CN116323658A

CN116323658A - Bifunctional protein targeting PD-1 or PD-L1 and TGF-β and its medical application

Info

Publication number: CN116323658A
Application number: CN202180066168.0A
Authority: CN
Inventors: 冯辉; 刘洪川; 姚盛; 何丽; 周岳华; 张静; 刘丹丹; 吴纯; 刘辉; 赵强
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Jinyu Bowo Runze Biotechnology Co ltd
Priority date: 2020-09-28
Filing date: 2021-09-28
Publication date: 2023-06-23
Also published as: WO2022063314A1

Abstract

The present invention relates to a bifunctional protein targeting PD‑1 or PD‑L1 and TGF‑β and its medical use. The bifunctional protein comprises (i) an antigen-binding fragment of an anti-PD-1 or PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) a TGF-βRII extracellular domain (ECD). The bifunctional protein of the present invention has strong affinity with PD‑1 or PD‑L1 protein, has good thermal stability, and can significantly promote the release of IFNγ and inhibit tumor growth.

Description

Bifunctional protein targeting PD-1 or PD-L1 and TGF-beta and medical application thereof

Technical Field

The invention relates to the technical field of medical biology. In particular, the present invention relates to bifunctional proteins targeting programmed death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) and transforming growth factor-beta (TGF- β), polynucleotides encoding the bifunctional proteins of the present invention, vectors and host cells for expressing the bifunctional proteins of the present invention, and the use of the bifunctional proteins of the present invention in the treatment, prevention and/or diagnosis of diseases associated with PD-1 or PD-L1 activity and TGF- β family activity.

Background

Immune checkpoints (immune checkpoint) are a class of inhibitory signaling molecules present in the immune system, avoid tissue damage by modulating the persistence and intensity of immune responses in peripheral tissues, and are involved in maintaining tolerance to self-antigens. It was found that one of the reasons why tumor cells can evade the immune system in vivo and proliferate uncontrollably is to use the inhibitory signaling pathway of immune checkpoints, thereby inhibiting the activity of T lymphocytes, so that T lymphocytes cannot effectively exert the killing effect on tumors.

Programmed death receptor 1 (PD-1) is a member of the CD28 superfamily. PD-1 is an important immune checkpoint protein and is currently also an important target for tumor immunotherapy. PD-1 is expressed in activated T cells, B cells and myeloid cells, which have two ligands, programmed death ligand-1 (programmed death ligand 1, PD-L1) and programmed death ligand-2 (PD-L2). PD-L1 interacts with the receptor PD-1 on T cells, playing an important role in the negative regulation of immune responses. The expression of PD-L1 protein can be detected in many human tumor tissues, the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells, and the expressed PD-L1 is favorable for the generation and growth of tumors and induces the apoptosis of anti-tumor T cells. The PD-1/PD-L1 pathway inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, and enable T cells to recover activity, so that immune response is enhanced, and therefore, the immunoregulation with PD-1/PD-L1 as a target point has important significance for tumor inhibition.

Programmed cell death protein ligand 1 (PD-L1), also known as cluster of differentiation 274 (cluster of differentiation, CD 274) or B7 homologous protein 1 (B7 homolog1, B7-H1), belongs to the tumor necrosis factor superfamily, is a type I transmembrane glycoprotein consisting of 290 amino acid residues, comprises an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and a 30 amino acid intracellular tail, and has a complete molecular weight of 40kDa1.PD-L1mRNA is expressed in almost all tissues, but PD-L1 protein is continuously expressed in only a small number of tissues, including liver, lung, tonsils, and immune-privileged tissues such as eyes, placenta, etc. PD-L1 is also expressed in activated T cells, B cells, monocytes, dendritic cells, macrophages and the like. The receptor of PD-L1 is PD-1, and is mainly expressed on the surfaces of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes.

Transforming growth factor-beta (transforming growth factor-beta, TGF-beta) belongs to the TGF-beta superfamily that regulates cell growth and differentiation. TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors.

Transforming growth factor-beta (tgfβ) is a pleiotropic cytokine that has immunomodulatory properties such as the limitation and termination of inflammatory and allergic immune responses. Tgfβ has a link to inflammatory, malignant, infectious and autoimmune diseases, and osteoporosis and fibrosis including cirrhosis and systemic sclerosis. In particular, sustained high levels of tgfβ in tumors are associated with increased immune tolerance, angiogenesis, metastasis and deposition of tumor extracellular matrix, all of which may drive cancer progression and resistance to therapy.

In an immune microenvironment, the PD-1/PD-L1 channel is blocked by an antibody targeting PD-1 or PD-L1, so that negative regulation signals are blocked, and T cells are enabled to restore activity, thereby enhancing immune response and more effectively improving the effect of inhibiting tumor occurrence and development.

Although antibody/TGF-beta receptor fusion proteins are currently disclosed, some fusion proteins still suffer from instability or low expression levels, and there remains a need for novel fusion proteins that include improved stability and activity over known antibodies in various respects.

Disclosure of Invention

The present invention provides bifunctional proteins targeting programmed death protein-1 (PD-1)/programmed death protein ligand 1 (PD-L1) and transforming growth factor-beta (TGF-beta) and their use in the treatment, prevention and/or diagnosis of diseases associated with PD-1 activity and TGF-beta activity. The bifunctional proteins provided herein may be used as stand alone therapies or in combination with other therapies and/or other anti-cancer agents, such as for the treatment of cancer.

In one aspect, the invention provides a bifunctional protein targeting PD-1 or PD-L1 and TGF-beta comprising

(i) An antigen binding fragment of an antibody that blocks the PD-1/PD-L1 pathway;

(ii) An immunoglobulin Fc domain; and

(iii) TGF-. Beta.RII extracellular domain (ECD).

In some embodiments, the antigen binding fragment of an antibody that blocks the PD-1/PD-L1 pathway of the present invention is an antigen binding fragment of an anti-PD-1 or PD-L1 antibody.

In some embodiments, the antigen-binding fragment of the anti-PD-1 antibodies of the invention comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences set forth in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

In some embodiments, the antigen-binding fragment of the anti-PD-1 antibodies of the invention comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the light chain variable region having a heavy chain variable region as set forth in SEQ ID NO. 7 as set forth in SEQ ID NO. 8.

In some embodiments, the antigen-binding fragment of the anti-PD-L1 antibodies of the invention comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as set forth in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as set forth in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively.

In some embodiments, the antigen-binding fragment of the anti-PD-L1 antibodies of the invention comprises a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 24 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 25.

In some embodiments, the antigen-binding fragment of the anti-PD-L1 antibodies of the invention comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 24 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 25.

In some embodiments, the antigen binding fragment of the anti-PD-1 or PD-L1 antibodies of the invention is a Fab, fab ', F (ab') 2, fv, scFv, or sdAb.

In some embodiments, the immunoglobulin Fc domain of the invention is a human IgG1, igG2, igG3, or IgG4 Fc domain; an Fc domain, preferably human IgG 4; preferably, the Fc domain of IgG4 comprises 1, 2, 3, 4 or 5 amino acid differences, preferably with an S228P amino acid substitution in the Fc domain; it is further preferred to have a C-terminal deletion of both K and G amino acids in the Fc domain.

In some embodiments, the immunoglobulin Fc domain of the present invention comprises an amino acid sequence as set forth in SEQ ID NO. 9, or an Fc domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 9.

In some embodiments, the bifunctional proteins of the invention, wherein the immunoglobulin Fc domain comprises an Fc domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 9.

In some embodiments, the immunoglobulin Fc domain of the present invention comprises an amino acid sequence as set forth in SEQ ID NO. 9.

In some embodiments, the TGF- βRII extracellular domain of the present invention has the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10.

In some embodiments, the TGF- βRII extracellular domain of the present invention comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10.

In some embodiments, the TGF- βRII extracellular domain of the present invention comprises the amino acid sequence set forth in SEQ ID NO. 10.

