CN114634567B - Development and application of immunomodulator - Google Patents

Abstract

The present disclosure relates to the development of an immunomodulatory agent that is an antibody or antigen-binding portion thereof that binds PD-L1, and uses thereof. The PD-L1 antibody has high blocking activity on the combination of PD-L1 and PD-1, and the novel bifunctional antibody formed by the PD-L1 antibody and TGFBR2 can release the anti-tumor activity of T cells by blocking the interaction of the PD-L1 and the PD-1, and simultaneously effectively blocks the immunosuppression caused by activating a smad signal channel by TGF-beta in a tumor microenvironment, thereby being beneficial to inducing effective and durable anti-tumor immune response in tumors which are not reacted originally.

Description

Development and application of immunomodulator

Technical Field

The present disclosure relates to the development and use of immunomodulators, in particular, antibodies that bind to programmed death ligand 1 (PD-L1) and uses thereof.

Background

PD-L1 is a cell surface glycoprotein, one of two known ligands for programmed death 1 (PD-1), which is thought to play an important role in immunomodulation and maintenance of peripheral tolerance. Expression of PD-L1 has been observed on the surface of many immune cells, including untreated lymphocytes and activated B and T cells, monocytes and dendritic cells (supra). In addition, PD-L1 mRNA is expressed in non-lymphoid tissues (including vascular endothelial cells, epithelial cells, muscle cells) and in tonsil and placenta tissues.

Although immune checkpoint therapy has achieved long lasting relief in a fraction of patients, currently marketed immune checkpoint inhibitors (including CTLA-4, PD-1 or PD-L1 mab) are effective (Ribas,A.and J.D.Wolchok,Cancer immunotherapy using checkpoint blockade.Science,2018.359(6382):p.1350-1355). studies only in about 15% -40% of patients, and generally have higher levels of tumor antigen-specific cd8+ T cell infiltration in tumor tissue responsive to immunotherapy; tumors that do not respond to immunotherapy, however, often lack internally activated T lymphocytes (Gajewski,T.F.,The Next Hurdle in Cancer Immunotherapy:Overcoming the Non-T-Cell-Inflamed Tumor Microenvironment.Semin Oncol,2015.42(4):p.663-71).

It was found that tgfβ pathway related genes (TGFB 1 and TGFBR 2) expression in tumor tissues were up-regulated in patients with metastatic urothelial cancer who were not responsive to treatment with the PD-L1 antibody atezolizumab. The TGF beta neutralizing antibody and the PD-L1 antibody are combined, so that in a mouse EMT6 breast cancer and MC38 colon cancer model, the CD8+ T cell infiltration and tumor regression can be induced, and the curative effect is obviously better than that of the PD-L1 antibody alone (Mariathasan,S.,et al.,TGFbeta attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.Nature,2018.554(7693):p.544-548).

Another study showed that homozygous mice with WNT, EGFR, p and mutations in the TGF-beta pathway produced colon cancer tumors resembling human immune rejection, with high levels of TGF-beta around tumor cells being produced primarily by tumor-associated fibroblasts. The TGF-beta receptor 1 kinase inhibitor can reduce the number of metastasis of tumor and increase the infiltration of CD4+ T cells and T-bet+ lymphocytes of tumor tissues. The TGF beta inhibitor and the PD-L1 antibody are treated in a combined way, so that obvious immune response can be induced, and the survival time of tumor-bearing mice can be obviously prolonged (Tauriello,D.V.F.,et al.,TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis.Nature,2018.554(7693):p.538-543).

TGF beta is a group of cytokines with a variety of biological functions, including TGF beta 1, TGF beta 2, and TGF beta 3, and has important regulatory effects on cell growth, proliferation, differentiation, apoptosis, migration, and immunity. After TGF-beta dimer binds with type II receptor TGFBR2 and type I receptor TGFBR1 on cell membrane, it induces receptor kinase activation, recruits downstream Smad protein, and gathers in cell nucleus to play a role of transcriptional regulation.

Tgfβ is thought to inhibit tumor growth in the early stages of tumorigenesis and to be converted to tumor promoting factors in the late stages. The signal transduction molecule Smad at the downstream of the TGF-beta receptor can regulate and control the expression of apoptosis genes such as DAPK, GADD45 beta, BIM, SHIP and the like, trigger apoptosis and can also inhibit the generation of tumor blood vessels. However, as mutations accumulate in tumor cells, particularly mutations in the tgfβ receptor or Smad gene downstream thereof, they can lead to reduced inhibition; TGF beta can influence the fibrosis, the vascular production and the immune cell infiltration (Neuzillet,C.,et al.,Perspectives of TGF-beta inhibition in pancreatic and hepatocellular carcinomas.Oncotarget,2014.5(1):p.78-94); of the microenvironment through TGF beta receptors on the surfaces of mesenchymal cells, endothelial cells, immune cells, stem cells derived from bone marrow and other cells in the tumor microenvironment, and can block the differentiation of immature T cells to Th1 cells and promote the transformation of the immature T cells to Treg subgroups, so that the immune escape of tumor cells is caused; tgfβ can also promote epithelial mesenchymal transition, thereby gaining the ability to invade and migrate.

Because of the wide range of biological functions of tgfβ, a new strategy is to utilize tumor targeting antibodies to form fusion proteins with tgfβ inhibitors (e.g., tgfβ receptors), which can target the tumor microenvironment while avoiding serious side effects caused by systemic inhibition of tgfβ.

In chinese patent application CN104334573a, antibodies against EGFR1 and the like are linked to the extracellular domain of type II receptor TGFBR2 to form a fusion protein, and targeting antibodies are used to localize TGFBR2 to tumors to counteract immune tolerance by tgfβ action in the tumor microenvironment.

In chinese patent application CN106103488a, anti-PD-L1 antibodies are linked to the extracellular domain of TGFBR2 via flexible linker G4S, and by counteracting tgfβ immunosuppression, the PD-1/PD-L1 checkpoint is blocked to further release the anti-tumor activity of T cells.

PCT application WO2018205985A1 discloses a method for truncating the N-terminus of the extracellular domain of TGFBR2 linked to a PD-L1 antibody by 14-26 consecutive amino acids to increase the stability or expression of the fusion protein during production.

Disclosure of Invention

The inventors have found a novel murine PD-L1 antibody with high blocking activity and its humanized antibody, and linked the PD-L1 antibody to a more stable TGFBR2 isomer (mutant) via a flexible linker to form a novel bifunctional antibody. The novel bifunctional antibody can release the anti-tumor activity of T cells by blocking the interaction of PD-L1 and PD-1, effectively block the immunosuppression caused by activating a smad signal channel by TGF-beta in a tumor microenvironment, and is beneficial to inducing effective and durable anti-tumor immune response in tumors which do not react originally.

In one aspect, the disclosure provides an antibody or antigen-binding portion thereof that binds PD-L1.

In one aspect, the disclosure provides a bispecific or multispecific antibody molecule comprising an antibody, or antigen-binding portion thereof, as described previously that binds PD-L1.

In one aspect, the present disclosure provides an antibody fusion protein comprising:

a) At least one heavy chain variable domain of an antibody selected from the group consisting of the aforementioned antibodies that bind PD-L1; and

B) Human TGFbRII or a fragment thereof capable of binding tgfβ.

In one aspect, the disclosure provides nucleic acids encoding the aforementioned antibodies or antigen-binding portions thereof that bind PD-L1, bispecific or multispecific antibody molecules, or antibody fusion proteins.

In one aspect, the disclosure provides a vector comprising the aforementioned nucleic acid.

In one aspect, the present disclosure provides a cell comprising the aforementioned vector.

In one aspect, the present disclosure provides a composition comprising the foregoing PD-L1-binding antibody or antigen-binding portion thereof, a bispecific or multispecific molecule, an antibody fusion protein, a nucleic acid, a vector, and/or a cell, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides an antibody-drug conjugate comprising the foregoing PD-L1-binding antibody or antigen-binding portion thereof, a bispecific or multispecific molecule, or an antibody fusion protein that is covalently linked to a therapeutic moiety.

In one aspect, the disclosure provides a kit comprising the foregoing binding PD-L1 antibody or antigen-binding portion thereof, a bispecific or multispecific molecule, an antibody fusion protein, a nucleic acid, a vector, and/or a cell.

In one aspect, the present disclosure provides a method of treating a PD-L1-related disorder comprising the steps of: administering to the subject a therapeutically effective amount of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, and/or compositions.

In one aspect, the present disclosure provides the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells and/or pharmaceutical compositions in the manufacture of a medicament or kit for treating a PD-L1-related disorder in a subject.

Drawings

FIG. 1 shows the binding of humanized antibodies to PD-L1.

FIG. 2 shows the blocking of PD-1 and PD-L1 binding by humanized antibodies.

FIG. 3 shows the binding of humanized antibodies to PD-L1 on cell membranes.

FIG. 4 shows that humanized antibodies block the binding of human PD-L1 to PD-1 on cell membranes.

FIG. 5 shows that humanized antibodies block PD-L1 signaling pathways.

Figure 6 shows a memory response test of humanized antibodies.

FIG. 7 shows the binding of PD-L1 humanized antibodies to human PD-L1.

FIG. 8 shows binding of PD-L1 humanized antibodies to cynomolgus monkey PD-L1.

FIG. 9 shows a sequence comparison of TGFbRII different splice isomers.

FIG. 10 shows the results of mass spectrometry detection of CHO stable cell expression.

FIG. 11 shows the trap end sequence of PDL1-TGFbRII fusion proteins.

FIG. 12 shows the mass spectrum detection results of PDL1-TGFbRII antibody fusion proteins.

FIG. 13 shows that PDL1-TGFbRII antibody fusion proteins bind both PD-L1 and TGF-beta.

FIG. 14 shows the binding of PDL1-TGFbRII antibody fusion proteins to PD-L1.

FIG. 15 shows blocking of PD-1 and PD-L1 binding by PDL1-TGFbRII antibody fusion proteins.

FIG. 16 shows the binding of PDL1-TGFbRII antibody fusion proteins to PD-L1.

FIG. 17 shows FACS detection of PDL1-TGFbRII blocking binding of human PD-1/PD-L1.

FIG. 18 shows the blocking of PD1/PD-L1 signaling pathway by PDL1-TGFbRII antibody fusion proteins.

FIG. 19 shows HEK293-SBE luc luciferase reporter cells.

FIG. 20 shows inhibition of TGF-beta 1-induced Smad activation by PDL 1-TGFbRII.

FIG. 21 shows the inhibition of TGF-beta 3 induced NRK-49F collagen production by PDL 1-TGFbRII.

FIG. 22 shows the inhibition of antibody fusion proteins PDL1-TGFbRII in a humanized mouse engrafting tumor model.

Detailed Description

I. Definition of the definition

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.

The term "anti-PD-L1 antibody", "anti-PD-L1", "PD-L1 antibody" or "PD-L1-binding antibody" refers to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of the anti-PD-L1 antibody to an unrelated non-PD-L1 protein is less than about 10% of the binding of the antibody to PD-L1, as measured by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds PD-L1 has a dissociation constant (K _D) of ∈1 μM, ∈100nM, +.10 nM, +.1 nM, +.0.1 nM, +.0.01 nM, or +.0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-PD-L1 antibodies bind PD-L1 epitopes that are conserved among PD-L1 from different species.

The term "TGF-beta receptor" or "tgfβr" is used herein to encompass all three subtypes of the tgfβr family (i.e., tgfβri, tgfβrii, tgfβriii). Tgfβ receptors are characterized by serine/threonine kinase activity and exist in several different isoforms (which may be homodimers or heterodimers).

The term "TGF-beta RII" refers to any natural TGF-beta RII (transforming growth factor beta receptor 2; also referred to as TGFR-2 or TGF-beta RII) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length", unprocessed TGFBR2 as well as any form of TGFBR2 derived from processing in a cell. The term also encompasses naturally occurring variants of TGFBR2, such as splice variants or allelic variants.

As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

The phrase "substantially identical" in reference to an antibody chain polypeptide sequence is understood to mean an antibody chain that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. In terms of nucleic acid sequences, the term is understood to mean nucleotide sequences which exhibit at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

Sequence "identity" or "identity" has art-recognized meanings and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., ：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Pres,New York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991). although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrilo, H. & Lipman, d., SIAM J APPLIED MATH: 1073 (1988)).

"Substitution type" variants are those in which at least one amino acid residue in the natural sequence has been removed and a different amino acid has been inserted at the same position. The substitution may be single, wherein only one amino acid in the molecule is substituted; or may be plural, in which the same molecule has two or more amino acids substituted. Multiple substitutions may be located at successive positions. Also, an amino acid may be substituted with multiple residues, where such variants include both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted at a particular position immediately adjacent to a native sequence. By immediately adjacent amino acid is meant a linkage to the alpha-carboxyl or alpha-amino functionality of the amino acid. A "deleted" variant is a variant in which one or more amino acids in the natural amino acid sequence have been removed. Typically, a deletion variant has one or two amino acids deleted in a particular region of its molecule.

With respect to the variable domains of antibodies, the term "variable" refers to portions of the relevant molecules that have wide sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. But the variability is not evenly distributed throughout the variable domains of the antibodies. Variability is focused on three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR 3) or hypervariable regions, all located within the variable domains of the light and heavy chains. The more conserved portions of the variable domains are referred to as Framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains comprises four FR regions, which adopt predominantly a β -sheet configuration, joined by three CDRs, which form a loop that connects the β -sheet structure and in some cases forms part of the β -sheet structure. The CDRs of each chain are typically joined in proximity by FR regions and aid in the formation of antibody target binding sites (epitopes or determinants) by means of CDRs from other chains (see Kabat et al Sequences of Proteins of Immunological Interest, nationalInstitute of Health, bethesda, MD (1987)). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. One CDR may have the ability to specifically bind to a cognate epitope.

As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. examples of antibody fragments include, but are not limited to, fab ', F (ab ') ₂, single chain Fv (scFv), fv, dsFv, diabodies, fd, and Fd ' fragments, and other fragments, including modified fragments (see, e.g., ,Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov)., which fragments may include multiple chains linked together, e.g., by disulfide bonds and/or by peptide linkers. antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacement of the corresponding region), obtains antibodies that immunospecifically bind (i.e., exhibit a Ka of at least or at least about 10 ⁷-10⁸ M-1) to an antigen. A "functional fragment" or "analog of an anti-PD-L1 antibody" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. as used herein, a functional fragment is generally synonymous with an "antibody fragment" and, in the case of an antibody, may refer to a fragment, such as Fv, fab, F (ab') ₂, and the like, that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. The "Fv" fragment consists of a dimer (V _H-V_L dimer) of one heavy chain variable domain and one light chain variable domain formed by non-covalent binding. In this configuration, the three CDRs of each variable domain interact to determine the target binding site on the V _H-V_L dimer surface, as is the case with intact antibodies. The six CDRs together confer target binding specificity to the intact antibody. But even a single variable domain (or half of an Fv comprising only 3 target-specific CDRs) can have the ability to recognize and bind a target.

As used herein, the term "bispecific" (Bispecific antibody, bsAb) refers to an antibody and/or antigen-binding molecule that is capable of specifically binding to two different antigenic determinants, typically, a bispecific antibody and/or antigen-binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, in particular two antigenic determinants expressed on two different cells.

As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies comprising antibodies having a plurality of different sequences. Monoclonal antibodies can be prepared by a number of well known methods (Smith et al (2004) J.Clin. Pathol.57,912-917; and Nelson et al, J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to produce antibodies from clonal populations of host cells in vitro by transforming the host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.

As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) produced by fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell. As known to those of ordinary skill in the art, hybridomas can proliferate and continue to supply for the production of specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, e.g., harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of a hybridoma.

As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and a hinge region, e.g., an antibody naturally produced by B cells by antibody secretion and an antibody synthetically produced with the same domains.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

By "humanized" antibody is meant a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., fv, fab, fab ', F (ab') ₂ or other antigen-binding subsequence of the antibody) that contains minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

Furthermore, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations, such as PCR-mediated mutations, can be introduced, and their effect on antibody binding or other functional properties can be assessed using in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions or deletions. In addition, mutations within the CDRs typically do not exceed one or two. Thus, humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations within the CDRs.

