EA043231B1 - ANTIBODIES AGAINST PD-L1 AND THEIR APPLICATIONS - Google Patents

Description

Technical field

Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homologue 1 (B7-H1), is a 40 kDa type 1 transmembrane protein thought to play a significant role in suppressing the immune system during specific events. such as pregnancy, tissue allograft, autoimmune disease, and other disease states such as hepatitis. When PD-L1 binds to PD-1 or B7.1, an inhibitory signal is transmitted that reduces the proliferation of CD8+ T cells in the lymph nodes; in addition, PD-1 is also able to control the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis, which is further mediated by less regulation of the Bcl-2 gene.

It has been shown that upregulation of PD-L1 can allow cancer to elude the host's immune system. Analysis of tumor samples from patients with renal cell carcinoma showed that high expression of PD-L1 in tumors is associated with increased tumor aggressiveness and an increased risk of death. Many PD-L1 inhibitors are in development for immuno-oncological therapy and are showing good results in clinical trials.

Inhibition of PD-L1 has also shown promise for the treatment of infectious diseases, along with the treatment of cancer. In a mouse intracellular infection model, L. monocytogenes induced PD-L1 protein expression in T cells, NK cells, and macrophages. Blockade of PD-L1 (eg using blocking antibodies) resulted in increased mortality in infected mice. The blockade reduced the production of TNF-α and nitric oxide by macrophages, reduced the production of granzyme B by NK cells, and reduced the proliferation of L. monocytogenes antigen-specific CD8 T cells (but not CD4 T cells). These data suggest that PD-L1 acts as a positive costimulatory molecule in intracellular infection.

Brief description of the invention

The present invention provides anti-PD-L1 antibodies having high binding affinity for human PD-L1 proteins and capable of effectively blocking the interaction between PD-L1 and its PD-1 receptor. It is also important that these examples demonstrate the stimulation of the specified antibodies against PD-L1 T-cell immune response and inhibition of tumor growth. Unlike known antibodies against PD-L1, which bind to the immunoglobulin V domain of the extracellular portion of the PD-L1 protein, these antibodies bind to the immunoglobulin C domain, in particular the amino acid residues Y134, K162 and N183. Said anti-PD-L1 antibodies are suitable for therapeutic use, for example in the treatment of various types of cancer as well as infections, and may also be used for diagnostic and prognostic purposes.

In one embodiment, the present invention provides an anti-PD-L1 antibody or fragment thereof that can specifically bind to the immunoglobulin C domain (Ig C) of a human programmed death ligand 1 (PD-L1) protein. In some embodiments, said IgC domain consists of amino acid residues 133-225. In some embodiments, said antibody or said antibody fragment can bind to at least one of amino acid residues Y134, K162, or N183 of the PD-L1 protein. In some embodiments, said antibody or said antibody fragment can bind to at least one of amino acid residues Y134, K162, and N183 of the PD-L1 protein. In some embodiments, said antibody or said antibody fragment does not bind to the immunoglobulin V domain (Ig V) of the PD-L1 protein, wherein said IgV domain consists of amino acid residues 19-127.

In one embodiment, the present invention provides an anti-PD-L1 antibody or fragment thereof, characterized in that said antibody or antibody fragment has specificity for human programmed death ligand 1 (PD-L1) protein and contains a VH CDR1 from SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, VL CDR1 of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5, and VL CDR3 of SEQ ID NO: 6. In some embodiments, said antibody or antibody fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, said light chain constant region is a kappa or lambda chain constant region. In some embodiments, said antibody or said antibody fragment is of the isotype IgG, IgM, IgA, IgE, or IgD. In some embodiments, said isotype is IgG1, IgG2, IgG3, or IgG4. Without limitation, said antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or fragment thereof is a humanized antibody.

According to the present invention, using mutagenesis, hotspots for mutations of residues in VH CDR3 (see, for example, antibodies A1, A2, C3, C4, C6, B1 and B6 in examples 13-17) and VL CDR3 (see, for example, for example, antibodies B3, C4 and A3 in examples 13-17). Accordingly, the present invention also provides antibodies comprising one or more mutations at these hotspots.

In some embodiments, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for the human PD-L1 protein and comprises (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 117 or a variant of SEQ ID NO: 117 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 117, with the second amino acid residue VH CDR3 is Leu;

(d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 6.

In one embodiment, said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in SEQ ID NO: 117, indicated VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

In one embodiment, said antibody or said antibody fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 149 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 150.

Also according to one embodiment, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for the human PD-L1 protein and contains (a) a VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 3;

(d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 140 or a variant of SEQ ID NO: 140 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 140, with at least least (i) amino acid residue 4 in VL CDR3 is Ser, (ii) amino acid residue 5 in VL CDR3 is Asp, or (iii) amino acid residue 6 in VL CDR3 is Ala.

In one embodiment, said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in SEQ ID NO: 3, indicated VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: 140.

In one embodiment, said antibody or said antibody fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 159 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 160.

In one embodiment, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for human PD-L1 protein and comprises (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 3;

(d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 6.

In some embodiments, said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in SEQ ID NO: 3, indicated VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, said antibody or said antibody fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 141 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 142.

Also, in some embodiments, there is provided a composition comprising an antibody or fragment thereof as described herein and a pharmaceutically acceptable carrier. Additionally, in some embodiments, an isolated cell is provided that contains one or more polynucleotides encoding an antibody or fragment thereof as described herein.

Methods of treatment and uses are also provided. In one embodiment, there is provided a method of treating cancer or infection in a patient in need thereof, comprising administering to said patient an effective amount of an antibody or fragment thereof as described herein. In some embodiments, said cancer is a solid tumor. In some embodiments, said cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer. In some embodiments, said cancer is selected from the group consisting of bladder cancer, liver cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, urethral cancer, colon and rectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cells, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer and small cell lung cancer. In some embodiments, said method further comprises administering to said patient a second cancer therapy agent. In some embodiments, said infection is a viral infection, a bacterial infection, a mycotic infection, or a parasitic infection.

In another embodiment, there is provided a method of treating cancer or infection in a patient in need thereof, comprising

- 3 043231 (a) treatment of cells in vitro with an antibody or fragment thereof according to the present description; and (b) administering the treated cell to said patient.

In some embodiments, said method further comprises isolating said cell from the subject prior to step (a). In some embodiments, said cell is isolated from the patient's body. In some embodiments, said cell is isolated from a non-patient donor individual. In some embodiments, said cell is a T cell, non-limiting examples of which include a tumor infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination of the above.

Diagnostic methods and applications are also provided. In one embodiment, a method is provided for detecting PD-L1 expression in a sample, comprising bringing said sample into contact with an antibody or fragment thereof under conditions allowing said antibody or fragment thereof to bind to PD-L1 and detecting said binding that is indicative of PD expression. -L1 in the sample. In some embodiments, said sample contains a tumor cell, tumor tissue, infected tissue, or a blood sample.

Antibodies and fragment according to the present description can be used to obtain bespecifically antibodies. In one embodiment, a bispecific antibody is provided, comprising a fragment as described herein and a second antigen-binding fragment having specificity for a molecular structure on an immune cell. In some embodiments, said molecule is selected from the group consisting of PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA, CD47 and CD73. In some embodiments, both said fragment and the second fragment are independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, said bispecific antibody further comprises an Fc fragment.

Brief description of the drawings

In FIG. 1 shows that HL1210-3 can bind to human PD-L1 with high affinity.

In FIG. 2 shows that HL1210-3 can effectively inhibit the binding of human PD-L1 to human PD1.

In FIG. 3 shows that the HL1210-3 antibody can highly effectively inhibit the binding of PD-1 to PD-L1 expressed on mammalian cells.

In FIG. 4 shows that the anti-PD-L1 antibodies tested can stimulate a human T cell response.

In FIG. 5 shows the binding kinetics of HL1210-3 to recombinant PD-L1.

In FIG. 6 shows that all humanized antibodies tested had comparable binding efficiency to human PD-L1 in contact with the chimeric antibody.

In FIG. 7 shows that all humanized antibodies tested can bind highly efficiently to mammalian cell-expressed PD-L1, comparable to a chimeric antibody.

In FIG. 8 shows that the humanized Hu1210-41 antibody is able to bind to rhesus monkey PD-L1 with lower affinity and is unable to bind to rat and mouse PD-L1.

In FIG. 9 shows that the Hu1210-41 antibody is only able to specifically bind to B7-H1 (PD-L1), but not to B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4, ICOS and BTLA.

In FIG. 10 shows that Hu1210-41 can effectively inhibit the binding of human PD-L1 to human PD1 and B7-1.

In FIG. 11 shows that Hu1210-41 can effectively inhibit the binding of human PD-L1 to human PD1 and B7-1.

In FIG. 12 shows that the humanized antibodies Hu1210-8, Hu1210-9, Hu1210-16, Hu1210-17, Hu1210-21, and Hu1210-36 can dose-dependently stimulate the production of IFN-γ and IL-2 in a mixed lymphocyte culture response.

In FIG. 13 shows that the humanized antibodies Hu1210-40, Hu1210-41, and Hu1210-17 can dose-dependently stimulate IFN-γ production in a CMV antigen panel assay.

In FIG. 14 shows that Hu1210-31 at 5 mg/kg can inhibit tumor growth by 30% in the HCC827-NSG xenograft model.

In FIG. 15 shows that Hu1210-41 antibody can dose-dependently inhibit tumor growth in the HCC827-NSG xenograft model, while Hu1210-41 antibody also reduced tumor mass in a dose-dependent manner.

On the graphs in Fig. 16 for each PD-L1 mutant shows the mean binding as a function of expression (anti-PD-L1 control monoclonal antibody (mAb) reactivity).

Fig. 17 illustrates the localization of Y134, K162 and N183, residues (spheres) involved in binding to anti-PD-L1 antibody Hu1210-41.

- 4 043231

In FIG. 18 compares the S60R mutant with the parental Hu1210-41 antibody in terms of binding efficiency to mammalian cell-expressed PD-L1.

In FIG. 19 shows the results of the binding assay (to human PD-L1) for the resulting antibodies.

In FIG. 20 shows that antibody B6 binds more efficiently to PD-L1 expressed on mammalian cells compared to parental antibody and tecentriq (Tecentriq™, atezolizumab).

In FIG. 21 shows the effect of these antibodies on IL-2 production in Jurkat cells, in which B6 also showed higher activity.

In FIG. 22 shows the IFN-γ production-stimulating activity of antibodies in vitro under mixed lymphocyte culture conditions.

In FIG. 23 shows the tumor growth inhibitory activity of antibodies in vivo.

Detailed description of the invention

Definitions.

It should be noted that the terms in the singular refer to one or more units of the specified object; for example, an antibody corresponds to one or more antibodies. Accordingly, the singular terms, the expressions one or more, and at least one may be used interchangeably herein.

As used herein, the term polypeptide encompasses a single polypeptide as well as a plurality of polypeptides, and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific product length. Accordingly, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term referring to a chain or chains of two or more amino acids are included in the definition of a polypeptide, and the term polypeptide may be used instead of any of the above terms or interchangeably with any of them. . The term polypeptide also refers to the products of post-expression modifications to a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with unnatural amino acids. The polypeptide may be from a natural biological source or obtained using recombinant technology, but is not necessarily translated from the specified nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

The term isolated herein in relation to cells, nucleic acids such as DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, that are present in the natural source of the specified macromolecule. The term isolated herein also refers to a nucleic acid or peptide that is substantially free of (free of) cellular material, viral material or culture medium when produced using recombinant DNA techniques, or chemical precursors or other chemicals if they have been chemically synthesized. . In addition, the term isolated nucleic acid includes nucleic acid fragments not naturally occurring as fragments and not found in the natural state. The term isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides.

As used herein, the term recombinant in relation to polypeptides or polynucleotides means a form of a polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be created by combining polynucleotides or polypeptides that do not normally occur together.

Homology or identity or similarity refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the position in each sequence that can be aligned for comparison purposes. If a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions in the sequences. The unrelated or non-homologous sequence has less than 40% identity, but preferably less than 25% identity, to one of the sequences as described herein.

If a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) shares a certain percentage (e.g., 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99%) sequence identity with another sequence, this means that after alignment at

- 5 043231 comparing two sequences they contain the indicated percentage of identical bases (or amino acids). Said alignment and percent homology or sequence identity can be determined using software known in the art, such as described in Ausubel et al. eds. (2007), Current Protocols in Molecular Biology. Preferably, the default settings are used for alignment. One of the alignment programs is BLAST with default settings. In particular, the programs are represented by BLASTN and BLASTP with the following default parameters:

genetic code=standard;

filter=no filter;

chain=both chains;

cutoff=60;

wait=10;

matrix=BLO SUM62;

descriptions=50 sequences;

sorting=highest score (HIGH SCORE);

databases=non-redundant, GenBank+EMBL+DDBJ+PDB+translated CDS sequences GenBank+SwissProtein+SPupdate+PIR.

Biologically equivalent polynucleotides are nucleotides having the above percent homology and encoding a polypeptide having the same or similar biological activity.

The term equivalent nucleic acid or equivalent polynucleotide refers to a nucleic acid having a nucleotide sequence characterized by a certain degree of homology or nucleotide sequence identity to said nucleic acid or its complement. A double-stranded nucleic acid homologue is intended to include nucleic acids having a nucleotide sequence with a certain degree of homology to said nucleic acid or its complement. In one aspect, nucleic acid homologues are capable of hybridizing to said nucleic acid or its complement. Similarly, an equivalent polypeptide refers to a polypeptide having a certain degree of homology or sequence identity relative to the amino acid sequence of the reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide contains 1, 2, 3, 4, or 5 additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.

Hybridization reactions can be carried out under conditions of varying severity. In general, the low stringency hybridization reaction is carried out at about 40° C. in about 10xSSC or equivalent ionic strength/temperature solution. Hybridization under moderate stringency conditions is typically carried out at about 50°C in about 6xSSC, and the hybridization reaction under high stringency conditions is typically carried out at about 60°C in about 1xSSC. Hybridization reactions may also be carried out under physiological conditions well known to those skilled in the art. A non-limiting example of physiological conditions are temperature, ionic strength, pH, and Mg ²⁺ concentration commonly found in a cell.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) instead of thymine in the case of an RNA polynucleotide. Accordingly, the term polynucleotide sequence refers to the literal representation of a polynucleotide molecule. Said letter representation may be entered into databases on a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology search. The term polymorphism refers to the coexistence of more than one form of a gene or part of it. A portion of a gene that has at least two different forms, i.e. represented by two different sequences of nucleotides, is called the polymorphic region of the gene. The polymorphic region may be a single nucleotide, different in different alleles.

The terms polynucleotide and oligonucleotide are used interchangeably and refer to the polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are: genome or or gene fragment (e.g. probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA,

- 6 043231 miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotide may contain modified nucleotides such as methylated nucleotides and nucleotide analogs. If there are modifications in the structure of the nucleotide, they can be carried out before or after the assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling moiety. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise indicated or required, any embodiment of the present invention providing for a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to form the double-stranded form.

The term encode in the context of polynucleotides refers to a polynucleotide which is said to encode a polypeptide if, in its natural state or after being manipulated using methods well known to those skilled in the art, it can be transcribed and/or translated to produce mRNA for the polypeptide and /or its fragment. The antisense strand is the complement of such a nucleic acid and the coding sequence can be derived from it.

As used herein, an antibody or antigen-binding polypeptide refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody may be a complete antibody and any antigen-binding fragment or single chain thereof. Accordingly, the term antibody includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having the biological activity of binding to an antigen. Examples of such molecules include, but are not limited to, a heavy or light chain complementarity determining region (CDR) or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one part of the binding protein.

The terms antibody fragment or antigen-binding fragment as used herein refers to a portion of an antibody such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of structure, an antibody fragment binds to the same antigen as recognized by an intact antibody. The term antibody fragment includes aptamers, spiegelmers and diabodies. The term antibody fragment also includes any synthetic or genetically engineered protein that acts as an antibody by binding a specific antigen to form a complex.

A single chain variable fragment, or scFv, refers to a fusion protein from the variable regions of heavy (VH) and light chains (V _L ) of immunoglobulins. In some aspects, these regions are connected by a short linker peptide of 10 to about 25 amino acids in length. Said linker may be enriched in glycine for flexibility and serine or threonine for solubility, and may connect either the N-terminus of VH to the C-terminus of V _L or vice versa. The specified protein retains the specificity of the original immunoglobulin, despite the removal of constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides that can be distinguished by biochemical methods. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), including some subclasses (eg, γ1-γ4). The nature of this chain determines the class of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG ₃ , IgG ₄ , IgG ₅ and the like, are described in detail and are known to provide functional specialization. The specialist will be able to easily determine the modified variants of each of these classes and isotypes after reading the present description and, accordingly, such variants are included in the scope of the present invention. All classes of immunoglobulins are expressly included within the scope of the present invention; the description below generally refers to the IgG class of immunoglobulin molecules. In the case of IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Da and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. These four chains are typically linked by disulfide bonds in a Y-shaped configuration, with the light chains spanning the heavy chains starting at the Y notch and extending through the variable region.

Antibodies, antigen-binding polypeptides, variants or derivatives thereof as used herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F( ab')2, Fd, Fv, Fv single chain (scFv), Fv disulfide linked single chain antibodies (sdFv), fragments containing a VK or VH domain, fragments produced by a Fab expression library; and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies as described herein). Molecules of immunoglobulins or antibodies according to the present description can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1,

IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass of immunoglobulin molecules.

Light chains are classified as kappa or lambda (κ, λ). Any class of heavy chains can be linked to either the kappa light chain or the lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the tails of the two heavy chains are linked together by covalent disulfide bonds or non-covalent bonds in immunoglobulin production by both hybridomas and B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from the N-terminus on the forked part of the Y-shaped configuration to the C-terminus at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used functionally. In this sense, it is to be understood that the variable domain portions of both the light (VK) and heavy (VH) chains determine antigen recognition and antigen specificity. In contrast, the light chain (CK) and heavy chain (CH1, CH2 or CH3) constant domains provide important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, and the like. It is conventionally considered that the numbering of the constant region of domains increases with distance from the antigen binding site or the amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terminus of the heavy and light chains, respectively.

