MONOCLONAL ANTIBODY FOR PD-L1.

Abstract

The present invention relates to the field of biotechnology and proposes antibodies that bind specifically to PD-L1. Furthermore, the discovery relates to the DNA encoding these antibodies, the corresponding expression vectors and production methods, as well as treatment methods that utilize these antibodies.

Description

[0001] Monoclonal antibody to PD-L1

[0002] Field of technology

[0003] The present invention relates to the field of biotechnology, namely to antibodies or antigen-binding fragments thereof, and their use. More specifically, the present invention relates to a monoclonal antibody that specifically binds to PD-L1 (CD274, B7-H1, programmed cell death receptor ligand 1). The invention also relates to a nucleic acid encoding this antibody or antigen-binding fragment thereof, an expression vector, a method for producing the antibody, and the use of the antibody to enhance T-cell function for upregulating cell-mediated immune responses and for the treatment of disorders associated with T-cell dysfunction, such as tumor immunity and for the treatment of cancer.

[0004] State of the art

[0005] Development and activation of lymphocytes

[0006] The two main types of lymphocytes in humans are T-type (thymocytes) and B-type (bone marrow-derived). These cells arise from hematopoietic stem cells in the bone marrow and embryonic liver, which are programmed for lymphoid development. The progeny of these stem cells mature through various pathways into B- or T-lymphocytes. Human B-lymphocyte development occurs exclusively in the bone marrow. T-cells, on the other hand, develop from immature precursors that leave the bone marrow and travel through the bloodstream to the thymus, where they proliferate and differentiate into mature T-lymphocytes.

[0007] Mature lymphocytes originating from the thymus or bone marrow exist in an inactive or "resting" state—that is, they are mitotically inactive. When they disperse into the bloodstream, these "untrained" or "naive" lymphocytes travel to various secondary or peripheral lymphoid organs, such as the spleen, lymph nodes, or tonsils. Most naive lymphocytes have a short lifespan and die within a few days of leaving the bone marrow or thymus. However, if such a cell receives signals indicating the presence of an antigen, it can become activated and undergo successive cycles of cell division. Some of the resulting progeny then return to a resting state and become memory lymphocytes—B and T cells that are essentially trained for the next encounter with a stimulating allergen.Another progeny of activated naive lymphocytes are effector cells, which live only a few days but perform certain types of protective activity.

[0008] Lymphocyte activation is an ordered sequence of events that a resting lymphocyte undergoes after being stimulated to divide and produce progeny, some of which become effector cells. The complete response includes both the induction of cell proliferation (mitogenesis) and the manifestation of immunological functions. Lymphocytes are activated when specific ligands bind to receptors on their surfaces. The ligands differ from those of T cells and B cells, but the final intracellular physiological mechanisms are similar.

[0009] Some foreign antigens themselves can induce lymphocyte activation, particularly large polymeric antigens that cross-link surface immunoglobulins on B cells or other glycoproteins on T cells. However, most antigens are not polymeric, and even direct binding to B cells in large quantities does not result in activation. These more common antigens activate B cells when they are stimulated in concert with nearby activated helper T cells. Such stimulation can occur from lymphokines secreted by the T cell, but it is most effectively mediated by direct contact of the B cell with T cell surface proteins, which interact with specific B cell surface receptors to generate a secondary signal. T cells

[0010] T lymphocytes do not express immunoglobulins, but they detect the presence of foreign substances using surface proteins called T cell receptors (TCRs). These receptors recognize antigens either through direct contact or by influencing the activity of other immune cells. Along with macrophages, T cells are the main cell type involved in cell-mediated immunity.

[0011] Unlike B cells, T cells can detect foreign substances only under specific conditions. Specifically, T lymphocytes recognize a foreign protein only if it is cleaved into small peptides, which are 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 certain conditions, but certain types are more specifically adapted for this purpose and are particularly important in regulating T cell activity, including macrophages and other B cells. Antigen presentation depends in part on specific proteins, called major histocompatibility complex (MHC) proteins, on the surface of APCs. 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.

[0012] There are two major subsets of T cells: cytotoxic T lymphocytes (Tc cells or CTLs) and helper T cells (Th), which are tentatively identified by the expression of cell surface markers CD8 and CD4. Tc cells are important in viral defense and are capable of directly killing viruses by recognizing certain viral peptides expressed on the cell surface. Th cells promote the proliferation, maturation, and immunological function of other cell types, such as the secretion of lymphokines to regulate the activity of B cells, macrophages, and cytotoxic T cells. Both naive and memory T lymphocytes typically remain in a quiescent state, in which state they do not exhibit significant helper or cytotoxic activity. Once activated, these cells undergo several rounds of mitotic division to produce daughter cells.Some of these daughter cells return to a resting state as memory cells, while others become effector cells, actively displaying helper or cytotoxic activity. These daughter cells are similar to their parent cells: CD4+ cells can only produce CD4+ progeny, and CD8+ cells produce only CD8+ progeny. Effector T cells express cell surface markers not expressed on resting T cells, such as CD25, CD28, CD29, CD40L, transferrin receptors, and MHC class II proteins. Upon removal of the activating stimuli, cytotoxic or helper activity gradually declines over several days as the effector cells either die or return to a resting state.

[0013] Similar to B cell activation, the T cell response to most antigens also requires two types of simultaneous stimuli. The first is the antigen, which, when appropriately displayed by MHC proteins on the antigen-presenting cell, can be recognized and bound by T cell receptors. Because this antigen-MHC complex does not send a signal into the cell, it is usually insufficient to lead to T cell activation. Full activation, such as that occurring with helper T cells, requires costimulation by other specific ligands, called costimulators, which are expressed on the surface of the antigen-presenting cell. On the other hand, cytotoxic T cell activation typically requires IL-2, a cytokine secreted by activated helper T cells.

[0014] PD-1 pathway

[0015] An important negative costimulatory signal regulating T cell activation is provided by the programmed death receptor 1 (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 was revealed using PD-1 knockouts (Pdcdl _ / ~ ) that are prone to autoimmunity and . (Nishimura et al, Immunity JJ: 141–51 (1999); Nishimura et al, Science 291: 319–22 (2001)). PD-1 is associated with CD28 and CTLA-4 but lacks the membrane-near cysteine ​​that allows for homodimerization . The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-binding inhibitory motif (ITIM, V / IxYxxL / V). PD-1 binds only to 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)).

[0016] PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs). PD-1 is expressed by activated, not unstimulated, human CD4 T cells. + and CD8 +, B cells, and myeloid cells. This differs 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 4 PD-1 variants 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 to 4 (Nielsen et al, Cell. Immunol. 235: 109–16 (2005)). With the exception of PD-1Dex3, all variants are expressed to the same extent as full-length PD-1 in resting peripheral blood mononuclear cells (PBMCs). Expression of all variants is significantly induced following activation of human T cells with anti-CD3 and anti-CD28 antibodies. PD-1Aex3 variants lack the transmembrane domain and are similar to soluble CTLA-4, which plays an important role in autoimmunity (Ueda et al, Nature 423: 506–11 (2003)).This variant 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 differ in their expression patterns. PD-L1 is constitutively expressed on murine T and B cells, CD40, macrophages, mesenchymal stem cells, and bone marrow mast cells (Yamazaki et al, J. Immunol. 169: 5538–45 (2002)). PD-L1 is expressed on many different non-hematopoietic cells (e.g., corneal, lung, vascular epithelial, non-parenchymal liver 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 following activation. Both type I and type II IFNs induce increases in PD-Ll (Eppihimer et al, Microcirculation 9: 133–45 (2002)); Schreiner et al, J. Neuroimmunol 155: 172–82 (2004).PD-L1 expression in cell lines is reduced by inhibition of MyD88, TRAF6, and MEK (Liu et al, Blood HO: 296–304 (2007)). JAK2 is also involved 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 modifies phosphatidylinositol 3-kinase,

[0017] (PI3K) and Akt signaling, increased post-transcriptional expression of PD-L1 in cancer (Parsa et al, Nat. Med. 13: 84–88 (2007)).

[0018] PD-L2 expression is more restricted than PD-L1. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow 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. 37: 2405–10 (2007)). PD-L2+ B1 cells bind phosphatidylcholine and may be important for the innate immune response to bacterial antigens. PD-L2 induction by IFN-γ is partly dependent on NF-κB (Liang et al, Eur. J. Immunol. 33: 2706–16

[0019] (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)).

[0020] The PD-1 signal generally influences cytokine production more than 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 the TCR signal, with greater inhibition being achieved at lower levels of TCR stimulation. This reduction can be overcome by costimulation via CD28 [Freeman et al, J. Exp. Med. 192: 1027

[0021] - 34 (2000)] or the presence of IL-2 [Carter et al, Eur. J. Immunol. 32 : 634–43 (2002) ].

[0022] There is growing evidence that signaling through PD-L1 and PD-L2 may be bidirectional. That is, in addition to modifying the TCR or BCR signal, the signal may also be delivered back to cells expressing PD-L1 and PD-L2. Although 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 class II or costimulatory B7 molecules, these cells produced higher amounts of proinflammatory cytokines, particularly TNF-a and IL-6, and stimulated T cell proliferation (Nguyen et al, J. Exp. Med. 196: 1393–98 (2002)). Treatment of mice with this antibody also (1) increased resistance to transplanted Nb 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

[0023] - 35 (2007)); (2) blocked the development of inflammatory airway disease in a mouse model of allergic asthma

[0024] (Radhakrishnan et al, J. Immunol. 173: 1360 - 65 (2004); Radhakrishnan et al, J. Allergy Clin. Immunol. UJy. 668 - 74

[0025] (2005) ) .

[0026] Further evidence for signaling back into dendritic cells (“DCs”) came from studies of bone marrow-derived DCs cultured with soluble PD-1 (the PD-1 EC domain fused to the 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 by administration of an anti-PD-1 antibody. Furthermore, some studies have identified a receptor 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 show that PD-L1 and B7.1 can interact through their IgV-like domains. B7.1:PD-L1 interaction can induce an inhibitory signal in T cells. Cross-linking of PD-L1 on CD4+ T cells by B7.1 or cross-linking of B7.1 on CD4+ T cells by PD-L1 mediates the inhibitory signal.T cells lacking CD28 and CTLA-4 exhibit reduced proliferation and cytokine production when stimulated with beads coated with anti-CD3 antibody plus B7.1. In T cells lacking all B7.1 receptors (i.e., CD28, CTLA-4, and PD-L1), inhibition of T cell proliferation and cytokine production by beads coated with anti-CD3 antibody plus B7.1 was abrogated. This indicates that B7.1 specifically acts through PD-L1 on T cells in the absence of CD28 and CTLA-4. Similarly, PD-1-null T cells exhibited reduced proliferation and cytokine production when stimulated with anti-CD3 plus PD-L1-coated beads, demonstrating the inhibitory effect of PD-L1 cross-linking on B7.1 on T cells. When T cells lack all known PD-L1 receptors (i.e., in the absence of PD-1 and B7.1), the attenuation of T cell proliferation by anti-CD3 plus PD-L1-coated beads is abolished.Thus, PD-L1 may exert inhibitory effects on T cells through B7.1 or PD-1.

[0027] The direct interaction between B7.1 and PD-L1 suggests that the current understanding of costimulation is incomplete and highlights the importance of the expression of these molecules on T cells. Studies of T cell PD-L1 indicate that PD-L1 on T cells can reduce T cell cytokine production (Latchman et al, Proc. Natl. Acad. Sci. USA 101: 10691–96 (2004)). Because both PD-L1 and B7.1 are expressed on T cells, B cells, DCs, and macrophages, there is potential for targeted interactions between B7.1 and PD-L1 on these cell types. Furthermore, PD-L1 on nonhematopoietic cells can interact with B7.1 as well as PD-1 on T cells, raising the question of whether PD-L1 is involved in their regulation. One possible explanation for the inhibitory effect of the B7.1:PD-L1 interaction is that T-cell PD-L1 may trap or sequester B7.1 APCs from interacting with CD28. This results in antagonism of the PD-L1 signal, which involves blocking the interaction of PD-L1 with PD-1, B7.1, or both, thereby preventing PD-L1 from sending a negative costimulatory signal to T cells and other antigen-presenting cells, possibly enhancing immunity in response to infection (e.g., acute or chronic) and tumor immunity. Furthermore, the anti-PD-L1 antibodies of the present invention can be combined with antagonists of other components of the PD-1:PD-L1 signal, such as an antagonist of anti-PD-1 and anti-PD-L2 antibodies.

[0028] In particular, inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for cancer treatment (e.g., tumor immunity) and infections, including both acute and chronic (e.g., resistant) infections.

[0029] Inhibitors blocking the PD-L1:PD-1 interaction are known, among other sources, from documents W02001014557, W02002086083, W0200 005874, W02010036959, W02010077634 and WO2011066389.

[0030] Currently, more than 10 mono- and bispecific drugs with an anti-PD-Ll component are in the early stages of clinical trials.

[0031] One monospecific anti-PD-LL antibody, MPDL3280A (atezolizumab, Roche), has successfully completed clinical trials (CTs) and is being used in clinical practice. Atezolizumab has been approved by the FDA for use in patients with metastatic urothelial cancer, and phase III CTs are ongoing in patients with non-small cell lung cancer (NSCLC), renal cell carcinoma, colorectal cancer (CRC), and breast cancer (BC). The drug is an IgL antibody with a modified Fc region (to eliminate the ADCC effect). Atezolizumab is described in document WO2010077634.

[0032] Other anti-PD-LL drugs in the final phase of clinical trials include avelumab (Pfizer) and durvalumab (Z). Durvalumab (MEDI-4736) is described in document WO2011066389. Avelumab is described in document WO2013079174. The main difference between avelumab and PD-LL is that the antibody exhibits ADCC effects, which can be enhanced by IFNγ or IL12 (NCT01772004). Moreover, the safety profile of avelumab is consistent with that of other anti-PD-l / PD-Ll drugs (Cancer Immunol Res; 3(10) October 2015; Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-Ll Antibody Avelumab on Human Tumor Cells; Benjamin Boyerinas).

[0033] Thus, there is a need to create an effective inhibitor of PD-L1 (programmed cell death receptor ligand 1)

[0034] In connection with the above, the creation of new antibodies that effectively bind to PD-L1 is relevant.

[0035] The BCD-135 antibody selectively binds to PD-L1 and is an effective inhibitor of programmed cell death receptor ligand 1.

[0036] Brief description of the invention

[0037] The present invention relates to binding molecules, in particular antibodies, targeted to bind to PD-L1. Such antibodies can be used to treat a disease or disorder mediated by PD-L1.

[0038] In one aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-L1, comprising a heavy chain variable domain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 3 and a light chain variable domain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 7.

[0039] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 3.

[0040] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises amino acid sequences that are at least 90% homologous to the sequences of SEQ ID NO: 1-3.

[0041] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises amino acid sequences represented by SEQ ID NOs: 1-3.

[0042] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises amino acid sequences that are at least 90% homologous to the sequences of SEQ ID NO: 5-7.

[0043] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises amino acid sequences represented by SEQ ID NOs: 5-7.

[0044] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NOs: 1-3, and a light chain variable domain that comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NOs: 5-7.

[0045] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequences of SEQ ID NOs: 1-3 and a light chain variable domain that comprises the amino acid sequences of SEQ ID NOs: 5-7.

[0046] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4.

[0047] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 8.

[0048] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 8.

[0049] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4, and a light chain variable domain that comprises an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 8.

[0050] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 8.

[0051] In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 9, and a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 10.

[0052] In some 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-length IgG antibody.

[0053] In some embodiments, the full-length IgG antibody is of the human IgG1, IgG2, IgG3, IgG4 isotype.

[0054] In some embodiments, the monoclonal antibody is of the human IgG1 isotype.

[0055] In one aspect, the present invention relates to a nucleic acid that encodes any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0056] In some embodiments, the nucleic acid is DNA.

[0057] In one aspect, the present invention relates to an expression vector comprising any of the above nucleic acids.

[0058] In one aspect, the present invention relates to a method for producing a host cell for producing any of the above-mentioned antibodies or antigen-binding fragments thereof, which comprises transforming the cell with the above-mentioned vector.

[0059] In one aspect, the present invention relates to a host cell for producing any of the above-mentioned antibodies or antigen-binding fragments thereof, which comprises any of the above-mentioned nucleic acid.

[0060] In one aspect, the present invention relates to a method for producing any of the above-mentioned antibodies or antigen-binding fragments thereof, which comprises culturing the above-mentioned host cell in a culture medium under conditions sufficient to produce the said antibody, if necessary, followed by isolating and purifying the resulting antibody.

[0061] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1, which comprises any of the above-mentioned antibodies or antigen-binding fragments thereof in combination with one or more pharmaceutically acceptable excipients. 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: HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC.

[0062] In one aspect, the present invention relates to a pharmaceutical combination for the prevention or treatment of a disease or disorder mediated by PD-L1, which comprises any of the above-mentioned antibodies or antigen-binding fragments thereof and at least one therapeutically active antitumor compound.