In some embodiments, the bifunctional proteins of the invention are in the order of (i), (ii) and (iii) from the N-terminus to the C-terminus; the order of (iii), (i) and (ii); or (iii), (ii) and (i) in the order of active linkage; preferably, the (i) and (ii) are connected by a hinge region, and the (i) and (iii) and (ii) and (iii) are connected by a connecting peptide, the amino acid sequence of the connecting peptide contains or consists of glycine (G) residues and serine (S) residues, and the length of the connecting peptide is 2-31 amino acid residues; preferably each independently is (GGGGS) nG, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.

In some embodiments, the bifunctional proteins of the invention are operably linked from the N-terminus to the C-terminus in the order of (i), (ii) and (iii), preferably, the linkage between (i) and (ii) is via a hinge region, and the linkage between (ii) and (iii) is via a linking peptide, the amino acid sequence of which contains or consists of glycine residues and serine residues, and is 2-31 amino acid residues in length; preferably each independently is (GGGGS) nG, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.

In some embodiments, the bifunctional proteins of the invention comprise an anti-PD-1 or PD-L1 antibody and one TGF- βRII ECD operably linked at the C-terminus of each of the two heavy chains of the antibody.

In some embodiments, the antibodies of the invention are IgG1, igG2, igG3, or IgG4 class antibodies, preferably IgG4 class antibodies; more preferably, the IgG4 class antibody comprises a 1, 2, 3, 4 or 5 amino acid difference in the Fc domain, preferably has an S228P amino acid substitution in the Fc domain; further preferred is a deletion of both the C-terminal K and G amino acids in the Fc domain.

In some embodiments, the bifunctional proteins of the invention, wherein the IgG4 comprises 1, 2, 3, 4 or 5 amino acid differences in the Fc domain, preferably with an S228P amino acid substitution in the Fc domain (according to the EU numbering system); further preferred is one having both C-terminal K and G amino acid deletions in the Fc domain (according to the EU numbering system).

In some embodiments, the anti-PD-1 antibodies of the invention have a heavy chain amino acid sequence as set forth in SEQ ID NO. 11 and a light chain amino acid sequence as set forth in SEQ ID NO. 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO. 11 and/or 12.

In some embodiments, the anti-PD-L1 antibodies of the invention comprise a heavy chain amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 26 or 31 and a light chain amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 27.

In some embodiments, the anti-PD-L1 antibodies of the invention comprise a heavy chain amino acid sequence as set forth in SEQ ID NO. 26 or 31 and a light chain amino acid sequence as set forth in SEQ ID NO. 27.

In some embodiments, the bifunctional proteins of the present invention comprise a first subunit of the bifunctional protein having an amino acid sequence shown in SEQ ID NO. 13, and a second subunit of the bifunctional protein having an amino acid sequence shown in SEQ ID NO. 12.

In some embodiments, the bifunctional proteins of the present invention comprise a first subunit of the bifunctional protein having an amino acid sequence shown in SEQ ID NO. 28, and a second subunit of the bifunctional protein having an amino acid sequence shown in SEQ ID NO. 27.

In some embodiments, the bifunctional proteins of the invention have one or more of the following properties:

(1) Has stronger binding to TGF-beta 1 and TGF-beta 3;

(2) Binds with high affinity to PD-1 or PD-L1;

(3) Blocking PD-1/PD-L1 interactions;

(4) Inhibit tgfβ1-induced activation of pSMAD 3;

(5) Has stronger endocytic activity;

(6) Promoting the release of IL-2 and/or IFNgamma.

In yet another aspect, the invention provides a polynucleotide encoding a bifunctional protein, or an antigen-binding fragment thereof, as described herein. Preferably, the polynucleotide has a first subunit nucleotide sequence as set forth in SEQ ID NO. 16 and/or a second subunit nucleotide sequence as set forth in SEQ ID NO. 17.

In yet another aspect, the invention provides a polynucleotide encoding a bifunctional protein, or an antigen-binding fragment thereof, as described herein. Preferably, the polynucleotide has a first subunit nucleotide sequence as set forth in SEQ ID NO. 29 and/or a second subunit nucleotide sequence as set forth in SEQ ID NO. 30.

In a further aspect, the invention provides an expression vector comprising a polynucleotide as described herein, preferably the expression vector is a eukaryotic expression vector.

In a further aspect, the invention provides a host cell comprising a polynucleotide or expression vector as described herein, preferably the host cell is a mammalian cell.

In yet another aspect, the invention provides a method of preparing a bifunctional protein as described herein, comprising expressing the bifunctional protein in a host cell as described herein under conditions suitable for expression of the bifunctional protein, and recovering the expressed bifunctional protein from the host cell.

In yet another aspect, the invention provides a pharmaceutical composition comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.

In a further aspect, the invention provides the use of a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition associated with PD-1, PD-L1 or TGF- β activity, preferably the disease or condition is a cancer, more preferably the cancer is selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer and rectal cancer.

In a further aspect, the invention provides a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein and a pharmaceutical composition as described herein for use in the treatment and/or prevention of a disease or disorder associated with PD-1, PD-L1 or TGF- β activity, preferably the disease or disorder is a cancer, more preferably the cancer is selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer and rectal cancer.

In a further aspect, the invention provides a method of treating and/or preventing a disease or disorder associated with PD-1, PD-L1 or TGF- β activity, comprising administering to a subject in need thereof a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein; preferably the disease or disorder is cancer, more preferably the cancer is selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer and rectal cancer.

In yet another aspect, the invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof described herein conjugated to a therapeutic or diagnostic agent.

In yet another aspect, the invention provides a combination product comprising an antibody or antigen-binding fragment thereof as described herein, an immunoconjugate as described herein or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

In yet another aspect, the invention provides a kit comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein.

In yet another aspect, the invention provides a method of detecting the presence of PD-1, PD-L1 or TGF- β in a sample using a bifunctional protein as described herein.

The bifunctional protein has stronger affinity with PD-1 or PD-L1 protein, has good thermal stability, and can obviously promote release of IFN gamma and inhibit growth of tumors.

Drawings

Fig. 1: schematic of the molecular structure of bifunctional proteins targeting PD-1 and TGF-beta.

Fig. 2: ELISA detects binding of bifunctional proteins targeting PD-1 and TGF-beta to human TGF-beta 1.

Fig. 3: ELISA detects binding of bifunctional proteins targeting PD-1 and TGF-beta to human TGF-beta 2.

Fig. 4: ELISA detects binding of bifunctional proteins targeting PD-1 and TGF-beta to human TGF-beta 3.

Fig. 5: ELISA detects binding of bifunctional proteins targeting PD-1 and TGF-beta to human PD-1.

Fig. 6: the reporter gene method detects the blocking effect of the bifunctional proteins targeting PD-1 and TGF-beta on the PD-1/PD-L1 channels.

Fig. 7: reporter gene methods detect the blocking effect of bifunctional proteins targeting PD-1 and TGF-beta on TGF-beta pathways.

Fig. 8: endocytosis experiments of bifunctional proteins targeting PD-1 and TGF- β.

Fig. 9: in vitro mixed lymphocyte responses targeting bifunctional proteins of PD-1 and TGF-beta. 9a: effects of bifunctional proteins targeting PD-1 and TGF- β on IL2 cytokine release levels following T cell activation; 9b: effects of bifunctional proteins targeting PD-1 and TGF- β on ifnγ cytokine release levels following T cell activation.

Fig. 10: inhibition of human melanoma A375 tumor growth in mice by bifunctional proteins targeting PD-1 and TGF-beta.

Fig. 11: schematic of the molecular structure of bifunctional proteins targeting PD-L1 and TGF-beta.

Fig. 12: ELISA detects binding of bifunctional proteins targeting PD-L1 and TGF-beta to human TGF-beta 1.

Fig. 13: ELISA detects binding of bifunctional proteins targeting PD-L1 and TGF-beta to human TGF-beta 2.

Fig. 14: ELISA detects binding of bifunctional proteins targeting PD-L1 and TGF-beta to human TGF-beta 3.

