X12-X13 (Sequence number 95), During the ceremony, X1 is selected from C, T, G, W, I, S, E, and K; X2 is selected from Y, P, V, W, L, A, and G; X3 is selected from S, T, Q, G, M, E, R, and K; X4 is selected from A, V, S, and T; X5 is selected from I, L, T, and V; X6 is selected from S, T, E, D, and R; X7 is selected from I, A, M, and V; X8 is selected from S, T, N, Q, I, G, E, K, and R; X9 is selected from V, L, and I; X10 is selected from N, T, I, and L; X11 is selected from V, A, and I; X12 is selected from Q, L, G, K, and R; and X13 is selected from A, D, and E.

[0136] In some embodiments, the bifunctional protein comprises an antigen binding site that binds to PD-L1, and comprises an engineered IL-2, wherein the engineered IL-2 polypeptide comprises: (a) an IL-2Rα binding region 1 comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 124-147; and / or (b) an IL-2Rβ binding region 2 motif comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 96-123. In some embodiments, the bifunctional protein comprises an antigen binding site that binds to PD-L1, and comprises an engineered IL-2, wherein (a) the IL-2Rα binding region 1 comprises an amino acid sequence selected from SEQ ID NOs: 124-147; and / or (b) the IL-2Rβ binding region 2 motif comprises an amino acid sequence selected from SEQ ID NOs: 96-123. In some embodiments, the antigen binding site that binds to PD-L1 is an scFV or Fab.

[0137] In certain embodiments, the bifunctional protein is a fusion protein comprising (a) a first subunit comprising an antigen-binding site that binds PD-L1 fused to (b) a second subunit comprising a wild-type or engineered IL-2 polypeptide, or a functional fragment or variant thereof. In some embodiments, the antigen-binding site that binds PD-L1 is an scFv or Fab. In some embodiments, the bifunctional protein comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 160-164. In some embodiments, the bifunctional protein comprises an amino acid sequence selected from SEQ ID NOs: 160-164 (EP199, EP200, EP201, and EP461, respectively).

[0138] In certain embodiments, the bifunctional protein is a heterodimer comprising (a) a first subunit comprising an antigen-binding site that binds to PD-L1 and a first antibody heavy chain constant region; and (b) a second subunit comprising a wild-type or engineered IL-2 polypeptide, or a functional fragment or variant thereof, and a second antibody heavy chain constant region. In some embodiments, the antigen-binding site that binds to PD-L1 is an scFv or Fab. In some embodiments, the bifunctional protein comprises a heterodimer, wherein the first subunit is an anti-PD-L1-scFv-Fc fusion protein and the second subunit is an IL-2-Fc fusion protein. In some embodiments, the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO: 176 (EP326) and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 165-175 (EP290, EP291, EP297, EP412, EP413, EP414, EP415, EP416, EP417, EP418, EP419, respectively). In some embodiments, the first subunit comprises or consists of the amino acid sequence of SEQ ID NO: 176 and the second subunit comprises or consists of an amino acid sequence selected from SEQ ID NOs: 165-175. In some embodiments, the bifunctional protein comprises a heterodimer of proteins comprising: (a) SEQ ID NO:176 and SEQ ID NO:165; (b) SEQ ID NO:176 and SEQ ID NO:166; (c) SEQ ID NO:176 and SEQ ID NO:167; (d) SEQ ID NO:176 and SEQ ID NO:168; (e) SEQ ID NO:176 and SEQ ID NO:169; (f) SEQ ID NO:176 and SEQ ID NO:170; (g) SEQ ID NO:176 and SEQ ID NO:171; (h) SEQ ID NO:176 and SEQ ID NO:172; (i) SEQ ID NO:176 and SEQ ID NO:173; (j) SEQ ID NO:176 and SEQ ID NO:174; or (k) SEQ ID NO:176 and SEQ ID NO:175.

[0139] In some embodiments, the bifunctional protein comprises a heterodimer, wherein the first subunit is an anti-PD-L1-Fab-Fc fusion protein and the second subunit is an IL-2-Fc fusion protein. In some embodiments, the first subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 177-180 (EP325, EP462, EP463, EP464 respectively), the first subunit further comprises an LC domain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 181 (EP205), and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 165-175 (EP290, EP291, EP297, EP412, EP413, EP414, EP415, EP416, EP417, EP418, EP419 respectively). In some embodiments, the first subunit comprises or consists of an amino acid sequence selected from SEQ ID NOs: 177-180, the LC domain comprises or consists of the amino acid sequence of SEQ ID NO: 181, and the second subunit comprises or consists of an amino acid sequence selected from SEQ ID NOs: 165-175. In some embodiments, the bifunctional protein comprises a protein heterodimer comprising: (a) a first subunit comprising SEQ ID NOs: 177 and 181, SEQ ID NOs: 178 and 181; SEQ ID NOs: 179 and 181; or SEQ ID NOs: 180 and 181; and (b) a second subunit comprising an amino acid sequence selected from SEQ ID NOs: 165-175.In some embodiments, the bifunctional protein is selected from the group consisting of: (a) SEQ ID NO:165, SEQ ID NO:177, and SEQ ID NO:181; (b) SEQ ID NO:166, SEQ ID NO:177, and SEQ ID NO:181; (c) SEQ ID NO:167, SEQ ID NO:177, and SEQ ID NO:181; (d) SEQ ID NO:168, SEQ ID NO:177, and SEQ ID NO:181; (e) SEQ ID NO:169, SEQ ID NO:177, and SEQ ID NO:181; (f) SEQ ID NO:170, SEQ ID NO:177, and SEQ ID NO:181; (g) SEQ ID NO:171, SEQ ID NO:177, and SEQ ID NO:181; (h) SEQ ID NO:172. (i) SEQ ID NO:173, SEQ ID NO:177, and SEQ ID NO:181; (j) SEQ ID NO:174, SEQ ID NO:177, and SEQ ID NO:181; (k) SEQ ID NO:175, SEQ ID NO:177, and SEQ ID NO:181; (l) SEQ ID NO:171, SEQ ID NO:178, and SEQ ID NO:181; (m) SEQ ID NO:171, SEQ ID NO:179, and SEQ ID NO:181; and (n) a heterodimer of a protein comprising a polypeptide having an amino acid sequence selected from SEQ ID NO:171, SEQ ID NO:180, and SEQ ID NO:181.

[0140] In certain embodiments, the bifunctional protein comprises (a) a first subunit that comprises an antigen-binding site that binds to PD-L1 and a first antibody heavy chain constant region; and (b) a second subunit that comprises an antigen-binding site that binds to PD-L1 and an engineered IL-2 polypeptide or a functional fragment or variant thereof and a second antibody heavy chain constant region. In some embodiments, the antigen-binding site that binds to PD-L1 is an scFv or Fab. In some embodiments, the bifunctional protein comprises a heterodimer, wherein the first subunit is an anti-PD-L1-Fab-Fc fusion protein and the second subunit is an anti-PD-L1-Fab-IL-2-fusion protein. In some embodiments, the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO: 182 (EP362), and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 183-185 (EP363, EP364, EP365); optionally wherein the first and second subunits each independently further comprise an amino acid sequence at least 90% identical to SEQ ID NO: 181 (EP205). In some embodiments, the first subunit comprises or consists of the amino acid sequence of SEQ ID NO: 182, and the second subunit comprises or consists of an amino acid sequence selected from SEQ ID NOs: 183-185; optionally wherein the first and second subunits each independently further comprise the amino acid sequence of SEQ ID NO: 181. In some embodiments, the bifunctional protein comprises a protein heterodimer comprising a polypeptide having an amino acid sequence selected from: (a) SEQ ID NO:182; SEQ ID NO:183; and SEQ ID NO:181; (b) SEQ ID NO:182; SEQ ID NO:184; and SEQ ID NO:181; and (c) SEQ ID NO:182; SEQ ID NO:185; and SEQ ID NO:181.

[0141] In some embodiments, the bifunctional protein binds to human PD-L1 with a KD of less than about 1 nM as measured by SPR, or with a comparable or even lower KD compared to a constructed antigen-binding site that binds to PD-L1, hi some embodiments, the bifunctional protein binds to IL-2Rβ with a KD of less than about 65 nM or less than about 50 nM as measured by SPR.

[0142] In some embodiments, the bifunctional protein binds to IL-2Rα with a KD of less than about 40 nM as measured by SPR. In some embodiments, the bifunctional protein binds to IL-2Rβ with a KD of less than about 100 nM, less than about 80 nM, less than about 50 nM, less than about 10 nM, or less than about 5 nM as measured by SPR. In some embodiments, the bifunctional protein binds to PD-L1 with a KD of less than about 0.5 nM or less than about 0.1 nM as measured by SPR. In some embodiments, the bifunctional protein binds to IL-2Rα with an EC50 of less than about 1 nM as measured by ELISA. In some embodiments, the bifunctional protein binds to IL-2Rβ with an EC50 of less than about 5 nM, less than about 2.5 nM, less than about 1.5 nM, less than about 1 nM, or less than about 0.6 nM as measured by ELISA. In some embodiments, the bifunctional protein binds to PD-L1 with an EC50 of less than about 0.4 nM as measured by ELISA.

[0143] In some embodiments, the bifunctional protein competes with PD-1 for binding to PD-L1 or inhibits binding of PD-L1 to PD-1.

[0144] In some embodiments, the bifunctional protein induces p-STAT5 expression in immune cells. In some embodiments, the bifunctional protein induces p-STAT5 expression in immune cells with an EC50 of less than about 1 nM, less than about 0.6 nM, less than 0.5 nM, or less than about 0.1 nM when measured in isolated human peripheral blood mononuclear cells (PBMCs), where the immune cells are T cells, NK cells, or Tregs. In certain embodiments, the bifunctional protein induces p-STAT5 expression in immune cells when measured in mouse splenocytes, where the immune cells are T cells, NK cells, or Tregs.

[0145] In some embodiments, the bifunctional protein inhibits tumor growth in vivo.

[0146] In some embodiments, the bifunctional protein induces proliferation of immune cells in vivo. In some embodiments, the immune cells are T cells or NK cells. In some embodiments, the T cells are CD8+ T cells. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

[0147] Examples of various antibodies, bifunctional proteins, and alternative formats of the present disclosure are shown in FIG. [Table 7-1] [Table 7-2]

[0148] Vectors and production methods In certain embodiments, the present disclosure further comprises an isolated polynucleotide encoding an antigen binding site, a protein and / or an antibody, and / or a bifunctional protein and / or an antibody as disclosed herein, or any fragment, variant, or combination thereof. In certain embodiments, the present disclosure further comprises an expression vector comprising a polynucleotide of the present disclosure. In certain embodiments, the present disclosure further comprises an engineered cell comprising an isolated polynucleotide of the present disclosure or an expression vector of the present disclosure.

[0149] In some embodiments, the disclosure provides an isolated polynucleotide encoding any of the PD-L1-antigen binding sites, specific antibodies, antigen-binding fragments, or variants thereof of the disclosure. In certain embodiments, the polynucleotide is codon optimized for expression in a host cell. Once the coding sequence is known or identified, codon optimization can be performed using known techniques and tools, e.g., the GenScript OptimiumGene tool; see also Scholten et al., Clin. Immunol. 119:135, 2006). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons are optimized for expression in a host cell) and fully codon-optimized sequences.

[0150] It will also be understood that a polynucleotide encoding a PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof of the present disclosure may have a different nucleotide sequence due to, for example, degeneracy of the genetic code, splicing, etc., while still encoding the same PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof.

[0151] Vectors are also provided, where the vector comprises or contains a polynucleotide as disclosed herein (i.e. a polynucleotide encoding a PD-L1 antigen-binding site, a specific antibody, an antigen-binding fragment, or variants thereof). The vector may comprise any one or more of the vectors disclosed herein.

[0152] In a further aspect, the present disclosure also provides a host cell that expresses a PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof according to the present disclosure, or comprises or contains a vector or polynucleotide according to the present disclosure.

[0153] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cell is a mammalian cell. In certain such embodiments, the cell is a CHO cell (e.g., DHFR-CHO cell (Urlaub et al., PNAS, 77:4216 (1980)), human embryonic kidney cell (e.g., HEK293T cell), PER.C6 cell, Y0 cell, Sp2 / 0 cell, NS0 cell, human hepatocyte cell, such as Hepa cell, or the like. The host cell line may be a mammalian cell line, such as RG cells, myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells (e.g., TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human hepatocytes (HepG2); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); mouse mammary tumor cells (MMT060562); TRI cells; MRC5 cells, and FS4 cells. Suitable mammalian host cell lines for antibody production include, for example, those described in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0154] In one embodiment, the host cell is a prokaryotic cell, such as E. coli. Expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio / Technology, 9:545-551 (1991)). For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523.

