HK40068405A - Humanized anti-pd-l1 antibodies - Google Patents

Description

Humanized anti-PD-L1 antibodies

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 62/835,764, filed 2019, 4/18, the disclosure of which is hereby incorporated by reference in its entirety.

Incorporation of sequence listing this application includes a sequence listing submitted via EFS-Web in ASCII format, entitled "QLSF 003PCT _ st25. txt", which is 158KB in size, and has a creation date of 2020, 4, 17. The contents of this sequence listing are hereby incorporated by reference in their entirety.

Background

Programmed cell death protein 1(PD-1) is a 288 amino acid cell surface protein molecule and is encoded by the PDCD1 gene in humans. PD-1 is a type I transmembrane protein comprising an immunoglobulin variable amino ectodomain terminus, a transmembrane region, and a cytoplasmic tail bearing an immunoreceptor tyrosine inhibition motif and an immunoreceptor tyrosine switch motif (Shitian et al, 1992). PD-1 is expressed in pro-B cells and activated T cells, but not on resting T cells in vivo. Programmed death-1 (PD-1) acts as a checkpoint protein on immune cells. It negatively regulates the immune system by inhibiting potent T cell function through binding to PD-L1, a ligand for the PD-1 receptor.

The PD-1/PD-L1 interaction plays an important role in autoimmunity as well as in cancer immunology. PD-L1 expression has been observed in a variety of cancers, including melanoma and non-small cell lung cancer. Many studies have shown that cancer cells overexpress PD-L1, which helps them escape immune attack by using the PD-1/PD-ligand (PDL) pathway. Based on the conclusions of these studies, a variety of checkpoint blockade inhibitors have been developed against the PD-1/PDL pathway.

The PD-1/PD-L1 blockade has enjoyed great clinical success in combating cancer. Pembrolizumab (Keytruda) and Nivolumab (Opdivo) targeting PD-1 and Atezolizumab (Tecntriq), Avelumab (Bavencio) and Durvalumab (Imfinzi) targeting PD-L1 are effective in blocking the binding between PD-1 and PD-L1 and thus reversing T cell dysfunction. However, the objective remission rate of monotherapy is only 20% in many advanced cancers (Xu-Monette et al, 2017).

Thus, improved therapies for PD-1 and PD-L1 are an area of great interest. In addition, a variety of other immunotherapies (including monotherapy and combination therapy) are now being tested in clinical trials. Thus, there remains a need for more effective immunotherapies targeting the PD-1/PD-L1 pathway, including use alone and in combination with other therapeutic agents in a drug treatment regimen.

Disclosure of Invention

The present disclosure provides isolated anti-PD-L1 monoclonal antagonist antibodies and antigen-binding portions thereof that specifically bind human PD-L1.

In one aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO 43. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:31, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 37. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) (ii) a light chain variable region CDR1 comprising SEQ ID No. 13; (b) comprises the light chain variable region CDR2 of SEQ ID NO. 19; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 25.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No.1 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 7. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 7.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises the heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO: 44. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:32, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 38. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) comprises the light chain variable region CDR1 of SEQ ID NO. 14; (b) comprises the light chain variable region CDR2 of SEQ ID NO. 20; and (c) a light chain variable region CDR3 comprising SEQ ID NO 26.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 2 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 8. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 8.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO: 45. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:33, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 39. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) comprises the light chain variable region CDR1 of SEQ ID NO. 15; (b) comprises the light chain variable region CDR2 of SEQ ID NO: 21; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 27.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 3 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 9. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 9.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO: 46. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:34, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 40. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) comprises the light chain variable region CDR1 of SEQ ID NO. 16; (b) comprises the light chain variable region CDR2 of SEQ ID NO. 22; and (c) a light chain variable region CDR3 comprising SEQ ID NO 28.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 4 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 10. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 10.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO: 47. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:35, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 41. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) comprises the light chain variable region CDR1 of SEQ ID NO. 17; (b) comprises the light chain variable region CDR2 of SEQ ID NO. 23; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 29.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 5 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 11. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 11.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises heavy chain variable region CDR3, which heavy chain variable region CDR3 comprises SEQ ID NO: 48. In some embodiments, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises heavy chain variable region CDR1, said heavy chain variable region CDR1 comprises SEQ ID NO:36, and heavy chain variable region CDR2, said heavy chain variable region CDR2 comprises SEQ ID NO: 42. In a preferred embodiment, the anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof further comprises: (a) 18, comprising the light chain variable region CDR1 of SEQ ID NO; (b) 24, comprising the light chain variable region CDR2 of SEQ ID NO; and (c) a light chain variable region CDR3 comprising SEQ ID NO 30.

In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 6 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 12. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID No. 12.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 65 through 67; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 49 through SEQ ID NO 51.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO 68 to SEQ ID NO 70; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:55 to SEQ ID NO: 58.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:71 to SEQ ID NO: 76; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO 59 to SEQ ID NO 61.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO 77 to SEQ ID NO 78; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:62 to SEQ ID NO: 63.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:79 to SEQ ID NO: 80; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 64.

In another aspect of the invention, an isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:81 to SEQ ID NO: 83; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO 52 to SEQ ID NO 54.

In another aspect of the invention, an isolated monoclonal antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 84 to SEQ ID NO 99, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 100 to SEQ ID NO 118.