Fig. 15: ELISA detects binding of bifunctional proteins targeting PD-L1 and TGF-beta to human PD-L1.

Fig. 16: the reporter gene method detects the blocking effect of the bifunctional proteins targeting PD-L1 and TGF-beta on the PD-1/PD-L1 channels.

Fig. 17: reporter gene methods detect the blocking effect of bifunctional proteins targeting PD-L1 and TGF-beta on TGF-beta pathways.

Fig. 18: endocytosis experiments of bifunctional proteins targeting PD-L1 and TGF- β.

Fig. 19: in vitro mixed lymphocyte reaction of bifunctional proteins targeting PD-L1 and TGF-beta. 19a: effects of bifunctional proteins targeting PD-L1 and TGF- β on IL2 cytokine release levels following T cell activation; 19b: effect of bifunctional proteins targeting PD-L1 and TGF-beta on IFNgamma cytokine release levels following T cell activation.

Detailed Description

Definition of the definition

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order that the invention may be more readily understood, certain technical and scientific terms are defined as follows. Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For definitions and terms in the art, the expert may refer specifically to Current Protocolsin Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including the claims), the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

"programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed primarily on previously activated T cells in vivo and binds to two ligands PD-L1 and PD-L2. The term "PD-1" as used herein includes variants, isoforms and species homologs of human PD-1 (hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1.

"programmed cell death protein ligand 1 (PD-L1)", which may also be referred to as cluster of differentiation274 (cluster of differentiation, CD 274) or B7 homologous protein 1 (B7 homolog1, B7-H1), belongs to the tumor necrosis factor superfamily. The term "PD-L1" as used herein includes variants, isoforms and species homologs of human PD-L1 (hPD-L1), hPD-L1, and analogs having at least one common epitope with hPD-L1.

"transforming growth factor-beta (TGF-beta)" belongs to the TGF-beta superfamily that regulates cell growth and differentiation. TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors. These type I and type II receptors are transmembrane proteins, which consist of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity. Type I receptors are critical for signaling, and type II receptors are necessary for binding ligand and expression of type I receptors. The type I and type II receptors form stable complexes upon ligand binding, thereby causing phosphorylation of the type I receptor by the type II receptor. Tgfβ has three mammalian isoforms, tgfβ1, tgfβ2 and tgfβ3, each with different functions in vivo. Binding of tgfβ to tgfβrii is a key step in initiating activation of tgfβ signaling pathways, causing SMAD2 phosphorylation, and translocation of the activated SMAD2/SMAD4 complex to the nucleus to regulate gene expression.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "immune response" refers to the action of soluble macromolecules (including antibodies, cytokines and complements) produced by, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes and by the above cells or liver, which results in the selective destruction, destruction or clearance of invasive pathogens, cells or tissues of infected pathogens, cancer cells or normal human cells or tissues in the case of autoimmune or pathological inflammation from the human body.

The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship typically initiated by protein-protein interactions such as binding of growth factors to receptors, which results in the transfer of signals from one part of a cell to another part of the cell. In general, delivery involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that cause signal transduction. The penultimate process typically involves a nuclear event, resulting in a change in gene expression.

The terms "activity" or "biological activity", or the terms "biological property" or "biological feature" are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize PD-1 activity in vivo or in vitro, IC50, in vivo stability of an antibody, and immunogenic properties of an antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeting peptide in general, or with other proteins or tissues), and the ability to maintain high levels of expression of the protein in mammalian cells. The aforementioned properties or characteristics are observed, assayed or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and/or secretion, signal transduction, and immunohistochemistry of tissue sections of different origin (including human, primate or any other source).

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

The term "isolated antibody" refers to a purified state of the bound compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean that such materials are completely absent or that water, buffer or salt are absent unless they are present in amounts that would significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The term "full length antibody" refers to an immunoglobulin molecule that when naturally occurring comprises four peptide chains: two heavy (H) chains (about 50-70kDa in full length) and two light (L) chains (about 25kDa in full length) are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) of high variability and regions spaced apart by more conserved regions called Framework Regions (FR). Each VH or VL region consists of, in order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of the antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody). The term "binding compound" refers to both antibodies and binding fragments thereof.

The term "single chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides generally also comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

The term "Fc" or "Fc region" or "Fc fragment" or "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH 2 domain and CH3 domain) derived from the two heavy chains of IgG, igA, and IgD class antibodies; or a second, third and fourth constant domain (CH 2 domain, CH3 domain and CH4 domain) comprising two heavy chains derived from IgM and IgE class antibodies. The Fc region herein may comprise a hinge region; the amino acid sequence of the Fc domain of the human IgG1 immunoglobulin is SEQ ID NO. 32, and the amino acid sequence of the Fc domain of the human IgG4 immunoglobulin is SEQ ID NO. 33.

The term "hinge region" refers to a polypeptide chain of an antibody that is located between CH1 and CH2, is proline-rich, and is easily stretched and bent.

The term "domain antibody" is an immunologically functional immunoglobulin fragment that contains only either a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The 2 VH regions of a bivalent domain antibody may target the same or different antigens.

The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, igG class immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH 1, CH2, and CH 3), also known as heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain, also known as a light chain constant region. The heavy chain of an immunoglobulin may belong to one of 5 classes, called α (IgA), δ (IgD), epsilon (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subclasses, such as γ1 (IgG 1), γ2 (IgG 2), γ3 (IgG 3), γ4 (IgG 4), α1 (IgA 1), and α2 (IgA 2). The light chains of immunoglobulins can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.

A "human immunoglobulin" is an immunoglobulin that possesses an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or that is derived from a non-human source that utilizes a human immunoglobulin repertoire or other sequences encoding human immunoglobulins.

The term "bivalent antibody" comprises 2 antigen binding sites. In some cases, 2 binding sites have the same antigen specificity. However, the bivalent antibody may be bispecific.

The term "diabody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is short enough not to allow pairing between two domains of the same strand, the domain is forced to pair with the complementary domain of the other strand and create two antigen binding sites.

The term "chimeric antibody" is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domain is obtained from an antibody ("parent antibody") of a rodent or the like, while the constant domain sequence is obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject as compared to the parent rodent antibody.

The term "humanized antibody" refers to a form of antibody that contains sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).

The term "fully human antibody" refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains, as produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, "mouse antibody" refers to an antibody that comprises only mouse immunoglobulin sequences. Alternatively, the fully human antibody may contain a rat sugar chain if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Likewise, "rat antibody" refers to an antibody comprising only rat immunoglobulin sequences.

"PD-1 antibody" refers to any chemical compound or biological molecule that binds to the PD-1 receptor, blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. Alternative nouns or synonyms for PD-1 and its ligands include: for PD-1 there are PDCD1, PD1, CD279 and SLEB2; examples of PD-L1 include PDCD1L1, PDL1, B7-H1, B7-4, CD274 and B7-H; and for PD-L2 there are PDCD1L2, PDL2, B7-DC and CD273. In any of the methods of treatment, medicaments and uses of the invention for treating a human individual, the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD 1. The human PD-1 amino acid sequence can be found in NCBI locus numbering: np_005009. Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbering: np_054862 and np_079515.

Herein, when referring to an "anti-PD-1 antibody," the term includes antigen-binding fragments thereof unless otherwise indicated or described.

"PD-L1 antibody" refers to any chemical compound or biological molecule that binds to the PD-L1 receptor, blocking the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells). Alternative nouns or synonyms for PD-1 and its ligands include: for PD-1 there are PDCD1, PD1, CD279 and SLEB2; examples of PD-L1 include PDCD1L1, PDL1, B7-H1, B7-4, CD274 and B7-H; and for PD-L2 there are PDCD1L2, PDL2, B7-DC and CD273. In any of the methods of treatment, medicaments and uses of the invention for treating a human subject, the PD-L1 antibody blocks the binding of human PD-1 to human PD-L1.

Herein, when referring to an "anti-PD-L1 antibody", the term includes antigen-binding fragments thereof unless otherwise indicated or described.