[0155] In certain embodiments, cells may be transfected with a vector according to the present description by an expression vector. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, such as a eukaryotic cell. In the context of the present description, the term "transfection" includes any method known to those skilled in the art for introducing a nucleic acid molecule into a cell, such as a eukaryotic cell, including a mammalian cell. Such methods include, for example, electroporation, lipofection, such as based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection, such as DEAE-dextran or polyethyleneimine. In certain embodiments, the introduction is non-viral.

[0156] Additionally, host cells of the present disclosure may be stably or transiently transfected with a vector of the present disclosure, for example to express a PD-L1 antigen-binding site, a PD-L1 antigen-specific antibody, antigen-binding fragment, or variants thereof of the present disclosure. In such embodiments, the cells may be stably transfected with a vector as described herein. Alternatively, the cells may be transiently transfected with a vector of the present disclosure encoding an antibody or antigen-binding fragment disclosed herein. In any of the presently disclosed embodiments, the polynucleotide may be heterologous to the host cell.

[0157] Thus, the present disclosure also provides recombinant host cells that heterologously express the PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variants thereof of the present disclosure. For example, the cell may be of a species different from the species from which the antibody was derived, fully or partially (e.g., a CHO cell expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not naturally express the PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variants thereof. Furthermore, the host cell may impart post-translational modifications (PTMs; e.g., glycosylation or fucosylation) to the PD-L1 antigen-binding site, PD-L1 antigen-specific antibody, antigen-binding fragment, or variants thereof that are not present in the native state of the antibody or antigen-binding fragment (or the native state of the parent antibody from which the antibody or antigen-binding fragment was engineered or derived). Such PTMs may result in potential differences (e.g., reduced immunogenicity). Thus, a PD-L1 antigen-binding site of the disclosure, specific antibody, antigen-binding fragment, or variant thereof produced by the host cells disclosed herein may contain one or more post-translational modifications that differ from the naturally occurring antibody (or parent antibody) (e.g., a human antibody produced by CHO cells may contain additional post-translational modifications that differ from the antibody when isolated from a human and / or when produced by a naturally occurring human B cell or plasma cell).

[0158] In a related aspect, the present disclosure provides a method for producing a PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof, comprising culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof. Methods useful for isolating and purifying recombinantly produced PD-L1 antigen-binding site, specific antibody, antigen-binding fragment, or variant thereof may include, for example, obtaining a supernatant from a suitable host cell / vector system that secretes the recombinant antibody into culture medium, and then concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a suitable purification matrix, or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps may be employed to further purify the recombinant polypeptide. These purification methods may also be employed when isolating immunogens from their natural environment. One or more large-scale production methods of isolated / recombinant antibodies described herein include batch cell cultures that are monitored and controlled to maintain appropriate culture conditions. Purification of soluble PD-L1 antigen-binding sites, PD-L1 antigen-specific antibodies, antigen-binding fragments, or variants thereof may be carried out according to methods described herein and known in the art and that comply with the laws and guidelines of domestic and foreign regulatory authorities.

[0159] Pharmaceutical Compositions In certain embodiments, the present disclosure further provides pharmaceutical compositions comprising the antigen-binding site of the present disclosure, the protein and / or antibody of the present disclosure, or the bifunctional protein and / or antibody of the present disclosure, and a pharma- ceutically acceptable diluent(s), excipient(s), or carrier(s). In some embodiments, the pharmaceutical composition comprises an additional therapeutic agent (e.g., combination therapy). The pharmaceutical compositions may be formulated in a conventional manner using one or more pharma- ceutical acceptable carriers, including excipients and adjuvants that facilitate processing of the antigen-binding site of the present disclosure, the protein and / or antibody of the present disclosure, or the bifunctional protein and / or antibody of the present disclosure into a pharma- ceutical usable formulation. The appropriate formulation depends on the route of administration selected. Any pharma- ceutically acceptable techniques, carriers, and excipients may be used as appropriate for formulating the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). Examples of IL-2 compositions are described in U.S. Patent Nos. 4,604,377 and 4,766,106, which are incorporated herein by reference.

[0160] As used herein, "pharmaceutical acceptable carrier" and "physiologically acceptable carrier" are used interchangeably and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption agents, retardants, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar materials, and combinations thereof, as known to those skilled in the art, and are generally non-toxic to recipients at the dosages and concentrations employed, i.e., molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, e.g., humans, as appropriate. (See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0161] The pharmaceutical composition may contain different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form and whether it needs to be sterile for the route of administration, such as injection. The antigen binding sites of this disclosure, the proteins and / or antibodies of this disclosure, or the bifunctional proteins and / or antibodies of this disclosure (and any additional therapeutic agents) can be administered intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrasplenic, intrarenal, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, by local irrigation directly bathing the target cells, via catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations of any of the above as known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used to administer polypeptide molecules such as the antigen binding sites of this disclosure, the proteins and / or antibodies of this disclosure, or the bifunctional proteins and / or antibodies of this disclosure.

[0162] Methods of Treatment and Use In some embodiments, provided herein is a method of treatment or use comprising administering a therapeutically effective amount of an antigen binding site disclosed herein, a protein or antibody disclosed herein, or a bifunctional protein or antibody disclosed herein to a subject in need thereof, thereby modulating an immune response. In some embodiments, modulating the immune response comprises at least one of enhancing T cell activity or enhancing NK cell activity. In some embodiments, the method of treatment or use is treating a disease in a subject in need thereof. In certain embodiments, the disease is cancer. In some embodiments, the cancer comprises breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, or melanoma. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, gastric cancer, glioblastoma, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, skin cancer, melanoma, bone cancer, renal cell carcinoma, and kidney cancer. Also included are precancerous conditions or lesions and cancer metastases.Other cell proliferation disorders include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system.Similarly, other cell proliferation disorders, such as hypergammaglobulinemia, lymphoproliferative disorders, dysproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, histiocytosis, and any other cell proliferation disease other than neoplasms located in the above organ systems, can also be treated.

[0163] In some embodiments, the use or method of treating or modulating an immune response further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent (e.g., combination therapy). In certain embodiments, the additional therapeutic agent is an anti-cancer agent. Examples of anti-cancer agents include checkpoint inhibitors (e.g., anti-PD1 antibodies), chemotherapeutic agents, agents that suppress the tumor microenvironment, cancer vaccines (e.g., Sipuleucel-T), oncolytic viruses (e.g., talimogene laherparepvec), immune cells expressing chimeric antigen receptors, and tumor-infiltrating lymphocytes. In certain embodiments, the additional therapeutic agent is a molecule comprising an antigen-binding moiety. In certain specific embodiments, the antigen-binding moiety is selected from a single domain antibody, a Fab molecule, a scFv, a diabody, a nanobody, a bispecific T cell engager, or an immunoglobulin. In certain embodiments, the antigen-binding moiety is specific for a tumor antigen (e.g., carcinoembryonic antigen, fibroblast activation protein-alpha, CD20) or a checkpoint protein (e.g., CTLA-4, PD-1). In some embodiments, the additional therapeutic agent comprises immune cells expressing a chimeric antigen receptor, immune cells expressing an engineered T cell receptor, or tumor infiltrating lymphocytes.

[0164] Other embodiments will become apparent from the following disclosure. EXAMPLES

[0165] The following examples are offered by way of illustration and not by way of limitation.

[0166] Example 1. Development of anti-PD-L1 antibodies This example describes the development and characterization of an antibody that specifically binds human PD-L1.

[0167] Screening and selection of scFv mRNA display 10 Using mRNA Display Technology 12~13PD-L1 binders were identified from a natural human scFv library of . Briefly, similar to a reported procedure (US Pat. No. 6,258,558), the DNA library was first transcribed into an mRNA library and then translated into an mRNA-scFv fusion library by covalent linkage via a puromycin linker. The fusion library was first counter-selected with human IgG (a negative protein) to remove non-specific binders, and then selected against recombinant PD-L1-Fc fusion protein, followed by capture onto protein G magnetic beads. To enrich for scFvs that block the interaction between PD-1 and PD-L1, PD-1 was utilized to compete out PD-L1 binders from protein G beads, and binders were enriched by PCR amplification with library-specific oligos. A total of four rounds of selection were performed to generate a highly enriched PD-L1 binding pool for screening.

[0168] Identification and characterization of anti-PD-L1 scFv After four rounds of selection, the PD-L1 enriched scFv library was cloned into the bacterial periplasmic expression vector pET22b and transformed into TOP10 competent cells. Each of the scFv molecules was engineered to have a C-terminal FLAG and 6xHIS tag for purification and assay detection. Clones from TOP10 cells were pooled and miniprep DNA was prepared and subsequently transformed into the bacterial Rosetta II strain for expression. Single clones were picked, expanded and induced with 0.1mM IPTG in 96-well plates for expression. Supernatants were harvested after 16-24 hours of induction at 30°C for assays to identify anti-PD-L1 scFv.

[0169] A PD-L1 binding screening ELISA was developed to identify individual anti-PD-L1 scFv. Briefly, human Fc and human PD-L1-Fc were immobilized in 384-well plates at a final concentration of 2μg / mL in 1×PBS in a total volume of 25μL per well. The plates were incubated overnight at 4℃, followed by blocking with 80μL per well of Superblock for 1 hour. 25μL of supernatant was added to the wells immobilized with human Fc and human PD-L1-Fc and incubated with shaking for 1 hour. PD-L1 binding was detected by adding 25μL of anti-FLAG HRP diluted 1:5000 in 1×PBST. Between each step, the plates were washed 3 times with 1×PBST on a plate washer. The plates were then developed with 20μL of TMB substrate for 5 minutes and stopped by adding 20μL of 2N sulfuric acid. Plates were read on a Biotek plate reader at OD450nm and binding and selectivity analyzed with Excel bar graphs. Clones with greater than 2-fold PD-L1 target binding compared to human Fc control were subjected to DNA sequencing. Superior clones were generated and purified for further characterization.

[0170] scFv production in E. coli Specific anti-PD-L1 clones were picked from glycerol stock plates and grown overnight in 5 mL cultures in Thomson 24-well plates with breathable membranes. This culture, and all subsequent cultures described below, were grown at 37°C with shaking at 225 rpm in Terrific Broth Complete supplemented with 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol, which also contained a 1:5,000 dilution of Antifoam 204, unless otherwise specified. This overnight starter culture was then used to inoculate larger cultures, and the starter cultures were diluted 1:100 into the designated production cultures and grown to an OD600 of 0.5-0.8. At this point, the cultures were induced with IPTG to a final concentration of 0.1 mM and incubated overnight at 30°C. The following day, the cultures were centrifuged at 5,000 x g for 30 minutes to pellet the cells, and the supernatant was then filter sterilized through a 0.2 μm sterile PES membrane.

[0171] For purification, 3 μL of Ni Sepharose Excel resin (GE Healthcare) was used per mL of filtered supernatant. Disposable 10 mL or 20 mL BioRad Econo-Pac columns were used. The resin was equilibrated with at least 20 column volumes (CV) of Buffer A (1×PBS, pH 7.4 with additional NaCl to 500 mM). Filter-sterilized supernatant was purified by gravity flow, either with a controlled flow rate of 1 mL / min or by pouring in two portions onto the same packed resin bed. The column was then washed with 10 CV of Buffer A and 20 CV of Buffer B (1×PBS, pH 7.4 with additional NaCl to 500 mM and 30 mM imidazole). Two Detox buffers were used to remove endotoxins as an optional step, if required. For 250 mL expression culture purification, the antibody binding column was washed successively with 20 CV of Buffer C (1×PBS, pH 7.4, with additional NaCl to 500 mM, 1% TX114), 20 CV of Buffer D (1×PBS, pH 7.4, with additional NaCl to 500 mM, 1% TX100 + 0.2% TNBP), and 40 CV of Buffer E (1×PBS, pH 7.4, with additional NaCl to 500 mM). Protein was eluted with elution buffer F (1×PBS, pH 7.4 with additional NaCl to 500 mM and 500 mM imidazole) in a total of 6 fractions (0.5 CV pre-elution, 5×1 CV elution). Fractions were analyzed by Bradford assay (100 μL of diluted Bradford solution + 10 μL of sample). Light blue fractions were pooled. Protein concentration was measured by A280 extension factor. SDS-PAGE gels were used to analyze the purity of the purified antibodies. In most cases, thermal shift assays were performed to measure the thermal stability of the purified antibodies.