As used herein, the term "CDR" refers to the complementarity determining region (complementarity-DETERMINING REGION), known as an antibody molecule, having 3 CDRs per heavy and light chain. CDRs are also known as hypervariable regions and are found in the variable regions of each of the heavy and light chains of antibodies with very high variability sites in the primary structure of the CDRs. In the present specification, CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the heavy chain, and CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the light chain. These sites are adjacent to each other in tertiary structure and determine the specificity of the antigen to which the antibody binds.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants generally comprise chemically active surface groupings of molecules such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody and the antibody binding site of the antigen. The antigen may be an isolated antigen or present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen binds the antigen with an affinity constant Ka (or dissociation constant (Kd) of 1×10 ^-7 M or 1×10 ^-8 M or less) of about or 1×10 ⁷M^-1 or 1×10 ⁸M^-1 or more. Affinity constants can be determined by standard kinetic methods of antibody reaction, e.g., immunoassays, surface Plasmon Resonance (SPR) (Rich and Myszka (2000) curr. Opin. Biotechnol 11:54; englebiene (1998) analysis 123:1599), isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., paul, ed., fundamental Immunology,2nd ed., RAVEN PRESS, new York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619) describing exemplary SPR and ITC methods for calculating binding affinity of antibodies. Instruments and methods for detecting and monitoring binding rates in real time are known and commercially available (see ,BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences;Malmqvist(2000)Biochem.Soc.Trans.27:335).

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules as well as RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen binding fragments.

As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

Also provided are "conservative sequence modifications" of the sequences described in the sequence listing herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of antibodies encoded by or containing the nucleotide sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the predicted nonessential amino acid residue in an anti-PD-L1 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, e.g.) Brummell et al.,Biochem.32:1180-1187(1993);Kobayashi et al.,Protein Eng.12(10):879-884(1999);Burks et al.,Proc.Natl.Acad.Sci.USA 94:412-417(1997)).

Alternatively, in another embodiment, mutations may be randomly introduced along all or a portion of the anti-PD-L1 antibody coding sequence, for example, by saturation mutagenesis, and the resulting modified anti-PD-L1 antibodies may be screened for improved binding activity.

As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, coomassie blue staining after gel electrophoresis, lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, heLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acids encoding polypeptides. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplifying the nucleic acid or for expressing/displaying the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

As used herein, vectors also include "viral vectors" or "viral vectors". The vector of the virus is an engineered virus operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

As used herein, an "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of a host cell.

As used herein, "treating" an individual with a disease or condition means that the symptoms of the individual are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also includes any antibody or antigen-binding fragment thereof provided, and any pharmaceutical use of the compositions provided herein.

As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that alters, generally improves or ameliorates symptoms of, or cures a disease or condition.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder.

As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "patient" refers to a mammal, such as a human.

Detailed description of the preferred embodiments

In one aspect, the disclosure provides an antibody or antigen binding portion thereof that binds PD-L1 comprising a light chain CDR selected from the amino acid sequences SEQ ID NO:2, 3, 4, 15 or any variant thereof and/or a heavy chain CDR selected from the amino acid sequences SEQ ID NO:7, 8, 9, 20 or any variant thereof.

An antibody or antigen binding portion thereof according to the previous aspect comprising a light chain CDR1 selected from the amino acid sequences SEQ ID No. 2, 15 or any variant thereof, a light chain CDR2 selected from the amino acid sequences SEQ ID No. 3 or any variant thereof, a light chain CDR3 selected from the amino acid sequences SEQ ID No. 4 or any variant thereof; and/or a heavy chain CDR1 selected from the amino acid sequences SEQ ID NO. 7 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequences SEQ ID NO. 8, 20 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequences SEQ ID NO. 9 or any variant thereof.

An antibody or antigen-binding portion thereof according to the previous aspect comprising a CDR combination of a heavy chain and a light chain selected from the group consisting of:

(1) Light chain CDR, CDR and CDR sequences comprising SEQ ID NO 2,3,4, respectively, and/or heavy chain CDR, CDR and CDR sequences comprising SEQ ID NO 7, 8, 9, respectively;

(2) Light chain CDR, CDR and CDR sequences comprising SEQ ID NO 2,3, 4, respectively, and/or heavy chain CDR, CDR and CDR sequences comprising SEQ ID NO 7, 20, 9, respectively;

(3) Light chain CDR, CDR and CDR sequences comprising SEQ ID NO 15, 3, 4, respectively, and/or heavy chain CDR, CDR and CDR sequences comprising SEQ ID NO 7, 8, 9, respectively;

(4) Light chain CDR, CDR and CDR sequences comprising SEQ ID NO 15, 3, 4, respectively, and/or heavy chain CDR, CDR and CDR sequences comprising SEQ ID NO 7, 20, 9, respectively.

The antibody or antigen binding portion thereof according to the previous aspect, comprising a light chain variable region selected from the amino acid sequences SEQ ID NOs 1, 11, 13 and/or a heavy chain variable region selected from the amino acid sequences SEQ ID NOs 6, 16, 18 or any variant thereof.

In some preferred embodiments of the present disclosure, it comprises a light chain variable region of amino acid sequence SEQ ID NO. 1 or any variant thereof, and a heavy chain variable region of amino acid sequence SEQ ID NO. 6 or any variant thereof.

In some preferred embodiments of the present disclosure, it comprises a light chain variable region of amino acid sequence SEQ ID NO. 11 or any variant thereof, and a heavy chain variable region of amino acid sequence SEQ ID NO. 16 or any variant thereof.

In some preferred embodiments of the present disclosure, it comprises a light chain variable region of amino acid sequence SEQ ID NO. 11 or any variant thereof, and a heavy chain variable region of amino acid sequence SEQ ID NO. 18 or any variant thereof.

In some preferred embodiments of the present disclosure, it comprises a light chain variable region of amino acid sequence SEQ ID NO. 13 or any variant thereof, and a heavy chain variable region of amino acid sequence SEQ ID NO. 16 or any variant thereof.

In some preferred embodiments of the present disclosure, it comprises a light chain variable region of amino acid sequence SEQ ID NO. 13 or any variant thereof, and a heavy chain variable region of amino acid sequence SEQ ID NO. 18 or any variant thereof.

In one aspect, the disclosure provides a bispecific or multispecific molecule comprising the antibody, or antigen-binding portion thereof, that binds PD-L1.

In one aspect, the present disclosure provides a PDL1-TGFbRII antibody fusion protein comprising:

a) A fusion polypeptide portion comprising a heavy chain portion of an antibody comprising a heavy chain or an antigen binding portion of the heavy chain, and a TGFbRII portion, said TGFbRII portion comprising a tgfβ binding portion of TGFbRII or said TGFbRII; and

B) A light chain portion of an antibody comprising a light chain of the antibody or an antigen binding portion of the light chain.

Wherein the antibody is the PD-L1-binding antibody of any one of claims 1-4.

In some preferred embodiments of the present disclosure, the C-terminus of the heavy chain portion of the antibody is linked to the N-terminus of the TGFbRII portion. In some preferred embodiments of the present disclosure, the connection is through a linker. In some preferred embodiments of the present disclosure, the linker is G4S or G (G4S) 3G.

In some preferred embodiments of the present disclosure, the TGFbRII comprises a sequence selected from the amino acid sequences SEQ ID NO. 21, 23, 25, 27, 29 or any variant thereof.

In some preferred embodiments of the present disclosure, the tgfβ binding portion of TGFbRII comprises a TGFbRII ectodomain fragment. In some preferred embodiments of the present disclosure, the ectodomain fragment comprises a fragment selected from amino acids 1-200 of the ectodomain. In some preferred embodiments of the present disclosure, the extracellular domain fragment is an amino acid fragment at positions 24-159 shown in SEQ ID NO.23 or a variant thereof. In some preferred embodiments of the present disclosure, the variant has a Lys30Glu mutation and/or an Asn34Ser mutation. In some preferred embodiments of the present disclosure, the ectodomain fragment is an amino acid fragment at positions 1-124 shown in SEQ ID NO.27 or a variant thereof. In some preferred embodiments of the disclosure, the variant has an Asn7Ser mutation.

In some preferred embodiments of the present disclosure, the fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises a sequence selected from the amino acid sequences SEQ ID NO. 31, 35, 37, 39, 41 or any variant thereof; the light chain portion of the antibody comprises the amino acid sequence SEQ ID NO. 33.

In a specific embodiment, the fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises the amino acid sequence SEQ ID NO. 31 and the light chain portion of the antibody comprises the amino acid sequence SEQ ID NO. 33.

In a specific embodiment, the fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises the amino acid sequence SEQ ID NO. 35 and the light chain portion of the antibody comprises the amino acid sequence SEQ ID NO. 33.

The fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises an amino acid sequence SEQ ID NO. 37 and the light chain portion of the antibody comprises an amino acid sequence SEQ ID NO. 33.

In a specific embodiment, the fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises the amino acid sequence SEQ ID NO. 39 and the light chain portion of the antibody comprises the amino acid sequence SEQ ID NO. 33.

In a specific embodiment, the fusion polypeptide of the PDL1-TGFbRII antibody fusion protein comprises the amino acid sequence SEQ ID NO. 41 and the light chain portion of the antibody comprises the amino acid sequence SEQ ID NO. 33.

In some preferred embodiments of the present disclosure, the antibody or antigen binding portion thereof is humanized.

In some embodiments, an antibody or antigen-binding portion thereof that binds PD-L1 has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an antibody or antigen-binding portion thereof of any of the foregoing aspects.

In one aspect, the disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof, a bispecific or multispecific molecule, an antibody fusion protein, or having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto of any one of the preceding aspects.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of antibody light chain variable region nucleotide sequences SEQ ID NO. 5, 12, 14 or any variant thereof, and/or a sequence selected from the group consisting of heavy chain variable region nucleotide sequences SEQ ID NO. 10, 17, 19 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises the sequence of the antibody light chain variable region nucleotide sequence SEQ ID NO. 5 or any variant thereof, and the sequence of the heavy chain variable region nucleotide sequence SEQ ID NO. 10 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises the sequence of the antibody light chain variable region nucleotide sequence SEQ ID NO. 12 or any variant thereof, and the sequence of the heavy chain variable region nucleotide sequence SEQ ID NO. 17 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises the sequence of the antibody light chain variable region nucleotide sequence SEQ ID NO:12 or any variant thereof, and the sequence of the heavy chain variable region nucleotide sequence SEQ ID NO:19 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises the sequence of the antibody light chain variable region nucleotide sequence SEQ ID NO. 14 or any variant thereof, and the sequence of the heavy chain variable region nucleotide sequence SEQ ID NO. 17 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises the sequence of the antibody light chain variable region nucleotide sequence SEQ ID NO. 14 or any variant thereof, and the sequence of the heavy chain variable region nucleotide sequence SEQ ID NO. 19 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequences SEQ ID NO. 32, 36, 38, 40, 42, or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34, or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequence of SEQ ID NO. 32 or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequence of SEQ ID NO. 36 or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequence of SEQ ID NO. 38 or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequence of SEQ ID NO. 40 or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34 or any variant thereof.

In some preferred embodiments of the present disclosure, the nucleic acid comprises a sequence selected from the group consisting of the fusion polypeptide partial nucleotide sequence of SEQ ID NO. 42 or any variant thereof, and the light chain partial nucleotide sequence of the antibody comprises a sequence of SEQ ID NO. 34 or any variant thereof.

In one aspect, the disclosure provides a vector comprising a nucleic acid of any one of the preceding aspects.

In one aspect, the disclosure provides a cell comprising the vector of any one of the preceding aspects.

In one aspect, the present disclosure is a composition comprising the aforementioned antibody or antigen-binding portion thereof, a bispecific or multispecific molecule, an antibody fusion protein, a nucleic acid, a rights vector, and/or a cell, and a pharmaceutically acceptable carrier.

The antibodies of the present disclosure are useful for treating, preventing and/or ameliorating a disease or disorder or condition, such as cancer, autoimmune disease or viral infection, and/or ameliorating at least one symptom associated with such a disease, disorder or condition. In some embodiments of the present disclosure, the antibodies described herein are useful for treating subjects having primary or recurrent cancers, including, for example, renal cell carcinoma, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, gastric cancer, breast cancer, head and neck cancer, non-small cell lung cancer, brain cancer, multiple myeloma, and melanoma. The antibodies are useful for treating early or late symptoms of cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure are useful for treating metastatic cancer. Antibodies can be used to reduce or inhibit or reduce tumor growth in both solid tumors and hematological cancers. In certain embodiments, the antibodies can be used to prevent recurrence of a tumor. In certain embodiments, treatment with an antibody or antigen-binding fragment thereof of the present disclosure can result in greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of a tumor in a subject. In certain embodiments, the antibodies can be used to increase survival of a subject suffering from cancer.

In certain embodiments, the antibodies of the disclosure are useful for treating a subject suffering from a chronic viral infection. In some embodiments, the antibodies of the disclosure can be used to reduce viral titers and/or rescue depleted T cells in a host. In one embodiment, the antibodies of the present disclosure, or antigen binding fragments thereof, may be administered at therapeutic doses to a patient infected with Human Immunodeficiency Virus (HIV) or Human Papilloma Virus (HPV) or hepatitis b/c virus (HBV/HCV). In a related embodiment, the antibodies of the present disclosure, or antigen binding fragments thereof, are useful for treating an infection caused by a Simian Immunodeficiency Virus (SIV) in a simian subject (e.g., cynomolgus monkey). In another embodiment, the antibodies or fragments thereof of the present disclosure are useful for treating chronic viral infections caused by lymphocytic choriomeningitis virus (LCMV).

In certain embodiments, blocking antibodies of the present disclosure may be administered in a therapeutically effective amount to a subject suffering from cancer or a viral infection.

In certain embodiments, the antibodies of the present disclosure are useful for treating autoimmune diseases, including but not limited to: alopecia areata, autoimmune hepatitis, celiac disease, graves 'disease, guillain-barre syndrome, hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, sjogren's syndrome, systemic lupus erythematosus, white spots, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and wegener's granulomatosis. In certain embodiments, the activated antibodies of the present disclosure are useful for treating a subject suffering from an autoimmune disease.

One or more antibodies of the present disclosure may be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

Also contemplated herein is prophylactic use of one or more antibodies of the disclosure to patients at risk of developing a disease or disorder (e.g., cancer and chronic viral infection).

In one embodiment of the present disclosure, the antibodies of the present disclosure are used to prepare a pharmaceutical composition for treating a patient suffering from cancer, an autoimmune disease, or a viral infection. In another embodiment of the present disclosure, the antibodies of the present disclosure are used as an adjunct therapy with any other agent or any other therapy known to those of skill in the art for treating cancer, autoimmune disease, or viral infection.

Anti-PD-L1 antibodies of the present disclosure and any other therapeutic agent that may be advantageously combined with the antibodies of the present disclosure or biologically active fragments of the antibodies of the present disclosure.

The antibodies of the present disclosure can be synergistically combined with one or more anti-cancer drugs or therapies for treating cancers, including, for example, renal cell carcinoma, ovarian cancer, prostate cancer, colorectal cancer, non-small cell lung cancer, and melanoma. Contemplated herein are the use of an anti-PD-L1 antibody of the present disclosure in combination with an immunostimulatory and/or immune-supporting therapy to inhibit tumor growth and/or increase survival of a cancer patient. The immunostimulatory therapies include direct immunotherapy that activates an immune response by "releasing the brake (RELEASING THE brake)" or "stepping on the accelerator (stepping on the gas)" on the suppressed immune cells to boost immune cell activity. Examples include targeting other checkpoint receptors, vaccination and adjuvants. The immune-supported form may enhance the antigenicity of a tumor by promoting immunogenic cell death, inflammation, or have other indirect effects of promoting an anti-tumor immune response. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

The PDL1-TGFbRII antibody fusion proteins described in the present disclosure can be used to treat cancer or reduce tumor growth in a patient. Exemplary cancers include: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharynx, testicle, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, fibrosarcoma of the carinius skin, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

Cancers or tumors to be treated with PDL1-TGFbRII antibody fusion proteins can be selected based on the expression or elevated expression of PD-L1 and tgfβ in the tumor, the correlation of the expression levels thereof with prognosis or disease progression, and preclinical and clinical experience with respect to the sensitivity of the tumor to treatment targeting PD-L1 and tgfβ. Such cancers or tumors include, but are not limited to, colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bladder, head-neck, liver, non-small cell lung cancer, melanoma, merkel cell carcinoma, and mesothelioma.

