Anti-PD-L1 monoclonal antibodies

Abstract

To provide antibodies that specifically bind to PD-L1, DNA encoding the antibodies, corresponding expression vectors, and methods of producing the antibodies and methods of treatment using the antibodies.SOLUTION: The present invention provides monoclonal antibodies that specifically bind to PD-L1 and antigen binding fragments thereof, comprising a heavy chain variable domain comprising CDR1, CDR2, and CDR3 comprising specific amino acid sequences respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 comprising specific amino acid sequences respectively.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to an antibody or an antigen-binding fragment thereof and its use. More specifically, the present invention relates to a monoclonal antibody that specifically binds to PD-L1 (CD274, B7-H1, programmed cell death ligand-1). The present invention also relates to a nucleic acid encoding the antibody or an antigen-binding fragment thereof, an expression vector, a method for producing the antibody, and the use of the antibody to upregulate cell-mediated immune responses and enhance T cell function to treat T cell dysfunction-related disorders, such as tumor immunity and to treat cancer. [Background technology]

[0002] Lymphocyte Development and Activation The two major types of lymphocytes in humans are T (thymocytes) and B (bone marrow derived). These cells originate from hematopoietic stem cells that are programmed into lymphoid developmental pathways in the bone marrow and fetal liver. The progeny of these stem cells follow divergent pathways to mature into either B or T lymphocytes. Human B lymphocyte development occurs entirely within the bone marrow. T cells, on the other hand, develop from immature precursor cells that leave the bone marrow and migrate through the bloodstream to the thymus where they proliferate and differentiate into mature T lymphocytes.

[0003] Mature lymphocytes arising from the thymus or bone marrow are in a quiescent or "resting" state, i.e., they are mitotically inactive. Upon dispersal in the bloodstream, these "virgin" or "naive" lymphocytes migrate to various secondary or peripheral lymphoid organs, such as the spleen, lymph nodes, or tonsils. Most virgin lymphocytes are characterized by an inherently short life span and die within a few days after leaving the bone marrow or thymus. However, if these cells receive a signal indicating the presence of an antigen, they can become activated and undergo successive cycles of cell division. Some of the resulting progeny then return to a quiescent state and become memory lymphocytes, essentially B and T cells that are primed for the next encounter with an irritating allergen. Other progeny of activated naive lymphocytes are effector cells that only survive for a few days but carry out specific defense activities.

[0004] Lymphocyte activation is an ordered series of events through which resting lymphocytes, once stimulated, undergo division and produce progeny, some of which become effector cells. The overall response includes both the induction of cell proliferation (mitogenesis) and the expression of immunological functions. Lymphocytes are activated when specific ligands bind to receptors on the surface of the lymphocyte. The ligands are different for T cells and B cells, but the intracellular physiological mechanisms that result are similar.

[0005] Some foreign antigens, especially large polymeric antigens that cross-link surface immunoglobulins on B cells or other glycoproteins on T cells, can themselves induce lymphocyte activation. However, most antigens are not polymeric, and even direct binding to large numbers of B cells fails to produce activation. These more common antigens activate B cells when costimulated with nearby activated helper T lymphocytes. Such stimulation can come from lymphokines secreted by T cells, but is most efficiently delivered by direct contact of B cells with T cell surface proteins that interact with specific B cell surface receptors to produce secondary signals.

[0006] T cells T lymphocytes do not express immunoglobulins, but instead detect the presence of foreign substances through surface proteins called T cell receptors (TCRs). These receptors recognize antigens by direct contact or by affecting the activity of other immune cells. Along with macrophages, T cells are the main cell type involved in cell-mediated immunity.

[0007] Unlike B cells, T cells can only detect foreign substances under certain conditions. In particular, T lymphocytes recognize foreign proteins only when they are first cleaved into small peptides and then displayed on the surface of a second host cell, called an antigen-presenting cell (APC). Many types of host cells can present antigens under some conditions, but certain types are more specifically adapted for this and are particularly important in controlling T cell activity, including macrophages and other B cells. Antigen presentation depends in part on certain proteins, called major histocompatibility complex (MHC) proteins, on the surface of the presenting cell. Thus, to stimulate cell-mediated immunity, foreign peptides must be presented to T cells in combination with MHC peptides, and this combination must be recognized by the T cell receptor.

[0008] There are two important T cell subsets: cytotoxic T lymphocytes (Tc cells or CTLs) and T helper (T H ) cells, which can be roughly identified based on cell surface expression of the markers CD8 and CD4. Tc cells are important in viral defense and are capable of directly killing viruses by recognizing specific cell surface expressed viral peptides. HThey promote the proliferation, maturation and immunological functions of other cell types, for example by secreting lymphokines to control the activity of B cells, macrophages and cytotoxic T cells. Both naive and memory T lymphocytes normally remain in a resting state, and in this state they do not display significant helper or cytotoxic activity. In the activated state, these cells undergo several cycles of mitosis to produce daughter cells. Some of these daughter cells return to a resting state as memory cells, while others become effector cells that actively display helper or cytotoxic activity. These daughter cells are similar to their parents: CD4+ cells can only produce CD4+ progeny, and CD8+ cells give rise to only CD8+ progeny. Effector T cells express cell surface markers that are not expressed on resting T cells, such as CD25, CD28, CD29, CD40L, transferrin receptor and class II MHC proteins. Without an activating stimulus, cytotoxic or helper activity gradually subsides over a period of days as effector cells die or revert to a quiescent state.

[0009] Similar to B cell activation, T lymphocyte responses to most antigens also require two types of costimulation. The first is the antigen, which is appropriately displayed by MHC proteins on antigen-presenting cells and can be recognized and bound by the T cell receptor. This antigen-MHC complex does not send a signal to the cell interior and is usually insufficient to cause T cell activation. Full activation, such as occurs with helper T cells, requires costimulation with other specific ligands, called costimulators, expressed on the surface of antigen-presenting cells. On the other hand, cytotoxic T cell activation generally requires IL-2, a cytokine secreted by activated helper T cells.

[0010] PD-1 pathway An important negative costimulatory signal controlling T cell activation is provided by the programmed cell death-1 receptor (PD-1, CD279) and its ligand binding partners, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The negative regulatory role of PD-1 has been demonstrated by PD-1 knockout (Pdcd1 - / - ) and the knockouts were prone to autoimmunity (Nishimura et al., Immunol. munity JJ: 141-51(1999); Nishimura et al., Science 291: 319-22(2001)). PD-1 associates with CD28 and CTLA-4 but lacks the membrane-proximal cysteine ​​that allows homodimerization. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-binding inhibitory motif (ITIM, V / IxYxxL / V). PD-1 binds only PD-L1 and PD-L2 (Freeman et al., J. Exp. Med. 192: 1-9(2000); Dong et al., Nature Med. 5: 1365-1369(1999); Latchman et al., Nature Immunol 2: 261-268(2001); Tseng et al., J. Exp. Med. 193: 839-846(2001)).

[0011] PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs). PD-1 binds activated human CD4 + and CD8 + It is expressed by T cells, B cells, and myeloid cells, but not by unstimulated ones. This is distinct from the more restricted expression of CD28 and CTLA-4 (Nishimura et al., Int. Immunol. 8: 773-80(1996); Boettler et al., J. Virol. 80: 3532-40(2006)). There are at least four variants of PD-1 that have been cloned from activated human T cells, including transcripts lacking (i) exon 2, (ii) exon 3, (iii) exons 2 and 3, or (iv) exons 2-4 (Nielsen et al., Cell. Immunol. 235: 109-16(2005)). With the exception of PD-1Δex3, all variants are expressed at levels similar to full-length PD-1 in resting peripheral blood mononuclear cells (PBMCs). The expression of all mutants is significantly induced upon activation of human T cells with anti-CD3 and anti-CD28 antibodies. The PD-1Δex3 mutant lacks the transmembrane domain and is similar to soluble CTLA-4, which plays an important role in autoimmunity (Ueda et al., Nature 423: 506-11(2003)). This mutant is enriched in synovial fluid and serum of patients with rheumatoid arthritis (Wan et al., J. Immunol. 177: 8844-50(2006)). The two PD-1 ligands have different expression patterns. PD-L1 is constitutively expressed on mouse T and B cells, CD4+, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells (Yamazaki et al., J. Immunol. 169: 5538-45(2002)). PD-L1 is expressed on many non-hematopoietic cells (e.g., corneal, lung, vascular epithelium, liver non-parenchymal cells, mesenchymal stem cells, pancreatic islets, placental syncytiotrophoblasts, keratinocytes, etc.) [Keir et al., Annu. Rev. Immunol. 26: 677-704(2008)] and is upregulated on many cell types after activation. Both type I and type II interferons, IFNs, upregulate PD-L1 (Eppihimer et al., Microcirculation 9: 133-45(2002)); Schreiner et al., J. Neuroimmunol 155: 172-82(2004)).PD-L1 expression in cell lines is decreased when MyD88, TRAF6, and MEK are inhibited (Liu et al., Blood HO: 296-304(2007)). JAK2 has also been implicated in PD-L1 induction (Lee et al., FEBS Lett, 580: 755-62(2006); Liu et al., Blood HO: 296-304(2007)). Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modulates phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increased post-transcriptional PD-L1 expression in cancer (Parsa et al., Nat. Med. 13: 84-88(2007)).

[0012] PD-L2 expression is more restricted than PD-L1. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells. PD-L2 is also expressed on approximately half to two-thirds of resting peritoneal B1 cells, but not on conventional B2 B cells (Zhong et al., Eur. J. Immunol. 2014;11:111-112). nol. 37: 2405-10(2007)). PD-L2+B1 cells bind phosphatidylcholine and may be important in the innate immune response to bacterial antigens. Induction of PD-L2 by IFN-γ is partially dependent on NF-KB (Liang et al., Eur. J. Immunol. 33_: 2706-16(2003)). PD-L2 can also be induced on monocytes and macrophages by GM-CF, IL-4, and IFN-γ (Yamazaki et al., J. Immunol. 169: 5538-45(2002); Loke et al., PNAS 100: 5336-41(2003)).

[0013] PD-1 signaling typically has a greater effect on cytokine production than on cell proliferation, with significant effects on IFN-γ, TNF-α and IL-2 production. PD-1-mediated inhibitory signaling also depends on the strength of TCR signaling, with lower levels of TCR stimulation providing greater inhibition. This reduction can be overcome by co-stimulation through CD28 [Freeman et al., J. Exp. Med. 192: 1027-34(2000)] or the presence of IL-2 [Carter et al., Eur. J. Immunol. 32: 634-43(2002)].

[0014] There is growing evidence that signaling through PD-L1 and PD-L2 can be bidirectional; that is, in addition to modulating TCR or BCR signaling, signals can also be delivered back to cells expressing PD-L1 and PD-L2. Treatment of dendritic cells with a natural human anti-PD-L2 antibody isolated from a patient with Waldenström's macroglobulinemia did not result in upregulation of MHC II or B7 costimulatory molecules, whereas these cells produced greater amounts of proinflammatory cytokines, particularly TNF-α and IL-6, and stimulated T cell proliferation (Nguyen et al., J. Exp. Med. 196:133-134). Treatment of mice with this antibody also (1) promoted resistance to transplanted b16 melanoma and rapidly induced tumor-specific CTL (Radhakrishnan et al., J. Immunol. 170: 1830-38 (2003); Radhakrishnan et al., Cancer Res. 64: 4965-72 (2004); Heckman et al., Eur. J. Immunol. 37: 1827-35(2007)); (2) blocked the development of airway inflammatory disease in a mouse model of allergic asthma (Radhakrishnan et al., J. Immunol. 173: 1360-65(2004); Radhakrishnan et al., J. Allergy Clin. Immunol. UJy. 668-74(2005)).

[0015] Further evidence of reverse signaling to dendritic cells ("DCs") came from studies of bone marrow-derived DCs cultured with soluble PD-1 (PD-1 EC domain fused to an Ig constant region - "s-PD-1") (Kuipers et al., Eur. J. Immunol. 36: 2472-82(2006)). This sPD-1 inhibited DC activation and increased IL-10 production in a reversible manner through anti-PD-1 administration. Furthermore, some studies have identified receptors for PD-L1 or PD-L2, independent of PD-1. B7.1 has already been identified as a binding partner for PD-L1 (Butte et al., Immunity 27: 111-22(2007)). Chemical cross-linking studies have shown that PD-L1 and B7.1 can interact through their IgV-like domains. B7.1:PD-L1 interactions can induce inhibitory signaling to T cells. Ligation of PD-L1 on CD4+ T cells by B7.1, or ligation of B7.1 on CD4+ T cells by PD-L1, provides an inhibitory signal. T cells lacking CD28 and CTLA-4 show reduced proliferation and cytokine production when stimulated with anti-CD3 plus B7.1-coated beads. T cells lacking all receptors for B7.1 (i.e., CD28, CTLA-4, and PD-L1) show reduced proliferation and cytokine production. In T cells, T cell proliferation and cytokine production were no longer inhibited by beads coated with anti-CD3 plus B7.1. This indicates that B7.1 acts specifically through PD-L1 on T cells in the absence of CD28 and CTLA-4. Similarly, T cells lacking PD-1 showed reduced proliferation and cytokine production when stimulated in the presence of beads coated with anti-CD-3 plus PD-L1, demonstrating the inhibitory effect of PD-L1 ligation on B7.1 on T cells. When T cells lacked all known receptors for PD-L1 (i.e., no PD-1 and no B7.1), T cell proliferation was no longer impaired by beads coated with anti-CD3 plus PD-L1. Thus, PD-L1 may exert an inhibitory effect on T cells through either B7.1 or PD-1.

[0016] The direct interaction between B7.1 and PD-L1 indicates that our current understanding of costimulation is incomplete and highlights the importance of expression of these molecules on T cells. - / - Studies of T cells have shown that PD-L1 on T cells can downregulate cytokine production by T cells (Latchman et al., Proc. Natl. Acad. Sci. USA 101: 10691-96 (2004)). Both PD-L1 and B7.1 are expressed on T cells, B cells, DCs and macrophages, so there is the possibility of directional interactions between B7.1 and PD-L1 on these cell types. Furthermore, PD-L1 on non-hematopoietic cells can interact with B7.1 as well as PD-1 on T cells, raising the question of whether PD-L1 is involved in its regulation. One possible explanation for the inhibitory effect of B7.1:PD-L1 interaction is that T cell PD-L1 can capture or separate APC B7.1 from its interaction with CD28.

[0017] The result is likely to be antagonism of signaling through PD-L1, including blocking PD-L1 from interacting with either PD-1, B7.1, or both, thereby preventing PD-L1 from sending negative co-stimulatory signals to T cells and other antigen presenting cells, which enhances immunity in response to infection (e.g., acute and chronic) and tumor immunity. Additionally, the anti-PD-L1 antibodies of the invention may be combined with antagonists of other components of PD-1:PD-L1 signaling, such as antagonists of anti-PD-1 and anti-PD-L2 antibodies.

[0018] In particular, inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (e.g., tumor immunity) and infections, including acute and chronic (e.g., persistent) infections.

[0019] Inhibitors that block PD-L1:PD-1 interaction are known, inter alia, from WO2001014557, WO2002086083, WO2007005874, WO2010036959, WO2010077634, and WO2011066389.

[0020] There are currently more than 10 single- and bispecific agents containing an anti-PD-L1 component in early-phase clinical trials. One monospecific anti-PD-L1 antibody, MPDL3280A (atezolizumab, Roche), has successfully passed clinical trials (CT) and is being used in clinical practice. Atezolizumab has been approved by the FDA for use in patients with metastatic urothelial carcinoma; Phase III CT is ongoing in patients with non-small cell lung cancer (NSCLC), renal cell carcinoma, colorectal cancer (CRC), and breast cancer (BC). The preparation is an Ig1 antibody with a modified Fc region (to eliminate ADCC effects). Atezolizumab is described in WO2010077634.