[0172] PD-L1 binding ELISA An ELISA assay was developed to determine the EC50 of anti-PD-L1 scFv. Briefly, 384-well plates were immobilized with human PD-L1-Fc at a final concentration of 2μg / mL in 1x PBS in a total volume of 25μL per well. Plates were incubated overnight at 4℃, followed by blocking with 80μL of Superblock per well for 1 hour. Purified anti-PD-L1 scFv was titrated 2-fold starting from 200nM. 25μL was added to the human PD-L1 immobilized wells and incubated with shaking for 1 hour. PD-L1 binding was detected by adding 25μL of anti-FLAG HRP diluted 1:5000 in 1x PBST. Between each step, plates were washed 3 times with 1x PBST on a plate washer. Plates were then developed with 20μL of TMB substrate for 5 minutes and stopped by adding 20μL of 2N sulfuric acid. Plates were read on a Biotek plate reader at OD450nm and then plotted using Prism 8.1 software. EC50 was calculated and shown in Table 8. Figure 1 shows the PD-L1 binding ELISA curves for selected scFvs. [Table 8]

[0173] Binding of scFv to PD-L1 in SPR The kinetic analysis of anti-PD-L1 scFv was assessed by surface plasmon resonance (SPR) technique using Biacore T200. The assay was performed using Biacore T200 control software version 2.0. Anti-human Fc antibody was immobilized on flow cells 1 and 2 of a CM5 sensor chip. For each cycle, 1μg / mL of human PD-L1-Fc protein was captured on flow cell 2 in 1× HBSP buffer on the anti-hFc sensor chip at a flow rate of 10μL / min for 60 seconds. Two-fold serially diluted HIS-tagged purified anti-PD-L1 scFv were injected on both reference flow cell 1 and PD-L1-Fc capture flow cell 2 at a flow rate of 30μL / min for 150 seconds, followed by washing for 300 seconds. The flow cells were then regenerated with antibody regeneration buffer (GE Healthcare) at a flow rate of 30μl / min for 30 seconds. Eight concentration points ranging from 300 nM to 0 nM were assayed for each anti-PD-L1 scFv in a 96-well plate. The kinetics of scFv binding to PD-L1 protein was analyzed using Biacore T200 evaluation software version 3.0. Specific binding response units were obtained by subtracting binding to the reference flow cell 1 from the PD-L1 capture flow cell 2. The binding kinetics (K a , K d , and K D ) were determined from sensorgram analysis and are shown in Table 9. [Table 9]

[0174] Binding of scFv to cell surface PD-L1 in FACS K562 cells (ATCC) were transfected with a construct encoding full-length human PD-L1 with C-terminal FLAG and Myc tags in pCMV6-Entry vector. A polyclonal drug-resistant pool of PD-L1 target-expressing cells was obtained by a G418 drug selection process. In parallel, an empty vector-transfected parental line was generated as a negative control. PD-L1 target-expressing cells were sorted by FACS to generate a PD-L1 target-expressing polyclonal pool. This pool was expanded under G418 drug selection. Single cell sorting was then performed, followed by further drug selection to form clonal cell lines. Clonal lines were screened for PD-L1 expression by FACS. Highly expressing PD-L1 cell lines were then used for screening and assays.

[0175] To determine whether anti-PD-L1 scFv binds to PD-L1 expressing cells, 200nM purified anti-PD-L1 scFv was diluted in complete medium and incubated with PD-L1 / K562 cells and K562 cells in a 96-well plate on ice for 1 hour. The cells were centrifuged at 1200rpm at 4℃ for 5 minutes to remove the primary antibody. The cells were then washed once with 200μL complete medium per well. Samples were detected with premixed anti-His biotin streptavidin Alexa Fluor647 by adding 100μL of diluted secondary antibody and incubating in the dark at 4℃ for 30 minutes. Samples were centrifuged at 1200rpm at 4℃ for 5 minutes and washed twice with 200μL 1×PBS per well. Samples were reconstituted in 200μL 1×PBS and read on an Attune NxT cytometer. Analysis was performed by Attune NxT software, which plotted overlay histograms of anti-PD-L1 scFv binding to both negative and target cell lines. The EC50 of PD-L1 binding on the cell surface for selected scFvs was calculated and is shown in Table 10. Figures 2A-B show the FACS curves of PD-L1 / K562 cell surface binding for selected scFvs. [Table 10]

[0176] Competition of scFv with PD-L1 and PD-1 interactions in HTRF The PD-1 / PD-L1 TR-FRET (HTRF) Assay Kit (BPS, 72038) was used to evaluate the neutralization activity of PD-1 by anti-PD-L1 scFv. Briefly, 1× Immuno Buffer 1 was made. Dye-labeled acceptor, PD-1-Eu, PD-L1 biotinylated and scFv were diluted in Buffer 1. Serially diluted scFv and reagents were added to each well of a 384-well plate according to the manufacturer's recommended volumes. Samples were incubated at room temperature for 1.5 hours, after which the fluorescence intensity was read on a Biotek Neo2 plate reader using the manufacturer's instrument settings for the assay. Data analysis was performed using the TR-FRET ratio of 665 nm emission / 620 nm emission and plotted in Prism 8.0. The competition IC50 of PD-1 and PD-L1 for selected scFvs was calculated and is shown in Table 11. Figure 3A-B shows the competition results of PD-1 and PD-L1 for selected scFvs. [Table 11]

[0177] scFv in NFAT reporter assay in Jurkat cells The PD-1 neutralizing activity of anti-PD-L1 scFv was also assessed in a cell assay using a PD-1 / PD-L1 Blockade Bioassay Kit (Promega, J1250). PD-L1 aAPC / CHOK1 cells were thawed and seeded into white flat-bottom assay plates according to the manufacturer's protocol. Cells were incubated at 37°C, 5% CO 2The cells were incubated at 37°C overnight. The following day, the medium was removed from each well. PD-L1 scFv clones were serially diluted 5-fold starting at 500 nM and 40 μL of 2× concentration of diluted scFv and controls were added to the cells. The plates were incubated at 37°C for 30 minutes to allow the scFv to bind to PD-L1 on the cell surface. PD-1 effector cells were thawed according to the manufacturer's protocol and 40 μL was added to each well containing PD-L1 aAPC / CHOK1 cells with anti-PD-L1 scFv. The plates were incubated at 37°C, 5% CO 2 The plates were incubated at RT for 16 hours. The assay plates were then equilibrated to room temperature for 10 minutes. 80 μL of room temperature Bio-Glo reagent was added to each well and to the control wells. The plates were incubated at room temperature with shaking for 30 minutes and protected from light. Luminescence was quantified on a Biotek Neo2 plate reader and plotted with Prism 8.0 software. The NFAT reporter assay EC50s of selected scFvs were calculated and are shown in Table 12. Figure 4 shows the results of the reporter assay. [Table 12]

[0178] Affinity maturation of anti-PD-L1 scFv 2018EP171-H02 To further improve the affinity and biological activity of anti-PD-L1 scFv, a rationally designed HCDR3 mutagenized scFv library of 2018EP171-H02 was constructed and subjected to stringent selection conditions. Residues in HCDR3 (SEQ ID NO: 46) were mutagenized at a WT to mutant ratio of 70% to 30%, gtg tat tac tgt gcg aga gat aaa ggg tat ggc agt ggc tgg agg ggt gac tac tgg ggc cag gga (SEQ ID NO: 190). The mutagenized library was constructed by overlap PCR with framework and VL. mRNA display was used to further enrich for high affinity scFv binders through two rounds of selection. The first round was selected on PD-L1 / CHOK1 cell line to ensure that the molecules bind to the native epitope of PD-L1. The second round was coupled with 10 nM PD-L1-Fc protein followed by a 16-hour off-rate selection in the presence of 500 nM immobilized PD-L1 as competitor. The immobilized PD-L1 was removed along with the weaker binders. The higher affinity scFv binders, PD-L1 Fc, were then captured with Protein G beads, eluted and amplified by PCR.

[0179] After a second round of mutagenic library selection, pools were cloned into the pET22b vector and screened as described above.

[0180] Generation, binding, and characterization assays for selected affinity matured scFv clones were all performed as described above. The data were analyzed and provided in Table 13 for ELISA binding, Table 14 for SPR kinetic analysis, Table 15 for PD-L1 / K6562 cell surface FACS binding, and Table 16 for Jurkat NFAT reporter assay surface PD-L1 binding results. Figure 5 shows the ELISA binding results. Figure 6 shows PD-L1 / K6562 cell surface FACS binding. Figure 7 shows the competitive HTRF results of PD-1 and PD-L1. Figure 8 shows the Jurkat NFAT reporter assay results for selected optimized scFvs. [Table 13] [Table 14] [Table 15] [Table 16]

[0181] Example 2. Antibody and bifunctional antibody structures The variable VH and VL sequences of 2019EP69-F03 were fused to the constant frame sequences of the human heavy chain IgG1 backbone and light chain lambda backbone (EP206 and EP205, respectively) to generate the anti-PD-L1 monoclonal antibody.

[0182] To generate the anti-PD-L1 / IL-15 fusion bifunctional antibody, the C-terminus of one heavy chain of anti-PD-L1 was modified to (G 4 S) 4 The heavy chain of anti-PD-L1 was fused to human IL-15(50-162) [P40933] by a linker. S354C, T366W, and K409A mutations (Wei et al., Oncotarget, 2017; Xu et al., mAbs, 2015) were introduced to create a chain as a knob molecule (EP203). The C-terminus of another heavy chain of anti-PD-L1 was (G 4 S) 4 It was fused to human IL-15Rα(31-97)[Q13261] by a linker. The following mutations (Wei et al., Oncotarget, 2017; Xu et al., mAbs, 2015) were introduced to generate the chain as the whole molecule (EP204). The light chain EP205 was used to pair with the knob anti-PD-L1 chain and the hole anti-PD-L1 chain, respectively.

[0183] In another case, the entire scFv chain of 2019EP69-F03 was fused to the constant Fc frame sequence of the human heavy chain IgG1 backbone. The C-terminus of one of the scFv-Fc molecules was (G 4S) 4 The C-terminus of another scFv-Fc molecule was fused to human IL-15(50-162) [P40933] via a linker. This molecule carried the S354C, T366W, and K409A mutations as the knob molecule. 4 S) 4 It was fused to human IL-15Rα(31-97)[Q13261] by a linker (EP207). This molecule carried the mutations Y349C, T366S, L368A, F405K, and Y407V as the hole molecule (sequence EP208). The mutations L234A, L235A, and P329G in both the knob and hole molecules were introduced to abolish the complement binding and Fc-γ-dependent antibody-dependent cellular cytotoxicity (ADCC) effects (Lo et al., JBC, 2017).

[0184] In another example, S354C, T366W, and K409A mutations were introduced into the heavy chain of an anti-PD-L1 sequence (e.g., EP205) to generate a knob molecule (e.g., EP362). The protein sequence encoding the engineered IL-2 polypeptide is 4 S) 4 The linker was fused to the C-terminal site of the constant frame sequence of the heavy chain of the anti-PD-L1 sequence (e.g., EP205). The following mutations were introduced to create the chain as a whole molecule (e.g., EP363, EP364, EP365): Y349C, T366S, L368A, F405K, and Y407V. In a specific embodiment, the following mutations were introduced in the Fc: L234A, L235A, and P329G to eliminate complement fixation and Fc-γ-dependent antibody-dependent cellular cytotoxicity (ADCC) effects (Lo et al., JBC, 2017).

[0185] In various formats, the C-terminus of one heavy chain of anti-PD-L1 is modified to (G 4 S) 4 The heavy chain of anti-PD-L1 was fused to IL-15 (50-162) via a linker. The S354C, T366W, and K409A mutations were introduced to create a chain as a knob molecule (EP203). The C-terminus of another heavy chain of anti-PD-L1 was (G 4 S) 4It was fused to human IL-15Rα(31-97) by a linker. Mutations of Y349C, T366S, L368A, F405K, and Y407V were introduced to create the chain as a whole molecule (e.g., EP204). Mutations of L234A, L235A, and P329G in both the knob and hole molecules were introduced to eliminate complement binding and Fc-γ-dependent antibody-dependent cellular cytotoxicity (ADCC) effects (Lo et al., JBC, 2017). The light chain EP205 was used to pair with the knob anti-PD-L1 chain and the hole anti-PD-L1 chain, respectively.