As stated above, the variable region allows an antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and the VH domain, or a subset of complementarity determining regions (CDRs) of an antibody, are combined to form a variable region defining a three-dimensional antigen binding site. Said quaternary antibody structure forms an antigen binding site present at the end of each Y arm. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VK chains (i.e. L1, CDR-L2 and CDR-L3). In some cases, for example, in the case of certain immunoglobulin molecules derived from the camelid species or engineered from camelid immunoglobulins, the complete immunoglobulin molecule may consist solely of heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature, 363:446-448 (1993).

In naturally occurring antibodies, the six complementarity determining regions, or CDRs, present in any antigen-binding domain are short, non-contiguous sequences of amino acids arranged specifically to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, called framework regions, exhibit less intermolecular variability. The framework regions generally adopt the β-sheet conformation, and the CDR regions form loops that connect the specified β-sheet structure and in some cases are part of it. Accordingly, the framework regions form a scaffold, which ensures the positioning of the CDR regions in the correct orientation due to interstrand non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface complementary to an epitope on the immunoreactive antigen. Said complementary surface facilitates non-covalent binding of the antibody to the cognate epitope. Amino acids containing CDRs and framework regions, in any heavy or light chain variable region, respectively, can be readily identified by one of skill in the art as they have been well defined (see Sequences of Proteins of Immunological Interest, Kabat, E. , et al., U.S., Department of Health and Human Services, (1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

In the event that a term that is used and/or accepted in the art has two or more definitions, the definition of the term herein includes all such meanings unless expressly stated otherwise. A specific example is provided by the use of the term complementarity determining region (CDR) to describe the non-contiguous antigen-binding sites found in the variable region of both heavy and light chain polypeptides. This particular area has been described by Kabat et al. and Chothia et al. in Kabat et al., U.S., Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983); and Chothia et al., J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety. When compared, the Kabat and Chothia CDR definitions include overlapping residues or subsets of amino acid residues. However, the use of any of the definitions for the CDR of an antibody or variants thereof is encompassed by the term defined and used herein. Suitable amino acid residues, which include the CDR regions according to each of the above references, are listed in the table below for comparison. The exact number of residues that include a particular CDR, vary depending on the sequence and size of the specified CDR. Those skilled in the art can routinely determine the residues that contain a particular CDR region based on the amino acid sequence of an antibody variable region.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. have also defined a variable domain sequence numbering system that is applicable to any antibody. One skilled in the art can unambiguously apply this Kabat numbering system to any variable domain sequence without relying on any experimental data other than the sequence itself. In this document, Kabat numbering refers to the numbering system described in the source: Kabat et al., U.S., Dept. of Health and Human Services, Sequence of Proteins of Immunological Interest (1983).

Along with the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), spans approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at residue 15 after the end of CDR-H1, is approximately 16-19 amino acids long, and ends at the next arginine or lysine residue. CDR-H3 starts at about 33 amino acids past the end of CDR-H2; includes 3-25 amino acids; and ends with the sequence W-G-X-G, where X is any amino acid. CDR-L1 starts at approximately residue 24 (i.e. beyond the cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins approximately 16 residues after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e. after a cysteine residue); contains approximately 7-11 residues and ends in the sequence F or W-G-X-G, where X is any amino acid.

Antibodies as described herein can be derived from any animal, including birds and mammals. Preferably said antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks).

As used herein, the term heavy chain constant region includes amino acid sequences derived from an immunoglobulin heavy chain. The heavy chain constant region-containing polypeptide comprises at least one of a CH1 domain, a hinge domain (eg, an upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use according to the present invention may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain and a CH3 domain. According to another implementation variant of the polypeptide according to the present description contains a polypeptide chain containing a CH3 domain. In addition, an antibody for use according to the present invention may lack at least a portion of the CH2 domain (eg, all or part of the CH2 domain). As described above, one of skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from that in a naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody as described herein can be derived from different immunoglobulin molecules. For example, a polypeptide heavy chain constant region may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG ₃ molecule. In another example, the heavy chain constant region may comprise a hinge region derived partly from an IgG1 molecule and partly from an IgG ₃ molecule. In another example, the heavy chain portion may comprise a chimeric hinge derived partly from an IgG1 molecule and partly from an IgG ₄ molecule.

As used herein, the term light chain constant region includes amino acid sequences derived from the light chain of an antibody. Preferably, the light chain constant region comprises at least one of the kappa chain constant domain or the lambda chain constant domain.

The light chain/heavy chain pair refers to a light chain/heavy chain assembly that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As stated previously, the subunit structures and three-dimensional configuration of the constant regions of various classes of immunoglobulins are well known. As used herein, the term VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term CC domain includes the first (closest to the amino-terminus) domain of an immunoglobulin heavy chain constant region. The CH1 domain is located adjacent to the VH domain and in the direction of the amino terminus relative to the hinge region of the heavy chain of the immunoglobulin molecule.

As used herein, the term CH2 domain includes the portion of a heavy chain molecule that extends, for example, from approximately residue 244 to residue 360 of an antibody using standard numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S., Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) The CH2 domain is unique in that it does not closely pair with another domain. two N-linked branched carbohydrate chains are introduced into the IgG molecule.It is also conclusively confirmed that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be subdivided into three distinct domains: superior, middle and inferior hinge domains (Roux et al., J. Immunol., 161:4083 (1998)).

As used herein, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term chimeric antibody means any antibody characterized in that the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be intact, partial, or modified in accordance with the present invention) is derived from a second species. kind. In certain embodiments, the target binding region or site is not from a human-derived source (eg, derived from a mouse or primate), but the constant region is derived from a human.

According to the present invention, the percentage of humanization is calculated by determining the number of amino acid differences in the framework sequence (i.e., differences not in the CDR) of the humanized domain and the germline domain, subtracting the resulting number from the total number of amino acids, then dividing the result by the total number of amino acids and multiplying by 100.

The terms "specifically binds" or "is characterized by specificity" generally imply that the antibody binds to an epitope via an antigen-binding domain, and that said binding implies some complementarity between the antigen-binding domain and the epitope. Under this definition, an antibody is said to specifically bind to an epitope if it binds to said epitope via the antigen-binding domain more easily than it would to a random unrelated epitope. The term specificity is used herein to qualitatively measure the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be considered to have higher specificity for a given epitope than antibody B, or antibody A may be said to bind epitope C with higher specificity than cognate epitope D.

As used herein, the terms treat or treatment refer to both therapeutic treatment and prophylactic or preventive measures, the purpose being to prevent or slow down (alleviate) an unwanted physiological change or disorder, such as the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, improvement in disease severity, stabilization (i.e., no worsening) of disease status, delay or slowing of disease progression, alleviation or temporary amelioration of the disease state, and remission (partial or complete) , detectable or undetectable. Treatment may also mean an increase in the duration of survival compared to the expected survival in the absence of treatment. Those in need of treatment include those who already have said condition or disorder, as well as those who are predisposed to said condition or disorder; or those in whom it is necessary to prevent the development of said condition or disorder.

- 10 043231

The term subject or individual or animal or patient or mammal refers to any subject, in particular a mammalian subject, in need of diagnosis, prognosis or therapy. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals used in sports or companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc. d.

As used herein, expressions such as a patient in need of treatment or a subject in need of treatment include subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition as described herein, used, for example, for detection, for a diagnostic procedure. and/or for treatment.

Antibodies against PD-L1.

The present invention provides anti-PD-L1 antibodies with high affinity for human PD-L1 protein. The tested antibodies showed strong binding and inhibitory activity, and are suitable for therapeutic and diagnostic applications.

The PD-L1 protein is a type 1 transmembrane protein of 40 kDa. Its extracellular portion includes the N-terminal immunoglobulin V domain (IgV) (amino acids 19-127) and the C-terminal immunoglobulin C-domain (IgC) (amino acids 133-225). PD-1 and PD-L1 interact through the conserved anterior and lateral portions of their IgV domains, as do the IgV domains of antibodies and T cell receptors. As expected, all currently known antibodies against PD-L1 bind to the IgV domain, which can disrupt the binding between PD-1 and PD-L1. Accordingly, surprising and unexpected was the discovery described herein that antibodies, for example, many of the antibodies as described herein, which bind to the IgC domain of the PD-L1 protein, are also able to effectively and perhaps even more inhibit PD-L1, resulting in even more improved therapeutic effects.

In one embodiment, the present invention accordingly provides an anti-PD-L1 antibody or fragment thereof capable of specifically binding to the immunoglobulin C domain (IgC) of a human programmed death ligand 1 (PD-L1) protein. In some embodiments, said IgC domain consists of amino acid residues 133-225.

In some embodiments, said antibody or said antibody fragment can bind to at least one of amino acid residues Y134, K162, or N183 of the PD-L1 protein. In some embodiments, said antibody or said antibody fragment can bind to at least two of the Y134, K162, or N183 amino acid residues of the PD-L1 protein. In some embodiments, said antibody or said antibody fragment can bind to at least one of amino acid residues Y134, K162, and N183 of the PD-L1 protein. In some embodiments, said antibody or said antibody fragment does not bind to the immunoglobulin V domain (IgV) of the PD-L1 protein, said IgV domain consisting of amino acid residues 19-127.

In accordance with one embodiment of the present invention, an antibody is provided that includes heavy chain and light chain variable domains with CDR regions as defined in SEQ ID NOs: 1-6.

Table 1

CDR region sequences

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 VHCDR2 TISDGGGYIYYSDSVKG 2 VH CDR3 EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VL CDR2 STSSRYT 5 VL CDR3 QQHYTTPLT 6

As demonstrated in experimental examples, antibodies containing these CDR regions - both murine and humanized or chimeric - showed pronounced PD-L1 binding and inhibitory activity. Using additional computer modeling, it was shown that certain residues within the CDR can be modified to maintain or improve the properties of antibodies. Such residues, which are underlined in Table. 1 are called hot spots. In some embodiments, an anti-PD-L1 antibody as described herein includes the VH and VL CDRs shown in Table 1. 1, with one, two, or three additional modifications. Such modifications may be additions, deletions or substitutions of amino acids.

In some embodiments, said modification is a replacement in at most one hot spot in each of the CDRs. In some embodiments, said modification is a replacement at one, two, or three of these hot spots. According to one implementation variant, the specified modification is a replacement in one of the hot spots.

- 11 043231

Such substitutions, in some embodiments, are conservative substitutions.

A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue containing the same side chain. Families of amino acid residues containing similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine ) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Accordingly, a non-essential amino acid residue in the immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a segment of the amino acid sequence may be replaced with a structurally similar segment that differs in the order and/or composition of the side chain family members.

Non-limiting examples of conservative amino acid substitutions are shown in the table below, with a similarity score of 0 or greater indicating a conservative substitution between two amino acids.

table 2

Amino acid similarity matrix

WITH G R S A T D E N Q H TO R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 m -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 TO -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 R -3 -1 6 G -3 5 WITH 12

- 12 043231

Table 3

Conservative amino acid substitutions

Replaceable amino acid: Change to Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Om D-Om Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Om, D-Om Methionine D-Met, S-Me-Cys, lie, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D Pro Serene D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, lie, D-Ile, Met, D-Met

Specific examples of CDRs with suitable substitutions are provided in SEQ ID NOs: 61-111 of Example 11. In some embodiments, respectively, an antibody according to the present disclosure comprises a VH CDR1 from SEQ ID NOs: 1 or any of sequences 61-67. In some embodiments, an antibody as described herein comprises a VH CDR2 of SEQ ID NO: 2 or any of 68-77. In some embodiments, an antibody as described herein comprises a VH CDR3 of SEQ ID NO: 1 or any of sequences 78-90. In some embodiments, an antibody as described herein comprises the VL CDR1 of SEQ ID NO: 4 or any of sequences 91-92. In some embodiments, an antibody as described herein comprises the VL CDR2 of SEQ ID NO: 5 or any of sequences 93-105. In some embodiments, an antibody as described herein comprises the VL CDR3 of SEQ ID NO: 6 or any of sequences 106-110.

In some embodiments, the antibody or fragment thereof includes no more than one, no more than two, or no more than three of the above substitutions. In some embodiments, said antibody or said antibody fragment comprises VH CDR1 of SEQ ID NO: 1 or any of SEQ ID NO: 61-67, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, VL CDR1 from SEQ ID NO: 4, VL CDR2 from SEQ ID NO: 5 and VL CDR3 from SEQ ID NO: 6.

In some embodiments, said antibody or said antibody fragment comprises VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2 or any of SEQ ID NO: 68-77, VH CDR3 of SEQ ID NO: 3, VL CDR1 from SEQ ID NO: 4, VL CDR2 from SEQ ID NO: 5 and VL CDR3 from SEQ ID NO: 6.

In some embodiments, said antibody or said antibody fragment comprises VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, or any of SEQ ID NO: 78-90, VL CDR1 from SEQ ID NO: 4, VL CDR2 from SEQ ID NO: 5 and VL CDR3 from SEQ ID NO: 6.

In some embodiments, said antibody or said antibody fragment comprises VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, VL CDR1 of SEQ ID NO: 4, or any of SEQ ID NO: 91-92, VL CDR2 of SEQ ID NO: 5 and VL CDR3 of SEQ ID NO: 6.

In some embodiments, said antibody or antibody fragment comprises VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, VL CDR1 of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5 or any of SEQ ID NO: 93-105 and VL CDR3 of SEQ ID NO: 6.

In some embodiments, said antibody or antibody fragment comprises VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3, VL CDR1 of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5 and VL CDR3 of SEQ ID NO: 6, or any of SEQ ID NO: 106-111.

Non-limiting examples of VH are shown in SEQ ID NOs: 7-26 and 113, with SEQ ID NOs: 113

- 13 043231 is a mouse VH and SEQ ID NOs: 7-26 are humanized sequences. In addition, among the humanized VHs, SEQ ID NOs: 9-15, 17-21, and 23-26 include one or more backmutations to a mouse variant. Similarly, non-limiting examples of VL (VK) are shown in SEQ ID NOs: 27-33. SEQ ID NOs: 28 and 30 are the original humanized sequences obtained with grafted CDRs as shown in the examples. SEQ ID NOs: 29 and 31-33 are backmutated humanized VLs.

Back mutations have been shown to be useful in maintaining certain characteristics of anti-PD-L1 antibodies. Accordingly, in some embodiments, anti-PD-L1 antibodies as described herein, particularly human or humanized antibodies, comprise one or more back mutations. In some embodiments, the VH backmutation (i.e., amino acid included at a specific position) is one or more of (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position 53 , (d) Ile at position 91, (e) Glu at position 1, (f) Val at position 37, (g) Thr at position 40, (h) Val at position 53, (i) Glu at position 54, ( j) Asn at position 77, (k) Arg at position 94, and (l) Thr at position 108, according to Kabat numbering, and combinations thereof.

In some embodiments, said backmutations are selected from (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d) Ile at position 91, according to Kabat numbering. , and their combinations.

In some embodiments, the VL backmutation is one or more of (a) Ser at position 22, (b) Gln at position 42, (c) Ser at position 43, (d) Asp at position 60, and ( e) Thr at position 63, according to Kabat numbering, and combinations thereof.

In some embodiments, an anti-PD-L1 antibody as described herein comprises VH of SEQ ID NOs: 7-26, VL of SEQ ID NOs: 27-33, or their corresponding biological equivalents. A VH or VL biological equivalent is a sequence that includes the designated amino acids while being 80%, 85%, 90%, 95%, 98%, or 99% identical overall. The biological equivalent of SEQ ID NO: 20, for example, may be a VH whose sequence is generally 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:20, but retains CDR regions (SEQ ID NO: 1- 6 or variants thereof) and optionally retains one or more or all of the backmutations. In one embodiment, VH has the amino acid sequence shown in SEQ ID NO: 20 and VL has the amino acid sequence shown in SEQ ID NO: 28.

Further improved anti-PD-L1 antibodies.

Through the use of mutation-controlled random mutagenesis in Examples 13-17, a number of hotspots for residue mutations were identified, particularly in CDR3 from the variable regions of both the heavy (e.g., B6, C3, C6, and A1) and light chains ( e.g. A3) (see tables 14 and 15). Mutagenesis was performed on a template antibody derived from Hu1210-41 (as indicated in the footnote under Table 14, the WT template antibody contains the substitution S60R (Kabat numbering) in the heavy chain CDR2). Hu1210-41 also included a G53A substitution relative to the chimeric antibody (see SEQ ID NO: 20) in VH CDR2. Antibody B6 from the tested mutant antibodies showed significantly improved binding affinity for human PD-L1 and biological activity.

In one embodiment, respectively, antibodies and an antigen-binding fragment are provided that include the following CDRs (from an S60R mutant) and variants thereof.

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 VHCDR2 TISDAGGYIYYRDSVKG 116 VH CDR3 EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VL CDR2 STSSRYT 5 VL CDR3 QQHYTTPLT 6

In one embodiment, respectively, antibodies and an antigen-binding fragment are provided that include the following CDRs (from B6) and variants thereof.

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 VHCDR2 TISDAGGYIYYRDSVKG 116 VH CDR3 ELPWRYALDY 117 VL CDR1 KASQDVTPAVA 4 VL CDR2 STSSRYT 5 VL CDR3 QQHYTTPLT 6

In one embodiment, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for the human PD-L1 protein and contains

- 14 043231 (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to

SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 3, with the second amino acid residue VH CDR3 is Leu;

(d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 6.

In one embodiment, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for human PD-L1 protein and comprises (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 117 or a variant of SEQ ID NO: 117 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 117, with the second amino acid residue VH CDR3 is Leu;

(d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or a variant of SEQ ID NO: 6 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 6.

Example variants of SEQ ID NO: 1 contain one amino acid substitution at one of the positions of amino acid residues 1, 2 and 5, for example, SEQ ID NO: 61-67.

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 TYDMS 61 CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66 SYDMC 67

Example variants of SEQ ID NO: 116 contain one or more amino acid substitutions, for example, SEQ ID NO: 118-127, 2 and 68-77. In some embodiments, said embodiments are represented by SEQ ID NOs: 118-127.