[0021] Other anti-PD-L1 preparations in late-stage CT are the avelumab (Pfizer) and durvalumab (AZ) preparations. Durvalumab (MEDI-4736) is described in WO2011066389. Avelumab is described in WO2013079174. The main difference of the avelumab preparation is the presence of an ADCC effect on the antibody, which can be further enhanced with IFNg or IL12 (NCT01772004). Furthermore, the safety profile of the avelumab preparation corresponds to that of other anti-PD-1 / PD-L1 preparations (Cancer Immunol Res; 3(10) October 2013). 2015; Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab on Human Tumor Cells; Benjamin Boyerinas). [Prior art documents] [Patent documents]

[0022] [Patent Document 1] WO2001014557 [Patent Document 2] WO2002086083 [Patent Document 3] WO2007005874 [Patent Document 4] WO2010036959 [Patent Document 5] WO2010077634 [Patent Document 6] WO2011066389 [Patent Document 7] WO2010077634 [Patent Document 8] WO2011066389 [Patent Document 9] WO2013079174 [Non-patent literature]

[0023] [Non-Patent Document 1] Nishimura et al., Immunity JJ: 141-51(1999) [Non-Patent Document 2] Nishimura et al., Science 291: 319-22(2001) [Non-Patent Document 3] Freeman et al., J. Exp. Med. 192: 1-9(2000) [Non-Patent Document 4] Dong et al., Nature Med. 5: 1365-1369(1999) [Non-Patent Document 5] Latchman et al., Nature Immunol 2: 261-268 (2001) [Non-Patent Document 6] Tseng et al., J. Exp. Med. 193: 839-846 (2001) [Non-Patent Document 7] Nishimura et al., Int. Immunol. 8: 773-80(1996) [Non-Patent Document 8] Boettler et al., J. Virol. 80: 3532-40 (2006) [Non-Patent Document 9] Nielsen et al., Cell. Immunol. 235: 109-16 (2005) [Non-Patent Document 10] Ueda et al., Nature 423: 506-11 (2003) [Non-Patent Document 11] Wan et al., J. Immunol. 177: 8844-50(2006) [Non-Patent Document 12] Yamazaki et al., J. Immunol. 169: 5538-45 (2002) [Non-Patent Document 13] Keir et al., Annu. Rev. Immunol. 26: 677-704 (2008) [Non-Patent Document 14] Eppihimer et al., Microcirculation 9: 133-45(2002) [Non-Patent Document 15] Schreiner et al., J. Neuroimmunol 155: 172-82 (2004) [Non-Patent Document 16] Liu et al., Blood HO: 296-304(2007) [Non-Patent Document 17] Lee et al., FEBS Lett, 580: 755-62(2006) [Non-Patent Document 18] Liu et al., Blood HO: 296-304(2007) [Non-Patent Document 19] Parsa et al., Nat. Med. 13: 84-88(2007) [Non-Patent Document 20] Zhong et al., Eur. J. Immunol. 37: 2405-10 (2007) [Non-Patent Document 21] Liang et al., Eur. J. Immunol. 33_: 2706-16(2003) [Non-Patent Document 22] Yamazaki et al., J. Immunol. 169: 5538-45(2002) [Non-Patent Document 23] Loke et al., PNAS 100: 5336-41 (2003) [Non-Patent Document 24] Freeman et al., J. Exp. Med. 192: 1027-34(2000) [Non-Patent Document 25] Carter et al., Eur. J. Immunol. 32: 634-43 (2002) [Non-Patent Document 26] Nguyen et al., J. Exp. Med. 196: 1393-98 (2002) [Non-Patent Document 27] Radhakrishnan et al., J. Immunol. 170: 1830-38 (2003) [Non-Patent Document 28] Radhakrishnan et al., Cancer Res. 64: 4965-72(2004) [Non-Patent Document 29] Heckman et al., Eur. J. Immunol. 37: 1827-35 (2007) [Non-Patent Document 30] Radhakrishnan et al., J. Immunol. 173: 1360-65 (2004) [Non-Patent Document 31] Radhakrishnan et al., J. Allergy Clin. Immunol. UJy. 668-74(2005) [Non-Patent Document 32] Kuipers et al., Eur. J. Immunol. 36: 2472-82 (2006) [Non-Patent Document 33] Butte et al., Immunity 27: 111-22 (2007) [Non-Patent Document 34] Latchman et al., Proc. Natl. Acad. Sci. USA 101: 10691-96 (2004) [Non-Patent Document 35] Cancer Immunol Res; 3(10) October 2015 Summary of the Invention [Problem to be solved by the invention]

[0024] Thus, there is a need to provide effective inhibitors of PD-L1 (programmed cell death ligand-1). In light of the above, it is of great importance to provide novel antibodies that effectively bind to PD-L1. It is.

[0025] The BCD-135 antibody selectively binds to PD-L1 and is a potent inhibitor of programmed cell death ligand-1. [Means for solving the problem]

[0026] The present invention relates to binding molecules, in particular antibodies directed to binding to PD-L1. Such antibodies may be used to treat diseases or disorders mediated by PD-L1.

[0027] In one aspect, the invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-L1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:3, and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7.

[0028] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:3. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO:7.

[0029] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NOs: 1-3. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:1-3.

[0030] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NOs:5-7. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO:5-7.

[0031] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:1-3, and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:5-7.

[0032] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:1-3, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:5-7.

[0033] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:4. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:4.

[0034] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:8. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO:8.

[0035] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof has at least one sequence similar to SEQ ID NO:4. The invention comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:8, and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:8.

[0036] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:4, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:8.

[0037] In certain embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:9, and a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:10.

[0038] In certain embodiments, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9, and a light chain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the monoclonal antibody specific for PD-L1 is a full IgG antibody.

[0039] In certain embodiments, the full length IgG antibody is of the human IgG1, IgG2, IgG3, IgG4 isotype. In certain embodiments, the monoclonal antibody is of the human IgG1 isotype.

[0040] In one aspect, the invention relates to a nucleic acid encoding any of the above-described antibodies or antigen-binding fragments thereof. In certain embodiments, the nucleic acid is DNA.

[0041] In one aspect, the invention relates to an expression vector comprising any of the above-mentioned nucleic acids. In one aspect, the invention relates to a method of producing a host cell adapted to produce any of the above-mentioned antibodies or antigen-binding fragments thereof, comprising the step of transforming a cell with the above-mentioned vector.

[0042] In one aspect, the invention relates to a host cell for producing any of the above-described antibodies or antigen-binding fragments thereof, comprising any of the above-described nucleic acids. In one aspect, the invention relates to a method for producing any of the above-mentioned antibodies or antigen-binding fragments thereof, comprising incubating the host cells in a culture medium under conditions sufficient to obtain the antibody, optionally followed by isolation and purification of the obtained antibody.

[0043] In one aspect, the invention relates to a pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1, comprising any of the above-mentioned antibodies or antigen-binding fragments thereof, and one or more pharma- ceutically acceptable excipients.

[0044] In some embodiments, the pharmaceutical composition is intended for the prevention or treatment of a disease or disorder mediated by PD-L1 selected from the group of: HNSCC, cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, CRC MSI.

[0045] In one aspect, the invention relates to a pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by PD-L1, comprising an antibody or antigen-binding fragment thereof as described above, and at least one therapeutically active anti-tumor compound.

[0046] In some embodiments, the pharmaceutical combination is intended for the prevention or treatment of a PD-L1 mediated disease or disorder selected from the group of: HNSCC, cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, CRC MSI.

[0047] In certain embodiments, the pharmaceutical combination comprises a therapeutically active anti-tumor compound selected from a chemotherapeutic agent, an antibody, or an anti-hormonal agent. In one aspect, the invention relates to a method for inhibiting the biological activity of PD-L1 in a subject in need thereof, comprising administering to the subject an effective amount of any of the above-described antibodies or antigen-binding fragments thereof.

[0048] In one aspect, the invention relates to the use of any of the above-described antibodies or antigen-binding fragments thereof, or the above-described pharmaceutical compositions, for the treatment of a PD-L1 mediated disease or disorder in a subject in need of such treatment.

[0049] In one embodiment, the invention relates to the use of any of the above-mentioned antibodies or antigen-binding fragments thereof, or the above-mentioned pharmaceutical compositions for the treatment of a PD-L1 mediated disease or disorder selected from the group of: HNSCC, cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin lymphoma, CRC MSI. [Brief description of the drawings]

[0050] [Figure 1] Scheme for human combinatorial naive library synthesis. [Diagram 2] Phagemid map for Fab phage display library cloning. [Diagram 3] Map of the expression plasmid for Fab production. [Figure 4] Electropherograms of BCD-135 under reducing (4A, 12% SDS-PAGE) and non-reducing (4B, 8% SDS-PAGE) conditions. [Diagram 5] Enzyme-linked immunosorbent assay of BCD-135 interaction with PD-L1 and other antigens. [Figure 6] Reactivation of NFAT signaling with anti-PD-L1 antibody in Jurkat-NFAT-PD-1 reporter cell line. [Figure 7] Analysis of BCD-135 interactions with FcRn and Fcγ receptors on an Octet RED 96 instrument. [Figure 8] Immunoenzymatic analysis of BCD-135 interaction with PD-L1 from different species. [Figure 9]Analysis of BCD-135 interaction with human and cynomolgus monkey PD-L1 on an Octet RED 96 instrument. [Figure 10] Conformational stability analysis of BCD-135. [Figure 11] Colloidal stability analysis of BCD-135. [Figure 12] Thermal stability analysis of BCD-135 in phosphate (A), acetate (B), and histidine (C) buffers. The x-axis indicates time and the y-axis indicates absorbance. [Figure 13] Stability analysis of BCD-135 in human serum. A: Calibration curve showing the optical density dependence on the concentration of BCD-135 added to the wells. B: Summary table showing the dependence of BCD-135 concentration upon incubation in human serum on incubation time. [Figure 14] Three-dimensional model of the complex between the N-terminal Ig domains of BCD-135 and PD-L1 antigens. A is a general overview of the 3D model; B is a detailed model in the area of ​​direct antigen-antibody contact (see table in Example 18). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Definitions and general methods Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation is not an admission that any of these documents form part of the common general knowledge in the art.

[0052] Further, unless the context requires otherwise, singular terms include plural terms and plural terms include the singular terms. Typically, the classifications and methods used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known to those of skill in the art and are widely used in the art. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.

[0053] In the present description and embodiments, the words "have" and "contain" or variations thereof, such as "has," "having," "contains," or "containing," shall be understood to include the indicated integer or group of integers but not to the exclusion of any other integer or group of integers.

[0054] Antibody Related Definitions PD-L1 (Programmed Death Ligand-1), also known as surface clustering antigen 274 (CD274) or homologue B7 (B7-H1), is a 40 kDa type 1 transmembrane protein. The protein consists of three domains: extracellular (220), transmembrane (21) and intracellular (31) represented by Ig V and C-like domains. The protein plays an important role in suppressing the immune system during pregnancy, transplantation of foreign tissues, and certain diseases, such as hepatitis. Under normal conditions, in response to self-antigens, a certain amount of antigen-specific CD8+ T effector cells accumulate in lymph nodes and spleen, and to prevent autoimmune processes, PD-1 / PD-L1 or B7-1 / PD-L1 complexes are formed, resulting in inhibitory signaling that reduces CD8+ T cell proliferation in lymph nodes. Therefore, PD-1 / PD-L interaction is one of the key events in the development of immune tolerance.

[0055] "Dysfunction" refers to a state of reduced immune responsiveness to antigenic stimulation in the context of immune dysfunction. The term includes the common elements of both exhaustion and / or anergy, in which antigen recognition may occur but the subsequent immune response is insufficient to control infection or tumor growth.

[0056] "Enhancing T cell function" means inducing, causing or stimulating T cells to have sustained or amplified biological function, or to regenerate or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include: increasing CD8 + These include increased secretion of gamma interferon from T cells, increased proliferation, and increased antigen responsiveness (e.g., viral or pathogen clearance). In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. This enhancement is measured by The manner in which this is determined is known to one of ordinary skill in the art.

[0057] "T cell dysfunction disorder" is a disorder or condition of T cells characterized by decreased responsiveness to antigenic stimulation. In certain embodiments, the T cell dysfunction disorder is associated with inappropriately increased signaling, particularly through PD-1. In another embodiment, the T cell dysfunction disorder is one in which T cells are anergic or have a decreased ability to secrete cytokines, proliferate, or perform cytolytic activity. In certain aspects, the decreased responsiveness results in ineffective control of pathogens or tumors that express immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction disorders include unresolved acute infections, chronic infections, and tumor immunity.

[0058] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity attenuates such evasion and is "treatable" when tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0059] The term "vaccine" as used herein includes any non-pathogenic immunogen that, when inoculated into a host, induces protective immunity against a particular pathogen. Vaccines may take many forms. A vaccine may be a whole organism that shares antigens with the pathogen but is not itself pathogenic (e.g., cowpox). Vaccines may also be prepared from killed (e.g., Salk polio vaccine) or attenuated (lost the ability to cause disease, e.g., Sabin polio vaccine) pathogens. Vaccines may also be prepared from purified macromolecules isolated from pathogenic organisms. For example, toxoid vaccines (e.g., tetanus and diphtheria), which contain inactive forms of soluble bacterial toxins and result in the production of antitoxin antibodies rather than immunity to intact bacteria. Subunit vaccines (e.g., hepatitis B) contain only a single immunogenic protein isolated from the pathogen of interest. Hapten conjugate vaccines attach specific carbohydrate or polypeptide epitopes isolated from the pathogen of interest to an immunogenic carrier, such as tetanus toxoid. These strategies essentially use the epitope as a hapten to induce antibody production, which then recognizes the same epitope in the natural pathogen. However, to be maximally effective, such vaccines must incorporate both B and T cell epitopes, and the epitopes must be selected to ensure that the T cell epitopes can be recognized, presented, and reacted to by the immune system of the host individual. DNA vaccines take advantage of the possibility that host cells will take up and express DNA that codes for pathogenic proteins injected intramuscularly. The host response to the immunogen can be enhanced if the immunogen is administered as a mixture with an adjuvant. Immune adjuvants function in one or more of the following ways: (1) prolonging retention of the immunogen; (2) increasing the effective size of the immunogen (and thus promoting phagocytosis and presentation to macrophages); (3) stimulating the influx of macrophages or other immune cells to the injection site; or (4) promoting local cytokine production and other immunological activity.Examples of adjuvants include complete Freund's adjuvant (CFA), aluminum salts, and mycobacteria-derived proteins such as muramyl dipeptide or tripeptide.

[0060] Amplification of this gene and / or overexpression of its protein has been found in many cancers, including HNSCC, cervical cancer, cancer of unknown primary site, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin lymphoma, and CRC MSI.

[0061] The term "binding molecule" includes antibodies and immunoglobulins. The term "antibody" (Ab) or "immunoglobulin" (Ig) as used herein refers to any It includes antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or individual chains thereof. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0062] The term "antigen-binding protein" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH / VHH domains (Ward et al. (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be linked using recombinant methods by a synthetic linker that allows them to be made as a single continuous chain (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as intact antibodies.

[0063] Preferably, the CDRs or the entire antigen-binding portion of the antigen-binding region of the antibody of the invention are obtained from a mouse, llama or donor human library or are substantially of human origin, with certain amino acid residues modified, e.g. replaced with different amino acid residues, to optimize certain antibody properties, e.g. KD, koff, IC50, EC50, ED50. Preferably, the antibody framework regions according to the invention are of human origin or are substantially of human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

[0064] In other embodiments, the antibody antigen-binding regions of the invention may be derived from other non-human species, including, but not limited to, mouse, llama, rabbit, rat or hamster, or alternatively, the antigen-binding regions may be derived from a human species.

[0065] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, variability is evident across the 110 amino acid span of the variable domains. They are not uniformly distributed. Instead, V regions consist of relatively invariant segments of 15-30 amino acids called framework regions (FRs) separated by shorter stretches called "hypervariable regions" or CDRs or "HVRs" or "HVs" that are highly variable. Each variable domain of a native heavy and light chain contains four FRs, given a mostly beta-sheet configuration, connected by three hypervariable regions that form loops that join and in some cases are part of the beta-fold structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding the antibody to an antigen, but exhibit different effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0066] The term "hypervariable region" ("HVR" or "HV"), as used herein, refers to the amino acid residues of an antibody which are involved in antigen binding. Typically, a hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" and / or those residues from a "hypervariable loop".

[0067] In some cases, it may also be preferable to modify one or more amino acid residues in the CDR region to increase the binding affinity for the target epitope. This is known as "affinity maturation" and may be performed in some cases in conjunction with humanization, for example, when antibody humanization results in a decrease in binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. A variety of affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method suggested by Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0068] "Framework Regions" (FR) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. When the CDRs are defined according to Kabat, the light chain FR residues are located at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located at about residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain. When a CDR includes amino acid residues from a hypervariable loop, the light chain FR residues are located in the light chain at about residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4), and the heavy chain FR residues are located in the heavy chain at about residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4). In some instances, when a CDR includes amino acids from both a CDR and a hypervariable loop as defined by Kabat, the FR residues are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are positions 1-25 and the FR2 residues are positions 36-49.

[0069] The antibodies of the present invention that "bind" to a target antigen allow the antibody to be used as a diagnostic and / or therapeutic agent when targeted to a protein or antigen-expressing cell or tissue. An antibody that binds to an antigen with sufficient affinity and is only slightly cross-reactive with other proteins, such that the antibody is capable of binding to the antigen with sufficient affinity to be detected by the antibody. Based on the analytical methods: fluorescence activated cell sorting (FACS), radioimmunoprecipitation (RIA) or ELISA, in such an embodiment, the degree of binding of the antibody to non-target proteins ("off-target proteins") is less than 10% of the antibody binding to the specific target protein. With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds" or "specific for" a specific polypeptide or epitope on a specific polypeptide target refer to binding that is detectably (measurably) different from non-specific interactions (e.g., with respect to bH1-44 or bH1-81, non-specific interactions are binding to bovine serum albumin, casein, fetal bovine serum, or neuravidin). For example, specific binding may be measured by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding may be determined by a competitive reaction with another molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.As used herein, the term "specific binding" to a specific polypeptide or epitope on a specific polypeptide target, or the phrase "specifically binds" or "is specific" to a specific polypeptide, can be indicated by a molecule having a Kd for the target of, for example, at least about 200nM, or at least about 150nM, or at least about 100nM, or at least about 60nM, or at least about 50nM, or at least about 40nM, or at least about 30nM, or at least about 20nM, or at least about 10nM, or at least about 8nM, or at least about 6nM, or at least about 4nM, or at least about 2nM, or at least about 1nM, or more.In one embodiment, the term "specific binding" refers to binding when a molecule binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0070] The term "Ka" as used herein refers to the association rate of a particular antibody-antigen interaction, while the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. "Binding affinity" generally refers to the strength of the totality of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic (native, true) binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Desirably, Kd is about 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM, or less. Affinity may be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present invention.

[0071] In one embodiment, the BIAcore was run at 25° C. using an immobilized antigen CM5 chip with ∼10 response units (RU). TM -2000 or BIAcore TM "Kd" or "Kd value" according to the present invention is measured by using a surface plasmon resonance assay on a BIAcore-3000 instrument (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is loaded with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide according to the manufacturer's instructions. The Fab is activated with NHS. The antigen is diluted to a concentration of 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, and then loaded (injected) at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of binding protein. After antigen injection, 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of the Fab (e.g., 0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at 25°C, at a flow rate of approximately 25 μl / min. Association and dissociation rates (kоff) are calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software, version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio kоff / kоn. For example, Chen, Y. et al. (1999) J. Mol. Biol. 293:865-881. The surface plasmon resonance assay described above demonstrated an on-rate of 10 6 M -1 s -1 may be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM concentration solution of anti-antigen antibody (Fab type) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured using a spectrometer, for example a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0072] The term "koff" refers to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. TM The system may be used to measure the dissociation rate constant, kоff+, by biolayer interferometry.