In various embodiments, one or more antibodies of the disclosure can be used in combination with: a second antibody directed against PD-L1, an antibody directed against PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of other T cell co-inhibitors or ligands (e.g., an antibody directed against CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), a second antibody directed against PD-L1, an antibody directed against PD-L1, a second antibody directed against PD-L4, a third antibody directed against iar 1, a fourth antibody directed against iar 1, a fifth antibody directed against iar 2B4, a sixth antibody directed against iar 2B, Indoleamine-2, 3-dioxygenase (IDO) inhibitors, vascular Endothelial Growth Factor (VEGF) antagonists [ e.g., "VEGF-traps", such as aflibercept or other VEGF-inhibiting fusion proteins as described in US 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab) or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib) ], ang2 inhibitors (e.g., nesvacumab), transforming growth factor beta (tgfβ) inhibitors, epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., Erlotinib, cetuximab), agonists of co-stimulatory receptors (e.g., agonists of glucocorticoid-induced TNFR-related proteins), antibodies to tumor-specific antigens (e.g., CA9, CA125, melanoma-related antigen 3 (MAGE 3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA 19-9), vaccines (e.g., BCG, cancer vaccines), adjuvants that enhance antigen presentation (e.g., granulocyte-macrophage colony stimulating factor), bispecific antibodies (e.g., CD 3X CD20 bispecific antibodies), PSMA x CD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), antibody-drug conjugates (ADC) (e.g., anti-CD 19-DM4 ADC and anti-DS 6-DM4 ADC), anti-inflammatory agents (e.g., corticosteroids and non-steroidal anti-inflammatory agents), food additives (e.g., antioxidants), or any palliative treatment. In certain embodiments, the anti-PD-L1 antibodies of the present disclosure can be used in combination with cancer vaccines (including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, and the like) to boost anti-tumor responses. Examples of cancer vaccines that can be used in combination with the anti-PD-L1 antibodies of the present disclosure include: a MAGE3 vaccine for melanoma and bladder cancer, a MUC1 vaccine for breast cancer, EGFRv3 (e.g., rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancer). In certain embodiments, the anti-PD-L1 antibodies of the present disclosure can be combined with a food additive (e.g., an antioxidant) or any palliative treatment to treat cancer.

In some embodiments, the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs".

In some embodiments, the antibody is conjugated to a cytotoxic moiety. The cytotoxic moiety may for example be selected from the following: paclitaxel; cytochalasin B; bacitracin D; ethidium bromide; ipecac; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracene diketones; tubulin inhibitors such as maytansine or an analogue or derivative thereof; antimitotics such as monomethyl auristatin E or F or analogues or derivatives thereof; dolastatin 10 or 15 or an analog thereof; irinotecan or an analog thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; antimetabolites, such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabine, 5 fluorouracil, decdiazine, hydroxyurea, asparaginase, gemcitabine or cladribine; alkylating agents, such as dichloromethyldiethylamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; docarmycin a, docarmycin SA, rapamycin (CC-1065) or an analogue or derivative thereof; antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, photomycin, mitomycin, mitoxantrone, plicin, diazepam (AMC); pyrrolo [2,1-c ] [1,4] -benzodiazepine (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments and hybrid molecules thereof, ricin such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, shiga-like toxins such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, pseudomonas exotoxin, arolin, saporin, kaplan, gelonin, abrin A chain, kaplan A chain, alpha-sarcin, aleurites fordii protein, caryophyllin protein, pokeweed proteins such as PAPI, PAPII and PAP-S, balsam pear (momordica charantia) inhibitor, bixin, crotonin, feverfew (sapaonaria officinalis) inhibitor, gelonin, mitomycin, restrictocin, phenomycin and enomycin toxin; ribonuclease (RNase); DNase I, staphylococcal endotoxin a; pokeberry antiviral protein; diphtheria toxin and pseudomonas endotoxin.

In some embodiments, the antibody is conjugated to a cytotoxic moiety selected from the group consisting of: anthracyclines, maytansine, calicheamicin, duocarmycin, rapamycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

In some embodiments, the antibody is conjugated to an anthracycline or analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to a docamycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to rapamycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E, or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin F, or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo [2,1-c ] [1,4] -benzodiazepine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan, or an analog, derivative, or prodrug thereof.

In some embodiments, the antibody is conjugated to a cytokine (e.g., IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、IL-28b、IL-29、KGF、IFNa、IFN3、IFNy、GM-CSF、CD40L、Flt3 ligand, stem cell factor, ansetron, and TNFa).

In some embodiments, the antibody is conjugated to a radioisotope or radioisotope-containing chelate. For example, the antibody may be coupled to a chelator linker (e.g., DOTA, DTPA, or thiacetam) that allows the antibody to complex with a radioisotope. Antibodies may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include ³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹²⁵I、¹³¹I、¹⁸⁶Re、²¹³Bi、²²⁵Ac and ²²⁷ Th. For therapeutic purposes, radioisotopes that emit beta or alpha particle radiation, such as ¹³¹I、⁹⁰Y、²¹¹At、²¹²Bi、⁶⁷Cu、¹⁸⁶Re、¹⁸⁸ Re and ²¹² Pb, may be used.

Techniques for coupling molecules to antibodies are well known in the art. Typically, the nucleic acid molecule is covalently linked to lysine or cysteine on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively. It has been reported that conjugation methods using engineered cysteines or integrated unnatural amino acids can improve the homogeneity of conjugates. In particular, those skilled in the art can also contemplate Fc-containing polypeptides engineered with acyl donor glutamine-containing tags (e.g., gin peptide-containing tags or Q-tags) or by polypeptide engineering (e.g., by amino acid deletion, insertion, substitution, or mutation on the polypeptide) to produce reactive endogenous glutamine. The transglutaminase can then be covalently crosslinked with an amine donor agent (e.g., a small molecule comprising or linked to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, wherein the amine donor agent is site-specifically coupled to the Fc-containing polypeptide by an acyl donor-containing glutamine tag or accessible/exposed/reactive endogenous glutamine (WO 2012059882).

It will be appreciated that therapeutic agents according to the embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A number of suitable formulations can be found in the pharmacopoeias known to all pharmaceutical chemists: remington's Pharmaceutical Sciences (15 th edition, mack Publishing Company, easton, pa. (1975)), particularly chapter 87 thereof Blaug, seymour. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., lipofectin ^TM), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerates the route of administration.

The antibodies and derivatives, fragments, analogs and homologs thereof described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions and guidelines for selecting the components are well known in the art, see, for example, remington's Pharmaceutical Sciences: THE SCIENCE AND PRACTICE Of Pharmacy, 19 th edition (Alfonso r. Gennaro et al, editions )Mack Pub.Co.,Easton,Pa.:1995;Drug Absorption Enhancement:Concepts,Possibilities,Limitations,And Trends,Harwood Academic Publishers,Langhorne,Pa.,1994; and PEPTIDE AND Protein Drug Delivery (ADVANCES IN PARENTERAL SCIENCES, volume 4), 1991,M.Dekker,New York.

Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. When antibody fragments are used, minimal inhibitory fragments that specifically bind to the target protein binding domain may be preferred. For example, based on the variable region sequence of an antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. Such peptides may be chemically synthesized and/or produced by recombinant DNA techniques (see, e.g., marasco et al, proc. Natl. Acad. Sci. USA,90:7889-7893 (1993)).

As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable pharmaceutically acceptable carriers are described in the latest version of Remington's Pharmaceutical Sciences, a standard reference in the art, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the antibody, its use in the composition is contemplated.

The pharmaceutical compositions of the embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates, and agents for regulating the osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, cremophor EL ^TM (BASF, parsippany, N.J.), or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents delaying absorption, for example, aluminum monostearate and gelatin.

If desired, sterile injectable solutions can be prepared by incorporating the antibodies in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle which contains an alkaline dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum-drying and freeze-drying of a powder containing the active ingredient plus any additional desired ingredient from a sterile filtered solution thereof as described previously.

For administration by inhalation, the compound is delivered as an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant, such as a gas such as carbon dioxide.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, one or more of the antibodies may be formulated as a cream, ointment, gel, or ointment as is commonly known in the art.

The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

In one embodiment, the antibodies may be prepared with carriers that prevent their rapid elimination by the body, such as slow/controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art.

It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to a physically separable unit suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of one or more of the antibodies calculated to produce the desired therapeutic effect in combination with the desired drug carrier. The specifications for the dosage unit forms of the embodiments are indicated below and directly depend on: the unique characteristics of antibodies and the particular therapeutic effect to be achieved, and limitations inherent in the field of formulation of such antibodies for treating individuals.

The pharmaceutical composition may be placed in a container, package, or dispenser together with instructions for administration.

In certain embodiments, the anti-PD-L1 antibodies of the present disclosure may be administered in combination with radiation therapy in a method to produce a long-lasting anti-tumor response and/or to increase survival of a cancer patient. In some embodiments, the anti-PD-L1 antibodies of the present disclosure may be administered prior to, concurrent with, or subsequent to administration of radiation therapy to a cancer patient. For example, radiation therapy may be administered to a tumor lesion at one or more doses followed by one or more doses of an anti-PD-L1 antibody of the present disclosure. In some embodiments, radiation therapy may be administered locally to the tumor lesion to increase the local immunogenicity of the patient's tumor (auxiliary radiation) and/or kill tumor cells (ablative radiation (ablative radiation)), followed by systemic administration of an anti-PD-L1 antibody of the present disclosure. For example, intracranial radiation as well as systemic administration of an anti-PD-L1 antibody of the present disclosure can be administered to a patient suffering from brain cancer (e.g., glioblastoma multiforme). In certain embodiments, the anti-PD-L1 antibodies of the present disclosure can be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., albesii).

The antibodies or fragments thereof of the present disclosure may be administered in combination with one or more antiviral agents known in the art, including, but not limited to: zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine and corticosteroids. In some embodiments, the anti-PD-L1 antibodies of the present disclosure can be administered in combination with any antagonist of LAG3 inhibitors, CTLA-4 inhibitors, PD-1 inhibitors, or other T cell co-inhibitors to treat chronic viral infections.

The antibodies or fragments thereof of the present disclosure may be used in combination with any drug or therapy known in the art (e.g., corticosteroids and other immunosuppressants) to treat autoimmune diseases or disorders including, but not limited to: alopecia areata, autoimmune hepatitis, celiac disease, graves 'disease, guillain-barre syndrome, hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, sjogren's syndrome, systemic lupus erythematosus, white spots, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and wegener's granulomatosis.

Additional therapeutically active components may be administered prior to, concurrently with, or after administration of the anti-PD-L1 antibodies of the present disclosure. For the purposes of this disclosure, such an administration regimen is considered to be administration of the anti-PD-L1 antibody "in combination" with the second therapeutically active component.

Additional therapeutically active components may be administered to the subject prior to administration of the anti-PD-L1 antibodies of the present disclosure. For example, a first component may be considered to be administered "before" a second component if the first component is administered 1 week, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before the second component is administered. In other embodiments, additional therapeutically active components may be administered to the subject after administration of the anti-PD-L1 antibodies of the present disclosure. For example, a first component may be considered to be administered "after" a second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours after administration of the second component. In still other embodiments, additional therapeutically active components may be administered to a subject concurrently with administration of the anti-PD-L1 antibodies of the present disclosure. For the purposes of this disclosure, "simultaneous" administration includes, for example, administration of an anti-PD-L1 antibody and an additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated), or in separate dosage forms within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered by the same route (e.g., the anti-PD-L1 antibody and additional therapeutically active component may be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered by a different route (e.g., the anti-PD-L1 antibody may be administered intravenously, and the additional therapeutically active component may be administered subcutaneously). In any case, for the purposes of this disclosure, administration of components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes is considered "simultaneous administration". For the purposes of this disclosure, administration of an anti-PD-L1 antibody "before", "simultaneously with" or "after" administration of an additional therapeutically active component (as those terms are defined herein above) is considered to be administration of the anti-PD-L1 antibody "in combination" with the additional therapeutically active component.

The present disclosure includes pharmaceutical compositions in which an anti-PD-L1 antibody of the present disclosure is co-formulated with one or more additional therapeutically active components as described elsewhere herein using a variety of dosage combinations.

In exemplary embodiments wherein an anti-PD-L1 antibody of the present disclosure is administered in combination with a VEGF antagonist (e.g., a VEGF trap, such as aflibercept), which comprises administering a co-formulation comprising the anti-PD-L1 antibody and the VEGF antagonist, the individual components may be administered to the subject and/or co-formulated using multiple dose combinations. For example, the amount of anti-PD-L1 antibody that can be administered to a subject and/or included in a co-formulation is selected from ：0.01mg、0.02mg、0.03mg、0.04mg、0.05mg、0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg、1.0mg、1.5mg、2.0mg、2.5mg、3.0mg、3.5mg、4.0mg、4.5mg、5.0mg、6.0mg、7.0mg、8.0mg、9.0mg and 10.0mg; the amount of VEGF antagonist (e.g., a VEGF trap, such as aflibercept) that can be administered to a subject and/or included in a co-formulation is selected from ：0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg、1.0mg、1.1mg、1.2mg、1.3mg、1.4mg、1.5mg、1.6mg、1.7mg、1.8mg、1.9mg、2.0mg、2.1mg、2.2mg、2.3mg、2.4mg、2.5mg、2.6mg、2.7mg、2.8mg、2.9mg and 3.0mg. The combination/co-formulation may be administered to a subject according to any of the administration regimens disclosed elsewhere herein, including, for example, twice weekly, once every 2 weeks, once every 3 weeks, once monthly, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, and the like.

According to certain embodiments of the present disclosure, multiple doses of an anti-PD-L1 antibody (or a pharmaceutical composition comprising a combination of an anti-PD-L1 antibody and any additional therapeutically active agent mentioned herein) may be administered to a subject over a defined time course. Methods according to this aspect of the disclosure include sequentially administering a plurality of doses of an anti-PD-L1 antibody of the disclosure to a subject. As used herein, "sequentially administered" means that each dose of anti-PD-L1 antibody is administered to a subject at different time points (e.g., on different days) at predetermined intervals (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering a single initial dose of an anti-PD-L1 antibody to a patient, followed by one or more second doses of an anti-PD-L1 antibody, and optionally followed by one or more third doses of an anti-PD-L1 antibody. The anti-PD-L1 antibody may be administered at a dose of 0.1mg/kg to about 100 mg/kg.

The terms "initial dose", "second dose" and "third dose" refer to the time sequence of administration of an anti-PD-L1 antibody of the present disclosure. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is a dose administered after the initial dose; and "third dose" is the dose administered after the second dose. The initial dose, the second dose, and the third dose may all contain the same amount of anti-PD-L1 antibody, but generally may differ from one another in the frequency of administration. However, in certain embodiments, the amounts of anti-PD-L1 antibody included in the initial dose, the second dose, and/or the third dose are different from one another (e.g., adjusted upward or downward as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen at a "loading dose" followed by subsequent doses (e.g., a "maintenance dose") at a lower frequency.

Methods according to this aspect of the disclosure may include administering any number of second and/or third doses of an anti-PD-L1 antibody to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6,7, 8, or more) second doses are administered to the patient. Likewise, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6,7, 8, or more) third doses are administered to the patient.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments of the present disclosure, the frequency of administration of the second and/or third dose to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment according to the needs of the individual patient after the clinical examination.

The present disclosure includes administration regimens in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once weekly, biweekly, three weekly, monthly, bi-monthly, etc.), followed by two or more maintenance doses administered to the patient at a lower frequency. For example, if loading doses are administered at a frequency such as once a month (e.g., two, three, four, or more loading doses administered once a month), a maintenance dose may be administered to a patient once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks, etc., in accordance with this aspect of the disclosure.

In one aspect, the antibodies of the disclosure have diagnostic uses. For example, for diagnostic purposes, the anti-PD-L1 antibodies of the present disclosure may be used to detect and/or measure PD-L1 in a sample. Some embodiments contemplate the use of one or more antibodies of the present disclosure in an assay to detect a disease or disorder, such as cancer, autoimmune disease, or chronic viral infection. Exemplary diagnostic assays for PD-L1 may include, for example: contacting a sample obtained from a patient with an anti-PD-L1 antibody of the present disclosure, wherein the anti-PD-L1 antibody is labeled with a detectable label or reporter or is used as a capture ligand to selectively isolate PD-L1 from the patient sample. Alternatively, unlabeled anti-PD-L1 antibodies may be used in combination with a secondary antibody that is itself detectably labeled in diagnostic applications. The detectable label or reporter may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; fluorescent or chemiluminescent moieties, such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure PD-L1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples useful in PD-L1 diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a patient that contains a detectable amount of PD-L1 protein or fragment thereof under normal or pathological conditions. Generally, the level of PD-L1 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from cancer or an autoimmune disease) will be measured to initially establish a baseline or standard level of PD-L1. This baseline level of PD-L1 may then be compared to the level of PD-L1 measured in a sample obtained from an individual suspected of having a cancer-related disorder or a symptom associated with such a disorder.

Antibodies specific for PD-L1 may contain no additional tag or moiety, or they may contain an N-terminal or C-terminal tag or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if any) may be determined after the peptide has bound to the surface, the orientation of the peptide relative to the surface. For example, if the surface is coated with avidin, the N-terminal biotin-containing peptide will be oriented such that the C-terminal portion of the peptide is distal to the surface.