An "isotype" antibody refers to the class of antibodies provided by the heavy chain constant region gene (e.g., igM, igE, igG such as IgGl, igG2, or IgG 4). Isoforms also include modified forms of one of these species, wherein modifications have been made to alter Fc function, e.g., to enhance or attenuate effector function or binding to Fc receptors.

The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "non-binding" protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e. the binding protein or cell has a KD of 1.0X10 ^-6 M or higher, more preferably 1.0X10 ^-5 M or higher, more preferably 1.0X10 ^- ⁴ M or higher, 1.0X10 ^-3 M or higher, more preferably 1.0X10 ^-2 M or higher.

The term "high affinity" for IgG antibodies refers to a KD of 1.0X10 for antigen ^-6 M or less, preferably 5.0X10 ^-8 M or less, more preferably 1.0X10 ^-8 M or less, 5.0X10 s ^-9 M or less, more preferably 1.0X10 ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtype refers to KD of 10 ^-6 M or less, preferably 10 ^-7 M or less, more preferably 10 ^-8 M or lower.

The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse cell membrane surface antigens from target cells, such as cancer cells, to which antibodies, such as the claudin18.2 antibody, bind.

The term "complement-dependent cytotoxicity" or "CDC" refers to the effector functions of IgG and IgM antibodies, which when bound to surface antigens trigger the classical complement pathway, including the formation of membrane attack complexes and target cell lysis. The antibodies of the invention, when bound to Claudin18.2, elicit CDC against cancer cells.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8,278,036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes 8:91-98 (1994)).

"construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-or double-stranded DNA or RNA polynucleotide molecule) derived from any source capable of integrating with the genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (i.e., operably linked). Recombinant constructs will typically comprise a polynucleotide of the invention operably linked to transcriptional initiation regulatory sequences that direct the transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters may be used to direct expression of the nucleic acids of the invention.

"vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes that are operably linked. Such vectors are referred to herein as "expression vectors".

The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired.

"activation", "stimulation" and "treatment" for a cell or receptor may have the same meaning, e.g., activation, stimulation or treatment of the cell or receptor with a ligand, unless the context indicates otherwise or clearly. "ligand" includes natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins and binding compounds derived from antibodies. "ligand" also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies. "activation" may refer to cell activation mediated by internal mechanisms and external or environmental factors. "response/reaction", such as a response of a cell, tissue, organ or organism, includes a change in a biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), wherein the change is associated with activation, stimulation or treatment, or with an internal mechanism such as genetic programming.

As used herein, the term "treatment" or "treatment" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (i.e., slowing or preventing or reducing at least one of the progression of the disease or its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernable by the patient. In another embodiment, "treating" or "treatment" refers to modulating a disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art unless explicitly described herein.

"subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. As used herein, the term "cyno" or "cynomolgus monkey" refers to cynomolgus monkeys.

Administration of one or more other therapeutic agents "in combination" includes simultaneous (co) administration and sequential administration in any order.

"therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody or antigen-binding fragment thereof or bifunctional protein of the invention that is effective to prevent or ameliorate a symptom of one or more diseases or conditions, or the development of the disease or condition, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents. A therapeutically effective dose also refers to an amount of an antibody or antigen binding fragment thereof sufficient to result in an improvement in symptoms, e.g., to treat, cure, prevent, or ameliorate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When an active ingredient is administered to an individual, a therapeutically effective dose refers to that ingredient alone. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.

"cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are benign and malignant cancers, and dormant tumors or micrometastases. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, advanced immunocompetent NHL, advanced small non-nucleated NHL, storage disease (busy blast), mantle cell lymphoma, AIDS and Waldenstrom's lymphoma, and capillary lymphomatosis (Waldenstrom), and leukemia (Waldenstrom's) and leukemia (lymphomatosis), and vascular disorders (lymphomas), and chronic leukemia (lymphomas), and lymphomatosis (lymphomas) associated with vascular disorders (lymphomas, such as those of the vascular diseases.

Bifunctional proteins

The present invention provides a bifunctional protein targeting PD-1 or PD-L1 and TGF-beta comprising

(ii) An immunoglobulin Fc domain; and

(iii) TGF-. Beta.RII extracellular domain (ECD).

In some embodiments, the bifunctional proteins of the invention are in the order of (i), (ii) and (iii) from the N-terminus to the C-terminus; the order of (iii), (i) and (ii); or (iii), (ii) and (i) in the order of active linkage; preferably, the (i) and (ii) are connected through a hinge region, and the (i) and (iii) and (ii) and (iii) are connected through a connecting peptide, wherein the amino acid sequence of the connecting peptide contains or consists of glycine residues and serine residues, and the length of the connecting peptide is 2-31 amino acid residues; preferably each independently is (GGGGS) nG, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.

In some embodiments, the bifunctional proteins of the invention are heterotetrameric glycoproteins composed of two bifunctional protein first subunits and two bifunctional protein second subunits that are disulfide-bonded.

The invention adopts the monoclonal antibody targeting PD-1 or PD-L1 as a molecular part of the bifunctional protein for blocking PD-1/PD-L1 passage, and is used for blocking negative regulation signals, so that T cells recover activity, and the immune response is enhanced. The invention adopts the TGF-beta RII extracellular domain as an immunoregulatory molecule part in the bifunctional protein, and is used for weakening the immune tolerance of cancer cells. The PD-1/PD-L1 pathway is inhibited on the basis of targeting and neutralizing the TGF-beta in the tumor microenvironment, so that the T cells can recover activity, enhance immune response and more effectively inhibit tumor occurrence and development.

In some embodiments, the bifunctional proteins of the invention are present at 10 ^-8 M or less, e.g. at 10 ^-9 M to 10 ^-12 The dissociation constant (KD) of M binds to PD-1 or PD-L1; and at 10 ^-8 M or less, e.g. at 10 ^-9 M to 10 ^-12 The dissociation constants (KD) of M bind specifically to TGF-beta 1/TGF-beta 2/TGF-beta 3 molecules, respectively.

anti-PD-1 antibodies and/or antigen-binding fragments of anti-PD-1 antibodies

The bifunctional proteins of the present invention comprise an anti-PD-1 antibody and/or an antigen-binding fragment of an anti-PD-1 antibody, which is capable of specifically binding to PD-1; or compete with the intact anti-PD-1 antibody and/or antigen-binding fragment thereof for binding to PD-1.

The "anti-PD-1 antibody or antigen-binding fragment thereof" described in the present invention may include any anti-PD-1 antibody or antigen-binding fragment thereof described in the art. The anti-PD-1 antibody may be a commercially available or published PD-1 antibody. Including but not limited to, e.g., PD-1 antibody nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, cemiplimab, etc. The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Antigen binding fragments include Fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv and ScFv fragments which bind to antibodies.

In some embodiments, the antigen binding fragment of an anti-PD-1 antibody in a bifunctional protein of the invention comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the heavy chain variable region shown in SEQ ID NO. 7 and to the light chain variable region having the amino acid sequence shown in SEQ ID NO. 8.

In some embodiments, the antigen-binding fragment of the anti-PD-1 antibodies of the invention comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 8.

In some embodiments, the antigen binding fragment of the anti-PD-1 antibodies of the invention comprises Fab, fab ', F (ab') 2, fv, scFv, or sdAb.

Methods and techniques for identifying CDRs in the amino acid sequences of the heavy chain variable region and the light chain variable region are known in the art and can be used to identify CDRs in the amino acid sequences of the specific heavy chain variable region and/or light chain variable region disclosed herein. Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, kabat definition, chothia definition, IMGT definition, and AbM definition. See, e.g., kabat, sequences of Proteins of Immunological Interest, national Institutes of Health, bethesda, md. (1991); al-Lazikani et Al Standard conformations for the canonical structures of immunoglobins, J.mol. Biol.273:927-948 (1997); martin AC et al Modeling antibody hypervariable loops: a combined algorithm, proc.Natl. Acad.Sci.USA 86:9268-9272 (1989); international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research,27,209-212).