- 15 043231

Name Subsequence SEQID NO: VHCDR2 TISDAGGYIYYRDSVKG 116 TISDAGAYIYYRDSVKG 118 TISDAGPYIYYRDSVKG 119 TISDAGGFIYYRDSVKG 120 TISDAGGHIYYRDSVKG 121 TISDAGGWIYYRDSVKG 122 TISDAGGYIYYRDTVKG 123 TISDAGGYIYYRDCVKG 124 TISDAGGYIYYRDSLKG 125 TISDAGGYIYYRDSIKG 126 TISDAGGYIYYRDSMKG 127 TISDGGGYIYYSDSVKG 2 TISDGGAYIYYSDSVKG 68 TISDGGPYIYYSDSVKG 69 TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71 TISDGGGWIYYSDSVKG 72 TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74 TISDGGGYIYYSDSLKG 75 TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDMKG 77

In some embodiments, said third amino acid residue of the VH CDR3 variant is Pro. In some embodiments, the fourth amino acid residue of the VH CDR3 variant is Trp.

Example variants of SEQ ID NO: 3 contain one or more amino acid substitutions at amino acid residue positions 1-6, eg SEQ ID NO: 78-90.

Name Subsequence SEQID NO: VH CDR3 EFGKRYALDY 3 QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80 EYGKRYALDY 81 EHGKRYALDY 82 EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY 85 EFGRRYALDY 86 EFGKKYALDY 87 EFGKRFALDY 88 EFGKRHALDY 89 EFGKRWALDY 90

Exemplary variants of SEQ ID NO: 117 contain one or more amino acid substitutions at amino acid residue positions 1, 5 and 6, eg SEQ ID NO: 128-139.

Name Subsequence SEQID NO: VH CDR3 ELPWRYALDY (Wb) 117 ELFNRYALDY (Bl) 128 ELHFRYALDY (C3) 129 ELYFRYALDY (C6) 130 ELLHRYALDY (Al) 131 ELRGRYALDY (A2) 132 QLPWRYALDY 133 DLPWRYALDY 134 NLPWRYALDY 135 ELPWKYALDY 136 ELPWRFALDY 137 ELPWRHALDY 138 ELPWRWALDY 139

- 16 043231

In some embodiments, said variant of SEQ ID NO: 4 contains one amino acid substitution at amino acid residue position 3, eg, SEQ ID NO: 91-92.

Name Subsequence SEQID NO: VL CDR1 KASQDVTPAVA 4 KATQDVTPAVA 91 KACQDVTPAVA 92

In some embodiments, said variant of SEQ ID NO: 5 contains one amino acid substitution at one of the positions of amino acid residues 1-6, for example, SEQ ID NO: 93-105.

Name Subsequence SEQID NO: VL CDR2 STSSRYT 5 TTSSRYT 93 CTSSRYT 94 SSSSRYT 95 SMSSRYT 96 SVSSRYT 97 STTSRYT 98 STCSRYT 99 STSTRYT 100 STSCRYT 101 STSSKYT 102 STSSRFT 103 STSSRHT 104 STSSRWT 105

Example variants of SEQ ID NO: 6 contain one amino acid substitution at one of the positions of amino acid residues 1 and 2, for example, SEQ ID NO: 106-111. Another exemplary variant is shown by SEQ ID NO: 140. _______________________________________________

Name Subsequence SEQID NO: VL CDR3 OOHYTTPLT 6 EQHYTTPLT 106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109 QDHYTTPLT 110 QNHYTTPLT 111 QQHSDAPLT(A3) 140

Mutant A3, which contains substitutions at three residue positions in CDR3 VL, also showed excellent binding affinity for human PD-L1. In one embodiment, respectively, antibodies and an antigen-binding fragment are provided which include the following CDRs and variants thereof.

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 VHCDR2 TISDAGGYIYYRDSVKG 116 VH CDR3 EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VLCDR2 STSSRYT 5 VL CDR3 QQHSDAPLT 140

In one embodiment, accordingly, an antibody or fragment thereof is provided, characterized or characterized in that said antibody or said antibody fragment has specificity for the human PD-L1 protein and comprises (a) a VH CDR1 comprising the amino acid sequence represented by in SEQ ID NO: 1 or a variant of SEQ ID NO: 1 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 1;

(b) a VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116 or a variant of SEQ ID NO: 116 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 116;

(c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 or a variant of SEQ ID NO: 3 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 3;

(d) a VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4 containing one, two or three substitutions, deletions or insertions compared to

- 17 043231

SEQ ID NO: 4;

(e) a VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a variant of SEQ ID NO: 5 containing one, two or three substitutions, deletions or insertions compared to

SEQ ID NO: 5; and (f) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 140 or a variant of SEQ ID NO: 140 containing one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 140, with at least least (i) amino acid residue 4 in VL CDR3 is Ser, (ii) amino acid residue 5 in VL CDR3 is Asp; or (iii) amino acid residue 6 in VL CDR3 is Ala.

Example variants of SEQ ID NO: 1 contain one amino acid substitution at one of the positions of amino acid residues 1, 2 and 5, for example, SEQ ID NO: 61-67.

Example variants of SEQ ID NO: 116 contain one or more amino acid substitutions, for example, SEQ ID NO: 118-127, 2 and 68-77.

Example variants of SEQ ID NO: 3 contain one or more amino acid substitutions, for example, SEQ ID NO: 117 and 128-139.________________________________________________

Name Subsequence SEQID NO: VH CDR3 ELPWRYALDY (Wb) 117 ELFNRYALDY (B1) 128 ELHFRYALDY (NW) 129 ELYFRYALDY (C6) 130 ELLHRYALDY (A1) 131 ELRGRYALDY (А2) 132 QLPWRYALDY 133 DLPWRYALDY 134 NLPWRYALDY 135 ELPWKYALDY 136 ELPWRFALDY 137 ELPWRHALDY 138 ELPWRWALDY 139

Exemplary variants of SEQ ID NO: 4 contain one amino acid substitution at amino acid residue position 3, eg SEQ ID NO: 91-92.

Example variants of SEQ ID NO: 5 contain one amino acid substitution at one of the positions of amino acid residues 1-6, for example, SEQ ID NO: 93-105.

In some embodiments, amino acid residue 4 in the VL CDR3 variant is Ser. In some embodiments, amino acid residue 5 in the VL CDR3 variant is Asp. In some embodiments, amino acid residue 6 in the VL CDR3 variant is Ala. Example variants of SEQ ID NO: 140 contain one amino acid substitution at one of the positions of amino acid residues 1 and 2, for example, SEQ ID NO: 161-166.

Name Subsequence SEQID NO: VL CDR3 QQHSDAPLT 140 EQHSDAPLT 161 DQHSDAPLT 162 NQHSDAPLT 163 QEHSDAPLT 164 QDHSDAPLT 165 QNHSDAPLT 166

Examples of antibodies resulting from the study of mutagenesis, or antigennegative fragments include containing variable regions of heavy chains and light chains, presented in table. 15. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 141 and the light chain variable region comprises SEQ ID NO: 142. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 143 and the light chain variable region comprises SEQ ID NO: 144. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 145 and the light chain variable region comprises SEQ ID NO: 146. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 147 and the light chain variable region the light chain region comprises SEQ ID NO: 148. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 149 and the light chain variable region comprises SEQ ID NO: 150. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 151 and the light chain variable region includes SEQ ID NO: 152. According to one

- 18 043231 embodiment, the heavy chain variable region comprises SEQ ID NO: 153 and the light chain variable region comprises SEQ ID NO: 154. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 155 and the light chain variable region comprises SEQ ID NO: 156. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 157 and the light chain variable region comprises SEQ ID NO: 158. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 159 and the light chain variable region light chain includes SEQ ID NO: 160.

One of skill in the art will also appreciate that antibodies as described herein may be modified such that their amino acid sequence differs from that of the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a certain protein may be similar, for example, be characterized by a certain percentage of identity of the starting sequence, for example, may be 60, 70, 75, 80, 85, 90, 95, 98 or 99 % is identical to the start sequence.

In some embodiments, said antibody comprises an amino acid sequence or one or more fragments not normally associated with the antibody. Modification examples are described in more detail below. For example, an antibody as described herein may contain a flexible linker sequence, or may be modified by the addition of a functional moiety (eg, PEG, drug, toxin, or label).

Antibodies, variants, or derivatives thereof, as used herein, include modified derivatives, that is, modified by covalently attaching any type of molecule to an antibody such that the covalent bond does not prevent said antibody from binding to an epitope. For example, but not limited to, these antibodies can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and the like. Any of the numerous chemical modifications may be carried out using known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more non-classical amino acids.

In some embodiments, said antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEGs.

Said antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an agent for improving the quality of ultrasound, a non-radioactive label, a combination of the above, and other such agents known in the art.

Detectable labeling of these antibodies can be carried out by conjugation with a chemiluminescent compound. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of specific compounds suitable for chemiluminescent labeling are luminol, isoluminol, acridine aromatic ester, imidazole, acridine salt and oxalic acid ester.

Detectable labeling of these antibodies can also be carried out using fluorescent metals such as ¹⁵² Eu or other lanthanide metals. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various fragments to an antibody are well known; see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), p. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., Antibodies For Drug Delivery, in: Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Marcel Dekker, Inc. ., p. 623-53 (1987) Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), p. -506 (1985) Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), Academic Press, p.303 -16 (1985); Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev. (52:119-58(1982)).

bifunctional molecules.

PD-L1 is an immune response checkpoint molecule and also a tumor antigen. To obtain a bispecific antibody, a PD-L1 specific antibody or

- 19 043231 specific for PD-L1 antigennegative fragment can be combined(o), as a tumor antigen targeting molecule, with a second antigennegative fragment specific for the immune cell.

In some embodiments, said immune cell is selected from the group consisting of T cell, B cell, monocyte, macrophage, neutrophil, dendritic cell, phagocyte, natural killer cell, eosinophil, basophil, and mast cell. Molecules suitable for targeting on an immune cell include, for example, CD3, CD16, CD19, CD28 and CD64. Other examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS /ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptors (KIR) and CD47. Specific examples of bispecificity include, without limitation, PD-L1/PD-1, PD-L1/LAG3, PD-L1/TIGIT, and PD-L1/CD47.

An antibody or antigen-binding fragment specific for PD-L1 can be combined, as an immune checkpoint inhibitor, with a second antigen-binding fragment specific for a tumor antigen to produce a bispecific antibody. A tumor antigen is an antigenic substance produced in tumor cells, ie. it triggers an immune response in the host. Tumor antigens are suitable for identifying tumor cells and are potential candidates for use in cancer therapy. Normal proteins in the body are not antigenic. Certain proteins, however, are produced or overexpressed during tumorigenesis and therefore appear foreign to the body.

These proteins may include well sequestered normal proteins inaccessible to the immune system, proteins normally produced in extremely low amounts, proteins normally produced only at certain developmental stages, or proteins whose structure has been modified due to mutation.

Many tumor antigens are known in the art, and new tumor antigens can be readily identified by screening. Non-limiting examples of tumor antigens include rEGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR (VEGF receptor), integrin, aVe3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin.

In some aspects, the monovalent unit has specificity for a protein that is overexpressed on a tumor cell relative to a corresponding non-tumor cell. The corresponding non-tumor cell herein refers to a non-tumor cell belonging to the same cell type as the original type for the tumor cell. Note that such proteins are not necessarily distinct from tumor antigens. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most carcinomas of the colon, rectum, breast, lung, pancreas, and gastrointestinal tract; heregulin receptors (HER-2, neu, or c-erbB-2), often overexpressed in breast, ovarian, colon, lung, prostate, and cervical cancers; epidermal growth factor receptor (eGF), highly expressed in a number of solid tumors, including tumors of the breast, head and neck, non-small cell lung and prostate cancer; asialoglycoprotein receptor; transferrin receptor; serpin-enzyme complex receptor expressed on hepatocytes; fibroblast growth factor receptor (FGFR) overexpressed on pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptor (VEGFR) for anti-angiogenic gene therapy; folic acid receptor, selectively overexpressed in 90% of non-cystic ovarian carcinomas; cell surface glycocalyx; carbohydrate receptors; and a polymeric immunoglobulin receptor suitable for gene delivery to airway epithelial cells and attractive for the treatment of lung diseases such as cystic fibrosis. Non-limiting examples of bispecificity in this case include PD-ShrEGFR, PD-L1/Her2, PD-L1/CD33, PD-L1/CD133, PD-LI/CEA, and PD-LI/VEGF.

Bispecific antibodies of another format have also been proposed. In some embodiments, the anti-PD-L1 fragment and the second fragment are both independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, said bispecific antibody further comprises an Fc fragment.

Also provided are bifunctional molecules that include more than just an antibody or antigen-binding fragment. An antibody or antigen binding fragment specific for PD-L1, such as described herein, can be combined as a tumor antigen targeting molecule with an immune cytokine or ligand, optionally via a peptide linker. Associated immune cytokines or ligands include, but are not limited to

- 20 043231 listed, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF -α,

CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bifunctional molecules may provide a combination of immune response checkpoint-blocking effect and local immunomodulation at the tumor site.

Polynucleotides encoding antibodies and methods for producing antibodies.

The present invention also provides isolated polynucleotides or nucleic acid molecules (eg, SEQ ID NOs: 34-60, 112 and 114) encoding antibodies, variants or derivatives thereof, as described herein. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof, on a single polynucleotide molecule or on individual polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof, on a single polynucleotide molecule or on separate polynucleotide molecules.

Methods for producing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptides described herein are fully human. Fully human antibodies can be obtained using techniques known in the art and described in this document. For example, fully human antibodies against a particular antigen can be generated by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, with its endogenous loci deactivated. Examples of techniques that can be used to obtain such antibodies are described in US patents 6150584; 6458592; 6420140, which are incorporated herein in their entirety by reference.

In some embodiments, the resulting antibodies do not elicit a detrimental immune response in the animal being treated, such as a human. In one embodiment, the antigen-binding polypeptides, variants or derivatives thereof, as described herein, are modified to reduce immunogenicity using techniques known in the art. For example, antibodies can be humanized, primatized, deimmunized; or chimeric antibodies can be generated. These types of antibodies are derived from a non-human antibody, typically a mouse or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but is less immunogenic in humans. This can be achieved in a variety of ways, including by (a) grafting complete non-human derived variable domains onto human constant regions to generate chimeric antibodies;

(b) grafting at least a portion of one or more non-human derived complementarity determining regions (CDRs) onto human framework and constant regions, with or without retaining critical framework residues; or by (c) transplantation of complete non-human derived variable domains, but masking them with a human-like region by replacing surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al. Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994); and US Pat. Nos. 5,585,089; 5,693,761;

Deimmunization may also be used to reduce the immunogenicity of an antibody. As used herein, the term deimmunization includes modifying an antibody to modify T cell epitopes (see, for example, WO/9852976 A1 and WO/0034317 A2). For example, the heavy chain variable sequences and light chain variable sequences of the parent antibody are analyzed, and a map of human T cell epitopes from each V region is generated, reflecting the location of the epitopes relative to complementarity determining regions (CDRs) and other key residues in the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with a low risk of altering the activity of the resulting antibody. A set of alternative heavy and light chain variable region sequences containing combinations of amino acid substitutions is designed and these sequences are then incorporated into a set of binding polypeptides. As a rule, get and test the binding and/or function of 12-24 variants of antibodies. Thereafter, complete heavy and light chain genes containing modified human variable and constant regions are cloned into expression vectors, and the resulting plasmids are introduced into cell lines to obtain a full-length antibody. These antibodies are then compared by appropriate biochemical and biological assays and the optimal variant is identified.

The binding specificity of antigen-binding polypeptides as described herein can be determined using in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

- 21 043231

As an option, the described methods for obtaining single-stranded units (US patent No. 4694778; Bird, Science, 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci., USA, 55:5879-5883 (1988 ); and Ward et al., Nature, 334:544-554 (1989)) can be adapted to produce single stranded units as described herein. Single chain units are formed by linking heavy and light chain fragments of the Fv region through an amino acid bridge to form a single chain fusion peptide. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science, 242:1038-1041 (1988)).

Examples of techniques that can be used to obtain single-chain Fv (scFv) and antibodies include those described in US patent No. 4946778 and No. 5258498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science, 240:1038-1040 (1988). For some applications, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric antibodies, humanized antibodies, or human antibodies. A chimeric antibody is a molecule in which different parts of an antibody are derived from different animal species, for example, antibodies containing a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science, 229:1202 (1985); Oi et al., BioTechniques, 4:214 (1986); Gillies et al., J. Immunol. Methods, 125:191-202 (1989); US patents No. 5807715; No. 4816567; and No. 4816397, which are incorporated herein in their entirety by reference.

Humanized antibodies are antibody molecules derived from an antibody derived from a non-human species that bind the desired antigen, containing one or more complementarity determining regions (CDRs) from a non-human species, and framework regions from a human immunoglobulin molecule. Frequently, framework residues in human framework regions are substituted with the corresponding CDR residue of the donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified using methods well known in the art, for example, modeling interactions between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparisons to identify unusual framework residues at specific positions. (See, for example, sources Queen et al., US patent No. 5585089; Riechmann et al., Nature, 332:323 (1988), which are incorporated herein in their entirety by reference). Antibodies can be humanized using various techniques known in the art including, for example, CDR grafting (EP 239400; PCT Publication WO 91/09967; US Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), masking, or surface modification. (EP 592106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska. et al ., Proc. Natl. Sci., USA, 91:969-973 (1994)), and strand permutation (US Pat. No. 5,565,332, which is incorporated herein by reference in its entirety).

The use of fully human antibodies is particularly desirable for the therapeutic treatment of human patients. Human antibodies can be generated by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also US Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; each of these references is incorporated herein in its entirety by reference.

Human antibodies can also be generated using transgenic mice incapable of expressing functional endogenous immunoglobulins but capable of expressing human immunoglobulin genes. For example, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells along with the human heavy and light chain genes. Mouse immunoglobulin heavy and light chain genes can be defunctionalized, either alone or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, a homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. These chimeric mice are then bred to produce homozygous offspring that express human antibodies. These transgenic mice are immunized in the usual way with the selected antigen, for example, the entire required target polypeptide or part thereof. Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using standard hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation and then go through class switching and somatic mutagenesis. Accordingly, the use of such a technique allows to obtain therapeutically useful antibodies IgG, IgA, IgM and IgE. About

- 22 043231 Detailed information regarding this technology for the production of human antibodies can be found in Lonberg and Huszar, Int. Rev. Immunol., 73:65-93 (1995). A detailed description of said technology for the production of human antibodies and human monoclonal antibodies, as well as protocols for the production of such antibodies, can be found, for example, in PCT publications WO 98/24893; W096/34096; WO 96/33735; US Pat. No. 5,413,923; No. 5625126; 5633425; 5569825; 5661016; 5545806; 5814318; and 5939598, which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, Inc. (Fremont, Calif.) and GenPharm (San Jose, Calif.) can be used to generate human antibodies directed against a selected antigen using a technique similar to that described above.