Some aspects of the disclosure relate to the use of the disclosed antibodies as markers for predicting prognosis of cancer or autoimmune disease in a patient. Antibodies of the disclosure can be used in diagnostic assays to assess prognosis of cancer and predict survival in patients.

For purposes of clarity and conciseness of description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include some embodiments having a combination of all or some of the features described.

Examples

Example 1 PD-L1 humanized antibodies

The PD-L1 antibody is derived from a murine hybridoma clone PDL1-3F2 obtained by immunizing an animal, and the variable region sequence of the antibody is as follows:

The amino acid sequence of the PDL1-3F2 light chain variable region is shown as SEQ ID NO.1, the CDR1, CDR2 and CDR3 are shown as SEQ ID NO.2, 3 and 4 respectively, and the encoding nucleic acid is shown as SEQ ID NO. 5.

<---------FR1--------->CDR1<-----FR2----->CDR2<-------------DIVLAQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGT--FR3------------->CDR3<--FR4-->

DFTLSINSVETEDFGMYFCQQSKSWPFTFGSGTRLEIK

Nucleic acid sequences

GATATTGTGCTAGCTCAGTCTCCAGCCACCCTGTCTGTGACTcCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCTAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAAAAGCTGGCCATTCACGTTCGGCTCGGGGACAAGGTTGGAAATAAAA

The amino acid sequence of the PDL1-3F2 heavy chain variable region is shown as SEQ ID NO.6, the CDR1, CDR2 and CDR3 are shown as SEQ ID NO.7, 8 and 9 respectively, and the encoding nucleic acid is shown as SEQ ID NO. 10.

<--------------FR1----------->CDR1<-----FR2----->CDR2<--DVQLQESGPDLVKPSQTLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIHHSSITNYNPSLKSRIS-----------FR3-------------->CDR3<---FR4--->

ITRDTSKNQFFLQLSSVTTEDTATFYCAREGYDYDWFAYWGQGTLVTVSA

Nucleic acid sequences

GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGACACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGGTATAACTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACCACAGTAGTATCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAGTTCTGTGACTACTGAGGACACAGCCACATTTTACTGTGCAAGAGAGGGGTATGATTACGACTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

Humanized modification is carried out on the murine monoclonal antibody PDL1-3F2, and light chain CDR is respectively transplanted to human light chain germ line gene Vk3-20 and heavy chain germ line gene VH4-59, so that the following different heavy chain derivatives and light chain derivatives are obtained:

TABLE 1 light chain variable region sequences of PD-L1 humanized antibodies

The amino acid sequence of YH23 VKv1 light chain variable region is shown as SEQ ID NO.11, the encoding nucleic acid is shown as SEQ ID NO.12, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.2, 3 and 4 respectively.

<---------FR1--------->CDR1<-----FR2----->CDR2<------------EIVLTQSPGTLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPDRFSGSGSGT--FR3------------->CDR3<--FR4-->

DFTLTISRLEPEDFAVYFCQQSKSWPFTFGQGTKLEIK

Nucleic acid sequences

GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCATCAGCAACAACCTGCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTAGACTGCTGATTAAGTACGCCAGCCAGTCTATCAGCGGCATCCCCGACAGATTCAGCGGCTCTGGCTCTGGCACAGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTTCTGCCAGCAGAGCAAGAGCTGGCCCTTCACATTTGGCCAGGGCACCAAGCTGGAAATCAAG

The YH23 VKv2 light chain variable region has an amino acid sequence shown as SEQ ID NO.13, the encoding nucleic acid is shown as SEQ ID NO.14, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.15, 3 and 4 respectively.

<---------FR1--------->CDR1<-----FR2----->CDR2<------------EIVLTQSPGTLSLSPGERATLSCRASQSISSNLHWYQQKPGQAPRLLIKYASQSISGIPDRFSGSGSGT--FR3------------->CDR3<--FR4-->

DFTLTISRLEPEDFAVYFCQQSKSWPFTFGQGTKLEIK

Nucleic acid sequences

GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCATCAGCAGCAACCTGCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTAGACTGCTGATTAAGTACGCCAGCCAGTCTATCAGCGGCATCCCCGACAGATTCAGCGGCTCTGGCTCTGGCACAGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTTCTGCCAGCAGAGCAAGAGCTGGCCCTTCACATTTGGCCAGGGCACCAAGCTGGAAATCAAG

TABLE 2 heavy chain variable region sequences of PD-L1 humanized antibodies

The amino acid sequence of YH23 VHv1 light chain variable region is shown as SEQ ID NO.16, the encoding nucleic acid is shown as SEQ ID NO.17, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.7, 8 and 9 respectively.

<--------------FR1----------->CDR1<-----FR2---->CDR2<--QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYNWHWIRQPPGKGLEWIGYIHHSSITNYNPSLKSRVT-----------FR3-------------->CDR3<---FR4--->

ISRDTSKNQFSLKLSSVTAADTAVYYCAREGYDYDWFAYWGQGTLVTVSS

Nucleic acid sequences

CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCTACTCTATCACCAGCGGCTACAACTGGCACTGGATCAGACAGCCTCCAGGCAAAGGCCTGGAATGGATCGGCTACATCCACCACAGCAGCATCACCAACTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCAGAGAGGGCTACGACTACGATTGGTTCGCTTACTGGGGCCAGGGCACCCTGGTTACAGTCTCTTCT

The amino acid sequence of YH23 VHv2 light chain variable region is shown as SEQ ID NO.18, the encoding nucleic acid is shown as SEQ ID NO.19, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.7, 20 and 9 respectively.

<--------------FR1----------->CDR1<-----FR2---->CDR2<--QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYNWHWIRQPPGKGLEWIGYIHHSSITYYNPSLKSRVT-----------FR3-------------->CDR3<---FR4--->

ISRDTSKNQFSLKLSSVTAADTAVYYCAREGYDYDWFAYWGQGTLVTVSS

Nucleic acid sequences

CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCTACTCTATCACCAGCGGCTACAACTGGCACTGGATCAGACAGCCTCCAGGCAAAGGCCTGGAATGGATCGGCTACATCCACCACAGCAGCATCACCTACTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCAGAGAGGGCTACGACTACGATTGGTTCGCTTACTGGGGCCAGGGCACCCTGGTTACAGTCTCTTCT

The DNA encoding the above amino acid sequence was subjected to full sequence synthesis and cloned into a vector containing the antibody kappa chain constant region or human IgG1 constant region CH1-CH3, respectively, to prepare a chimeric antibody. After light and heavy chain plasmids are combined and paired, HEK293E cells (SAFC Biosciences) are transfected, cell culture fluid is collected after 3-5 days of expression, and maternal 3F2 chimeric antibody, PD-L1 humanized antibody (YH 23-VH1/VK1, YH23-VH1/VK2, YH23-VH2/VK1 and YH23-VH2/VK 2) are obtained by purifying by Protein A affinity chromatography.

Example 2 binding and blocking Activity of PD-L1 humanized antibodies

1. ELISA detection of humanized antibody binding to human PD-L1

Human PD-L1 protein (Sinobiological # 10084-H08H) was coated on 96-well ELISA plates at 1. Mu.g/ml overnight at 4 ℃. After 2% milk blocking, 100 μl of the recombinant expressed PD-L1 humanized antibody prepared in example 1 was added to each well and incubated for 1 hour at room temperature; after 3 washes with PBST and PBS, 100. Mu.l of HRP-labeled anti-human IgG F (ab)' ₂ secondary antibody was added to each well and incubated for 60 min at room temperature. After three washes with PBST and PBS, 100. Mu.l of TMB substrate was added to each well and developed at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and the absorbance was read at a wavelength of 450 nm. Control antibody atezolizumab was synthesized according to IMGT published sequence and prepared by expression in HEK293E cells.

The detection results are shown in FIG. 1. FIG. 1 shows that humanized PD-L1 antibodies bind PD-L1 at the antigen level with comparable capacity to the parental 3F2 chimeric antibody and atezolizumab.

2. ELISA detection of humanized antibody blocking binding of human PD-1/PD-L1

Human PD-1 recombinant protein (Sinobiological # 10377-H05H) was coated on 96-well ELISA plates at 4℃overnight. Wash three times with PBS and add 200 μl of 2% mill per well for 1 hour. The PD-L1 humanized antibody prepared in example 1 was mixed with biotin-labeled human PD-L1 protein and incubated at room temperature for 30 minutes. The 96-well ELISA plate was washed three times with PBS, and 100. Mu.l of the incubated mixture was added to each well and incubated at room temperature for 1 hour. After three washes with PBST and PBS, 100. Mu.l SA-HRP was added to each well and incubated for 30 minutes at room temperature in the absence of light. After washing three times with PBST and PBS, respectively, 100. Mu.l of TMB substrate was added to each well and developed at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and the absorbance was read at a wavelength of 450 nm.

The detection results are shown in FIG. 2. FIG. 2 shows that humanized PD-L1 antibodies have comparable blocking ability to PD-1 binding to PD-L1 than atezolizumab at the antigen level.

3. FACS detection of binding of humanized antibodies to PD-L1 on cell membranes

Human PD-L1 full-length gene is transfected into CHO cells, 8 mu g/ml puromycin is added after 48 hours for pressurized screening, and CHO-PD-L1 cell strain which stably expresses human PD-L1 is obtained. Calculating the required cell number by 5X 10 ⁴ cells per well, taking 300g of CHO-PD-L1 stable transfer cells, centrifuging for 5 minutes, and absorbing and removing the supernatant; cells were washed once with 1ml of 1% BSA. 3% BSA was added and the cells were diluted to 1X 10 ⁶ cells/ml with 1% BSA on ice for 30 minutes, 100. Mu.l/well of the resuspended cells were plated in 96-well plates, centrifuged at 300g for 5 minutes, the supernatant was discarded, 50. Mu.l/well of the PD-L1 antibody prepared in example 1 was added in gradient dilution, and incubated on ice for 45 minutes. Cells were washed twice with 200. Mu.l/well of 1% BSA, 50. Mu.l of AF 647-goat anti-human IgG, fcgamma secondary antibody solution was added to each well and incubated on ice for 45 min. Cells were washed three times with 200. Mu.l/well of 1% BSA and stained with 60. Mu.l of 5. Mu.g/ml PI/PBS solution for each well and then run on a machine. The fluorescence values of PI negative cell populations were analyzed.

The detection result is shown in FIG. 3. FIG. 3 shows that the binding capacity of the humanized antibodies to PD-L1 on the cell membrane was comparable to that of the parental 3F2 chimeric antibody and atezolizumab at the cellular level.

4. FACS detection of blocking of PD-L1 and PD-1 binding by humanized antibodies on cell membranes

Calculating the required cell number by 5×10 ⁴ cells per well, taking CHO-PD-L1 cells, centrifuging at 300g for 5 min, and removing the supernatant; cells were washed once with 1ml of 1% BSA. Cells were diluted to 1X 10 ⁶ cells/ml with 1% BSA, plated in 96-well plates with 100. Mu.l/well of resuspended cells, centrifuged for 5 min at 300g, the supernatant discarded, 50. Mu.l/well of the PD-L1 antibody prepared in example 1 with gradient dilution added, incubated on ice for 45 min, 6. Mu.g/ml biotin-labeled PD-1 antigen added per well, and incubated on ice for 45 min. Cells were washed three times with 200. Mu.l wells of 1% BSA, 50. Mu.l 1:300 diluted SA-PE (Jackson immunoResearch, # 016-130-084) was added to each well and incubated on ice for 45 minutes. Cells were washed three times with 200. Mu.l/well of 1% BSA and stained with 60. Mu.l of 5. Mu.g/ml PI/PBS solution for each well and then run on a machine. The fluorescence values of PI negative cell populations were analyzed.

The detection results are shown in FIG. 4. FIG. 4 shows that the ability of humanized antibodies to block PD-L1 and PD-1 binding on cell membranes at the cellular level is comparable to that of the parental 3F2 chimeric antibody as well as atezolizumab.

Table 3 lists the binding or blocking results of PD-L1 antibodies to recombinant proteins.

TABLE 3 binding or blocking of PD-L1 antibodies to recombinant proteins

Example 3 humanized antibodies affinity assay for PD-L1

10. Mu.g/ml of human PD-L1 recombinant protein was coupled to a CM5 chip (GE HEALTHCARE) by an amino coupling reagent at a flow rate of 10. Mu.l/min and an antigen coupling amount of about 200RU was controlled by BiacoreT200,200. After the chip was rinsed with buffer to baseline plateau, the PD-L1 antibody prepared in example 1 was diluted in a gradient (7 gradients diluted 2 times from 10. Mu.g/ml) and flowed through the chip at a flow rate of 30. Mu.l/min, setting a binding time of 350 seconds and a dissociation time of 600 seconds. Kinetic constants were obtained by fitting with Biacore T200 evaluation software using a 1:1 binding model (Table 4).

TABLE 4 affinity of PD-L1 humanized antibodies to PD-L1 recombinant proteins

Example 4 blocking of PD-L1 signalling pathway by humanized antibodies

Human PD-1 gene is transfected into Jurkat cells (ATCC TIB-152), puromycin of 0.5 mu g/ml is added after 48 hours for pressurized screening, thus obtaining Jurkat cell strain Jurkat-PD1 which stably expresses human PD-1, then reporter plasmid pGL4.22-luc2P NFAT Hygro (Promega) is transfected into Jurkat-PD1 stably transfected cells, hygromycin B of 200 mu g/ml is added for screening, and finally Jurkat PD1 NFAT reporter gene cell strain is obtained.

The scFv CD3 antibody gene was transfected into CHO-PD-L1 cells to construct CHO-PD-L1-CD3 stably transformed cells stably expressing CD3 antibody and human PD-L1 (Wang L et al, J Pharm Biomed Anal 2017Oct 25; 145:447-453), F12 complete medium resuspended cell density to 5X 10 ⁵ cells/ml. 100. Mu.l/well of resuspended cells were plated into 96-well plates and the 96-well plates were incubated at 37℃in a 5% CO ₂ incubator for 18-20 hours. Jurkat PD1NFAT cells were collected, washed once with 10ml PBS, and resuspended to a cell density of 2X 10 ⁶ cells/ml with reaction buffer. The 96-well plate was removed, the supernatant was discarded, 50. Mu.l/well of resuspended cells was plated into the 96-well plate, 50. Mu.l/well of the PD-L1 antibody prepared in example 1 was added, and the 96-well plate was incubated at 37℃in a 5% CO ₂ incubator for 6 hours. Adding the prepared fluorescence detection substrate solution, gently oscillating the pore plate, standing for 3 minutes at room temperature, and detecting chemiluminescence by using an enzyme-labeled instrument.

The detection results are shown in FIG. 5. FIG. 5 shows that humanized PD-L1 antibodies are effective in blocking binding of Jurkat cells expressing PD-1 molecules to CHO-PD-L1-CD3 cells with blocking activity comparable to that of maternal 3F2 chimeric antibody and atezolizumab, with a drug concentration dose dependent effect and an IC50 of about 2-3nM (Table 5).

TABLE 5 blocking of PD-L1/PD1 Signal paths by PD-L1 humanized antibodies

Example 5 antigen memory response assay

PBMC density was adjusted to 2X 10 ⁶ cells/ml using X-VIVO15 medium (Lonza # 04-418Q) as buffer, 100. Mu.l/well was added to flat bottom 96 well plates, 8. Mu.g/ml CMV 50. Mu.l/well was added to the well plates, and then diluted PD-L1 antibody prepared in example 1 (final concentration 20. Mu.g/ml, 5-fold gradient dilution, total two dilutions). The culture supernatant was incubated at 37℃for 96 hours under 5% CO ₂, and the IFN-. Gamma.content of the culture supernatant was determined by using the Human IFN-. Gamma.ELISA Kit (BD, cat# 555142). The capture antibody anti-human IFN-gamma (1:250) was diluted with coating solution, and the ELISA plate was added at 100. Mu.l/well and coated overnight at 4 ℃. Three washes with 0.05% PBST, 100. Mu.l/well with 5% calf serum/PBS, and blocking at room temperature for 1 hour. Three washes with 0.05% PBST, 100. Mu.l/well of sample (96 well plates were removed from incubator, centrifuged at 1000rpm for 3 min, supernatant was taken, diluted 20-fold with 5% calf serum/PBS) and gradient diluted standard IFN-. Gamma.were added. Incubate at room temperature for 2 hours. Five washes with 0.05% PBST, 100. Mu.l/well of biotin anti-human IFN-. Gamma./SA-HRP, each diluted 250-fold with 5% calf serum/PBS, were added and incubated for 1h at room temperature. The solution was washed seven times with 0.05% PBST, 100. Mu.l/well of the solution was added thereto, and the solution was protected from light at room temperature for 10-15 minutes. The reaction was stopped by adding 2M concentrated sulfuric acid to 50. Mu.l/well, and the absorbance was read at a wavelength of 450 nm.