[0073] BIAcore was run at 25°C using immobilized antigen CM5 chips at ~10 response units (RU). TM -2000 or BIAcoreTM The "association rate" ("on rate") or "k on " according to the present invention may also be determined using the same surface plasmon resonance technique described above using a -3000 instrument (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen is diluted to a concentration of 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, and then loaded (injected) at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After injection of antigen, 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at 25° C. with a flow rate of approximately 25 μl / min. Association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software, version 3.2) by simultaneously fitting the association and dissociation sensorgrams. Equilibrium dissociation constants (K d ) are calculated as the ratio k off / k on . See, e.g., Chen, Y. et al. (1999) J. Mol. Biol. 293:865-881. However, the surface plasmon resonance assay described above shows that the on-rate is 10 6 M -1 s -1 may be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM concentration solution of anti-antigen antibody (Fab type) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured using a spectrometer, for example a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0074] Unless otherwise stated, the phrases "biologically active" and " "Biological activity" and "biological property" mean having the ability to bind to biological molecules.

[0075] The phrase "biological molecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biological molecule is naturally occurring. Antibody fragments, such as Fab and F(ab')2 fragments, may be obtained by pepsin or papain hydrolysis of whole antibodies by conventional methods. Moreover, antibodies, antibody portions, and immunoadhesion molecules may be obtained using standard recombinant DNA techniques, as described herein.

[0076] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line that contains a nucleotide sequence(s) encoding an antibody, where said nucleotide sequence(s) is not naturally associated with the cell.

[0077] The term "variant" antibody, as used herein, refers to an antibody having an amino acid sequence that differs from that of its "parent" antibody by the addition, removal, and / or substitution of one or more amino acid residues compared to the parent antibody sequence. In preferred embodiments, the variant antibody contains at least one or more (e.g., 1 to 12, e.g., 2, 3, 4, 5, 6, 7, 8 or 9, 10, 11 or 12, and in some embodiments of the invention, 1 to about 10) additions, deletions, and / or substitutions of amino acids compared to the parent antibody. In some embodiments, the additions, deletions, and / or substitutions of the present invention are made at CDR variant antibody sites. Identity or homology with respect to variant antibody sequences is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably epitope, as the parent antibody, and in some embodiments has at least one property or biological activity that is greater than the similar property of the parent antibody. For example, a variant antibody may have, for example, an expressed binding affinity, a longer half-life, a lower IC50, or an enhanced ability to inhibit the biological activity of an antigen, compared to the parent antibody. Of particular interest herein are variant antibodies that exhibit a biological activity that is at least 2-fold (preferably at least 5-fold, 10-fold, or 20-fold) greater than the biological activity of the parent antibody.

[0078] The term "bispecific antibody" refers to an antibody that contains one or more antigen-binding domains that are capable of specifically binding to two different epitopes on one biological molecule or that are capable of specifically binding to epitopes on two different biological molecules. Bispecific antibodies are also referred to herein as having or being "bispecific" antibodies.

[0079] The term "chimeric antibody" broadly refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically of partly human origin and partly non-human origin, i.e., partly obtained from a non-human animal, such as mouse, rat, or other rodent, or from a camelid, such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies to reduce the risk of immune responses directed against human antibodies, such as in the case of murine antibodies, directed against murine antibodies in humans. A typical example of a chimeric antibody is one in which the variable region sequences are murine, while the constant region sequences are human. In the case of chimeric antibodies, the non-human portion may be subjected to further changes for antibody humanization.

[0080] The term "humanized" refers to antibodies that are fully or partially of non-human origin, e.g., mouse or llama antibodies obtained by immunization of a mouse or llama, respectively, with an antigen of interest, or chimeric antibodies based on such mouse or llama antibodies, which are similar to those used in humans. This refers to the fact that some amino acids of the heavy and light chains, especially those in the framework and constant domains, may be substituted to avoid or minimize immune reactions caused by the antibody. The specificity of the interaction between the antibody and the target antigen is primarily inherent to the amino acid residues located in the six heavy and light chain CDR regions. Thus, the amino acid sequences within the CDR regions are much more diverse between different antibodies than sequences outside the CDR regions. Because the CDR region sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody, or more generally, any particular antibody with a given amino acid sequence, for example, by constructing an expression vector that expresses the CDR region sequence from a particular antibody within the framework sequence of another antibody. As a result, it is possible to "humanize" a non-human antibody and substantially retain the binding specificity and affinity of the parent antibody. Although it is not possible to accurately predict the immunogenicity, and therefore the immune response, to human antibodies, certain antibodies, non-human antibodies, are generally more immunogenic than human antibodies. Chimeric antibodies, in which foreign (e.g. camelid or rodent) constant regions are replaced by sequences of human origin, generally exhibit lower immunogenicity than antibodies of completely foreign origin, and there is a trend towards using humanized or fully human antibodies as therapeutic antibodies. Chimeric or other antibodies of non-human origin may therefore be humanized to reduce the risk of an immune response directed against the antibody in humans.

[0081] For chimeric antibodies, humanization generally involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity determining regions (CDR regions) are most likely not changed by humanization, but in some cases it may be desirable to change individual amino acid residues in the CDR regions, for example to remove glycosylation sites, deamidation sites, asparagine isomerization sections, or undesired cysteine ​​or methionine residues. N-linked glycosylation occurs by the addition of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutation, preferably conservative substitution, of the Asn or Ser / Thr residue with another residue. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, especially when they occur in Asn-Gly sequences, and to a lesser extent in other dipeptides, such as Asn-Ala. In such deamidated regions, especially Asn-Gly in CDR region sequences, it may be preferable to remove the region, typically by conservative substitution, to remove one of the residues involved.

[0082] Many methods for humanizing antibody sequences are known in the art; see, for example, the review by Almargo & Fransson, Front Biosci. 13:1619-1633(2008). One of the most commonly used methods is CDR region grafting, for example when chimeric antibodies of murine origin involve the identification of human germline gene equivalents for murine variable region genes and grafting mouse CDR region sequences into this framework. CDR region grafting may be based on the CDR region definition by Kabat, but later publications (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225(2007)) suggest that the definition by IGMT® (International ImMunoGeneTics Information System®, www.imgt.org) may improve humanization results (Lefranc et al., Dev. Comp Immunol. 27:55-77(2003)). In some cases, CDR region grafting may reduce the binding specificity and affinity, and therefore the biological activity, of the non-human CDR-grafted antibody compared to the parent antibody CDR regions from which it was derived. Backmutations (sometimes referred to as "framework region repair") may be applied to selected positions throughout the body, usually in framework regions. Information available in the literature and in antibody databases may be used to determine possible positions for backmutation. Amino acid residues that are candidates for backmutation are typically exposed on the surface of the antibody molecule, while residues that are located deep within or have a low degree of surface exposure will usually not be altered. An alternative to the CDR region grafting and backmutation methods of humanization is surface alteration, in which non-exposed residues of non-human origin are retained, while exposed residues are replaced with human residues.

[0083] There are two techniques for obtaining fully human antibodies: using in vitro constructed phage libraries or in vivo immunization of humanized animals (mice, rats, etc.).

[0084] Phage display is the first and most widely used in vitro technique for antibody identification. In 1985, Smith discovered that foreign DNA sequences could be cloned into the filamentous bacteriophage M13 so that the cloned gene sequences would be expressed on the phage particle surface as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315-1317). Thus, fusion proteins of interest may be selected based on their ability to bind to other proteins. This discovery was combined with PCR amplification technology, which allows the cDNA repertoire of immunoglobulin genes to be cloned to generate diverse phage libraries containing variable domains that can be used to rapidly search for target-specific monoclonal antibodies. The repertoire of the phage library reflects the repertoire of B lymphocyte antibodies of each human or animal whose blood was used to generate the library. In 1995, two papers reported the generation of genetically engineered mice expressing fully human antibodies whose repertoires could be matched to those produced by hybridoma technology (Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG et al.: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859; Green LL, Hardy MC, Maynard-Currie CE, Tsuda H, Louie DM, Mendez MJ, Abderrahim H, Noguchi M, Smith DH, Zeng Y et al.: Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 1994, 7:13-21). These animals have disrupted and targeted genes for the animal's own endogenous immunoglobulin heavy and kappa light chains, as well as introduced transgenes corresponding to segments of human heavy and kappa light chain genes. The human gene repertoire was found to be used by the mouse immune system to generate highly specific and high affinity antibodies against a greater variety of antigens. Despite the fact that human immunoglobulin transgenic mice express B cell receptors that are essentially hybrids of mouse and human components (e.g., human immunoglobulins, mouse Igα and Igβ, and other signaling molecules), their B cells develop and mature normally. In some cases, it may also be preferable to modify one or more amino acid residues in the CDR regions to increase binding affinity for the target epitope. This is known as "affinity maturation" and may in some cases be performed in conjunction with humanization, for example when humanization of an antibody results in a loss of binding specificity or affinity and sufficient improvement of binding specificity or affinity by back mutation alone is not possible. Various affinity maturation methods, such as those described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), are used. Methods such as in vitro scanning saturation mutagenesis and the stepwise in vitro affinity maturation method suggested by Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998) are known in the art.

[0085] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and secreted by an individual clonal population of cells. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells, hybridomas, typically produced by fusion of individual cells from a lymphocytic tumor with individual B lymphocytes from an immunized animal. Hybridomas are an engineered cell type and do not exist in nature.

[0086] A "natural antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0087] The definition of "isolated" ("separated") used to describe the various antibodies of this disclosure refers to an antibody that has been isolated and / or regenerated from the cell or cell culture in which it was identified and expressed. Contaminating components of its natural environment (contaminants) are usually substances that would interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous species. In a preferred embodiment, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue or preferably silver staining. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

[0088] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is distinct from the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from the nucleic acid molecule present in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that normally express the antibody, for example, when the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0089] The term "epitope" as used herein refers to that part (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, e.g., a bispecific binding molecule). Epitopic determinants are usually chemically active surface portions of molecules, e.g., amino acids or sugar side chains. Epitopes consist of groups and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be "linear" or "conformational". In a linear epitope, the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) all occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired antigen epitope has been identified, antibodies may be generated against this epitope using techniques well known in the art. Furthermore, the generation and characterization of antibodies or other binding molecules may shed light on information about the desired epitope. Based on this information, binding molecules may then be competitively screened for binding to the same or similar epitopes, for example by competition studies to find binding molecules that compete for binding to an antigen.

[0090] The term "peptide linker" as used herein means any peptide capable of linking domains, containing any amino acid sequence, with a length depending on the domains to be linked together. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0091] The term "effector function" of an antibody refers to a biological activity that is associated with the Fc region of an antibody (either a native Fc region sequence or a variant Fc region amino acid sequence) and varies depending on the antibody isotype. Examples of antibody effector functions include C1 q binding; complement dependent cytotoxicity; Fc receptor binding; antibody dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor, BCR) and B cell activation.

[0092] "Cellular antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, leukocytes, and macrophages, recognize bound antibodies on target cells and subsequently induce target cell lysis. The primary cells mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0093] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells are preferred. As described herein, effector cells may be isolated from their native source, for example from blood or PBMCs.

[0094] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Further, a preferred FcR is an FcR that binds an IgG antibody (gamma receptor), and preferred receptors include the FcγRI, FcγRII, and FcγRIII subclasses of receptors, including those These receptors include allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (Daeron, Annu. Rev. Immunol. 15: 203-234). (1997). Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995) provide general overviews of FcR. Other FcRs, including those that will be identified in the future, are included herein within the term "FcR". The term also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976), and Kim et al., J. Immunol. 24: 249 (1994)).

[0095] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with its antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

[0096] The term "identity" or "homology" should be taken to mean the percentage of residues in a candidate sequence that have corresponding sequences compared to the same amino acid residues, after aligning the sequences and introducing "gaps", if necessary, to achieve the maximum percent identity of the entire sequence, and not substituting any portion of the sequence identity for that which would be considered conserved. Neither N- nor C-terminal extensions and insertions shall be considered to reduce identity or homology. Methods and computer programs for alignment are well known. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is useful for such sequences by determining the degree of homology with respect to various substitutions, deletions (removals), and other modifications.

[0097] The term "homologous" in relation to an antibody polypeptide sequence shall refer to an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to the polypeptide sequence. The term in relation to a nucleic acid sequence shall refer to a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity to the nucleic acid sequence.

[0098] Modification(s) in the amino acid sequence of the antibodies described herein are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion of, and / or substitution of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. The amino acid modifications may also alter post-translational processes in the antibody, for example, changing the number or position of glycosylation sites.

[0099] Mutation to modify the amino acid sequence of antibody by amino acid substitution. Such mutation is the replacement of at least one amino acid residue in antibody molecule with another residue. The sites of interest for substitution mutagenesis include hypervariable regions or CDRs, but substitution is also contemplated in FR or Fc regions. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions cause changes in biological activity, more substantial changes may be introduced and the products screened, as referred to as exemplary substitutions in Table A, or as further described below in relation to amino acid classes.

[0100] [Table 1]

[0101] The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably and refer to a precise sequence of modified or unmodified nucleotides, determine nucleic acid fragments or regions that may or may not contain non-naturally occurring nucleotides, and are either double-stranded DNA or RNA or single-stranded DNA or RNA, or are a transcription product of said DNA.

[0102] It should be understood that the present invention does not relate to the nucleotide sequence in its natural chromosomal environment, i.e. in its natural state. The sequences of the present invention are isolated and / or purified, i.e. they are collected directly or indirectly, for example by copying, and the environment is at least partially modified. Thus, isolated nucleic acids obtained by genetic recombination, for example by receiving cells (host cells), or obtained by chemical synthesis, are also intended herein.

[0103] A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand, with its complementary sequence.

[0104] The expression "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0105] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA sequence is operably linked to a polypeptide DNA if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and contiguous and in reading phase with respect to the secretory leader. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at existing restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used, in accordance with conventional practice.

[0106] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In certain embodiments of the invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In certain embodiments of the invention, the vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. In certain embodiments of the invention, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). In other embodiments of the invention, the vector (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into a host cell, and thereby replicated along with the host genes. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0107] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to a host cell, which may, for example, comprise the vector described above according to the present invention. The present invention also relates to a host cell, which comprises, for example, a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding a light chain or an antigen-binding portion thereof, or both the first binding domain and / or the second binding domain in the trispecific binding molecule of the present invention. It should be understood that "recombinant host cell" and "host cell" are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not in fact be identical to the parent cell, since modifications may be retained in subsequent generations, either due to mutation or environmental influences, but such cells are still included within the scope of the term "host cell" as used herein.

[0108] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. "Pharmaceutical composition" means a composition comprising an antibody of the present invention and at least one component selected from the group consisting of pharma- ceutically acceptable and pharmacologically compatible vehicles, solvents, diluents, carriers, adjuvants, dispersing and receptive agents, delivery agents, such as preservatives, stabilizing agents, fillers, dispersing agents, humectants, emulsifying agents, suspending agents, thickening agents, sweeteners, fragrances, flavoring agents, antibacterial agents, fungicides, lubricants, and extended delivery modulating agents, the selection and appropriate ratio of which depends on the nature and method of administration and dosing. Exemplary suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. Protection against microbial action may be provided by various antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and similar compounds. The composition may also include isotonic agents, such as sugars, sodium chloride, and the like. The action of the composition may be extended by using absorption retardants such as monostearate and gelatin. Examples of suitable carriers, solvents, diluents, or delivery agents are water, ethanol, polyalcohols and their mixtures, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate). Exemplary fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, etc. Examples of dispersing and distributing agents are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycols. Pharmaceutical compositions for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical, or rectal administration of the agent, alone or in combination with another agent, may be administered to animals and humans in standard dosage forms in admixture with conventional pharmaceutical carriers. Suitable standard administration forms include oral forms, such as tablets, gelatin capsules, pills, powders, granules, chewing gum and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal, or intraocular administration forms and rectal administration forms.

[0109] "Drug (pharmaceutical product)" means a substance in tablet, capsule, injection, ointment type and other ready-to-use type (or mixture of substances in pharmaceutical composition type) intended to restore, correct or modify physiological function in humans and animals, and also for the treatment and prevention of disease, diagnosis, anesthesia, contraception, cosmetology, and so forth.

[0110] The term "PD-L1-mediated disease or disorder" intends any disease or disorder that is either directly or indirectly associated with PD-L1, including the etiology, occurrence, progression, persistence or pathology of the disease or disorder.

[0111] "Treating", "treatment" and "therapy" refer to a method of attenuating or eliminating at least one of a biological disorder and / or its associated symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or occurrence of the symptoms of the disease, disorder or condition. Furthermore, references to "treatment" herein include references to curative, symptomatic and prophylactic therapy.

[0112] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject. The subject may be male or female of any age. The term "disorder" refers to any condition that can be improved by the treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the manifestation of the disorder. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemia and lymphoid malignancies, especially breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoelic disorders; inflammatory, angiogenic and immunological disorders. A preferred disorder to be treated according to the present invention is cancer.

[0113] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Included in this definition are benign and malignant cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric (gastric or stomach) cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal cell carcinoma), prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various types of head and neck cancer.