The antibodies of the disclosed invention may be further engineered into a form suitable for human therapy by modifications that minimize immunogenicity. Suitable antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. The affinity, stability and specificity of the disclosed antibodies can also be further optimized by techniques known to those skilled in the art. Other forms may involve oligomerization, drug conjugation, and fusion of the disclosed antibodies with other functional proteins.

The antibodies of the disclosed invention mayIs, for example, a full length antibody, such as an IgG1, IgG2, IgG3 or IgG4 isotype. Alternatively, the disclosed antibodies can be antibody fragments, such as Fab fragments, Fab 'fragments, and F (ab')₂Fragments, diabodies, triabodies, tetrabodies, single chain variable region fragments (scFv), disulfide stable variable region fragments (dsFv) and half antibodies. Alternatively, the disclosed antibodies can be bispecific antibodies.

In another aspect of the invention, the antibody or antigen binding fragment thereof has a 5 x 10 to PD-L1^-8M to 1X 10^-10M or 1.32X 10^-9M to 2.68X 10^-10Affinity (KD) in the range of M.

In some embodiments, the anti-PD-L1 antagonist antibody or antigen-binding portion thereof binds to and blocks human PD-L1. Thus, the antibody or antigen binding portion can stimulate an anti-tumor immune response. In some embodiments, the anti-PD-L1 antagonist antibody or antigen-binding portion thereof binds to and blocks non-human primate PD-L1.

In another aspect of the invention, there is also provided a composition comprising the isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof.

In another aspect of the invention, there is also provided a pharmaceutical composition comprising the isolated anti-PD-L1 monoclonal antagonist antibody or antigen-binding portion thereof and a pharmaceutically acceptable transporter. Also provided are compositions comprising the immunoconjugates of the invention and a pharmaceutically acceptable transporter.

In another aspect of the invention, there is also provided a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, and a host cell comprising an expression vector comprising the nucleic acid molecule.

The invention further provides a method of stimulating an immune response using an anti-PD-L1 antagonist antibody of the disclosed invention. For example, in one embodiment, the disclosed invention provides a method of treating a subject in need thereof comprising the step of administering to the subject an effective amount of an antibody or antigen-binding portion of the disclosed invention.

In another aspect, the disclosed invention provides a method of treating cancer in a human comprising the step of administering to the human an anti-PD-L1 antagonist antibody or antigen-binding portion of the disclosed invention in an amount effective to treat the cancer.

In another aspect, the disclosed invention provides a method of treating an infectious disease in a human comprising the step of administering to the human an anti-PD-L1 antagonist antibody or antigen-binding portion of the disclosed invention in an amount effective to treat the infectious disease.

Other features and advantages of the disclosure will be apparent from the following description and examples, which should not be construed as limiting. The contents of all references, GenBank entries, patents, and published patent applications cited in this application are expressly incorporated herein by reference.

Drawings

Exemplary embodiments are shown in the referenced figures. The embodiments and figures disclosed herein are to be regarded as illustrative rather than restrictive.

Figure 1 shows that the anti-PD-L1 lead candidate binds to PD-L1 on a431 cells.

FIG. 2 shows that the anti-PD-L1 lead candidate blocks the PD-1 interaction on HEK293/huPD-L1 cells.

FIG. 3 shows that anti-PD-L1 antibody enhanced IL-2 release by stimulation with SEB.

Figure 4 shows that the PD-L1 antibody enhances IFN γ secretion in a mixed lymphocyte reaction assay, indicating that the lead anti-PD-L1 antibody blocks PD-1 mediated inhibition.

Figure 5 shows that blocking of PD-L1 by lead antibodies enhances interferon gamma production by T cells in a CMV peptide-specific recall response assay.

Figure 6 shows the thermostability of the humanized anti-PD-L1 leader antibody.

Detailed Description

The embodiments and aspects thereof are described and illustrated below in conjunction with systems, compositions, and methods, which are meant to be exemplary and illustrative, not limiting in scope.

Definition of

As used herein, the terms "comprises" or "comprising" are used to indicate compositions, methods, and respective components thereof that are useful for the embodiments but may include unspecified elements, whether or not useful. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

The use of the terms "a" and "an" and "the" and similar referents in the context of describing particular embodiments of the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation "e.g." is derived from latin-exempli gratia and is used herein to indicate a non-limiting example. The abbreviation "e.g." is therefore synonymous with the word "e.g.". No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

The term "about" as used herein refers to a measurable value, such as a quantity, duration, etc., and encompasses variations of the specified values ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or ± 0.1%.

The term "epitope" as used herein may include any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants are typically composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the equilibrium dissociation constant is ≦ 1 μ M (preferably ≦ 100nM, most preferably ≦ 10 nM).

The term "K_D"may refer to the equilibrium dissociation constant for a particular antibody-antigen interaction.

The term "immune response" as used herein may refer to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement), which cause selective damage to an organism, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal body cells or tissues (in the case of autoimmune or pathological inflammation) in an organism.

As used herein, "antigen-specific T cell response" may refer to a T cell response elicited by stimulation of T cells by a T cell-specific antigen. Non-limiting examples of T cell responses to antigen-specific stimuli include proliferation and cytokine production (e.g., IL-2 production).