[0063] In some embodiments, the pharmaceutical combination is intended for the prevention or treatment of a disease or disorder mediated by PD-L1 selected from the group: HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC.

[0064] In some embodiments, the pharmaceutical combination comprises a therapeutically active antitumor compound that is selected from a chemotherapeutic agent, an antibody, or an antihormonal agent.

[0065] In one aspect, the present invention relates to a method for inhibiting the biological activity of PD-L1 in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0066] In one aspect, the present 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 disease or disorder mediated by PD-L1 in a subject in need of such treatment. In some embodiments, 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 disease or disorder selected from the group: HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC.

[0067] Brief description of the drawings

[0068] Fig. 1. Scheme of synthesis of combinatorial naive human library.

[0069] Fig. 2. Phagemid map for cloning Fab phage display libraries.

[0070] Fig. 3. Map of the expression plasmid for Fab production.

[0071] Fig. 4A. Electropherogram of BCD-135 under reducing conditions, 12% SDS-PAGE.

[0072] Fig. 4B. Electropherogram of BCD-135 under non-reducing conditions, 8% SDS-PAGE.

[0073] Fig. 5. Enzyme-linked immunosorbent assay of BCD-135 interaction with PD-L1 and other antigens.

[0074] Fig. b. Reactivation of NFAT signaling by anti-PD-L1 antibodies in the Jurkat-NFAT-PD-1 reporter cell line.

[0075] Fig. 7. Analysis of BCD-135 interactions with FcRn and Fcy receptors using the Octet RED 96 instrument.

[0076] Fig. 8. Enzyme-linked immunosorbent assay of BCD-135 interactions with PD-L1 from different species.

[0077] Fig. 9. Analysis of interactions of BCD-135 with human and cynomolgus monkey PD-L1 using the Octet RED 96 instrument.

[0078] Fig. 10. Conformational stability analysis of BCD-135.

[0079] Fig. 11. Colloidal stability analysis of BCD-135.

[0080] Fig. 12A. Thermal stability analysis of BCD-135 in phosphate buffer. X-axis shows time, Y-axis shows absorbance.

[0081] Fig. 12B. Thermal stability analysis of BCD-135 in acetate buffer. X-axis – time, Y-axis – absorbance. Fig. 12C. Thermal stability analysis of BCD-135 in histidine buffer. X-axis – time, Y-axis – absorbance.

[0082] Fig. 13A. Analysis of the stability of BCD-135 in human serum. The calibration curve reflects the dependence of optical density on the concentration of BCD-135 added to the well.

[0083] Fig. 13B. Analysis of BCD-135 stability in human serum. The resulting table shows the dependence of BCD-135 concentration during incubation in human serum on the incubation time.

[0084] Fig. 14A. 3D spatial model of the complex of BCD-135 and the N-terminus Ig domain of the PD-L1 antigen. General view of the 3D model.

[0085] Fig. 14B. 3D spatial model of the complex of BCD-135 and the N-terminus Ig domain of the PD-L1 antigen. Detailed model in the region of direct antigen-antibody contacts (see abl. in Example 18).

[0086] Description of the invention

[0087] Definitions and general methods

[0088] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will prevail. Although a number of documents are cited herein, such citation does not constitute an admission that any of these documents form part of the common knowledge in the art.

[0089] Furthermore, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. The generally used classification and methods of cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry, described herein are well known to those skilled in the art and are widely used in the art. Enzyme reactions and purification methods are carried out in accordance with the manufacturer's instructions, as commonly practiced in the art, or as described herein.

[0090] In this description and embodiments of the invention, the words "have" and "contain" or variations thereof such as "has," "having," "contains," or "comprising" should be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes.

[0091] Antibody-related definitions

[0092] PD-L1 (programmed cell death receptor ligand 1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa transmembrane protein of type 1. It consists of three domains: an extracellular domain, represented by IgV and C-like domains (220), a transmembrane domain (21), and an intracellular domain (31). It plays an important role in suppressing the immune system during pregnancy, transplantation of foreign tissue, and certain diseases, such as hepatitis. Under normal conditions, in response to self-antigens, a certain number of antigen-specific CD8+ T effector cells accumulate in the lymph nodes and spleen. To prevent autoimmunity, PD-1 / PD-L1 or B7-1 / PD-L1 complexes are formed. This leads to the transmission of an inhibitory signal that reduces the proliferation of these CD8+ T cells in the lymph nodes. Thus, the PD-1 / PD-L interaction is a key factor in the development of immune tolerance.

[0093] "Dysfunction" in the context of immune dysfunction refers to a state of reduced immune response to antigen stimulation. The term includes general elements of exhaustion and / or anergy, in which antigen recognition may occur, but the resulting immune response is ineffective in controlling infection or tumor growth.

[0094] "Enhancement of T cell function" means the induction, elicitation, or stimulation of sustained or enhanced biological function of a T cell or the restoration or reactivation of exhausted or inactive T cells. Examples of enhanced T cell function include: increased secretion of γ-interferon from CD8 +T-cells, increased proliferation, and an increased response to an antigen (e.g., virus or pathogen clearance) relative to pre-intervention values. In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. The method for measuring this enhancement is known to those skilled in the art.

[0095] A "T cell dysfunction-associated disorder" is a disorder or condition of T cells characterized by a reduced response to antigen stimulation. In a specific embodiment, the T cell dysfunction-associated disorder is a disorder specifically associated with inappropriately elevated signaling via PD-1. In another embodiment, the T cell dysfunction-associated disorder is a disorder in which T cells are energetic or have a reduced capacity for cytokine secretion, proliferation, or cytolytic activity. In a specific aspect, the reduced response results in failure to control a pathogen or tumor expressing an immunogen. Examples of T cell dysfunction-associated disorders characterized by T cell dysfunction include unresolved acute infection, chronic infection, and tumor immunity.

[0096] "Tumor immunity" refers to the process by which tumors evade immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "treatable" when such evasion is attenuated and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination. The term "vaccine," as used herein, includes any nonpathogenic immunogen that, when inoculated into a host, elicits protective immunity against a specific pathogen. Vaccines can take various forms. Vaccines can be whole organisms that share antigens with the pathogen but are not themselves pathogenic (e.g., cowpox). Vaccines can also be prepared from killed organisms.

[0097] (e.g., Salk polio vaccine) or attenuated (having lost the ability to cause disease—e.g., Sabin polio vaccine). Vaccines can also be prepared from purified macromolecules isolated from the pathogen. For example, toxoid vaccines (e.g., tetanus and diphtheria) contain an inactive form of a soluble bacterial toxin and result in the production of antibodies against the toxin, but not immunity to the intact bacterium. Subunit vaccines

[0098] (e.g., hepatitis B) contain only a single immunogenic protein isolated from the desired pathogen. Hapten conjugate vaccines attach certain carbohydrate or polypeptide epitopes isolated from the desired pathogen to immunogenic carriers, such as tetanus toxoid. These techniques primarily use epitopes, such as haptens, to induce antibody production, which then recognize a single epitope on the natural pathogen. However, for maximum effectiveness, such vaccines must include both B- and T-cell epitopes, and T-cell epitopes must be selected to ensure that they can be recognized, presented, and responded to by the host immune system. DNA vaccines utilize the ability of host cells to take up and express DNA encoding pathogenic proteins, which is administered intramuscularly. The host response to immunogens can be enhanced when administered in a mixture with adjuvants.Immune adjuvants function by one or more of the following means: (1) prolonging the retention of the immunogen, (2) increasing the effective size of the immunogen (and hence activating phagocytosis and presentation to macrophages).

[0099] (3) stimulating the influx of macrophages and other immune cells to the injection site; or (4) activating local cytokine production and other immunological activity. Examples of adjuvants include complete Freund's adjuvant (CFA), aluminum salts, and mycobacterial-derived proteins such as muramyl dipeptides or tripeptides.

[0100] Amplification of this gene and / or overexpression of its protein have been detected in many cancers, including HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cell carcinoma, ovarian cancer, Hodgkin's lymphoma, and MSI CRC.

[0101] The term "binding molecule" includes antibodies and immunoglobulins.

[0102] The term "antibody" or "immunoglobulin" (Ig), as used herein, includes whole antibodies and any antigen-binding fragment (i.e., the "antigen-binding portion") 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 an antigen-binding portion 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 consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists 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 consists 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 the binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0103] The term "antigen-binding portion" of an antibody or "antigen-binding fragment" (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 included within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH 1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment, consisting of the VH and CH 1 domains; (iv) the Fv fragment, which consists of the VL and VH domains in a single arm of the antibody, (v) the dAb fragment (Ward et al., (1989) Nature 341:544–546), which consists of the VH / VHH domain; and (vi) the isolated complementarity determining region (CDR).Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by different genes and can be joined by recombinant techniques using a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also intended to be included within the term "antigen-binding portion" of an antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art and the fragments are screened in the same manner as intact antibodies.

[0104] Preferably, the CDRs of the antigen-binding region or the entire antigen-binding region of the antibodies of the invention are of mouse, llama, or donor human library origin, or are of substantially human origin with certain amino acid residues altered, for example, substituted with different amino acid residues, in order to optimize specific properties of the antibody, for example, KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or are of substantially human origin (at least 80, 85, 90, 95, 96, 97, 98, or 99% human origin).

[0105] In other embodiments, the antigen-binding region of the antibody of the invention may be from other non-human species, including, but not limited to, mouse, llama, rabbit, rat, or hamster. Alternatively, the antigen-binding region may be from a human species.

[0106] The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, variability is unevenly distributed across the 110-amino acid stretch of the variable domains. In contrast, V regions consist of invariant fragments called framework regions (FRs) of 15-30 amino acids, separated by shorter regions of extreme variability called hypervariable regions (CDRs, HVRs, or HVs). Each variable domain of native heavy and light chains contains four FRs, generally adopting a beta-sheet configuration, linked by three hypervariable regions that form loops that link and, in some cases, are part of a beta-sheet structure.The hypervariable regions in each chain are held together in close proximity by the FR and, with the hypervariable regions of 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 directly involved in antibody-antigen binding but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

[0107] The term "hypervariable region" ("HVR" or "HV") as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, the hypervariable region contains amino acid residues from the "complementarity-determining region" or "CDR" and / or those from the "hypervariable loop."

[0108] In some cases, it may also be advantageous to alter one or more amino acid residues of the CDR regions to enhance 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, in situations where humanization of an antibody results in decreased binding specificity or affinity, and it is not possible to sufficiently improve binding specificity or affinity using backmutations alone. Various methods for affinity maturation 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.

[0109] (1997) , and the stepwise in vitro affinity maturation method proposed by u et al., Proc Natl Acad Sci USA 95:6037 6042

[0110] (1998) .

[0111] Framework regions (FRs) are the residues of a variable domain other than the CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3, and FR4. When CDRs are defined according to Kabat, the light chain FR residues are located approximately in the region of residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located approximately in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113

[0112] (HCFR4) in the heavy chain. While the CDR regions contain amino acid residues from hypervariable loops, the FR residues of the light chain are located approximately at residues 1-25 (LCFR1), 33-49

[0113] (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, and the FR residues of the heavy chain are located approximately at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some examples, when a CDR contains amino acids from both the Rabat CDR and the hypervariable loop, the FRs are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the FR1 residues of the heavy chain are at positions 1-25, and the FR2 residues are at positions 36-49.

[0114] An antibody of the present invention that "binds" a target antigen is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting a protein or a cell or tissue expressing the antigen, and cross-reacts only slightly with other proteins. In such embodiments, the degree of binding of the antibody to a non-target protein (an "off-target protein") is less than 10% of the binding of the antibody to the specific target protein, as determined by fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA), or ELISA.With respect to the binding of an antibody to a target molecule, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide means binding that is measurably different from non-specific interaction.

[0115] (e.g., for LB1-44 or LB1-81, nonspecific interaction is binding to bovine serum albumin, casein, fetal bovine serum, or neutravidin). Specific binding can be quantified, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can 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 if the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target.In this specification, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide may be characterized by reference to a molecule having a Ko! to the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or higher.In one embodiment of the invention, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

[0116] The term "Ka" as used herein refers to the rate of association of a particular antibody-antigen interaction, whereas the term or "Kd" refers to the rate of dissociation of a particular antibody-antigen interaction.

[0117] "Binding affinity" generally refers to the strength of the combined noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity, which reflects the 1:1 interaction between the members of the binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can typically be represented by the dissociation constant (Kd). It is desirable that the Kd value is approximately 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. The affinity can be measured by conventional methods known in the art, including the methods described herein.Low-affinity antibodies typically bind to antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention.

[0118] In one embodiment of the invention, the "Kd" or "Kd value" of the present invention is measured by surface plasmon resonance methods on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using chips with immobilized CM5 antigen at -10 relative units.

[0119] (response units, RU). In short, biosensor chips with carboxymethyldextran (CM5, BIAcore Inc.) are activated by hydrochloride (3-dimethylaminopropyl)-carbodiimide

[0120] (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to a concentration of 5 μg / mL (~0.2 μM) and then injected at a flow rate of 5 μL / min until approximately 10 relative units (RU) of bound protein are achieved. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., from 0.78 nM to 500 nM) are injected in PBS with 0.05% T een 20 (PBST) at 25°C at a flow rate of approximately 25 μL / min. The association rate (kop) and association rate (koff) values ​​are calculated using the simple Langmuir model for one-plus-one binding.

[0121] (BIAcore Evaluation Software version 3.2), by simultaneously obtaining a sensorgram of association and dissociation. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol . Biol. 293: 865–881. If the association rate exceeds 106 M−1 sec−1 according to the above surface plasmon resonance method, then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescence emission

[0122] (excitation = 295 nm; emission = 340 nm, band 16 nm) at 25°C of a 20 nM solution of antibody against the antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured with a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco (ThermoSpectronie) 8000 series spectrophotometer with a stirred cuvette.

[0123] The term "Koff" refers to the dissociation rate constant of a specific interaction between a binding molecule and an antigen. The dissociation rate constant koff+ can be measured using biolayer interferometry, such as the Octet™ system.

[0124] The "on-rate" or "on-rate" of the present invention can also be determined by the same surface plasmon resonance method described above on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using chips with immobilized CM5 antigen at -10 relative units (RU). Briefly, biosensor chips with carboxymethyldextran (CM5, BIAcore Inc.) are activated with hydrochloride '-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 mM sodium acetate solution, pH 4.8, to a concentration of 5 μg / ml (~0.2 μM) and then injected

[0125] (injection) at a flow rate of 5 μl / min until approximately 10 relative units (RU) of bound protein are achieved. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., from 0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rate (kop) and association rate (koff) values ​​are calculated using the simple Langmuir model for one-plus-one binding.

[0126] (BIAcore Evaluation Software version 3.2), simultaneously obtaining association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y . , et al., (1999) J. Mol . Biol. 293: 865–881. However, if the association rate is greater than 106 M-1 sec-1 as determined by the surface plasmon resonance method described above, then it can be determined by the fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band 16 nm) at 25°C of a 20 nM solution of antibody to the antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, measured with a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco (ThermoSpectronic) 8000 series spectrophotometer with a stirred cuvette.

[0127] Unless otherwise specifically stated, the terms "biologically active," "biological activity," and "biological characteristics," when used with respect to a polypeptide of the present invention, mean having the ability to bind to a biological molecule.

[0128] The term "biological molecule" refers to a nucleic acid, protein, carbohydrate, lipid, or combination thereof. In one embodiment, the biological molecule exists in nature.

[0129] Antibody fragments, such as Fab and F(ab')2 fragments, can be obtained from whole antibodies using traditional methods such as papain or pepsin digestion of whole antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques, such as those described herein.

[0130] The term "recombinant antibody" means an antibody that is expressed from a cell or cell line containing nucleotide sequence(s) that encodes antibodies, wherein said nucleotide sequence(s) are not associated with the cell in nature.

[0131] The term "variant" antibody, as used herein, refers to an antibody having an amino acid sequence that differs from the amino acid sequence of its "parent" antibody by the addition, deletion, and / or substitution of one or more amino acid residues relative to the sequence of the parent antibody. In a preferred embodiment, a variant antibody comprises at least one or more (e.g., one to twelve, such as two, three, four, five, six, seven, eight or nine, ten, eleven, or twelve; and in some embodiments, one to about ten) amino acid additions, deletions, and / or substitutions relative to the parent antibody. In some embodiments, the additions, deletions, and / or substitutions are made in the CDR regions of the variant antibody.Identity or homology with respect to the sequence of a variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to residues of the parent antibody, after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably the epitope, to which the parent antibody binds, and in some embodiments, has at least one property or biological activity superior to that of the parent antibody. For example, a variant antibody may have, for example, a more pronounced binding affinity, a longer half-life, a lower IC50 value, or an increased ability to inhibit the biological activity of an antigen compared to the parent antibody.Of particular interest herein is a variant antibody that exhibits 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.

[0132] The term "bispecific antibody" means an antibody comprising an antigen-binding domain or domains that are capable of specifically binding to two different epitopes on a single biological molecule or capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having "dual specificity" or as being an antibody with "dual specificity."