[0114] The terms "immune response," "autoimmune response," and "autoimmune inflammation" refer to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by these cells or hepatocytes, including antibodies, cytokines, and complement, that result from the selective damage to, destruction of, or elimination from the human body of invasive pathogens, pathogen-infected cells or tissues, cancerous cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0115] A "therapeutically effective amount" is considered to be that amount of a therapeutic agent administered during treatment that will relieve to some extent one or more symptoms of the disease being treated. The term "chronic" application refers to continuous (extended) use of an agent(s) in contrast to acute (short-term) administration, such that the initial therapeutic effect (activity) is maintained for an extended period of time.

[0116] "Intermittent" use refers to treatment that is cyclic in nature rather than carried out consistently without interruption. In this specification and in the claims which follow, unless the context otherwise requires, the words "have," "contain," and "comprise" or variations thereof, such as "has," "having," "contains," "containing," "comprises," or "comprising," are to be understood to include a reference integer or group of integers but not to the exclusion of any other integer or group of integers.

[0117] Detailed Description of the Invention antibody The present invention relates to antibodies or antigen-binding fragments that bind to PD-L1 (Programmed Cell Death Ligand 1 protein).

[0118] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a) a heavy chain variable region comprising a CDR3 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of APLLLAMTFGVGS (SEQ ID NO: 3); and (b) a light chain variable region comprising a CDR3 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of ALYMGNGGHM (SEQ ID NO: 7); The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising:

[0119] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a) a heavy chain variable region comprising a CDR3 having the amino acid sequence of APLLLAMTFGVGS (SEQ ID NO: 3); and (b) a light chain variable region comprising a CDR3 having the amino acid sequence of ALYMGNGGHM (SEQ ID NO: 7) The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising:

[0120] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a) (i) a CDR1 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of DYAMS (SEQ ID NO: 1); (ii) a CDR2 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence DISWSGSNTNYADSVKG (SEQ ID NO: 2); (iii) a CDR3 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of APLLLAMTGVGS (SEQ ID NO: 3); and a heavy chain variable region comprising (b) (i) a CDR1 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of GLSSGTVTAINYPG (SEQ ID NO: 5); (ii) a CDR2 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of NTNTRHS (SEQ ID NO: 6); (iii) a CDR3 having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of ALYMGNGGHM (SEQ ID NO: 7); A light chain variable region comprising The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising:

[0121] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a) (i) a CDR1 having the amino acid sequence of DYAMS (SEQ ID NO: 1); (ii) C having the amino acid sequence of DISWSGSNTNYADSVKG (SEQ ID NO: 2) DR2, (iii) a heavy chain variable region comprising a CDR3 having the amino acid sequence of APLLLAMTFGVGS (SEQ ID NO: 3); and (b) (i) CDR1 having the amino acid sequence of GLSSGTVTAINYPG (SEQ ID NO: 5); (ii) CDR2 having the amino acid sequence of NTNTRHS (SEQ ID NO: 6); (iii) CDR3 having the amino acid sequence of ALYMGNGGHM (SEQ ID NO: 7) A light chain variable region comprising The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising:

[0122] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a)

[0123] [ka]

[0124] a heavy chain variable region having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of (SEQ ID NO: 4); and (b)

[0125] [ka]

[0126] A light chain variable region having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of (SEQ ID NO:8). The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising:

[0127] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a)

[0128] [ka]

[0129] A heavy chain variable region having the amino acid sequence of (SEQ ID NO: 4), and (b)

[0130] [ka]

[0131] A light chain variable region having the amino acid sequence of (SEQ ID NO: 8) The present invention relates to an isolated antibody or antigen-binding fragment thereof comprising: In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a)

[0132] [ka]

[0133] a heavy chain variable region having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of (SEQ ID NO: 9); (b)

[0134] [ka]

[0135] A light chain variable region having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the sequence of (SEQ ID NO: 10). The present invention relates to an isolated antibody comprising:

[0136] In one embodiment, the present invention provides an antibody that binds to PD-L1 and: (a)

[0137] [ka]

[0138] A heavy chain variable region having the amino acid sequence of (SEQ ID NO:9); (b)

[0139] [ka]

[0140] A light chain variable region having the amino acid sequence of (SEQ ID NO: 10) The present invention provides an isolated antibody comprising: In one embodiment of the invention, the isolated antibody that binds PD-L1 is a monoclonal antibody.

[0141] In one embodiment of the invention, the monoclonal antibody that binds to PD-L1 is a full length IgG antibody. In one embodiment of the invention, the full length IgG antibody is of the human IgG1, IgG2, IgG3, IgG4 isotype.

[0142] In one embodiment of the invention, the full length IgG antibody is of the human IgG1 isotype. In one embodiment of the invention, the isolated antibody is a BCD-135 antibody that binds to PD-L1 and contains a heavy chain variable region comprising a CDR3 with the amino acid sequence of SEQ ID NO:3, and a light chain variable region comprising a CDR3 with the amino acid sequence of SEQ ID NO:7.

[0143] In one embodiment of the invention, the isolated antibody is a BCD-135 antibody that binds to PD-L1 and contains a heavy chain variable region comprising CDRs 1-3 having the corresponding amino acid sequences of SEQ ID NOs: 1-3, and a light chain variable region comprising CDRs 1-3 having the corresponding amino acid sequences of SEQ ID NOs: 5-7.

[0144] In one embodiment of the invention, the isolated antibody is the BCD-135 antibody, which binds to PD-L1 and contains a heavy chain variable region having the amino acid sequence of SEQ ID NO:4, and a light chain variable region having the amino acid sequence of SEQ ID NO:8.

[0145] In one embodiment of the invention, the isolated antibody is the BCD-135 antibody, which binds to PD-L1 and contains a heavy chain having the amino acid sequence of SEQ ID NO:9, and a light chain having the amino acid sequence of SEQ ID NO:10.

[0146] nucleic acid molecule The present invention also relates to nucleic acid molecules and sequences encoding the anti-PD-L1 antibodies of the invention described herein. In some embodiments, the amino acid sequences of the first and second domains of the anti-PD-L1 antibodies are encoded by multiple nucleic acid molecules. Where the first and / or second domains comprise a heavy chain and a light chain, in some embodiments the heavy and light chain amino acid sequences are encoded by multiple nucleic acids. In other embodiments, the heavy and light chain amino acid sequences are encoded by the same nucleic acid molecule. In certain embodiments, the nucleic acid molecule may encode any combination of amino acid sequences of the first and second domains (e.g. heavy and light chain sequences). In certain embodiments, the nucleic acid molecule may encode the amino acid sequence of the first binding domain and the light chain amino acid sequence of the second binding domain, optionally including any sequence of a peptide linker linking them. The nucleic acid A reference to a strand sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand with its complementary sequence. The term "polynucleotide" as used herein means a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide, of at least 10 bases in length. The term includes single- and double-stranded forms.

[0147] The present invention also relates to a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more of the above nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 5-7. In a particular embodiment, the nucleotide sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 or 8. The present invention also relates to a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more of the above nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-10.

[0148] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 1-10. The nucleic acid molecule may also comprise any combination of these nucleotide sequences. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7. In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1-3 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5-7. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10.

[0149] In any of the above aspects, the nucleic acid molecule may be isolated. The nucleic acid molecules of the invention may be isolated from any source that produces anti-PD-L1 antibodies or portions thereof, hi certain embodiments, the nucleic acid molecules of the invention are synthesized, but not isolated.

[0150] In some embodiments, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding the VH domain of the first or second domain from an anti-PD-L1 antibody of the invention coupled in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding the VL domain of the first or second domain from an anti-PD-L1 antibody of the invention combined in-frame to a nucleotide sequence encoding a light chain constant domain from any source.

[0151] In another aspect of the invention, nucleic acid molecules encoding the variable domains of the heavy (VH) and light (VL) chains of the first or second binding domain may be "converted" into full-length antibody genes. In one embodiment, a nucleic acid molecule encoding a VH or VL domain is converted into a full-length antibody gene by inserting it into an expression vector already encoding a heavy chain constant (CH) or light chain constant (CL) domain, respectively, such that the VH segment is operably linked to a CH segment(s) in the vector and / or the VL segment is operably linked to a CL segment in the vector. In another embodiment, standard molecular biology techniques are used to link a nucleic acid molecule encoding a VH and / or VL domain to a nucleic acid molecule encoding a CH and / or CL domain, e.g. Nucleic acid molecules encoding the VH and / or VL domains are converted into full-length antibody genes, for example by ligation. The nucleic acid molecules encoding the full-length heavy and / or light chains may then be expressed from a cell into which they have been introduced.

[0152] The nucleic acid molecules may be used to express large amounts of recombinant anti-PD-L1 antibodies.The nucleic acid molecules may be used to produce human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives as described herein.

[0153] vector In yet another aspect, the invention relates to vectors suitable for the expression of any of the nucleotide sequences described herein.

[0154] The present invention relates to vectors containing a nucleic acid molecule encoding any of the amino acid sequences of an anti-PD-L1 antibody or portions thereof (e.g. the first binding domain heavy chain and second binding domain heavy chain and / or light chain sequences) as described herein.The present invention further relates to vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments.

[0155] In another embodiment of the invention, the nucleic acid molecules and vectors may be used to produce mutated anti-PD-L1 antibodies. The antibodies may be mutated within the variable domains of the first and / or second binding domain heavy and / or light chains, for example to alter the binding affinity of the anti-PD-L1 antibody. For example, mutations may be made in one or more CDR regions to increase the K D Increase or decrease k оff In another embodiment, the one or more mutations are made at amino acid residues known to be modified compared to germline in antibodies corresponding to the first or second binding domain of the anti-PD-L1 antibody of the invention. These mutations may be made in the CDR or framework regions in the variable or constant domain. In a preferred embodiment of the invention, the mutations are made in the variable domain. In another embodiment of the invention, the one or more mutations are made at amino acid residues known to be modified compared to germline in the CDR or framework regions of the variable domain in the anti-PD-L1 antibody of the invention.

[0156] In some embodiments, the anti-PD-L1 antibody of the present invention is expressed by inserting DNA encoding partially or completely the sequences of the first and second binding domains (e.g., heavy and light chain sequences, where the binding domains comprise heavy and light chain sequences), obtained as described above, in an expression vector, such that the gene is operably linked to the necessary expression control sequences, e.g., transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule may be ligated into a vector such that the transcriptional and translational control sequences in the vector provide the intended function of DNA transcriptional and translational control. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. The DNA molecules encoding partial or full-length sequences of the first and second binding domains (e.g., heavy and light chain sequences, where the binding domains comprise heavy and light chain sequences) may be inserted into separate vectors. In one embodiment of the present invention, any combination of the above DNA molecules is introduced into the same expression vector. The DNA molecules may be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

[0157] A suitable vector is one that encodes a functionally complete sequence of a human CH or CL immunoglobulin, as described above, and is designed with suitable restriction sites so that any VH or VL sequence can be easily incorporated and expressed. The NA and LC coding genes in such vectors may contain intron sequences that lead to an overall increase in protein antibody product by stabilizing the corresponding mRNA. The intron sequences are surrounded by splice donor and splice acceptor sites, which determine where RNA splicing occurs. When several introns are used, the intron sequences may be located either in the variable or constant regions of the antibody chain, or in both the variable and constant regions. Polyadenylation and transcription termination may be downstream of the region coding for the native chromosomal site. The recombinant expression vector may also encode a signal peptide that facilitates production of the antibody chain by the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked to the reading frame amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide that is not that of a native immunoglobulin protein).

[0158] In addition to the antibody chain genes, the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. One of skill in the art will recognize that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of expression of the protein desired, and the like. Preferred regulatory sequences for mammalian expression host cells include promoters and / or enhancers from viral elements, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., large adenovirus late promoter (AdMLP)), polyoma virus, and strong mammalian promoters, such as the native immunoglobulin and actin promoters, that ensure high levels of protein expression in mammalian cells. See, e.g., U.S. Patent Nos. 5,168,062; 4,510,245; and 4,968,615 for further description of viral regulatory elements, and sequences thereof. Methods for the expression of binding molecules, such as antibodies, in plants are known in the art, including promoter and vector descriptions, and plant transformation. See, for example, U.S. Patent No. 6,517,529. Methods for the expression of polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0159] In addition to the antibody chain genes and control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase gene (DHFR) (for use in dhfr host cells with selection / MTX amplification), the ne о gene (for selection with G418), and the glutamate synthetase gene.

[0160] The term "expression control sequences" as used herein refers to polynucleotide sequences necessary to effect expression and processing of coding sequences to which they are ligated. Expression control sequences 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 enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" includes at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fused cellular sequences.

[0161] host cell A further aspect of the invention relates to methods for producing the anti-PD-L1 antibodies of the invention. One embodiment of the invention relates to a method for producing an anti-PD-L1 antibody as defined herein comprising the steps of providing a recombinant host cell capable of expressing the anti-PD-L1 antibody, culturing said host cell under conditions suitable for the production of the anti-PD-L1 antibody, and recovering the anti-PD-L1 antibody produced. The anti-PD-L1 antibody produced by such expression in such a recombinant host cell is referred to herein as a "recombinant anti-PD-L1 antibody". The invention also relates to progeny of such host cells, and to anti-PD-L1 antibodies obtained by the same methods.

[0162] Nucleic acid molecules encoding the anti-PD-L1 antibodies of the present invention, and vectors containing these nucleic acid molecules, may be used to transfect suitable mammalian, plant, bacterial or yeast host cells. Transformation may occur by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, nucleic acid and positively charged polymer complex transfection, nucleic acid and calcium phosphate precipitation transfection, polybrene-mediated transfection, protoplast fusion, transfection with polynucleotides encapsulated in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461; and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0163] Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, among others, Chinese hamster ovary cells (CHO), NS0 cells, SP2, HEK-293T cells, Freestyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. The cell line is selected through determining which cell line has a high expression level of the protein to be produced and provides the desired characteristics. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. If a recombinant expression vector encoding an anti-PD-L1 antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or more preferably, release of the antibody into the medium in which the host cells are grown. The anti-PD-L1 antibody may be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, These include Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0164] Additionally, many known methods may be used to enhance the production levels of the anti-PD-L1 antibodies of the invention from a production cell line. For example, the glutamine synthetase gene expression system (GS system) is very popular for enhancing expression under certain conditions. The GS system is generally or in part discussed in connection with EP Patent Nos. 0216846, 0256055, 0323997, and 0338841.

[0165] Anti-PD-L1 antibodies from different cell lines or transgenic animals may have different glycosylation profiles, however, all anti-PD-L1 antibodies encoded by the nucleic acid molecules described herein or comprising the amino acid sequences set forth herein, regardless of the glycosylation state of the binding molecule, and generally, regardless of the presence or absence of post-translational modifications, are part of the present invention.

[0166] Antibody preparation The present invention also relates to methods and processes for producing the anti-PD-L1 antibodies and antigen-binding fragments thereof.

[0167] Monoclonal antibodies The monoclonal antibodies may be made using the hybridoma-based method first described by Kohler et al., Nature, 256, 1975, p. 495, or may be made by recombinant DNA methods (US 4,816,567).

[0168] In hybridoma-based methods, a mouse or other suitable host animal, such as a hamster, is immunized as described herein above to elicit lymphocytes that produce or are capable of producing antibodies capable of specifically binding to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 2003). Press, 1986, pp. 59-103).

[0169] The hybridoma cells thus prepared are plated and grown in an appropriate medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parent myeloma. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0170] Myeloma cells that are preferred for use as components of cell fusion are those that fuse easily, maintain stable high levels of antibody production by the selected antibody-producing cells, and are sensitive to selection media that selects for unbound parent cells. Preferred myeloma cell lines are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and the American Type Culture Collection. and SP-2 or X63-Ag8-653 cells available from the Cellular Immunology Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133, 1984, p. 3001; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker Inc., New York, 1987, pp. 51-63).

[0171] Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0172] The binding affinity of a monoclonal antibody, or portion thereof, may also be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107, 1980, p. 220.

[0173] Upon identification of hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity, the clones may be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103). Media useful for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, the hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by intraperitoneal (ip) injection of the cells into mice.

[0174] The monoclonal antibodies secreted by the subclones may be separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as affinity chromatography (e.g., Protein A or Protein Q-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0175] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to provide for monoclonal antibody synthesis in recombinant host cells. For a review article on recombinant expression in bacteria of antibody-encoding DNA, see Skerra et al., Curr. Opinion in Imunol., 5, 1993, pp. 256-262, and Pliickthun, Immunol. Revs. 130, 1992, pp. 151-188.

[0176] In a further embodiment of the invention, monoclonal antibodies or antibody fragments may be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348, 1990, pp. 552-554. Clackson et al., Nature, 352, 1991, pp. 624-628 and Marks et al., J. Mol. Biol., 222, 1991, pp. 581-597, describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications have demonstrated the isolation of high affinity (nM range) human antibodies by chain shuffling. We describe the production of antibodies (Marks et al., Bio / Technology, 10, 1992, pp. 779-783), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21, 1991, pp. 2265-2266). These techniques are therefore promising alternatives to traditional monoclonal antibody hybridoma-based techniques for the isolation of monoclonal antibodies.

[0177] Also, for example, heavy and light chains (C H and C L The DNA encoding the antibody may be modified, for example, to obtain chimeric or fusion antibody polypeptides, by substituting constant region sequences for homologous murine sequences (US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851), or by covalently linking immunoglobulin coding sequences to all or part of a sequence encoding a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequence may be substituted with an antibody constant region or with the variable region of an antibody antigen-binding site to generate a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for another antigen.