The term "antibody" as used herein refers to an intact immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or FcRn binding fragment of an Fc region, referred to herein as an "Fc fragment" or "Fc region. Antigen-binding fragments can be produced by recombinant DNA techniques or enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, inter alia, Fab ', F (ab')₂Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer a specific antigen to which the polypeptide binds. The Fc region includes portions of the two heavy chains that make up the two or three classes of antibodies. The Fc region may be produced by recombinant DNA techniques or by enzymatic (e.g., papain cleavage) or by chemical cleavage of intact antibodies.

The term "antibody fragment" as used herein refers to a fragment comprising only a portion of an intact antibodyA partial protein fragment, typically includes the antigen binding site of an intact antibody and thus retains the ability to bind antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having a VL region, a CL region, a VH region and a CH1 region; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 region; (iii) an Fd fragment having the VH region and the CH1 region; (iv) an Fd' fragment having the VH and CH1 regions and one or more cysteine residues C-terminal to the CH1 region; (v) (ii) an Fv fragment having a VL region and a VH region of a single arm of an antibody; (vi) dAb fragments consisting of VH regions (Ward et al, Nature 341, 544-546 (1989)); (vii) an isolated CDR region; (viii) f (ab')₂Fragments (bivalent fragments) comprising two Fab' fragments linked by a disulfide bond at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al, Science 242: 423-; (x) "diabodies" having two antigen-binding sites, comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) in the same polypeptide chain (see, e.g., EP404,097; WO 93/11161; and Hollinger et al, Natl. Acad. Sci. USA,90: 6444-; (xi) "Linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that form, together with a complementary light chain polypeptide, a pair of antigen binding regions (Zapata et al, Protein Eng.8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

As used herein, a "single chain variable fragment," "single chain antibody variable fragment," or "scFv" antibody refers to a form of antibody comprising only the variable regions of the heavy chain (VH) and light chain (VL) linked by a linker peptide. The scFv can be expressed as a single chain polypeptide. The scFv retains the specificity of the intact antibody from which it was derived. The light and heavy chains may be in any order, e.g., VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is maintained.

As used herein, an "isolated antibody" can refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds PD-L1 protein can be substantially free of antibodies that specifically bind antigens other than PD-L1 protein). However, isolated antibodies that specifically bind human PD-L1 protein may be cross-reactive with other antigens (such as PD-L1 protein from other species). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

Cells, such as hybridomas, that produce anti-PD-L1 antagonist antibodies can be selected, cloned, and further screened for desired characteristics, including robust growth, high antibody production, and desired antibody characteristics. Hybridomas can be expanded in syngeneic animals, in animals lacking the immune system (e.g., nude mice), or in cell culture in vitro. Methods for selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein may refer to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "recombinant human antibody" as used herein may refer to all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) transgenic or transchromosomal for a human immunoglobulin gene or a hybridoma prepared therefrom (as described below), (b) antibodies isolated from a host cell transformed to express a human antibody, e.g., an antibody isolated from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing of a human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in particular embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animal transgenes for human Ig sequences, to in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, may not be present in the in vivo human antibody germline repertoire under natural conditions.

The term "isotype" can refer to the class of antibodies (e.g., IgM or IgGl) encoded by the heavy chain constant region gene. The antibody may be, or be derived from, an immunoglobulin g (igg), IgM, IgE, IgA or IgD molecule.

Herein, the phrases "an antibody recognizing an antigen" and "an antibody specific to an antigen" are used interchangeably with the term "an antibody specifically binding to an antigen".

As used herein, an antibody that "specifically binds human PD-L1" may refer to an antibody that binds human PD-L1 protein (and possibly PD-L1 protein from one or more non-human species) but does not substantially bind non-PD-L1 protein. Preferably, the antibody binds human PD-L1 protein with "high affinity", i.e. with 1 × 10^-7M is less than or equal to, more preferably 5X 10^-8M is equal to or less, more preferably 3X 10^-8M is equal to or less, more preferably 1X 10^-8M is less than or equal to, more preferably 5X 10^-9M or less or even more preferably 1X 10^-9M or K below_D。

The term "substantially not binding" a protein or cell as used herein may mean that it is not able to bind or bind with high affinity to a protein or cell, i.e. at 2x 10^-6M or more, more preferably 1X 10^-5M or more, more preferably 1X 10^-4M or more, more preferably 1X 10^-3M or more, even more preferably 1X 10^-2M or more K_DBinding to proteins or cells.

The term "high affinity" for an IgG antibody can refer to 1 × 10 for the target antigen^-6M or less, preferably 1X 10^-7M or less, more preferably 1X 10^-8M or less, even more preferably 1X 10^-9M or less, even more preferably 1X 10^-10M or K below_DThe antibody of (1). However, "high affinity" binding may vary for other antibody isotypes. The term "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and that is free of additional ingredients that have unacceptable toxicity to the subject to which the formulation is to be administered.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical agent or cell) refers to an amount effective to achieve the desired therapeutic result, e.g., a pharmacokinetic or pharmacodynamic effect for the treatment of a disease, condition, or disorder and/or therapy, at the necessary dosage and for the necessary period of time. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the cell population administered. In some embodiments, the methods provided herein involve administering the cells and/or compositions in an effective amount (e.g., a therapeutically effective amount).