[0133] The term "chimeric antibody" refers broadly to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody that is partly human and partly non-human, that is, derived partly from a non-human animal, such as a mouse, rat, or other rodent, or camelids such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies to reduce the risk of an immune response directed against human antibodies, such as an anti-mouse response in humans in the case of a murine antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine, while the constant region sequences are human.In the case of a chimeric antibody, the non-human portions may be further modified to humanize the antibody.

[0134] The term "humanization" refers to the fact that when an antibody is of non-human origin, in whole or in part, such as a murine or llama antibody obtained by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a murine or llama antibody, certain amino acids can be substituted, particularly in the framework regions and constant domains of the heavy and light chains, to avoid or minimize the immune response in humans. The specificity of antibody interaction with the target antigen is primarily determined by the amino acid residues located within the six CDR regions of the heavy and light chains. Therefore, amino acid sequences within the CDR regions are much more variable between individual antibodies than sequences outside the CDR regions.Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific natural antibody, or more generally, any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express CDR sequences from a specific antibody into the framework sequences of another antibody. As a result, it is possible to "humanize" a non-human antibody while largely preserving the binding specificity and affinity of the original antibody. Although it is impossible to accurately predict immunogenicity and thus the immune response directed against an antibody in humans to a specific antibody, non-human antibodies are generally more immunogenic than human antibodies.Chimeric antibodies, in which foreign (e.g., rodent or camel) constant regions have been replaced with human-derived sequences, have generally shown lower immunogenicity than antibodies of completely foreign origin, and there is a trend toward using humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other non-human antibodies can thus be humanized to reduce the risk of an anti-antibody immune response in humans.

[0135] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity-determining regions (CDRs) will most often not be altered by humanization, although in some cases it may be desirable to alter individual amino acid residues of a CDR, for example, to remove a glycosylation site, a deamidation site, an aspartate isomerization site, or an unwanted cysteine ​​or methionine residue. N-linked glycosylation occurs by attaching 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 the N-linked glycosylation site can be achieved by mutating the Asn or Ser / Thr residue with another residue, preferably by conservative substitution.Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily if they are present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences, such as Asn-Ala. When such a deamidated site is present, particularly Asn-Gly in a CDR sequence, it may be preferable to remove this site, typically by a conservative substitution to remove one of the involved residues.

[0136] Numerous methods for humanizing an antibody sequence are known in the art; see, for example, the review by Almagro & Fransson, Front Biosci. 13:1619–1633 (2008). One of the most commonly used methods is CDR grafting, for example, where chimeric antibodies of murine origin involve identifying human germline gene equivalents to murine variable region genes and grafting murine CDR sequences into this framework. CDR transplantation can be based on Rabat CDR definitions, although a more recent publication (Magdelaine-Beuzelin et al ., Crit Rev. Oncol Hematol . 64:210 225 (2007)) suggests that IMGT® (the international ImMunoGeneTics information system®, www.imgt.org) definition may improve the humanization outcome (see Lefranc et al., Dev. Comp Immunol. 27:55–77 (2003)).In some cases, CDR grafting can reduce the binding specificity and affinity, and hence biological activity, of the CDR-grafted non-human antibody compared to the parent antibody from which the CDRs are derived. Backmutations (sometimes referred to as "framework repairs") can be applied at selected CDR positions of the grafted antibody, typically in the framework regions, to restore the binding specificity and affinity of the parent antibody. Positions for potential backmutations can be determined using information available in the literature and antibody databases. Amino acid residues that are candidates for backmutations are typically located on the surface of the antibody molecule, while residues that are recessed or have a low degree of surface exposure will generally not be affected.An alternative method of humanization to CDR transplantation and back mutation is surface modification in which non-human surface-exposed residues are retained while surface-exposed residues are modified to human residues.

[0137] There are two technologies for producing fully human antibodies: using lp vit-assembled phage libraries or in vivo immunization of humanized animals (mice, rats, etc.).

[0138] Phage display is the first and most widely used in vitro technology for antibody discovery. In 1985, Smith discovered that foreign DNA sequences could be cloned into the filamentous bacteriophage M13 such that the cloned gene sequences were expressed on the surface of the phage particles 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 could be selected based on their ability to bind other proteins. This discovery was combined with PCR amplification techniques to clone cDNA repertoires of immunoglobulin genes to generate diverse phage libraries containing variable domains that could be used to rapidly screen for target-specific monoclonal antibodies.The repertoire of phage libraries reflects the repertoire of antibodies of B lymphocytes of each person or animal whose blood was used to create the library. In 1995, two papers reported the creation of genetically engineered mice that expressed fully human antibodies whose repertoire could be compared with those obtained 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 had their own endogenous immunoglobulin heavy and light chain genes deliberately disrupted and were given transgenes representing segments of the human heavy and light chain genes. It turned out that the human gene repertoire can be used by the mouse immune system to generate highly specific and high-affinity antibodies to a wider variety of antigens. Although the transgenic mice express B-cell receptors that are essentially hybrids of mouse and human ones (human immunoglobulin, mouse IgA, IgG, and other signaling molecules), their B cells develop and mature normally. In some cases, it may also be preferable to alter one or more amino acid residues of 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, such as in situations where humanization of an antibody results in a decrease in binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by backmutations alone. Various methods of affinity maturation 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 proposed by Wu et al., Proc Natl Acad Sci USA 95:6037–6042 (1998).

[0139] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and secreted by a distinct 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, or hybridomas, which are typically produced by fusing individual B lymphocytes from immunized animals with individual lymphocytic tumor cells. Hybridomas are a type of engineered cell and do not occur naturally.

[0140] Native antibodies are typically heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, consisting 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 bonds between heavy chains varies among immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain 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 at one end (VL) and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.Specific amino acid residues are thought to form the interface between the variable domains of the light chain and heavy chain.

[0141] The term "isolated" as used to describe various antibodies described herein means an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Contaminants from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning glass cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or, preferably, silver staining.An isolated antibody includes antibodies produced in situ within recombinant cells, as at least one component of the polypeptide's natural environment is absent. Typically, an isolated polypeptide is obtained through at least one purification step.

[0142] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been 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 composition in which it is naturally found. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule naturally present in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule present in cells that normally express the antibody, for example, if the nucleic acid molecule has a chromosomal location different from its natural cellular location.

[0143] The term "epitope," as used herein, refers to the portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, such as a bispecific binding molecule). Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or carbohydrates, or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be "linear" or "conformational." In a linear epitope, all points of interaction between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) 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 each other in the primary amino acid sequence.Once a desired epitope of an antigen is identified, antibodies to that epitope can be generated using techniques well known in the art. Furthermore, the generation and characterization of antibodies or other binding molecules can provide insight into the desired epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example, by conducting competition studies to find binding molecules that compete for binding to the antigen.

[0144] The term "peptide linker" as used herein means any peptide capable of connecting domains of a length depending on the domains it links together, comprising any amino acid sequence.

[0145] Preferably, the peptide linker is more than 5 amino acids in length and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0146] The term "effector function" of an antibody refers to the biological activities associated with the Fc region (either the native Fc region sequence or Fc region amino acid sequence variants) of an antibody, and varies depending on the antibody isotype. Examples of antibody effector functions include: Cl 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.

[0147] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and then cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express only FCYRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. 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 the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patents W-5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al. PNAS (USA) 95: 652–656 (1998).

[0148] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform 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. Effector cells can be isolated from their natural source, such as blood or PBMCs, as described herein.

[0149] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds the Fc region of an antibody. A preferred FcR is the native-sequence human FcR. Additionally, a preferred FcR is the FcR that binds IgG antibody (the gamma receptor), and preferred receptors include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains a tyrosine-based immunoreceptor inhibitory motif (ITIM) in its cytoplasmic domain (see review in Daeron, Annu. Rev. Immunol.15: 203–234 (1997) . A review of FcRs is presented in Ravetch and Kinet, Annu. Rev. Immunol 9: 457–92.

[0150] (1991); Capel et al., Immunomethods 4: 25–34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330–41 (1995). Other FcRs, including FcRs that will be identified in the future, are included herein under the term "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).

[0151] "Complement-dependent cytotoxicity" and "CDC" refer 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 (Clq) to the molecule.

[0152] (e.g., an antibody) in complex with its antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

[0153] The term "identity" or "homology" should be interpreted to mean the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding sequence to which it is compared, after sequence comparison and the introduction of gaps as necessary to achieve the maximum percentage identity for the entire sequence, and without considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertion segments should be construed as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined 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 suitable for such sequences by determining the degree of homology for various substitutions, deletions (eliminations) and other modifications.

[0154] The phrase "homologous" when used with respect to a polypeptide sequence of an antibody should be interpreted as an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity with respect to the polypeptide sequence. The term when used with respect to a nucleic acid sequence should be interpreted as a sequence of nucleotides exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% sequence identity with respect to the nucleic acid sequence.

[0155] Modification(s) of the amino acid sequences of the antibodies described in this publication are proposed. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Variants of the amino acid sequence of the antibody are obtained by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions is carried out to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

[0156] A variant of modifying the amino acid sequences of antibodies using amino acid substitutions. This variant involves replacing at least one amino acid residue in the antibody molecule with another residue. Locations of greatest interest for substitution mutagenesis include the hypervariable regions or CDRs, but changes in the FR or Fc region are also contemplated. Conservative substitutions are shown in Table 1 under the heading "preferred substitutions." If such substitutions result in altered biological activity, additional significant changes, termed "example substitutions" in Table A, or changes further described below under the amino acid classes, can be introduced, and product screening can be performed.

[0157]

[0158]

[0159] Norleucine

[0160] The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide",

[0161] "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, mean a distinct sequence of nucleotides, modified or unmodified, defining a fragment or section of nucleic acid, whether or not containing unnatural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNAs.

[0162] It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state.

[0163] The sequences of the present invention have been isolated and / or purified, i.e., taken directly or indirectly, for example by copying, while their environment has been at least partially modified. Thus, this also includes isolated nucleic acids obtained by genetic recombination, for example, using host cells, or obtained by chemical synthesis.

[0164] A reference to a nucleotide sequence includes its complement unless otherwise stated. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand and its complementary sequence.

[0165] The term "control sequences" refers to DNA sequences required for the expression of a functionally linked coding sequence in a given host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator, and a ribosome binding site. Eukaryotic cells are known to contain promoters, polyadenylation signals, and enhancers.

[0166] A nucleic acid is "operably linked" if it is in a functional relationship with another nucleotide sequence. For example, the DNA of a presequence or secretory leader sequence is operably linked to the DNA of a polypeptide 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; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Typically, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and in the reading phase. However, enhancers do not necessarily have to be contiguous. Linking occurs by ligation into existing restriction sites.If such sites do not exist, then, according to known practice, synthetic oligonucleotide adapters or linkers are used.

[0167] The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. In some embodiments, vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors).In other embodiments of the invention, vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, thereby replicating along with the host gene. Furthermore, some 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").

[0168] The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells that may comprise, for example, a vector according to the present invention, as described above. The present invention also relates to host cells that comprise, for example, a nucleotide sequence encoding the heavy chain or its antigen-binding portions, a nucleotide sequence encoding the light chain or its antigen-binding portions, or both, of the first binding domain and / or the second binding domain of the trispecific binding molecule of the present invention. It should be understood that "recombinant host cell" and "host cell" mean not only the specific cell claimed, but also the progeny of such a cell.Because modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are still included within the scope of the term "host cell" when used herein. The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention.

[0169] "Pharmaceutical composition" means a composition comprising an antibody according to the invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and receiving means, delivery means, such as preservatives, stabilizers, fillers, grinders, humectants, emulsifiers, suspending agents, thickeners, sweeteners, flavorings, aromatizers, antibacterial agents, fungicides, lubricants, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures of these substances.Protection against the action of microorganisms can be provided by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged action of the composition can be achieved by using agents that delay the absorption of the active ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery vehicles include water, ethanol, polyalcohols, and mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Examples of fillers include lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Examples of disintegrating and dispensing agents include starch, alginic acid and its salts, and silicates.Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethyleneglycol. A pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical, or rectal administration of the active ingredient, alone or in combination with another active ingredient, can be administered to animals and humans in unit administration form as a mixture with traditional pharmaceutical carriers. Suitable unit administration forms include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gums, 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.

[0170] "Medicine (drug)" - a substance (or mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules, injections, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, as well as for the treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology, etc.

[0171] The term "PD-L1-mediated disease or disorder" means all diseases or disorders that are either directly or indirectly associated with PD-L1, including the etiology, development, progression, persistence, or pathology of the disease or disorder.

[0172] "Treat," "treatment," and "therapy" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviate" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and prophylactic therapy. In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. The subject may be male or female of any age.

[0173] The term "disorder" means any condition that can be improved by treatment according to the present invention. This term is defined to include chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to developing the disorder. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemias and lymphoid malignancies, in particular cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, prostate or bladder; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoele disorders; inflammatory, angiogenic and immunological disorders. A preferred disorder to be treated according to the invention is cancer.

[0174] The terms "cancer" or "cancerous" refer to or describe a physiological condition in mammals typically characterized by unregulated (i) cell growth / proliferation. This definition encompasses both benign and malignant cancers. Examples of cancers 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, squamous cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary gland carcinoma, kidney cancer (renal cell carcinoma), prostate cancer (prostate cancer), cancer of the external female genitalia, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, and various types of head and neck cancer.

[0175] The terms "immune response", "autoimmune reaction", "autoimmune inflammation" refer, for example, to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by these cells or liver cells (including antibodies, cytokines and complement resulting from the selective damage, destruction or elimination from the human body of invasive pathogens, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

[0176] A "therapeutically effective amount" is defined as the amount of a therapeutic agent administered during treatment that will relieve, to a specified degree, one or more symptoms of the disease being treated.

[0177] The term "chronic" administration refers to continuous (long-term) administration of the agent(s), as opposed to acute (short-term) administration, so as to maintain the initial therapeutic effect (activity) over an extended period of time.

[0178] "Intermittent" use refers to treatment that is not administered consecutively without interruption, but is rather periodic in nature.

[0179] In the present description and in the following claims, unless the context otherwise requires, the words "have," "include," and "contain," or variations thereof such as "has," "having," "includes," "including," "contains," or "containing," are to be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes. Detailed Description of the Invention

[0180] Antibody

[0181] The present invention relates to antibodies or an antigen-binding fragment that bind PD-L1 (programmed death protein ligand 1).

[0182] In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1, and comprises:

[0183] (a) a heavy chain variable region comprising a CDR3 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence APLLLAMTFGVGS (SEQ ID NO: 3), and

[0184] (b) a light chain variable region comprising a CDR3 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the sequence ALYMGNGGHM (SEQ ID NO: 7).

[0185] In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1, and comprises:

[0186] (a) a heavy chain variable region comprising CDR3 with the amino acid sequence APLLLAMTFGVGS (SEQ ID NO: 3), and

[0187] (b) a light chain variable region comprising CDR3 with the amino acid sequence ALYMGNGGHM (SEQ ID NO: 7).

[0188] In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1, and comprises:

[0189] (a) a heavy chain variable region comprising:

[0190] (i) a CDR1 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the DYAMS sequence (SEQ ID NO: 1),

[0191] (ii) a CDR2 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence

[0192] DISWSGSNTNYADSVKG (SEQ ID NO: 2),

[0193] (iii) a CDR3 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence APLLLAMTFGVGS (SEQ ID NO: 3), and

[0194] (b) a light chain variable region comprising:

[0195] (i) a CDR1 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence GLSSGTVTAINYPG (SEQ ID NO: 5),

[0196] (ii) a CDR2 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence of NTNTRHS (SEQ ID NO: 6),

[0197] (iii) a CDR3 with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence ALYMGNGGHM (SEQ ID NO: 7).

[0198] In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1, and comprises:

[0199] (a) a heavy chain variable region comprising:

[0200] (i) CDR1 with the amino acid sequence DYAMS (SEQ ID NO: 1),

[0201] (ii) CDR2 with the amino acid sequence DISWSGSNTNYADSVKG (SEQ ID NO: 2),

[0202] (iii) CDR3 with the amino acid sequence APLLLAMTFGVGS (SEQ ID NO: 3), and

[0203] (b) a light chain variable region comprising:

[0204] (i) CDR1 with the amino acid sequence GLSSGTVTAINYPG (SEQ ID NO: 5),

[0205] (ii) CDR2 with the amino acid sequence NTNTRHS (SEQ ID NO: 6),

[0206] (iii) CDR3 with the amino acid sequence ALYMGNGGHM (SEQ ID NO: 7). In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1 and comprises:

[0207] (a) a heavy chain variable region with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence

[0208] EVQLVESGGGWRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSDISWSGSNTNYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARAPLLLAMTFGVGSWGQGTLVTVSS

[0209] (SEQ ID NO: 4) , and

[0210] (b) a light chain variable region with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence

[0211] QTWTQEPSLSVSPGGTVTLTCGLSSGTVTAINYPGWYQQTPGQAPRTLIYNTNTRHSGVPD RFSGS ISGNKAAL I GAQAEDEADYYCALYMGNGGHMFGGGTK (SEQ ID NO: 8) .