[0178] Humanized antibodies Methods for generating "humanized" antibodies in non-human animals are known in the art. Preferably, a humanized antibody has one or more amino acid residues incorporated herein from a non-human source. These amino acid residues from the non-human source are often referred to as "imported" residues, since they are usually obtained from the "imported" variable region. Humanization may be essentially carried out according to the method of Winter and coworkers (Jones et al., Nature, 321, 1986, pp. 522-525; Riechmann et al., Nature, 332, 1988, pp. 323-327; Verhoeyen et al., Science, 239, 1988, pp. 1534-1536) by replacing the hypervariable region sequences with the sequences of the corresponding human antibody. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species (US 4,816,567). In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous regions in rodent antibodies.

[0179] The choice of human variable domains, both light and heavy, to be used in making a humanized antibody is very important to reduce antigenicity and HAMA reactions (human anti-murine antibodies) when the antibody is intended for human therapy. According to the so-called "best-fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable region sequences. The human V-region sequence closest to that of the rodent is identified, and a suitable human framework (FR) is selected therein for use in the humanized antibody (Sims et al., J. Immunol., 151, 1993, p. 2296; Chothia et al., J. Mol. Biol., 196, 1987, p. 901). Another method uses a specific framework derived from the consensus sequence of a specific subgroup of all human antibody light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA: 89, 1992, p. 4285; Presta et al., J. Immunol., 151, 1993, p. 2623).

[0180] While retaining high binding affinity for the antigen and other favorable biological properties, It is further important to humanize the antibody. To this end, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various humanized products using conceptual three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and import sequences may be selected and combined such that the desired antibody characteristic, e.g. increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0181] The humanized antibody may be an antibody fragment, e.g., a Fab fragment, that is optionally conjugated with one or more cytotoxic agent(s) to generate an immunoconjugate. Alternatively, the humanized antibody may be a full-length antibody, e.g., a full-length IgG1 antibody.

[0182] Human antibodies and phage display library technology As an alternative to humanization, human antibodies may be obtained. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing the full range of human antibodies in the absence of endogenous immunoglobulin production. For example, in chimeric and germ-line mutant mice, the antibody heavy chain joining region (J H It has been described that homozygous deletion of the IgG1 (IgG2 segment) gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into these germ-line mutant mice results in the production of human antibodies upon antigen challenge (e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA: 90, 1993, p. 2551; Jakobovis et al., Nature, 362, 1993, pp. 25-258; Bruggemann et al., Year in Immuno., 7, 1993, p. 33; US 5,545,806; 5,569,825; 5,591,669 (all to GenPharm); US 5,545,807; and WO 97 / 17852).

[0183] Alternatively, phage display technology may be used to produce human antibodies and antibody fragments in vitro from a range of immunoglobulin variants (V) from immunized donors (McCafferty et al., Nature, 348, 1990, pp. 552-553). According to this technique, antibody V region genes are cloned in the same reading frame as either the major or minor envelope protein genes of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody having said properties. Thus, the phage mimics certain properties of B cells. Phage display may be carried out in a variety of formats (for review, see, e.g., Johnson Kevin S. and Chiswell David J., Current Opinion in Structural Biology, 3, 1993, (See, e.g., pp. 564-571). Different sources of V gene segments may be used for phage display. Clackson et al., Nature, 352, 1991, pp. 624-628, isolated a diverse set of anti-oxazolone antibodies from a small arbitrary combinatorial library of V genes derived from the spleens of immunized mice. It is possible to design diverse V genes obtained from immunized human donors and to generate antibodies with different Antibodies against a set of antigens (including self-antigens) may be isolated generally according to the methods described in Marks et al., J. Mol. Biol., 222, 1991, pp. 581-597, or Griffith et al., EMBO J., 12, 1993, pp. 725-734 (see also US 5,565,323; and 5,537,905).

[0184] Human antibodies may also be produced in vitro by activated B cells as discussed above (see US 5,567,610 and US 5,229,275). antibody fragment In certain cases, it may be desirable to use antibody fragments rather than whole antibodies: the smaller size of the fragments allows for rapid clearance and may allow for better penetration into solid tumors.

[0185] A variety of techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained through proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24, 1992, pp. 107-117, and Brennan et al., Science, 229, 1985, p. 81). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv fragments of antibodies can be expressed in and secreted from E. coli, which allows for the facile production of large amounts of these fragments. Antibody fragments can be isolated from phage antibody libraries. In another embodiment, Fab'-SH fragments can be directly isolated from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10, 1992, pp. 163-167). According to another approach, F(ab')2 fragments may be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life, in which the epitope-binding receptor residues are retained, are described in US 5,869,046. Other methods of preparing antibody fragments should be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93 / 16185, US 5,571,894, and US 5,587,458). Fv and sFv are the only species with intact binding sites that lack constant regions; thus, they may be used to reduce non-specific binding when used in vivo. Fusion proteins bearing sFv may be designed to result in fusion of effector proteins at either the N- or C-terminus of the sFv (Antibody Engineering, Borrebaeck, Ed., see above). An antibody fragment may also be a "linear antibody", for example as described in US 5,641, 870. Such fragments of linear antibodies may be monospecific or bispecific.

[0186] bispecific antibody Bispecific antibodies are antibodies capable of specifically binding to two different epitopes. For example, a bispecific antibody may bind to two different epitopes of the PD-L1 protein. Other bispecific antibodies may possess a binding site for PD-L1 in combination with a binding site for another protein. Bispecific antibodies may be obtained as full length antibodies or antibody fragments (e.g., F(ab')2 fragments of bispecific antibodies).

[0187] Methods for making bispecific antibodies are well known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where both chains have different specificities (Millstein et al., Nature, 305, 1983, pp. 537-539). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) have the potential for 10 different antibody molecules. This produces a mixture of antibodies, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually done by several affinity chromatography steps, is rather cumbersome and the product yields are low. Similar methods are disclosed in WO 93 / 08829, and in Traunecker et al., EMBO J., 10, 1991, pp. 3655-3659.

[0188] According to a different approach, antibody variable domains with the desired binding specificities (antibody antigen-binding sites) are fused to immunoglobulin constant region sequences. The fusion is preferably H 2 and C H The first heavy-chain constant region (C) containing the site necessary for light-chain binding, present in at least one of the fusions, is an Ig heavy-chain constant region that contains at least part of the three regions. HIt is preferred to have 1). The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in the embodiment where an unequal ratio of the three polypeptide chains is used in the construct to provide optimal yields. However, it is also possible to insert the coding sequences of two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important.

[0189] In a preferred embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific molecule from undesired immunoglobulin chain combinations, since to facilitate separation, an immunoglobulin light chain is present in only one half of the bispecific molecule. This approach is disclosed in WO 94 / 04690. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121, 1986, Please see p. 210.

[0190] According to another approach described in US 5,731,168, the interface between a pair of antibody molecules may be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is HThe antibody molecule comprises at least a portion of the three regions. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain(s) is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0191] Bispecific antibodies include cross-linked antibodies or "heteroconjugates". For example, one of the antibodies in the heteroconjugate may be coupled to avidin, and the other to biotin. For example, such antibodies may be used to target immune system cells to unwanted cells (US 4,676,980) and to treat HIV infection (WO 91 / 00360, WO 92 / 200373 and EP 03089). Any suitable cross-linking method may be used to make heteroconjugate antibodies. Suitable cross-linking agents are well known in the art and are described in US 4,676,980, along with a number of cross-linking techniques.

[0192] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical linkage may be used to prepare bispecific antibodies. Brennan et al., Science, 229, 1985, p. 81, describes a method for proteolytic cleavage of intact antibodies to generate F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, such as sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide bond formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to generate the bispecific antibody. The bispecific antibody produced may be used as an agent for the selective immobilization of enzymes.

[0193] Current advances can facilitate the direct recovery of Fab'-SH fragments from E. coli, which may be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175, 1992, pp. 217-225, described the production of a fully humanized bispecific antibody molecule, F(ab')2 fragment. Each Fab' fragment was isolated separately from E. coli and subjected to direct chemical coupling in vitro to form bispecific antibodies. The bispecific antibodies thus obtained had the ability to bind to cells characterized by overexpression of the ErbB2 receptor and normal human T cells, as well as to stimulate the lytic activity of human cytotoxic lymphocytes targeted by human breast tumors.

[0194] A variety of techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, a leucine "zipper" may be used to produce bispecific antibodies (Kostelny et al., J. Immunol., 148(5), 1992, pp. 1547-1553). The leucine "zipper" peptides from the Fos and Jun proteins were linked to the Fab' fragments of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method may also be used to produce antibody homodimers. Hollinger et al., Proc. Natl. Acad. The "diabody" technology described in Sci. USA, 90, 1993, pp. 6444-6448 is an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable (VH) region linked to a light chain variable (VL) region by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL regions of one fragment are forced to pair with the complementary VL and VH regions of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments has also been reported, based on the use of single chain Fv (sFv) dimers (see Gruber et al., J. Immunol, 152, 1994, p. 5368).

[0195] Antibodies with more than two valencies are also within the scope of the present invention. For example, triabodies may be prepared (Tutt et al., J. Immunol., 147, 1991, p. 60).

[0196] Multivalent antibodies Multivalent antibodies can be internalized (and / or catabolized) by cells expressing the antigen, and they bind to the antigen more rapidly than bivalent antibodies. The antibodies of the present invention may be multivalent antibodies (non-IgM class) that contain three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily prepared by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody may contain a dimerization domain and three or more antigen binding sites. A preferred dimerization domain comprises (or consists of) an Fc fragment or hinge region. In such a case, the antibody may contain an Fc fragment and three dimerization domains N-terminal to the Fc fragment. A multivalent antibody should contain one or more antigen-binding sites. As used herein, preferred multivalent antibodies contain (or consist of) three to about eight, but preferably four, antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain(s) comprises two or more variable regions. For example, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain(s) may comprise the following chain: VH-CH1-flexible linker-VH-CH1-Fc region; or VH-CH1-VH-CH1-Fc region. The multivalent antibodies provided herein preferably further comprise at least two (and preferably four) light chain variable region polypeptides. The multivalent antibodies provided herein comprise, for example, about two to about eight light chain variable region polypeptides. As used herein, a light chain variable region polypeptide comprises a light chain variable region, and optionally further comprises a CL region.

[0197] Pharmaceutical Compositions Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) an antibody specific for PD-L1. The pharmaceutical composition may include any anti-PD-L1 antibody as described herein. In some embodiments of the invention, the composition is for ameliorating, preventing or treating a disorder that may be associated with PD-L1 activity.

[0198] Generally, the anti-PD-L1 antibodies of the invention are suitable for use as a dosage form in combination with one or more pharma- ceutically acceptable excipients, for example as described herein below.

[0199] Pharmaceutical compositions of the invention may contain at least one anti-PD-L1 antibody and one or more additional binding molecules (e.g. antibodies) that target one or more respective surface receptors.

[0200] A pharmaceutical composition is "sterile" if it is aseptic, ie, free of viable microorganisms and their spores. A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity over its potential shelf life at a storage temperature, e.g., 2-8°C. It is desirable for the active agent to retain its physical and chemical stability, as well as its biological activity. The shelf life is selected based on the results of stability studies under accelerated and native storage conditions.

[0201] The term "excipient" as used herein describes any ingredient other than the compound(s) of the present invention. The choice of an inert excipient will depend in large part on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutical acceptable excipient" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and similar physiologically compatible substances. Some examples of pharmaceutical acceptable excipients are water, saline, phosphate buffer, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferable to include an isotonicity agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Further examples of pharmaceutical acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

[0202] By "buffer" is meant a solution capable of maintaining a pH value due to the interaction between the acidic and alkaline components that make up the composition. In general, the pH of a pharmaceutical composition is preferably between 4.5 and 7.0. Examples of buffers known to those skilled in the art and that can be found in the literature include, but are not limited to, histidine, citric acid, succinic acid, acetic acid, phosphoric acid, phosphate-salts, citrate-phosphate buffers, and tromethamine-based buffers, etc., or suitable mixtures.

[0203] By "isotonic agent" is meant an auxiliary substance, or a mixture of two or more auxiliary substances, that provides an isotonic osmolality of the solution. "Isotonic" is considered to be a solution that produces an osmolality of about 250-350 mOsm / kg. As isotonic agents, polyols, mono- or disaccharides, amino acids, metal salts, such as sodium chloride, etc. may be used, but are not limited to these. The term "hypotonic" describes a composition that has an osmolality lower than that of human blood. Correspondingly, the term "hypertonic" describes a composition that has an osmolality higher than that of human blood.

[0204] The term "surfactant" (also referred to as detergent or surface active agent or SAS) herein refers to an excipient that can alter the surface tension of a liquid antibody formulation. In certain embodiments, the surfactant reduces the surface tension of the liquid antibody formulation. In other embodiments, the "surfactant" can also contribute to an improvement in the colloidal stability or solubility of any antibody in the formulation. The surfactant can also reduce aggregation of the produced antibody preparation and / or minimize the formation of particulates in the preparation and / or reduce adsorption. The surfactant can also improve the stability of the antibody during or after freeze / thaw cycles and during shaking. The surfactant can be ionic or non-ionic. Exemplary non-ionic surfactants that can be included in the formulations of the invention include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Particular non-ionic surfactants that may be included in the formulations of the invention include, for example, polysorbates, such as polysorbate 20 (Tween 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 (Tween 80), polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188 (Kolliphor P188), poloxamer 407; polyethylene-polypropylene glycols; or polyethylene glycol (PEG), ethylene and propylene glycol copolymers (such as, for example, Pluronic® PF68).

[0205] By "stabilizer" is meant an auxiliary substance or a mixture of two or more auxiliary substances that provide physical and / or chemical stability of an active agent.As a stabilizer, amino acids may be used, including, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, including, but not limited to, polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade name Poloxamer), Pluronic (registered trademark), sodium dodecyl sulfate (SDS); antioxidants, including, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of sulfuric acid, etc.; chelating agents, including, but not limited to, EDTA, DTPA, sodium citrate, etc.

[0206] "Pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic at the concentrations and forms formulated. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfuric acid, sulfanilic acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include linear and branched chain alkyl, aromatic, cyclic, cyclic aliphatic, aryl aliphatic, and aryl cyclic aliphatic acids. These include fatty acids, heterocyclic acids, saturated, unsaturated, mono, di, and tricarboxylic acids, such as formic acid, acetic acid, 2-hydroxyacetic acid, trifluoroacetic acid, phenylacetic acid, trimethylacetic acid, t-butylacetic acid, anthranilic acid, propanoic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid, propanedioic acid, cyclopentanepropionic acid, cyclopentanepropionic acid, 3-phenylpropionic acid, butanoic acid, butanedioic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, 2-acetoxy-benzoic acid, ascorbic acid, cinnamic acid, lauryl sulfuric acid, stearic acid, muconic acid, mandelic acid, succinic acid, embonic acid, fumaric acid, malic acid, maleic acid, hydroxymaleic acid, malonic acid, lactic acid, citric acid, tartaric acid, glycosaminoglycan, glyceryl ester ... These include carboxylic acid, glyconic acid, gluconic acid, pyruvic acid, glyoxalic acid, oxalic acid, mesylic acid, succinic acid, salicylic acid, phthalic acid, palmoic acid, palmeic acid, thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chorobenzenesulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-3-(hydroxy-2-ene-1-carboxylic acid), and hydroxynaphthoic acid.

[0207] "Pharmaceutically acceptable bases" include inorganic and organic bases that are non-toxic at the concentrations and forms formulated. For example, suitable bases include inorganic base-forming metals, such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine, and organic non-toxic bases including primary, secondary and tertiary amines, substituted amines, cyclic amines, and basic ion exchange resins [e.g., N(R')4+, where R' is independently H or C, 1-4Alkyl, e.g., ammonium, Tris)], such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine. Additional pharma- ceutically acceptable salts and bases useful in the present invention include those derived from amino acids, such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, and asparagine.

[0208] Herein, the "diluent" of interest is one that is pharma- ceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid compositions, such as compositions that are reconstituted after lyophilization.Exemplary diluents include water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.In alternative embodiments, the diluent may include an aqueous solution of salt and / or buffer.

[0209] A "preservative" is a compound that may be added to the compositions provided herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple dose) composition. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, where the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butyl and benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and and m-cresol. The most preferred preservative provided herein is benzyl alcohol.

[0210] The term "lyophilized" as used herein refers to a preparation that has been subjected to a process known in the art as freeze-drying, which involves freezing of the preparation and then sublimation of ice from the frozen contents.

[0211] The term "amino acid" as used herein refers to an amino acid (a free amino acid, i.e., not an amino acid in a peptide or protein sequence). Amino acids, as used herein, include, but are not limited to, for example, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tryptophan, serine, methionine, and proline.

[0212] The pharmaceutical compositions of the present invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation are described, for example, in Remington, The Science and Practice of Pharmacy, 21st ed., Troy, Beringer., Lippincott Williams and Wilkins., Philadelphia, PA 2006. Production of pharmaceutical compositions preferably meets Good Manufacturing Practice (GMP).

[0213] The pharmaceutical composition of the present invention may be manufactured, packaged, or generally distributed as a single unit dose, or as a plurality of single unit doses.The term "single unit dose" as used herein means a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a suitable fraction of the dosage, such as half or one-third of such dosage.

[0214] Any art-accepted method for administering a peptide, protein, or antibody may be suitably used in connection with the anti-PD-L1 antibodies of the invention. The pharmaceutical composition of the present invention is generally suitable for parenteral administration. As used herein, the term "parenteral administration" of a pharmaceutical composition includes any administration route characterized by administration of the pharmaceutical composition into tissue through physical disruption of the integrity of the subject's tissue and tissue damage, generally resulting in direct contact with the bloodstream, intramuscularly, or internal organs. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is envisaged to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intraarticular injection or infusion; as well as kidney dialysis and infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be useful. Also contemplated is local perfusion. Preferred embodiments include intravenous and subcutaneous routes.