An "antagonistic antibody" as used herein is an antibody that blocks or inhibits a biological response by binding and blocking a ligand (e.g., PD-L1) that binds to the antibody. For example, antagonists may bind to PD-L1 and block the binding of PD-L1 to PD-1, thereby inhibiting the phosphorylation of PD-1 receptors or may inhibit signal/cell activation. In one embodiment, the antibody of the invention is an anti-PD-L1 antagonist antibody.

A "CDR-grafted antibody" is an antibody that comprises one or more CDRs derived from a particular species or isotype and the framework of another antibody of the same or a different species or isotype.

A "humanized antibody" has a sequence that differs from an antibody derived from a non-human species by one or more amino acid substitutions, deletions and/or additions such that the humanized antibody is less likely to induce an immune response and/or induce a less severe immune response when administered to a human subject than an antibody of a non-human species. In one embodiment, specific amino acids in the framework and constant regions of the heavy and/or light chain of an antibody of a non-human species are mutated to produce a humanized antibody. In another embodiment, the constant region from a human antibody is fused to a variable region of a non-human species. In another embodiment, the humanized antibody is a CDR-grafted antibody comprising one or more CDRs derived from an antibody of a particular species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when administered to a human subject, wherein the altered amino acid residues are not critical for immunospecific binding of the antibody to its antigen or the alterations to the amino acid sequence are conservative such that binding of the humanized antibody to the antigen is not significantly inferior to binding of the non-human antibody to the antigen. For an example of how to make humanized antibodies, see U.S. Pat. nos. 6,054,297, 5,886,152 and 5,877,293.

The term "chimeric antibody" (cAb) refers to an antibody comprising one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the PD-L1 is derived from a human anti-PD-L1 antibody. In another embodiment, all CDRs are derived from a human anti-PD-L antibody. In another embodiment, the CDRs from more than one human anti-PD-L1 antibody are mixed and matched in a chimeric antibody. For example, the chimeric antibody may comprise a CDR1 from the light chain of a first human anti-PD-L1 antibody, a CDR2 and a CDR3 from the light chain of a second human anti-PD-L1 antibody, and a CDR from the heavy chain of a third anti-PD-L1 antibody. In the context of the present disclosure, cabs represent the variable regions of a mouse monoclonal antibody fused to the Fc region of a human antibody. In another embodiment, other combinations are possible.

The term "subject" may refer to any human or non-human animal. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects. The term "non-human animal" includes all vertebrates, e.g. mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, rabbits, mice, rats, amphibians and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

Binding of the antibodies of the disclosed invention to PD-L1 can be assessed by using one or more established techniques in the art. For example, in a preferred embodiment, the antibody may be tested by an ELISA assay, e.g., using recombinant PD-L1 protein. Other suitable binding assays include, but are not limited to, flow cytometry assays in which an antibody is reacted with a cell line expressing human PD-L1, such as Expi that has been transfected to express PD-L1 (e.g., human PD-L1) on its cell surface293 cells or expihho cells. Additionally or alternatively, binding of the antibody can be tested in a BIAcore binding assay, Octet Red96(Pall), and the like, including binding kinetics (e.g., K)_DValue).

Preferably, the antibody binding of the disclosed invention has a size of 5 × 10^-8M or K below_DHuman PD-L1 protein with a binding of 2X 10^-8M or K below_DHuman PD-L1 protein with a binding of 2X 10^-8M or K below_DHuman PD-L1 protein having a binding of 5X 10^-9M or K below_DHuman PD-L1 protein having a binding of 4X 10^-9M or K below_DHuman PD-L1 protein having a binding of 3X 10^-9M or K below_DHuman PD-L1 protein having a binding of 2X 10^-9M or K below_DHuman PD-L1 protein having a binding of 1X 10^-9M or K below_DHuman PD-L1 protein.

The present disclosure relates to isolated monoclonal antibodies, or antigen-binding portions thereof, that bind to and block PD-L1 and uses thereof. In particular embodiments, the antibodies of the disclosed invention are derived from identified heavy and light chain germline sequences and/or comprise identified structural features, such as CDR regions comprising identified amino acid sequences. The present disclosure provides isolated antibodies, methods of making such antibodies, and antigen-binding portions thereof, of the disclosed invention. The disclosure also relates to methods of using antibodies, such as stimulating an immune response using the anti-PD-L1 antibodies of the disclosed invention alone or in combination with other immunostimulatory antibodies. Thus, methods of using the anti-PD-L1 antagonist antibodies of the disclosed invention are also provided, for example, including but not limited to, the treatment of human cancer. Various aspects of the invention relate to antibodies and antibody fragments, pharmaceutical compositions, nucleic acids, recombinant expression vectors, and host cells for making such antibodies and fragments. The invention also includes methods of using the antibodies of the invention to detect human PD-L1, inhibit PD-L1 activity in vitro or in vivo, and prevent or treat diseases such as cancer.

Complementarity Determining Regions (CDRs) are referred to as hypervariable regions in the light chain variable region and the heavy chain variable region. The more highly conserved portions of the variable regions are called the Framework (FR). The Complementarity Determining Regions (CDRs) and Framework Regions (FRs) of a given antibody may be determined using Kabat et al, supra; lefranc et al and/or honeygger and Pluckthun. The person skilled in the art is also familiar with the numbering system described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In this regard, Kabat et al defines a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region amino acid sequence without relying on any experimental data other than the sequence itself.