[0186] DNA encoding the entire sequence designed above was then synthesized with codons optimized for mammalian cell expression and subcloned into pCDNA3.4 (Invitrogen).Figure 9 shows a schematic diagram of the formats of selected anti-PD-L1 antibodies.

[0187] Example 3. Generation of IL-2-Fc and antibodies Anti-PD-L1 monoclonal antibodies were transiently expressed in ExpiHEK293-F cells in a Freestyle system (Invitrogen) following standard protocols with a 1:2 ratio of heavy and light chain plasmid DNA. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2 μm PES membrane. Antibodies were purified by MabSelect PrismA Protein A resin (GE Health). Proteins were eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM citric acid (pH 5.0) + 300 mM NaCl. Antibodies were then further purified using a Superdex 200 16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10 EU / mg and was kept in 20 mM histidine pH 6.0 + 150 mM NaCl.

[0188] For the generation of anti-PD-L1 / IL-15 bifunctional fusion antibodies, the respective IgG1 backbone format "knob" and "hole" constructs were transfected into ExpiHEK293-F cells in a Freestyle system (Invitrogen) according to standard protocols. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2μm PES membrane. Antibodies were purified by MabSelect PrismA Protein A resin (GE Health). Proteins were eluted with 100mM Gly (pH2.5)+150mM NaCl and quickly neutralized with 20mM citric acid (pH5.0)+300mM NaCl. Antibodies were then further purified by a Superdex 200 Increase16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10EU / mg and was kept in 1x PBS buffer.

[0189] For the generation of monovalent IL-2-Fc fusion proteins, the "knob" and "hole" constructs in the respective IgG1 backbone formats were transfected in a 1:1 ratio into ExpiHEK293-F cells. Cells were grown for 5 days, and the supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. First, the Fc fusion agonist was purified by MabSelect PrismA Protein A resin (GE Health). The protein was eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM citrate (pH 5.0) + 300 mM NaCl. The agonist protein was then concentrated to 1 mL and further purified by a Superdex 200 16 / 600 gel filtration column. The monomer peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10 EU / mg and was kept in 1x PBS. Purified monovalent IL-2-Fc fusion agonist was run on an SDS gel (4-12% Bis-Tris Bolt gel, containing MES running buffer).

[0190] For the generation of monovalent (Fab format) anti-PD-L1 / IL-2 bifunctional antibodies, each IgG1 backbone format "knob" and "hole" construct was transfected with the corresponding light chain construct into ExpiHEK293-F cells in the Freestyle system (Invitrogen) following standard protocols with a knob:hole:light chain ratio of 1:4:4. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2 μm PES membrane. Antibodies were purified by MabSelect PrismA Protein A resin (GE Health). Proteins were eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM Histidine (pH 5.0) + 150 mM NaCl. Antibodies were then further purified by a Superdex 200 16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10 EU / mg and was maintained in 20 mM histidine, 150 mM NaCl buffer.

[0191] For the generation of monovalent (scFv format) anti-PD-L1 / IL-2 bifunctional antibodies, the respective IgG1 backbone format "knob" and "hole" constructs were transfected into ExpiHEK293-F cells in the Freestyle system (Invitrogen) following standard protocols with a knob:hole ratio of 1:2. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2 μm PES membrane. Antibodies were purified by MabSelect PrismA Protein A resin (GE Health). Proteins were eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM Histidine (pH 5.0) + 150 mM NaCl. Antibodies were then further purified by a Superdex 200 16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10 EU / mg and was maintained in 20 mM histidine, 150 mM NaCl buffer.

[0192] For the generation of bivalent (Fab format) anti-PD-L1 / IL-2 bifunctional antibodies, each IgG1 backbone format "knob" and "hole" construct was transfected with the corresponding light chain construct into ExpiHEK293-F cells in the Freestyle system (Invitrogen) following standard protocols with a knob:hole:light chain ratio of 1:2:2. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2 μm PES membrane. Antibodies were purified by MabSelect PrismA Protein A resin (GE Health). Proteins were eluted with 100 mM Gly (pH 2.5) + 150 mM NaCl and quickly neutralized with 20 mM Histidine (pH 5.0) + 150 mM NaCl. Antibodies were then further purified by a Superdex 200 16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10 EU / mg and was maintained in 20 mM histidine, 150 mM NaCl buffer.

[0193] For generation of anti-PD-L1 / IL-2 fusion antibodies, constructs were transfected into ExpiHEK293-F cells in a Freestyle system (Invitrogen) according to standard protocols. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2μm PES membrane. Antibodies were purified by Ni-Sepharose (GE Healthcare) affinity column according to the manufacturer's protocol. Antibodies were further purified by a Superdex 200 16 / 600 column. Highly homogenous monomer peak fractions of agonists were pooled and concentrated, respectively. Final endotoxin levels were less than 10EU / mg. Proteins were stored in 1xPBS buffer for binding and functional analysis, respectively.

[0194] Example 4. Characterization of anti-PD-L1 antibody IgG Anti-PD-L1 IgG antibodies that bind to PD-L1 in ELISA An ELISA assay was developed to determine the EC50 of anti-PD-L1 IgG antibodies. Briefly, 384-well plates were immobilized with human PD-L1 HIS-tagged recombinant protein at a final concentration of 2μg / mL in 1x PBS in a total volume of 25μL per well. Plates were incubated overnight at 4℃, followed by blocking with 80μL of Superblock per well for 1 hour. Purified PD-L1 IgG was titrated in two-fold serial dilutions starting at 200nM, and 25μL was added to the human PD-L1 immobilized wells and incubated with shaking for 1 hour. PD-L1 binding was detected by adding 25uL of anti-hFc HRP diluted 1:5000 in 1x PBST. Between each step, plates were washed 3 times with 1x PBST on a plate washer. Plates were then developed with 20μL of TMB substrate for 5 minutes and stopped by adding 20μL of 2N sulfuric acid. Plates were read at OD450nm using a Biotek plate reader and then plotted in Prism 8.1 software.Table 17 shows the ELISA binding EC50 for anti-PD-L1 IgG antibodies. [Table 17]

[0195] Binding kinetics of anti-PD-L1 IgG antibody to PD-L1 in SPR The kinetic analysis of anti-PD-L1 IgG was evaluated by SPR technology with Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1μg / mL of anti-hPD-L1-IgG was captured on flow cell 2 in 1×HBSP buffer on a protein A sensor chip at a flow rate of 10μL / min for 60 seconds. Two-fold serial dilutions of hPD-L1-HIS tagged protein were injected on both reference flow cell 1 and anti-PD-L1 IgG capture flow cell 2 at a flow rate of 30μL / min for 150 seconds, followed by washing for 300 seconds. The flow cells were then regenerated with glycine pH 2 at a flow rate of 30μl / min for 60 seconds. Eight concentration points from 100nM-0nM per anti-PD-L1 IgG were assayed in a 96-well plate. The kinetics of anti-PD-L1 IgG binding to PD-L1 protein was analyzed by Biacore T200 evaluation software version 3.0. Specific binding response units were obtained by subtracting the binding to reference flow cell 1 from the antibody capture flow cell 2. Table 18 below shows the binding kinetics of anti-PD-L1 IgG antibodies by SPR. [Table 18]

[0196] Binding of anti-PD-L1 IgG antibody to cell surface PD-L1 in FACS 200nM purified anti-PD-L1 IgG antibody was diluted in complete medium and incubated with PD-L1 / K562 cells and K562 cells in a 96-well plate on ice for 1 hour. The cells were centrifuged at 1200rpm at 4℃ for 5 minutes to remove the primary antibody. The cells were then washed once with 200μL complete medium per well. Samples were detected with anti-hFc Alexa Fluor647 by adding 100μL of diluted secondary antibody and incubating in the dark at 4℃ for 30 minutes. Samples were centrifuged at 1200rpm at 4℃ for 5 minutes and washed twice with 200μL 1xPBS per well. Samples were reconstituted in 200μL 1xPBS and read on an Attune NxT cytometer. Analysis was performed with Attune NxT software, which plotted overlay histograms of anti-PD-L1 antibody binding to both negative and target cell lines.

[0197] Binding competition of anti-PD-L1 IgG antibodies for PD-L1 and PD-1 interactions in HTRF assays, and activation of immune cells in Jurkat cell NFAT reporter assays were the same as described for scFv characterization. Table 19 below shows the EC50 in the Jurkat cell reporter assay, PD-L1 / K562 cell surface binding EC50, and HTRF PD-1 and PD-L1 competition results for IgG antibodies. Figure 10 shows the results of FACS binding of PD-L1 / K562 cell surface. [Table 19]

[0198] Example 5. Characterization of anti-PD-L1 / IL-15 fusion antibodies Anti-PD-L1 / IL-15 binding to PD-L1 and IL-15 receptors in ELISA An ELISA assay was developed to determine the EC50 of anti-PD-L1 antibodies. Briefly, 384-well plates were immobilized with human IL-15Rα-HIS-tagged protein or human PD-L1 HIS-tagged protein at a final concentration of 2μg / mL in 1×PBS in a total volume of 25μL per well. Plates were incubated overnight at 4℃, followed by blocking with 80μL of Superblock per well for 1 hour. Purified anti-PD-L1 / IL-15 was titrated three-fold starting from 200nM. 25μL was added to the wells immobilized with human PD-L1 / IL-15 and incubated with shaking for 1 hour. IL-15 or PD-L1 binding was detected by adding 25μL of anti-human HRP diluted 1:10000 in 1×PBST. Between each step, plates were washed three times with 1×PBST on a plate washer. Plates were then developed with 20μL of TMB substrate for 5 minutes and stopped by adding 20μL of 2N sulfuric acid. Plates were read on a Biotek plate reader at OD450nm and plotted in Prism 8.1 software to calculate EC50. Table 20 shows the ELISA binding EC50 of PD-L1 for the antibodies. [Table 20]

[0199] Binding kinetics of anti-PD-L1 / IL-15 to IL-2Rb and PD-L1 in SPR The kinetic analysis of the bifunctionality of anti-PD-L1-IgG / IL-15 and anti-PD-L1-scFv-Fc / IL-15 towards IL-15Rβ and PD-L1 was assessed by SPR technology using a Biacore T200. It should be noted that the IL-15 receptor shares the same beta subunit with the IL-2 receptor. Therefore, IL-15Rβ is also called IL-2Rβ and CD122. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1μg / mL of anti-hPD-L1-IgG / IL-15 or anti-PD-L1-scFv-Fc / IL-15 was captured on flow cell 2 in 1× HBSP buffer on a Protein A sensor chip for 60 seconds at a flow rate of 10μl / min. Two-fold serially diluted IL-15Rβ-HIS or hPD-L1-HIS tagged proteins were injected onto both the reference flow cell 1 and the anti-PD-L1 / IL-15 bifunctional capture flow cell 2 at a flow rate of 30μl / min for 150 seconds, followed by a 300 second wash. The flow cells were then regenerated with glycine pH 2 for 60 seconds at a flow rate of 30μl / min. Eight concentration points were assayed per anti-PD-L1 IgG, ranging from 100nM-0nM (IL-15Rβ-HIS) or 300nM-0nM (PD-L1-HIS) in a 96-well plate. The kinetics of bifunctional binding of anti-PD-L1 / IL-15 to IL-15Rβ and PD-L1 proteins were analyzed by Biacore T200 Evaluation Software version 3.0. Specific binding response units were obtained by subtracting the binding to the reference flow cell 1 from the antibody-captured flow cell 2. Table 21 shows the results of the binding kinetics of anti-PD-L1 / IL-15 to IL-2Rβ and PD-L1. [Table 21]

[0200] Anti-PD-L1 / IL-15 antibodies in NFAT reporter assays Activation of immune cells in Jurkat cell NFAT reporter assay (Promega, J1250) was the same as described in the characterization of the scFv. The EC50 for the bifunctional antibodies is shown in Table 22. Figure 11 shows the results of the NFAT reporter assay. [Table 22]

[0201] P-STAT5 activity of anti-PD-L1 / IL-15 antibodies Human PBMCs were isolated from leukoreduction system (LRS) cones of two separate donors and seeded at 250,000 cells / well in 90 μL of medium in 96-well plates. Cells were left at 37°C for 1 hour. Cells were stimulated with anti-PD-L1 / IL-15 antibodies at 10x concentration in 10 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The % of cells that were p-STAT5+ was determined and graphed against the dose titration of each antibody or IL-15 (Peprotech). EC50 values ​​for P-STAT5 activation were determined using Prism software. The EC50 for P-STAT5 is shown in Table 23. Figure 12 shows the results of p-STAT5 activation in CD4+ T cells, CD8+ T cells, NK cells, and regulatory T cells. [Table 23]