Fully human antibodies that recognize the selected epitope can also be obtained using a technique called guided selection. In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., Bio/Technology, 72:899-903 (1988). See also US Pat. No. 5,565,332, which is incorporated by reference in its entirety).

In another embodiment, DNA encoding the desired monoclonal antibodies can be easily isolated and sequenced using standard procedures (eg, by using oligonucleotide probes that are capable of specifically binding to genes encoding mouse antibody heavy and light chains). Isolated and subcloned hybridoma cells serve as the preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, COS monkey cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. More specifically, isolated DNA (which may be synthetic as described herein) can be used to clone constant and variable region sequences to generate antibodies as described in US Patent No. 5,658,570 (Newman et al.), filed Jan. included by reference. Essentially, this involves extracting RNA from selected cells, converting to cDNA, and amplifying by PCR using Ig-specific primers. Suitable primers for this purpose are also described in US Pat. No. 5,658,570. As discussed in more detail below, transformed cells expressing the desired antibody can be grown in relatively large numbers to provide clinical and commercial immunoglobulin supplies.

In addition, one or more CDR regions of the antigen-binding polypeptides of the present disclosure may be inserted, using conventional recombinant DNA techniques, into framework regions, eg human framework regions, to humanize the non-human antibody. Framework regions can be represented by naturally occurring or consensus framework regions, preferably human α-framework regions (see, for example, a list of human framework regions in Chothia et al., J. Mot Biol., 278:457-479 (1998)) . Preferably, the polynucleotide obtained by combining the framework regions and the CDRs encodes an antibody that specifically binds to at least one epitope of the desired polypeptide, eg, LIGHT. Preferably, one or more amino acid substitutions can be introduced into the framework regions, and preferably said amino acid substitutions improve the binding of said antibody to its antigen. In addition, such methods can be used to produce amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bond formation, resulting in antibody molecules lacking one or more intrachain disulfide bonds. Other polynucleotide modifications are also contemplated by the present invention and are known in the art.

In addition, techniques developed for the production of chimeric antibodies can be used (Morrison et al., Proc. Natl. Acad. Sci., USA, 851-855 (1984); Neuberger et al., Nature, 372:604-608 (1984 ); Takeda et al., Nature, 314:452-454 (1985)) by splicing genes from a mouse antibody molecule with suitable antigen specificity together with genes from a human antibody molecule with suitable biological activity. As used herein, a chimeric antibody is a molecule whose different parts are derived from different animal species, for example, molecules containing a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

Another highly efficient method for producing recombinant antibodies is described in Newman, Biotechnology, 10:1455-1460 (1992). In particular, this technique provides for the production of primatized antibodies that contain simian variable domains and human constant sequences. The cited source is incorporated herein by reference in its entirety. In addition, this technique is also described in U.S. Patents Nos. 5,658,570, 5,693,780, and 5,756,096 owned by the same owner, each of which is incorporated herein by reference.

- 23 043231

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in various laboratory manuals and major publications. However, methods suitable for the use of the present invention as described below are provided in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates and Wiley-Interscience, New York (1991), which is incorporated herein in its entirety by links, including attachments.

In addition, standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR- L1, CDR-L2 or CDR-L3. Alternatively, mutations can be introduced randomly throughout all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

Cancer treatment.

As described herein, the antibodies, variants or derivatives thereof of the present invention may be used in certain therapeutic and diagnostic methods.

The present invention also relates to antibody-based therapy, comprising the introduction of antibodies according to the present description to a patient, for example, an animal, a mammal and a human, for the treatment of one or more of the disorders or conditions described herein. Therapeutic compounds according to the present description include, but are not limited to, antibodies according to the present description (including their variants and derivatives as described herein), as well as nucleic acids or polynucleotides encoding antibodies according to the present description (including their variants and derivatives). as described in this document).

Antibodies according to the present description can also be used to treat or inhibit cancer. PD-L1 may be overexpressed in tumor cells. Tumor-derived PD-L1 can bind to PD-1 on immune cells, thus limiting antitumor T-cell immunity. The results of using small molecule inhibitors or monoclonal antibodies targeting PD-L1 in mouse tumor models show that PD-L1-targeted therapy is an important alternative and a realistic approach to effectively control tumor growth. As demonstrated in experimental examples, anti-PD-L1 antibodies activated adaptive immune response mechanisms, which may lead to improved survival in cancer patients.

Accordingly, in some embodiments, methods are provided for treating cancer in a patient in need thereof. Said method, in one embodiment, comprises administering to said patient an effective amount of an antibody as described herein. In some embodiments, at least one of the cancer cells (eg, stromal cells) in the patient expresses, overexpresses, or is induced to express PD-L1. Induction of the expression of PD-L1, for example, can be carried out by the introduction of a tumor vaccine or radiation therapy.

Tumors that express the PD-L1 protein include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colon and rectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, and gastrointestinal cancer. intestinal tract, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer and small cell lung cancer. Accordingly, the antibodies of the present invention may be used to treat any one or more of these cancers.

Also according to the present invention proposed cellular therapy, for example, therapy using T-cell chimeric antigen receptors (CAR). A suitable cell may be used that is contacted with an anti-PD-L1 antibody as described herein (or alternatively engineered to express an anti-PD-L1 antibody as described herein). Following such contact or construction, the cell may be appropriately administered to a cancer patient in need of treatment. Said cancer patient may have any of the types of cancer described herein. The cell (eg, T cell) may be, for example, a tumor infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination of these cells, without limitation.

In some embodiments, said cell has been isolated from the body of the cancer patient itself. In some embodiments, said cell was obtained from a donor or cell bank. If the cell is isolated from a cancer patient, unwanted immune responses can be minimized.

Additional diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and/or predicted using antibodies or variants or derivatives thereof as described herein include, but are not limited to, progression and/or metastasis of malignant neoplasms and related disorders such as leukemia (including acute leukemias (eg, acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia)) and chronic leukemias (eg, chronic myelocytic (granulocytic) ) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer glands, breast cancer, thyroid cancer, cancer endometrium, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pin ealoma, hemangioblastoma , acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

Combination therapy options.

In a further embodiment, the compositions of the present disclosure are administered in combination with an antineoplastic agent, an antiviral agent, an antibacterial or antibiotic agent, or antimycotic agents. Any of these agents known in the art may be incorporated into the compositions of the present invention.

According to another implementation variant of the composition according to the present description is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with compositions as described herein include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogen (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5-FU, methotrexate, floxuridine, interferon alfa-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (eg medroxyprogesterone, estramustine sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlortrianisene and testolactone); mustargen derivatives (eg mephalen, chlorambucil, mechlorethamine (mustargen) and thiotepa); steroids and their combinations (for example, betamethasone sodium phosphate); and others (eg, dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

According to an additional implementation variant of the composition according to the present description is administered in combination with cytokines. Cytokines that can be administered with compositions according to the present description include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-10, IL-12, IL- 13, IL-15, anti-CD40, CD40L and TNF-α.

In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens such as, for example, radiation therapy.

Combination therapy options are also provided that include the use of one or more of the anti-PD-L1 antibodies as described herein along with a second anti-cancer (chemotherapeutic) agent. Chemotherapeutic agents can be classified according to their mechanism of action, for example, into the following groups:

antimetabolites/anticancer agents such as the pyrimidine analogs floxuridine, capecitabine and cytarabine;

purine analogs, folate antagonists and related inhibitors;

antiproliferative/antimitotic agents, including natural products such as vinca alkaloid (vinblastine, vincristine) and microtubule acting agents such as taxane (paclitaxel, docetaxel), vinblastine, nocodazole, epothilones, vinorelbine (NAVELBIN®), and epipo - 25 043231 dophyllotoxins (etoposide, teniposide);

DNA damaging agents such as actinomycin, amsacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide (Cytoxan, CYTOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea ina, procarbazine, taxol, taxotere, teniposide, etoposide and triethylenethiophosphoramide;

antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mitramycin) and mitomycin;

enzymes such as L-asparaginase, which systemically metabolizes L-asparagine and deprives it of cells that do not have the ability to synthesize their own asparagine;

antiplatelet agents;

antiproliferative/antimitotic alkylating agents such as the mustargens cyclophosphamide and analogs (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkylnitrosoureas (carmustine) and analogs, streptozocin and triazenes (dacarbazine);

antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate);

platinum coordination complexes (cisplatin, oxaliplatin and carboplatin), procarbazine, hydroxyurea, mitotane and aminoglutethimide;

hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide and nilutamide), and aromatase inhibitors (letrozole and anastrozole);

anticoagulants such as heparin, synthetic heparin salts and other thrombin inhibitors;

fibrinolytic agents such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine and clopidogrel;

anti-migration agents;

antisecretory agents (brefeldin);

the immunosuppressive agents tacrolimus, sirolimus, azathioprine, and mycophenolate;

compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors);

angiotensin receptor blockers, nitric oxide donors;

antisense oligonucleotides;

antibodies such as trastuzumab and rituximab;

cell cycle inhibitors and differentiation inducers such as tretinoin;

inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone);

growth factor signaling kinase inhibitors;

inducers of dysfunction;

toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase activators; and chromatin.

Additional examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);

alkylsulfonates such as busulfan, improsulfan and piposulfan;

aziridines such as benzodopa, carbokwon, meturedopa and uredopa;

ethyleneamines and methyleneamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine;

acetogenins, in particular bullatacin and bullatacinone;

camptothecin, including a synthetic analogue of topotecan;

bryostatin;

callistatin;

CC-1065, including its synthetic analogues adoselesin, carzelesin and bizelesin;

cryptophycins, in particular cryptophycin 1 and cryptophycin 8;

dolastatin;

duocarmycin, including synthetic analogues of KW-2189 and CBI-TMI;

eleutherobin;

pancratistatin;

sarcodictyin;

spongistatin;

mustargens such as chlorambucil, chlornaphasine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide and uramustine;

nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine;

- 26 043231 antibiotics such as enediyne antibiotics (for example, calicheamycin, in particular calicheamycin gamma-II and calicheamicin phiI1), dynemycin, including dynemycin A, bisphosphonates such as clodronate, esperamycin, chromophore neocarzistatin, and related enediine antibiotic chromophore chromoproteins, aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, sactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino -doxorubicin , 2-pyrrolin-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex , zinostatin and zorubicin;

antimetabolites such as methotrexate and 5-fluorouracil (5-FU);

folic acid analogs such as demopterin, methotrexate, pteropterin and trimetrexate;

purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin and thioguanine;

pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine and floxuridine;

androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane and testolactone;

adrenal depressants such as aminoglutethimide, mitotane and trilostane;

folic acid supplements such as frolinic acid;

trichothecins, in particular T-2 toxin, verracurin A, roridin A and anhidine;

taxoids such as paclitaxel (TAXOL®) and docetaxel (TAXOTHER®);

platinum analogues such as cisplatin and carboplatin;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcin; diaziquon; eflornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; losoxantrone; fluoropyrimidine; folinic acid; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; chlorambucil;

gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (navelbin, NAVELBINE®); novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin and irinotecan); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Also encompassed by the definition of a chemotherapeutic agent are antihormonal agents such as antiestrogen and selective estrogen receptor modulators (SERMs), aromatase enzyme inhibitors, antiandrogens, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing that regulate or inhibit the action of hormones on tumors.

Examples of antiestrogens and SERMs include, for example, tamoxifen (including Nolvadex, NOLVADEX™), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapriston, and toremifene (Fareston, FARESTON®).

Aromatase enzyme inhibitors regulate the production of estrogen by the adrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megeis, MEGACE®), exemestane, formestane, fadrozole, vorozol (RIVISOR, RIVISOR®), letrozole (Femara, FEMARA®), and anastrozole (Arimidex, ARIMIDEX®).

Examples of antiandrogens include flutamide, nilutamide, bicalutamide, leuprolide, and goserelin.

Examples of chemotherapeutic agents also include anti-angiogenic agents, including, but not limited to, retinoic acid and its derivatives, 2-methoxyestradiol, angiostatin (ANGIOSTATIN®), endostatin (ENDOSTATIN®), suramin, squalamine, tissue inhibitor of metalloproteinase-1, tissue metalloproteinase-2 inhibitor, plasminogen activator-1 inhibitor, plasminogen activator-2 inhibitor, cartilage inhibitor, paclitaxel (nab-paclitaxel), platelet factor 4, protamine sulfate (lupein), sulfated derivatives of chitin (derived from snow crab shells), complex sulfated polysaccharides with peptidoglycans (sp-pg), staurosporine, matrix metabolism modulators, including proline analogs ((1-acetidine2-carboxylic acid (LACA)), cishydroxyproline, d,I-3,4-dehydroproline, thiaproline, α, α'-dipyridyl, beta-aminopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3N)-oxazolone, methotrexate, mitoxantrone, heparin, interferons, serum 2-macroglobulin, chicken metalloproteinase-3 inhibitor (ChIMP-3 ), chymostatin, beta-cyclodextrintetradecasulfate, eponemycin, fumagillin, aurothiomalate

- 27 043231 sodium, d-penicillamine, serum beta-1-anticollagenase, alpha-2-antiplasmin, bisantrene, disodium lobenzarite, n-2-carboxyphenyl-4-chloroanthranilic acid disodium salt, or CCA, thalidomide, angiostatic steroid, carboxyaminoimidazole and metalloproteinase inhibitors such as BB-94. Other anti-angiogenic agents include antibodies, preferably monoclonal antibodies, against these angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, isoforms of VEGF, VEGF-C, FRG/SF and Ang-1/Ang-2.

Examples of chemotherapeutic agents also include antifibrotic agents, including but not limited to compounds such as beta-aminopropionitrile (BAPN) as well as compounds disclosed in U.S. Patent No. 4,965,288 (Palfreyman, et al.) relating to lysyl oxidase inhibitors. and their use in the treatment of diseases and conditions associated with abnormal collagen deposition and in US Pat. Additional examples of inhibitors are described in US Pat. 5059714 (Palfreyman et al.), no. 5120764 (McCarthy et al.), no. 5182297 (Palfreyman et al.), no. and US Publication No. 2004/0248871 (Farjanel et al.), which are incorporated herein by reference.

Examples of antifibrotic agents also include primary amines that react with the carbonyl group of the active site of lysyl oxidases, and more specifically those that produce, after binding to the carbonyl, a resonance-stabilized product, such as the following primary amines: ethylenediamine, hydrazine, phenylhydrazine, and their derivatives; semicarbazide and urea derivatives; aminonitriles such as BAPN or 2-nitroethylamine; unsaturated or saturated haloamines such as 2-bromoethylamine, 2-chloroethylamine, 2-trifluoroethylamine, 3-bromopropylamine and p-halobenzylamines; and selenohomocystein lactone.

Other antifibrotic agents are copper chelating agents, cell-penetrating or non-penetrating. Examples of compounds include indirect inhibitors that block aldehyde derivatives obtained by oxidative deamination of lysyl and hydroxylysyl residues with lysyl oxidases. Examples include thiolamines, in particular D-penicillamine and its analogues such as 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3methyl-3-((2-acetamidoethyl)dithio)butanoic acid, p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid, sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butanesulfurate, 2-acetamidoethyl -2-acetamidoethanethiolsulfanate and sodium-4-mercaptobutanesulfinate trihydrate.

Examples of chemotherapeutic agents also include, but are not limited to, immunotherapeutic agents, including therapeutic antibodies suitable for the treatment of patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumab, bavituximab, bektumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citutuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab , drozitumab, duligotumab, duzigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, encituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritutomab, igovomab, imgatuzumab, indatuximab, inotuzumab , intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumumab, mitumomab, moxetumumab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab , ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab , panitumumab, parsatuzumab, patritumab, pemtumumab, pertuzumab, pintumumab, pritumumab, racotumomab, radretumumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, takatuzumab, taplitumomab, tenatumomab, teprotu mumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab , veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8. Rituximab can be used to treat indolent B-cell cancers, including marginal zone lymphoma, Waldenström macroglobulinemia (WM), CLL, and small lymphocytic lymphoma. In particular, the combination of rituximab and chemotherapy agents is effective.

These examples of therapeutic antibodies can be further labeled or combined with a radioisotope particle such as indium-111, yttrium-90, or iodine-131.

In one embodiment, the additional therapeutic agent is an alkylating agent, mustargen. Non-limiting examples of mustargen alkylating agents include chlorambucil.

In one embodiment, the compounds and compositions described herein may be used together or combined with one or more additional therapeutic agents. These one or more therapeutic agents include, but are not limited to, Abl inhibitor, activated CDC kinase (ACK), A2B adenosine receptor

- 28 043231 (A2B), apoptosis signal regulating kinase (ASK), aurora kinase, Bruton's tyrosine kinase (BTK), BET bromodomain (BRD) such as BRD4, c-Kit, c-Met, CDK-activating kinase (CAK ), calmodulin-dependent protein kinase (CaMK), cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domain receptor (DDR), epidermal growth factor receptors (eGF), focal adhesion kinase (FAK), Flt-3, FYN , glycogen synthase kinase (GSK), HCK, histone deacetylase (HDAC), IKK such as IKKβε, isocitrate dehydrogenase (IDH) such as IDH1, Janus kinase (JAK), KDR, lymphocyte-specific tyrosine protein kinase (LCK), lysyl oxidase-like protein protein (LOXL), LYN, matrix metalloproteinase (MMP), MEK, mitogen-activated protein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor (TPF), phosphorylase kinase (PK), Polo-like kinase ( PLK), phosphatidylinositol 3-kinase (PI3K), protein kinase (PK) such as protein kinase A, B and/or C, PYK, spleen tyrosine kinase (SYK), TPL2 serine/threonine kinase, STK serine/threonine kinase, transfer factor signal and transcription (STAT), SRC, serine/threonine protein kinase (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascular endothelial growth factor receptor (VEGFR), YES, or any combination of the above.

ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors include but are not limited to those described in WO 2011/008709 (Gilead Sciences) and WO 2013/112741 (Gilead Sciences).

Examples of BTK inhibitors include, but are not limited to, ibrutinib, HM71224, ONO-4059, and CC-292.

DDR inhibitors include DDR1 and/or DDR2 inhibitors. Examples of DDR inhibitors include, but are not limited to, those disclosed in WO 2014/047624 (Gilead Sciences), US 2009/0142345 (Takeda Pharmaceutical), US 2011/0287011 (Oncomed Pharmaceuticals), WO 2013/027802 (Chugai Pharmaceutical) and WO 2013/ 034933 (Imperial Innovations).

Examples of HDAC inhibitors include, but are not limited to, pracinostat and panobinostat.

JAK inhibitors inhibit JAK1, JAK2 and/or JAK3. Examples of JAK inhibitors include, but are not limited to, filgotinib, ruxolitinib, fedratinib, tofacitinib, baricitinib, lestaurtinib, pacritinib, XL019, AZD1480, INCB039110, LY2784544, BMS911543, and NS018.

LOXL inhibitors include LOXL1, LOXL2, LOXL3, LOXL4 and/or LOXL5 inhibitors. Examples of LOXL inhibitors include, but are not limited to, the antibodies described in WO 2009/017833 (Arresto Biosciences).

Examples of LOXL2 inhibitors include, but are not limited to, the antibodies described in WO 2009/017833 (Arresto Biosciences), WO 2009/035791 (Arresto Biosciences) and WO 2011/097513 (Gilead Biologies).

MMP inhibitors include MMP1-10 inhibitors. Examples of MMP9 inhibitors include, but are not limited to, marimastat (BB-2516), cypemastat (Ro 32-3555), and those described in WO 2012/027721 (Gilead Biologics).

PI3K inhibitors include PI3Ky, PI3Kδ, PI3Ke, PI3Ka and/or pan-PI3K inhibitors. Examples of PI3K inhibitors include, but are not limited to, wortmannin, BKM120, CH5132799, XL756, and GDC-0980.

Examples of PI3Ky inhibitors include, but are not limited to, ZSTK474, AS252424, LY294002, and TG100115.

Examples of PI3Kδ inhibitors include, but are not limited to, PI3K II, TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443, and the compounds described in WO 2005/113556 (ICOS), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO 2014/100765 (Gilead Calistoga), WO 2014/100767 (Gilead Calistoga) and WO 2014/20 1409 ( Gilead Sciences).

Examples of PI3Ke inhibitors include, but are not limited to, GSK2636771, BAY 10824391, and TGX221.

Examples of PI3Ka inhibitors include, but are not limited to, buparlisib, BAY 806946, BYL719, PX-866, RG7604, MLN1117, WX-037, AEZA-129, and PA799.

Examples of pan-PI3K inhibitors include, but are not limited to, LY294002, BEZ235, XL147 (SAR245408), and GDC-0941.

Examples of SYK inhibitors include, but are not limited to, tamatinib (R406), fostamatinib (R788), PRT062607, BAY-61-3606, NVP-QAB 205 AA, R112, R343; and described in US patent No: 8450321 (Gilead Connecticut).

TKIs can target epidermal growth factor receptors (eGF) and fibroblast growth factor (FGF), platelet-derived growth factor (TPF), and vascular endothelial growth factor (VEGF) receptors. Examples of TKIs targeting rEGFR include, but are not limited to, gefinitib and erlotinib. Sunitinib is a non-limiting example of a TKI targeting FGF, TRF, and VEGF receptors.

Anti-PD-L1 antibodies according to the present description can be used, according to some

- 29 043231 embodiments, together with an immune checkpoint inhibitor. Immune response checkpoints are immune system molecules that either turn on a signal (co-stimulatory molecules) or turn off a signal (coinhibitor molecules). Many cancers are protected from the immune system by inhibiting T-cell signaling through an agonist for co-inhibitor molecules or an antagonist for co-stimulatory molecules. An agonist or antagonist of immune response checkpoints can help stop such a defense mechanism in cells. The immune checkpoint agonist or antagonist can target any one or more of the following checkpoint molecules: PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40/OX40L, CD40/CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272).

Programmed T cell death 1 (PD-1) protein is a transmembrane protein found on the surface of T cells, binding of which to the programmed T cell death ligand 1 (PD-L1) on tumor cells results in downregulation of T cell activity and mediated T cell cytotoxicity. Accordingly, PD-1 and PD-L1 are down-regulators of the immune system, or immune response checkpoint switches. Examples of PD-1 inhibitors include, without limitation, nivolumab, (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001, MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C .

CTLA-4 is a protein receptor that down-regulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206).

Lymphocyte activation gene 3 (LAG-3) is a cell surface immune response checkpoint receptor that functions to suppress the immune response through its action on Treg cells as well as direct effects on CD8+ T cells. LAG-3 inhibitors include, without limitation, LAG525 and BMS-986016.

CD28 is expressed constitutively on almost all human CD4+ T cells and on about half of all CD8 T cells, causing T cell proliferation. Non-limiting examples of CD28 inhibitors include TGN1412.

CD122 increases the proliferation of CD8+ effector T cells. Non-limiting examples include NKTR-214.

4-1BB (also known as CD137) is involved in T cell proliferation. CD137 mediated signaling is also known to protect T cells, and in particular CD8+ T cells, from activation-induced cell death. Examples of CD137 inhibitors are PF-05082566, urelumab (BMS-663513) and lipocalin.

When using any of the combination treatment options listed above, the anti-PD-L1 antibody can be administered simultaneously with or separately from another anticancer agent. When administered alone, the anti-PD-L1 antibody may be administered before or after another anti-cancer agent.

Treatment of infection.

As demonstrated in the experimental examples, antibodies according to the present description can activate an immune response, which, accordingly, can be useful in the treatment of infection.

Infection is the invasion of the tissues of the body by disease-causing agents, their reproduction and the reaction of the host tissues to these organisms and the toxins produced by them. Infection can be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks and ticks, fleas and lice, fungi such as trichophytosis pathogens, and other macroparasites. such as tapeworms and other helminths. In one aspect, said infectious agent is a bacterium, such as a Gram-negative bacterium. In one aspect, said infectious agent is a virus, such as DNA viruses, RNA viruses, and reverse transcription viruses. Non-limiting examples of viruses include adenovirus, Coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, HIV, influenza virus , measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella zoster virus.

Antibodies of the present disclosure may also be used to treat an infectious disease caused by a microorganism or to kill the microorganism by targeting said microorganism and an immune cell to effect elimination of said microorganism. In one aspect, said microorganism is a virus, including RNA and DNA viruses, a gram positive bacterium, a gram negative bacterium, a protozoan, or a fungus. Non-limiting examples of infectious diseases and related microorganisms are given in table. 4 below.

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Table 4

Infectious diseases and related microorganism sources

Infection Source microorganism Acinetobacter infections Acinetobacter baumannii Actinomycosis Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus African sleeping sickness (African trypanosomiasis) Trypanosoma brucei AIDS (Acquired Immune Deficiency Syndrome) HIV (Human Immunodeficiency Virus) Amoebiasis Entamoeba histolytica Anaplasmosis Genus Anaplasma anthrax Bacillus anthracis Arcanobacterium haemolyticum infection Arcanobacterium haemolyticum Argentine hemorrhagic fever Junin virus Ascariasis Ascaris lumbricoides Aspergillosis Genus Aspergillus Astrovirus infection Family Astroviridae babesiosis Genus Babesia Bacillus cereus infection Bacillus cereus bacterial pneumonia Row of bacteria Bacterial vaginosis (BV) Row of bacteria Bacteroides infection Genus Bacteroides Balantidiasis Balantidium coli Baylisascaris infection Genus Baylisascaris VK virus infection VK virus black piedra Piedraia hortae Blastocystis hominis infection Blastocystis hominis Blastomycosis Blastomyces dermatitidis Bolivian hemorrhagic fever Machupo virus Borrelia infection Genus Borrelia Botulism (and infant botulism) Clostridium botulinum Brazilian hemorrhagic fever Sabia virus Brucellosis Genus Brucella Burkholderia infection Typically Burkholderia cepacia and other Burkholderia species Buruli ulcer Mycobacterium ulcerans Calicivirus (Norovirus and Sapovirus) infection Family Caliciviridae campylobacteriosis Genus Campylobacter Candidiasis (moniliosis; thrush) Typically Candida albicans and other Candida species cat scratch disease Bartonella henselae Cellulite As a rule, Streptococcus from group A and Staphylococcus Chagas disease (American trypanosomiasis) Trypanosoma cruzi chancroid Haemophilus ducreyi Chicken pox Varicella zoster virus (VZV) Chlamydia Chlamydia trachomatis Chlamydophila pneumoniae infection Chlamydophila pneumoniae Cholera Vibrio cholerae Chromoblastomycosis As a rule, Fonsecaea pedrosoi Clonorchiasis Clonorchis sinensis Clostridium difficile infection Clostridium difficile coccidioidomycosis Coccidioides immitis and Coccidioides posadasii Colorado tick fever (CTF) Colorado tick fever virus (CTFV)

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Colds (acute viral nasopharyngitis; coryza) As a rule, rhinoviruses and coronaviruses. Creutzfeldt-Jakob disease (CJD) Prion CJD Crimean-Congo hemorrhagic fever (CCHF) Crimean-Congo hemorrhagic fever virus Cryptococcosis Cryptococcus neoformans Cryptosporidiosis Genus Cryptosporidium Larva migrans syndrome, cutaneous form (CLM) As a rule, Ancylostoma braziliense; a number of other parasites Cyclosporiasis Cyclospora cayetanensis Cysticercosis Taeniasolium Cytomegalovirus infection Cytomegalovirus Dengue fever Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) Flaviviruses Dientamobiasis Dientamoeba fragilis Diphtheria Corynebacterium diphtheriae Diphyllobothriasis Diphyllobothrium Dracunculiasis Dracunculus medinensis Ebola hemorrhagic fever Ebolavirus (EBOV) Echinococcosis Genus Echinococcus erlichiosis Genus Ehrlichia Enterobiasis (pinworm infection) Enterobius vermicularis Enterococcal infection Genus Enterococcus Enteroviral infection Genus Enterovirus epidemic typhus Rickettsia prowazekii Erythema infectiosum (fifth disease) Parvovirus B19 Sudden exanthema (sixth disease) Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7) Fasciolopsiosis Fasciolopsis buski Fascioliasis Fasciola hepatica and Fasciola gigantica Fatal familial insomnia (FFI) Prion FFI Filariasis Superfamily Filarioidea Clostridium perfringens food poisoning Clostridium perfringens Free-living amoeba infection A number of pathogens Fusobacterial infection Genus Fusobacterium Gas gangrene (clostridial muscle necrosis) As a rule, Clostridium perfringens; other Clostridium species Geotrichosis Geotrichum candida Gerstmann-Straussler Scheinker Syndrome (GSS) Prion GSS Giardiasis Giardia intestinalis Glanders Burkholderia mallei Gnathostomiasis Gnathostoma spinigerum and Gnathostoma hispidum Gonorrhea Neisseria gonorrhoeae Inguinal granuloma (donovanosis) Klebsiella granulomatis Streptococcal infection, group A Streptococcus pyogenes Streptococcal infection, group B Streptococcus agalactiae Haemophilus influenzae infection haemophilus influenzae

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Hand-foot-mouth syndrome (HFMD) Enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71) Hantavirus Pulmonary Syndrome (HPS) Virus Sin Nombre Helicobacter pylori infection Helicobacter pylori Hemolytic uremic syndrome (HUS) Escherichia coli O157.H7, 0111 ή. Ο104:Η4 Hemorrhagic fever with renal syndrome (HFRS) Family Bunyaviridae Hepatitis A Hepatitis A virus Hepatitis B Hepatitis B virus Hepatitis C Hepatitis C virus Hepatitis D hepatitis D virus Hepatitis E Hepatitis E virus Herpes simplex Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) Histoplasmosis Histoplasma capsulatum hookworm infection Ancylostoma duodenale and Necator americanus Bocavirus infection in humans Human bocavirus (HBoV) Human ehrlichiosis caused by Ehrlichia ewingii Ehrlichia ewingii Human granulocytic anaplasmosis (HGA) Anaplasma phagocytophilum human metapneumovirus infection Human metapneumovirus (hMPV) Human monocytic ehrlichiosis Ehrlichia chaffeensis Human papillomavirus (HPV) infection Human papillomavirus (HPV) Human parainfluenza virus infection Human parainfluenza virus (HPIV) G imenolipedosis Hymenolepis dad and Hymenolepis diminuta Epstein-Barr virus infectious mononucleosis (mononucleosis) Epstein-Barr virus (EBV) Flu Family Orthomyxoviridae isosporosis Isospora belli Kawasaki disease Unknown; there is evidence of contagiousness Keratitis A number of pathogens Kingella kingae infection Kingella kingae Kuru Prion Kuru Lassa fever Lassa virus Legionellosis (Legionnaires' disease) Legionella pneumophila Legionellosis (Pontiac fever) Legionella pneumophila Leishmaniasis Genus Leishmania Leprosy Mycobacterium leprae and Mycobacterium lepromatosis Leptospirosis Genus Leptospira Listeriosis Listeria monocytogenes Lyme disease (Lyme borreliosis) Typically Borrelia burgdorferi and other Borrelia species Lymphatic filariasis (elephantiasis) Wuchereria bancrofti and Brugia malayi Lymphocytic choriomeningitis Lymphocytic choriomeningitis virus (LCMV) Malaria Genus Plasmodium Marburg hemorrhagic fever (MHF) Marburg virus

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Measles measles virus Melioidosis (Whitmore's disease) Burkholderia pseudomallei Meningitis A number of pathogens Meningococcal infection Neisseria meningitidis Metagonimiasis Typically Metagonimus yokagawai microsporidiosis Microsporidia phylum Molluscum contagiosum (MS) Molluscum contagiosum virus (MCV) Piggy mumps virus Rat typhus (endemic typhus) Rickettsia typhi Mycoplasma pneumonia Mycoplasma pneumoniae Mycetoma Numerous species of bacteria (actinomycetoma) and fungi (eumycetoma) miaz Larvae of parasitic flies from the Diptera order Neonatal Conjunctivitis (Neonatal Ophthalmia) Most common Chlamydia trachomatis and Neisseria gonorrhoeae (New) variant of Creutzfeldt-Jakob disease (vCJD, nvCJD) Prion vCJD Nocardiosis Typically Nocardia asteroides and other Nocardia species Onchocerciasis (river blindness) Onchocerca volvulus Paracoccidioidomycosis (South American Blastomycosis) Paracoccidioides brasiliensis Paragonimiasis Typically Paragonimus westermani and other Paragonimus species pasteurellosis Genus Pasteurella Head lice (head lice) Pediculus humanus capitis Body lice (body lice) Pediculus humanus corporis Pubic pediculosis (Pubic lice, flatheads) Phthirus pubis Pelvic inflammatory disease (PID) A number of pathogens Whooping cough (whooping cough) Bordetella pertussis Plague Yersinia pestis pneumococcal infection Streptococcus pneumoniae Pneumocystis pneumonia (1111) Pneumocystis jirovecii Pneumonia A number of pathogens Polio poliovirus Prevotella infection Genus Prevotella Primary amoebic meningoencephalitis (PAM) Usually Naegleria fowleri Progressive multifocal leukoencephalopathy JC virus Psittacosis Chlamydophila psittaci Q fever Coxiella burnetii Rabies Rabies virus Rat bite fever Streptobacillus moniliformis and Spirillum minus respiratory syncytial virus infection Respiratory syncytial virus (RSV) Rhinosporidiosis Rhinosporidium seeberi

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Rhinovirus infection Rhinovirus Rickettsial infection Genus Rickettsia Smallpox rickettsiosis Rickettsia akari Rift Valley Fever (RVF) Rift Valley fever virus Rocky Mountain spotted fever (RMSF) Rickettsia rickettsii Rotavirus infection Rotavirus Rubella rubella virus salmonellosis Genus Salmonella SARS (severe acute respiratory syndrome) SARS coronavirus Scabies Sarcoptes scabiei Schistosomiasis Genus Schistosoma Sepsis A number of pathogens Shigellosis (bacillary dysentery) Genus Shigella Shingles (Herpes zoster) Varicella zoster virus (VZV) Smallpox (variola) Variola major or Variola minor Sporotrichosis Sporothrix schenckii Staphylococcal food poisoning Genus Staphylococcus staph infection Genus Staphylococcus Strongyloidiasis Strongyloides stercoralis Syphilis Treponema pallidum teniasis genus Taenia Tetanus (tetanus) Clostridium tetani Tinea barbae - ringworm of the beard and mustache (hairdresser's rash) Typically the genus Trichophyton Tinea capitis (tinea capitis) As a rule, Trichophyton tonsurans Tinea corporis (smooth skin tinea) Typically the genus Trichophyton Tinea cruris (inguinal ringworm) Typically, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes Tinea manuum (dermatomycosis of the skin of the hands) Trichophyton rubrum Tinea nigra (black lichen) usually Hortaea werneckii Dermatophytosis of the feet (athlete's foot) usually the genus Trichophyton Tinea unguium (onychomycosis) usually the genus Trichophyton Tinea versicolor (pityriasis versicolor) Genus Malassezia Toxocariasis (Ocular form of larva migrans syndrome (OLM)) Toxocara canis or Toxocara cati Toxocariasis (Visceral form of larva migrans syndrome (VLM)) Toxocara canis or Toxocara cati Toxoplasmosis Toxoplasma gondii Trichinosis Trichinella spiralis Trichomoniasis Trichomonas vaginalis Trichuria (whipworm infection) Trichuris trichiura Tuberculosis Typically, mycobacterial tuberculosis Tularemia Francisella tularensis Ureaplasma urealyticum infection Ureaplasma urealyticum Venezuelan equine encephalitis Venezuelan equine encephalitis virus Venezuelan hemorrhagic fever Guanarito virus Viral pneumonia Various viruses West Nile fever West Nile virus White Piedra (Tinea nuance) Trichosporon beigelii Yersinia pseudotuberculosis infection Yersinia pseudotuberculosis Yersiniosis Yersinia enterocolitica Yellow fever yellow fever virus Zygomycosis Order Mucorales (mucormycosis) and Entomophthorales (entomophoramycosis)

The specific dosage and treatment regimen for a particular patient will depend on various factors, including the particular antibody, variant or derivative thereof used, age, body weight, general health, sex, and diet of the patient; time of administration, rate of elimination, drug combinations, and the severity of the particular disease being treated. The evaluation of such factors by medical professionals is within the ordinary skill of the art. The amount also depends on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount to be used can be determined using pharmacological and pharmacokinetic principles well known in the art.