[0212] In one embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds PD-L1, and comprises:

[0213] (a) the variable region of the heavy chain with the amino acid sequence

[0214] EVQLVESGGGWRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSDISWSGSNTNYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARAPLLLAMTFGVGSWGQGTLVTVSS

[0215] (SEQ ID NO: 4) , and

[0216] (b) a light chain variable region with an amino acid sequence

[0217] QTWTQEPSLSVSPGGTVTLTCGLSSGTVTAINYPGWYQQTPGQAPRTLIYNTNTRHSGVPD RFSGS ISGNKAALTITGAQAEDEADYYCALYMGNGGHMFGGGTK (SEQ ID NO: 8) .

[0218] In one embodiment, the present invention relates to an isolated antibody that binds PD-L1, and comprising:

[0219] (a) a heavy chain with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence

[0220] EVQLVESGGGWRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSDISWSGSNTNYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARAPLLLAMTFGVGSWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO: 9), and

[0221] (b) a light chain with an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the sequence

[0222] QTWTQEPSLSVSPGGTVTLTCGLSSGTVTAINYPGWYQQTPGQAPRTLIYNTNTRHSGVPD RFSGSISGNKAALTITGAQAEDEADYYCALYMGNGGHMFGGGTKLTVLGQPKAAPSVTLFPP SEEELQANKATLVCLISDFYРGAVTVAWKADSSRVKAGVETTTRSKQS NKYAASSYLSLTP EQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10) .

[0223] In one embodiment, the present invention relates to an isolated antibody that binds PD-L1, and comprising:

[0224] (a) heavy chain with the amino acid sequence EVQLVESGGGWRPGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSDISWSGSNTNYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARAPLLLAMTFGVGSWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO: 9), and

[0225] (b) a light chain with the amino acid sequence QTWTQEPSLSVSPGGTVTLTCGLSSGTVTAINYPGWYQQTPGQAPRTLIYNTNTRHSGVPD RFSGS ISGNKAALTITGAQAEDEADYYCALYMGNGGHMFGGGTKLTVLGQPKAAPSVTLFPP

[0226] SSEELQANKATLVCLISDFYРGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP EQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10) .

[0227] In one embodiment of the present invention, the isolated antibody that binds PD-L1 is a monoclonal antibody.

[0228] In one embodiment of the present invention, a monoclonal antibody that binds PD-L1 is a full-length IgG antibody. In one embodiment of the present invention, the full-length IgG antibody is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0229] In one embodiment of the present invention, the full-length IgG antibody is of the IgG1 isotype.

[0230] In one embodiment of the present invention, the isolated antibody is the BCD135 antibody, which binds PD-L1 and comprises 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.

[0231] In one embodiment of the present invention, the isolated antibody is the BCD135 antibody, which binds PD-L1 and comprises a heavy chain variable region comprising CDRs 1-3 with the corresponding amino acid sequences of SEQ ID NOs: 1-3, and a light chain variable region comprising CDRs 1-3 with the corresponding amino acid sequences of SEQ ID NOs: 5-7.

[0232] In one embodiment of the present invention, the isolated antibody is the BCD135 antibody, which binds PD-L1 and comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 4 and a light chain variable region with the amino acid sequence of SEQ ID NO: 8.

[0233] In one embodiment of the present invention, the isolated antibody is the BCD135 antibody, which binds PD-L1 and comprises a heavy chain with the amino acid sequence of SEQ ID NO: 9 and a light chain with the amino acid sequence of SEQ ID NO: 10.

[0234] Nucleic acid molecules

[0235] The present invention also provides nucleic acid molecules and sequences encoding the anti-PD-L1 antibody of the invention, as described herein. In some embodiments, different nucleic acid molecules encode the first domain and the second domain of the amino acid sequence of the anti-PD-L1 antibody. Where the first domain and / or the second domain comprises a heavy chain and a light chain, in some embodiments, different nucleic acids encode the heavy chain and the light chain amino acid sequences. In other embodiments, the same nucleic acid molecule encodes the heavy chain and the light chain amino acid sequence. In certain embodiments, a nucleic acid molecule can encode any combination of the amino acid sequences (e.g., the heavy and light chain sequences) of the first and second domains.In a specific embodiment of the invention, a nucleic acid molecule may encode the amino acid sequence of a first binding domain and the amino acid sequence of the light chain of a second binding domain, optionally including any sequence of a peptide linker connecting them. Reference to a nucleotide sequence encompasses its complement unless otherwise indicated. Thus, reference to a nucleic acid having a particular sequence should be understood as encompassing its complementary strand with its complementary sequence. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms.

[0236] The present invention also relates to nucleotide sequences that are at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% homologous or identical to one or more nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1 _ 3, 5-7. In certain embodiments, the nucleotide sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or 8. The present invention also relates to nucleotide sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more of the aforementioned nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 9-10.

[0237] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NOs: 1-10. The nucleic acid molecule may also comprise any combination of said 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 nucleotide sequences encoding the amino acid sequence of SEQ ID NOs: 1-3 and nucleotide sequences encoding the amino acid sequence of SEQ ID NOs: 5-7.In one embodiment of the invention, 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 of the invention, 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.

[0238] In any of the above embodiments of the invention, the nucleic acid molecules may be isolated.

[0239] The nucleic acid molecule of the present invention can be isolated from any source that produces an antibody to PD-L1 or a portion thereof. In certain embodiments, the nucleic acid molecule of the present invention can be synthesized rather than isolated.

[0240] In some embodiments, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a VH domain from a first or second domain of an anti-PD-L1 antibody of the invention, linked 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 a VL domain from a first or second region of an anti-PD-L1 antibody of the invention, linked in frame to a nucleotide sequence encoding a light chain constant domain from any source.

[0241] In another aspect of the present invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and / or light (VL) chains of the first or second binding domain can be "converted" along the entire length of the antibody genes. In one embodiment of the invention, nucleic acid molecules encoding the VH or VL domains are converted into the entire length of the antibody genes by insertion into an expression vector already encoding the constant domains of the heavy chain (CH) or light chain (CL), respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and / or the VL segment is operably linked to the CL segment in the vector.In another embodiment of the invention, nucleic acid molecules encoding the VH and / or VL domains are converted into genes along the entire length of the antibody by joining, for example, by ligation, a nucleic acid molecule encoding the VH and / or VL domains to a nucleic acid molecule encoding the CH and / or CL domains using standard molecular biology techniques. Nucleic acid molecules encoding the entire length of the heavy and / or light chains can then be expressed from the cell into which they have been introduced.

[0242] Nucleic acid molecules can be used to express large numbers of recombinant antibodies to PD-L1. The nucleic acid molecules can 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.

[0243] Vector

[0244] In another aspect, the present invention relates to a vector suitable for expressing any of the nucleotide sequences described herein.

[0245] The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of antibodies to PD-L1 or portions thereof (e.g., the heavy chain sequences of the first and / or heavy and / or light chain of the second binding domain) as described herein. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, and antibody fragments.

[0246] In another embodiment, nucleic acid molecules and vectors can be used to produce mutated antibodies to PD-L1. The antibodies can be mutated in the variable domains of the heavy and / or light chains of the first and / or heavy and / or light chains of the second binding domain, for example, to alter the binding affinity of the antibodies to PD-L1. For example, the mutation can occur in one or more CDR regions to increase or decrease K Dantibodies to PD-L1 to increase or decrease k0ff or alter the binding specificity of the antibody for PD-L1. In another embodiment of the invention, one or more mutations are made to an amino acid residue that is known to be altered compared to the germline in an antibody corresponding to the first or second binding domain of an anti-PD-L1 antibody of the invention. These mutations can be made in the CDR region or framework region of the variable domain, or in the constant domain. In a preferred embodiment of the invention, the mutations are made in the variable domain. In another embodiment of the invention, one or more mutations are made to an amino acid residue that is known to be altered compared to the germline in the CDR region or framework region of the variable domain of an anti-PD-L1 antibody of the invention.

[0247] In some embodiments, the anti-PD-L1 antibodies of the invention are expressed by inserting DNA encoding part or all of the first and second binding domain sequences (e.g., heavy and light chain sequences, wherein the binding domain comprises heavy and light chain sequences), prepared as described above, into expression vectors such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YACs, EBV-derived episomes, and the like.DNA molecules can be ligated into a vector such that transcription and translation control sequences in the vector perform the intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules encoding part or all of the first and second binding domain sequences (e.g., heavy and light chain sequences, where the binding domain comprises a heavy and light chain sequence) can be introduced into separate vectors. In one embodiment, any combination of the aforementioned DNA molecules is introduced into the same expression vector.DNA molecules can be introduced into the expression vector by standard methods (e.g., by ligation of complementary restriction sites on the antibody gene fragment and the vector, or by blunt-end ligation if restriction sites are not present).

[0248] A suitable vector is one that encodes functionally complete CH or CL sequences of human immunoglobulin with an appropriate restriction site engineered so that any VH or VL sequence can be readily incorporated and expressed as described above. The HC and LC-encoding genes in such vectors may contain intronic sequences, which results in an overall increase in antibody protein products by stabilizing the corresponding mRNA. Intronic sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing will occur. The location of intronic sequences can be either in the variable or constant regions of the antibody chains, or in both variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosomal site of the encoded regions.A recombinant expression vector can also encode a signal peptide that facilitates production of the antibody chain by the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the reading frame of the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than an immunoglobulin).

[0249] In addition to the antibody gene sequence, the recombinant expression vectors of the present invention may carry regulatory sequences that control the expression of the antibody gene sequence in the host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the expression level of the desired protein, and so on.Preferred regulatory sequences for a mammalian expression host cell include viral elements that provide high-level expression of proteins in mammalian cells, such as promoters and / or enhancers derived from the retroviral LTR, cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus large late promoter (AdMLP)), polyoma virus, and strong mammalian promoters such as native immunoglobulin and actin promoters. For further descriptions of viral regulatory elements and sequences thereof, see, e.g., U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules such as plant antibodies, including the description of promoters and vectors, as well as plant transformation, are known in the art. See, for example, U.S. Patent 6,517,529.Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

[0250] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, a selectable marker gene typically 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 (DHFR) gene (for use in dhfr host cells for methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0251] The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to influence the 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., the Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion.The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include the ribosomal binding site promoter and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes, at a minimum, 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 fusion sequences.

[0252] Host cells

[0253] An additional aspect of the present invention relates to methods for producing anti-PD-L1 antibodies according to the invention. One embodiment of the invention relates to a method for producing anti-PD-L1 antibodies as defined herein, comprising obtaining a recombinant host cell capable of expressing an anti-PD-L1 antibody, culturing said host cell under conditions suitable for producing an anti-PD-L1 antibody, and isolating the resulting anti-PD-L1 antibody. An anti-PD-L1 antibody obtained by such expression in such recombinant host cells is referred to herein as "recombinant anti-PD-L1 antibodies". The invention also relates to progeny cells of such host cells and anti-PD-L1 antibodies obtained in a similar manner.

[0254] Nucleic acid molecules encoding the anti-PD-L1 antibodies of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or its cell, a plant or its cell, or a bacterial or yeast host cell. Transformation can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a complex of a nucleic acid and a positively charged polymer, transfection with a nucleic acid-calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into nuclei. Additionally, nucleic acid molecules can be introduced into mammalian cells via viral vectors.Methods for transfecting cells are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art, including, 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.

[0255] Mammalian cell lines used as transformation hosts are well known in the art and include a variety of immortalized cell lines readily available. These include, but are not limited to, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Ir itrogen), NIH-3T3 cells, HeLa cells, hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by identifying which cell lines have high expression levels and provide the desired characteristics of the protein produced. Other cell lines that can be used include insect cell lines such as Sf9 or Sf21 cells.When recombinant expression vectors encoding anti-PD-L1 antibodies are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells or, preferably, by releasing the antibodies into the growth medium in which the host cells are grown. Anti-PD-L1 antibodies can be isolated from the growth medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe.

[0256] Saccharomyces cerevisiae and Pichia pastoris.

[0257] Furthermore, the production level of the PD-L1 antibodies of this invention from the producing cell line can be enhanced using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is widely used to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846, 0256055, 0323997, and 0338841.

[0258] It is likely that anti-PD-L1 antibodies from different cell lines or transgenic animals will differ from one another in their glycosylation profiles. However, all anti-PD-L1 antibodies encoded by the nucleic acid molecules described herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation state of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

[0259] The invention also relates to methods and processes for producing antibodies to PD-L1 and their antigen-binding fragments. Monoclonal antibodies

[0260] Monoclonal antibodies can be produced, for example, by the hybridoma method first described by Kohler et al., Nature, 256, 1975, p. 495, or by recombinant DNA techniques (US 4816567).

[0261] Using the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to induce the formation of lymphocytes that produce or can produce antibodies capable of specifically binding to the protein used for immunization. Alternatively, lymphocytes can be obtained by in vitro immunization. Following immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable coupling agent, such as polyethylene glycol, to produce a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103).

[0262] The resulting hybridoma cells are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), substances that inhibit the growth of HGPRT-deficient cells.

[0263] Preferred myeloma cell fusion components are those that readily fuse, maintain stable, high levels of antibody production by the selected antibody-producing cells, and are sensitive to the selective medium used to select unrelated parental cells. Preferred myeloma cell lines include murine myeloma lines, such as the mouse tumor cell-derived MOPC-21 and MPC-11 lines, available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and the SP-2 or X63-Ag8-653 lines, available from the American Type Culture Collection, Rockville, Maryland, USA. The use of human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (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).

[0264] Preferably, the binding specificity of monoclonal antibodies produced using hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0265] The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard analysis described by Munson et al., Anal. Biochem., 107, 1980, p. 220.

[0266] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or potency have been identified, the clones can be subcloned using limiting dilutions and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103). Suitable media include, for example, DMEM or RPMI-1640. Hybridoma cells can also be grown in vivo as ascites tumors in animals, for example, by intraperitoneal (ip) injection of cells into mice.

[0267] Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascites fluid, or serum using conventional antibody purification methods, such as affinity chromatography (e.g., using protein A or protein Q-Sepharose), or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0268] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that bind specifically to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be incorporated into expression vectors, which are then transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not produce antibody protein without transfection, resulting in the synthesis of monoclonal antibodies in recombinant host cells. For a review of articles on the recombinant expression of antibody-encoding DNA in bacteria, see Skerra et al., Curr. Opinion in Immunol., 5 (1993), pp. 256-262 and Pliickthun, Immunol. Revs. 130, 1992, cc. 151-188.

[0269] According to another embodiment of the invention, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the methods described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10 (1992), pp. 779–783) and combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21 (1991), pp. 2265–2266). These methods thus represent a viable alternative to traditional monoclonal antibody hybridoma-based isolation methods.

[0270] The DNA encoding the antibody may also be modified, for example, to produce chimeric or fusion antibody polypeptides, for example by replacing the heavy and light chain constant region sequences (CH and CL) with homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851) or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can be replaced with constant regions of an antibody or replaced with variable regions of the antigen-binding site of an antibody, creating a chimeric bivalent antibody that contains one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0271] Humanized antibodies

[0272] Methods for creating "humanized" non-human antibodies are known in the art. Preferably, a humanized antibody has incorporated one or more amino acid residues derived from a non-human source. These non-human amino acid residues are often referred to as "import" residues because they are typically derived from an "import" variable region. Humanization can generally be accomplished according to the method of Winter et al. (Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-327; Verhoeyen et al., Science, 1988, 239:1534-1536) by replacing hypervariable region sequences with the corresponding sequences of a human antibody.Thus, "humanized" antibodies are chimeric antibodies (US 4816567) in which a region substantially smaller than the intact human variable region is replaced by the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous regions of rodent antibodies.

[0273] The selection of human variable regions of both the light and heavy chains to be used in humanized antibodies is critical to reducing antigenicity and the HAMA (human anti-mouse antibody) response when the antibody is intended for human use. In the so-called "best-fit" approach, the variable region sequence of a rodent antibody is screened against a complete library of known human variable region sequences. The human V region sequence that is most similar to the rodent sequence is identified, and a human framework region (FR) suitable for use in a humanized antibody is selected from within it (Sims et al., J. Immunol., 1993, 151, p. 2296; Chothia et al., J. Mol. Biol., 1987, 196, p. 901).Another method uses a specific framework region derived from the consensus sequence of a specific subset of the light or heavy chains of all human antibodies. The same framework region can 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).

[0274] It is also important that antibodies be humanized while maintaining high antigen binding affinity and other important biological properties. To achieve this, the preferred method involves generating humanized antibodies by analyzing the parental sequences and various humanized products using conceptual three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Studying these images allows for analysis of the potential role of residues in the function of a candidate immunoglobulin sequence, i.e., analysis of the residues that influence the ability of the candidate immunoglobulin to bind to an antigen.In this way, FR residues can be selected and combined with recipient and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen(s). Typically, hypervariable region residues have the most direct and significant impact on antigen binding.