The antigen binding fragment amino acid sequence of the anti-PD-1 antibody constructed by the bifunctional protein JS-TZO3 is as follows (the CDR boundary adopts a Kabat scheme):

amino acid sequence of the heavy chain variable region of PD-1 antibody: (SEQ ID NO: 7)

HCDR1 DYEMH SEQ ID NO:1

HCDR2 VIESETGGTAYNQKFKG SEQ ID NO:2

HCDR3 EGITTVATTYYWYFDV SEQ ID NO:3

Amino acid sequence of the light chain variable region of the PD-1 antibody: (SEQ ID NO: 8)

LCDR1 RSSQSIVHSNGNTYLE SEQ ID NO:4

LCDR2 KVSNRFS SEQ ID NO:5

LCDR3 FQGSHVPLT SEQ ID NO:6

anti-PD-L1 antibodies and/or antigen-binding fragments of anti-PD-L1 antibodies

The bifunctional proteins of the present invention comprise an anti-PD-L1 antibody and/or an antigen-binding fragment of an anti-PD-L1 antibody, which is capable of specifically binding to PD-L1; or compete with the intact anti-PD-L1 antibody and/or antigen-binding fragment thereof for binding to PD-L1.

The "anti-PD-L1 antibody or antigen-binding fragment thereof" described in the present invention may include any anti-PD-L1 antibody or antigen-binding fragment thereof described in the art. The anti-PD-L1 antibody may be a commercially available or published PD-L1 antibody. The antibodies of the invention may be monoclonal, chimeric, humanized or fully human. Antigen binding fragments include Fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv and ScFv fragments which bind to antibodies.

In some embodiments, the antigen binding fragment of the anti-PD-L1 antibodies of the invention comprises Fab, fab ', F (ab') 2, fv, scFv, or sdAb.

The antigen binding fragment amino acid sequence of the anti-PD-L1 antibody constructed by the bifunctional protein JS-TZO4 is as follows (the CDR boundary adopts an IMGT scheme):

amino acid sequence of the heavy chain variable region of an anti-PD-L1 antibody: (SEQ ID NO: 24)

HCDR1 GDSITRGY SEQ ID NO:18

HCDR2 ISYTGST SEQ ID NO:19

HCDR3 ATSTGWLDPVDY SEQ ID NO:20

Amino acid sequence of the anti-PD-L1 antibody light chain variable region: (SEQ ID NO: 25)

LCDR1 QNVDTS SEQ ID NO:21

LCDR2 SAS SEQ ID NO:22

LCDR3 QQYYGYPFT SEQ ID NO:23

Immunoglobulin Fc domain

An "immunoglobulin Fc domain" in a bifunctional protein of the invention comprises all amino acid residues of a naturally occurring immunoglobulin Fc domain or comprises a portion of amino acid residues of a naturally occurring immunoglobulin Fc domain. Immunoglobulin Fc domains provide advantageous pharmacokinetic properties to the bifunctional proteins of the invention, including but not limited to long serum half-life.

Immunoglobulin Fc domains are typically dimeric molecules that can be produced by papain digestion or trypsin digestion of an intact (full length) immunoglobulin or can be produced recombinantly, comprising a CH2 domain, a CH3 domain, and optionally a CH4 domain.

In some embodiments, the IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the immunoglobulin Fc domain has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO. 9. In some embodiments, the immunoglobulin Fc domain has an amino acid sequence as shown in SEQ ID NO. 9.

In some embodiments, the immunoglobulin Fc domain of the invention is a human IgG1, igG2, igG3, or IgG4 Fc domain; preferably human IgG4 Fc domain.

In some embodiments, the immunoglobulin Fc domains of the invention further comprise peptide sequences obtained by subjecting the amino acid sequences to one or more amino acid substitutions, deletions or derivatizations.

In some embodiments, an amino acid substitution, particularly an amino acid substitution S228P, is included in the IgG Fc domain at position S228 that prevents arm exchange from occurring.

In some embodiments, terminal amino acid deletions, preferably both C-terminal K and G amino acid deletions, are included in the IgG Fc domain to prevent cleavage at the C-terminus.

The amino acid sequence of the immunoglobulin Fc domain constructed by the bifunctional proteins JS-TZO3 and JS-TZO4 can be selected from the following: (SEQ ID NO: 9)

anti-PD-1 antibodies

In some embodiments, the anti-PD-1 antibodies of the invention have an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO. 11 and/or SEQ ID NO. 12.

In some embodiments, the anti-PD-1 antibodies of the invention have a heavy chain amino acid sequence as set forth in SEQ ID NO. 11 and a light chain amino acid sequence as set forth in SEQ ID NO. 12.

The PD-1 antibody heavy chain sequence constructed by the bifunctional protein JS-TZO3 can be selected from the following: (SEQ ID NO: 11)

The PD-1 antibody light chain sequence constructed by the bifunctional protein JS-TZO3 can be selected from the following: (SEQ ID NO: 12)

anti-PD-L1 antibodies

The PD-L1 antibody heavy chain sequence constructed by the bifunctional protein JS-TZO4 can be selected from the following: (SEQ ID NO: 31)

The PD-L1 antibody light chain sequence constructed by the bifunctional protein JS-TZO4 can be selected from the following: (SEQ ID NO: 27)

TGF- βRII extracellular domain

The "extracellular domain (ECD) of TGF- βRII" in the bifunctional proteins of the invention comprises all or a portion of the amino acid residues of a naturally occurring TGF- βRII ECD.

In some embodiments, the TGF- βRII extracellular domain of the present invention has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO. 10.

In some embodiments, the TGF- βRII extracellular domain of the present invention has the amino acid sequence shown in SEQ ID NO 10.

In addition to the sequences defined as SEQ ID NO. 10, the TGF-. Beta.RII ECD may comprise peptide sequences obtained by additional sequence modifications of SEQ ID NO. 10, such as those obtained by one or more conservative substitutions, deletions or derivations of amino acid residues in SEQ ID NO. 10, provided that they have substantially the same activity or function as the unmodified peptide. The modified peptide will retain the activity or function associated with the unmodified peptide. The modified peptide typically has an amino acid sequence that is substantially homologous to the amino acid sequence of the unmodified sequence.

The amino acid sequence of the TGF-beta RII extracellular domain constructed by the bifunctional protein JS-TZO3 or JS-TZO4 is as follows:

TGF-beta RII extracellular domain (1-136 polypeptide, SEQ ID NO: 10):

bifunctional proteins

The present invention uses a linker peptide (G4S) 4G to link the heavy chain portion of the PD1 or PD-L1 antibody to the tgfbetarii extracellular domain as a bifunctional protein.

The amino acid sequence of the first subunit of the bifunctional protein JS-TZO3 is as follows: (heavy chain, SEQ ID NO: 13):

the second subunit amino acid of the bifunctional protein JS-TZO3 is the same as the PD-1 antibody light chain sequence, and is the amino acid sequence shown in SEQ ID NO. 12.

The amino acid sequence of the first subunit of the bifunctional protein JS-TZO4 is as follows: (heavy chain, SEQ ID NO: 28):

The second subunit amino acid of the bifunctional protein JS-TZO4 is the same as the PD-L1 antibody light chain sequence, and is the amino acid sequence shown in SEQ ID NO. 27.

In some embodiments, the amino acid change comprises an amino acid deletion, insertion, or substitution. In some embodiments, the bifunctional proteins of the invention include those having an amino acid sequence that has been mutated by amino acid deletion, insertion, or substitution, but still has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the bifunctional proteins described above.

In some embodiments, polynucleotides encoding antibodies of the invention include polynucleotides that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60%, 70%, 80%, 90%, 95%, or 100% identity to the CDR-corresponding coding regions depicted in the sequences described above.

In some embodiments, the bifunctional proteins provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Antibody expression

In yet another aspect, the invention provides a polynucleotide encoding a bifunctional protein as described herein. The polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of an antibody, or a polynucleotide encoding the amino acid sequence of the first subunit and/or the second subunit of a bifunctional protein.