Methods for administering antibodies or variants thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption

- 35 043231 ions through epithelial or mucocutaneous membranes (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered together with other biologically active agents. Accordingly, pharmaceutical compositions comprising the antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (eg, by application of powders, ointments, drops, or transdermal patch), buccally, or as an oral or nasal spray. .

The term parenteral as used herein refers to routes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

The introduction can be systemic or local. Additionally, it may be desirable to administer the antibodies of the present disclosure to the central nervous system by any suitable route, including intraventricular and intrathecal injections; to facilitate intraventricular injection, an intraventricular catheter may be used, for example attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be used, for example by inhaler or nebulizer, and in formulations with an aerosol agent.

It may be desirable to administer antibody polypeptides or antibody compositions as described herein locally to the area where treatment is desired; this can be achieved, for example, but not limited to, by local infusion during surgery, topical application, for example, in combination with a wound dressing after surgery, by injection, through a catheter, in the form of a suppository or implant, and the specified implant can be represented by a porous, non-porous or gel-like material, including membranes such as silastic membranes, or fibers. Preferably, when introducing a protein according to the present description, including antibodies, materials that do not absorb said protein should be used.

In another embodiment, said antibody or composition can be delivered in a vesicle, such as a liposome (see Langer, 1990, Science, 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see ibid. in general).

In yet another embodiment, said antigen-binding polypeptide or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery, 88:507; Saudek et al., 1989, N. Engl J. Med., 321:574). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984), Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem., 23:61; see also Levy et al., 1985, Science, 228:190; During et al., 1989, Ann. Neurol., 25:351; Howard et al., 1989, J. Neurosurg., 71:105). In yet another embodiment, the controlled release system may be placed in the vicinity of the therapeutic target, i. e. the brain, therefore, requires only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are reviewed by Langer (1990, Science, 249:1527-1533).

In a specific embodiment, if a composition according to the present disclosure comprises a nucleic acid or polynucleotide encoding a protein, said nucleic acid may be administered in vivo to promote expression of the encoded protein by constructing it as part of a suitable nucleic acid expression vector and administering it in such a manner, that it becomes intracellular, for example, using a retroviral vector (see US patent No. 4980286), or by direct injection, or the use of microparticle bombardment (for example, using a gene gun; biolist, Dupont), or coating with lipids, or using cellular receptors surface, or transfection agents, or by administration bound to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci., USA, 88 :1864-1868), etc. Alternatively, the nucleic acid may be introduced intracellularly and incorporated into the DNA of the host cell for expression by homologous recombination.

The number of antibodies according to the present description, which will be effective in the treatment, inhibition and prevention of inflammatory, immune or malignant diseases, disorders or conditions can be determined using standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The exact dosage to be used in the formulation also depends on the route of administration and the severity of the disease, disorder or condition, and should be selected based on the judgment of the attending physician and the circumstances of each patient. Effective doses can be derived by extrapolation from dose-response curves obtained in vitro or in model animal test systems.

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In general, the dose of antigen-binding polypeptides according to the present disclosure administered to a patient is typically 0.1 to 100 mg/kg patient body weight, 0.1 to 20 mg/kg patient body weight, or 1 to 10 mg/kg body weight of the patient. Typically, human antibodies have a longer half-life in the human body compared to antibodies from other species due to the immune response to foreign polypeptides. Accordingly, for human antibodies, lower dosages and less frequent administration are often possible. In addition, the dosage and frequency of administration of antibodies as described herein can be reduced by enhancing uptake and penetration of antibodies into tissues (eg, the brain) by modifications such as, for example, lipidization.

Methods for treating an infectious or malignant disease, condition, or disorder comprising administering an antibody, variant, or derivative thereof as described herein are generally tested for the desired therapeutic or prophylactic activity in vitro and then in vivo in a suitable animal model prior to use in humans. Suitable animal models, including transgenic animals, are well known to those skilled in the art. For example, in vitro assays to demonstrate the therapeutic utility of an antigen-binding polypeptide described herein include assaying the effect of the antigen-binding polypeptide on a patient's cell line or tissue sample. The effect of an antigen-binding polypeptide on a cell line and/or tissue sample can be determined using techniques known to those skilled in the art, such as the assays described in various sections of this document. As used herein, in vitro assays that can be used to determine whether administration of a specific antigen-binding polypeptide is indicated include in vitro cell culture assays where a patient tissue sample is cultured and exposed to a compound or otherwise administered with a compound and then observed. the effect of such a compound on the tissue sample.

Various delivery systems are known and can be used to administer an antibody as described herein or a polynucleotide encoding an antibody as described herein, e.g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing said compound, receptor-mediated endocytosis Wu and Wu, 1987, J. Biol. Chem., 262:4429-4432), constructing the nucleic acid as part of a retroviral or other vector, and the like.

Diagnostic methods.

PD-L1 is overexpressed in certain tumor samples, and patients with PD-L1 overexpressing cells are expected to respond to treatment with anti-PD-L1 antibodies as described herein. Accordingly, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

The sample, which preferably includes a cell, may be obtained from a patient, which may be a cancer patient or a patient to be diagnosed. Said cell is a cell from a tumor tissue or tumor block, a blood sample, a urine sample, or any sample from a patient. After optional sample pretreatment, said sample may be incubated with an antibody as described herein under conditions that allow said antibody to interact with a PD-L1 protein potentially present in the sample. Techniques such as ELISA using an anti-PD-L1 antibody can be used to detect the presence of a PD-L1 protein in a sample.

The presence of PD-L1 protein in a sample (optionally quantified or concentrated) can be used to diagnose cancer, as an indication that a patient is suitable for treatment with said antibody, or as an indication that a patient has responded (or has not responded) to cancer treatment . In the case of the diagnostic method, the detection may be performed once, twice or more times, at certain stages, after the start of cancer treatment to determine the progress of the treatment.

Compositions.

The present invention also provides pharmaceutical compositions. Such compositions contain an effective amount of the antibody and a suitable carrier. In some embodiments, said composition further comprises a second anticancer agent (eg, an immune checkpoint inhibitor).

In a particular embodiment, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency, or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more specifically, in humans. In addition, the pharmaceutically acceptable carrier is usually a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material, or any type of prescription auxiliary agent.

The term carrier refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame

- 37 043231 oil, etc. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and etc. Said composition, if required, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Also included are antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for correcting tonicity such as sodium chloride or dextrose. Said compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Said composition may be formulated in a suppository with conventional binders and carriers such as triglycerides. The oral formulation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences, ed. E. W. Martin, incorporated herein by reference. Such compositions contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of carrier to provide a form for appropriate administration to said patient. The specified composition should correspond to the method of administration. The original composition can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

In an embodiment, the formulation of said composition is prepared according to conventional procedures for a pharmaceutical composition intended for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, said composition may also include a solubilizing agent and a local anesthetic such as lignocaine to reduce pain at the injection site. Typically, these ingredients are either presented separately or mixed in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the amount of active agent. If the composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

The compounds of the present disclosure may be formulated in neutral form or in salt form. Pharmaceutically acceptable salts include salts formed by anions, for example, derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc., and formed by cations, for example, derived from sodium, potassium, ammonium, calcium, iron (III) hydroxide , isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Examples

Example 1 Preparation of human monoclonal antibodies against human PD-L1.

Received mouse monoclonal antibodies against human PD-L1 using hybridoma technology.

Antigen: highly expressing human PDL1-Fc protein and human PD-L1 cell line CHOK1 (PDL1-CHOK1 cell line).

Immunization: To obtain mouse monoclonal antibodies to human PD-L1, 6-8 week old female BALB/c mice were first immunized with 1.5 x 10 ⁷ PDL1-CHOK1 cells. On day 14 and day 33 after the first immunization, immunized mice were boosted with 1.5 x 10 ⁷ PDL1-CHOK1 cells, respectively. To select mice producing antibodies that bind the PD-L1 protein, sera from immunized mice were tested using ELISA ELISA. Briefly, microtiter plates were coated with human PD-L1 protein at 1 μg/ml in PBS at 100 μl/well at room temperature (RT) overnight, after which they were blocked with 100 μl/well 5% BSA. Plasma dilutions from immunized mice were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP) conjugated anti-mouse IgG for 1 h at RT. After washing, the plates were developed with ABTS substrate and analyzed on a spectrophotometer at OD 405 nm. Mice with sufficient anti-PDL1 IgG titers were given a booster dose of 50 μg human PDL1-Fc protein on day 54 post-immunization. The final selected mice were used for fusion. Hybridoma supernatants were tested for anti-PD-L1 IgG using ELISA ELISA.

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Hybridoma clones HL1210-3, HL1207-3, HL1207-9 and HL1120-3 were selected for further analysis. The amino acid and polynucleotide sequences of the HL1210-3 variable regions are shown in Table 1. 5 below.

Table 5

HL1210-3 variable sequences

Name Subsequence SEQID NO: HL1210-3 vh GAAGTGAAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGC CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATT 112 CACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACT CCGGAGAAGTCTGGAGTGGGTCGCAACCATTAGTGATG GTGGTGGTTACATCTACTATTCAGACAGTGTGAAGGGGCG AT T TAC CAT CTC CAGAGACAAT G C CAAGAACAAC S T GTAC CTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGT ATATTTGTG CAAGAGAATTTGGTAAGCGCTATGCTTTGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA HL1210-3 vh EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQT PEKSLEWVATISDGGGYIYYSDSVKGRFTISRDNAKNNLY LQMSSLRSEDTALYICAREFGKRYALDYWGQGTSVT 113 HL1210-3 VL GACAT T GT GAT GAC C CAGT ST CACAAAAT T CAT GT C CACAT CGTAGGAGACAGGGTCAGCATCTCCTGCAAGGCCAGTCA GGATGTGACTCCTGCTGTCGCCTGGTATCAACAGAAGCCA GGACAATCTCCTAAACTACTGATTTACTCCACATCCTCCC GGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATC TGGGACGGA TTTCACTTTCACCATCAGCAGTGTGCAGGCT GAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTA CTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAA A 114 HL1210-3 VL DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKP GQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTFTISSVQA EDLAVYYCQQHYTTPLTFGAGTKLELK 115

Example 2 Human PD-L1 binding activity of mouse monoclonal antibody HL1210-3.

To assess the binding activity of the hybridoma clone HL1210-3, the mAb purified from the specified clone was tested by ELISA ELISA. Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.1 μg/ml in PBS at 100 μl/well at 4° C. overnight followed by blocking with 100 μl/well 5% BSA. 3-fold dilutions of HL1210-3 antibodies, starting at 0.2 μg/ml, were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at RT. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm. As shown in FIG. 1, HL1210-3 can bind to human PD-L1 with high activity ( _EC50 =5.539 ng/mL).

Example 3 Mouse mAb HL1210-3 blocked the binding of human PD-L1 to its PD-1 receptor.

Receptor blocking assay using recombinant human PD-L1.

An ELISA-based receptor blocking assay was used to evaluate the blocking effect of the mouse mAb HL1210-3 on the binding of recombinant human PD-L1 to its PD-1 receptor. Briefly, microtiter plates were coated with 1 μg/ml human PD-L1-Fc protein in PBS at 100 μl/well at 4° C. overnight followed by blocking with 100 μl/well 5% BSA. 50 µl of biotin-labeled human PD-1-Fc protein and 50 µl of 3-fold dilutions of HL1210-3 antibodies, starting at 2 µg/ml, were added to each well and incubated for 1 hour at 37°C. The plates were washed with PBS/Tween and then incubated with streptavidin-HRP for 1 h at 37°C. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm. As shown in FIG. 2, HL1210-3 can effectively inhibit the binding of human PD-L1 to human PD1 with an IC ₅₀ =0.7835 nM.

Receptor blocking assay using mammalian-expressed human PD-L1.

To assess the blocking effect of the mouse mAb HL1210-3 on the binding of human PD-L1 expressed on mammalian cells to its PD-1 receptor, a FACS-based receptor blocking assay was used. Briefly, PDL1-CHOK1 cells were first incubated with 3-fold serial dilutions of mouse HL1210-3 mAb starting at 20 μg/mL at RT for 1 h. After washing with FACS buffer (PBS with 2% PBS), labeled huPD-1 biotin and incubated at RT for 1 h. Then, after washing twice with FACS buffer, streptavidin-PE was added to each well for 0.5 h. Mean fluorescence intensity (MFI) of PE was assessed using FACSAriaIII. As shown in FIG. 3, the HL1210-3 antibody can highly efficiently inhibit PD-1 binding to mammalian cell-expressed PD-L1 with an IC ₅₀ of 2.56 nM with 92.6% maximum inhibition.

_ /, MFI of test antibody \ .

% inhibition = I 1-- ¹² ---------- x 100% \ MFI control-base /

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Example 4 Mouse mAb HL1210-3 stimulated human T cell immune response.

To evaluate the effect of mouse mAb HL1210-3, the response of human T cells was evaluated under mixed lymphocyte reaction conditions. Human DC were differentiated from CD14+ monocytes in the presence of GM-CSF and IL-4 for 7 days. Then, CD4+ T cells isolated from another donor were co-cultured with DC and serial dilutions of a blocking antibody against PD-L1. On day 5 after inoculation, the production of IFN-γ in the culture supernatant was analyzed. The results showed that HL1210-3 antibodies can dose-dependently stimulate IFN-γ production, suggesting the ability of the anti-PD-L1 antibody to stimulate human T cell response (FIG. 4).

Example 5 Binding affinity of mouse mAb HL1210-3.

Binding of HL1210-3 antibodies to recombinant PD-L1 protein (His-tagged human PD-L1) was tested in the BIACORE™ system using the capture method. The mouse HL1210-3 mAb was captured using an anti-mouse Fc antibody coated on the CM5 chip. Serial dilutions of the His-tagged human PD-L1 protein were injected over the captured antibody for 3 min at a flow rate of 25 μg/mL. The antigen was allowed to dissociate for 900 s. This experiment was carried out entirely on Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The result is shown in Fig. 5 and in table. 6 below.

Table 6

Binding Kinetics of HL1210-3 to Recombinant Human PD-L1

Antibody ka (1/ms) kd (1/s) KD (M) HL1210-3 1.61E+05 4.69Е-05 2.93Е-10

Example 6 Humanization of mouse mAb HL1210-3.

The variable region genes of the HL1210-3 mAb were used to create a humanized mAb. In the first step of this process, the amino acid sequences of VH and VK from MAb HL1210-3 were compared with human Ig gene sequences from the available database to find overall best matched human Ig germline gene sequences. For the light chain, the best match among human sequences was found for the O18/Jk2 and KV1-39*01/KJ2*04 genes, and for the heavy chain, the best match among human sequences was found for the VH3-21 gene. The VH3-11, VH3-23, VH3-7*01 and VH3-48 genes were also selected due to their significant overlap.

Humanized variable domain sequences were then designed in which CDR1 (SEQ ID NO: 4), 2 (SEQ ID NO: 5) and 3 (SEQ ID NO: 6) of the HL1210-3 light chain were grafted onto the framework sequences of the O18/Jk2 and KV1-39*01/KJ2*04, a sequences of CDR1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3) from HL1210-3 VH were grafted onto the framework sequences of the VH3 gene -21, VH3-11, VH3-23, VH3-48 or VH3-7*01. A 3D model was then created to determine if substitution of a mouse amino acid for a human amino acid at any position in the framework could affect CDR binding and/or conformation. For the light chain, 22S, 43S, 60D, 63T and 42Q (Kabat numbering; see Table 7) were identified in the framework. In the case of the heavy chain, 1E, 37V, 40T, 44S, 49A, 77N, 91I, 94R and 108T were backmutated in the framework.

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Table 7

Humanization design

Design VH I: VH3-21/JH6 Design Mutation Hul210VH Chimera Hul210VH. 1 CDR inoculation Hul210VH. 1a S49A Hul210VH.lb S49A, G44S, Y91I Design VH II: VH3-11/JH6 Hul210VH.2 Inoculation CDR, Q1E Hul210VH.2a Q1E, S49A Hul210VH.2b Q1E, I37V, S49A, G44S, Y91I Design VH III: VH3-23/JH6 Hul210VH.3 Graft CDR, K94R Hul210VH.3a G44S, S49A, Y91I, K94R Design VH IV: VH3-48/JH6 Hul210VH.4 CDR inoculation Hul210VH.4a S49A Hul210VH.4b S49A, G44S, Y91I Hul210VH.4c D52E, S49A, G44S, Y91I Hul210VH.4d G53A, S49A, G44S, Y91I Hul210VH.4e G53V, S49A, G44S, Y91I VHV design: VH3-7*01/ HJl*01 Hul210VH.5 CDR inoculation Hul210VH.5a H91I Hul210VH.5b H9P, H108T Hul210VH.5c H91I, H77N Hul210VH.5d H91I, H77N, H40T Design VK I: 018/Jk2 Design Mutation Hul210Vk Chimera Hul210Vk.l CDR inoculation Hul210Vk.la A43S Design VK II: KVl-39*01/KJ2*04 Hul210Vk.2 CDR inoculation Hul210Vk.2a L60D, L63T Hul210Vk.2b L60D, L63T, L42Q, L43S Hul210Vk.2c L60D, L63T, L42Q, L43S, T22S

The amino acid and nucleotide sequences of some of the humanized antibodies are listed in Table 1. 8 below.