[0215] Dosage forms of pharmaceutical compositions suitable for parenteral administration suitably comprise the active ingredient in association with a pharma- ceutically acceptable carrier / excipient, such as sterile water or sterile isotonic saline. Such dosage forms may be prepared, packaged, or distributed in a form suitable for bolus or continuous administration. Injectable dosage forms may be prepared, packaged, or distributed in unit dosage form, for example in ampoules or multi-dose containers containing a preservative. Dosage forms for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like. Such dosage forms may also contain one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of the composition of the invention for parenteral administration, the active ingredient is dissolved in a suitable base (e.g., sterile water or sterile isotonic saline) prior to parenteral administration of the reconstituted formulation. The parenteral dosage forms may be provided in a dry form (i.e., powder or granules) for dissolution in a suitable carrier, such as sterile pyrogen-free water. Parenteral dosage forms also include aqueous solutions that may contain fillers, such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9, and more preferably at a pH of 4.5 to 7), although for certain applications, more suitable dosage forms may be sterile non-aqueous solutions, or dry forms for use in combination with a suitable carrier, such as sterile pyrogen-free water. Examples of parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms may be buffered if desired. Other suitable dosage forms for parenteral administration may include those that contain the active ingredient in microcrystalline form or in a liposomal preparation. Dosage forms for parenteral administration may be made for immediate and / or modified release. Dosage forms with modified release include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0216] For example, in one aspect, sterile injectable solutions may be prepared by incorporating the trispecific anti-PD-L1 antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile solvent containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation is freeze-drying (lyophilization), which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and with the use of surfactants. Prolonged absorption of the injectable compositions may be achieved by including in the composition an absorption-retarding agent, such as monostearate salts and gelatin, and / or by modified release coatings (e.g., slow release coatings).

[0217] The anti-PD-L1 antibodies of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder inhaler (alone, in a mixture, or in the form of particles of the mixed components, e.g. mixed with a suitable pharmaceutically acceptable bulking agent) with or without a suitable propellant, from a dry powder inhaler, such as an aerosol pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydraulic principles to produce a fine mist) or nebulizer, or as nasal drops.

[0218] The pressurized container, pump, spray, atomizer, or nebulizer will generally contain a solution or suspension of a binding molecule of the invention, e.g., with a propellant as a solvent, an agent suitable for dispersing, dissolving, or prolonging the release of the active agent.

[0219] Before use as a dry powder or suspension, the drug is usually micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be accomplished by any suitable comminuting method, such as milling in a spiral jet mill, a jet mill fluidized bed, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0220] Capsules, blisters and cartridges for use in an inhaler or insuffloator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base and active modifier.

[0221] A suitable formulation of solution for use in an atomizer that produces a fine mist using electrohydraulic principles may contain a suitable dose of the anti-PD-L1 antibodies of the invention per actuation, and the volume per actuation may vary, for example from 1 μl to 200 μl, more preferably from 1 μl to 100 μl.

[0222] For dosage forms of the invention intended for inhaled / intranasal administration, suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added.

[0223] Dosage forms for parenteral administration may be made to be immediate and / or modified release. Dosage forms with modified release include delayed, sustained, pulsed, controlled, targeted and programmed release.

[0224] In the case of dry powder inhalers and aerosols, the dosage unit is established by a valve which delivers a measured amount. Units according to the invention are usually configured to administer a measured dose or "injection" of the binding molecule of the invention. The total daily dose will generally be administered in a single dose or more frequently as divided doses over the day.

[0225] The anti-PD-L1 antibodies of the invention may also be formulated in dosage forms for oral administration, which may involve swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration, whereby the compound enters the blood stream directly from the mouth.

[0226] Dosage forms suitable for oral administration include solid, semi-solid and liquid systems, such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids, or powders; lozenges (including liquid-filled); chewable forms; gels; fast dissolving dosage forms; films; suppositories; sprays; and buccal / mucoadhesive patches.

[0227] Liquid dosage forms include suspensions, solutions, syrups and elixirs.These formulations can be used as fillers in soft or hard capsules (e.g., made of gelatin or hydroxypropylmethylcellulose), and typically contain a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or suitable oil, and one or more emulsifiers and / or suspending agents.Liquid dosage forms can also be prepared by reduction of solids, for example, from sachets.

[0228] Therapeutic Uses of the Anti-PD-L1 Antibodies of the Invention In one aspect, the anti-PD-L1 antibodies of the invention are used to treat diseases and disorders associated with PD-L1 activity, such as a disease or disorder selected from the group of: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary site, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, CRC MSI (microsatellite instability high colorectal cancer).

[0229] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject. The subject may be male or female of any age. In the case of a tumor (e.g., cancer), a therapeutically effective amount of an antibody or antibody fragment (e.g., an antibody or antibody fragment that specifically binds to PD-L1) may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. The antibody or antibody fragment may also prevent to some extent the proliferation of existing cancer cells and / or kill said cancer cells, providing a cytostatic and / or cytotoxic effect. For cancer therapy, efficacy may be measured, for example, in terms of life span, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life. It is also possible to measure it by

[0230] As used herein, the terms "co-formulation," "co-formulated," and "in combination," referring to an anti-PD-L1 antibody and one or more other therapeutic agents, are deemed to mean, refer to, or include the following: 1) simultaneous administration to a patient in need of treatment of such a combination of an anti-PD-L1 antibody of the invention and a therapeutic agent, wherein such components are formulated together in a single dosage form such that said components are released to said patient substantially simultaneously; 2) simultaneous administration of such a combination of an anti-PD-L1 antibody of the invention and a therapeutic agent to a patient in need of such treatment, wherein such components are separately formulated into different dosage forms such that, upon about simultaneous introduction thereof into the patient, said components are released to the patient substantially simultaneously; 3) sequential administration of such a combination of an anti-PD-L1 antibody of the invention and a therapeutic agent to a patient in need of treatment, where such components are formulated separately from one another in distinct dosage forms which, when ingested sequentially by the patient with a sufficient time interval between each administration, result in the components being released to the patient at substantially different times; and 4) Sequential administration of such combinations of anti-PD-L1 antibodies of the invention and therapeutic agents to a patient in need of treatment, where such components are formulated together in a single dosage form from which the release of the components is controlled so that they are released to the patient simultaneously, sequentially or together at the same and / or different time points, and where each part may be administered by one route or by different routes.

[0231] The anti-PD-L1 antibodies of the present invention may be prescribed without additional therapeutic treatment, i.e., as an individual therapy. Furthermore, the treatment of the anti-PD-L1 antibodies of the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the anti-PD-L1 antibodies may be administered or combined with another drug / drug therapy for the treatment of cancer.

[0232] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , and radioactive isotopes of Lu), chemotherapeutic agents, and fragments and / or variants thereof, are intended to include toxins of bacterial, fungal, plant or animal origin, such as small molecule toxins or enzymatically active toxins.

[0233] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelarnine; acetogenins, particularly bullatacin and and blattacinone; delta-9-tetrahydrocannabinol (dronabinol, MARENOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs, topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podof phosphate; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as the enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma II and calicheamicin omega II (e.g., Angew, Chem. Intl. Ed. Engl., 33: 183-186 (1994))); the dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (including MYOCET®, PEGylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafer (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate;Hydroxyurea; Lentinan; Lonidainine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; schizofiran; spirofermalium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A and anguidine); urethanes; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANE), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; metriotrexate; platinum agents such as ci; suplatin, oxaliplatin, and carboplatin; the vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®, and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopeptide phosphonates such as clodronate (e.g. BENEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®).alendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOOPE® vaccine and gene therapy vaccines, such as the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which is an abbreviation for a treatment schedule with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN).

[0234] Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, e.g., antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxyphene, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyphene, keoxifene, and selective estrogen receptor modulators (SERMs), e.g., SERM3; pure antiestrogens without agonist properties, e.g., fulvestrant (FASLODEX®), and EM800 (SEQ ID NO: 1). These agents may also block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels; aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrazole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide, and other aromatase inhibitors, including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazoles; leuprolide (LUPRON® and and ELIGARD®), goserelin, buserelin, and tripterelin; progestins, such as megestrol acetate and medroxyprogesterone acetate, estrogens, such as diethylstilbestrol and premarin, and androgens / retinoids, such as sex steroids, including fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens, such as flutamide, nilutamide, and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; as well as combinations of two or more of the above.

[0235] Other therapeutic agents that may be used in combination with the anti-PD-L1 antibodies of the invention may be growth factor function inhibitors, for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (e.g. the anti-erbB2 antibody trastuzumab [Herceptin], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab [Erbitux, C225], and any of the growth factor or growth factor receptor antibodies disclosed in Stern et al. Critical reviews in oncology / haematology, 2005, Vol. 54, pp11-29); anti-angiogenic agents, such as the inhibitory effect of vascular endothelial growth factor (e.g. the anti-vascular endothelial growth factor antibody, bevacizumab (Avastin)), anti-vascular endothelial growth factor receptor antibodies, such as anti-KDR antibodies and anti-flt1 antibodies; antisense therapies, such as those directed against the above targets, for example ISIS 2503, an anti-ras antisense agent or G3139 (Genasense), an anti-bcl2 antisense agent; approaches involving replacement of abnormal genes, such as abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches using cytosine deaminase, thymidine kinase or bacterial nitroreductase enzymes, and approaches aimed at increasing a patient's tolerance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy; gene therapy approaches, including monoclonal antibodies directed against CD52, such as anti-CD22 directed antibodies, treatment with alemtuzumab (Campus-1H), or treatment with antibodies directed against CD22, ex vivo and in vivo approaches that increase the immunogenicity of the patient's tumor cells, transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony-stimulating factor, approaches that aim to reduce T cell anergy, for example treatment with monoclonal antibodies that inhibit CTLA-4, approaches using transfected immune cells, for example dendritic cells transfected with cytokines, approaches using tumor cell lines transfected with cytokines, and approaches using anti-idiotypic antibodies, ex vivo approaches These include immunotherapeutic approaches, including adoptive transfer of T cells using T cells that have been exposed to non-specific activation in vivo or targeted to a specific antigen of interest; protein degradation inhibitors, e.g., proteasome inhibitors, e.g., Velcade (bortezomib); and biotherapeutic approaches using peptides or proteins (e.g., antibodies or soluble constructs of external receptor domains) that, for example, sequester ligand receptors, block ligand binding to receptors, or attenuate receptor signaling (e.g., by increasing receptor degradation or decreasing expression levels).

[0236] Dosage and Route of Administration The anti-PD-L1 antibodies of the invention are administered in an amount effective to treat the subject's condition, i.e., at a dosage and for a period of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition, age, sex, and weight of the patient to be treated, and whether administration of the anti-PD-L1 antibody is an individual treatment or is performed in combination with one or more additional anti-autoimmune or anti-inflammatory therapies.

[0237] Dosage schedules may be adjusted to provide the optimum desired response. A single dose may be administered, or several separate doses may be administered over a period of time, or the dose may be proportionally reduced or increased depending on the acuteness of the therapeutic situation. Particularly useful is the production of parenteral compositions in unit dosage forms for simplicity of administration and uniformity of dosage. Herein, unit dosage forms refer to physically discrete units suitable as unitary doses for the patient / subject to be treated: each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. Typically, the specifications of the unit dosage forms of the present invention are defined and are directly dependent on (a) the unique properties of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the formulation technology for such active compounds to treat susceptibility in the subject.

[0238] Therefore, based on the disclosure provided herein, those skilled in the art will recognize that the dose and dosing schedule will be adjusted according to methods well known in the therapeutic art. That is, the maximum tolerated dose can be easily established, and the effective amount that provides a detectable therapeutic benefit to the patient can also be determined, as can the time requirements for administration of each agent that provides a detectable therapeutic benefit to the patient. Thus, some doses and dosing schedules are provided herein as examples, but these examples do not in any way limit the doses and dosing schedules that may be required for a patient when implementing the present invention.

[0239] It is noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include one or more doses. Furthermore, it is to be understood that for any particular patient, the particular dosing schedule should be adjusted over time according to the individual needs and at the discretion of the physician administering or supervising the administration of the composition, and that the concentration ranges herein are provided by way of example only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, dosing schedules for the compositions of the invention may be based on different factors, including the type of disease, age, weight, sex, health status of the patient, severity of the condition, route of administration, and the particular anti-PD-L1 antibody used. Thus, dosing regimens may vary widely, but are routinely determinable using standard methods. For example, doses may be determined based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects, e.g., toxic effects, or laboratory values. Thus, the present invention includes individual dose escalation, as determined by one of skill in the art. Determination of the necessary doses and regimens is well known to the relevant art, and will be recognized by one of skill in the art upon insight into the disclosure herein.

[0240] Examples of suitable routes of administration are provided above. Suitable doses of the anti-PD-L1 antibodies of the invention are in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, intended to include about 0.5-50 mg / kg, such as about 1-20 mg / kg. For example, the anti-PD-L1 antibodies may be administered at at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, such as at least 3 mg / kg, including at least 4 mg / kg, such as at least 5 mg / kg; and for example up to 50 mg / kg, including up to 30 mg / kg, such as up to 20 mg / kg, including up to 15 mg / kg. Administration will usually be at appropriate time intervals, such as once weekly, once every two weeks, once every three weeks or Once every four weeks, and repeated for as long as the physician deems advisable, and if necessary, the dose may be increased or decreased as desired by the physician.

[0241] Articles (products) and kits Further aspects of the present invention relate to the treatment of cancer, particularly HNSCC, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, The article includes a product for the treatment of bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, kidney cancer, ovarian cancer, Hodgkin's lymphoma, CRC MSI. The product is a container and a label or package insert that may be placed on or inserted into the container. Acceptable containers are, for example, cans, vials, syringes, and the like. The container may be made of a variety of materials, such as glass or plastic. The container contains a composition effective for treating a particular condition and may have a sterile inlet channel (e.g., the container may be an intravenous solution bag or vial provided with a stopper pierceable by a needle for hypodermic injection). At least one active agent in the composition is an anti-PD-L1 antibody of the present invention. The label or package insert indicates the use of the composition to treat a particular condition. The label or package insert should further contain instructions for administering the antibody composition to a patient.

[0242] The package insert contains the usual instructions for use that accompany the packaging of therapeutic products offered for sale, including exemplary information on indications, use, dosage, route of administration, contraindications and / or precautions for the use of such therapeutic products. In one embodiment of the invention, the package leaflet indicates that the composition is used for the treatment of cancer, particularly HNSCC, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, CRC MSI.

[0243] Additionally, the article may further include a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWI), phosphate-buffered saline, Ringer's solution, and dextrose solution.In addition, it may include other products necessary from a commercial and user standpoint, particularly other buffers, diluents, filters, needles, and syringes.

[0244] The present invention also relates to kits that may be used to detect PD-L1 for a variety of purposes, for example in tissues, cells or body fluids of mammalian organs. Such kits may be suitable for screening for PD-L1 disease. The kits include a specific binding agent or antibody of the invention and a means for indicating the reactive behavior, if any, of the specific binding agent or antibody with PD-L1. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the PD-L1 binding antibody is labeled. In another embodiment, the antibody is an unlabeled primary antibody and the kit further includes a primary antibody detection agent. In one embodiment, the detection agent includes a labeled secondary antibody that is an anti-immunoglobulin. The antibody may be labeled with a marker selected from the group consisting of a fluorochrome, an enzyme, a radionuclide and a radio-opacifier. The kit may include an antibody for detecting and quantifying PD-L1 in vitro, for example in an ELISA or Western blotting. As with the article, the kit may also include a container and a label or package insert to be placed on or within the container. The container holds a composition comprising at least one anti-PD-L1 antibody according to the invention. Additional containers may contain, for example, diluents and buffers, control antibodies. The label or package insert may include a description of the composition and instructions for use in vitro or for diagnostic purposes.

[0245] Diagnostic Uses and Compositions The anti-PD-L1 antibodies of the invention may also be used in diagnostic processes (e.g. in vitro or in vivo). For example, the anti-PD-L1 antibodies may be used to detect or measure the levels of PD-L1 in a sample obtained from a patient (e.g. a tissue sample or a body fluid sample, such as inflammatory exudate, blood, serum, intestinal fluid, saliva or urine). Suitable detection and measurement techniques include immunological techniques, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence analysis, radioimmunoassay, and immunohistology. The present invention further provides kits (e.g. diagnostic kits) comprising the anti-PD-L1 antibodies as described herein. We provide a service that provides a

[0246] In order that the present invention may be best understood, the following examples are provided, which are provided for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any manner.

[0247] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of avoiding ambiguity, those skilled in the art will clearly recognize that certain changes and modifications may be made based on the concepts disclosed herein without departing from the scope of the basic principles and attendant aspects of the invention. EXAMPLES

[0248] Example 1 Production of recombinant antigens and antibodies in suspension mammalian cell cultures Antibodies and antigens were produced in established cell lines derived from Chinese hamster ovary cells (CHO-K1) according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91(6):670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun;28(11):843-848; Biotechnol Bioeng. 2003 Nov 5;84(3):332-342]. Cells constitutively expressing the gene for EBNA1 protein (Epstein-Barr virus nuclear antigen 1) were used. Suspension cultures were performed in flasks on an orbital shaker using serum-free medium from Life Technologies and according to the manufacturer's instructions. For transient expression, linear polyethyleneimine (PEI MAX, Polyscienecs) was used to culture the EBNA1 protein in 200 µL of 1 ... * 10 6 Cells were transfected at a concentration of 1000 μg / ml. The DNA / PEI ratio was 1:3 / 1:10. Five to seven days after transfection, the cell culture was centrifuged at 2000 g for 20 min and filtered through a 0.22 μm filter. The target protein was isolated from the culture medium by affinity chromatography.