The detection results are shown in FIG. 6. FIG. 6 shows that the PD-L1 humanized antibody can significantly stimulate the massive proliferation of T cells, promote the production of IFN-gamma, and has the activity equivalent to that of female parent 3F2 and atezolizumab.

Example 6 Cross-species reaction

Human and cynomolgus PD-L1 proteins were coated on 96-well ELISA plates at 1. Mu.g/ml, respectively, overnight at 4 ℃. After 2% milk blocking, 100 μl of anti-PD-L1 antibody was added to each well and incubated for 1 hour at room temperature; after 3 washes with PBST and PBS, 100. Mu.l of HRP-labeled anti-human IgG F (ab)' ₂ secondary antibody was added to each well and incubated for 60 min at room temperature. After three washes with PBST and PBS, 100. Mu.l of TMB substrate was added to each well and developed at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and the absorbance was read at a wavelength of 450 nm.

The detection results are shown in fig. 7 and 8. FIG. 7 shows the binding of the humanized antibody YH23-VH1/VK1 to human PD-L1 recombinant antigen; FIG. 8 shows the binding of the humanized antibody YH23-VH1/VK1 to cynomolgus PD-L1 recombinant antigen. Humanized antibody YH23-VH1/VK1 was comparable to human and cynomolgus monkey PD-L1 in affinity, binding to cynomolgus monkey PD-L1 was slightly stronger than Atezolizumab (Table 6).

TABLE 6 binding of humanized antibodies to human and cynomolgus monkey PD-L1

EXAMPLE 7 construction of PDL1-TGFbRII antibody fusion proteins

The TGFBR2 gene is located on human chromosome 3, 3p24.1, containing 7 exons, and the TGFbRII receptor exists in multiple isoforms due to alternative splicing of the gene, genebank showed TGFbRII isoforms to include: isomer A (NP-001020018.1, the amino acid sequence of which is shown in SEQ ID NO.21, the coding nucleic acid sequence of which is shown in SEQ ID NO. 22), isomer B (NP-003233.4, the amino acid sequence of which is shown in SEQ ID NO.23, the coding nucleic acid sequence of which is shown in SEQ ID NO. 24), isomer X1 (XP-011532345.1, the amino acid sequence of which is shown in SEQ ID NO. 25), and isomer X2 (XP-011532347.1, the amino acid sequence of which is shown in SEQ ID NO.27, the coding nucleic acid sequence of which is shown in SEQ ID NO.28, XP-016862595.1, the amino acid sequence of which is shown in SEQ ID NO.29, and the coding nucleic acid sequence of which is shown in SEQ ID NO. 30) differ in the initial translation sites of the extracellular domain of the N-terminus (FIG. 9).

TABLE 7 different TGF-beta RII isomer sequences

The gene fragment encoding the TGFbRII isoform B extracellular domain (24-159) was first ligated to the C-terminus of the heavy chain (YH 23 VHv 1) of the PD-L1 humanized antibody (YH 23-VH1/VK 1) prepared in example 1 via a flexible linker G (G4S) 3G, cloned into a MERCK PCGS expression plasmid vector along with the sequence encoding the light chain (YH 23 VKv 1), linearized, transfected into CHO cells (Merck) and screened under pressure to obtain mini pool. After the expression of 5% CO ₂ rpm at 37 ℃ for 8 days, harvesting cell culture supernatant, purifying by utilizing protein A affinity chromatography to obtain recombinant antibody fusion protein PDL1-TGFbRII-01, wherein the amino acid sequence of polypeptide fused by PD-L1 antibody heavy chain and TGFbRII is shown as SEQ ID NO.31, and the coding sequence is shown as SEQ ID NO. 32; the PD-L1 antibody light chain has the amino acid sequence shown as SEQ ID NO.33 and the coding sequence shown as SEQ ID NO. 34. Mass spectrometry found that in addition to the intact PD-L1-TGFbRII protein, there was a degradation fragment lacking around 15kDa in the purified protein (fig. 10), with cleavage sites at heavy chains 469 and 473 (corresponding to Lys at position 30, asn at position 34 of TGFbRII receptor isomer B). Interestingly, no degradation fragments were found in the same plasmid when expressed in HEK293E cells, and forced degradation experiments with purified proteins showed that no degradation fragments were found also after incubation at 37 ℃ for 10 days (results not shown).

Since TGFbRII isomer X2 does not contain a site where degradation occurs, novel antibody fusion proteins PDL1-TGFbRII-02 and PDL1-TGFbRII-03 are constructed by using the extracellular domain (1-124) of isomer X2, or after mutation of a site where cleavage may occur in the original isomer B, antibody fusion proteins PDL1-TGFbRII-04 and PDL1-TGFbRII-05 are constructed (FIG. 11). CHO-S cells (Thermofisher, R80007) are transfected, the expression is carried out for 8 days at 37 ℃ with 5% CO ₂ rpm, when the cell activity rate is reduced to 80%, cell culture supernatant is harvested, and recombinant antibody fusion protein PD-L1-TGFbRII is obtained by utilizing protein A affinity chromatography purification.

TABLE 8 PDL1-TGFbRII antibody fusion protein sequences

Wherein, the amino acid sequence of the polypeptide fused with the heavy chain of the PD-L1 antibody in PDL1-TGFbRII-01 and TGFbRII is shown as SEQ ID NO.31, and the coding sequence is shown as SEQ ID NO. 32; the amino acid sequence of the PD-L1 antibody light chain in PDL1-TGFbRII-01 is shown as SEQ ID NO.33, and the coding sequence is shown as SEQ ID NO. 34;

wherein, the amino acid sequence of the polypeptide fused with the heavy chain of the PD-L1 antibody in PDL1-TGFbRII-02 and TGFbRII is shown as SEQ ID NO.35, and the coding sequence is shown as SEQ ID NO. 36; the amino acid sequence of the PD-L1 antibody light chain in PDL1-TGFbRII-02 is shown as SEQ ID NO.33, and the coding sequence is shown as SEQ ID NO. 34;

Wherein, the amino acid sequence of the polypeptide fused with the heavy chain of the PD-L1 antibody in PDL1-TGFbRII-03 and TGFbRII is shown as SEQ ID NO.37, and the coding sequence is shown as SEQ ID NO. 38; the amino acid sequence of the PD-L1 antibody light chain in PDL1-TGFbRII-03 is shown as SEQ ID NO.33, and the coding sequence is shown as SEQ ID NO. 34;

Wherein, the amino acid sequence of the polypeptide fused with the heavy chain of the PD-L1 antibody in PDL1-TGFbRII-04 and TGFbRII is shown as SEQ ID NO.39, and the coding sequence is shown as SEQ ID NO. 40; the amino acid sequence of the PD-L1 antibody light chain in PDL1-TGFbRII-04 is shown as SEQ ID NO.33, and the coding sequence is shown as SEQ ID NO. 34.

Wherein, the amino acid sequence of the polypeptide fused with the heavy chain of the PD-L1 antibody in PDL1-TGFbRII-05 and TGFbRII is shown as SEQ ID NO.41, and the coding sequence is shown as SEQ ID NO. 42; the amino acid sequence of the PD-L1 antibody light chain in PDL1-TGFbRII-05 is shown as SEQ ID NO.33, and the coding sequence is shown as SEQ ID NO. 34.

Recombinant proteins were identified using an Agilent 1100 high performance liquid chromatograph, TOSOH gel column (G3000 SWXL,7.8 x 300mm,5 um). The results showed that the content of the PD-L1-TGFbRII-03, PD-L1-TGFbRII-04 and PD-L1-TGFbRII-05SEC-HPLC fragments expressed by CHO cells was around 1%, which was significantly lower than that of PD-L1-TGFbRII-01 (about 20%, see Table 9). Post-desugaring mass spectrometry detection showed that PD-L1/TGF-beta RII-01 exhibited a TGFbRII shed fragment peak around 163kDa, whereas none of the new constructs PD-L1-TGFbRII-03, PD-L1-TGFbRII-04 and PD-L1-TGFbRII-05 detected any TGFbRII shed-formed degradation fragments (FIG. 12).

TABLE 9 SEC-HPLC of antibody fusion protein PD-L1-TGFbRII

Example 8 affinity of antibody fusion proteins PDL1-TGFbRII with TGF-beta

The kinetic binding activity of the intact antibody fusion proteins PDL1-TGFbRII to human tgfβ1, tgfβ2 and tgfβ3 was determined using Surface Plasmon Resonance (SPR). 5. Mu.g/ml of antibody fusion Protein PDL1-TGFbRII was captured onto the sensor chip Protein A at a flow rate of 10. Mu.l/min, and then human TGF-beta 1, TGF-beta 2 and TGF-beta 3 (initial concentration 3.9nM, 2-fold gradient dilution) were passed over the chip surface at a flow rate of 30. Mu.l/min, respectively. After each cycle, the Protein A chip surface was regenerated with 10mM glycine (pH 2.0). Apparent binding constants KD were obtained by fitting using Langmuir 1:1kinetic model using BIAcore BIAevaluation software from BIAcore T200.

The results show that the antibody fusion proteins PDL1-TGFbRII-01, PDL1-TGFbRII-03, PDL1-TGFbRII-04 and PDL1-TGFbRII-05 all bind human TGF-beta 1, TGF-beta 2 and TGF-beta 3 homodimers with high affinity (Table 10), consistent with literature reports (DERYNCK AND Budi, sci.Signal.12, eaav5183 (2019)), heavy chain Fc-end fused TGFbRII can bind TGF-beta dimer simultaneously.

Table 10, affinity of PDL1-TGFbRII fusion proteins to TGF-beta

Example 9 Activity of antibody fusion proteins PDL1-TGFbRII to bind PD-L1 and TGF-beta simultaneously

Human PD-L1 recombinant proteins were coupled to CM5 chip (GE HEALTHCARE) via amino groups, and the complete antibody fusion proteins PDL1-TGFbRII-01, PDL1-TGFbRII-03 and PDL1-TGFbRII-05 were captured, respectively, and human TGF-beta 1, TGF-beta 2 and TGF-beta 3 were gradient diluted samples, respectively, at 30 μl/min flow rates across the chip surface.

The results show that the intact antibody fusion protein PDL1-TGFbRII can recognize and bind to the PD-L1 protein immobilized on the chip via the Fab end, while Fc-end fused TGFbRII binds human tgfβ1, tgfβ2 and tgfβ3 homodimers with high apparent affinity (2:2), with comparable binding activity compared to PDL1-TGFbRII-01 (fig. 13).

Example 10 binding and blocking Activity of antibody fusion proteins PDL1-TGFbRII

1. ELISA detection of binding of antibody fusion proteins PDL1-TGFbRII to human PD-L1

Human PD-L1 recombinant protein of 1 μg/ml was coated on 96-well ELISA plates overnight at 4 ℃. After 2% milk is blocked, 100 μl of recombinant expressed antibody fusion protein PDL1-TGFbRII is added to each well and incubated for 1 hour at room temperature; after 3 washes with PBST and PBS, 100. Mu.l of HRP-labeled anti-human IgG F (ab)' ₂ secondary antibody was added to each well and incubated for 60 min at room temperature. After three washes with PBST and PBS, 100. Mu.l of TMB substrate was added to each well and developed at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and the absorbance was read at a wavelength of 450 nm. anti-PD-L1/TGF-beta trap control molecules were synthesized according to document WO2015118175, expressed and prepared in HEK 293E.

The detection results are shown in FIG. 14. FIG. 14 shows that antibody fusion proteins PDL1-TGFbRII are capable of binding to PD-L1 with an EC50 of 0.18nM, and that the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05 have no significant difference in affinity compared to PDL1-TGFbRII-01, with EC50 of 0.16nM and 0.18nM, respectively, both of which are comparable to the affinity of the control molecule against the PD-L1/TGF-beta trap.

Table 11 shows the binding results of the antibody fusion proteins PDL1-TGFbRII with PD-L1.

TABLE 11 binding of antibody fusion proteins PDL1-TGFbRII to PD-L1

2. ELISA detection antibody fusion protein PDL1-TGFbRII blocks binding of human PD-1/PD-L1

2. Mu.g/ml human PD-1 recombinant protein was coated on a 96-well ELISA plate at 4℃overnight. Wash three times with PBS and add 200 μl of 2% mill per well for 1 hour. Antibody fusion proteins PDL1-TGFbRII were mixed with biotin-labeled human PD-1 protein and incubated for 30 minutes at room temperature. The 96-well ELISA plate was washed three times with PBS, and 100. Mu.l of the incubated mixture was added to each well and incubated at room temperature for 1 hour. After three washes with PBST and PBS, 100. Mu.l of anti-mouse IgG Fc-HRP was added to each well and incubated for 30 minutes at room temperature in the absence of light. After washing three times with PBST and PBS, respectively, 100. Mu.l of TMB substrate was added to each well and developed at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and the absorbance was read at a wavelength of 450 nm.

The detection results are shown in FIG. 15. FIG. 15 shows that antibody fusion proteins PDL1-TGFbRII are capable of blocking PD-1/PD-L1 binding, with an IC50 of 11.1nM, and that the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05 have no significant difference in affinity compared to PDL1-TGFbRII-01, with an IC50 of 11.4nM, comparable to the anti-PD-L1/TGF-beta trap blocking activity of the control molecule.

Table 12 shows the blocking results of the binding of PD-1 and PD-L1 by antibody fusion proteins PDL 1-TGFbRII.

TABLE 12 blocking of PD-1 and PD-L1 binding by antibody fusion proteins PDL1-TGFbRII

3. FACS detection of binding of antibody fusion proteins PDL1-TGFbRII to human PD-L1

Calculating the required cell number by 5X 10 ⁴ per well, taking CHO-PD-L1 stable transfer cells, centrifuging for 5 minutes at 300g, and absorbing and discarding the supernatant; cells were washed once with 1ml of 1% BSA. 3% BSA was added and the cells were diluted to 1X 10 ⁶ cells/ml with 1% BSA on ice, 100. Mu.l/well of the resuspended cells were plated in 96-well plates, centrifuged at 300g for 5 min, the supernatant was discarded, 50. Mu.l/well of primary antibody solution was added and incubated on ice for 45 min. Cells were washed twice with 200. Mu.l/well of 1% BSA and 50. Mu.l of AF 647-goat anti-human IgG, fcγ (1:300) secondary antibody was added to each well. Incubate on ice for 45 minutes. Cells were washed three times with 200. Mu.l/well of 1% BSA and stained with 60. Mu.l of 5. Mu.g/ml PI/PBS solution for each well and then run on a machine. The fluorescence values of PI negative cell populations were analyzed.

The results show that antibody fusion proteins PDL1-TGFbRII are capable of binding to human PD-L1 on the cell surface with an EC50 of 1.88nM, and that the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05 have no significant difference in affinity compared to PDL1-TGFbRII-01, with EC50 of 2.31nM and 1.77nM, respectively, comparable to the anti-PD-L1/TGF-beta trap binding activity of the control molecule (FIG. 16).

Table 13 shows the results of binding of antibody fusion proteins PDL1-TGFbRII to PD-L1.

TABLE 13 binding of PDL1-TGFbRII antibody fusion proteins to PD-L1

4. FACS detection of PDL1-TGFbRII antibody fusion protein blocking binding of human PD-1/PD-L1

Calculating the required cell number by 5X 10 ⁴ cells per well, taking cell CHO-PD-L1 stable transfer cells, centrifuging at 300g for 5 minutes, and absorbing and removing the supernatant; cells were washed once with 1ml of 1% BSA. Cells were diluted to 1X 10 ⁶ cells/ml with 1% BSA, 100. Mu.l/well of resuspended cells were plated in 96-well plates, centrifuged for 5 min at 300g, the supernatant was discarded, 50. Mu.l/well was added with gradient diluted antibody, incubated on ice for 45 min, 6. Mu.g/ml biotin-labeled PD-1 antigen was added per well, and incubated on ice for 45 min. Cells were washed three times with 200. Mu.l/well of 1% BSA, 50. Mu.l of SA-PE (1:3000) secondary antibody was added to each well and incubated on ice for 45 minutes. Cells were washed three times with 200. Mu.l/well of 1% BSA and stained with 60. Mu.l of 5. Mu.g/ml PI/PBS solution for each well and then run on a machine. The fluorescence values of PI negative cell populations were analyzed.