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Table 8

Humanized antibody sequences (CDRs in bold)

Name Subsequence SEQID NO: HL1210-VH EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYICAREF GKRYALDYWGQGTSVTVSS 7 Hul210 VH.1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST ISDGGGYIYYSDSVKGRFTIS RDNAKN S LYLQMNS LRAEDTAVYYCAREF GKRYALDYWGQGTTVTVS S 8 Hul210 VH.la EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTIS RDNAKN S LYLQMNS LRAEDTAVYYCAREF GKRYALDYWGQGTTVTVS S 9 Hul210 VH.lb EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVS S 10 Hul210 VH.2 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVST ISDGGGYIYYSDSVKGRFTIS RDNAKN S LYLQMNS LRAEDTAVYYCAREF GKRYALDYWGQGTTVTVS S eleven Hul210 VH.2a EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTIS RDNAKN S LYLQMNS LRAEDTAVYYCAREF GKRYALDYWGQGTTVTVS S 12 Hul210 VH.2b EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVS S 13 Hul210 VH.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTTVTVS S 14 Hul210 VH.3a EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVS S 15 Hul210 VH.4 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF GKRYALDYWGQGTTVTVS S 16 Hul210 VH.4a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 17

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ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF GKRYALDYWGQGTTVTVS S Hul210 VH.4b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVS S 18 Hul210 VH.4c EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISEGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVS S 19 Hul210 VH.4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVS S 20 Hul210 VH.4e EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT IS DVGGYIYYS DSVKGRFTIS RDNAKNSLYLQMNS LRDEDTAVYICAREF GKRYALDYWGQGTTVTVS 21 Hul210 VH.5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTIS RDNAKN S LYLQMNS LRAEDTAVYYCAREF GKRYALDYWGQGTLVTVS S 22 HU1210 VH.5a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVS S 23 HU1210 VH.5b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVS S 24 HU1210 VH.5C EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVS S 25 HU1210 VH.5d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVS S 26 HL1210-VK DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKPGQSPKLLIYS TSSRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTTPLTFGA GTKLELK 27 Hul210 VK.l DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 28 Hul210 VK.la DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKSPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 29 Hul210 Vk.2 DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR thirty Hul210 Vk.2a DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR 31 Hul210 Vk.2b DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGQSPKLLIYS TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR 32 Hul210 Vk.2c DIQMTQSPSSLSASVGDRVTISCKASQDVTPAVAWYQQKPGQSPKLLIYS TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR 33

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HL1210 VH GAGGTGAAGCTGGTGGAGAGCGGCGGAGATCTGGTGAAGCCTGGCGGCAGCCTGAAGCTG AGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGGCAGACC CCCGAGAAGAGCCTGGAGTGGGTGGCCACCATCAGCGATGGCGGCGGCTACATCTACTAC AGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC GCCAAGAACAACCTGTAC CTGCAGATGAGCAGCCTGAGGAGCGAGGACACCGCCCTGTACATCTGCGCCAGGGAGTTC GGCAAAGAGGTACGCCCTGGACTACTGGGGACAGGGCACCAGCGTGACCGTGAGCAGC 34 Hul210 VH.l GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 35 Hul210 VH.la GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 36 Hul210 VH.lb GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 37 Hul210 VH.2 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTAC CAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTGCGCCAGGGAGTTC GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 38 Hul210 VH.2a GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 39 Hu 1210 VH.2b GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 40 Hul210 VH.3 GAGGTGCAAGCTGCTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTGCGCCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 41 Hul210 VH.3a GAGGTGCAGCTGCTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC 42

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GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hul210 VH.4 GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTGCGCCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 43 Hu 1210 VH.4a GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTGCGCCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 44 Hu 1210 VH.4b GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 45 Hu 1210 VH.4c GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGAAGGCGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 46 Hul210 VH.4d GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGCGGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAAAGGTACGCCCTGGACTACTGGGCCAGGGCACAACCGTGACCGTGAGCAGC 47 Hul210_VH.4 e GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAACCCGGAGGCAGCCTGAGACTG AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGTTGGCGGCTACATCTATTAC TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC GGCAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC 48 Hul210 VH.5 GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGTGTGAGGCAGGCT CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCG GGACAACGCCAAGAACTCCCTGTAC CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTGTATTACTGCGCCAGGGAGTTT GGCAAGAGGTACGCCCTGGATTACTGGGCCAGGGCACACTGGTGACAGTGAGCTCC 49 Hul210 VH.5a GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGTGTGAGGCAGGCT CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCG GGACAACGCCAAGAACTCCCTGTAC CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT GGCAAGAGGTACGCCCTGGATTACTGGGCCAGGGCACACTGGTGACAGTGAGCTCC 50 Hul210 VH.5b GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCT CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC 51

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TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTAC Hul210 VH.5c GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGTGAGGCAGACC CCTGAGAAGAGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGA CAACGCCAAGAACAACCTGTAC CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC 52 Hul210 VH.5d GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGTGTGAGGCAGGCT CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCG GGACAACGCCAAGAACTCCCTGTAC CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT GGCAAGAGGTACGCCCTGGATTACTGGGCCAGGGCACAACCGTGACAGTGAGCTCC 53 HL1210 VK GACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGATAGGGTGAGC ATCAGCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC GGCCAGAGCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCGAC GCAGCGTGCAGGCC GAGGACCTGGCCGTGTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCGCC GGCACCAAGCTGGAGCTGAAG 54 Hul210 VK.l GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC GGCAAGGCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGC AGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCAT CAGCAGCCTGCAGCCC GAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAG GGCACCAAGCTGGAGATCAAG 55 Hul210 VK.la GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC GGCAAGTCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGC AGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCAT CAGCAGCCTGCAGCCC GAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAG GGCACCAAGCTGGAGATCAAG 56 Hul210 VK.2 GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT GGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCTCC AGGTTTAGCGGCAGCGGCTCCGGCACCGATTTCACCCTG ACCATTTCCTCCCTGCAGCCC GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG GGCACCAAGCTGGAGATCAAGCGG 57 Hu 1210 VK.2a GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT GGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC AGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTG ACCATTTCCTCCCTGCAGCCC GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG GGCACCAAGCTGGAGATCAAGCGG 58 Hu 1210 VK.2b GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT GGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC AGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGAC CATTTCCTCCCTGCAGCCC GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG GGCACCAAGCTGGAGATCAAGCGG 59 Hu 1210 VK.2c GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 60 ATCAGCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAAGAAGCCT GGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC CTTCGGCCAG GGCACCAAGCTGGAGATCAAGCGG

The humanized VH and VK genes were synthetically generated and then respectively cloned into vectors containing the human gamma 1 and human kappa constant domains. Pairing of human VH and human VK generated 40 humanized antibodies (see Table 9).

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Table 9

Humanized antibodies with VH and VL regions

vh Vk Hul210 VH.1 Hul210 VH.la Hul210 VH.lb Hul210 VH.2 Hul210 VH.2a Hul210 VH 2.b Hul210 VH Hul210 Vk.l Hul210-l Hul210-2 Hul210-3 Hul210-4 Hul210-5 Hul210 Vk.la Hul210-7 Hul210-8 Hul210-9 Hul210-10 Hul210-ll Hul210Vk Chimera H1210

vh Vk Hul210 VH.3 Hul210 VH.3a Hul210 VH.4 Hul210 VH.4a Hul210 VH.4b Hul210 Vk.l Hul210-13 Hul210-14 Hul210-15 Hul210-16 Hul210-17 Hul210 Vk.la Hul210-18 Hul210-19 Hu 1210-20 Hul210-21 Hu 1210-22

vh VK Hul210 VH.5 HU1210 VH.5a HU1210 VH.5b HU1210 VH.5c HU1210 VH.5d Hul210 Vk.2 Hu 1210-23 Hu 1210-27 Hul210-31 hul210-32 Hul210-36 Hul210 Vk.2a Hu 1210-24 Hul210-28 Hul210-33 Hul210-37 Hul210 Vk.2b Hu 1210-25 Hu 1210-29 Hul210-34 Hul210-38 Hul210 Vk.2c Hu 1210-26 hul210-30 hul210-35 Hul210-39

vh Vk Hul210 VH.4c Hul210 VH.4d Hul210 VH.4e Hul210 Vk.l Hu 1210-40 Hul210-41 Hu 1210-42

Example 7 Antigen-binding properties of humanized anti-PD-L1 antibodies.

Binding capacity of recombinant human PD-L1.

To assess antigen-binding activity, humanized antibodies were tested by ELISA ELISA. Briefly, microtiter plates were coated with human PD-L1-Fc protein at 0.1 μg/ml in PBS, 100 μl/well at 4° C. overnight, followed by blocking with 100 μl/well 5% BSA. Five-fold dilutions of humanized antibodies, starting at 10 μg/ml, were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at RT. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm. As shown in FIG. 6, all humanized antibodies show comparable binding efficiency to human PD-L1 compared to the chimeric antibody.

The ability to bind to mammalian expressed human PD-L1.

To assess antigen-binding capacity, the binding of humanized antibodies to mammalian-expressed PD-L1 was analyzed using FACS. Briefly, PDL1-CHOK1 cells were first incubated with 5-fold serial dilutions of humanized antibodies, starting at 2 μg/ml, at RT for 1 h. After washing with FACS buffer (PBS with 2% PBS), Alexa-conjugated 488 anti-human IgG antibody and incubated at RT for 1 h. MFI Alexa 488 was evaluated using FACSAriaIII. As shown in FIG. 7, all humanized antibodies can bind highly efficiently to PD-L1 expressed on mammalian cells in a manner comparable to the chimeric antibody.

To study the binding kinetics of the humanized antibody in this example, affinity ranking was performed using Octet Red 96. As shown in table. 10, Hu1210-3, Hu1210-8, Hu1210-9, Hu1210-14, Hu1210-17, Hu1210-1 and Hu1210-22 show the best affinity comparable to the chimeric antibody.

- 47 043231

Table 10

Affinity ranking of humanized antibodies

Antibody KD (M) to _op (1/Ms) k _dis (l/c) Antibody KD (M) to _op (1/Ms) k _d is(l/c) Ni1210 7D6E-09 3.94E+05 2.83Е-03 Ni1210-11 4D8E-09 7.54E+04 ZD5E-04 (mlgG) H1210 chimera 1.07Е-09 1.62E+05 1.73Е-04 Ni1210-13 4.36Е-09 8.38E+04 3.66E-04 Ni1210-1 4.25Е-09 7D0E+04 3.02E-04 Ni1210-14 2.34Е-09 8.41E+04 1.97Е-04 Ni1210-2 3.23E-09 7.78E+04 2.51Е-04 Ni1210-15 4.45Е-09 7.87E+04 3.50E-04 Ni1210-3 2.64Е-09 8.62E+04 2.28E-04 Ni1210-16 ZD4E-09 8.41E+04 2.64Е-04 Ni1210-4 7.68Е-09 7D2E+04 5.46Е-04 Ni1210-17 2.20E-09 8D7E+04 1.80E-04 Ni1210-5 4.83Е-09 7.93E+04 3.83E-04 Ni1210-18 4.50Е-09 7.92E+04 3.57Е-04 Ni1210-7 4.78Е-09 8.45E+04 4.04Е-04 Ni1210-19 2.50E-09 9.03E+04 2.25Е-04 Ni1210-8 1.64Е-09 7.72E+04 1.27E-04 Ni1210-20 4.51Е-09 8.87E+04 4.00Е-04 Ni1210-9 2,ZZE-09 8.37E+04 1.95Е-04 Ni1210-21 ZD2E-09 9.39E+04 2.93E-04 Ni1210-10 7.03Е-09 8.59E+04 6.04Е-04 Ni1210-22 2.56Е-09 9.00E+04 2,ZOE-04 Determination of the total kinetic affinity of humanized antibodies using

Biacore®.

Binding of humanized antibodies to recombinant PD-L1 protein (His-tagged human PD-L1) was tested using the Biacore™ capture method. Mouse mAb HL1210-3 was captured using an anti-mouse Fc antibody coated on a CM5 chip. Serial dilutions of the his-tagged human PD-L1 protein were injected over the captured antibody for 3 min at a flow rate of 25 μg/mL. The antigen was allowed to dissociate for 900 s. The experiment was carried out entirely on Biacore T200. Data analysis was performed using Biacore T200 analytical software; the results are presented in table. 11 below.

Table 11

Determination of affinity using Biacore

Antibody ka (1/ms) kd (1/s) KD (M) Ni1210-8 9.346E+4 7D69E-5 7.671E-10 Ni1210-9 9.856E+4 4D28E-5 4.594E-10 Ni1210-14 1.216E+5 5D93E-5 4.352E-10 Ni1210-16 9.978E+4 6.704E-5 6.720E-10 Ni1210-17 1D01E+5 2D28E-5 1.933E-10 Neither 1210-28 1D89E+5 1.080E-4 8.378E-10 Ni1210-31 E486E+5 1D68E-4 7.862E-10 Ni1210-36 E461E+5 7.852E-5 5.376E-10 Neither 1210-40 8.77E+04 1.31Е-04 1.49Е-09 Ni1210-41 9D7E+04 3.46E-05 3.78E-10 Neither 1210-42 8.68E+04 7.53Е-05 8.67E-10 Chimera 1210 Е236Е+5 3.265E-5 2.642E-10

interspecies activity.

To assess the binding of humanized antibodies to huPD-L1, mouse PD-L1, rat PD-L1, rhesus monkey PD-L1, antibodies were tested by ELISA. Briefly, microtiter plates were coated with human, mouse, rat, and rhesus monkey PD-L1-Fc protein at 1 μg/ml in PBS, 100 μl/well at 4°C overnight, followed by blocking with 100 μl/well 5% BSA . Three-fold dilutions of humanized antibodies, starting at 1 μg/ml, were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at RT. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm.

The Hu1210-41 antibody can bind to rhesus monkey PD-L1 with lower affinity and is unable to bind to rat and mouse PD-L1 (FIG. 8).

Human rhesus monkey Rat Mouse EU50 0.215 nM 0.628 nM No binding No binding

Specificity for members of the family.

To assess the binding of the humanized anti-PD-L1 antibody to the human B7 family and other immune response checkpoints, the binding of the indicated antibody to B7-H1 (PD-L1), B7-DC, B7-1, B7-2, B7-H2, PD -1, CD28, CTLA4, ICOS and BTLA using ELISA ELISA. As shown in FIG. 9, the Hu1210-41 antibody is only able to specifically bind to B7-H1 (PD-L1).

- 48 043231

Example 8 Humanized antibodies blocked human PD-L1 activity against PD-1.

Cellular receptor blocking assay.

A FACS-based receptor blocking assay was used to evaluate the blocking effect of humanized antibodies on the binding of human PD-L1 expressed on mammalian cells to its PD-1 receptor. Briefly, PDL1-CHOK1 cells were first incubated with 3-fold serial dilutions of mouse HL1210-3 mAb starting at 20 μg/mL at RT for 1 h. After washing with FACS buffer (PBS with 2% PBS), biotin-labeled huPDs were added to each well. -1 and incubated at RT for 1 h. After that, streptavidin-FE was added to each well for 0.5 h after washing twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was assessed using FACSAriaHI.

|„, - ( _l MFI of test antibody \ % inhibition = 1------------- x 100% \ MFI of base control /

As shown in Table. 12 below, antibodies Hu1210-3, Hu1210-9, Hu1210-8, Hu1210-14, Hu1210-17, Hu1210-19, and Hu1210-22 show comparable efficacy to the chimeric antibody in blocking PD-L1 binding to PD-1.

Table 12

PD-1 receptor blocking assay

Bio-PDl (30 µg/ml) TOR EU50 Chimera H1210 87.16 3.961 Ni1210-8 86.35 4.194 Ni1210-9 85.7 4.038 Ni1210-16 88.02 5.436 Ni1210-17 80.88 4.424 Ni1210-3 84.28 3.693 Ni1210-14 79.56 3.572 Ni1210-19 87.45 4.52 Neither 1210-22 85.83 4.505 Neither 1210-27 103.9 11.48 Ni1210-31 92.91 6.179 Ni1210-36 91.75 8.175

Receptor blocking assay using recombinant human PD-L1.

There are two human PD-L1 receptors, namely PD-1 and B7-1. To study the blocking properties of a humanized anti-PD-L1 antibody to these two proteins, a receptor blocking protein assay was used in this work. Briefly, microtiter plates were coated with human PD-L1-Fc protein at 1 μg/mL in PBS at 100 μL/well at 4°C overnight, followed by blocking with 200 μL/well 5% BSA at 37°C at for 2 h. 50 µl of biotin-labeled human protein PD-1-Fc or B7-lv and 50 µl of 5-fold dilutions of antibodies to PD-L1, starting from 100 nM, were added to each well and incubated for 1 h at 37 °C The plates were washed with PBS/Tween and then incubated with streptavidin-HRP for 1 hour at 37°C. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450 nm. As shown in FIG. 10 and 11, Hu1210-41 can effectively inhibit the binding of human PD-L1 to human PD1 and B7-1.

Example 9 Humanized Antibody Stimulated Human T Cell Immune Response.

Analysis using the reaction of mixed culture of lymphocytes.

To assess the function of humanized antibodies in vitro, the response of human T cells was evaluated under reaction conditions of a mixed culture of lymphocytes. Human DC were differentiated from CD14+ monocytes in the presence of GM-CSF and IL-4 for 7 days. Then, CD4+ T cells isolated from another donor were co-cultured with DC and serial dilutions of a blocking antibody against PD-L1. On day 5 after inoculation, the production of IL-2 and IFN-γ in the culture supernatant was analyzed. The results showed that antibodies Hu1210-8, Hu1210-9, Hu1210-16 and Hu1210-17 can dose-dependently stimulate the production of IL-2 and IFN-γ, indicating the ability of antibodies against PD-L1 to stimulate the response of human T cells.

Analysis with a panel of CMV antigens.

To assess the function of humanized antibodies in vitro, the response of human T cells in the analysis with a panel of CMV antigens was evaluated. Human PBMCs were stimulated with 1 μg/ml of CMV antigen in the presence of serial dilutions of humanized antibodies. As shown in FIG. 12 and 13, Hu1210-40, Hu1210-41, and Hu1210-17 can stimulate IFN-γ production in a dose-dependent manner.

Example 10 Inhibition of tumor growth by anti-PD-L1 mAb.