[0202] Example 6. Characterization of monovalent IL-2-Fc IL-2Rα and IL-2Rβ receptor binding ELISA For monovalent IL-2Fc fusion proteins, recombinant His-tagged human IL-2Rα and IL-2Rβ were added to wells of a 384-well plate in 25 μL of 1×PBS and incubated overnight at 4° C. to coat the plate. The plate was washed three times with 0.05% Tween20 / 1×PBS. The plate was blocked with 100 μL of SuperBlock for 1 hour at room temperature and then washed three times with 0.05% Tween20 / 1×PBS. IL-2 mutants were diluted from 1000 nM to 0 nM in 0.05% Tween20 / 1×PBS and added to the plate for 2 hours at room temperature. The plate was then washed six times with 0.05% Tween20 / 1×PBS. Anti-His tag-HRP was diluted 1:5000 in 0.05% Tween20 / 1×PBS and added to the plate for 1 hour at room temperature. Plates were then washed six times with 0.05% Tween 20 / 1x PBS and TMB was added to develop a blue color. The reaction was stopped with 2N hydrogen sulfide and the light absorbance at 450 nm was read on a BioTek plate reader. Absorbance is plotted versus IL-2 concentration for human IL-2Rα and IL-2Rβ. ELISA binding EC50 values ​​are summarized in Table 24. Figures 13A and 13B show the binding of monovalent IL-2 Fc protein to the IL-2Rα and IL-2Rβ receptors, respectively. [Table 24]

[0203] SPR of binding to IL-2Rα and IL-2Rβ receptors The binding kinetics of monovalent IL-2Rβ Fc fusion protein has been analyzed by SPR technology using Biacore T200. Briefly, anti-hFc antibody was immobilized on flow cells 1 and 2. In each cycle, 1 μg / mL of IL-2 Fc fusion protein was captured on flow cell 2 in 1×HBSP buffer on the anti-hFc immobilized chip at a flow rate of 10 μl / min for 60 s. 100 nM of IL-2Rα-His tagged or IL-2Rβ-His tagged were serially diluted 2-fold and injected on both reference flow cell 1, and IL-2Fc fusion protein was captured on flow cell 2 at a flow rate of 30 μl / min for 150 s, with a 300 s wash after the last injection. The assay was set up with 8 serial dilution concentration points in a 96-well format. Kinetic data was analyzed using Biacore T200 Evaluation Software 3.0. Specific binding response units were obtained by subtracting binding to reference flow cell 1 from target flow cell 2. A summary of the binding kinetics of monovalent IL-2-Fc fusion proteins to the IL-2 receptor is shown in Table 25. [Table 25] *Steady state affinity ND: undetectable binding

[0204] P-STAT5 profile Human PBMCs were isolated from LRS cones of two separate donors and seeded at 250,000 cells / well in 90 μL of media in 96-well plates. Cells were left to rest for 1 hour at 37°C. Cells were stimulated with human IL-2-Fc WT and engineered IL-2-Fc mutants at 10× concentration in 10 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The % of cells that were p-STAT5+ was determined and graphed for each IL-2 dose setting. EC50 values ​​for P-STAT5 activation were determined using Prism software and are shown in Table 26. Figures 14A-D show p-STAT5 profiling curves for CD4+ T cells, CD8+ T cells, NK cells, and regulatory T cells from donor 126. Figures 14E-H show p-STAT5 profiling curves for CD4+ T cells, CD8+ T cells, NK cells, and regulatory T cells from donor 359. [Table 26]

[0205] Example 7. Characterization of IL-2 / anti-PD-L1-scFv bifunctionality Binding activity of IL-2 / anti-PD-L1 scFv antibodies to IL-2 receptor and PD-L1 in ELISA A 384-well plate was immobilized with anti-human Fc at a final concentration of 2μg / mL in 1x PBS in a total volume of 25μL per well. The plate was incubated overnight at 4℃, followed by blocking with 80μL of Superblock per well for 1 hour. A titration of IL-2 / anti-PD-L1 scFv starting at 50nM in a volume of 25μL was added to the wells and incubated with shaking for 1 hour. After washing, 25μL of 30nM biotinylated PD-L1 was added to each well. PD-L1 binding by IL-2 / anti-PD-L1 scFv was detected by adding 25μL of streptavidin-HRP diluted 1:10000 in 1x PBST. Between each step, the plate was washed 3 times with 1x PBST on a plate washer. The plate was then developed with 25μL of TMB substrate for 5 minutes and stopped by adding 25μL of 2N sulfuric acid. Plates were read on a Biotek plate reader at OD 450 nm and binding correlations were plotted by Prism 8.1 software. ELISA binding assays of antibodies against IL-2Rα and IL-2Rβ receptors were performed as described in Example 6. ELISA binding EC50 values ​​are shown in Table 27. Figures 15A, 15B, and 15C show the ELISA binding curves of antibodies against IL-2Rα, IL-2Rβ, and PD-L1, respectively. [Table 27]

[0206] Binding competition of anti-PD-L1 IgG antibodies for PD-L1 and PD-1 interactions in HTRF assays and activation of immune cells in Jurkat cell NFAT reporter assays were performed as described in scFv characterization. The IC50 values ​​for HTRF and EC50 values ​​for NFAT reporter assays are shown in Table 28. Figure 16 shows the results of the NFAT reporter assay. [Table 28]

[0207] Binding to IL-2Rα, IL-2Rβ receptors and PD-L1 in SPR The kinetic analysis of anti-PD-L1 / IL-2 bifunctional agents against IL-2Rα, IL-2Rβ receptors and PD-L1 was evaluated by SPR technology using Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1μg / mL of anti-hPD-L1 / IL-2 was captured on flow cell 2 in 1×HBSP buffer on a Protein A sensor chip at a flow rate of 10μL / min for 60 seconds. Two-fold serially diluted IL-2Rα-HIS or IL-2Rβ-HIS or hPD-L1-HIS tagged proteins were injected on both reference flow cell 1 and anti-PD-L1 / IL-2 bifunctional agent capture flow cell 2 at a flow rate of 30μL / min for 150 seconds, followed by washing for 300 seconds. The flow cells were then regenerated with glycine pH 2 at a flow rate of 30μl / min for 60 seconds. Eight concentration points were assayed per anti-PD-L1 IgG, ranging from 100nM-0nM (IL-2Rα-HIS and IL-2Rβ-HIS) or 300nM-0nM (PD-L1-HIS) in a 96-well plate. The bifunctional binding kinetics of anti-PD-L1 / IL-2 to IL-2Rα, IL-2Rβ and PD-L1 proteins were analyzed by Biacore T200 Evaluation Software version 3.0. Specific binding response units were obtained by subtracting the binding to the reference flow cell 1 from the antibody captured flow cell 2. The binding kinetics of the antibodies to IL-2 and PD-L1, respectively, are shown in Table 29. [Table 29]

[0208] IL-2 / anti-PD-L1 scFv activates P-STAT5 in human PBMCs Human PBMCs were isolated from LRS cones of two separate donors and seeded at 250,000 cells / well in 90 μL of media in 96-well plates. Cells were left to rest for 1 hour at 37°C. Cells were stimulated with human IL-2 / anti-PD-L1 scFv WT and engineered IL-2 / anti-PD-L1 scFv mutants at 10× concentration in 10 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL-2 dose setting. EC50 values ​​for P-STAT5 activation were determined using Prism software and are shown in Table 30. Figures 17A-D show p-STAT5 results for PBMC cells from donor 359. Figures 17E-H show p-STAT5 results for PBMC cells from donor 126. [Table 30]

[0209] Example 8. Characterization of IL-2 / anti-PD-L1-Fab bifunctional and anti-PD-L1 / IL-2 fusions ELISA, HTRF competition and Jurkat reporter assays of IL-2 / anti-PD-L1 Fab binding to PD-L1 A 384-well plate was immobilized with anti-human Fc at a final concentration of 2μg / mL in 1×PBS in a total volume of 25μL per well. The plate was incubated overnight at 4℃, followed by blocking with 80μL of Superblock per well for 1 hour. A titration of IL-2 / anti-PD-L1 FAB starting at 50nM in a volume of 25μL was added to the wells and incubated with shaking for 1 hour. After washing, 25μL of 30nM biotinylated PD-L1 was added to each well. PD-L1 binding by IL-2 / anti-PD-L1 FAB was detected by adding 25μL of streptavidin-HRP diluted 1:10000 in 1×PBST. Between each step, the plate was washed 3 times with 1×PBST on a plate washer. The plate was then developed with 25μL of TMB substrate for 5 minutes and stopped by adding 25μL of 2N sulfuric acid. The ELISA binding assays of antibodies to IL-2Rα and IL-2Rβ receptors were the same as described in Example 6. Plates were read on a Biotek plate reader at OD450nm and binding correlations were plotted with Prism 8.1 software. EC50 values ​​of ELISA binding were shown in Table 31. Figure 18A, Figure 18B, and Figure 18C show the ELISA binding curves of IL-2 Fc / anti-PD-L1 Fab variants to IL-2Rα, IL-2Rβ, and PD-L1, respectively. [Table 31]

[0210] The binding competition of the bifunctional proteins for PD-L1 and PD-1 interaction in HTRF assay and the activation of immune cells in Jurkat cell NFAT reporter assay were the same as those described in the characterization of scFv. The IC50 of HTRF and EC50 of NFAT reporter assay are shown in Table 32. Figure 19 shows the curves of NFAT reporter assay. [Table 32]

[0211] SPR of binding to IL-2Rα, IL-2Rβ receptors and PD-L1 The kinetic analysis of anti-PD-L1 / IL-2 bifunctional agents against IL-2Rα, I2Rβ and PD-L1 was evaluated by SPR technology using Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1μg / mL of anti-hPD-L1 / IL-2 was captured on flow cell 2 in 1×HBSP buffer on a Protein A sensor chip at a flow rate of 10μL / min for 60 seconds. Two-fold serially diluted IL-2Rα-HIS or IL-2Rβ-HIS or hPD-L1-HIS tagged proteins were injected on both reference flow cell 1 and anti-PD-L1 / IL-2 bifunctional agent capture flow cell 2 at a flow rate of 30μL / min for 150 seconds, followed by washing for 300 seconds. The flow cells were then regenerated with glycine pH 2 at a flow rate of 30μl / min for 60 seconds. Eight concentration points were assayed per anti-PD-L1 IgG ranging from 100nM-0nM (IL-2Rα-HIS and IL-2Rβ-HIS) or 300nM-0nM (PD-L1-HIS) in a 96-well plate. The bifunctional binding kinetics of anti-PD-L1 / IL-2 to IL-2Rα, IL-2Rβ and PD-L1 proteins were analyzed by Biacore T200 Evaluation Software version 3.0. Specific binding response units were derived from the binding to the antibody capture flow cell 2 minus the binding to the reference flow cell 1.