[0275] A humanized antibody may be an antibody fragment, such as a Fab fragment, optionally conjugated to one or more cytotoxic agents to create an immunoconjugate. Alternatively, a humanized antibody may be a full-length antibody, such as a full-length IgG1 antibody.

[0276] Human antibodies and a phage display library-based approach

[0277] As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that, after immunization, can produce a full spectrum of human antibodies without producing endogenous immunoglobulin. For example, it has been described that homozygous deletion of the antibody heavy chain gene junction regions (jH segment) in chimeric mice and mice with a germline mutation leads to complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin germline gene complement into such a mutant mouse germline results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovis et al., Proc. Natl. Acad. Sci. USA: 90 (1993), p. 2551; Jakobovis et al., Nature, 362 (1993), pp. 25–258; Bruggermann et al., Year in Immune, 7 (1993), p. 33; and US 5545806, 5569825, 5591669 (all to GenPharm); 5545807; and W0 97 / 17852).

[0278] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable region (V) gene repertoire from immunized donors (McCafferty et al., Nature, 1990, 348, pp. 552–1553). In this technique, antibody V region genes are cloned in frame with either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as functional antibody fragments on the surface of a phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for functional antibody properties also selects a gene encoding an antibody that exhibits these properties. The phage thus mimics some of the properties of a B cell. Phage presentation can be accomplished in a variety of formats.

[0279] (for a review of which see, for example, Johnson Kevin S. and Chiswell David J., Current Opinion in Structural Biology, 3 (1993), pp. 564–571). Various sources of V-gene segments can be used for phage presentation. Clackson et al., Nature, 352 (1991), pp. 624–628, isolated different repertoires of antibodies to oxazolone from a small, random combinatorial library of V-genes obtained from the spleens of immunized mice. A repertoire of V-genes obtained from the body of immunized human donors can be constructed, and antibodies to a diverse repertoire of antigens (including self-antigens) can be isolated generally according to the methods described by Marks et al., J. Mol. Biol., 222 (1991), pp. 581-597 or Griffith et al., EMBO J. , 12, 1993, pp. 725-734)

[0280] (see also US 5565323 and 5537905).

[0281] As described above, human antibodies can also be produced in vitro by activated B cells (see US 5,567,610 and 5,229,275).

[0282] Antibody fragments

[0283] In certain circumstances, it may be appropriate to use antibody fragments rather than whole antibodies. The smaller size of fragments facilitates their rapid clearance and may improve penetration into solid tumors.

[0284] Various methods have been developed for producing antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., 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 using recombinant host cells. Fab, Fv, and scFv antibody fragments can be expressed and secreted from E. coli, facilitating the production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. According to another variant, Fab'-SH fragments can be directly isolated from E. coli and chemically cross-linked to obtain F(ab')2 fragments (Carter et al., Bio / Technology, 10, 1992, pp. 163-167). According to another approach, F(ab')2 fragments can be isolated directly from the culture of recombinant host cells.Fab and F(ab')2 fragments with increased in vivo half-life that retain epitope-binding receptor residues are described in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will 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; U.S. Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458). Fv and sFv are the only species with intact binding sites, lacking constant regions; as a result, they can be used for reduced non-specific binding in vivo applications. Fusion proteins bearing sFv can be engineered to fusion the effector protein to either the N- or C-terminus of sFv (see Antibody Engineering, Borrebaeck, ed., supra). An antibody fragment may also be a "linear antibody", such as described in US 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0285] Bispecific antibodies

[0286] Bispecific antibodies are antibodies that exhibit binding specificity to two different epitopes. For example, bispecific antibodies can bind to two different epitopes of the PD-L1 protein. Other bispecific antibodies may carry a binding site for PD-L1 in combination with a binding site for another protein. Bispecific antibodies can be produced as full-length antibodies or antibody fragments (e.g., F(ab')2 fragments of bispecific antibodies).

[0287] Methods for creating bispecific antibodies are known in the art. The conventional production of full-length bispecific antibodies is based on the coexpression of two pairs of immunoglobulin heavy and light chains, where the two chains have different specificities (Millstein et al., Nature, 305 (1983), pp. 537–539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) can potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually accomplished in several steps using affinity chromatography, is quite labor-intensive, and the yield of the product is low. Similar processes are described in WO 93 / 08829 and by Traunecker et al., EMBO J., 10 (1991), pp. 3655–3659.

[0288] According to another approach, the variable regions of an antibody with the desired binding specificity (antigen-binding sites of the antibody) are fused to immunoglobulin constant region sequences. The fusion is preferably performed with the constant region of the Ig heavy chain, which includes at least part of the CH2 and CH3 hinge regions. Preferably, at least one of the fusions contains the first constant region of the heavy chain (CH1), which contains the site necessary for light chain binding. DNAs encoding the fusions of the immunoglobulin heavy chain and, optionally, the immunoglobulin light chain are inserted into various expression vectors and co-transfected into a suitable host organism. This provides greater flexibility in selecting the overall proportions of the three polypeptide fragments in variants where unequal ratios of the three polypeptide chains are used in the construct to optimize yields.However, it is also possible to insert coding sequences into two or all three polypeptide chains in a single expression vector when expression of at least two polypeptide chains in equal proportions provides high yields or when the ratios are not critical.

[0289] In a preferred embodiment of this approach, the bispecific antibodies are a hybrid of an immunoglobulin heavy chain providing the first binding specificity in the first arm and a hybrid of an immunoglobulin heavy chain-light chain pair (providing the second binding specificity) in the second arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific molecule from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule facilitates separation. This approach is described in WO 94 / 04690. For further detailed description of the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121 (1986), p. 210.

[0290] According to a further approach, described in US Patent 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region. According to this method, one or more small amino acids with side chains from the interface of the first antibody molecule are replaced by molecules with larger side chains (e.g., tyrosine or tryptophan). Equilibrating "cavities" identical to or similar in size to the large side chain(s) are created in the interface of the second antibody molecule by replacing amino acids with large side chains with amino acids with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer relative to other undesirable end products, such as

[0291] Bispecific antibodies include cross-linked antibodies or "heteroconjugates." For example, one antibody in a heteroconjugate may be cross-linked to avidin and the other to biotin. Such antibodies can be used, for example, 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). Heteroconjugate antibodies can be created using any of the conventional cross-linking methods. Suitable cross-linkers are well known in the art and are described in US 4,676,980, along with various cross-linking methods.

[0292] Methods for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be produced by chemical coupling. Brennan et al., Science, 229, 1985, p. 81, described a procedure in which intact antibodies are subjected to proteolytic cleavage to yield F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, such as sodium arsenite, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted to the thionitrobenzoate (TNB) derivative. 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 yield a bispecific antibody. The resulting bispecific antibodies can be used as agents for selective immobilization of enzymes.

[0293] Recent progress has made it possible to facilitate the direct isolation of Fab'-SH fragments from E. coli, which can be chemically linked to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175 (1992), pp. 217–225, described the production of the F(ab')2 fragment of a fully humanized bispecific antibody molecule. Each Fab' fragment was individually secreted from E. coli and subjected to direct chemical coupling in vitro to yield the bispecific antibody. The bispecific antibody thus obtained was capable of binding to cells overexpressing the ErbB2 receptor and to normal human T cells, and of stimulating the lytic activity of human cytotoxic lymphocytes targeting human breast tumor.

[0294] Various methods for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies were produced using leucine zippers (Kostelny et al., J. Immunol., 148(5), 1992, pp. 1547–1553). Leucine zipper peptides from the Fos and Jun proteins were linked to Fab' fragments of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to yield monomers, and then reoxidation yielded antibody heterodimers. This method can also be used to produce antibody homodimers. The double antibody technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90, 1993, pp. 6444-6448, represents another mechanism for producing bispecific antibody fragments.The fragments contain a VH region linked to a VL region by a linker that is too short to allow pairing of the two domains of the same chain. Thus, the VH and VL regions of one fragment must pair with the complementary VL and VH regions of the other fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments has also been described, based on the use of single-chain (Fv)-(sFv) dimers (see Gruber et al., J. Immunol., 152 (1994), p. 5368).

[0295] The invention also encompasses antibodies having more than two valencies. For example, trispecific antibodies can be produced (Tutt et al., J. Immunol., 147, 1991, p. 60).

[0296] Polyvalent antibodies

[0297] A multivalent antibody can be internalized (and / or dissimilate) by a cell expressing the antigen to which the antibody binds more rapidly than a bivalent antibody. The antibodies of the present invention can be multivalent antibodies (other than the IgM class) with three or more antigen-binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of a nucleic acid encoding the polypeptide chains of the antibody. A multivalent antibody can comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In such a case, the antibody should comprise an Fc region and three or more antigen-binding sites located at the N-terminus relative to the Fc region.According to the present description, a preferred multivalent antibody comprises (or consists of) from 3 to about 8, but preferably 4, antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise(s) two or more variable regions. For example, the polypeptide chain(s) may comprise(s) VD1-(XI)n-VD2-(X2)n-Fc, where VD1 is the first variable region, VD2 is the second variable region, Fc is one polypeptide chain of the Fc fragment, XI and X2 are an amino acid or a polypeptide, and n is 0 or 1. For example, the polypeptide chain(s) may comprise(s) the following chain: VH-CH1-flexible linker-VH-CH1-Fc fragment; or VH-CHl-VH-CHl-Fc-fragment.The multivalent antibody of the present invention preferably further comprises at least 2 (and preferably 4) light chain variable region polypeptides. The multivalent antibody of the present invention may, for example, comprise from about 2 to about 8 light chain variable region polypeptides. In the context of the present description, the light chain variable region polypeptides are understood to comprise a light chain variable region and optionally further comprise a CL region. Pharmaceutical compositions.

[0298] Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the sole active ingredient), an antibody that is specific to PD-L1. The pharmaceutical composition may include any PD-L1 antibody as described herein. In some embodiments, the compositions are intended to ameliorate, prevent, or treat disorders that may be associated with PD-L1 activity.

[0299] Typically, the anti-PD-L1 antibodies of the present invention are suitable for use in dosage forms in combination with one or more pharmaceutically acceptable excipients, for example as described below.

[0300] The pharmaceutical compositions of the present invention may comprise at least one antibody to PD-L1 and one or more additional binding molecules (e.g., antibodies) that target one or more corresponding surface receptors.

[0301] A pharmaceutical composition is "sterile" if it is aseptic, i.e. free from microorganisms and their spores.

[0302] A pharmaceutical composition is considered "stable" if the active ingredient maintains its physical stability and / or chemical stability and / or biological activity throughout its shelf life at a storage temperature, e.g., 2-8°C. Preferably, the active ingredient maintains both physical and chemical stability, as well as biological activity. The shelf life is determined based on the results of stability studies under accelerated and natural storage.

[0303] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of inert excipient will depend largely on factors such as the particular route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and similar physiologically compatible substances. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffer, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.Additional examples of pharmaceutically acceptable substances include wetting agents or minor amounts of excipients such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or potency of the antibody.

[0304] A "buffering agent" is a solution capable of maintaining its pH value through the interaction of its acidic and alkaline components. Generally, pharmaceutical compositions have pH values ​​ranging from 4.5 to 7.0. Examples of buffers known to those skilled in the art and found in the literature include, but are not limited to, histidine, citrate, succinate, acetate, phosphate, phosphate-saline, citrate-phosphate, tromethamine-based buffers, and the like, or suitable mixtures thereof.

[0305] "Isotonic agents" refers to an excipient or a mixture of two or more excipients that provide an isotonic osmotic pressure to a solution. An "isotonic" solution is considered to have an osmotic pressure of approximately 250 to 350 mOsm / kg. Polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, and the like can be used as isotonic agents, but are not limited to them. The term "hypotonic" characterizes a composition with an osmotic pressure lower than the osmotic pressure of human blood. Accordingly, the term "hypertonic" characterizes a composition with an osmotic pressure higher than the osmotic pressure of human blood.

[0306] The term "surfactant" (also known as a surfactant or detergent) as used 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 help improve the colloidal stability or solubility of any antibody in the formulation. The surfactant can reduce aggregation of the formulated antibody formulation and / or minimize particle formation in the formulation and / or reduce adsorption. The surfactant can also improve the stability of the antibody during, including after, freezing / thawing and upon shaking. Surfactants can be ionic or nonionic.Illustrative nonionic surfactants that can be included in the compositions of the present 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. Specific nonionic surfactants that can be included in the compositions of the present 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 glycol or polyethylene glycol (PEG), copolymers of ethylene and propylene glycol (for example, Pluronics PF68, etc.).

[0307] By "stabilizer" is meant an auxiliary substance or a mixture of two or more auxiliary substances that ensure physical and / or chemical stability of the active agent. Amino acids such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants such as, but not limited to, polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxamer (Poloxaner), Pluronic (Pluronic) ), sodium dodecyl sulfate (SDS); antioxidants such as, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of sulfur acids, etc.; chelating agents such as, but not limited to, EDTA, DTPA, sodium citrate, etc.

[0308] "Pharmaceutically acceptable acid" includes inorganic and organic acids that are non-toxic in the concentration and form in which they are formulated. For example, suitable inorganic acids include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric, sulfonic, sulfanilic, phosphoric, carboxylic, etc. Suitable organic acids include linear or branched alkyl, aromatic, cyclic, cycloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono-, di- and tricarboxylic acids, including, for example, formic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, t-butylacetic, anthranilic, propanoic, 2-hydroxypropanoic, 2-oxopropanoic, malonic, cyclopentanepropionic, 3-phenylpropionic, butanoic, butanedioic, benzoic, 3-(4-hydroxybenzoyl) benzoic, 2-acetoxybenzoic, ascorbic, cinnamic, lauryl sulfuric, stearic,muconic, almond, succinic, emboneic, fumaric, malic, maleic, hydroxymaleic, malonic, lactic, citric, tartaric, glycolic, gluconic, gluconic, pyruvic, glyoxalic, oxalic, mesyl, succinic, salicylic, phthalic, palmic, palmeic, thiocyanic, methanesulfonic, ethanesulfonic, 1,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, naphthalene-2-sulfonic, p-toluenesulfonic, camphorsulfone, 4-methylbicyclo I 2.2.2 ] -oct-2-ene-1 -carboxylic, glucoheptone, 4,4'- 4, 4' -methylenebis-3- (hydroxy-2-ene-1 -carboxylic), hydroxynaphthoic.,

[0309] "Pharmaceutically acceptable bases" include inorganic and organic bases that are non-toxic in the concentration and form in which they are formulated.For example, suitable bases include bases formed from 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 alkyl, e.g., ammonium, Tris)], e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc.Particularly preferred organic non-toxic bases include isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine. Additional pharmaceutically acceptable acids and bases useful with the present invention include those derived from amino acids, such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine, and asparagine.

[0310] A "diluent" of interest according to the present invention is a diluent that is pharmaceutically acceptable (safe and non-toxic for human administration) and suitable for preparing a liquid composition, such as a composition reconstituted after lyophilization. Typical diluents include water, bacteriostatic water for injection (BWFI), a pH-buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution. In an alternative embodiment, diluents may include aqueous solutions of salts and / or buffers.

[0311] A "preservative" is a compound that can be added to the compositions provided herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the preparation of a multiple-dose composition. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl dimethyl ammonium chlorides in which 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 m-cresol. The most preferred preservative provided herein is benzyl alcohol.

[0312] The term "lyophilized" as used herein refers to a formulation that has been subjected to a process known in the art as freeze-drying, which involves freezing the formulation and then removing the ice from the frozen contents.

[0313] The term "amino acid" as used herein means an amino acid (a free amino acid, i.e., not an amino acid in a peptide or protein sequence). Amino acids used in the present invention include, but are not limited to, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tryptophan, serine, cysteine, methionine, and proline.

[0314] The pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington, The Science and Practice of Pharmacy, 21st Edition, Troy, Beringer, Lippincott Williams and Wilkins, Philadelphia, PA 2006. The manufacture of pharmaceutical compositions should preferably comply with GMP (good manufacturing practice) requirements.

[0315] The pharmaceutical composition of this invention may be manufactured, packaged, or commercially distributed as a single unit dose or multiple unit doses. As used herein, the term "unit dose" refers to a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0316] Any method of administering peptides, proteins or antibodies accepted in the art can be suitably used for the anti-PD-L1 antibody of the present invention.

[0317] The pharmaceutical compositions of the present invention are generally suitable for parenteral administration. As used herein, the term "parenteral administration" of a pharmaceutical composition includes any route of administration that involves physically breaking the integrity of a subject's tissue and introducing the pharmaceutical composition through the tissue breach, typically resulting in direct entry into the bloodstream, muscle, or internal organ. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by introducing the composition through a surgical incision, by applying the composition through a tissue-penetrating non-surgical wound, and the like.In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intra-articular injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be useful. Regional perfusion is also contemplated. Preferred embodiments of the invention include intravenous and subcutaneous routes.