Human lung adenocarcinoma HCC827 cells are grafted into NOD Scid Gamma (NSG) mice. NSG mice are immunodeficient NOD Scid Gamma mice with the most

- 49 043231 female immunodeficiency, which makes them ideal recipients of human tumor cells and PBMC inoculation. 10 days after inoculation, tumor-bearing mice are transplanted with human PBMC. Approximately 20 days after inoculation, when the tumor volume reaches 100-150 mm ³ , mice begin to receive PD-L1 antibody every other day at a dosage of 5 mg/kg. Tumor volume is monitored every other day when the antibody is administered. As shown in FIG. 14, Hu1210-31 can inhibit tumor growth by 30% at 5mg/kg. The Hu1210-41 antibody can dose-dependently inhibit tumor growth, while the Hu1210-41 antibody also reduces tumor weight in a dose-dependent manner (FIG. 15).

Example 11 Computer Simulation of Additional Options and Optimization of Humanized Antibodies.

It was envisaged that certain amino acid residues within the CDR regions or framework regions can be changed to further improve or maintain the activity and/or stability of antibodies. Using a computational tool (VectorNTI, available at www.ebi.ac.uk/tools/msa/clustalo/) the structural, conformational and functional properties of the variants were tested; the prospects discovered (within the CDR regions) are listed in the tables below.

Table 13

VH and VL CDR regions and their variants suitable for inclusion in humanized antibodies

Name Subsequence SEQID NO: VH CDR1 SYDMS 1 TYDMS 61 CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66 SYDMC 67

Name Subsequence SEQID NO: VHCDR2 TISDGGGYIYYSDSVKG 2 TISDGGAYIYYSDSVKG 68 TISDGGPYIYYSDSVKG 69 TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71 TISDGGGWIYYSDSVKG 72 TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74 TISDGGGYIYYSDSLKG 75 TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDMKG 77

Name Subsequence SEQID NO: VH CDR3 EFGKRYALDY 3 QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80 EYGKRYALDY 81 EHGKRYALDY 82 EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY 85 EFGRRYALDY 86 EFGKKYALDY 87 EFGKRFALDY 88 EFGKRHALDY 89 EFGKRWALDY 90

Name Subsequence SEQID NO:

- 50 043231

VL CDR1 KASQDVTPAVA 4 KATQDVTPAVA 91 KACQDVTPAVA 92

Name Subsequence SEQID NO: VL CDR2 STSSRYT 5 TTSSRYT 93 CTSSRYT 94 SSSSRYT 95 SMSSRYT 96 SVSSRYT 97 STTSRYT 98 STCSRYT 99 STSTRYT 100 STSCRYT 101 STSSKYT 102 STSSRFT 103 STSSRHT 104 STSSRWT 105

Name Subsequence SEQID NO: VL CDR3 OOHYTTPLT 6 EQHYTTPLT 106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109 QDHYTTPLT 110 QNHYTTPLT 111

Hotspots for mutated residues and corresponding substitutions are underlined. Example 12 Identification of the PD-L1 epitope.

The specified study was carried out to identify amino acid residues involved in the binding of PD-L1 antibodies according to the present description.

A PD-L1 library was constructed using alanine scanning. Briefly, 217 PD-L1 mutant clones were generated on a protein engineering platform from Integral Molecular. The binding of Hu1210-41 Fab to each variant in the PD-L1 mutant library was determined in duplicate using high throughput flow cytometry. Background fluorescence was subtracted from each raw data point and normalized for reactivity to wild type PD-L1 (WT). For each PD-L1 variant, a plot of mean binding as a function of expression (reactivity against anti-PD-L1 control mAb) was plotted. Thresholds (dashed lines) corresponding to >70% WT binding to control MAb and <30% WT reactivity to Hu1210-41 Fab were used for preliminary identification of critical clones (crossed circles) (FIG. 16). Y134, K162 and N183 from PDL1 were identified as residues required for Hu1210-41 binding. The low reactivity of clone N183A towards Hu1210-41 Fab suggests that it is the main energetic participant in Hu1210-41 binding, with Y134 and K162 contributing less.

Key residues (spheres) were identified in the 3D PD-L1 structure (PDB ID# 5JDR, Zhang et al., 2017) shown in Fig. 17. These residues, Y134, K162 and N183, respectively, constitute the PD-L1 epitope responsible for binding to antibodies according to various embodiments of the present invention.

Interestingly, all of these residues, Y134, K162, and N183, are localized within the IgC domain of the PD-L1 protein. The extracellular portions of both PD-1 and PD-L1 contain an IgV domain and an IgC domain. It is well known that PD-L1 binds to PD-1 by linking their IgV domains to each other. However, Hu1210-41, unlike such standard antibodies, binds to the IgC domain, which is expected to be ineffective in inhibiting PD-1/PD-L1 binding. This other epitope of Hu1210-41, in an unexpected way, is expected to contribute to the outstanding activity of Hu1210-41.

Example 13 Construction of Anti-PDL1 Antibody.

Examples 13-17 describe work to identify further improved Hu1210-41 antibodies using mutagenesis.

Fusion protein of inducible cytidine deaminase (AID) activation and lacking nuclease

- 51 043231 the activity of CRISPR-associated Clustered Short Palindromic Repeat (CRISPR) protein 9 (dCas9) was used for high throughput screening of functional variants in T cells 293. Briefly, single guide (og)RNAs recognizing 6 CDR antibodies can direct the fusion protein dCas9-AID to CDR 6 antibodies and induce mutations in the CDR region. The mutated antibodies were exposed on the surface of 293 cells. The resulting cells showed better binding activity than the unmutated equivalent; they were sorted by FACS for subsequent sequencing. A potentially antibody-positive mutation from S60 to R in CDRH2 has been identified.

To assess the antigen-binding properties of the S60R (CDRH2) mutant, the binding of these antibodies to mammalian-expressed PD-L1 was analyzed using FACS. Briefly, Raji PDL1 cells were first incubated with 5-fold serial dilutions of humanized antibodies starting at 2 μg/ml at RT for 1 h. After washing with FACS buffer (PBS with 2% PBS), Alexa 488 conjugated antibody was added to each well. against human IgG and incubated at RT for 1 h. MFI for Alexa 488 was evaluated using FACSAriaIII. As shown in FIG. 18, the S60R mutant binds highly efficiently to mammalian cell-expressed PD-L1 with greater activity than the parent Hu1210-41 antibody. This S60R mutant was then used as the parent antibody for further mutational analysis as described below and is designated WT.

For antibody construction, four anti-PD-L1 monoclonal antibody sub-libraries were created using any of the following strategies. According to strategy 1, mutagenesis of the heavy chain variable domain of VH CDR3 or VL-CDR3 was performed by highly randomized mutations. According to strategy 2, two combined CDR libraries consisting of (VH-CDR3, VLCDR3 and VL-CDR1) or (VH-CDR1, VH-CDR2 and VL-CDR2) were generated using mutation controlled walking CDR screening.

Biopanning: Phage panning methods have been adapted by shortening the duration of incubation/binding prior to washing under stringent conditions. Briefly, 100 μl of magnetic streptavidin beads (Invitrogen, USA) were blocked with 1 ml of MFBS for 1 h at room temperature. In another tube, the phage library was pre-incubated (5 x 10 ^L 11~12 for each round) with 100 μl of magnetic streptavidin beads in 1 ml of MPBS to remove unwanted binders. A magnetic particle concentrator was used to separate phage and pellets. Biotinylated PD-L1 protein was added to the phage, incubated for 2 h at room temperature, and gently mixed with a top-drive shaker. The phage-bearing beads from the solution were separated in a magnetic particle concentrator; the supernatant was discarded. The beads were washed with fresh wash buffer, washed 10 times with PBS and 10 times with PBS (pH 7.4). 0.8 ml of 0.25% trypsin in PBS (Sigma, USA) was added and incubated for 20 min at 37°C to elute the phage. The resulting phage was titrated and the phage was rescued for the next round of panning, with a decrease in antigen concentration from round to round.

ELISA screening and binding/dissociation rate ranking Clones were selected and induced from the desired panning products; carried out ELISA ELISA of phage for primary screening; positive clones were analyzed by sequencing; found unique hotspots. In table. 14 shows the identified mutations. As shown below, FGK residues in CDRH3 are hotspot residues that provide improved antibodies.

Table 14

Mutations in CDR

CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 WT* SYDMS TISDAGGYIYYRDSVKG EFGKRYALDY KASQDVTPAVA STSSRYT QQHYTTPLT SEQID NO: 1 116 3 4 5 6 VZ — — — - TO-------- — M-------- C4 — — --------- _s ------W---- ---S----- IN 1 — — -IFN------- — — — wb — — -LPW------ — — — NW — — -LHF------ — — — Sat — — -LYF------ — — — A1 — — -LLH------ — — — A2 — — -LRG------ — — — AZ — — — — — ---SDA---

* WT differs from Hu1210-41 by replacing S60R (Kabat numbering) in the heavy chain to increase affinity.

The amino acid sequences of the variable regions of these antibodies are shown in table. 15 below.

- 52 043231

Antibody sequences

Table 15

Name Subsequence SEQID NO: WT-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS 141 WT-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 142 B3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS 143 B3-Vk DIQMTQSPSSLSASVGDRVTITCKAKQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCMQHYTTPLTFGQ GTKLEIK 144 C4-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDSWGQGTTVTVSS 145 C4-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVWPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPLTFGQ 146 GTKLEIK Bl-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREI FNRYALDYWGQGTTVTVSS 147 Bl-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 148 B6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL PWRYALDYWGQGTTVTVS S 149 B6-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 150 C3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL HFRYALDYWGQGTTVTVS S 151 C3-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 152 C6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL YFRYALDYWGQGTTVTVS S 153 C6-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 154 Al-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL LHRYALDYWGQGTTVTVSS 155 Al-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 156 A2-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL RGRYALDYWGQGTTVTVSS 157 A2-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK 158 A3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVS S 159 A3-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSDAPLTFGQ GTKLEIK 160

Example 14 Antigen-binding properties of antibodies to PD-L1.

As shown in Table. 14 and 15, a total of 9 unique clones were characterized and converted to full length IgG.

The ability to bind to recombinant human PD-L1.

To assess antigen-binding activity, antibodies were tested by ELISA ELISA. Briefly, microtiter plates were coated with human PD-L1-Fc protein at a concentration of 2 µg/ml in PBS, added at 100 µl/well, at 4°C overnight, after which the additions were blocked.

- 53 043231 100 µl/well 5% BSA. 4-fold dilutions of humanized antibodies, starting at 10 μg/ml, were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at RT. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm. As shown in FIG. 19, all humanized antibodies showed superior binding efficiency to human PD-L1, with B6 and C3 showing better results than the original wild-type (WT) clone.

The ability to bind to mammalian expressed human PD-L1.

To assess the antigen-binding capacity of antibodies, their binding to mammalian-expressed PD-L1 was analyzed using FACS. Briefly, PDL1-Raji cells were first incubated with 5-fold serial dilutions of humanized antibodies, starting at 2 μg/ml, at RT for 1 h. After washing with FACS buffer (PBS with 2% PBS), Alexa-conjugated 488 anti-human IgG antibody and incubated at RT for 1 h. MFI Alexa 488 was evaluated using FACSAriaIII. As shown in FIG. 20, B6 binds highly efficiently to mammalian cell-expressed PD-L1 with greater activity than the original wild-type (WT) antibody.

Affinity ranking of humanized antibodies using Biacore.

To study the binding kinetics of the humanized antibody in this example, affinity ranking was performed using Biacore. As shown in Table. 16, B6, C3, C6, A1 and A3 showed higher affinity compared to the original wild-type (WT) antibody.

Table 16

Affinity ranking

Antibody ka (1/ms) kd (1/s) KD (M) wt C77E+05 4.64Е-04 2.63Е-09 VZ 1D9E+05 2.96E-04 2.49Е-09 C4 1DZE+05 5.06Е-04 4.50Е-09 IN 1 C63E+05 2.61E-04 Ts60E-09 wb 2.42E+05 2.46Е-04 Ts02E-09 sz 2D8E+05 2.99E-04 Ts37E-09 C6 2.06E+05 3.34Е-04 C63E-09 A1 2.03E+05 2.76Е-04 Ts36E-09 A2 C87E+05 4.75Е-04 2.55Е-09 AZ 2D8E+05 3.24Е-04 Ts49E-09

Example 15 Cellular function of anti-PDL1 antibodies.

hPD-1 expressing Jurkat cells were used to test the ability of anti-PDL1 antibodies to stimulate a T cell response. Briefly, Jurkat is a human T-cell leukemia cell line that can produce IL-2 upon TCR stimulation. In this assay, Jurkat cells transfected with the human PD-1 gene using lentivirus were used as responder cells. Raji-PDL1 cells were used as antigen presenting cells (APCs). Staphylococcal enterotoxins (SE) are used to stimulate the TCR signal. In this system, ectopically expressed huPDL1 can suppress SE-stimulated IL-2 production by Jurkat cells, while anti-PDL1 antibodies can prevent IL-2 production. Briefly, APCs (2.5x104) were co-cultured with PD-1 expressing Jurkat T cells (1x105) when stimulated with SE. Anti-PDL1 antibodies were added at the beginning of the culture (starting at 100 nM, serial dilutions 1:4 for 8 doses). After 48 hours, the production of IL-2 in the culture supernatant was assessed using ELISA ELISA. As shown in FIG. 21, the B6 monoclonal antibody was more active than the original wild-type (WT) antibody.

Example 16 Mixed culture reaction of lymphocytes.

To assess the function of antibodies to PDL1 in vitro, the response of human T cells was evaluated under reaction conditions of a mixed culture of lymphocytes. Briefly, human DC were differentiated from CD 14+ monocytes in the presence of GM-CSF and IL-4 for 7 days. Then, CD4+ T cells isolated from another donor were co-cultured with DC and serial dilutions of a blocking antibody against PD-L1. On day 5 after inoculation, the production of IFN-γ in the culture supernatant was analyzed. The results (FIG. 22) showed that the B6 antibody more actively stimulated IFN-γ production compared to the original wild-type (WT) antibody.

Example 17 In vivo efficacy of an anti-PDL1 antibody in a syngeneic MC38 model.

The humanized PDL1 syngeneic MC38 tumor model was used to evaluate the effect of PDL1 on tumor growth. In this model, the human PDL1 gene was expressed in mouse MC38 cells, and the extracellular domain of the mouse PDL1 gene was replaced with the human PDL1 gene. Accordingly, in this syngeneic model with the humanized MC38 PDL1 gene, the efficacy of an anti-human PDL1 antibody on tumor growth can be assessed. huPDL1 MC38 cells were inoculated subcutaneously

- 54 043231 humanized PDL1 mice. After the tumor had reached a volume of 100-150 m ³ , a total of 6 doses of the original WT antibody and B6 antibody were administered intraperitoneally at a dosage of 3 mg/kg twice a week. The result (FIG. 23) showed that the B6 antibody was more active than the original WT antibody from day 19 to day 26.

* * *

The scope of the present invention is not limited to the specific embodiments described, which are given as separate illustrative examples of individual aspects of the present invention, and any functionally equivalent compositions or methods are within the scope of the present invention. Those skilled in the art will appreciate that various modifications and variations of the methods and compositions of the present disclosure may be implemented without departing from the spirit or scope of the present invention. Accordingly, modifications and variations of the present invention are intended to be covered by the present description, provided that they fall within the scope of the appended claims, as well as their equivalents.

All publications and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually identified as being incorporated by reference.

Claims (16)

1. An antibody or fragment thereof, wherein said antibody or fragment thereof has specificity for human PD-L1 protein and contains a heavy chain variable region (VH) and a light chain variable region (VL), where VH contains (a) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 1; (b) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116; and (c) a VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 117; and VL contains (d) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4; (e) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5; and (f) VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; or VH contains (g) VH CDR1 containing the amino acid sequence shown in SEQ ID NO: 1; (h) VH CDR2 containing the amino acid sequence shown in SEQ ID NO: 116; and (i) VH CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; And VL contains (j) VL CDR1 containing the amino acid sequence shown in SEQ ID NO: 4; (k) VL CDR2 containing the amino acid sequence shown in SEQ ID NO: 5; and (l) a VL CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or 140. 2. An antibody or fragment thereof according to claim 1, wherein said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in in SEQ ID NO: 117, said VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: : 6. 3. An antibody or fragment thereof according to claim 2, wherein VH contains the amino acid sequence shown in SEQ ID NO: 149 and VL contains the amino acid sequence shown in SEQ ID NO: 150. 4. An antibody or fragment thereof according to claim 1, wherein said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence shown in in SEQ ID NO: 3, said VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: : 140. 5. An antibody or fragment thereof according to claim 4, wherein VH contains the amino acid sequence shown in SEQ ID NO: 159 and VL contains the amino acid sequence shown in SEQ ID NO: 160. 6. An antibody or fragment thereof according to claim 1, wherein said VH CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, said VH CDR2 contains the amino acid sequence shown in SEQ ID NO: 116, said VH CDR3 contains the amino acid sequence, - 55 043231 shown in SEQ ID NO: 3, said VL CDR1 contains the amino acid sequence shown in SEQ ID NO: 4, said VL CDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in SEQ ID NO: 5, and said VL CDR3 contains the amino acid sequence shown in in SEQ ID NO: 6. 7. An antibody or fragment thereof according to claim 6, wherein VH contains the amino acid sequence shown in SEQ ID NO: 141 and VL contains the amino acid sequence shown in SEQ ID NO: 142. 8. An antibody or fragment thereof according to any one of claims 1 to 7, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination of the above. 9. An antibody or fragment thereof according to any one of claims 1 to 8, wherein said antibody is a humanized antibody. 10. A bispecific antibody comprising a fragment according to any one of claims 1 to 9 and a second antigen-binding fragment having specificity for a molecular structure on an immune cell. 11. The bispecific antibody of claim 10, wherein said molecule is selected from the group consisting of PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40 , CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR and CD47. 12. A pharmaceutical composition for the treatment of cancer or infection, comprising an antibody or fragment thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier. 13. An isolated cell for the treatment of cancer or infection, comprising one or more polynucleotides encoding an antibody or fragment thereof according to any one of claims 1-10. 14. The use of an antibody or fragment thereof according to any one of claims 1 to 10 for the treatment of cancer or infection. 15. Use according to claim 14, characterized in that said cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer. 16. Use according to claim 14, characterized in that said infection is a viral infection, a bacterial infection, a fungal infection or a parasitic infection.