[0212] To assess whether the bifunctional antibodies can simultaneously bind IL-2 receptor and PD-L1, the anti-PDL1 / IL2 bifunctional antibodies were captured on flow cell 2 in 1×HBSP buffer on a Protein A sensor chip at a flow rate of 10 μL / min for 60 seconds. hPD-L1-HIS tagged protein was injected on both reference flow cell 1 and anti-PD-L1 / IL-2 bifunctional agent capture flow cell 2 for 60 seconds, and then IL-2Rα-HIS or IL-2Rβ-HIS protein was injected on both reference flow cell 1 and hPD-L1-bound anti-PD-L1 / IL-2 bifunctional agent capture flow cell 2 at a flow rate of 30 μL / min for 90 seconds, followed by a 120 second wash. Figure 20 shows the simultaneous binding of PD-L1 and IL-2Rα and IL-2Rβ to antibodies at a single concentration. The binding kinetics of the anti-PDL1 / IL2 bifunctional agent to IL-2 receptor and PD-L1 were measured as described above, and the data are shown in Table 33. [Table 33]

[0213] P-STAT5 activation by IL-2 / anti-PD-L1 Fab in human PBMCs Human PBMCs were isolated from LRS cones of two separate donors and plated at 250,000 cells / well in 90 μL of media in 96-well plates. Cells were left at 37°C for 1 hour. Cells were stimulated with human IL-2 / anti-PD-L1 Fab WT and engineered IL-2 / anti-PD-L1 Fab mutants at 10× concentration in 10 μL for 20 minutes at 37°C. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL-2 dose setting. EC50 values ​​for P-STAT5 activation were determined using Prism software and are shown in Table 34. Figures 21A-D show p-STAT5 activation curves for donor 857. Figures 21E-H show p-STAT5 activation curves for donor 359. [Table 34]

[0214] IL-2 / anti-PD-L1 Fab activates P-STAT5 in mouse splenocytes Purchased mouse splenocytes were seeded at 250,000 cells / well in 90μL of media in 96-well plates. Cells were left to rest for 1 hour at 37℃. Cells were stimulated with mouse IL-2, human IL-2 / anti-PD-L1 Fab WT and engineered IL-2 / anti-PD-L1 Fab mutants at 10x concentration in 10μL for 20 minutes at 37℃. Stimulated PBMCs were immediately fixed, permeabilized, stained for lineage markers (CD3, NKp46, CD4, CD8, FOXP3) and p-STAT5 and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+NKp46-CD4-CD8+. NK cells were defined as CD3-NKp46+. Regulatory T cells were defined as CD3+NKp46-CD4+CD8-FOXP3+. The % of cells that were p-STAT5+ was determined and graphed for each IL-2 dose setting. The EC50 values ​​of p-STAT5 activation were determined using Prism software and shown in Table 35. Mouse IL2 (PeproTech, mIL-2) was used as a reference. Figures 22A-D show p-STAT5 activation curves in mouse splenocytes. [Table 35]

[0215] Example 9. Tumor growth inhibition of anti-PD-L1 mAb (MB-231 tumor) Seven-week-old female NCG mice humanized with CD34+ umbilical cord blood from a single donor were injected subcutaneously in the dorsal flank with 500,000 MDA-MB-231 cells in 50% Matrigel. Tumors were measured with calipers. The average volume was 100 mm. 3 Once tumor growth rate reached 100%, mice were treated with 200μg of atezolizumab or engineered anti-PD-L1 mAb EP204 / EP206 every 3 days for 20 days. Tumor volumes and mouse body weights were measured 3 times a week. Tumor volumes and body weights were plotted over time relative to vehicle-treated groups. Figure 23 shows the tumor growth inhibition curves.

[0216] For cytokine profiling, mice were sacrificed by exsanguination and the blood was immediately centrifuged to separate plasma. Human IFNγ and TNFα concentrations in plasma were measured using Duoset's human TNFα and IFNγ ELISA kits. Figures 24A and 24B show human TNFα and IFNγ levels in plasma, respectively.

[0217] Example 10. Blood and splenocyte immune profiling of IL-2 / anti-PD-L1-Fab candidates in MC38 tumor-bearing mice Seven-week-old female C57BL / 6 mice were injected subcutaneously in the dorsal flank with 100,000 MC38 cells in 50% Matrigel. Tumors were measured with calipers. The average volume was 100 mm. 3 Once the IL-2 / anti-PD-L1 Fab reached 100% CI, mice were treated with 2μg of human IL-2 / anti-PD-L1 Fab WT (EP290 / EP325 / EP205) or engineered IL-2 / anti-PD-L1 Fab mutants (EP412 / EP325 / EP205; EP415 / EP325 / EP205; EP416 / EP325 / EP205; EP417 / EP325 / EP205; and EP418 / EP325 / EP205) every 4 days for 11 days. Mice were sacrificed on day 11 and peripheral blood was isolated via tail vein bleeding. Red blood cells were lysed and immune cells were analyzed using an Attune flow cytometer. CD4+ T cells were defined as CD45+CD3+NKp46-CD4+CD8-. CD8+ T cells were defined as CD45+CD3+NKp46-CD4-CD8+. NK cells were defined as CD45+CD3-NKp46+. Regulatory T cells NK cells were defined as CD45+CD3+NKp46-CD4+CD8-FOXP3+. The percentage of each subtype within the CD45+ population was plotted for each treatment group. Figures 25A-D show the results of immune cell profiling in blood. Figures 26A-D show the results of immune cell profiling in splenocytes.

[0218] Example 11. Tumor growth inhibition of IL-2 / anti-PD-L1-Fab candidates (B16F10-hPD-L1 tumors in hPD-1 transgenic mice) The following example describes an experiment to evaluate tumor growth inhibition by IL-2 / anti-PD-L1-Fab in an in vivo mouse model. Seven-week-old female hPD-1 transgenic mice were injected subcutaneously into the dorsal flank with 500,000 B16F10-hPD-L1 cells in 50% Matrigel. Tumors were measured with calipers. Tumors ranged in size from 70 to 90 mm in average volume. 3 At that time, mice were treated with 200μg atezolizumab every 3 days or 10μg engineered IL-2 / anti-PD-L1-Fab bifunctional protein (EP415 / EP325 / EP205; EP418 / EP325 / EP205) every 5 days for 20 days. Tumor volumes and mouse body weights were measured 3 times a week and tumor volumes and body weights were plotted over time compared to vehicle-treated groups. Figure 27 shows the tumor growth inhibition curves.

[0219] Example 12. Tumor growth inhibition of IL-2 / anti-PD-L1-Fab candidates (MB-231 tumors) The following example describes an experiment to evaluate tumor growth inhibition by IL-2 / anti-PD-L1-Fab in an in vivo mouse model. 500,000 MDA-MB-231 cells in 50% Matrigel were injected subcutaneously into the dorsal flank of 7-week-old female NCG mice humanized with CD34+ cord blood from a single donor. Tumors were measured with calipers. The mean volume was 80-100 mm. 3 Upon reaching this threshold, mice were treated with 200μg of engineered anti-PD-L1 mAb EP205 / EP206 every 3 days, or 5μg of IL-2 / anti-PD-L1-Fab bifunctional protein (EP290 / EP325 / EP205; EP412 / EP325 / EP205; EP415 / EP325 / EP205; EP418 / EP325 / EP205) every 5 days for 20 days. Tumor volumes and mouse body weights were measured three times a week. Tumor volumes and body weights were plotted over time compared to vehicle-treated groups. Figure 28 shows the tumor growth inhibition curves.

[0220] Example 13. Treatment of human PBMC with IL2 / anti-PD-L1-Fab antibody results in increased PD-1 expression on CD8+ T cells in vitro. Human PBMCs were isolated from LRS cones from a single donor and plated at 100,000 cells / well in 180μL complete medium in a 96-well plate. Cells were placed in a 5% CO2 incubator at 37℃ for 1 hour. Cells were stimulated with 20μL of IL-2 Fc / anti-PD-L1 Fab containing EP415 / EP325 / EP205 or EP290 / EP325 / EP205 conjugates diluted in medium to give a final IL2 concentration in the well between 100nM and 0.0001nM. Cells were placed in the incubator for 5 days. After 5 days, PBMCs were washed with PBS and incubated with a viability dye. After subsequent washing, cells were fixed, permeabilized, and then stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3) and PD-1 and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+ and TRegs were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of CD8+ T cells and TRegs expressing PD-1 was calculated and plotted against IL2 concentration. Figure 29A shows dose-dependent PD-1 positive CD8+ T cells after treatment with EP415 / EP325 / EP205 and EP290 / EP325 / EP205, respectively. Figure 29B shows PD-1 positive Treg cells after treatment with EP415 / EP325 / EP205 and EP290 / EP325 / EP205, respectively.

[0221] Example 14. EP415 / EP325 / EP205 are potent IL2Rβ receptor agonists HEK-Blue IL-2 cells were purchased from InvivoGen and cultured according to the manufacturer's instructions. To quantify surface expression of CD25, CD122, and PD-L1 by these cells, HEK Blue IL2 cells were seeded in 96-well plates at a density of 100,000 cells per well in 200 μL of medium. Cells were allowed to recover for at least 1 hour in an incubator. Cells were then washed and stained with fluorescent antibodies against CD25, CD122, or PD-L1. Concurrently, Quantum Simply Cellular microsphere standards (Bangs Laboratories, Inc.) were also stained with the same antibodies. After washing again, cells and microspheres were visualized on an Attune flow cytometer. The median fluorescence intensity (MFI) of each microsphere in the appropriate fluorescent channel was calculated, and a standard curve of MFI versus antibody binding content (ABC) was generated. The MFI from cells for each antibody was compared to the appropriate standard, and the median receptor count was calculated and plotted.

[0222] HEK-Blue IL-2 cells were seeded in 96-well plates at a density of 100,000 cells per well in 100 μL of medium. Cells were allowed to recover for at least 1 hour in an incubator. Plates were centrifuged, medium removed, and cells were resuspended in fresh medium containing a 1:100 dilution of anti-CD25 antibody or medium alone. Cells were incubated at 4°C for 30 minutes with shaking. After incubation, cells were centrifuged, medium discarded, and cells were resuspended in fresh medium containing titrated EP415 / EP325 / EP205 or EP290 / EP325 / EP205. Cells were then incubated at 4°C for 1 hour with shaking. Medium was then removed and replaced with medium containing anti-human Fc antibody conjugated with Alex Fluor647. Cells were incubated at 4°C for 30 minutes with shaking. Cells were washed and the presence of bound EP415 / EP325 / EP205 or EP290 / EP325 / EP205 was determined using an Attune flow cytometer. The percentage of HEK Blue IL2 cells bound by EP415 / EP325 / EP205 or EP290 / EP325 / EP205 was plotted against IL2 concentration. Figure 30A shows the expression levels of CD25, CD122, and PD-L1 on HEK Blue IL-2 cells. Figure 30B shows the FACS binding activity of EP415 / EP325 / EP205 and EP290 / EP325 / EP205 to HEK-Blue IL-2 cells with or without anti-CD25 antibody interference. CD25 antibodies reduced the binding of EP290 / EP325 / EP205 to HEK-Blue IL-2 cells, whereas they had no effect on the binding of EP415 / EP325 / EP205. This data suggests that EP415 / 325 / 205 prefers IL2Rβγ receptor binding, as anti-CD25 antibodies do not appear to interfere with EP415 / 325 / 205 interaction with the cell line. In contrast, EP290 / EP325 / EP205 exhibit high levels of binding to IL2Rαβγ due to the presence of the IL2-WT sequence, and binding is reduced due to interference from anti-CD25 antibodies.

[0223] Example 15. EP415 / EP325 / EP205 are tumor-localizing in vivo. C57BL / 6N and B6N albino mice were inoculated bilaterally subcutaneously with 250,000 MC38 cells (left flank) and 1,000,000 MC38-hPD-L1 cells (right flank). 3 Once tumor tissue had reached 100 nmol / kg, mice were administered 1 mg / kg of Alexa Fluor 750 fluorescently labeled EP415 / EP325 / EP205 by intraperitoneal injection (IP). The presence of fluorescently labeled EP415 / EP325 / EP205 at the tumor site was determined using an IVIS Lumina III LT (Perkin Elmer) system. During imaging, mice were kept on 3% isoflurane via a nose cone attached to an internal anesthesia manifold and placed on the heated (37°C) shelf of the imaging chamber for epi-illumination image acquisition. After scanning, mice were removed and returned to their respective cages for recovery. Mice were imaged at the following time points after administration: 15 min, 1 h, 2 h, 4 h, 6 h, and 24 h. The total luminescence of each tumor was calculated and plotted against time. Figure 31A shows representative imaging results of a tumor-bearing mouse 24 h after EP415 / EP325 / EP205 injection. Figure 31B shows the time-dependent enrichment of EP415 / EP325 / EP205 at the tumor sites of MC-38-hPD-L1 and MC-38, respectively. These results indicate that EP415 / EP325 / EP205 preferentially localize to MC-38-hPD-L1, but not MC-38, tumor sites.

[0224] Example 16. EP415 / EP325 / EP205 effective in MDA-MB-231 cell hot tumor model 28-29 week old female NCG mice humanized with CD34+ cord blood from 2-3 donors were injected subcutaneously in the dorsal flank with 500,000 MDA-MB-231 cells in 50% Matrigel. Tumors were measured with calipers. The mean tumor volume was 100 mm. 3Once tumor volume reached 100 mg / kg, mice were treated intraperitoneally with anti-PD-L1 EP205 / EP206 (200μg, 1x3 days), EP290 / EP325 / EP205 IL2 / anti-PD-L1 (5ug, 1x5 days), or EP415 / EP325 / EP205 IL2 / anti-PD-L1 (5ug, 1x5 days). Tumor volumes were measured 2-3 times weekly and plotted over time relative to vehicle control groups.