The detection results are shown in FIG. 17. FIG. 17 shows that antibody fusion proteins PDL1-TGFbRII are capable of blocking PD-1/PD-L1 binding, with an IC50 of 0.72nM, the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05, and PDL1-TGFbRII-01, have no significant difference in affinity, with IC50 of 0.70nM and 0.63nM, respectively, comparable to the anti-PD-L1/TGF-beta trap blocking activity of the control molecule.

Table 14 sets forth the results of FACS detection of PDL1-TGFbRII blocking human PD-1/PD-L1 binding.

TABLE 14 FACS detection of PDL1-TGFbRII blocking binding of human PD-1/PD-L1

Example 11 blocking of PD1/PD-L1 Signal pathway by antibody fusion proteins PDL1-TGFbRII

CHO-PDL1 cells were collected, washed once with 10ml PBS, and F-12 complete medium was resuspended to a cell density of 5X 10 ⁵ cells/ml. 100. Mu.l/well of resuspended cells were plated into 96-well plates and the 96-well plates were incubated at 37℃in a 5% CO ₂ incubator for 18-20 hours. Jurkat PD1 NFAT cells were collected, washed once with 10ml PBS, and resuspended to a cell density of 2X 10 ⁶ cells/ml with reaction buffer. The 96-well plate was removed, the supernatant was discarded, 50. Mu.l/well of resuspended cells was plated into the 96-well plate, 50. Mu.l/well of antibody was added in gradient dilution, and the 96-well plate was incubated at 37℃in a 5% CO ₂ incubator for 6 hours. 100 μl/well of the prepared fluorogenic detection substrate solution was added, the well plate was gently shaken, left at room temperature for 3 minutes, and the chemiluminescent was detected by an enzyme-labeled instrument.

The results show that antibody fusion proteins PDL1-TGFbRII are capable of blocking PD-1/PD-L1 binding, with an IC50 of 1.1nM, the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05, with no significant difference in affinity compared to PDL1-TGFbRII-01, with IC50 of 1.5nM and 1.1nM, respectively, comparable to the anti-PD-L1/TGF-beta trap blocking activity of the control molecule (FIG. 18).

Table 15 shows the results of blocking PD1/PD-L1 signaling pathway by antibody fusion proteins PDL 1-TGFbRII.

TABLE 15 blocking of PD1/PD-L1 Signal pathway by antibody fusion proteins PDL1-TGFbRII

EXAMPLE 12 inhibition of TGF-beta 1-induced Smad activation by antibody fusion proteins PDL1-TGFbRII

HEK293-SBE luc reporter gene cell strain

After the TGFbR receptor recognizes TGF- β, intracellular signaling can be activated, and transcription of the downstream target gene is regulated by activated transcription factor Smads, which enters the nucleus and binds to Smad Binding Elements (SBE) on the promoter. The SBE sequence element (5'-GGTACCTGAGCTCAGTATGTCTAGACTGAAGTATGTCTAGACTGAAGTATGTCTAGACTGACTCGAG-3') shown in SEQ ID No.43 on the promoter is connected into pGL4.22[ luc2CP/Hygro ] plasmid (Promega) through KpnI/XhoI double digestion, pGL4.22-luc2P SBE report plasmid is constructed, HEK293 cells (ATCC CRL-1573) are transfected, 150 mug/ml hygromycin B solution is added after 48 hours for pressurized screening, and the obtained stable transgenic cells are used as HEK293-SBE luc report gene cell strains for monoclonal screening. After 24 hours of stimulation with different concentrations of TGF-beta 1, the fluorescence signal of the positive monoclonal cell line was detected. The results showed that as the concentration of added TGF-beta 1 increased, the fluorescence signal expressed by the HEK293-SBE luc reporter cell was gradually increased, indicating that the cell line could be used to detect the activation of TGF-beta/Smad signaling pathway by TGF-beta 1 (FIG. 19).

HEK293-SBE luc reporter cells were cultured in complete medium at 37℃under 5% CO ₂ at a cell concentration of 3.0-6.0X10- ⁵ cells per 1 ml. After digestion with trypsin EDTA solution (0.25%), the cells were collected by centrifugation at 300 g/min at 4℃for 3-5 min, and the supernatant was discarded. Cells were resuspended in 0.5% FBS/DMEM medium to a cell suspension containing 3.0X10 ⁶ cells per 1ml, inoculated into 96 well sterile fluorescent assay white plates, 100 μl per well, and incubated at 37℃under 5% CO ₂. After 16-18 hours, the cell supernatant from the well plate was discarded and 50. Mu.l of 0.5% FBS/DMEM medium was added. After mixing and incubating antibody fusion proteins PDL1-TGFbRII with equal volumes of TGF-beta 1 of 4ng/ml for 15-30 minutes, 50 μl of antibody/TGF-beta 1 mixed solution is added into cells, and after culturing for 24 hours under the conditions of 37 ℃ and 5% CO ₂ in the total volume of each well, the fluorescence intensity is detected by using a luciferase detection kit, and the measurement result is recorded.

The results show that antibody fusion proteins PDL1-TGFbRII are capable of blocking TGF/Smad signaling pathway with an IC50 of 0.008nM, the novel constructs PDL1-TGFbRII-03, PDL1-TGFbRII-05 have no significant difference in affinity compared to PDL1-TGFbRII-01, with IC50 of 0.008nM and 0.007nM, respectively, comparable to the anti-PD-L1/TGF beta trap blocking activity of the control molecule (FIG. 20).

Table 16 shows inhibition of TGF-beta 1-induced Smad activation by antibody fusion proteins PDL 1-TGFbRII.

Table 16 inhibition of TGF-beta 1-induced Smad activation by PDL1-TGFbRII

EXAMPLE 13 inhibition of TGF-beta 3-induced production of collagen by antibody fusion proteins PDL1-TGFbRII

NRK-49F cells (ATCC, CRL-1570) were collected, washed once with 10ml PBS, and resuspended to a cell density of 1.3X10 ⁵ cells/ml in DMEM complete medium. The resuspended cells were plated at 600. Mu.l/well into 24-well plates, and the 24-well plates were incubated at 37℃in a 5% CO ₂ incubator for 18-20 hours. The supernatant was discarded, washed once with PBS, and starved overnight by adding 100. Mu.l/well of 0.1% DMEM,37℃and 5% CO ₂. The supernatant was discarded, 250. Mu.l/well of protein diluted in a gradient of antibody fusion protein PDL1-TGFbRII, 250. Mu.l/well of TGF-beta.3 (20 ng/ml) was added and the well plate was incubated at 37℃for 72 hours in 5% CO ₂. Taking out the 24-well plate, collecting supernatant and placing at 4 ℃ for detection. The collected supernatant was added to the ELISA plate at 100. Mu.l/well at 4℃overnight. Wash 1 time with PBS. 200 μl/well of 2% BSA was added to the plate and incubated for 2h at room temperature. The PBS was washed 4 times. Mu.l of primary anti-rabbit anti-mouse collagen antibody was added to each well and incubated for 2 hours at room temperature. The PBS was washed 4 times. Mu.l of two Goat Anti-Rabbit IgG-HRP was added to each well and incubated for 2 hours at room temperature. The PBS was washed 4 times. After adding 100. Mu.l of TMB substrate per well and developing at 37℃for 10 minutes, the reaction was stopped with 50. Mu.l of 2M sulfuric acid solution per well, and OD450 was measured by a microplate reader.

The results showed that antibody fusion proteins PDL1-TGFbRII were able to effectively inhibit tgfβ3-induced collagen production by NRK-49F and had a drug concentration dose dependent effect (figure 21).

EXAMPLE 14 inhibition of antibody fusion proteins PDL1-TGFbRII in humanized mouse transplantation tumor model

Female B-hPD-L1 mice (southern mode) were selected for 6-8 weeks and MC38 tumor cells stably transfected with human PD-L1 gene were inoculated subcutaneously at 5X 10 ⁵ per mouse. Anti-mouse CD20 antibody 18B2 mu IgG2a (Ahuja A et al J immunol.2007) was injected tail vein 3 consecutive days after tumor inoculation. When the tumor volume reaches-100 mm ³, random grouping administration is carried out. The administration groups were respectively set to 10mg/kg PDL1-TGFbRII-03, 10mg/kg PDL1-TGFbRII-05, anti-PD-L1/TGF-beta trap and Anti-KLH hIgG 410 mg/kg. The administration volume was 100. Mu.l/dose, and the administration interval was 5 times every 3 days. The tumor volume and body weight of the mice were recorded each time, and the mice were sacrificed by cervical scission after the end of the experiment. The results are shown in FIG. 22, where tumor growth was slow and tumor regressions were observed after administration of PDL1-TGFbRII-03 and PDL 1-TGFbRII-05.

SEQUENCE LISTING

<110> Kannoa biomedical technology (Chengdu) Co., ltd

<120> Development of an immunomodulator and use thereof

<130> MTI20184

<160> 43

<170> PatentIn version 3.5

<210> 1

<211> 107

<212> PRT

<213> Mus musculus

<400> 1

Asp Ile Val Leu Ala Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Lys Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 2

<211> 11

<212> PRT

<213> Mus musculus

<400> 2

Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His

1 5 10

<210> 3

<211> 7

<212> PRT

<213> Mus musculus

<400> 3

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 4

<211> 10

<212> PRT

<213> Mus musculus

<400> 4

Gln Gln Ser Lys Ser Trp Pro Phe Thr Phe

1 5 10

<210> 5

<211> 321

<212> DNA

<213> Mus musculus

<400> 5

gatattgtgc tagctcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 60

ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 120

catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctct 180

aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 240

gaagattttg gaatgtattt ctgtcaacag agtaaaagct ggccattcac gttcggctcg 300

gggacaaggt tggaaataaa a 321

<210> 6

<211> 119

<212> PRT

<213> Mus musculus

<400> 6

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Thr Phe Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 7

<211> 6

<212> PRT

<213> Mus musculus

<400> 7

Ser Gly Tyr Asn Trp His

1 5

<210> 8

<211> 16

<212> PRT

<213> Mus musculus

<400> 8

Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 9

<211> 10

<212> PRT

<213> Mus musculus

<400> 9

Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr

1 5 10

<210> 10

<211> 357

<212> DNA

<213> Mus musculus

<400> 10

gatgtgcagc ttcaggagtc aggacctgac ctggtgaaac cttctcagac actttcactc 60

acctgcactg tcactggcta ctccatcacc agtgggtata actggcactg gatccggcag 120

tttccaggaa acaaactgga atggatgggc tacatacacc acagtagtat cactaactac 180

aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga gttctgtgac tactgaggac acagccacat tttactgtgc aagagagggg 300

tatgattacg actggtttgc ttactggggc caagggactc tggtcactgt ctctgca 357

<210> 11

<211> 107

<212> PRT

<213> Homo sapiens

<400> 11

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 321

<212> DNA

<213> Homo sapiens

<400> 12

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60

ctgagctgta gagccagcca gagcatcagc aacaacctgc actggtatca gcagaagcct 120

ggacaggccc ctagactgct gattaagtac gccagccagt ctatcagcgg catccccgac 180

agattcagcg gctctggctc tggcacagac ttcaccctga ccatcagcag actggaaccc 240

gaggacttcg ccgtgtactt ctgccagcag agcaagagct ggcccttcac atttggccag 300

ggcaccaagc tggaaatcaa g 321

<210> 13

<211> 107

<212> PRT

<213> Homo sapiens

<400> 13

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 321

<212> DNA

<213> Homo sapiens

<400> 14

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60

ctgagctgta gagccagcca gagcatcagc agcaacctgc actggtatca gcagaagcct 120

ggacaggccc ctagactgct gattaagtac gccagccagt ctatcagcgg catccccgac 180

agattcagcg gctctggctc tggcacagac ttcaccctga ccatcagcag actggaaccc 240

gaggacttcg ccgtgtactt ctgccagcag agcaagagct ggcccttcac atttggccag 300

ggcaccaagc tggaaatcaa g 321

<210> 15

<211> 11

<212> PRT

<213> Homo sapiens

<400> 15

Arg Ala Ser Gln Ser Ile Ser Ser Asn Leu His

1 5 10

<210> 16

<211> 119

<212> PRT

<213> Homo sapiens

<400> 16

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 17

<211> 357

<212> DNA

<213> Homo sapiens

<400> 17

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttct 357

<210> 18

<211> 119

<212> PRT

<213> Homo sapiens

<400> 18

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 19

<211> 357

<212> DNA

<213> Homo sapiens

<400> 19

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat cacctactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttct 357

<210> 20

<211> 16

<212> PRT

<213> Homo sapiens

<400> 20

Tyr Ile His His Ser Ser Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 21

<211> 592

<212> PRT

<213> Homo sapiens

<400> 21

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp

20 25 30

Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn

35 40 45

Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr

50 55 60

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

65 70 75 80

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

85 90 95

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

100 105 110

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

115 120 125

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

130 135 140

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

145 150 155 160

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

165 170 175

Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val

180 185 190

Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile

195 200 205

Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser

210 215 220

Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His

225 230 235 240

Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys

245 250 255

Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp

260 265 270

Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu

275 280 285

Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe

290 295 300

Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser

305 310 315 320

Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu

325 330 335

Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe

340 345 350

His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser

355 360 365

Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala

370 375 380

His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile

385 390 395 400

Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu

405 410 415

Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr

420 425 430

Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg

435 440 445

Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val

450 455 460

Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp

465 470 475 480

Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu

485 490 495

Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met

500 505 510

Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe

515 520 525

Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu

530 535 540

Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala

545 550 555 560

Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser

565 570 575

Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys

580 585 590

<210> 22

<211> 1776

<212> DNA

<213> Homo sapiens

<400> 22

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggatgtgg aaatggaggc ccagaaagat 120