[0318] Dosage forms of pharmaceutical compositions suitable for parenteral administration typically comprise the active ingredient in association with a pharmaceutically acceptable carrier / excipient, such as sterile water or a sterile isotonic solution. Such dosage forms may be manufactured, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable dosage forms may be manufactured, packaged, or sold in unit dosage form, such as 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 ingredients, including, but not limited to, suspending, stabilizing, or dispersing agents.In one embodiment of the invention of a composition for parenteral administration, the active ingredient is provided in dry form (i.e., powder or granules) for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted formulation.

[0319] Parenteral dosage forms also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents (preferably a pH of 3 to 9, even more preferably a pH of 4.5 to 7). However, for some applications, a more suitable dosage form may be a sterile non-aqueous solution or a dry form for use in combination with a suitable vehicle 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 necessary. Other suitable parenteral dosage forms may include those containing the active ingredient in microcrystalline form or in a liposomal preparation. Parenteral dosage forms may be formulated for immediate and / or modified release.Modified release dosage forms include delayed, sustained, pulsatile, controlled, targeted and programmed release.

[0320] For example, in one aspect, sterile injectable solutions can be prepared by incorporating the anti-PD-L1 antibody in the required amount in an appropriate solvent with one or a combination of the ingredients listed above, as needed, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile solvent that contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, methods of preparation include freeze-drying (lyophilization), which yields a powder of the active ingredient plus any additional desired ingredient from its previously sterile-filtered solution.Proper fluidity of the solution can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearates and gelatin, and / or by modified-release coatings (e.g., slow-release coatings).

[0321] The anti-PD-L1 antibody of the present invention may also be administered intranasally or by inhalation, typically in the form of a dry powder (alone, as a mixture, or as particles with mixed components, such as mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler such as a pressurized aerosol container, pump, spray, nebulizer (preferably an atomizer that uses the principle of electrohydrodynamics to produce a fine mist) or nebulizer, with or without a suitable propellant, or as nasal drops.

[0322] The pressurized container, pump, spray, nebulizer or atomizer typically contains a solution or suspension of the binding molecule of the present invention, including, for example, a suitable substance for dispersing, solubilizing or extending the release of the active substance, a propellant as a solvent.

[0323] Before use as a dry powder or suspension, the drug is typically micronized to a size suitable for delivery by inhalation (usually less than 5 microns). This can be achieved by any suitable milling method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid purification to form nanoparticles, high-pressure homogenization, or spray drying.

[0324] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention, a suitable powder base and a potency modifier.

[0325] A suitable solution formula for use in a nebulizer that utilizes the principle of electrohydrodynamics to produce a fine mist may contain a suitable dose of the PD-L1 antibody of the present 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.

[0326] Suitable flavoring agents such as menthol and levomenthol, or sweeteners such as saccharin or sodium saccharin may be added to the dosage forms of the present invention intended for inhalation / intranasal administration.

[0327] Parenteral dosage forms can be designed for immediate and / or modified release. Modified-release dosage forms include delayed, sustained, pulsatile, controlled, targeted, and programmed release.

[0328] In the case of dry powder inhalers and aerosols, the dosage unit is set by a valve that delivers a metered amount. Units according to the present invention are typically set to deliver a metered dose or "puff" of the binding molecule according to the present invention. The total daily dose will typically be administered as a single dose or, more commonly, in divided doses throughout the day.

[0329] The anti-PD-L1 antibody of this invention can also be formulated for oral administration. Oral administration can involve swallowing, so that the compound enters the gastrointestinal tract and / or enters the bloodstream buccally, lingually, or sublingually directly from the oral cavity.

[0330] Dosage forms suitable for oral administration include solid, semi-solid, and liquid systems such as tablets; soft or hard capsules containing multi- or nanoparticulates, liquids, or powders; lozenges (including liquid-filled); chewable forms; gels; rapidly dissolving dosage forms; films; suppositories; sprays; and buccal / mucoadhesive patches.

[0331] Liquid dosage forms include suspensions, solutions, syrups, and elixirs. Such dosage forms can be used as fillers in soft or hard capsules (e.g., gelatin or hydroxypropyl methylcellulose) and typically contain a carrier, e.g., water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid dosage forms can also be prepared by reconstitution of a solid, e.g., from a sachet. Therapeutic use of the anti-PD-L1 antibody of the present invention

[0332] In one aspect, the PD-L1 antibody of the invention is used in the treatment of diseases and disorders that are associated with PD-L1 activity, such as a disease or disorder selected from the group: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC (microsatellite instability-high colorectal cancer).

[0333] In one aspect, the treatment subject or patient is a mammal, preferably a human subject. The said subject may be male or female and of any age.

[0334] In the case of a tumor (e.g., a cancer tumor), a therapeutically effective amount of an antibody or antibody fragment (e.g., an antibody or antibody fragment that specifically binds to PD-L1) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (i.e., slow to some extent and, preferably, stop) the infiltration of peripheral organs by cancer cells; inhibit (i.e., slow to some extent and, preferably, stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or alleviate, to some extent, one or more symptoms associated with the disorder. The antibody or antibody fragment can, to some extent, prevent the growth of and / or kill existing cancer cells, it can cause a cytostatic and / or cytotoxic effect.In cancer therapy, in vivo efficacy can be determined, for example, by assessing survival, time to disease progression (TTP), tumor response rate (RR), duration of response, and / or quality of life. As used herein, the terms "co-administration," "co-administered," and "in combination with" when referring to an anti-PD-L1 antibody with one or more other therapeutic agents are intended to mean, refer to, or include:

[0335] 1) the simultaneous administration of such a combination of an antibody to PD-L1 according to the present invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which said components are released substantially simultaneously to said patient,

[0336] 2) the simultaneous administration of such a combination of an antibody to PD-L1 according to the present invention and a therapeutic agent to a patient in need of treatment, wherein such components are formulated separately in different dosage forms, the administration of which occurs at substantially the same time to said patient, whereupon said components are released substantially simultaneously to said patient,

[0337] 3) sequentially administering such a combination of an anti-PD-L1 antibody of the invention and a therapeutic agent to a patient in need of treatment, wherein such components are formulated separately from one another in separate dosage forms that are administered sequentially in time to said patient with a significant time interval between each administration, wherein said components are released at substantially different times to said patient; and

[0338] 4) sequential administration of such a combination of an anti-PD-L1 antibody of the present invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which the release of said components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released at the same time and / or different times to said patient, wherein each part can be administered by the same or different routes. The anti-PD-L1 antibody of the present invention can be administered without additional therapeutic treatment, i.e., as a standalone therapy. In addition, treatment with an anti-PD-L1 antibody of the present invention can include at least one additional therapeutic treatment (combination therapy). In some embodiments of the invention, the anti-PD-L1 antibody can be co-administered or formulated with another drug / treatment agent for cancer.

[0339] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The term is intended to include 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 (Lu), chemotherapeutic agents and toxins, such as small molecular weight toxins or enzymatically active toxins, originating from bacteria, fungi, plants or animals, including fragments and / or variants thereof.

[0340] A "chemotherapeutic agent" is a chemical compound used to treat a malignant tumor. 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, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (particularly bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARIN0L ® ) ; beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN ® ), SRT-11 (irinotecan, CAMPTOSAR ®), acetyl camptothecin, scopolectin and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (in particular, riptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; esperamicin; as well as the neocarzinostatin chromophore and related chromoprotein chromophores enediyne antibiotics, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-3-norleucine, doxorubicin (including ADRIAMICIN. ® , morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicin HCl in injectable liposomes (DOXOL ® ), liposomal doxorubicin TLC D-99 (MYOCET ® ), pegylated liposomal doxorubicin (CAELYX ®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ® ) , tegafur (UFTORAL ® ), capecitabine (XELODA ® ), epothilone and

[0341] 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrex; purine analogs such as fludarabine, β-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine,

[0342] 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; adrenal suppressants such as aminoglutethimide, mitotane, trilostane; folate supplementer such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK ®(JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (such as T-2 toxin, verracurin A, roridin A, and anguidine); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("aga-C"); thiotepa; a taxoid such as paclitaxel

[0343] (TAX0L ® ), a paclitaxel preparation based on engineered albumin-linked nanoparticles (ABRAXANETM) and docetaxel

[0344] (TAXOTERE ® ) ; chlorambucil; b-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin, and carboplatin; vinca alkaloids that prevent tubulin polymerization from nascent microtubules, including vinblastine (VELBAN ® ), vincristine

[0345] (ONCOVIN ® ), vindesine (ELDISINE ® , FILDESIN ® ) and vinorelbine

[0346] (NAVELBINE ® ) ; etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine

[0347] (DMF0) ; retinoids such as retinoic acid, including bexarotene ( TARGRETIN ® ) ; bisphosphonates such as clodronate

[0348] (for example, BONEFOS ® or 0STAC ® ), etidronate (DIDROCAL ® ), NE-58095, zoledronic acid / zoledronate (ZOMETA ® ), alendronate

[0349] (F0SAMAX ® ), pamidronate (AREDIA ® ), tiludronate (SKELID ® ) or risendronate (ACTONEL ®) ; troxacitabine (1 , 3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, in particular oligonucleotides that inhibit the expression of genes in signal transduction pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine ® and gene therapy vaccines, such as the ALLOVECTIN vaccine ® , LEUVECTIN vaccine ® and the VAXID vaccine ® ; topoisomerase 1 inhibitor (eg, LURTOTECAN ® ) ; rmRH

[0350] (for example, ABARELIX ® ) ; BAY439006 (sorafenib; Bayer); SU-11248

[0351] (Pfizer); perifosin, COX-2 inhibitor (eg, celecoxib or etoricoxib), proteasome inhibitor (eg, PS341); bortezomib

[0352] (VELCADE ® ) ; CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor 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 foregoing; and combinations of two or more of the foregoing such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for the treatment regimen of oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovorin.

[0353] Also included in the definition are antihormonal agents that act by regulating or inhibiting the action of hormones on tumors, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX ® ), 4-hydroxytamoxifen, trioxifen, toremifene (FARESTON ® ) ; idoxifene, droloxifene, raloxifene (EVISTA ® ), trioxifene, keoxifene and selective estrogen receptor modulators

[0354] (SERM) such as SERM3; pure antiestrogens without agonist properties such as fulvestrant (FASLODEX ® ) and EM800 (such agents can block the dimerization of estrogen receptors

[0355] (ER), inhibit DNA binding, enhance ER metabolism, and / or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane

[0356] (AROMAS IN ® ), and nonsteroidal aromatase inhibitors such as anastrozole (ARIMIDEX ® ), letrozole (FEMARA ® ) and aminoglutethimide and other aromatase inhibitors, including vorozole (RIVIS0R ® ), megestrol acetate (MEGASE ® ), fadrozole, imidazole; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON ® and ELIGARD ®), goserelin, buserelin, and tripterelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and Premarin, and androgens / retinoids such as fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor down-regulators (ERDs); antiandrogens such as flutamide, nilutamide, and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

[0357] Other therapeutic agents that may be used in combination with the anti-PD-L1 antibodies of the invention may be inhibitors of growth factor function, such as 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 by Stern et al. Critical reviews in oncology / haematology, 2005, Vol. 54, pp. 11-29); antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor [eg, the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin)], antibodies against vascular endothelial growth factor receptors such as anti-KDR antibodies and anti-fltl antibodies;Antisense therapies, such as those directed at the above targets, such as ISIS 2503, anti-ras antisense agents or G3139 (Genasense), anti-bc12 antisense agents; Gene therapy approaches, including, for example, approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches using cytosine deaminase, thymidine kinase or bacterial nitroreductase enzyme, and approaches aimed at increasing patient tolerance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy;Immunotherapeutic approaches, including, for example, treatment with Alemtuzumab (campath-lH), a monoclonal antibody directed to CD52, or treatment with antibodies directed to CD22, ex vivo and in vivo approaches to enhance the immunogenicity of a patient's tumor cells, transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte-macrophage colony-stimulating factor, approaches aimed at reducing T cell anergy such as treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines, and approaches using anti-idiotypic antibodies, adoptive T cell transfer using T cells subjected to non-specific activation or targeted to a specific antigen of interest ex vivo;protein degradation inhibitors, such as a proteasome inhibitor such as Velcade (bortezamide); biotherapeutic therapeutic approaches, such as those using peptides or proteins (such as antibodies or soluble receptor external domain constructs) that sequester ligand receptors, block ligand binding to the receptor, or attenuate receptor signaling (e.g., due to increased receptor degradation or decreased expression levels).

[0358] Dosage and administration routes

[0359] The anti-PD-L1 antibody of the present invention will be administered in an amount effective to treat the condition in question, i.e., at doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the specific condition being treated, the patient's age, gender, and weight, and whether the anti-PD-L1 antibody is administered as a stand-alone treatment or in combination with one or more additional anti-autoimmune or anti-inflammatory therapies.

[0360] Dosage regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased depending on the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for the patients / subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specification for the unit dosage forms of the present invention is generally dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compound for the treatment of sensitivity in subjects.

[0361] Thus, those skilled in the art will appreciate, based on the disclosure provided herein, that doses and dosage regimens are adjusted according to methods well known in the therapeutic art. This means that the maximum tolerated dose can be readily established, and an effective amount providing a detectable therapeutic effect for a patient can also be determined, as can the timing of administration of each agent to achieve a detectable therapeutic effect for the patient. Thus, although certain doses and administration regimens are provided as examples herein, these examples in no way limit the doses and administration regimens that may be required for a patient practicing the present invention.

[0362] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated and may involve one or more doses. Furthermore, it should be understood that for any particular patient, specific administration regimens should be adjusted over time according to individual needs and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges provided herein are provided as an example only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, the dosage regimen for the compositions of the present invention may be based on various factors, including the type of disease, age, weight, gender, health status of the patient, severity of the condition, route of administration, and the specific anti-PD-L1 antibody used.Thus, the dosage regimen may vary widely but can be determined regularly using standard methods. For example, doses can be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects, such as toxic effects, or laboratory values. Thus, the present invention encompasses individualized dose escalation, as determined by a skilled artisan. Determining the required dose and regimens are well known in the art and will be readily apparent to those skilled in the art upon familiarity with the teachings disclosed herein.

[0363] Examples of suitable routes of administration are provided above.

[0364] It is contemplated that a suitable dose of the PD-L1 antibody of the present invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, such as about 1-20 mg / kg. The PD-L1 antibody can be administered, for example, at a dose of 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 a maximum of 50 mg / kg, including up to a maximum of 30 mg / kg, such as up to a maximum of 20 mg / kg, including up to a maximum of 15 mg / kg.The administration will usually be repeated at suitable intervals, such as once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsible physician, who may in some cases increase or decrease the dose if necessary.

[0365] Product(s) and sets

[0366] Another embodiment of the invention is an article of manufacture that contains products used to treat cancer, in particular HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC. The article is a container and a label or package insert that are placed on the container or inserted into it. Suitable containers are, for example, jars, vials, syringes, etc. Containers can be made of various materials, such as glass or plastic. The container contains a composition effective for treating a particular condition and can have a sterile inlet (for example, the container can be a bag for an intravenous solution or a vial provided with a stopper that can be pierced with a hypodermic needle).At least one active ingredient in the composition is an anti-PD-L1 antibody as proposed in the invention. The label or package insert indicates that the composition is used to treat a specific condition. The label or package insert must also contain instructions for administering the antibody composition to the patient.

[0367] The package insert contains the usual instructions included in the marketing package of therapeutic products, including exemplary information regarding the indications, use, dosage, route of administration, contraindications, and / or precautions related to the use of such therapeutic products. In one embodiment, the package insert indicates that the composition is used for the treatment of cancer, in particular HNSCC, cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, and MSI CRC.

[0368] The product may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also include other products required from a commercial and consumer perspective, including other buffers, diluents, filters, needles, and syringes.

[0369] The invention also relates to kits that can be used for various purposes, for example, for detecting PD-L1 in tissues, cells or body fluids of a mammal. Such a kit will be suitable for screening for PD-L1-associated diseases. The kit comprises a specific binding agent or antibody according to the invention and means for indicating the reaction of the specific binding agent or antibody with PD-L1, if present. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody that binds PD-L1 is labeled. In another embodiment, the antibody is an unlabeled primary antibody, and the kit further comprises means for detecting the primary antibody. In one embodiment, the means for detecting includes a labeled second 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-opaque material. The kit may be a kit containing antibodies for detecting and quantifying PD-L1 in vitro, such as by ELISA or Western blotting. As with the article of manufacture, the kit comprises a container and a label or package insert located on the surface of or within the container. The container contains a composition that includes at least one anti-PD-L1 antibody according to the invention. Additional containers may contain, for example, diluents and buffers, and control antibodies. The label or package insert may contain a description of the composition, as well as instructions for its use in vitro or for diagnostic purposes.

[0370] Diagnostic uses and compositions

[0371] The anti-PD-L1 antibody of the present invention is also used in diagnostic processes (e.g., in vitro, ex vivo). For example, the anti-PD-L1 antibody can be used to detect or measure the level of PD-L1 in samples obtained from a patient (e.g., a tissue sample or a body fluid sample such as inflammatory exudate, blood, blood serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The invention further includes kits (e.g., diagnostic kits) containing the anti-PD-L1 antibodies described herein.