In some embodiments, the polynucleotides of the invention comprise a first subunit nucleotide sequence as set forth in SEQ ID NO. 16 and/or a second subunit nucleotide sequence as set forth in SEQ ID NO. 17.

The first subunit nucleotide sequence of the bifunctional protein JS-TZO3 is as follows: (SEQ ID NO: 16):

the second subunit nucleotide sequence of the bifunctional protein JS-TZO3 is as follows: (SEQ ID NO: 17):

in some embodiments, the polynucleotides of the invention comprise a first subunit nucleotide sequence as set forth in SEQ ID NO. 29 and/or a second subunit nucleotide sequence as set forth in SEQ ID NO. 30.

The first subunit nucleotide sequence of the bifunctional protein JS-TZO4 is as follows: (SEQ ID NO: 29):

the second subunit nucleotide sequence of the bifunctional protein JS-TZO4 is as follows: (SEQ ID NO: 30):

In a further aspect, the invention provides an expression vector comprising a polynucleotide as described herein, preferably the vector is a eukaryotic expression vector. In some embodiments, a polynucleotide as described herein is contained in one or more expression vectors.

In a further aspect, the invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell.

In yet another aspect, the invention provides a method for preparing a bifunctional protein as described herein, comprising expressing the antibody in a host cell described herein under conditions suitable for expression of the antibody, and recovering the expressed bifunctional protein from the host cell.

The present invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, chinese Hamster Ovary (CHO) cells, NS0, SP2/0 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells, a549 cells, 293T cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

In one embodiment, the invention provides a method of preparing a bifunctional protein of the invention, wherein said method comprises, upon introducing an expression vector into a mammalian host cell, producing the bifunctional protein by culturing the host cell for a period of time sufficient to allow expression of the bifunctional protein in the host cell, or more preferably secretion of the bifunctional protein into the medium in which the host cell is grown. The bifunctional proteins can be recovered from the culture medium using standard protein purification methods.

It is likely that bifunctional proteins expressed by different cell lines or in transgenic animals have different glycosylation from each other. However, all bifunctional proteins encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibody. Also, in certain embodiments, nonfucosylated antibodies are advantageous because they generally have greater efficacy than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic because their sugar structures are normal components of natural human serum IgG.

Pharmaceutical composition and pharmaceutical formulation

In yet another aspect, the invention provides a pharmaceutical composition comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein or a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.

It will be appreciated that the bifunctional proteins, or pharmaceutical compositions thereof, provided herein may incorporate suitable carriers, excipients and other agents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerability, etc.

The term "pharmaceutical composition" refers to a formulation that allows for the biologically active effective form of the active ingredient contained therein to exist and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation is administered.

Pharmaceutical formulations comprising the bifunctional proteins of the invention as described herein may be prepared by mixing the bifunctional proteins of the invention with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Editions (1980)) in the desired purity, preferably in the form of an aqueous solution or a lyophilized formulation.

The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. In some embodiments, the other active ingredient is a chemotherapeutic agent, immune checkpoint inhibitor, growth inhibitor, antibiotic or known various anti-tumor or anti-cancer agent, which are present in a suitable combination in an amount effective for the intended use.

In some embodiments, the pharmaceutical compositions of the invention further comprise a composition of polynucleotides encoding a bifunctional protein.

In yet another aspect, the invention provides a pharmaceutical combination comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

In yet another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein.

Medical application

In some embodiments, the cancer or tumor described herein may be selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, and rectal cancer.

In some embodiments, modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intra-articular (e.g., in an arthritic joint), by inhalation, aerosol delivery, or intratumoral administration, and the like.

In some embodiments, the invention provides for co-administering to a subject a therapeutically effective amount of one or more therapies (e.g., therapeutic regimens and/or other therapeutic agents). In some embodiments, the therapy comprises surgical treatment and/or radiation therapy.

In some embodiments, the methods or uses provided herein further comprise administering one or more therapies (e.g., therapeutic regimens and/or other therapeutic agents) to the individual. The antibodies of the invention may be used alone or in combination with other therapeutic agents in therapy. For example, it may be co-administered with at least one additional therapeutic agent. For example, PD-1/PD-L1 antibodies, LAG-3 antibodies and/or CTLA-4 antibodies.

Method for diagnosis and detection

In yet another aspect, the invention provides a method of detecting the presence of PD-1, PD-L1 or TGF- β in a sample using a bifunctional protein as described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue.

The invention includes all combinations of the specific embodiments recited. Further embodiments and applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including references, are incorporated by reference in their entirety for all purposes.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.

The invention adopts the following abbreviations:

ECD represents an extracellular domain;

PEI stands for polyethylenimine;

BSA stands for bovine serum albumin;

PBS represents phosphate buffered saline;

FBS represents fetal bovine serum;

TMB stands for 3,3', 5' -tetramethylbenzidine;

PBST represents phosphate buffer with 0.02% tween added.

Examples

The invention is illustrated by the following examples, which are not intended to be limiting in any way. The invention has been described in detail herein, with particular embodiments thereof also disclosed. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

Example 1 preparation of the bifunctional protein JS-TZO3 targeting PD-1 and TGF-beta

1.1 construction of bifunctional protein expression vectors

The Nanjing Jinsri is entrusted to synthesize the nucleotide sequence of the anti-human PD-1 antibody (the heavy chain amino acid sequence is shown as SEQ ID NO:11, the light chain amino acid sequence is shown as SEQ ID NO: 12), and the gene synthesis fragment 1A6H2 is connected to the vector HXT4s by BSPQI restriction endonuclease to obtain the vector HXT4s-1A6H2. The nucleotide sequence encoding the extracellular domain (ECD) of TGF-beta RII (the amino acid sequence of which is shown in SEQ ID NO: 10) was delegated to Nanjing Jinsri. Connecting peptide ((G) ₄ S) ₄ G) The nucleotide sequence of (2) is obtained by means of a technique in the art. The primers TGF beta R2-G4SBSPQIFV2 and TGF beta R2-BSPQIRV2 are synthesized, human TGF-beta RII ECD gene fragments are obtained through PCR amplification, and the TGF-beta RII ECD gene fragments are connected to a vector HXT4s-1A6H2 through BSPQI restriction enzyme to obtain an expression vector HXT4s-1A6H2-TGF beta RII. The expression vector HXT4s-1A6H2-TGF beta RII expresses a bifunctional protein JS-TZO3, a second subunit amino acid sequence of the bifunctional protein JS-TZO3 is shown as SEQ ID NO. 12, and a first subunit amino acid sequence is shown as SEQ ID NO. 13.

Wherein,

the nucleotide sequence (SEQ ID NO: 14) of the primer TGF-beta R2-G4SBSPQIFV2 according to the 5'-3' sequence is as follows:

the nucleotide sequence of the primer TGF beta R2-BSPQIRV2 according to the 5'-3' sequence (SEQ ID NO: 15) is as follows:

1.2 expression and purification of bifunctional proteins

Liquid nitrogen stored 293F cells (FreeStyle) ^TM 293-F Cells, thermosipher) were resuscitated and added to expression medium (Freestyle ^TM 293 expression medium, gibco). After several generations of culture, 293F cells were diluted to (1.5-2.0). Times.10 the day before transfection ⁶ During transfection, 1/10 transfection volume of the culture medium is firstly used for incubating an expression vector HXT4s-1A6H2-TGF beta RII and a transfection reagent (PEI), the dosage of the expression vector is 0.5-2 mug/mL, and the dosage of the transfection reagent is 3-14 mug/mL. The incubation time is 20-30min, and after the incubation is completed, the transfection mixture is added into the 293F cells treated one day in advance, and the mixture is shaken while adding. Placing the transfected shake flask into a carbon dioxide shaking table for culturing at 36.5deg.C, 120rpm and 7% CO ₂ . After 7 days, the supernatant was collected by centrifugation and purified. Purification was performed using AKTA AVANT instrument using protein a affinity packing (MabSelect SuRe ^TM GE Healthcare) preloaded purification column, centrifuging the supernatant, filtering the supernatant by a 0.22 mu m filter, loading the supernatant, washing impurities by PBS, eluting by a citric acid buffer solution, and neutralizing by a Tris buffer solution after the elution is completed to obtain the bifunctional protein JS-TZO3. The prepared bifunctional protein JS-TZO3 can be used in each of the following examples.