gaaatcatct gccccagctg taataggact gcccatccac tgagacatat taataacgac 180

atgatagtca ctgacaacaa cggtgcagtc aagtttccac aactgtgtaa attttgtgat 240

gtgagatttt ccacctgtga caaccagaaa tcctgcatga gcaactgcag catcacctcc 300

atctgtgaga agccacagga agtctgtgtg gctgtatgga gaaagaatga cgagaacata 360

acactagaga cagtttgcca tgaccccaag ctcccctacc atgactttat tctggaagat 420

gctgcttctc caaagtgcat tatgaaggaa aaaaaaaagc ctggtgagac tttcttcatg 480

tgttcctgta gctctgatga gtgcaatgac aacatcatct tctcagaaga atataacacc 540

agcaatcctg acttgttgct agtcatattt caagtgacag gcatcagcct cctgccacca 600

ctgggagttg ccatatctgt catcatcatc ttctactgct accgcgttaa ccggcagcag 660

aagctgagtt caacctggga aaccggcaag acgcggaagc tcatggagtt cagcgagcac 720

tgtgccatca tcctggaaga tgaccgctct gacatcagct ccacgtgtgc caacaacatc 780

aaccacaaca cagagctgct gcccattgag ctggacaccc tggtggggaa aggtcgcttt 840

gctgaggtct ataaggccaa gctgaagcag aacacttcag agcagtttga gacagtggca 900

gtcaagatct ttccctatga ggagtatgcc tcttggaaga cagagaagga catcttctca 960

gacatcaatc tgaagcatga gaacatactc cagttcctga cggctgagga gcggaagacg 1020

gagttgggga aacaatactg gctgatcacc gccttccacg ccaagggcaa cctacaggag 1080

tacctgacgc ggcatgtcat cagctgggag gacctgcgca agctgggcag ctccctcgcc 1140

cgggggattg ctcacctcca cagtgatcac actccatgtg ggaggcccaa gatgcccatc 1200

gtgcacaggg acctcaagag ctccaatatc ctcgtgaaga acgacctaac ctgctgcctg 1260

tgtgactttg ggctttccct gcgtctggac cctactctgt ctgtggatga cctggctaac 1320

agtgggcagg tgggaactgc aagatacatg gctccagaag tcctagaatc caggatgaat 1380

ttggagaatg ttgagtcctt caagcagacc gatgtctact ccatggctct ggtgctctgg 1440

gaaatgacat ctcgctgtaa tgcagtggga gaagtaaaag attatgagcc tccatttggt 1500

tccaaggtgc gggagcaccc ctgtgtcgaa agcatgaagg acaacgtgtt gagagatcga 1560

gggcgaccag aaattcccag cttctggctc aaccaccagg gcatccagat ggtgtgtgag 1620

acgttgactg agtgctggga ccacgaccca gaggcccgtc tcacagccca gtgtgtggca 1680

gaacgcttca gtgagctgga gcatctggac aggctctcgg ggaggagctg ctcggaggag 1740

aagattcctg aagacggctc cctaaacact accaaa 1776

<210> 23

<211> 567

<212> PRT

<213> Homo sapiens

<400> 23

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn

180 185 190

Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys

195 200 205

Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg

210 215 220

Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu

225 230 235 240

Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala

245 250 255

Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu

260 265 270

Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys

275 280 285

Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile

290 295 300

Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln

305 310 315 320

Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr

325 330 335

Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser

340 345 350

Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys

355 360 365

Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn

370 375 380

Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu

385 390 395 400

Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser

405 410 415

Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser

420 425 430

Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr

435 440 445

Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val

450 455 460

Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu

465 470 475 480

His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly

485 490 495

Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met

500 505 510

Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg

515 520 525

Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu

530 535 540

Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp

545 550 555 560

Gly Ser Leu Asn Thr Thr Lys

565

<210> 24

<211> 1701

<212> DNA

<213> Homo sapiens

<400> 24

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcaacc 600

tgggaaaccg gcaagacgcg gaagctcatg gagttcagcg agcactgtgc catcatcctg 660

gaagatgacc gctctgacat cagctccacg tgtgccaaca acatcaacca caacacagag 720

ctgctgccca ttgagctgga caccctggtg gggaaaggtc gctttgctga ggtctataag 780

gccaagctga agcagaacac ttcagagcag tttgagacag tggcagtcaa gatctttccc 840

tatgaggagt atgcctcttg gaagacagag aaggacatct tctcagacat caatctgaag 900

catgagaaca tactccagtt cctgacggct gaggagcgga agacggagtt ggggaaacaa 960

tactggctga tcaccgcctt ccacgccaag ggcaacctac aggagtacct gacgcggcat 1020

gtcatcagct gggaggacct gcgcaagctg ggcagctccc tcgcccgggg gattgctcac 1080

ctccacagtg atcacactcc atgtgggagg cccaagatgc ccatcgtgca cagggacctc 1140

aagagctcca atatcctcgt gaagaacgac ctaacctgct gcctgtgtga ctttgggctt 1200

tccctgcgtc tggaccctac tctgtctgtg gatgacctgg ctaacagtgg gcaggtggga 1260

actgcaagat acatggctcc agaagtccta gaatccagga tgaatttgga gaatgttgag 1320

tccttcaagc agaccgatgt ctactccatg gctctggtgc tctgggaaat gacatctcgc 1380

tgtaatgcag tgggagaagt aaaagattat gagcctccat ttggttccaa ggtgcgggag 1440

cacccctgtg tcgaaagcat gaaggacaac gtgttgagag atcgagggcg accagaaatt 1500

cccagcttct ggctcaacca ccagggcatc cagatggtgt gtgagacgtt gactgagtgc 1560

tgggaccacg acccagaggc ccgtctcaca gcccagtgtg tggcagaacg cttcagtgag 1620

ctggagcatc tggacaggct ctcggggagg agctgctcgg aggagaagat tcctgaagac 1680

ggctccctaa acactaccaa a 1701

<210> 25

<211> 576

<212> PRT

<213> Homo sapiens

<400> 25

Met Leu Ser Thr Ser Gly Val Leu Thr Leu Lys Thr Thr Leu Glu Asp

1 5 10 15

Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn

20 25 30

Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr

35 40 45

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

50 55 60

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

65 70 75 80

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

85 90 95

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

100 105 110

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

115 120 125

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

130 135 140

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

145 150 155 160

Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val

165 170 175

Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile

180 185 190

Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser

195 200 205

Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His

210 215 220

Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys

225 230 235 240

Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp

245 250 255

Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu

260 265 270

Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe

275 280 285

Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser

290 295 300

Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu

305 310 315 320

Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe

325 330 335

His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser

340 345 350

Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala

355 360 365

His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile

370 375 380

Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu

385 390 395 400

Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr

405 410 415

Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg

420 425 430

Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val

435 440 445

Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp

450 455 460

Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu

465 470 475 480

Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met

485 490 495

Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe

500 505 510

Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu

515 520 525

Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala

530 535 540

Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser

545 550 555 560

Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys

565 570 575

<210> 26

<211> 1728

<212> DNA

<213> Homo sapiens

<400> 26

atgctgagta cttcaggtgt attaaccctc aaaacaactt tagaagatgt ggaaatggag 60

gcccagaaag atgaaatcat ctgccccagc tgtaatagga ctgcccatcc actgagacat 120

attaataacg acatgatagt cactgacaac aacggtgcag tcaagtttcc acaactgtgt 180

aaattttgtg atgtgagatt ttccacctgt gacaaccaga aatcctgcat gagcaactgc 240

agcatcacct ccatctgtga gaagccacag gaagtctgtg tggctgtatg gagaaagaat 300

gacgagaaca taacactaga gacagtttgc catgacccca agctccccta ccatgacttt 360

attctggaag atgctgcttc tccaaagtgc attatgaagg aaaaaaaaaa gcctggtgag 420

actttcttca tgtgttcctg tagctctgat gagtgcaatg acaacatcat cttctcagaa 480

gaatataaca ccagcaatcc tgacttgttg ctagtcatat ttcaagtgac aggcatcagc 540

ctcctgccac cactgggagt tgccatatct gtcatcatca tcttctactg ctaccgcgtt 600

aaccggcagc agaagctgag ttcaacctgg gaaaccggca agacgcggaa gctcatggag 660

ttcagcgagc actgtgccat catcctggaa gatgaccgct ctgacatcag ctccacgtgt 720

gccaacaaca tcaaccacaa cacagagctg ctgcccattg agctggacac cctggtgggg 780

aaaggtcgct ttgctgaggt ctataaggcc aagctgaagc agaacacttc agagcagttt 840

gagacagtgg cagtcaagat ctttccctat gaggagtatg cctcttggaa gacagagaag 900

gacatcttct cagacatcaa tctgaagcat gagaacatac tccagttcct gacggctgag 960

gagcggaaga cggagttggg gaaacaatac tggctgatca ccgccttcca cgccaagggc 1020

aacctacagg agtacctgac gcggcatgtc atcagctggg aggacctgcg caagctgggc 1080

agctccctcg cccgggggat tgctcacctc cacagtgatc acactccatg tgggaggccc 1140

aagatgccca tcgtgcacag ggacctcaag agctccaata tcctcgtgaa gaacgaccta 1200

acctgctgcc tgtgtgactt tgggctttcc ctgcgtctgg accctactct gtctgtggat 1260

gacctggcta acagtgggca ggtgggaact gcaagataca tggctccaga agtcctagaa 1320

tccaggatga atttggagaa tgttgagtcc ttcaagcaga ccgatgtcta ctccatggct 1380

ctggtgctct gggaaatgac atctcgctgt aatgcagtgg gagaagtaaa agattatgag 1440

cctccatttg gttccaaggt gcgggagcac ccctgtgtcg aaagcatgaa ggacaacgtg 1500

ttgagagatc gagggcgacc agaaattccc agcttctggc tcaaccacca gggcatccag 1560

atggtgtgtg agacgttgac tgagtgctgg gaccacgacc cagaggcccg tctcacagcc 1620

cagtgtgtgg cagaacgctt cagtgagctg gagcatctgg acaggctctc ggggaggagc 1680

tgctcggagg agaagattcc tgaagacggc tccctaaaca ctaccaaa 1728

<210> 27

<211> 532

<212> PRT

<213> Homo sapiens

<400> 27

Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys

1 5 10 15

Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys

20 25 30

Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val

35 40 45

Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr

50 55 60

Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp

65 70 75 80

Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu

85 90 95

Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile

100 105 110

Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val

115 120 125

Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala

130 135 140

Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln

145 150 155 160

Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu

165 170 175

Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile

180 185 190

Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro

195 200 205

Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr

210 215 220

Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala

225 230 235 240

Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys

245 250 255

Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe

260 265 270

Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu

275 280 285

Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg

290 295 300

His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala

305 310 315 320

Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro

325 330 335

Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val

340 345 350

Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg

355 360 365

Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val

370 375 380

Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn

385 390 395 400

Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala

405 410 415

Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val

420 425 430

Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys

435 440 445

Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu

450 455 460

Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu

465 470 475 480

Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala

485 490 495

Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu

500 505 510

Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu

515 520 525

Asn Thr Thr Lys

530

<210> 28

<211> 1596

<212> DNA

<213> Homo sapiens

<400> 28

atgatagtca ctgacaacaa cggtgcagtc aagtttccac aactgtgtaa attttgtgat 60

gtgagatttt ccacctgtga caaccagaaa tcctgcatga gcaactgcag catcacctcc 120

atctgtgaga agccacagga agtctgtgtg gctgtatgga gaaagaatga cgagaacata 180

acactagaga cagtttgcca tgaccccaag ctcccctacc atgactttat tctggaagat 240

gctgcttctc caaagtgcat tatgaaggaa aaaaaaaagc ctggtgagac tttcttcatg 300

tgttcctgta gctctgatga gtgcaatgac aacatcatct tctcagaaga atataacacc 360

agcaatcctg acttgttgct agtcatattt caagtgacag gcatcagcct cctgccacca 420

ctgggagttg ccatatctgt catcatcatc ttctactgct accgcgttaa ccggcagcag 480

aagctgagtt caacctggga aaccggcaag acgcggaagc tcatggagtt cagcgagcac 540

tgtgccatca tcctggaaga tgaccgctct gacatcagct ccacgtgtgc caacaacatc 600

aaccacaaca cagagctgct gcccattgag ctggacaccc tggtggggaa aggtcgcttt 660

gctgaggtct ataaggccaa gctgaagcag aacacttcag agcagtttga gacagtggca 720

gtcaagatct ttccctatga ggagtatgcc tcttggaaga cagagaagga catcttctca 780

gacatcaatc tgaagcatga gaacatactc cagttcctga cggctgagga gcggaagacg 840

gagttgggga aacaatactg gctgatcacc gccttccacg ccaagggcaa cctacaggag 900

tacctgacgc ggcatgtcat cagctgggag gacctgcgca agctgggcag ctccctcgcc 960

cgggggattg ctcacctcca cagtgatcac actccatgtg ggaggcccaa gatgcccatc 1020

gtgcacaggg acctcaagag ctccaatatc ctcgtgaaga acgacctaac ctgctgcctg 1080

tgtgactttg ggctttccct gcgtctggac cctactctgt ctgtggatga cctggctaac 1140

agtgggcagg tgggaactgc aagatacatg gctccagaag tcctagaatc caggatgaat 1200

ttggagaatg ttgagtcctt caagcagacc gatgtctact ccatggctct ggtgctctgg 1260

gaaatgacat ctcgctgtaa tgcagtggga gaagtaaaag attatgagcc tccatttggt 1320

tccaaggtgc gggagcaccc ctgtgtcgaa agcatgaagg acaacgtgtt gagagatcga 1380

gggcgaccag aaattcccag cttctggctc aaccaccagg gcatccagat ggtgtgtgag 1440

acgttgactg agtgctggga ccacgaccca gaggcccgtc tcacagccca gtgtgtggca 1500

gaacgcttca gtgagctgga gcatctggac aggctctcgg ggaggagctg ctcggaggag 1560

aagattcctg aagacggctc cctaaacact accaaa 1596

<210> 29

<211> 532

<212> PRT

<213> Homo sapiens

<400> 29

Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys

1 5 10 15

Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys

20 25 30

Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val

35 40 45

Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr

50 55 60

Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp

65 70 75 80

Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu

85 90 95

Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile

100 105 110

Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val

115 120 125

Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala

130 135 140

Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln

145 150 155 160

Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu

165 170 175

Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile

180 185 190

Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro

195 200 205

Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr

210 215 220

Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala

225 230 235 240

Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys

245 250 255

Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe

260 265 270

Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu

275 280 285

Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg

290 295 300

His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala

305 310 315 320

Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro

325 330 335

Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val

340 345 350

Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg

355 360 365

Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val

370 375 380

Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn

385 390 395 400

Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala

405 410 415

Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val

420 425 430

Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys

435 440 445

Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu

450 455 460

Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu

465 470 475 480

Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala

485 490 495

Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu

500 505 510

Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu

515 520 525

Asn Thr Thr Lys

530

<210> 30

<211> 1596

<212> DNA

<213> Homo sapiens

<400> 30

atgatagtca ctgacaacaa cggtgcagtc aagtttccac aactgtgtaa attttgtgat 60

gtgagatttt ccacctgtga caaccagaaa tcctgcatga gcaactgcag catcacctcc 120

atctgtgaga agccacagga agtctgtgtg gctgtatgga gaaagaatga cgagaacata 180

acactagaga cagtttgcca tgaccccaag ctcccctacc atgactttat tctggaagat 240

gctgcttctc caaagtgcat tatgaaggaa aaaaaaaagc ctggtgagac tttcttcatg 300

tgttcctgta gctctgatga gtgcaatgac aacatcatct tctcagaaga atataacacc 360

agcaatcctg acttgttgct agtcatattt caagtgacag gcatcagcct cctgccacca 420

ctgggagttg ccatatctgt catcatcatc ttctactgct accgcgttaa ccggcagcag 480

aagctgagtt caacctggga aaccggcaag acgcggaagc tcatggagtt cagcgagcac 540

tgtgccatca tcctggaaga tgaccgctct gacatcagct ccacgtgtgc caacaacatc 600

aaccacaaca cagagctgct gcccattgag ctggacaccc tggtggggaa aggtcgcttt 660

gctgaggtct ataaggccaa gctgaagcag aacacttcag agcagtttga gacagtggca 720

gtcaagatct ttccctatga ggagtatgcc tcttggaaga cagagaagga catcttctca 780

gacatcaatc tgaagcatga gaacatactc cagttcctga cggctgagga gcggaagacg 840

gagttgggga aacaatactg gctgatcacc gccttccacg ccaagggcaa cctacaggag 900

tacctgacgc ggcatgtcat cagctgggag gacctgcgca agctgggcag ctccctcgcc 960

cgggggattg ctcacctcca cagtgatcac actccatgtg ggaggcccaa gatgcccatc 1020

gtgcacaggg acctcaagag ctccaatatc ctcgtgaaga acgacctaac ctgctgcctg 1080

tgtgactttg ggctttccct gcgtctggac cctactctgt ctgtggatga cctggctaac 1140

agtgggcagg tgggaactgc aagatacatg gctccagaag tcctagaatc caggatgaat 1200

ttggagaatg ttgagtcctt caagcagacc gatgtctact ccatggctct ggtgctctgg 1260

gaaatgacat ctcgctgtaa tgcagtggga gaagtaaaag attatgagcc tccatttggt 1320

tccaaggtgc gggagcaccc ctgtgtcgaa agcatgaagg acaacgtgtt gagagatcga 1380

gggcgaccag aaattcccag cttctggctc aaccaccagg gcatccagat ggtgtgtgag 1440

acgttgactg agtgctggga ccacgaccca gaggcccgtc tcacagccca gtgtgtggca 1500

gaacgcttca gtgagctgga gcatctggac aggctctcgg ggaggagctg ctcggaggag 1560

aagattcctg aagacggctc cctaaacact accaaa 1596

<210> 31

<211> 598

<212> PRT

<213> Homo sapiens

<400> 31

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro

450 455 460

Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn

465 470 475 480

Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg

485 490 495

Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile

500 505 510

Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg

515 520 525

Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys

530 535 540

Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys

545 550 555 560

Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser

565 570 575

Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr

580 585 590

Asn Thr Ser Asn Pro Asp

595

<210> 32

<211> 1794

<212> DNA

<213> Homo sapiens

<400> 32

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttctgct 360

agcaccaagg gccccagcgt gttccccctg gccccttgca gcagaagcac cagcgagagc 420

acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aacagcggcg ctctgaccag cggcgtgcat accttccccg ccgtgctcca gagcagcgga 540