[0372] To better understand the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0373] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example to avoid ambiguity, those skilled in the art, based on the teachings disclosed in this invention, will readily understand that certain changes and modifications can be made without departing from the spirit and scope of the accompanying embodiments of the invention.

[0374] Examples

[0375] Example 1.

[0376] Production of recombinant antigens and antibodies in suspension culture of mammalian cells

[0377] Antibodies and antigens were produced in a constant cell line derived from Chinese hamster ovary cells (CHO-K1 line) 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 EBNA1 (Epstein-Barrvirus nuclear antigen 1) protein gene were used. Suspension cultivation was performed in flasks on an orbital shaker using serum-free media from Life Technologies Corporation and according to the manufacturer's instructions. For transient expression, cells at a concentration of 2*10 6 / ml were transfected using linear polyethyleneimine (PEI MAX, Polysciences). The DNA / PEI ratio was 1:3 / 1:10. Five to seven days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a 0.22 μm filter. Target proteins were isolated from the culture fluid using affinity chromatography.

[0378] Recombinant PD-L1 protein containing the EPEA tag (glutamic acid-proline-glutamic acid-alanine) at the C-terminus of the protein was isolated and purified from culture fluid using the CaptureSelect C-tag Affinity Matrix sorbent. The culture fluid was passed through a chromatography column pre-packed with 5 ml of C-tag sorbent, then washed with 25 ml of PBS to remove non-specifically bound components. Bound antigen was eluted under mild conditions using 20 mM Tris, 2 M MgCl2 pH 7.0-7.4. The protein was then converted to PBS (pH 7.4) by dialysis using a semipermeable dialysis membrane, filtered (0.22 μm), transferred to tubes, and stored at -70°C.

[0379] Recombinant PD-1 and PD-Ll-Fc proteins were isolated and purified from the culture fluid using a protein A affinity chromatography column. The clarified culture fluid was passed through a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash out nonspecific binding components. Bound antigen was eluted using 0.1 M glycine buffer pH 3. The main protein elution peak was collected and its pH was adjusted to neutrality with 1 M Tris buffer (pH 8). All steps were performed at a flow rate of 110 cm / h. The protein was then converted to PBS (pH 7.4) by dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 µm), transferred into test tubes and stored at -70 °C.

[0380] IgG1 antibodies were purified using a 1 ml Hi Trap rProteinA FF column (GE Healthcare) according to the method described above for antigens. The purity of the resulting protein solution was assessed by SDS gel electrophoresis (Figs. 4A and 4B).

[0381] Example 2.

[0382] Creation of a naive Fab library of human antibodies MeganLibTM

[0383] Total B-lymphocyte RNA was isolated from individual blood samples from over a thousand human donors using the RNeasy Mini Kit according to the suggested protocol (from QIAGEN). RNA concentration was determined using the Nanovue kit (from GE Healthcare), and the quality of the isolated RNA was verified by electrophoresis in a 1.5% agarose gel.

[0384] The reverse transcription reaction was performed using the MMLV RT kit (Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexameric oligonucleotides as primers.

[0385] The reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides according to the authors' protocols [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

[0386] The resulting VL-CK-VH DNA preparation (Fig. 1) was treated with restriction endonucleases NheI / Eco91I and ligated into the original pH5 phagemid (Fig. 2). The ligation products were transformed into electrocompetent cells of the SS320 strain, prepared according to the protocols [Methods Enzymol. 2000; 328: 333-63]. The repertoire of the combinatorial phage Fab-display library MeganLibTM was 10 11 transformants. Phage preparations from Fab libraries were prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5;222(3): 581–97].

[0387] Example 3.

[0388] Selection of Fab libraries of phage antibodies

[0389] Specific human phage Fab antibodies against PD-L1 were obtained from the MeganLibTM combinatorial phage Fab display library. Selection was performed on human PD-L1 using the phage display method [Nat Biotechnol . 1996 Mar; 14 ( 3 ): 309–14 ; J Mol Biol. 1991 Dec 5; 222(3): 581–97], but with the use of magnetic particles and a KingFisher Flex instrument, since the use of this technique allows for the parallel execution of up to 96 different biopanning schemes and variants.

[0390] In biopanning selection, biotinylated PD-Ll-Fc at a concentration of 10 μg / ml was immobilized on the surface of streptavidin magnetic particles by incubating the protein with the particles for 1 hour at room temperature on a rotator. The particles were then washed with PBS (pH 7.4), then blocked with a solution of 2% skim milk in PBS (pH 7.4) for 1 hour. A solution of phages in PBS (pH 7.4) with 2% skim milk was then added to the magnetic particles with bound antigen; the concentration of phage particles was 2.5 x 10 12per ml. This mixture was incubated for 40 min with stirring. Unbound phages were removed during several washes of magnetic particles with a PBS solution (pH 7.4) with 0.1% Tween 20. The number of washes was increased from round to round (10 washes in the 1st round, 20 in the 2nd and 30 in the 3rd). Phages bound to the antigen on the surface of magnetic particles were eluted from the particles with a 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring, after elution they were neutralized with a 1 M TRIS-HC1 solution (pH 7.6). Bacteria of the TGI strain E. coli were infected with the obtained phages, phages were produced in them, they were isolated and used in the next round of selection. After two or three rounds, DNA was isolated from the phages (phagemids), and the variable domain genes of the antibodies were cloned into expression vectors (Fig. 3) to produce Fabs in E. coli cells.

[0391] Example 4.

[0392] Screening of Fabs specifically binding to human PD-L1 ELISA was used to search for Fabs binding to human PD-L1. Fab with the published sequence of Atezolizumab (Genentech) was used as a positive control. For the specific binding assay, wells of ELISA plates (medium binding from Greiner bio one) were coated with 50 μl of PD-L1-FE (0.2 μg / ml in 1 χThe plates were then sealed and incubated overnight at 4°C. All subsequent steps were performed according to the standard ELISA protocol using a high-throughput automated platform based on the Genetix Qpix2xt robotic systems (Molecular Device) and Tecan Freedom EVO 200 (Tecan). To block non-specific binding, blocking buffer BB (200 μl of 0.5% non-fat milk in PBS) was added. The plates were incubated on a shaker for 1 hour at room temperature. After washes with PBS-Tween, 50 μl of the test cell supernatant containing the studied Fab mixed with an equal volume of BB were added per well. The plates were again incubated with shaking for 1 hour at room temperature, after which each well of the plates was washed three times with PBS-Tween buffer. After washing, 50 µl / well of anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added at a ratio of 1:5000 in PBS-Tween.The plates were shaken on a rotary shaker (50 min, room temperature) and washed three times with PBS-Tween buffer as described above. The colorimetric signal was developed by adding TMB (50 µl / well) to saturation (on average, 3-5 min), then further development was stopped by adding a stop solution (30 µl / well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (Tecap). The degree of antibody binding was proportional to the production of a color signal. Clones with a color signal exceeding the signal from the control antibody were tested in ELISA for nonspecific binding.

[0393] Example 5.

[0394] Analysis of non-specific binding of selected Fabs to other antigens

[0395] ELISA was also used to analyze the non-specific binding of the studied Fab fragments to other antigens. The study was carried out as described above, but IL6R-Fc, INFa2b, PCSK9-VG-FE, PD-1-Fc (2.5 μg / ml in 1 χ carbonate buffer). PD-Ll-Fc (0.2 μg / ml in 1 χ (carbonate buffer). All subsequent steps were performed according to the standard ELISA protocol using a high-throughput automated platform based on the Genetix Qpix2xt robotic systems (Molecular Device) and Tesap Freedom EVO 200 (Tesap). Clones in which the color signal of nonspecific binding did not exceed the signal from specific binding were tested in a competitive ELISA assay to identify antagonist Fabs that block the interaction of ligand and receptor.

[0396] Example b.

[0397] Competitive ELISA assay for blocking the interaction of PD-L1 with its receptor PD-1

[0398] A competitive ELISA was used to test previously selected specific Fabs against human PD-L1 for their ability to block interaction with the PD-1 receptor. A Fab with the published sequence of Atezolizumab (Genentech) was used as a positive control antagonist.

[0399] PD-1-Fc was immobilized in ELISA plates (medium binding from Greiner bio one) at a concentration of 1 μg / mL in 1% carbonate buffer (50 μL) and incubated overnight at 4°C. All subsequent steps were performed according to standard ELISA protocols using a high-throughput automated platform based on the Genetix Qpix2xt robotic systems (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). To block nonspecific binding, blocking buffer BB (200 μL of 0.5% nonfat milk in PBS) was added. The plates were incubated on a shaker for 1 hour at room temperature.

[0400] In parallel, the cell supernatant containing the tested Fab and PD-Ll-Fc (at a final concentration of 2 μg / ml in PBS-Tween) were mixed in a 1:1 ratio in non-sorbent plates and incubated for 45 minutes at room temperature and shaking at 500 rpm.

[0401] After washing the PD-1 receptor-containing plate from the BB, a mixture of Fab and PD-L1 was transferred there, incubated for 45 minutes at room temperature and shaken at 500 rpm. After that, each well of the plate was washed three times with PBS-Tween buffer, 50 μl / well of anti-human Fab HRP-conjugated secondary antibody (from Pierce-

[0402] ThermoScientific) at a ratio of 1:5000 in PBS-Tween. Incubation was performed for 45 min at room temperature with shaking at 500 rpm, after which each well of the plates was washed three times with PBS-Tween buffer, as described above. The colorimetric signal was developed by adding TMB (50 μl / well) to saturation (on average 3-5 min), then further development was stopped by adding stopping solution (30 μl / well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (Tecan). The degree of Fab binding was inversely proportional to the production of a color signal. Clones showing blocking at the level of the control Fab antibody Atezolizumab were marked as positive and used in further analyses. The variable domain genes of the positive clones were sequenced according to standard protocols on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and analyzed.

[0403] Example 7.

[0404] Comparative screening of human anti-PD-Ll Fab candidates by koff

[0405] Koff screening was performed using a Pall Forte Bio Octet Red 96 instrument. Anti-FABCH1 biosensors were rehydrated for 30 min in a working buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. The working buffer was added to the studied E. coli supernatant samples to a final concentration of 1*. Then, the anti-FABCH1 biosensors were immersed in E. coli supernatants containing Fab fragments of the candidate antibodies for 12 h at 4°C. Sensors with Fab fragments immobilized on the surface were transferred to wells with the working buffer, where the baseline (60 s) was recorded. The sensors were then transferred to wells containing the analyte solution (PD-L1, 30 μg / ml) to allow the antigen-antibody complex to associate (300 s). The sensors were then returned to wells containing the working buffer for a subsequent 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) and then used in the next experiment.The analysis of the obtained curves was performed using Octet Data Analysis software (version 7.0) according to the standard procedure using the 1:1 interaction model.

[0406] Example 8.

[0407] Enzyme-linked immunosorbent assay of the interaction of anti-PD-L1 antibodies with PD-L1 and other antigens

[0408] ELISA was used to measure the comparative affinity of antibodies to PD-L1 and other antigens. For the binding assay, wells of ELISA plates (medium binding from Greiner bio one) were coated with 50 μl of PD-Ll-Fc, fPCSK9-EPEA, Ang2-H6F, GM-CSF-FE, CD3-ED-Fc, IL17a, CD38-Fc, IL6R-Fc (1 μg / ml in 1 χThe plates were prepared in a carbonate buffer, sealed, and incubated overnight at 4°C. All subsequent steps were performed according to the standard ELISA protocol. To block nonspecific binding, buffer BB (200 μl of 0.5% nonfat milk in PBS) was added. The plates were incubated on a shaker for an hour at room temperature. After washing with PBS-Tween, 50 μl of the BCD-135 test antibody at a concentration of 5 μg / ml in PBS-Tween were added per well. The plates were again incubated, shaking, for one hour at room temperature, after which each well of the plates was washed three times with PBS-Tween buffer. After washing, anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added (50 µl / well) at a ratio of 1:5000 in PBS-Tween. The plates were shaken on a rotary shaker (50 min, room temperature) and washed three times with PBS-Tween buffer as described above.The colorimetric signal was developed by adding TMB (50 μl / well) to saturation (3-5 min on average), then further development was stopped by adding stopping solution (30 μl / well, 10% sulfuric acid). The color signal was measured at 450 nm using a suitable Tecan-Sunrise plate reader (Tecamp). The degree of antibody binding was proportional to the color signal production (Fig. 5). The anti-PD-L1 antibody specifically bound to PD-L1 and did not bind to other antigens tested.

[0409] Example 9.

[0410] Reactivation of NFAT signaling by anti-PD-LL antibodies in the Jurkat-NFAT-PD-1 reporter cell line

[0411] The Jurkat human T-cell line was engineered by introducing two genetic constructs into its genome. One construct encoded the human PD-1 receptor gene. The second construct encoded the luciferase gene, which is under the control of an NFAT-responsive genetic element. This resulted in the Jurkat-NFAT-PD-1 reporter cell line, which expressed the PD-1 receptor on the surface membrane and contained an NFAT-dependent promoter that directed transcription of the luciferase gene. Luciferase synthesis in this cell line was proportional to the level of NFAT activity, which, in turn, reflected the overall level of T-lymphocyte activation.

[0412] The activity of anti-PD-L1 antibodies using this cell line was analyzed as follows: activation of TCR receptors with anti-CD3 and anti-CD28 antibodies triggered an intracellular cascade that resulted in activation of the NFAT promoter. PD-L1 is expressed on the surface of interferon-activated MDA-MB-231 cells. The interaction of PD-L1 with PD-1 inhibited signaling from TCR receptors to the NFAT promoter. Anti-PD-L1 antibodies uncoupled the PD-L1-PD-1 interaction, reactivating intracellular signaling.

[0413] MDA-MB-231 cells were activated to produce PD-L1 with a solution of interferon gamma. For this purpose, 72 hours before the analysis, interferon gamma was added to the cell suspension to a concentration of 20 ng / ml, then the cells were seeded into 9-well culture plates at a rate of 10,000 cells / well.

[0414] After 72 hours of activation, the growth medium was removed from the plates with MDA-MB-231 cells and dilutions of the analyzed antibodies, control antibody and isotype control in the cell growth medium from 10 μg / ml to 0.001 μg / ml were added to them and incubated for 30 minutes at room temperature.

[0415] Next, a suspension of Jurkat-NFAT-PD-1 cells and a solution of activating antibodies aCD3 / aCD28 / a-mouseIgG were added to each well. The plate was placed in a CO2 incubator for 6 hours.

[0416] A pre-prepared Bio-Glo Luciferase assay system (Promega) luciferase substrate was added at a ratio of V cells to V substrate. Luminescence was measured on a Fluoroscan Ascent (Fig. 6). Anti-PD-L1 antibodies reactivated luminescence in the Jurkat-PD-1-NFAT reporter cell line.

[0417] Example 10.

[0418] Analysis of interactions of anti-PD-LL antibodies with FcRn and Fcγ receptors using Octet RED 96. A Fortebio Octet RED96 instrument was used to analyze the interaction of antibodies with FcgRIIIaV, FcgRIa, and FcRn receptors. Receptors biotinylated at the C-terminus and streptavidin-coated biosensors (SA-Streptavidin) were used.

[0419] Biotinylated receptors were immobilized on the sensor surface. Next, an association step was performed: the sensors with bound antigen were immersed in antibody solutions of varying concentrations (a series of antibody dilutions in the working buffer were prepared in advance 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 solution to wells containing the working buffer.

[0420] To evaluate the affinity constant of the antibody to FcgRIIIaV and FcgRIa, phosphate buffer pH7.4 was used, and for FcRn, phosphate buffer pH6.0.

[0421] The obtained curves were analyzed using the Forte Bio Data Analysis 8.2 software and a 1:1 binding model. The results are shown in Fig. 7. No binding to Fcg receptors was detected for the modified IgG1 antibody compared to the wild-type variant, suggesting the absence of effector functions for the analyzed antibody. The affinity constant for FcRn of the analyzed anti-PD-L1 antibody was 1.69E-08 1 / M.

[0422] Example 11.

[0423] Enzyme-linked immunosorbent assay of interactions between anti-PD-L1 antibodies and PD-L1 in different organisms

[0424] ELISA was used to measure the comparative affinity of antibodies to PD-L1 from different organisms. For the binding analysis, wells of ELISA plates (medium binding from Greiner bio one) were coated with 50 μl of PD-Ll-Fc from human, cynomolgus, mouse, rat, dog, and rabbit (0.5 μg / ml in 1 χcarbonate buffer), sealed and incubated overnight at 4°C. All subsequent steps were performed according to the standard ELISA protocol described above. The anti-PD-L1 antibody specifically bound to human and cynomolgus monkey PD-L1 and did not bind to the other receptors studied (Fig. 8). Example 12.