In particular embodiments, the invention provides anti-PD-L1 antagonist antibodies or antigen-binding portions thereof. In one embodiment, the mouse antibody or portion comprises (a) light chain variable region CDR1, which light chain variable region CDR1 comprises SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16; 17, 18, (b) a light chain variable region CDR2, the light chain variable region CDR2 comprising SEQ ID NO 19, 20, 21, 22, 23, SEQ ID NO: 24; (c) light chain variable region CDR3, the light chain variable region CDR3 comprising SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30; (d) heavy chain variable region CDR1, the heavy chain variable region CDR1 comprises SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36; (e) heavy chain variable region CDR2, the heavy chain variable region CDR2 comprises SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42; (f) heavy chain variable region CDR3, the heavy chain variable region CDR3 comprises SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48.

In one embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No.1, SEQ ID No. 49, SEQ ID No. 50, or SEQ ID No. 51; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 7, SEQ ID NO 65, SEQ ID NO 66 or SEQ ID NO 67.

In another embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds to the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 2, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, or SEQ ID No. 58; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 8, 68, 69 or 70.

In yet another embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 3, SEQ ID No. 59, SEQ ID No. 60, or SEQ ID No. 61; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 9, 71, 72, 73, 74, 75 or 76.

In yet another embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds to the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 4, SEQ ID No. 62, or SEQ ID No. 63; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 10, 77 or 78.

In yet another embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 5 or SEQ ID No. 64; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 11, 79 or 80.

In yet another embodiment, the present disclosure provides a monoclonal antibody, or antigen-binding portion thereof, that binds the PD-L1 epitope, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 6, SEQ ID No. 52, SEQ ID No. 53, or SEQ ID No. 54; and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 12, 81, 82 or 83.

Whereas each of these antibody fabs can bind to human PD-L1, the VH and VL sequences can be "mixed and matched" to produce other anti-PD-L1 binding molecules of the invention. Preferably, when the VH and VL chains are mixed and matched, the VH sequences from a particular VH/VL pairing are replaced with structurally similar VH sequences. Also, preferably, VL sequences from a particular VH/VL pairing are replaced with structurally similar VL sequences.

In some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:65 to SEQ ID NO: 67; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 49 through SEQ ID NO 51. Preferred heavy and light chain combinations include, but are not limited to:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 49;

(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:66 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50;

(c) (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 67 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 51;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:68 to SEQ ID NO:70 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:55 to SEQ ID NO: 58. Preferred heavy and light chain combinations include, but are not limited to:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 68 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 55;

(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:69 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56;

(c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 70 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 57;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:71 to SEQ ID NO:76 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:59 to SEQ ID NO: 61. Preferred heavy and light chain combinations include, but are not limited to:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 71 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 59;

(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 60;

(c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 73 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 61;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:77 to SEQ ID NO:78 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:62 to SEQ ID NO: 63. Preferred heavy and light chain combinations include, but are not limited to:

(a) (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62;

(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 63;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:79 to SEQ ID NO: 80; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 64. Preferred heavy and light chain combinations include, but are not limited to:

(a) (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:79 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:81 to SEQ ID NO:83 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:52 to SEQ ID NO: 54. Preferred heavy and light chain combinations include, but are not limited to:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 81 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 52;

(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO 53;

(c) (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:83 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54;

in some embodiments, the humanized anti-PD-L1 antibody or antigen-binding portion thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NO:84 to SEQ ID NO:99 and a heavy chain comprising an amino acid sequence selected from SEQ ID NO:100 to SEQ ID NO: 118.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID NO 43 and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence as set forth in any one of SEQ ID NO 7 or SEQ ID NO 65 to SEQ ID NO 67.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID No. 44 and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence as set forth in any one of SEQ ID No. 8 or SEQ ID No. 68 to SEQ ID No. 70.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID No. 45 and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence as set forth in any one of SEQ ID No. 9 or SEQ ID No. 71 to SEQ ID No. 76.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID No. 46 and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence as set forth in any one of SEQ ID No. 10 or SEQ ID No. 77 to SEQ ID No. 78.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID No. 47, and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence as set forth in any one of SEQ ID No. 11 or SEQ ID No. 79 to SEQ ID No. 80.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as set forth in SEQ ID No. 48 and comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence as set forth in any one of SEQ ID No. 12 or SEQ ID No. 81 to SEQ ID No. 83.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 25 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence as set forth in any one of SEQ ID No.1 or SEQ ID No. 49 through SEQ ID No. 51.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 26 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in any one of SEQ ID No. 2 or SEQ ID No. 55 to SEQ ID No. 58.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 27 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence as set forth in any one of SEQ ID No. 3 or SEQ ID No. 59 to SEQ ID No. 61.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 28 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence as set forth in any one of SEQ ID No. 4 or SEQ ID No. 62 to SEQ ID No. 63.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 29 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence as set forth in any one of SEQ ID No. 5 or SEQ ID No. 64.

In one embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as set forth in SEQ ID No. 30 and having a light chain variable region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence as set forth in any one of SEQ ID No. 6 or SEQ ID No. 52 to SEQ ID No. 54.