[0225] Mice were sacrificed 24 days after the start of treatment and tumors were harvested. Tumors were dissociated into single cell suspensions and incubated with viability dyes. After washing, tumor cells were fixed, permeabilized, and stained for lineage markers (CD3, CD56, CD4, CD8, FOXP3). The percentages of CD4+ T cells (CD3+CD56-CD4+CD8-FOXP3-), CD8+ T cells (CD3+CD56-CD4-CD8+), NK cells (CD3-CD56+), and TRegs (CD3+CD56-CD4+CD8-FOXP3+)) were calculated as a percentage of total viable cells for EP415 / EP325 / EP205 and EP290 / EP325 / EP205 treated groups and compared to vehicle treated mice. Figure 32A shows the tumor growth inhibition curves of EP415 / EP325 / EP205 against anti-PD1 / PD-L1 responsive cancer cells MDA-MB-231 in humanized NCG mice. EP415 / EP325 / EP205 show superior tumor growth inhibition activity to both anti-PD-L1 antibody and EP290 / EP325 / EP205. Accordingly, Figures 32B-E show the levels of immune cells at the tumor site. The activation of CD8 T cells and NK cells at the tumor site correlates with the observed efficacy.

[0226] Example 17. EP415 / EP325 / EP205 are effective in the COLO205 cell cold tumor model 5,000,000 COLO205 cells in 50% Matrigel were injected subcutaneously into the dorsal flank of 28-29 week old female NCG mice humanized with CD34+ cord blood from 2-3 donors. Tumors were measured with calipers. The average tumor volume was 100 mm. 3Once tumor volume reached 100 μg, mice were treated intraperitoneally with anti-PD-L1 (200 μg, EP205 / EP206, once every 3 days), EP415 / EP325 / EP205 (5 ug, once every 5 days), or EP415 / EP325 / EP205 (50 ug, single dose). Tumor volumes were measured 2-3 times per week and plotted over time relative to the vehicle control group. Mouse body weights were measured daily and plotted against time. Figure 33A shows the tumor growth inhibition curves of EP415 / EP325 / EP205 against cancer cells COLO205 in humanized mice. Figure 33B shows the corresponding body weight changes of the mice. The data in Figures 33A-D confirm that EP415 / EP325 / EP205 is more effective at suppressing tumor growth than anti-PD-L1 antibodies.

[0227] Example 18. EP415 / EP325 / EP205 is effective against anti-PD1 resistant NCI-H1975 cell tumors 20-24 week old female NOG mice humanized with CD34+ umbilical cord blood from 2-3 donors were inoculated with 8 × 10 cells in 100 μL of serum-free medium mixed with Matrigel (v / v 1:1) onto the dorsal right flank. 6 NCI-H1975 cells were injected subcutaneously. Tumors were measured with a caliper. The average tumor volume was 100 mm 3 Once tumor volume reached 100 mg / kg, mice were treated intraperitoneally with either EP415 / EP325 / EP205 (10 μg, once on day 10), EP415 / EP325 / EP205 (5 ug, once on day 6), or pembrolizumab (200 ug, once on day 4). Tumor volumes were measured twice weekly and plotted over time relative to vehicle control groups. Mouse body weights were measured 3–5 times weekly and plotted against time.

[0228] Mice were sacrificed 20 days after the start of treatment and tumors were harvested. Tumors were dissociated into single cell suspensions and incubated with viability dyes. After washing, tumor cells were fixed, permeabilized, and stained for lineage markers (mCD45, hCD45, hCD3, hCD56, hCD4, hCD8, hFOXP3). The number of live hCD45 cells (mCD45-hCD45+) and live hCD8+ T cells (mCD45-hCD45+hCD3+hCD56-hCD4-hCD8+) per mm3 of tumor was calculated for each treatment group and compared to the vehicle control group. The ratios of intratumoral hCD8+ T cells and NK cells (mCD45-hCD45+hCD3-hCD56+) and hTRegs (mCD45-hCD45+hCD3+hCD56-hCD8-hCD4+hFOXP3+) were also calculated for each treatment group and plotted relative to the vehicle control group. Figure 34A shows the tumor growth inhibition curves of EP415 / EP325 / EP205 against cancer cells H1975 in humanized mice. Figure 34B shows the corresponding body weight changes of the mice. Figures 34C-F show the profiling results of tumor-infiltrating immune cells. EP415 / EP325 / EP205 retain tumor growth inhibitory activity against H1975 cells resistant to the anti-PD1 antibody pembrolizumab.

[0229] Example 19. Cynomolgus monkey PK / PD study of Ep415 / Ep325 / Ep205 Non-naive cynomolgus monkeys aged 2-5 years, weighing in the range 3.0-5.0 kg, received a single IV bolus dose of either 0.1 mg / kg or 0.5 mg / kg EP415 / EP325 / EP205. Blood was collected at selected time points after dosing. Concentrations of constructs in plasma were quantified by ELISA assay and expressed in ng / mL. Concentrations of EP415 / EP325 / EP205 detected in monkey plasma were plotted against time. Plasma concentrations at each time point from each animal were used for pharmacokinetic (PK) calculations. Area under the curve (AUC) was estimated using the trapezoidal method. Other parameters were estimated using established equations.

[0230] For pharmacodynamic (PD) analysis, blood was isolated daily (pre-dose on days 0 and 7), stained for immune cell lineage markers (CD3, CD4, CD8, CD16, CD45, FOXP3, CD20) and quantified by flow cytometry. The following immune populations were quantified: CD4+FOXP3- T cells (CD45+CD3+CD16-CD4+CD8-FOXP3-), CD4+FOXP3+ regulatory T cells (CD45+CD3+CD16-CD4+CD8-Foxp3+), CD8+ T cells (CD45+CD3+CD16-CD4-CD8+), B cells (CD45+CD3-CD16-CD20+), and natural killer (NK) cells (CD45+CD3-CD16+CD20-). Raw numbers of each of these populations were normalized to the total number of cellular events observed and plotted against time. Figure 35A shows the concentrations of EP415 / EP325 / EP205 detected in monkey plasma by ELISA and plotted against time. Figures 35B-C show the percentage of immune cell populations in monkey blood after administration of EP415 / EP325 / EP205. An increase in the CD8+ T cell population was observed after each administration, while the levels of CD4+FOXP3+ Tregs remained at baseline.

[0231] Incorporation by Reference The entire disclosures of each of the patent documents and scientific articles referred to herein are incorporated by reference for all purposes.

[0232] Equivalent The antigen-binding site described in this application may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are not intended to limit the antigen-binding site described herein, but should be considered in all respects as illustrative. The scope of this application is therefore indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0233] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 140,749, filed January 22, 2021, which is incorporated herein by reference in its entirety.

Claims

1. An antigen-binding site that binds to PD-L1, (a) a heavy chain variable region (VH) comprising the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences of SEQ ID NOs: 11, 12, and 89, respectively; and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (b) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3, 4, and 5, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 8, respectively; (c) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 13, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 14, 15, and 16, respectively; (d) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 19, 20, and 21, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 22, 23, and 24, respectively; (e) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 19, 20, and 27, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30, respectively; (f) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 33, 34, and 35, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 36, 37, and 38, respectively; (g) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3, 41, and 5, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 42, and 43, respectively; (h) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 46, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (i) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 52, 53, and 54, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 55, 56, and 57, respectively; (j) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 46, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 60, and 49, respectively; (k) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 63, 64, and 65, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 66, 67, and 68, respectively; (l) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 71, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (m) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 74, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (n) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 77, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (o) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 80, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; (p) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 83, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively; or (q) a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 11, 12, and 86, respectively; and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 48, and 49, respectively. An antigen-binding site comprising:

2. (a) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:87 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:88; (b) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:2; (c) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:9 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:10; (d) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 18; (e) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:25 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:26; (f) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:31, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:32; (g) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 39, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 40; (h) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45; (i) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:50, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:51; (j) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:58, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:59; (k) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:61, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:62; (l) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:69, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:70; (m) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:72, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:73; (n) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:75, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:76; (o) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:78 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:79; (p) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 81, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 82; (q) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 84, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85; (r) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 87 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 88; (s) the VH comprises or consists of the amino acid sequence of SEQ ID NO:1 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:2; (t) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 9 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 10; (u) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 17 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 18; (v) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 25 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 26; (w) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 31 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 32; (x) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 39 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 40; (y) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 45; (z) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 50 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 51; (aa) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:58 and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:59; (bb) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 61 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 62; (cc) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 69 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 70; (dd) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 72 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 73; (ee) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 75 and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 76; (ff) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 78 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 79; (gg) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 82; or (hh) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 85; 2. An antigen-binding site according to claim 1.

3. 3. The antigen-binding site according to claim 1 or 2, wherein the antigen-binding site is a single chain fragment variable fragment (scFv) or an antigen-binding fragment (Fab). (a) the antigen-binding site has a K of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by surface plasmon resonance (SPR). Ｄ binds to human PD-L1 with (b) the antigen-binding site binds to human PD-L1 with an EC 50 of less than about 160 nM, less than about 10 nM, less than about 1.5 nM, less than about 1.2 nM, less than about 1.0 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, or less than about 0.4 nM, as measured by enzyme-linked immunosorbent assay (ELISA); (c) the antigen-binding site binds to cells expressing human PD-L1 with an EC 50 of less than about 40 nM, less than about 10 nM, less than about 8 nM, less than about 6 nM, less than about 4 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.3 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by fluorescence-activated cell sorting (FACS); and / or (d) the antigen-binding site competes with PD-1 for binding to PD-L1 or inhibits binding of PD-L1 to PD-1; An antigen-binding site according to any one of claims 1 to 3.

5. A protein comprising an antigen-binding site according to any one of claims 1 to 4, optionally comprising one or more antibody heavy chain constant regions, and further optionally comprising: (a) the antibody heavy chain constant region is a human IgG heavy chain constant region; (b) the antibody heavy chain constant region is a human IgG1 heavy chain constant region; (c) the antibody heavy chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:

90. (d) the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V, numbered according to the EU numbering system, relative to SEQ ID NO: 90; (e) the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 91 or SEQ ID NO: 92; and / or (f) the protein comprises a first antibody heavy chain constant region comprising one or more mutations selected from S354C, T366W and K409A, numbered according to the EU numbering system, relative to SEQ ID NO: 90, and a second antibody heavy chain constant region comprising one or more mutations selected from Y349C, T366S, L368A, F405K and Y407V, relative to SEQ ID NO: 90; A protein according to any one of claims 1 to 4.

6. wherein the protein is an antibody, and optionally the antibody comprises: (a) a heavy chain (HC) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 148, and a light chain (LC) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 149; (b) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 150 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 151; (c) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 152 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 153; (d) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 154 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 155; (e) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 156 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 157; (f) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 159 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 160; (g) a HC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 148 and a LC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 149; (h) a HC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 150 and a LC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 151; (i) a HC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 152 and a LC comprising, or consisting of, the amino acid sequence of SEQ ID NO: 153; (j) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 154 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 155; (k) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 156 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 157; or (l) a HC comprising or consisting of the amino acid sequence of SEQ ID NO: 159 and a LC comprising or consisting of the amino acid sequence of SEQ ID NO: 160; The protein described in claim 5. (a) the antibody has a K of less than about 10 nM, less than about 6 nM, less than about 3 nM, less than about 1 nM, or less than about 0.4 nM when measured by SPR. Ｄ binds to human PD-L1 with (b) the antibody binds to human PD-L1 with an EC 50 of less than about 0.2 nM as measured by ELISA; (c) the antibody binds to cells expressing human PD-L1 with an EC 50 of less than about 7 nM as measured by FACS; (d) the antibody inhibits tumor growth in vivo, and / or (e) the antibody induces in vivo secretion of IFNγ and TNFα at levels comparable to or even enhanced relative to atezolizumab; The protein described in claim 6.

8. (a)(i) a heavy chain variable domain (VH) comprising: a complementarity determining region 1 (CDR1) of SEQ ID NO: 11, 3, 19, 33, 52, or 63; a complementarity determining region 2 (CDR2) of SEQ ID NO: 12, 4, 20, 34, 41, 53, or 64; and a complementarity determining region 3 (CDR3) of SEQ ID NO: 89, 5, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, or 86; and (ii) a light chain variable domain (VL) comprising: a CDR1 sequence of SEQ ID NO: 47, 6, 14, 22, 28, 36, 55, or 66; a CDR2 sequence of SEQ ID NO: 48, 7, 15, 23, 29, 37, 42, 56, 60, or 67; and a CDR3 sequence of SEQ ID NO: 49, 8, 16, 24, 30, 38, 43, 57, or 68. an antigen-binding site that binds to PD-L1, comprising: (b) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Rα) polypeptide, a wild-type interleukin-2 (IL-2) polypeptide, or an engineered IL-2 polypeptide, or a functional fragment or variant thereof. A bifunctional protein comprising: Optionally, the bifunctional protein further comprises one or more antibody heavy chain constant regions.