[0249] Recombinant PD-L1 protein containing an EPEA tag (glutamic acid-proline-glutamic acid-alanine) at the C-terminus of the protein was isolated and purified from culture fluid using CaptureSelect C-tag affinity matrix adsorbent. Culture fluid was passed through a chromatography column pre-packed with 5 ml of C-tag adsorbent, and the column was then washed with 25 ml of PBS to wash away non-specific binding components. Bound antigen was eluted using mild conditions of 20 mM Tris, 2M MgCl2 pH 7.0-7.4. The protein was then dialyzed into PBS (pH 7.4) using a semi-permeable dialysis membrane, filtered (0.22 μm), transferred to tubing, and stored at -70°C.

[0250] Recombinant proteins PD-1 and PD-L1-Fc were isolated and purified from the culture fluid using a protein A affinity HPLC column. The clarified culture fluid was passed through a 5ml HiTrap rProtein A Sepharose FF column (GE Healthcare) equilibrated with phosphate buffered saline (PBS, pH 7.4). The column was then washed with 5 volumes of PBS to remove non-specifically bound components. The bound antigen was eluted with 0.1M glycine buffer pH 3. The main protein elution peak was collected and brought to neutral pH with 1M Tris buffer (pH 8). All steps were performed at a flow rate of 110cm / h. The isolated proteins were then dialyzed into PBS (pH 7.4) using a semipermeable dialysis membrane, filtered (0.22μm), transferred to tubes, and stored at -70℃.

[0251] The IgG1 antibody was purified on a 1 ml HiTrap rProtein A FF column (GE Healthcare) according to the method described above for the antigen. The purity of the resulting protein solution was assessed by SDS-PAGE (Figures 4A and 4B).

[0252] Example 2 Generation of a naive human antibody Fab library, MeganLibTM Total B lymphocyte RNA from blood samples collected from over 1000 human donors was isolated using the RNeasy mini kit (QIAGEN) according to the protocol provided. RNA concentration assays were performed using the Nanovue kit (GE Healthcare) and the quality of the isolated RNA was tested by 1.5% agarose gel electrophoresis.

[0253] Reverse transcription reactions were carried out using the MMLV RT kit (Evrogen) according to the recommended protocol, using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0254] The reverse transcriptase was used as a template in a two-step polymerase chain reaction to generate variable domain genes flanked by restriction sites using oligonucleotide sets according to the authors' protocol [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

[0255] The resulting DNA preparation VL-CK-VH (Figure 1) was treated with the restriction enzymes NheI / Eco91I and ligated to the original phagemid pH5 (Figure 2). The ligation product was transformed into electrocompetent SS320 cells prepared according to the protocol described in [Methods Enzymol. 2000;328:333-63.]. The repertoire of the combinatorial phage Fab display library MeganLibTM consists of 10 11 The phage preparation from the Fab library was prepared according to the method described previously [J Mol Biol. 1991 Dec 5;222(3):581-97].

[0256] Example 3 Phage antibody Fab library selection Specific phage human anti-PD-L1 Fab antibodies were obtained from the combinatorial phage Fab display library MeganLibTM. Selection was performed against human PD-L1 by phage display technology [Nat Biotechnol. 1996 Mar;14(3):309-14; J Mol Biol.1991 Dec 5;222(3): 581-97] using magnetic particles and a KingFisher Flex instrument, because the use of this technology allows up to 96 different schemes and variations of biopanning to be performed in parallel.

[0257] In biopanning selection, biotinylated PD-L1-Fc was immobilized on the surface of streptavidin magnetic particles at a concentration of 10 μg / ml by incubating the particles with the protein on a rotating device at room temperature for 1 hour. The particles were then washed with PBS (pH 7.4), and then the particles were blocked with 2% skim milk solution on PBS (pH 7.4) for 1 hour. A phage solution in PBS (pH 7.4) containing 2% skim milk was then added to the antigen-bound magnetic particles, and the concentration of phage particles was 2.5 * 10 12 The concentration of phage was 1 / ml. The mixture was incubated for 40 min with stirring. Unbound phages were removed during several washings of the magnetic particles with PBS solution (pH 7.4) containing 0.1% Tween 20. The number of washings was increased from cycle to cycle (10 times in the first cycle, 20 times in the second cycle, and 30 times in the third cycle). Phages that remained bound to the antigen on the magnetic particle surface were removed. The phages were eluted from the particles with 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring, and then neutralized with 1 M TRIS-HCl (pH 7.6). The resulting phages were used to infect E. coli TG1 bacteria; phages were produced in the E. coli and were isolated and used in the next selection round. After 2-3 rounds, DNA (phagemid) was isolated from the phages, and antibody variable domain genes were cloned into an expression vector (Figure 3) for producing Fab in E. coli cells.

[0258] Example 4 Screening of Fabs that specifically bind to human PD-L1 ELISA was used to screen for Fab binding to human PD-L1. As a positive control, a Fab with published sequence, atezolizumab (Genentech), was used. For specific binding assays, ELISA plate wells (Greiner bio one, medium binding) were coated with 50 μl of PD-L1-FE (0.2 μg / ml in 1x carbonate buffer), hermetically sealed, and incubated overnight at 4°C. All subsequent steps were carried out according to standard ELISA protocols using a high-throughput automation platform based on GenetixQ-Qpix2xt (Molecular Device) and Tecan Freedom EVO 200 (Tecan) robotic systems. To block non-specific binding, blocking buffer BB (0.5% skim milk in PBS, 200 μL) was added. Plates were incubated for 1 h at room temperature on a shaker. After washing with PBS-Tween, 50 μl of test cell supernatant containing test Fab mixed with an equal volume of BB was added per well. Again, the plate was incubated and shaken at room temperature for 1 h, after which each plate well was washed three times with PBS-Twin buffer. Once washed, (50 μL / well) anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added at a ratio of 1:5000 in PBS-Tween. The plate was shaken on a rotary shaker (50 min, room temperature) and washed three times with FSB-Twin buffer as above. The colorimetric signal was developed by adding TMB (50 μL / well) until saturation (average 3-5 min), and then development was stopped by adding stop solution (30 μL / well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan). The level of antibody binding was proportional to the color signal produced. Clones with color signals greater than that from the control antibody were tested for non-specific binding by ELISA.

[0259] Example 5 Analysis of non-specific binding of selected Fabs to other antigens ELISA is used to measure the non-specific binding of the studied Fab fragments with other antigens. The study was performed as described above, but using IL6R-Fc, INFα2b, PCSK9-VG-FE, PD-1-Fc (2.5 μg / ml in 1x carbonate buffer) as antigens for immobilization. PD-L1-Fc (0.2 μg / ml in 1x carbonate buffer) was used as a control for specific binding. All further steps were carried out according to standard ELISA protocols using a high-throughput automation platform based on GenetixQ-Qpix2xt (Molecular Device) and Tecan Freedom EVO 200 (Tecan) robotic systems. Clones with color signals of non-specific binding not greater than the signals from specific binding were tested in a competitive ELISA assay to identify antagonist Fabs that block the interaction between the ligand and the receptor.

[0260] Example 6 Competitive ELISA to block the interaction between PD-L1 and its PD-1 receptor A competitive ELISA was used to test preselected anti-human PD-L1 specific Fabs for their ability to block interaction with the PD-1 receptor. As a positive control antagonist, a Fab with published sequence, atezolizumab (Genentech), was used.

[0261] PD-1-Fc was immobilized in 50 μl of 1 μg / ml concentration in 1x carbonate buffer onto ELISA plate wells (Greiner bio one medium binding) and incubated overnight at 4°C. All subsequent steps were carried out according to standard ELISA protocols using a high-throughput automation platform based on GenetixQ-Qpix2xt (Molecular Device) and Tecan Freedom EVO 200 (Tecan) robotic systems. Blocking buffer BB (0.5% skim milk in PBS, 200 μL) was added to block non-specific binding. Plates were incubated on a shaker at room temperature for 1 h.

[0262] In parallel, cell supernatants containing test Fab and PD-L1-Fc (at a final concentration of 2 μg / ml in PBS-Twin) were mixed in a 1:1 ratio in a non-adsorbent plate and incubated at room temperature and with shaking at 500 rpm for 45 min.

[0263] After BB washing from the plate containing PD-1 receptor, the mixture of Fab and PD-L1 was added thereto and incubated at room temperature and with shaking at 500 rpm for 45 min. Then, each plate well was washed three times with PBS-Twin buffer and 50 μl / well of anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientifc) was added at a ratio of 1:5000 in PBS-Tween. These were incubated at room temperature and with shaking at 500 rpm for 45 min, after which each plate well was washed three times with PBS-Twin buffer as above. The colorimetric signal was developed by adding TMB (50 μL / well) until saturation (average 3-5 min), and then the color development was stopped by adding stop solution (30 μL / well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan). The level of Fab binding was inversely proportional to the color signal generated. Clones showing blocking at the level of the control Fab antibody, Atezolizumab, were scored as positive and used for further analysis. Variable domain genes from positive clones were sequenced and analyzed on an Applied Biosystems 3130 Genetic Analyzer instrument (Applied Biosystems) according to standard protocols.

[0264] Example 7 Koff-based comparative screening of human anti-PD-L1 Fab candidates Koff screening was performed using a Pall Forte Bio Octet Red 96 instrument. Anti-FABCH1 biosensors (SA) were rehydrated for 30 min in reaction buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. Reaction buffer was added to the study E. coli supernatant samples to a final concentration of 1x. Anti-FABCH1 biosensors were then immersed in E. coli supernatant containing Fab fragments of candidate antibodies for 12 h at 4°C. The sensors with the Fab fragments immobilized on their surface were transferred into wells containing reaction buffer where a baseline was recorded (60 s). The sensors were then transferred to wells containing analyte solution (PD-L1, 30 μg / mL) for association with antigen-antibody complexes (300 s). The sensors were then transferred back to wells containing reaction buffer for the next dissociation step (600 s). After each experiment, the used sensors were regenerated by placing them three times in regeneration buffer (Gly-HCl, pH 1.7) before they were used in the next experiment. Analysis of the resulting curves was performed using Octet data analysis (version 7.0) software according to standard methods using a 1:1 interaction model.

[0265] Example 8 Enzyme immunoassays of anti-PD-L1 antibody interactions with PD-L1 and other antigens ELISA was used to measure the relative affinity of anti-PD-L1 antibodies and other antigens. For binding assays, ELISA plate wells (Greiner bio one medium binding) were coated with 50μl of PD-L1-Fc, fPCSK9-EPEA, Ang2-H6F, GM-CSF-FE, CD3-ED-Fc, IL17a, CD38-Fc, IL6R-Fc (1μg / ml in 1x carbonate buffer), hermetically sealed, and incubated overnight at 4℃. All subsequent steps were performed according to standard ELISA protocols. Buffer BB (0.5% skim milk in PBS, 200μL) was added to block non-specific binding. Plates were incubated on a shaker at room temperature for 1 hour. After washing once with PBS-Tween, 50 μl of the test BCD-135 antibody at a concentration of 5 μg / ml in PBS-Tween was added per well. The plate was again incubated and shaken at room temperature for 1 hour, after which each plate well was washed three times with PBS-Tween buffer. After washing, (50 μL / well) anti-human Fab was added. HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added at a ratio of 1:5000 in PBS-Tween. Plates were shaken on a rotary shaker (50 min, room temperature) and washed three times with FSB-Twin buffer as above. Colorimetric signal was developed by adding TMB (50 μL / well) until saturation (average 3-5 min), and then color development was stopped by adding stop solution (30 μL / well, 10% sulfuric acid). Color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (Tecan). Antibody binding ratio was proportional to the resulting color signal (Figure 5). Anti-PD-L1 antibodies specifically bind to PD-L1 and not to other antigens studied.

[0266] Example 9 Reactivation of NFAT signaling with anti-PD-L1 antibody in Jurkat-NFAT-PD-1 reporter cell line The human T cell-derived line Jurkat was engineered by introducing two genetic constructs into its genome. One construct encoded the human PD-1 receptor gene. The other encoded the luciferase gene under the control of NFAT-sensitive genetic elements. The result was the Jurkat-NFAT-PD-1 reporter cell line, which expresses the PD-1 receptor on its surface membrane and contains an NFAT-dependent promoter directing luciferase gene transcription. The synthesis of luciferase enzyme in cells of this line is proportional to the level of NFAT activity, which in turn reflects the overall level of T-lymphocyte activation.

[0267] This cell line was used to analyze the activity of anti-PD-L1 antibodies as follows: Activation of TCR receptors by anti-CD3 and anti-CD28 antibodies triggered an intracellular cascade leading to NFAT promoter activation. PD-L1 is presented on the surface of MDA-MB-231 cells activated by interferon. The interaction between PD-L1 and PD-1 inhibited signaling from the TCR receptor to the NFAT promoter. Anti-PD-L1 antibodies cleaved the PD-L1-PD-1 interaction and reactivated intracellular signaling.

[0268] MDA-MB-231 cells were activated with interferon-gamma solution for PD-L1 production; for this purpose, interferon-gamma was added to the cell suspension to a concentration of 20 ng / ml 72 hours prior to the assay, and the cells were then plated in 96-well culture plates at a rate of 10,000 cells / well.

[0269] After 72 hours of activation, the growth medium was removed from the plates containing the MDA-MB-231 cells and the antibodies to be analyzed, controls, etc. were incubated in cell growth medium at 10 μg / ml to 0.001 μg / ml. Dilutions of antibodies and isotype controls were added and incubated for 30 minutes at room temperature.

[0270] In addition, a suspension of Jurkat-NFAT-PD-1 cells and a solution of activating antibody aCD3 / aCD28 / a-mouse IgG were added to each well. The plate was placed in a CO2 incubator for 6 hours.

[0271] For the Luciferase Bio-Glo Luciferase Assay System (Promega), pre-prepared substrate was added to V cells / V substrate. Luminescence levels were measured on a Fluoroscan Ascent (Figure 6). Anti-PD-L1 antibodies reactivated luminescence levels in the Jurkat-PD-1-NFAT reporter line.

[0272] Example 10 Assaying Anti-PD-L1 Antibody Interaction with FcRn and Fcγ Receptors Using Octet RED 96 To assay antibody interactions with FcgRIIIaV, FcgRIa, and FcRn receptors, a Fortebio Octet RED96 instrument was used, with C-terminally biotinylated receptors and streptavidin-coated biosensors (SA-streptavidin).

[0273] Biotinylated receptors were immobilized on the surface of the sensors. An association step was then performed: the sensors with bound antigens were immersed in antibody solutions of different concentrations (previously, a series of antibody dilutions in working buffer were prepared and placed in the appropriate wells of a 96-well plate). After this, a dissociation step was performed: the sensors were transferred from the antibody solutions to wells containing working buffer.

[0274] To assay the antibody affinity constants for FcgRIIIaV and FcgRIa, phosphate buffer pH 7.4 was used, and for FcRn, phosphate buffer pH 6.0 was used.

[0275] The resulting curves were analyzed using Forte Bio Data Analysis 8.2 software and a 1:1 binding model. The results are shown in Figure 7. No binding of the modified IgG1 antibody to Fcg receptors was detected compared to the wild-type variant, suggesting the absence of effector function in the analyzed antibody. The affinity constant for FcRn in the analyzed anti-PD-L1 antibody was 1.69E-08 1 / M.

[0276] Example 11 Enzyme-linked immunosorbent assay of the interaction between anti-PD-L1 antibodies and PD-L1 from different organisms ELISA was used to measure the relative affinity of anti-PD-L1 antibodies from different organisms. For the binding assay, ELISA plate wells (Greiner bio one medium binding) were coated with 50μl of human, cynomolgus, murine, rat, dog, rabbit PD-L1-Fc (0.5μg / ml in 1x carbonate buffer), hermetically sealed, and incubated overnight at 4℃. All subsequent steps were carried out according to the standard ELISA protocol described above. The anti-PD-L1 antibodies specifically bound to human and cynomolgus PD-L1, and did not bind to the other receptors studied (Figure 8).

[0277] Example 12 Analysis of anti-PD-L1 antibody interactions with human and cynomolgus PD-L1 on the Octet RED 96 instrument Antibody binding affinity constants to human and cynomolgus PD-L1 were measured with an OctetRed 96 instrument (ForteBio). BCD-135 antibody at a concentration of 30 μg / ml was non-specifically immobilized onto the surface of a second generation amino-reactive biosensor (ForetBio, AR2G) following the standard protocol, following the manufacturer's instructions for AR2G sensor preparation and immobilization. Assays were performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as reaction buffer. Binding of human and monkey PD-L1 solutions to the sensor-bound antibodies was analyzed in working buffers containing antigen concentrations from 10 μg / ml to 1 μg / ml.

[0278] Reference signal-subtracted binding curves were analyzed using Octet data analysis (version 8.2) software according to standard methods using a 1:1 interaction model. The anti-PD-L1 antibodies bind specifically and affinely to human and cynomolgus PD-L1 antigens with binding constants of <1.0E-12 and 5.55E-10 1\M, respectively (Figure 9).

[0279] Example 13 Anti-PD-L1 antibody stability determination Dynamic light scattering technique (DLS) was used to evaluate the conformational stability of BCD-135 based on the protein aggregation point. Protein aggregation point determination of the studied protein (1 mg / ml) was performed using a Zetasizer nano ZSP instrument. For this purpose, 0.5 ml of the solution was placed in a dust-free quartz cuvette, which was gradually heated from 50°C to 90°C in the instrument while the scattered light intensity was continuously measured. The BCD-135 antibody showed high conformational stability in 20 mM acetate buffer, with a melting point above 80°C (Figure 10).