[0425] Analysis of interactions of anti-PD-Ll antibodies with human and cynomolgus PD-L1 using the Octet RED 96 instrument

[0426] The binding affinity constants of the antibody to human and cynomolgus PD-L1 were measured using an OctetRed 96 (ForteBio). BCD-135 antibody at a concentration of 30 μg / mL was nonspecifically immobilized on the surface of second-generation amine-reactive sensors (ForteBio, AR2G) using the standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors. The assay was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. The binding of human and monkey PD-L1 solutions to the sensor-bound antibody was analyzed in a working buffer with antigen concentrations from 10 μg / mL to 1 μg / mL.

[0427] Baseline-subtracted binding curves were analyzed using Octet Data Analysis (version 8.2) according to the standard procedure using a 1:1 interaction model. The anti-PD-L1 antibody binds specifically and with affinity to human and cynomolgus monkey PD-L1 antigen (Fig. 9) with constants <1.0E-12 and 5.55E-10 1\M, respectively.

[0428] Example 13.

[0429] Determination of the stability of anti-PD-Ll antibodies

[0430] The conformational stability of BCD-135 was assessed by the protein aggregation point using dynamic light scattering (DLS). The aggregation point of the studied proteins (1 mg / ml) was determined 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 setup while continuously measuring the scattered light intensity. The BCD-135 antibody demonstrated high conformational stability in 20 mM acetate buffer, with a melting point greater than 80°C. ° C (Fig. 10).

[0431] The colloidal stability of the candidates was assessed using the PEG protein aggregation method. Samples with a protein concentration of 5 mg / mL were used for the experiment. The calculated amounts of sample, placebo solution, and PEG 6000 solution were transferred to UV spectrophotometry plates. All solutions obtained in the wells were thoroughly mixed by pipetting. The turbidity of the solutions was then assessed visually, and the optical density of the solutions was measured at λ = 320 nm. The BCD-135 antibody demonstrated high colloidal stability (Fig. 11).

[0432] Thermal stability of the antibody was assessed by thermal stress at 50 °C for 48 h in three different buffers: 20 mM phosphate buffer at pH 6.0 (Fig. 12A), 20 mM acetate buffer at pH 5.0 (Fig. 12B), and 20 mM histidine buffer at pH 5.5 (Fig. 12C). Homogeneity control was performed by SEC HPLC.

[0433]

[0434] The test samples at a protein concentration of ~5 mg / ml were divided into 2 parts and placed in separate test tubes: 1 tube for each composition was placed in a refrigerator for storage at 4°C, the rest were placed in a thermostat and incubated at 50°C for 72 hours. After heating was complete, the tubes were removed from the thermostat and transferred for analysis (in Figures 12A, 12B and 12C, the control stored at +4 is shown in red, and the sample after thermal stress is shown in blue). The anti-PD-LL antibody demonstrated high thermal stability in all three buffers (the difference between the aggregate content in the solution before and after thermal stress was no more than 5%).

[0435] The stability of BCD-135 in normal human serum was also assessed for 7 days at 37C. For this purpose, recombinant PDL1 (100 μl, 2.5 μg / ml in 1 χThe samples (0.5% nonfat milk in TBST) were added to the wells of a 96-well high-sorption ELISA plate. The plates were incubated at 4°C for 18 hours. The wells were then cleared of their contents and replaced with blocking buffer (200 μl of 0.5% nonfat milk in TBST). The plates were incubated at 37°C for 30 minutes and then washed twice with TBST.

[0436] 100 μl of solutions for constructing a calibration graph containing BCD-135 at a concentration of 0; 7.8; 15.6; 31.25; 62.5; 125.0; 250.0 ng / ml, diluted in blocking buffer, were added to the wells of the first vertical row.

[0437] The plates were incubated for 30 min at 37°C and then washed three times with TBST. Then, 100 µl of horseradish peroxidase-conjugated goat polyclonal antibodies to the Fc fragment of human IgG were added to each well. The plates were incubated for 30 min at 37°C. The plates were then washed 4-5 times with TBST. 100 µl of TMB solution were added to the washed and dried wells to develop color. The plates were placed in a dark place and incubated at 22°C for 20-25 min to develop color. The reaction was stopped by adding 50 µl of 0.9 M sulfuric acid stop solution to each well. The optical density of the solution in the wells was measured using a microplate spectrophotometer at a wavelength of 450 nm.

[0438] Based on the obtained data, a calibration curve (Fig. 13A) was constructed, reflecting the dependence of optical density on the concentration of BCD-135 added to the well. The arithmetic mean of the optical density values ​​of the solutions was used to construct the curve. Using the calibration curve, the BCD-135 concentration in the sample was determined, corresponding to the optical density values ​​obtained in the experiment.

[0439] According to the results of the study, after 7 days of storage at 37 °C, the determined concentration of BCD-135 in human serum did not differ significantly from the concentration determined in serum samples prepared immediately before the analysis (Fig. 13B), which indicated the stability of the antibody.

[0440] Example 14.

[0441] Construction of a library of BCD-135 mutant antibodies specific to PD-L1. To generate BCD-135 mutant antibodies specific to PD-L1, structural analysis was performed based on 3D modeling using the YLab software package from BIOCAD and the PD-L1 model (PDB 4ZQK, PDB 4Z18) (see also Example 18). Based on the calculation models, BCD-135 gene libraries were synthesized, having partially degenerate codons (FUH G ET AL. , Improving antibody binding affinity and specificity for therapeutic development, Methods Mol Biol., 2009, 525, 353-376) in positions in the first and third CDR regions of the variable domain of the heavy chain SEQ ID NO: 4 and the third CDR of the variable domain of the light chain SEQ ID NO: 8. The resulting DNA of the randomized gene was cloned into the phage display plasmid pH5 (Fig. 2), according to the protocol described in Example 2.Transformation of the indicated constructs into the SS320 strain yielded 5*10e7 independent transformants for the library, according to the procedure [Methods Enzymol. 2000; 328: 333–63]. Phage preparations of mutant VH BCD-135 libraries were prepared according to the procedure described previously [Mol Biol. 1991 Dec 5;222(3): 581–97].

[0442] Selections of the obtained phage mutant BCD-135 Fab libraries were carried out under conditions similar to those described above (Example 3).

[0443] Following the third round of selection of the libraries described above using a recombinant human PD-Ll-Fc preparation, ELISA analysis of the polyclonal phage preparations revealed significant enrichment, exceeding the nonspecific binding background by more than 10-fold. Gene pools from the enriched phage libraries of BCD-135 mutant Fab antibodies specific for human PD-Ll-Fc were recloned into the pLL expression plasmid (Fig. 3), containing a myc-tag peptide at the C-terminus for ELISA detection.

[0444] Example 15.

[0445] Comparative ELISA analysis of BCD-135 mutant Fab antibodies specific to PD-L1

[0446] ELISA is used to measure the binding of the studied mutant Fab antibodies to human PD-L1, similar to that described in Example 4. The number of clones producing BCD-135 mutant Fab antibodies tested was 400. The wild-type BCD-135 Fab antibody with the sequences SEQ ID NO: 4 and SEQ ID NO: 8 was used as a positive control.

[0447] As a result, 85 positive clones were selected that gave a signal higher or similar to the control wild BCD-135 Fab antibody (values ​​in the range of 0.7-1.2 relative units).

[0448] Example 1 b .

[0449] Characterization of BCD-135 mutant Fab antibodies specific for PD-L1

[0450] Eighty-five candidate positive clones selected through ELISA screening (Example 15) yielding a signal higher than or similar to the control wild-type BCD-135 Fab antibody were sequenced on an Applied Biosystems 3130 sequencer according to the manufacturer's recommended protocols. This yielded 25 unique clones. Eight mutant Fab antibodies from these unique clones were further analyzed using the quantitative kinetic dissociation constant method on an Octet Red 96 instrument, as described in Example 7.

[0451] Koff screening was performed using a Pall Forte Bio Octet Red 96 instrument, similar to that described in Example 7. Anti-FABCH1 biosensors were rehydrated for 30 minutes in a working buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. Working buffer was added to the studied E. coli supernatant samples to a final concentration of 1 χThen, the anti-FABCH1 biosensors were immersed in E. coli supernatants containing Fab fragments of the antibody candidates for 12 hours at 4°C. Sensors with Fab fragments immobilized on the surface were transferred to wells with a working buffer, where the baseline (60 s) was recorded. Next, the sensors were transferred to wells with an analyte solution (PD-L1, 30 μg / ml) for association of the antigen-antibody complex (300 s). Then, the sensors were returned to the wells containing the working buffer for the subsequent 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) and then used in the next experiment. Analysis of the obtained curves was performed using Octet Data Analysis software (version 7.0) according to the standard procedure for the 1:1 interaction model.

[0452] As a result, kinetic dissociation constants of mutant Fab antibodies were obtained, which demonstrated comparable characteristics with the wild-type BCD-135 Fab variant, and thus tolerance of up to 8% substitutions in three CDRs of the variable domains.

[0453] Anti-PDLl VK of BCD-135

[0454] BCD-135 VH

[0455] 10 20 30 40 50 70 80

[0456] 90 100 110 120

[0457] 12345678301234567890123456 890123456789012 567890123456783012345678901234567890123 56 789012345678901234567890123 56789012

[0458] EVQb ^SGGGVT EGGSbIULSCjyk3GFTFDDYi^SWVRQJLPGRGLEWVa DISWSGSMTNYJUDSVKGEFTISRDKi-K SLYbQMKSb

[0459] RftE DTAbYHCARaPbLb AMT FGVGSWGQGTLVWS 3

[0460] EVQbVES{-H-H_arVRPGGS:LRbS ^^

[0461] RAEDTJU^YHCA^EbiSxXTF^V SWGQGTL SS

[0462] BCD-135 VL

[0463] 10 20 30 40 50 BO 70 80

[0464] 90 100

[0465] 12345673901234567830123456 83012345678901234567890123456789012345678901234567890123 56 789012345678 90123456

[0466] QT TQEPSbSVSPGGTVTbTCGbSSGTVTAIHYPGWYQQTPGQAPRTLI^

[0467] DYYCALYMGNGGHMFGGGTK

[0468] QTWTQEFSbSVSEGGTV LTCGbSSGTVTAIHYFGmYQQTF^

[0469] DYYCiibY G GKHMFGGGT

[0470] Example 17.

[0471] Establishment of a stable cell line, production and purification of anti-PD-LL antibodies

[0472] A stable cell line producing the monoclonal antibody BCD-135 was obtained by electroporation transfection using the Neon Transfection System (Life Technologies) of the parental CH0-S suspension cell line with vector constructs containing the antibody's light and heavy chains in an optimized ratio. High-productivity clonal lines (over 1000 mg / L) were obtained using the ClonePix robotic platform (Molecular Devices) and preliminary minipool selection steps using antibiotics in different culture formats. Productivity analysis was performed using the Octet RED96 analytical system (Pall Life Sciences). DOE for the selection of the basal medium and the cultivation scheme was performed using the Biomek FX robotics automated system (Beckman Coulter). Serum-free media and feeders containing no animal proteins were used for culturing the producer.The production of BCD-135 for preclinical studies was carried out in a HyClone single-use bioreactor (Thermo Fisher Scientific) with a working volume of 50 L.

[0473] Clarification of the culture fluid was performed on a Millistak COHC depth filter (Merck-Millipore). Primary purification of the antibody from the clarified culture fluid was performed on a protein A affinity sorbent. Specific elution of the target protein was performed under acidic conditions of pH 3.3-3.8 in glycine buffer. The resulting eluate was maintained at an acidic pH for 30-60 minutes to inactivate viruses and then neutralized with 1 M Tris-OH solution to a pH of 6.5-7.0. Final chromatographic purification in breakthrough mode was performed on a CaptoAdhere sorbent (GE Healthcare LifeSciences) to remove residual DNA, producer cell proteins, cleaved affinity sorbent ligand, aggregates, and antibody fragments. For this purpose, the protein solution was passed through a prepared sorbent at pH 6.5-7.0 with a low conductivity value (<3 mS / cm2 ). The purified protein was subjected to antiviral filtration using a Viresolve PRO filter kit (Millipore), concentration, and diafiltration against a final buffer containing acetate buffer (pH 5.0-5.5) and trehalose. The concentration of the obtained protein was 50 mg / ml or more.

[0474] Example 18.

[0475] In silico modeling of the complex of BCD-135 antibody and human PD-L1.

[0476] To generate BCD-135 mutant antibodies specific for PD-L1, a 3D modeling-based structural analysis was performed using the Schrodinger Suite and YLab software packages from Schrodinger and BIOCAD. PDB 5C3T was chosen as the crystal structure of the target because it has more crystallized amino acids than the classical 4ZQK structure, which focuses on PD-1. Docking was performed using the HEDGE tool (part of the YLab package from BIOCAD). The optimal positions were selected using 10-ns free energy molecular dynamics simulations (Desmond tool, part of the Schrodinger Suite). The resulting structure was visualized using the PyMOL tool from Schrodinger. The model including the variable domains of BCD-135 is shown in Fig. 14A, while Fig.14 B shows the region of interaction between the antigen and antibody with the highlighted amino acid residues that form a tight interprotein contact.

[0477] Table B shows the key amino acid residues of both antibody and antigen that determine tight protein-protein interactions.

[0478] Table B. The middle column shows the amino acid residues of the BCD-135 antibody that interact with human PD-L1. The right column shows the corresponding amino acid residues of the PD-L1 antigen that interact with the BCD-135 antibody.

[0479]

Claims

Invention formula 1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-L1, comprising: a heavy chain variable domain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 3; a light chain variable domain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO:

7.

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

3.

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

7.

4. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the variable domain of the heavy chain comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NO: 1-3.

5. A monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the variable domain of the heavy chain comprises amino acid sequences represented by the sequences SEQ ID NO: 1-3.

6. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the variable domain of the light chain comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NO: 5-7.

7. A monoclonal antibody or antigen-binding fragment thereof according to item 6, wherein the variable domain of the light chain contains amino acid sequences represented by the sequences SEQ ID NO: 5-7.

8. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, where the heavy chain variable domain comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NO: 1-3; the light chain variable domain comprises amino acid sequences at least 90% homologous to the sequences of SEQ ID NO: 5-7.

9. A monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 1-3; the light chain variable domain comprises the amino acid sequences of SEQ ID NO: 5-7.

10. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the variable domain of the heavy chain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO:

4.

11. A monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the variable domain of the heavy chain comprises the amino acid sequence of SEQ ID NO:

4.

12. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the variable domain of the light chain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO:

8.

13. A monoclonal antibody or antigen-binding fragment thereof according to claim 12, wherein the variable domain of the light chain comprises the amino acid sequence of SEQ ID NO:

8.

14. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, where the heavy chain variable domain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4; the light chain variable domain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO:

8.

15. A monoclonal antibody or antigen-binding fragment thereof according to claim 14, where 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.

16. A monoclonal antibody according to claim 1, comprising: a heavy chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO: 9; a light chain comprising an amino acid sequence at least 90% homologous to the sequence of SEQ ID NO:

10.

17. A monoclonal antibody according to claim 16, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; a light chain comprising the amino acid sequence of SEQ ID NO:

10.

18. The monoclonal antibody of any one of claim 1, wherein the antibody specific to PD-L1 is a full-length IgG antibody.

19. The monoclonal antibody according to claim 18, wherein the full-length IgG antibody is of the human IgG1, IgG2, IgG3, IgG4 isotype.

20. The monoclonal antibody of claim 19, wherein the full-length IgG antibody is of the human IgG1 isotype.

21. A nucleic acid that encodes an antibody or antigen-binding fragment thereof according to any one of claims 1-20.

22. The nucleic acid of claim 21, wherein the nucleic acid is DNA.

23. An expression vector containing a nucleic acid according to any one of claims 21-22.

24. A method for producing a host cell for producing an antibody or an antigen-binding fragment thereof according to any of ππ. 1-20, including transformation of a cell by a vector according to p.

23.

25. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-20, containing a nucleic acid according to any one of claims 21-22.

26. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1-20, comprising culturing the host cell according to claim 25 in a culture medium under conditions sufficient to produce said antibody, if necessary, followed by isolating and purifying the resulting antibody.

27. A pharmaceutical composition 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 claims 1-20, in combination with one or more pharmaceutically acceptable excipients.

28. A pharmaceutical composition according to claim 27, intended for the prevention or treatment of a disease or disorder mediated by PD-L1, selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC (Colorectal cancer with high microsatellite instability).

29. 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 claims 1-20 and at least one therapeutically active antitumor compound.

30. A pharmaceutical combination according to claim 29, intended for the prevention or treatment of a disease or disorder mediated by PD-L1, selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC (microsatellite instability-high colorectal cancer).

31. The pharmaceutical combination according to paragraphs 29-30, wherein the therapeutically active antitumor compound is selected from a chemotherapeutic agent, an antibody, or an antihormonal agent.

32. A method for inhibiting the biological activity of PD-L1 in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-20.

33. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1-20 or a pharmaceutical composition according to claim 27 for the treatment of a disease or disorder mediated by PD-L1 in a subject in need of such treatment.

34. The use according to claim 33, wherein the disease or disorder is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer of unknown origin, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (Non-small cell lung cancer), kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC (Colorectal cancer with high microsatellite instability).