9. The antigen-binding site that binds to PD-L1 is (a) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:87 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:88; (b) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:2; (c) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:9 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:10; (d) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 18; (e) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:25 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:26; (f) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:31, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:32; (g) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 39, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 40; (h) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45; (i) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:50, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:51; (j) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:58, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:59; (k) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:61, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:62; (l) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:69, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:70; (m) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:72, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:73; (n) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:75, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:76; (o) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:78 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:79; (p) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 81, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 82; (q) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 84, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85; (r) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 87 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 88; (s) the VH comprises or consists of the amino acid sequence of SEQ ID NO:1 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:2; (t) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 9 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 10; (u) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 17 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 18; (v) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 25 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 26; (w) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 31 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 32; (x) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 39 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 40; (y) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 45; (z) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 50 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 51; (aa) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:58 and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:59; (bb) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 61 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 62; (cc) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 69 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 70; (dd) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 72 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 73; (ee) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 75 and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 76; (ff) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 78 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 79; (gg) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 82; or (hh) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 85; The bifunctional protein of claim 8.

10. (a) a first subunit comprising an antigen-binding site that binds to PD-L1, an interleukin-15 (IL-15) polypeptide, or a functional fragment or variant thereof, and a first antibody heavy chain constant region; and (b) a second subunit comprising the antigen-binding site that binds to PD-L1, an interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment or variant thereof, and a second antibody heavy chain constant region. The bifunctional protein of claim 8 or 9.

11. (a) the IL-15 polypeptide comprises amino acids 50 to 162 of SEQ ID NO: 93, or a functional fragment or variant thereof; and / or (b) the IL-15Rα polypeptide comprises amino acids 31 to 97 of SEQ ID NO: 94, or a functional fragment or variant thereof. The bifunctional protein according to any one of claims 8 to 10, comprising:

12. (a) the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:186, and the second subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:187, and optionally, the first subunit further comprises an amino acid sequence at least 90% identical to SEQ ID NO:181, optionally, the first subunit comprises the amino acid sequence of SEQ ID NO:186 and the second subunit comprises the amino acid sequence of SEQ ID NO:187, and optionally, the first subunit further comprises the amino acid sequence of SEQ ID NO:181, or (b) the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:188 and the second subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:189; optionally, the first subunit comprises or consists of the amino acid sequence of SEQ ID NO:188 and the second subunit comprises or consists of the amino acid sequence of SEQ ID NO:189; The bifunctional protein according to any one of claims 8 to 11.

13. the engineered IL-2 polypeptide comprising: (a) Compared to wild-type IL-2 A mutation at position K35 selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C; A mutation at position R38 selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, or R38N; A mutation at position F42 selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H; and A mutation at position Y45 selected from Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F. and / or an IL-2 receptor alpha (IL-2Rα) binding domain 1 comprising one or more mutations at one or more positions selected from (b) X １ -X ２ -X ３ -D-X ４ -X5-X ６ -N-X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ (Allocation number 95) Contains IL-2 receptor β (IL-2Rβ) binding domain 2モチーフ [In the formula, X １ is selected from C, T, G, W, I, S, E, and K; X ２ is selected from Y, P, V, W, L, A, and G; X ３ is selected from S, T, Q, G, M, E, R, and K; X ４ is selected from A, V, S, and T; X ５ is selected from I, L, T, and V; X ６ is selected from S, T, E, D, and R; X ７ is selected from I, A, M, and V; X ８ is selected from S, T, N, Q, I, G, E, K, and R; X ９ is selected from V, L, and I; X １０ is selected from N, T, I, and L; X １１ is selected from V, A, and I; X １２ is selected from Q, L, G, K, and R; and X １３ is selected from A, D, and E; Including, The bifunctional protein according to claim 8 or 9.

14. 4. The engineered IL-2 polypeptide comprising: (a) an IL-2Rα binding region 1 comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 124-147, optionally wherein said IL-2Rα binding region 1 comprises an amino acid sequence selected from SEQ ID NOs: 124-147; and / or (b) an IL-2Rβ binding region 2 motif comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 96-123, optionally wherein the IL-2Rβ binding region 2 motif comprises an amino acid sequence selected from SEQ ID NOs: 96-123; The bifunctional protein of claim 13 .

15. The bifunctional protein according to claim 8 or 9, comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 160-164 or an amino acid sequence selected from SEQ ID NOs: 160-164.

16. (a) a first subunit comprising the antigen-binding site that binds to PD-L1 and a first antibody heavy chain constant region; and (b) a second subunit comprising a wild-type interleukin-2 (IL-2) or an engineered IL-2 polypeptide or a functional fragment or variant thereof and a second antibody heavy chain constant region. and optionally (i) the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO: 176 and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 165-175, and further optionally, the first subunit comprises the amino acid sequence of SEQ ID NO: 176 and the second subunit comprises an amino acid sequence selected from SEQ ID NOs: 165-175; or (ii) the first subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 177-180, and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 165-175, and optionally, the first subunit further comprises an amino acid sequence at least 90% identical to SEQ ID NO: 181, and further optionally (A) the first subunit comprises an amino acid sequence selected from SEQ ID NOs: 177-180, and the second subunit comprises an amino acid sequence selected from SEQ ID NOs: 165-175, and optionally the first subunit further comprises the amino acid sequence of SEQ ID NO: 181; or (B) the first subunit comprises the amino acid sequence of SEQ ID NO: 177 and SEQ ID NO: 181, and the second subunit comprises the amino acid sequence of SEQ ID NO: 171; 15. A bifunctional protein according to any one of claims 8, 9, 13 and 14.

17. (a) a first subunit comprising the antigen-binding site that binds to PD-L1 and a first antibody heavy chain constant region; and (b) a second subunit comprising an antigen-binding site that binds to PD-L1, wild-type interleukin-2 (IL-2) or an engineered IL-2 polypeptide, or a functional fragment or variant thereof, and a second antibody heavy chain constant region. Including, Optionally, the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:182, and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs:183-185; optionally, the first and second subunits each independently further comprise an amino acid sequence at least 90% identical to SEQ ID NO:181, further optionally, the first subunit comprises the amino acid sequence of SEQ ID NO:182, and the second subunit comprises an amino acid sequence selected from SEQ ID NOs:183-185, and optionally, the first and second subunits each independently further comprise the amino acid sequence of SEQ ID NO:

181.

15. A bifunctional protein according to any one of claims 8, 9, 13 and 14.

18. The method of claim 1, further comprising administering to said subject an antibody or a method for treating said cancer, comprising administering to said subject a cancer cell line ... (a) the antibody heavy chain constant region is a human IgG1 heavy chain constant region; (b) the antibody heavy chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:

90. (c) the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V, numbered according to the EU numbering system, relative to SEQ ID NO: 90; (d) one of the first and second antibody heavy chain constant regions comprises one or more mutations selected from S354C, T366W and K409A, numbered according to the EU numbering system, relative to SEQ ID NO: 90; and the other antibody heavy chain constant region comprises one or more mutations selected from Y349C, T366S, L368A, F405K and Y407V, relative to SEQ ID NO:

90. (e) one of the first and second antibody heavy chain constant regions comprises the mutations S354C, T366W and K409A numbered according to the EU numbering system compared to SEQ ID NO: 90; and the other antibody heavy chain constant region comprises the mutations Y349C, T366S, L368A, F405K and Y407V compared to SEQ ID NO: 90; and / or (f) one of the first and second antibody heavy chain constant regions comprises the amino acid sequence of SEQ ID NO: 91, and the other antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 92; The bifunctional protein according to any one of claims 8 to 17. (a) the bifunctional protein has a K of less than about 1 nM as measured by SPR. Ｄ or a K that is comparable or even lower than a constructed antigen-binding site that binds to PD-L1 Ｄ binds to human PD-L1 with (b) the bifunctional protein binds to PD-L1 with a K D of less than about 0.5 nM or less than about 0.1 nM as measured by SPR; (c) the bifunctional protein binds to PD-L1 with an EC 50 of less than about 0.4 nM as measured by ELISA. (d) the bifunctional protein competes with PD-1 for binding to PD-L1 or inhibits binding of PD-L1 to PD-1; and / or (e) the bifunctional protein inhibits binding of PD-L1 to PD-1; The bifunctional protein according to any one of claims 8 to 18.

20. (a) The bifunctional protein has a K of less than about 100 nM, less than about 80 nM, less than about 50 nM, less than about 10 nM, or less than about 5 nM as measured by SPR. Ｄ binds to IL-2Rβ at (b) the bifunctional protein binds to IL-2Rβ with a K D of less than about 65 nM or less than about 50 nM as measured by SPR; (c) the bifunctional protein binds to IL-2Rα with a K D of less than about 40 nM as measured by SPR; (d) the bifunctional protein binds to IL-2Rα with an EC 50 of less than about 1 nM as measured by ELISA; and / or (e) the bifunctional protein binds to IL-2Rβ with an EC 50 of less than about 5 nM, less than about 2.5 nM, less than about 1.5 nM, less than about 1 nM, or less than about 0.6 nM as measured by ELISA; The bifunctional protein according to any one of claims 8, 9 and 13 to 19. (a) the bifunctional protein induces expression of P-STAT5 in immune cells; (b) the bifunctional protein induces expression of p-STAT5 in an immune cell with an EC 50 of less than about 1 nM, less than about 0.6 nM, or less than about 0.1 nM when measured in isolated human peripheral blood mononuclear cells (PBMCs), wherein the immune cell is a T cell, an NK cell, or a Treg; (c) the bifunctional protein induces expression of p-STAT5 in an immune cell with an EC 50 of less than about 0.5 nM, or less than about 0.1 nM, as measured in isolated PBMCs, wherein the immune cell is a T cell, an NK cell, or a Treg. (d) the bifunctional protein induces expression of p-STAT5 in immune cells as measured in mouse splenocytes, wherein the immune cells are T cells, NK cells, or Tregs. (e) the bifunctional protein inhibits tumor growth in vivo; and / or (f) the bifunctional protein induces proliferation of immune cells in vivo, optionally, the immune cells are T cells or NK cells, and further optionally, the T cells are CD8+ T cells. The bifunctional protein according to any one of claims 8 to 20.

22. An antibody comprising a VH and a VL, wherein the VH comprises the polypeptide sequence of SEQ ID NO: 87, and the VL comprises the polypeptide sequence of SEQ ID NO: 88, and optionally, the antibody comprises the polypeptide sequences of SEQ ID NO: 177 and SEQ ID NO:

181.

23. An isolated polynucleotide encoding the antigen-binding site of any one of claims 1 to 4, the protein of any one of claims 5 to 7, the bifunctional protein of any one of claims 8 to 21, or the antibody of claim 22.

24. An expression vector comprising the polynucleotide of claim 23.

25. 25. A modified cell comprising the isolated polynucleotide of claim 23 or the expression vector of claim 24.

26. A pharmaceutical composition comprising an antigen-binding site according to any one of claims 1 to 4, a protein according to any one of claims 5 to 7, a bifunctional protein according to any one of claims 8 to 21, or an antibody according to claim 22, and a pharma- ceutically acceptable carrier.

27. 23. A pharmaceutical composition for modulating an immune response comprising an antigen-binding site according to any one of claims 1 to 4, a protein according to any one of claims 5 to 7, a bifunctional protein according to any one of claims 8 to 21, or an antibody according to claim 22, optionally wherein said modulation of the immune response comprises at least one of enhanced T cell activity, or enhanced NK cell activity, and further optionally wherein the treated subject is treated with an additional therapeutic agent.

28. 23. A pharmaceutical composition for the treatment of a disease comprising an antigen-binding site according to any one of claims 1 to 4, a protein according to any one of claims 5 to 7, a bifunctional protein according to any one of claims 8 to 21, or an antibody according to claim 22, optionally wherein said disease is cancer, further optionally wherein said cancer comprises breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, or melanoma, further optionally wherein the subject to be treated is treated with an additional therapeutic agent.

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