Thus, in certain embodiments, the CDR3 regions remain constant, while variability can be introduced into the remaining CDRs and/or framework regions of the heavy and/or light chains, while the antibody or antigen-binding fragment thereof retains the ability to bind to PD-L1 and retains the functional properties of the parent, e.g., binding affinity.

In one embodiment, a substitution made within a heavy or light chain that is at least 95% identical (or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) is a conservative amino acid substitution. "conservative amino acid substitutions" are those to which one amino acid residue is replaced with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods mol. biol.24, incorporated by reference herein: 307-331. Examples of groups of amino acids with side chains of similar chemical nature include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) fatty hydroxyl side chain: serine and threonine; (3) amide-containing side chain: asparagine and glutamine; (4) aromatic side chain: phenylalanine, tyrosine, tryptophan; (5) basic side chain: lysine, arginine, histidine; (6) acidic side chain: aspartic acid and glutamic acid, and (7) the sulfur-containing side chains are cysteine and methionine. In addition to amino acids that are conserved at a chemical level, substitutions may include any amino acid that occurs at a similar position in related evolutionarily conserved human variable heavy chain sequences, human variable light chain sequences, and orthologous sequences from non-human species.

Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from the context, the following terms and phrases do not exclude the meaning of such terms or phrases as they have been in the art. These definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. There is no admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Working example:

the following examples are not intended to limit the scope of the claims of the present invention, but are intended to be exemplary of particular embodiments. Any variations in the example methods that occur to the skilled person are intended to fall within the scope of the invention.

Vector construction:

the vector pcDNA3.4TOPO (Invitrogen) was ligated to a short polylinker comprising EcoRI, XhoI and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, we assembled the prepared vector, the gblock encoding and VH region (IDT), and human IgG2 gblock encoding XhoI sites at the J chain and CH1 domain junctions using the Gibson assembly method. Plasmids were prepared and digested by EcoRI and XhoI to accommodate all humanized heavy Variable (VH) domains with IgG2 isotype. All assembly was done by Gibson method (NEB). Light variable regions were constructed by a similar method and using gblock to assemble Vkappa regions together by encoding gblock fragments of constant kappa (ck).

Protein expression, purification and binding characterization:

plasmids were prepared using a transient expression system (Thermo Fisher) and transfected into Expi293 or expihho cells. Briefly, plasmid was transfected into 3e6 cells/ml cells at 1. mu.g total plasmid DNA/ml culture. Heavy and light chain plasmids were run at 1:1, and mixing. The culture was cultured with shaking at 37 ℃. After 16 hours, we added transfection-enhancing agent 1 and transfection-enhancing agent 2 to the culture and continued the culture for 6 days. The supernatant was filtered and the protein titer was determined by the IgG quantification protocol using Octet Red96 (Pall). IgG was purified by Mab Select Sure Protein-A column purification on the ACTA PURE system and dialyzed overnight in PBS. The affinity of the purified antibody for the antigen was characterized by Octet Red96 by loading the purified antibody onto an anti-human heavy chain (AHC) capture sensor and measuring the binding and dissociation rates of the PD-L1 histidine-tagged target at three concentrations. (Table 1).

Table 1. The monovalent binding kinetics of the humanized anti-PD-L1 antibody was compared to a baseline control group as determined by Octet.

Flow cytometry binding analysis of anti-PD-L1 antibodies to A431 cancer cells expressing PD-L1

Two days prior to FACS analysis, a431 cancer cells were seeded into 24-well plates at 30% to 60% confluence to stimulate PD-L1 expression in the absence or presence of 1000U/ml recombinant human IFN- γ. On the day FACS analysis was performed, wells were washed with PBS and collected using Trypsin/EDTA. The isolated cells were washed twice, resuspended in FACS buffer at 5E6 cells/ml, and aliquoted into 96-well plates at 1E5 cells/well. Cells were stained with 5ug/ml anti-PD-L1 antibody, positive control antibody "C1-IgG 1" or isotype IgG1 on ice for 45 minutes, then washed and stained twice with 1:500 diluted goat anti-human IgG-AF 647. After the last wash and addition of 7-AAD, the cells were analyzed by flow cytometry (fig. 1).

PD-1His Tag blockade assay to detect humanized anti-PD-L1 on hPDL1/HEK293 cells

For screening of hybridomas, hPD-L1 expressing 293 cells were harvested by Accutase and resuspended at 4E6 cells/ml using FACS wash buffer. To compare the blocking capacity of the antibodies, we mixed 30ul cells/well (120,000), 30ul of 6ug/ml rhPD-1His Tag protein (2ug/ml final concentration) and 30ul of a continuous titration of anti-PDL 1 antibody and incubated on ice for 20 min. After culture, cells were washed and bound PD-1 was detected using anti-His Tag-APC mouse IgG1(R & D Cat # IC 050A). After the last wash and addition of 7-AAD, the cells were analyzed by flow cytometry. The blocking ability of the humanized antibody was compared to the same assay using rhPD-1Fc-biotin at a final concentration of 0.5ug/ml and detection using Streptavidin-APC (R & D cat # F0050) (FIG. 2) (Table 2).