The JS-TZO3 bifunctional protein molecular structure is schematically shown in FIG. 1.

Example 2: ELISA detection of binding of bifunctional protein JS-TZO3 and humanized TGF-beta

1. Purpose of testing

ELISA method is used for detecting the combination of the bifunctional protein JS-TZO3 and human TGF-beta 1, TGF-beta 2 and TGF-beta 3.

2. Test procedure and results

a. Recombinant human TGF-beta 1 (Novoprotein, CA 59) as antigen was coated in 96-well plates at a concentration of 0.5. Mu.l/well and incubated at 37℃for 1.5 hours.

300. Mu.l of 1 XPBST were washed 4 times, 200. Mu.l of 2% BSA were added and blocked at 37℃for 1.5 hours.

300 μl 1 XPBST was washed 4 times, and was incubated with a gradient of diluted bifunctional protein JS-TZO3 and negative control antibody (Anti-KLH IgG 4) (initial concentration 100 μg/ml, 2.5-fold gradient diluted 12 concentration spots) for 1 hour at 37 ℃.

d.300. Mu.l 1 XPBST were washed 4 times.

e. Mu.l of horseradish peroxidase (HRP) -conjugated murine anti-human antibody IgG4Fc (1:5000) (Southern Biotech, 9200-05) was added to each well and incubated for 1 hour at 37 ℃.

f. Mu.l of 0.1mg/ml TMB was added to each well, and after incubation at 37℃for 12 minutes, 100. Mu.l of 2M hydrochloric acid solution was added to terminate the reaction.

g. And detecting the absorption value of 450nm/620nm on an enzyme label instrument, and analyzing data by using Graphpad Prism 5.

As shown in figures 2, 3 and 4, the bifunctional protein JS-TZO3 of the invention has stronger binding with TGF-beta 1 and TGF-beta 3 and relatively weaker binding with TGF-beta 2.

Example 3: biacore detection of affinity of bifunctional protein JS-TZO3 with TGF-beta

1. Purpose of testing

The affinity of the bifunctional protein JS-TZO3 of the present invention with human TGF- β1, TGF- β2, TGF- β3 was examined using Biacore T200 instrument (GE medical life sciences).

2. Test procedure and results

The method for determining the binding affinity of the bifunctional protein and the humanized TGF-beta 1, TGF-beta 2 and TGF-beta 3 is as follows: 4 μg/ml bifunctional proteins were captured with Protein A chip (GE medical life sciences) and then gradient diluted human TGF- β1, TGF- β2, TGF- β3 (off-shore organisms) were flowed over the chip surface. The dilution gradient of TGF- β1 was 1.14nM,0.76nM,0.5057nM,0.3378nM,0.2252nM,0.1501nM, where 1.14nM is repeated. The dilution gradient of TGF- β2 was 1.8nM,1.2nM,0.8nM,0.5333nM,0.3556nM,0.2370nM, where 1.2nM is repeated. The dilution gradient of TGF-beta 3 was 1.2nM,0.8nM,0.5333nM,0.3556nM,0.2370nM, where 1.2nM is repeated. After each antigen gradient injection, the binding and dissociation signals were measured using the Biacore T200 system (GE Healthcare) to obtain binding and dissociation kinetics curves. Affinity KD values were determined using the software Biacore T200 Evaluation Software 3.0 fit to the binding dissociation curve.

As shown in Table 1, the bifunctional protein JS-TZO3 has stronger affinity with TGF beta 1 and TGF beta 3 and weaker affinity with TGF beta 2.

Table 1: affinity of bifunctional protein JS-TZO3 with TGF-beta

Example 4: ELISA (enzyme-Linked immuno sorbent assay) for detecting combination of bifunctional protein JS-TZO3 and PD-1

1. Test purpose:

ELISA detects the combination of the bifunctional protein JS-TZO3 and PD-1.

2. Test procedure

a. The 96-well plate was coated with human PD-1 (0.3. Mu.g/ml, junyua) as antigen at 100. Mu.l/well and incubated at 37℃for 1.5 hours.

300 μl 1 XPBST was washed 4 times, and incubated with a gradient of diluted bifunctional protein JS-TZO3, positive control PD-1 mAb (Torilaiimab, jundia) and negative control antibody (Anti-KLH IgG 4) (initial concentration 1 μg/ml, 2.5-fold gradient diluted 12 concentration points) for 1 hour at 37 ℃.

d.300. Mu.l 1 XPBST were washed 4 times.

f. Mu.l of 0.1mg/ml TMB was added to each well, and after incubation for 30 minutes at room temperature, 100. Mu.l of 2M hydrochloric acid solution was added to terminate the reaction.

As shown in FIG. 5, the bifunctional protein JS-TZO3 of the invention has stronger combination with PD-1, and is equivalent to a positive control antibody Toipalimab.

Example 5: biacore detection of affinity of bifunctional protein JS-TZO3 to PD-1

1. Purpose of testing

The affinity of the bifunctional protein JS-TZO3 of the present invention to human PD-1 protein was examined using a Biacore T200 instrument (GE medical life sciences).

2. Test procedure and results

The method for determining the binding affinity of the bifunctional protein and PD-1 comprises the following steps: 40 μg/ml of goat anti-human IgG-Fc fragment antibody (Jackson Immuno Research) was conjugated to the surface of a CM5 chip (GE Healthcare Life Sciences, cat. No. BR-1005-30) for capture of the antibody. After that, 5. Mu.g/ml of bifunctional protein was captured on the CM5 chip surface. Human PD-1 protein (Junzhen) was subjected to gradient dilution (140 nM,70nM,35nM,17.5nM,8.75nM,4.375nM, 140nM being a repeat sample), human PD-1 was sampled and passed through a CM5 chip, and binding and dissociation signals were measured using Biacore T200 system (GE Healthcare). Affinity KD values were determined using the software Biacore T200Evaluation Software 3.0 fit to the binding dissociation curve.

As shown in Table 2, the bifunctional protein JS-TZO3 has stronger affinity with PD-1 protein.

Table 2: affinity of bifunctional protein JS-TZO3 to PD-1

Example 6: reporter gene test for detecting blocking of bifunctional protein JS-TZO3 to PD-1/PD-L1 passage

1. Test purpose:

in order to study the blocking effect of the bifunctional protein JS-TZO3 on the PD-1/PD-L1 signal path, a PD-1/PD-L1 reporter gene system constructed by Promega company is adopted to detect the cell biological activity of the PD-1 end in the bifunctional protein.

2. Test procedure

CHO/PD-L1 cells (Promega) were taken, digested and resuspended in complete medium with F-12 nutrient mix, and cell density was adjusted to 5 using complete medium based on cell count results×10 ⁵ Cell suspensions were transferred to a loading well and 100. Mu.l/well into 96-well plates using a multichannel pipette and placed at 37℃in 5% CO ₂ Culturing in an incubator for 16-20 h; the next day a Jurkat/PD-1 (Promega) cell suspension was prepared, the cells were resuspended using analytical medium (RPMI 1640 Medium+2%FBS) according to the cell count results, and the cell density was adjusted to 2.5X10 ⁶ Individual cells/ml; the cell culture plate with the CHO/PD-L1 cells added thereto was removed from the incubator, and 100. Mu.l of the culture broth was removed per well using a multi-channel pipette. A gradient dilution (initial concentration 1650 nM) of bifunctional protein JS-TZO3, positive control PD-1 mab (Torilaiimab, jun.) and negative control antibody (Anti-KLH IgG 4) was added at 40. Mu.l/well. The Jurkat/PD-1 cell suspension was then transferred to a loading well and added to a cell culture plate at 40. Mu.l/well and placed at 37℃in 5% CO ₂ Culturing in an incubator for 5-6 h. During the protein incubation, one-Glo reagent (Promega, cat No. E6130) was removed to allow its temperature to return to room temperature. Taking out the cell culture plate, placing at room temperature for 5-10 min, adding 40 μl of One-Glo reagent (promega, E6130) into each hole, placing on a mixer, mixing for 5-10 min, and reading the luminescence signal value by using an M1000 pro multifunctional enzyme-labeled instrument (Tecan).