[0280] The colloidal stability of the candidates was evaluated by the PEG-protein aggregation method. For the experiment, samples with a protein concentration of 5 mg / ml were used. Calculated amounts of samples, placebo solutions, and PEG6000 solutions were added to the UV spectroscopy plate. All solutions in the wells were mixed well by pipetting. In addition, the solution turbidity was visually evaluated, and the solution optical density at λ=320 nm was also measured. The BCD-135 antibody showed high colloidal stability (Figure 11).

[0281] Antibody thermal stability was evaluated by heat stress at 50° C. for 48 hours in three different buffers: 20 mM phosphate buffer, pH 6.0 (FIG. 12A), 20 mM acetate buffer, pH 5.0 (FIG. 12B) and 20 mM histidine buffer, pH 5.5 (FIG. 12C). Homogeneity control was performed by HPLC method (SEC HPLC).

[0282] [Table 2]

[0283] Test samples with a protein concentration of ~5mg / ml were divided into two portions and placed in separate tubes. The anti-PD-L1 antibodies were placed in a thermostat: for each formulation, one tube was kept in a refrigerator at 4°C, the rest were placed in a thermostat and incubated at 50°C for 72 hours. Once warmed up, the tubes were removed from the thermostat and transferred for analysis (Figures 12A, 12B and 12C, red indicates the control kept at +4°C, blue indicates the sample after heat stress). The anti-PD-L1 antibodies showed high thermal stability in all three buffers (the difference between the aggregate content in the solution before and after heat stress did not exceed 5%): The stability of BCD-135 in normal human serum was also evaluated for 7 days at 37°C. For this purpose, recombinant PDL1 (100 μl, 2.5 μg / ml in 1x carbonate buffer) was added into the wells of a 96-well high-absorbency ELISA plate. This was incubated for 18 hours at 4°C. Furthermore, the contents of the wells were removed and blocking buffer (200 μl of 0.5% skim milk in TBST) was added. The plate was incubated for 30 minutes at 37°C and then washed twice with TBST solution.

[0284] To plot the standard curve, 100 μl of a solution containing BCD-135 diluted in blocking buffer at concentrations of 0; 7.8; 15.6; 31.25; 62.5; 125.0; 250.0 ng / ml was added to the wells of the first vertical row.

[0285] The plate was incubated at 37°C for 30 minutes, and then the plate was washed three times with TBST solution. Further, 100 μl of a solution of anti-human IgG Fc fragment goat polyclonal antibody conjugated with horseradish peroxidase was added to each well. The plate was incubated at 37°C for 30 minutes. The plate was then washed 4-5 times with TBST solution. 100 μl of TMB solution was added to the washed and dried wells to develop the color. The plate was placed in place, protected from light, and incubated at a temperature of 22°C for 20-25 minutes for color development. The reaction was stopped by adding 50 μl of a stop solution of 0.9 M sulfuric acid to the wells. The optical density of the solution in the wells was measured on a microplate reader at a wavelength of 450 nm.

[0286] Based on the data obtained, a calibration curve was plotted (FIG. 13A), showing the optical density dependence on the concentration of BCD-135 added to the wells. The solution optical density arithmetic mean was used to plot the curve. Based on the calibration curve, the BCD-135 concentration values ​​in the samples were found to correspond to the optical density values ​​obtained in the experiment.

[0287] Based on the study results, the BCD-135 concentrations determined in human serum after storage at 37° C. for 7 days were not significantly different from those determined in serum samples prepared immediately prior to analysis ( FIG. 13B ), demonstrating the stability of the antibody.

[0288] Example 14 Construction of a library of BCD-135 mutant antibodies specific for PD-L1 To construct BCD-135 mutant antibodies specific for PD-L1, structural analysis based on 3D modeling was performed using BIOCAD's YLab software package and PD-L1 models (PDB 4ZQK, PDB 4Z18) (see also Example 18). Based on the computational model, a library of BCD-135 genes with partially degenerate codons (FUH G et al., Improving antibody binding affinity and specificity for therapeutic development, Methods Mol Biol., 2009, 525, 353-376) was synthesized at positions in the first and third CDR regions of the heavy chain variable domain of SEQ ID NO: 4 and in the third CDR of the light chain variable domain of SEQ ID NO: 8. The resulting DNA of the randomized genes was cloned into the phage display plasmid pH5 (Figure 2) according to the protocol described in Example 2. Transformation of these constructs into the SS320 strain was performed according to the method [Methods Enzymol. 2000; 5 for libraries according to [328: 333-63] * 10e7 independent transformants were obtained. Phage preparations of the mutant VH BCD-135 library were prepared according to a method previously described [Mol Biol. 1991 Dec 5;222(3):581-97].

[0289] Selection of the resulting phage mutant BCD-135 Fab library was carried out under conditions similar to those described above (Example 3). After the third round of selection of the above library against human recombinant PD-L1 products, ELISA assays performed on the polyclonal phage products revealed significant enrichment and binding greater than 10-fold excess over non-specific binding background.The gene pool from the enriched phage library of BCD-135 mutant Fab antibodies specific for human PD-L1-Fc was recloned into the expression plasmid pLL (Figure 3) containing a myc tag peptide at the C-terminus for ELISA detection.

[0290] Example 15 Comparative ELISA assay of BCD-135 mutant Fab antibodies specific for PD-L1 ELISA is used to measure the binding of the test mutant Fab antibodies to human PD-L1, similar to that described in Example 4. The number of test clones producing BCD-135 mutant Fab antibodies was 400 units. Wild-type BCD-135 Fab antibodies with sequences of SEQ ID NO:4 and SEQ ID NO:8 were used as positive controls.

[0291] As a result, 85 positive clones were selected that gave signals higher or similar to those of the control wild-type BCD-135 Fab antibody (values ​​ranging from 0.7 to 1.2 relative units). Example 16 Characterization of BCD-135 mutant Fab antibodies specific for PD-L1 85 ELISA screened (Example 15) positive clone candidates that gave signals higher or similar to the control wild type BCD-135 Fab antibody were analyzed by PCR according to the protocol recommended by the manufacturer. Applied Biosystems 3130 The mutant Fab antibodies were sequenced on a sequencer, resulting in 25 sequence-unique clones. Eight mutant Fab antibodies from the unique clones were further analyzed by quantifying the kinetic dissociation constants on an Octet Red 96 instrument as described in Example 7.

[0292] Koff screening was performed using a Pall Forte Bio Octet Red 96 instrument, similar to that shown in Example 7. Anti-FABCH1 biosensors (SA) were rehydrated for 30 min in reaction buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. Reaction buffer was added to the study E. coli supernatant samples to a final concentration of 1x. Anti-FABCH1 biosensors were then immersed in E. coli supernatant containing the Fab fragments of the candidate antibodies for 12 h at 4°C. The sensors with the Fab fragments immobilized on their surface were transferred into wells containing reaction buffer, where a baseline was recorded (60 s). The sensors were then transferred to wells containing analyte solution (PD-L1, 30 μg / mL) for association with the antigen-antibody complex (300 s). The sensors were then transferred back to wells containing reaction buffer for the next dissociation step (600 s). After each experiment, the used sensors were regenerated by placing them three times in regeneration buffer (Gly-HCl, pH 1.7) before they were used in the next experiment. Analysis of the resulting curves was performed using Octet data analysis (version 7.0) software according to standard methods using a 1:1 interaction model.

[0293] As a result, the kinetic dissociation constants of the mutant Fab antibodies were obtained, which showed comparable properties to the wild type of BCD-135 Fab and thus a tolerance of up to 8% of substitutions for the three CDRs of the variable domain.

[0294] Anti-PDL1 VH of BCD-135

[0295] [ka]

[0296] [Table 3]

[0297] Example 17 Generation of stable cell lines, production and purification of anti-PD-L1 antibodies A stable cell line producing the monoclonal antibody BCD-135 was obtained by electroporating the parent suspension cell line CHO-S with a vector construct containing the light and heavy chains of the antibody in an optimized ratio using a Neon transfection system (Life Technologies) device. Clonal lines with high levels of productivity (higher than 1000 mg / L) were generated using the ClonePix robotic platform (Molecular Devices) and a preliminary stage of minipool selection with antibiotics in different culture formats. Productivity assays were performed using an Octet RED96 analytical system (Pall Life Sciences). DOE of basal medium selection and incubation schedules was performed using a Biomek FX robotics computer-based system (Beckman Coulter). Serum-free medium and animal protein-free feeding were used to incubate the producing cells. HyLone disposable bioreactors (Thermo Fisher Scientific) with a working volume of 50 l were used. We prepared BCD-135 for preclinical studies in a fermenter (Bio-Rad Scientific). Ta.

[0298] The culture fluid was clarified on Millistak COHC depth filters (Merck-Millipore). Primary purification of antibodies was performed from the clarified CL on affinity sorbents containing protein A. Specific elution of the target protein was performed under acidic conditions at pH 3.3-3.8 in glycine buffer. The resulting eluate was kept at acidic pH for 30-60 min to inactivate viruses and then neutralized to pH 6.5-7.0 with 1 M Tris-OH solution. Final chromatographic purification was performed in breakthrough mode on CaptoAdhere sorbents (GE HealthCare LifeSciences) to remove residual DNA, producer cell proteins, cleaved ligands of the affinity sorbent, antibody aggregates and fragments. For this purpose, a pH of 6.5-7.0, low conductivity (<3 mS / cm 2The protein solution was passed through the prepared adsorbent at 100° C. for 24 hours. The purified protein was subjected to antiviral filtration using a Viresolve PRO filter set (Millipore), concentration, and diafiltration against a final buffer containing acetate buffer (pH 5.0-5.5) and trehalose. The resulting protein concentration was 50 mg / ml or higher.

[0299] Example 18 In silico modeling of the BCD-135 antibody and human PD-L1 complex To generate a BCD-135 mutant antibody specific for PD-L1, structural analysis based on 3D modeling was performed using Schrodinger's Schrodinger Suite software and BIOCAD's YLAB package. Focusing on PD-1 as the target crystal structure, PDB 5C3T was selected because it has more crystallized amino acids than the classical 4ZQK structure. Docking was performed using the HEDGE apparatus (part of BIOCAD's YLab package). Selection of optimal positions was performed by estimating the free energy at 10 nanosecond molecular dynamics intervals (Desmond apparatus, part of Schrodinger Suite package). Visualization of the resulting structure was generated using Schrodinger's PyMOL apparatus. The model including the variable domain BCD-135 is shown in Figure 14A, while Figure 14B shows the regions of antigen and antibody interaction with isolated amino acid residues that form tight protein-protein contacts.

[0300] Table B presents important amino acid residues of both the antibody and the antigen that mediate tight protein-protein interactions.

[0301] [Table 4]

[0302] Table B. The center column shows the amino acid residues of the BCD-135 antibody that interact with human PD-L1. The right column lists the corresponding amino acid residues of the PD-L1 antigen that interact with the BCD-135 antibody. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] A monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-L1: - a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:3; - a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:7 The monoclonal antibody or antigen-binding fragment thereof comprising: [Aspect 2] 2. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:3. [Aspect 3] 2. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:7. [Aspect 4] 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain of the heavy chain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 1 to 3. [Aspect 5] 5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain variable domain comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 3. [Aspect 6] 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 5 to 7. [Aspect 7] 7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein the light chain variable domain comprises an amino acid sequence of any one of SEQ ID NOs: 5 to 7. [Aspect 8] 2. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, - the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NOs: 1-3; - the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5-7 The monoclonal antibody or antigen-binding fragment thereof. [Aspect 9] 9. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 8, - the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1-3; - the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5 to 7 The monoclonal antibody or antigen-binding fragment thereof. [Aspect 10] 2. The monoclonal antibody or antigen-binding fragment thereof of embodiment 1, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:4. [Aspect 11] 11. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 10, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4. [Aspect 12] 2. The monoclonal antibody or antigen-binding fragment thereof of embodiment 1, wherein the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:8. [Aspect 13] 13. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 12, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:8. [Aspect 14] The monoclonal antibody or antigen-binding fragment thereof according to embodiment 1. - the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:4; - the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:8 The monoclonal antibody or antigen-binding fragment thereof. [Aspect 15] 15. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 14. - the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4; the light chain variable domain comprises the amino acid sequence of SEQ ID NO:8 The monoclonal antibody or antigen-binding fragment thereof. [Aspect 16] 2. The monoclonal antibody of embodiment 1, comprising: - a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 9; a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10 The monoclonal antibody comprising: [Aspect 17] 17. The monoclonal antibody according to embodiment 16, comprising: - the heavy chain comprises the amino acid sequence of SEQ ID NO: 9; the light chain comprises the amino acid sequence of SEQ ID NO: 10 The monoclonal antibody. [Aspect 18] The monoclonal antibody of embodiment 1, wherein the antibody that specifically binds to PD-L1 is a full length IgG antibody. [Aspect 19] 20. The monoclonal antibody of embodiment 18, wherein the full length IgG antibody is of the human antibody IgG1, IgG2, IgG3, IgG4 isotype. [Aspect 20] 20. The monoclonal antibody of embodiment 19, wherein the full length IgG antibody is of the human antibody IgG1 isotype. [Aspect 21] A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 20. [Aspect 22] 22. The nucleic acid of embodiment 21, which is DNA. [Aspect 23] 23. An expression vector comprising the nucleic acid of embodiment 21 or embodiment 22. [Aspect 24] A method for producing a host cell adapted to produce the antibody or antigen-binding fragment thereof according to any one of aspects 1 to 20, the method comprising the step of transforming the host cell with an expression vector according to aspect 23. [Aspect 25] A host cell for producing the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 20, comprising a nucleic acid of embodiment 21 or embodiment 22. [Aspect 26] A method for producing the antibody or antigen-binding fragment thereof of any one of aspects 1 to 20, comprising incubating a host cell according to aspect 25 in a culture medium under conditions sufficient to obtain the antibody, and optionally followed by isolation and purification of the obtained antibody. [Aspect 27] 21. A pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1, comprising the antibody or antigen-binding fragment thereof of any one of aspects 1 to 20, and one or more pharma- ceutically acceptable excipients. [Aspect 28] 28. The pharmaceutical composition according to embodiment 27, for the prevention or treatment of a disease or disorder mediated by PD-L1, wherein the disease or disorder mediated by PD-L1 is one of: SCCHN (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI-H CRC (microsatellite instability high colorectal cancer). [Aspect 29] 21. A pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by PD-L1, comprising an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 20, and at least one therapeutic anti-tumor compound. [Aspect 30] 30. The pharmaceutical combination according to embodiment 29 for the prevention or treatment of a disease or disorder mediated by PD-L1, wherein the disease or disorder mediated by PD-L1 is one of: SCCHN (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI-H CRC (colorectal cancer with high microsatellite instability). [Aspect 31] 31. The pharmaceutical combination according to any one of aspects 29 to 30, wherein the therapeutic anti-tumor compound is selected from the group consisting of a chemotherapeutic agent, an antibody or an anti-hormonal agent. [Aspect 32] 21. A method for inhibiting the biological activity of PD-L1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in any one of aspects 1 to 20. [Aspect 33] Use of the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 20, or the pharmaceutical composition of embodiment 27, for treating a disease or disorder mediated by PD-L1 in a subject in need thereof. [Aspect 34] The use according to embodiment 33, wherein the disease or disorder is one of: SCCHN (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI-H CRC (colorectal cancer with high microsatellite instability).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-L1: a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, a CDR3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6 and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; The monoclonal antibody or antigen-binding fragment thereof comprising:

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

4.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 , wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

8.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1. - the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4; - the light chain variable domain comprises the amino acid sequence of SEQ ID NO:8 The monoclonal antibody or antigen-binding fragment thereof.

5. 2. The monoclonal antibody of claim 1, comprising: - the heavy chain comprises the amino acid sequence of SEQ ID NO:9; the light chain comprises the amino acid sequence of SEQ ID NO: 10 The monoclonal antibody.

6. The monoclonal antibody of claim 1, wherein the antibody that specifically binds to PD-L1 is a full-length IgG antibody, wherein the full-length IgG antibody is a human antibody of the isotype IgG1, IgG2, IgG3, or IgG4.

7. A nucleic acid encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 6, said nucleic acid being DNA.

8. An expression vector comprising the nucleic acid of claim 7.

9. 9. A method for producing a host cell adapted to produce the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, comprising transforming the host cell with an expression vector according to claim 8.

10. A host cell for producing the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, comprising the nucleic acid of claim 7.

11. A method for producing an antibody or antigen-binding fragment thereof of any one of claims 1 to 6, comprising incubating a host cell of claim 10 in a culture medium under conditions sufficient to obtain the antibody.

12. The method of claim 11, further comprising isolating and purifying the antibody obtained.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, and one or more pharma- ceutically acceptable excipients.

14. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and at least one therapeutic anti-tumor compound.

15. 15. The pharmaceutical combination of claim 14, wherein the therapeutic anti-tumor compound is selected from the group consisting of a chemotherapeutic agent, an antibody or an anti-hormonal agent.

16. A pharmaceutical composition according to claim 13 or a pharmaceutical combination according to any one of claims 14-15 for the prevention or treatment of a disease or disorder mediated by PD-L1.

17. 16. A pharmaceutical composition according to claim 13 or a pharmaceutical combination according to any one of claims 14-15 for the prevention or treatment of a disease or disorder mediated by PD-L1, wherein the disease or disorder mediated by PD-L1 is one of: SCCHN (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI-H CRC (microsatellite instability high colorectal cancer).

18. 10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 in the manufacture of a pharmaceutical composition for treating a disease or disorder mediated by PD-L1.

19. 19. The use according to claim 18, wherein the disease or disorder is one of: SCCHN (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI-H CRC (microsatellite instability high colorectal cancer).

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