SEB stimulation assay

Fresh PBMC were diluted to 2E6 cells/ml in X-Vivo15 medium (Lonza cat # 04-744Q). 200ng/ml (2X) of SEB (Millipore cat #324798) was added to PBMCs. Cells were treated with antibody by adding 100ul of diluted antibody and 100ul of PBMC/SEB antigen mixture to a 96-well flat bottom plate. After incubation at 37 ℃ for 48 hours, the supernatants were analyzed for IL-2 using ELISA kits (R & D Systems) (FIG. 3).

Mixed lymphocyte reaction assay

PBMCs were separated from human buffy coats using density gradient centrifugation (Miltenyi Biotec) and washed 4 times with PBS. CD4+ T cells (Miltenyi Biotec cat #130-096-533) were isolated from PBMC and resuspended at 4E6 cells/ml in X-Vivo15 medium.

Monocyte-derived dendritic cells were generated from positively selected CD14+ monocytes (monocyte isolation kit II, Cat No.130-091-153, Miltenyi Biotec). Cells were seeded at 5E5 cells/ml in whole RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and cultured for 7 days. Recombinant human (rh-) IL-4(1000U/ml) (R & D Systems) and rh granulocyte-macrophage colony-stimulating factor (rh-GMCSF) (500U/ml) (R & D Systems) were supplemented to the cultures on days 0, 2 and 5. Immature DCs were collected on day 7 and resuspended in 5ml of RPMI-1640, 10% FBS medium. Cells were mixed occasionally with 20ug/ml mitomycin C (Roche REF 10107409001) and incubated at 37 ℃ for 1 hour, then washed and resuspended to 4E5 cells/ml in X-Vivo15 medium.

In 96-well flat-bottom plates, we mixed 50ul of DC cells (2E4 DCs per well), 100ul of serially diluted anti-PD-L1 antibody and control at 2-fold concentration, and 50ul of purified CD4+ T cells (2E5 CD4+ T cells per well) (DC: T cells) at a ratio of 1: 10. Plates were incubated for 5 days and supernatants were analyzed for IFNg release (R & D Systems) by ELISA (fig. 4).

In vitro CMV antigen recall response using anti-PDL 1 treatment

Thawed PBMCs from CMV + donors (Astarte Biologics) were counted and resuspended at 2E6 cells/ml in AIM-V medium containing β -mercaptoethanol (1: 1000). In a 96-well flat-bottom plate, we mixed 100ul CMV + PBMC (2E5 cells/well), 50ul of 5ug/ml CMV antigen (Astarte cat #1004), and 50ul of serial dilutions of anti-PDL 1 antibody, along with a control. Plates were incubated for 4 days and supernatants were analyzed for IFNg release (R & D Systems) by ELISA (fig. 5).

Protein heat transfer of humanized anti-PDL 1 antibody

Ten (10) ug/ml anti-PD-L1 antibody was mixed with 2ul 50X protein heat transfer dye and PBS to a final volume of 100 ul. Samples were aliquoted into PCR 96 tube plates in four aliquots (25 ul/well). Protein heat transfer reactions were measured on an Applied Biosystems StepOne Real-Time PCR instrument using a continuous temperature gradient varying 1 ℃ from 22 ℃ to 95 ℃ every 1 minute and 5 seconds. Tm was analyzed by the derivative method (FIG. 6).

Claims (47)

1. The method comprises the following steps:

an isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID NO 43.

2. An isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID No. 44.

3. An isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising SEQ ID NO 45.

4. An isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising SEQ ID NO 46.

5. An isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising SEQ ID NO: 47.

6. An isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region CDR3, said heavy chain variable region CDR3 comprising SEQ ID NO 48.

7. The monoclonal antibody, or antigen binding portion thereof, of claim 1, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID No. 31 and a heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising SEQ ID No. 37, respectively.

8. The monoclonal antibody, or antigen binding portion thereof, of claim 2, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID NO:32 and a heavy chain variable region CDR2, respectively, said heavy chain variable region CDR2 comprising SEQ ID NO: 38.

9. The monoclonal antibody, or antigen binding portion thereof, of claim 3, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID NO:33, and a heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising SEQ ID NO:39, respectively.

10. The monoclonal antibody, or antigen binding portion thereof, of claim 4, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID NO:34, and a heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising SEQ ID NO:40, respectively.

11. The monoclonal antibody, or antigen binding portion thereof, of claim 5, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID NO:35, and a heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising SEQ ID NO:41, respectively.

12. The monoclonal antibody, or antigen binding portion thereof, of claim 6, further comprising a heavy chain variable region CDR1, said heavy chain variable region CDR1 comprising SEQ ID NO:36, and a heavy chain variable region CDR2, said heavy chain variable region CDR2 comprising SEQ ID NO:42, respectively.

13. The monoclonal antibody, or antigen binding portion thereof, of claim 7, further comprising

(a) (ii) a light chain variable region CDR1 comprising SEQ ID No. 13;

(b) comprises the light chain variable region CDR2 of SEQ ID NO. 19; and

(c) comprises the light chain variable region CDR3 of SEQ ID NO. 25;

wherein the antibody or portion specifically binds to human PD-L1.

14. The monoclonal antibody, or antigen binding portion thereof, of claim 8, further comprising

(a) Comprises the light chain variable region CDR1 of SEQ ID NO. 14;

(b) comprises the light chain variable region CDR2 of SEQ ID NO. 20; and

(c) 26 comprising the light chain variable region CDR3 of SEQ ID NO;

wherein the antibody or portion specifically binds to human PD-L1.