ctgtactccc tgagcagcgt ggtgaccgtg ccttccagca gcctgggcac caagacctac 600

acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag 660

tacggccctc cctgcccccc ttgccctgcc cccgagttcg aaggcggacc tagcgtgttc 720

ctgttccccc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 780

gtggtggtgg acgtgtccca ggaggacccc gaggtccagt ttaattggta cgtggacggc 840

gtggaagtgc ataacgccaa gaccaagccc agagaggagc agttcgccag cacctacaga 900

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 960

aaggtctcca acaagggcct gcctagcagc atcgagaaga ccatcagcaa ggccaagggc 1020

cagccacggg agccccaggt ctacaccctg ccacctagcc aagaggagat gaccaagaac 1080

caggtgtccc tgacctgtct ggtgaaaggc ttctatccca gcgatatcgc cgtggagtgg 1140

gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1200

ggcagcttct tcctgtactc caagctgacc gtggacaagt ccagatggca ggagggcaac 1260

gtcttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320

agcctgagcc tgggcggcgg aggcggagga tctggtggcg gaggaagcgg tggcggcgga 1380

tccggaattc ctccacacgt gcagaaaagc gtgaacaacg acatgatcgt gaccgacaac 1440

aacggggccg tgaagttccc tcagctgtgc aagttctgcg acgtgcggtt cagcacctgt 1500

gacaaccaga aaagctgcat gagcaactgc agcatcacca gcatctgcga gaagccccaa 1560

gaagtgtgcg tcgccgtttg gagaaagaac gacgagaaca tcaccctgga aaccgtgtgt 1620

cacgacccca agctgcccta ccacgacttc atcctggaag atgccgcctc tcctaagtgc 1680

atcatgaagg aaaagaagaa gcccggcgag acattcttca tgtgcagctg ctctagcgac 1740

gagtgcaacg acaacatcat cttcagcgaa gagtacaaca caagcaaccc cgac 1794

<210> 33

<211> 214

<212> PRT

<213> Homo sapiens

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 34

<211> 642

<212> DNA

<213> Homo sapiens

<400> 34

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60

ctgagctgta gagccagcca gagcatcagc aacaacctgc actggtatca gcagaagcct 120

ggacaggccc ctagactgct gattaagtac gccagccagt ctatcagcgg catccccgac 180

agattcagcg gctctggctc tggcacagac ttcaccctga ccatcagcag actggaaccc 240

gaggacttcg ccgtgtactt ctgccagcag agcaagagct ggcccttcac atttggccag 300

ggcaccaagc tggaaatcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 35

<211> 586

<212> PRT

<213> Homo sapiens

<400> 35

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Met Ile

450 455 460

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

465 470 475 480

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

485 490 495

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

500 505 510

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

515 520 525

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

530 535 540

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

545 550 555 560

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

565 570 575

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

580 585

<210> 36

<211> 1758

<212> DNA

<213> Homo sapiens

<400> 36

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttctgct 360

agcaccaagg gccccagcgt gttccccctg gccccttgca gcagaagcac cagcgagagc 420

acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aacagcggcg ctctgaccag cggcgtgcat accttccccg ccgtgctcca gagcagcgga 540

ctgtactccc tgagcagcgt ggtgaccgtg ccttccagca gcctgggcac caagacctac 600

acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag 660

tacggccctc cctgcccccc ttgccctgcc cccgagttcg aaggcggacc tagcgtgttc 720

ctgttccccc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 780

gtggtggtgg acgtgtccca ggaggacccc gaggtccagt ttaattggta cgtggacggc 840

gtggaagtgc ataacgccaa gaccaagccc agagaggagc agttcaacag cacctacaga 900

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 960

aaggtctcca acaagggcct ggccagcagc atcgagaaga ccatcagcaa ggccaagggc 1020

cagccacggg agccccaggt ctacaccctg ccacctagcc aagaggagat gaccaagaac 1080

caggtgtccc tgacctgtct ggtgaaaggc ttctatccca gcgatatcgc cgtggagtgg 1140

gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1200

ggcagcttct tcctgtactc caagctgacc gtggacaagt ccagatggca ggagggcaac 1260

gtcttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320

agcctgagcc tgggcggcgg aggcggagga tctggtggcg gaggaagcgg tggcggcgga 1380

tccggaatga tcgtgaccga caacaacggg gccgtgaagt tccctcagct gtgcaagttc 1440

tgcgacgtgc ggttcagcac ctgtgacaac cagaaaagct gcatgagcaa ctgcagcatc 1500

accagcatct gcgagaagcc ccaagaagtg tgcgtcgccg tttggagaaa gaacgacgag 1560

aacatcaccc tggaaaccgt gtgtcacgac cccaagctgc cctaccacga cttcatcctg 1620

gaagatgccg cctctcctaa gtgcatcatg aaggaaaaga agaagcccgg cgagacattc 1680

ttcatgtgca gctgctctag cgacgagtgc aacgacaaca tcatcttcag cgaagagtac 1740

aacacaagca accccgac 1758

<210> 37

<211> 586

<212> PRT

<213> Homo sapiens

<400> 37

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Met Ile

450 455 460

Val Thr Asp Asn Ser Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

465 470 475 480

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

485 490 495

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

500 505 510

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

515 520 525

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

530 535 540

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

545 550 555 560

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

565 570 575

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

580 585

<210> 38

<211> 1758

<212> DNA

<213> Homo sapiens

<400> 38

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttctgct 360

agcaccaagg gccccagcgt gttccccctg gccccttgca gcagaagcac cagcgagagc 420

acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aacagcggcg ctctgaccag cggcgtgcat accttccccg ccgtgctcca gagcagcgga 540

ctgtactccc tgagcagcgt ggtgaccgtg ccttccagca gcctgggcac caagacctac 600

acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag 660

tacggccctc cctgcccccc ttgccctgcc cccgagttcg aaggcggacc tagcgtgttc 720

ctgttccccc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 780

gtggtggtgg acgtgtccca ggaggacccc gaggtccagt ttaattggta cgtggacggc 840

gtggaagtgc ataacgccaa gaccaagccc agagaggagc agttcaacag cacctacaga 900

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 960

aaggtctcca acaagggcct ggccagcagc atcgagaaga ccatcagcaa ggccaagggc 1020

cagccacggg agccccaggt ctacaccctg ccacctagcc aagaggagat gaccaagaac 1080

caggtgtccc tgacctgtct ggtgaaaggc ttctatccca gcgatatcgc cgtggagtgg 1140

gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1200

ggcagcttct tcctgtactc caagctgacc gtggacaagt ccagatggca ggagggcaac 1260

gtcttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320

agcctgagcc tgggcggcgg aggcggagga tctggtggcg gaggaagcgg tggcggcgga 1380

tccggaatga tcgtgaccga caacagcggg gccgtgaagt tccctcagct gtgcaagttc 1440

tgcgacgtgc ggttcagcac ctgtgacaac cagaaaagct gcatgagcaa ctgcagcatc 1500

accagcatct gcgagaagcc ccaagaagtg tgcgtcgccg tttggagaaa gaacgacgag 1560

aacatcaccc tggaaaccgt gtgtcacgac cccaagctgc cctaccacga cttcatcctg 1620

gaagatgccg cctctcctaa gtgcatcatg aaggaaaaga agaagcccgg cgagacattc 1680

ttcatgtgca gctgctctag cgacgagtgc aacgacaaca tcatcttcag cgaagagtac 1740

aacacaagca accccgac 1758

<210> 39

<211> 598

<212> PRT

<213> Homo sapiens

<400> 39

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro

450 455 460

Pro His Val Gln Glu Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn

465 470 475 480

Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg

485 490 495

Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile

500 505 510

Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg

515 520 525

Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys

530 535 540

Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys

545 550 555 560

Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser

565 570 575

Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr

580 585 590

Asn Thr Ser Asn Pro Asp

595

<210> 40

<211> 1794

<212> DNA

<213> Homo sapiens

<400> 40

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttctgct 360

agcaccaagg gccccagcgt gttccccctg gccccttgca gcagaagcac cagcgagagc 420

acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aacagcggcg ctctgaccag cggcgtgcat accttccccg ccgtgctcca gagcagcgga 540

ctgtactccc tgagcagcgt ggtgaccgtg ccttccagca gcctgggcac caagacctac 600

acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag 660

tacggccctc cctgcccccc ttgccctgcc cccgagttcg aaggcggacc tagcgtgttc 720

ctgttccccc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 780

gtggtggtgg acgtgtccca ggaggacccc gaggtccagt ttaattggta cgtggacggc 840

gtggaagtgc ataacgccaa gaccaagccc agagaggagc agttcaacag cacctacaga 900

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 960

aaggtctcca acaagggcct ggccagcagc atcgagaaga ccatcagcaa ggccaagggc 1020

cagccacggg agccccaggt ctacaccctg ccacctagcc aagaggagat gaccaagaac 1080

caggtgtccc tgacctgtct ggtgaaaggc ttctatccca gcgatatcgc cgtggagtgg 1140

gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1200

ggcagcttct tcctgtactc caagctgacc gtggacaagt ccagatggca ggagggcaac 1260

gtcttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320

agcctgagcc tgggcggcgg aggcggagga tctggtggcg gaggaagcgg tggcggcgga 1380

tccggaattc ctccacacgt gcaggagagc gtgaacaacg acatgatcgt gaccgacaac 1440

aacggggccg tgaagttccc tcagctgtgc aagttctgcg acgtgcggtt cagcacctgt 1500

gacaaccaga aaagctgcat gagcaactgc agcatcacca gcatctgcga gaagccccaa 1560

gaagtgtgcg tcgccgtttg gagaaagaac gacgagaaca tcaccctgga aaccgtgtgt 1620

cacgacccca agctgcccta ccacgacttc atcctggaag atgccgcctc tcctaagtgc 1680

atcatgaagg aaaagaagaa gcccggcgag acattcttca tgtgcagctg ctctagcgac 1740

gagtgcaacg acaacatcat cttcagcgaa gagtacaaca caagcaaccc cgac 1794

<210> 41

<211> 598

<212> PRT

<213> Homo sapiens

<400> 41

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His His Ser Ser Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Tyr Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro

450 455 460

Pro His Val Gln Glu Ser Val Asn Ser Asp Met Ile Val Thr Asp Asn

465 470 475 480

Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg

485 490 495

Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile

500 505 510

Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg

515 520 525

Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys

530 535 540

Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys

545 550 555 560

Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser

565 570 575

Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr

580 585 590

Asn Thr Ser Asn Pro Asp

595

<210> 42

<211> 1794

<212> DNA

<213> Homo sapiens

<400> 42

caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60

acctgtaccg tgtccggcta ctctatcacc agcggctaca actggcactg gatcagacag 120

cctccaggca aaggcctgga atggatcggc tacatccacc acagcagcat caccaactac 180

aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cagagagggc 300

tacgactacg attggttcgc ttactggggc cagggcaccc tggttacagt ctcttctgct 360

agcaccaagg gccccagcgt gttccccctg gccccttgca gcagaagcac cagcgagagc 420

acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aacagcggcg ctctgaccag cggcgtgcat accttccccg ccgtgctcca gagcagcgga 540

ctgtactccc tgagcagcgt ggtgaccgtg ccttccagca gcctgggcac caagacctac 600

acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag 660

tacggccctc cctgcccccc ttgccctgcc cccgagttcg aaggcggacc tagcgtgttc 720

ctgttccccc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 780

gtggtggtgg acgtgtccca ggaggacccc gaggtccagt ttaattggta cgtggacggc 840

gtggaagtgc ataacgccaa gaccaagccc agagaggagc agttcaacag cacctacaga 900

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga atacaagtgc 960

aaggtctcca acaagggcct ggccagcagc atcgagaaga ccatcagcaa ggccaagggc 1020

cagccacggg agccccaggt ctacaccctg ccacctagcc aagaggagat gaccaagaac 1080

caggtgtccc tgacctgtct ggtgaaaggc ttctatccca gcgatatcgc cgtggagtgg 1140

gagagcaacg gccagcccga gaacaactac aagaccaccc cccctgtgct ggacagcgac 1200

ggcagcttct tcctgtactc caagctgacc gtggacaagt ccagatggca ggagggcaac 1260

gtcttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1320

agcctgagcc tgggcggcgg aggcggagga tctggtggcg gaggaagcgg tggcggcgga 1380

tccggaattc ctccacacgt gcaggagagc gtgaacagcg acatgatcgt gaccgacaac 1440

aacggggccg tgaagttccc tcagctgtgc aagttctgcg acgtgcggtt cagcacctgt 1500

gacaaccaga aaagctgcat gagcaactgc agcatcacca gcatctgcga gaagccccaa 1560

gaagtgtgcg tcgccgtttg gagaaagaac gacgagaaca tcaccctgga aaccgtgtgt 1620

cacgacccca agctgcccta ccacgacttc atcctggaag atgccgcctc tcctaagtgc 1680

atcatgaagg aaaagaagaa gcccggcgag acattcttca tgtgcagctg ctctagcgac 1740

gagtgcaacg acaacatcat cttcagcgaa gagtacaaca caagcaaccc cgac 1794

<210> 43

<211> 67

<212> DNA

<213> Homo sapiens

<400> 43

ggtacctgag ctcagtatgt ctagactgaa gtatgtctag actgaagtat gtctagactg 60

actcgag 67

Claims (13)

1. An antibody or antigen-binding portion thereof that binds PD-L1, the CDR sequences of the heavy and light chains of the antibody or antigen-binding portion thereof being one of the following combinations:

(1) Light chain CDR1, CDR2 and CDR3 with sequences shown as SEQ ID NO 2,3 and 4 respectively, and heavy chain CDR1, CDR2 and CDR3 with sequences shown as SEQ ID NO 7, 20 and 9 respectively;

(2) Light chain CDR1, CDR2 and CDR3 with sequences shown as SEQ ID NO 15, 3 and 4 respectively, and heavy chain CDR1, CDR2 and CDR3 with sequences shown as SEQ ID NO 7, 8 and 9 respectively;

(3) Light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO 15, 3 and 4, and heavy chain CDR1, CDR2 and CDR3 shown in SEQ ID NO 7, 20 and 9.

2. The antibody or antigen binding portion thereof of claim 1, having a light chain variable region and a heavy chain variable region of one of the following combinations:

(1) A light chain variable region with an amino acid sequence shown as SEQ ID NO.11, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 16;

(2) A light chain variable region with an amino acid sequence shown as SEQ ID NO.11, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 18;

(3) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 16;

(4) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 18.

3. A bispecific or multispecific antibody molecule comprising the antibody or antigen-binding portion thereof of claim 1 or 2 that binds PD-L1.

4. A PDL1-TGFbRII antibody fusion protein, wherein:

The amino acid sequence of the polypeptide fused with TGFbRII of the PD-L1 antibody heavy chain in the antibody fusion protein is shown as SEQ ID NO. 37 or SEQ ID NO. 41, and the amino acid sequence of the PD-L1 antibody light chain in the antibody fusion protein is shown as SEQ ID NO. 33.

5. The antibody or antigen-binding portion thereof of claim 1 or 2 or the bispecific or multispecific antibody molecule of claim 3, wherein the antibody or antigen-binding portion thereof is humanized.

6. A nucleic acid encoding the antibody or antigen binding portion thereof of claim 1 or 2, the bispecific or multispecific antibody molecule of claim 3, the antibody fusion protein of claim 4, the antibody or antigen binding portion thereof of claim 5, or the bispecific or multispecific antibody molecule.

7. The nucleic acid of claim 6, comprising one selected from the group consisting of:

(1) The nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 12, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 17;

(2) The nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 12, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 19;

(3) The nucleotide sequence of the light chain variable region is shown as SEQ ID NO.14, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 17;

(4) The nucleotide sequence of the light chain variable region is shown as SEQ ID NO.14, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 19;

(5) The nucleotide sequence of the fusion polypeptide is shown as SEQ ID NO. 38, and the nucleotide sequence of the antibody light chain is shown as SEQ ID NO. 34;

(6) The nucleotide sequence of the fusion polypeptide is shown as SEQ ID NO. 42, and the nucleotide sequence of the antibody light chain is shown as SEQ ID NO. 34.

8. A vector comprising the nucleic acid of claim 6.

9. A cell comprising the nucleic acid of claim 6 or the vector of claim 8.

10. A composition comprising the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific or multispecific antibody molecule of claim 3, the antibody fusion protein of claim 4, the antibody or antigen-binding portion or bispecific or multispecific antibody molecule of claim 5, the nucleic acid of claim 6, the vector of claim 8 and/or the cell of claim 9, and a pharmaceutically acceptable carrier.

11. An antibody-drug conjugate comprising the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific or multispecific antibody molecule of claim 3, the antibody fusion protein of claim 4, or the antibody or antigen-binding portion thereof of claim 5, or the bispecific or multispecific antibody molecule, covalently bound to a therapeutic moiety.

12. A kit comprising the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific or multispecific antibody molecule of claim 3, the antibody fusion protein of claim 4, the antibody or antigen-binding portion or bispecific or multispecific antibody molecule of claim 5, the nucleic acid molecule of claim 6, the vector of claim 8, the cell of claim 9, the composition of claim 10, and/or the antibody-drug conjugate of claim 11.

13. Use of an antibody or antigen binding portion thereof according to claim 1 or 2, a bispecific or multispecific antibody molecule according to claim 3, an antibody fusion protein according to claim 4, an antibody or antigen binding portion or bispecific or multispecific antibody molecule according to claim 5, a nucleic acid molecule according to claim 6, a vector according to claim 8, a cell according to claim 9, a composition according to claim 10 and/or an antibody-drug conjugate according to claim 11 in the manufacture of a medicament or kit for the diagnosis, treatment or prevention of colorectal cancer.