15. The monoclonal antibody, or antigen binding portion thereof, of claim 9, further comprising

(a) Comprises the light chain variable region CDR1 of SEQ ID NO. 15;

(b) comprises the light chain variable region CDR2 of SEQ ID NO: 21; and

(c) comprises the light chain variable region CDR3 of SEQ ID NO. 27;

wherein the antibody or portion specifically binds to human PD-L1.

16. The monoclonal antibody, or antigen binding portion thereof, of claim 10, further comprising

(a) Comprises the light chain variable region CDR1 of SEQ ID NO. 16;

(b) comprises the light chain variable region CDR2 of SEQ ID NO. 22; and

(c) comprises the light chain variable region CDR3 of SEQ ID NO. 28;

wherein the antibody or portion specifically binds to human PD-L1.

17. The monoclonal antibody, or antigen binding portion thereof, of claim 11, further comprising

(a) Comprises the light chain variable region CDR1 of SEQ ID NO. 17;

(b) comprises the light chain variable region CDR2 of SEQ ID NO. 23; and

(c) comprises the light chain variable region CDR3 of SEQ ID NO. 29;

wherein the antibody or portion specifically binds to human PD-L1.

18. The monoclonal antibody, or antigen binding portion thereof, of claim 12, further comprising

(a) 18, comprising the light chain variable region CDR1 of SEQ ID NO;

(b) 24, comprising the light chain variable region CDR2 of SEQ ID NO; and

(c) comprises the light chain variable region CDR3 of SEQ ID NO. 30;

wherein the antibody or portion specifically binds to human PD-L1.

19. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No.1 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 7.

20. The monoclonal antibody, or antigen binding portion thereof, of claim 1, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO. 7.

21. The monoclonal antibody, or antigen-binding portion thereof, of claim 2, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 2 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 8.

22. The monoclonal antibody, or antigen binding portion thereof, of claim 2, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO. 8.

23. The monoclonal antibody, or antigen-binding portion thereof, of claim 3, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID NO. 3 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO. 9.

24. The monoclonal antibody, or antigen binding portion thereof, of claim 3, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO 9.

25. The monoclonal antibody, or antigen-binding portion thereof, of claim 4, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 4 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID No. 10.

26. The monoclonal antibody, or antigen binding portion thereof, of claim 4, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO 10.

27. The monoclonal antibody, or antigen-binding portion thereof, of claim 5, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID NO. 5 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO. 11.

28. The monoclonal antibody, or antigen binding portion thereof, of claim 5, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO. 11.

29. The monoclonal antibody, or antigen-binding portion thereof, of claim 6, comprising a light chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 6 and a heavy chain variable region amino acid sequence having at least 95% identity to SEQ ID NO 12.

30. The monoclonal antibody, or antigen binding portion thereof, of claim 6, comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO 12.

31. The monoclonal antibody, or antigen binding portion thereof, of claim 1, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO 65 to SEQ ID NO 67 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO 49 to SEQ ID NO 51.

32. The monoclonal antibody, or antigen binding portion thereof, of claim 2, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:68 to SEQ ID NO:70, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:55 to SEQ ID NO: 58.

33. The monoclonal antibody, or antigen binding portion thereof, of claim 3, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:71 to SEQ ID NO:76 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:59 to SEQ ID NO: 61.

34. The monoclonal antibody, or antigen binding portion thereof, of claim 4, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:77 to SEQ ID NO:78, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:62 to SEQ ID NO: 63.

35. The monoclonal antibody, or antigen binding portion thereof, of claim 5, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:79 to SEQ ID NO:80, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 64.

36. The monoclonal antibody, or antigen binding portion thereof, of claim 6, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:81 to SEQ ID NO:83 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:52 to SEQ ID NO: 54.

37. An isolated monoclonal antibody, or antigen-binding portion thereof, comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 84 to SEQ ID NO 99, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 100 to SEQ ID NO 118.

38. The monoclonal antibody, or antigen binding portion thereof, of any one of claims 1-37, which is an Fab fragment, F (ab')₂Fragments, Fv fragments, single chain antibodies or bispecific antibodies.

39. The monoclonal antibody of any one of claims 1-37, which is a chimeric antibody or a humanized antibody.

40. The monoclonal antibody of any one of claims 1-37, which is an immunoglobulin g (igg), IgM, IgE, IgA, or IgD molecule.

41. The monoclonal antibody of claim 39, which is IgG1, IgG2, IgG3, or IgG 4.

42. An immunoconjugate comprising the antibody or antigen binding portion thereof of any one of claims 1-41 linked to a therapeutic agent.

43. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-41 and a pharmaceutically acceptable transporter.

44. A method of stimulating an immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 43 to stimulate an immune response in the subject.

45. A method of treating an infectious disease in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 43 to treat the infectious disease.

46. A method of treating cancer in a subject comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 43 to treat the cancer.

47. The antibody or antigen-binding fragment thereof of any one of claims 1-41, wherein the antibody or antigen-binding fragment thereof has an affinity (KD) for PD-L1 of 5 x 10^-8M to 1X 10^-10M。