3. Results

As shown in FIG. 6, the bifunctional protein JS-TZO3 and the anti-PD-1 monoclonal antibody Torpalimab can effectively block the interaction of PD-1/PD-L1, promote the enhancement of NFAT signals and improve EC ₅₀ The values are 11.01nM and 10.4nM respectively, and have remarkable drug concentration dose-dependent effect, and the activity levels of the two are equivalent.

Example 7: test for detecting blocking of TGF beta channel by bifunctional protein JS-TZO3 through reporter gene method

1. Purpose of testing

This experiment investigated the inhibition of TGF- β1-induced Smad3 activation by the bifunctional protein JS-TZO3 of the invention by 4T1 cells expressing Smad3 binding original (SBE) with luciferase reporter gene.

2. Test procedure

4T1-SBE cells (Junychia) containing 10% FBS1640 complete medium (Gibco, 11875-093) was cultured and passaged every 2 days. Experiment first day at a density of 100,000 cells per well, seeded in 96-well plates (Costar, 3917), at 37 ℃, 5% co ₂ Culturing for 16-20 hours under the condition. The following day, the medium in the cell culture plates was discarded and a gradient of 50. Mu.l/well of a bifunctional protein diluted at an initial concentration of 1650nM and a positive control G4Fc TGF beta R2ecd (TGF beta Trap, jun. RTM.) were added. Cells were incubated at 37℃with 5% CO ₂ Culturing for 0.5-1 hr. 50. Mu.L of recombinant humanized TGF-beta.1 (Novoprotein, cat. CA 59) was added to each well. Cells were incubated at 37℃with 5% CO ₂ Culturing for 5-6 hr. Then 50 μl of luciferase substrate ONE-Glo was added per well ^TM The reagent (promega, E6130) was placed on a mixer and mixed for 5-10 min, and then the luminescence signal value was read using an M1000 pro multifunctional enzyme-labeled instrument (Tecan). IC of sample to be tested ₅₀ The values were calculated using the data processing software Graphpad prism 7.0.

3. Results

As shown in FIG. 7, the bifunctional proteins JS-TZO3 and G4Fc TGF beta R2ecd (TGF beta Trap) of the present invention are both capable of inhibiting TGF beta 1-induced activation of pSMAD3 in a dose-dependent manner.

Example 8: endocytosis test of bifunctional protein JS-TZO3

1. Purpose of testing

Endocytic activity of the bifunctional protein JS-TZO3 of the present invention was detected at the in vitro level using an antibody endocytic assay.

2. Test procedure

The CypHer 5 Mono NHS Ester dye labels the sample to be tested: using Label buffer (labelling buffer, PBS: naCO) ₃ =9:1, ph: 8.3) the concentration of bifunctional protein JS-TZO3 to be tested was adjusted to 1.0mg/ml. The amount of bifunctional protein was calculated, cypHer 5 Mono NHS Ester dye (GE, PA 15401) was added, and mixed well at room temperature in the dark for 1h. Transferring the mixed solution of the reacted bifunctional protein JS-TZO3 and the dye into a 0.5ml ultrafiltration tube (30 KD), centrifuging for 10min, discarding the waste liquid, supplementing Label buffer into the ultrafiltration tube, centrifuging for 4 times, and washing away the unbound superfluous dye.

CHO/PD-L1 cells (Promega) were taken, digested and resuspended in complete medium of F-12 nutrient mixture, 293F JS1E 4 cells (Junychia) were taken, digested and resuspended in complete medium of 1640, and cell density was adjusted to 2X 10 according to cell count using complete medium of 1640 ⁶ Individual cells/ml.

Endocytic assay (antibody co-incubation with target cells): (temperature, 37 ℃ C. With antibody endocytosis, 4 ℃ C. With inhibition of antibody endocytosis) 96-well ultra-low adsorption U bottom plate was used, cells were plated in 96-well plates, 50. Mu.l of cell suspension was added per well (cell number per well was 1X 10) using a multichannel pipette ⁵ Individual/holes); then, bifunctional protein, PD-1 monoclonal antibody (Torilimab) and negative control (Anti-KLH IgG 4) were combined with cells at different concentrations (initial concentration of 100. Mu.g/ml) in CO ₂ The incubator was incubated for 4h (37 ℃). Taking out the 96-well cell plate after incubation, centrifuging at 1000rpm for 5 minutes, and discarding the supernatant; cells were then resuspended in staining buffer (PBS+1% BSA) and centrifuged at 1000rpm for 5 minutes, the supernatant discarded, and unbound excess bifunctional protein was washed away by repeating the procedure twice, 150ul of the above staining buffer was added to each well of a 96 well cell plate, examined on a FACS machine and analyzed by FlowJo software.

3. Results

As shown in FIG. 8, the bifunctional protein JS-TZO3 and anti-PD-1 monoclonal antibody (Torilimab) have strong endocytosis activity and are dose-dependent. The negative control Anti-KLH IgG4 had no endocytosis.

Example 9: in vitro mixed lymphocyte reaction of bifunctional protein JS-TZO3

1. Purpose of testing

The invention researches the release level of IL2 and IFN gamma cytokines after T cell activation by the bifunctional protein JS-TZO3 by using a mixed lymphocyte reaction experiment at an in vitro level.

2. Test procedure

The adherent cells were stimulated with commercial human PBMC (SAILY BIO, cat#SLB-HP 050B) with serum-containing medium at 37℃for 2h, after which the suspension cells were washed out, and then with 10ng/ml GM-CSF (Sino Biological, cat#GMP-11846-HNAE) and 10ng/ml IL-4 (Sino Biological, cat#GMP-10015-HNAH), respectively, for 5 days, followed by 5ng/ml IL-1β (Sino Biological, cat#10139-HNAE), 10ng/ml IL-6 (Sino Biological, cat#GMP-10395-HNAE), 10ng/ml TNF-a (Sino Biological, cat#10602-HNAE) and 1. Mu. Mol/L PGE2, respectively, for 2 days to induce dendritic cells (mDC). Commercial Human PBMC (SAILY BIO, cat#SLB-HP050B, lot# 190100) were purified with CD4T microblades, human (Miltenyi Biotec, cat#130-045-101) to give CD4T cells.

Different concentrations (0.15 nM-150. Mu.M, 10-fold dilution) of bifunctional protein JS-TZO3, negative control antibody (anti-KLH IgG 4) +TGF-beta Trap (monad) control, PD-1 mab (Torpalimab) and PD-1 mab and TGF-beta Trap combination (PD-1 antibody+TGF-beta Trap control) were combined with 100ng/ml of TGF-beta 1 recombinant protein (novoprotein, cat#CA 59) and mDC cells (10000 cells per well) and purified CD4T cells (100000 cells per well) as described above, respectively, in 96-well round plates at CO at 37 DEG C ₂ Incubate for 5 days. Part of the reaction supernatant was collected from each well of 96-well plates on day 3 of co-incubation, IL-2 release was detected with BD CBA human IL2Flex set (BD, cat# 558270), and the IFNγ content in the supernatant was detected with BD CBA human IFNγ Flex set (BD, cat# 558269) on day 5 of co-incubation.

3. Results

As can be seen from the results of FIG. 9, the combined group of the bifunctional proteins JS-TZO3 and PD-1 monoclonal antibodies and the TGF-beta Trap of the present invention can promote the release of IL-2 in the mixed lymphocyte reaction, but the difference is not obvious (FIG. 9 a). The bifunctional protein JS-TZO3 can obviously promote release of IFN gamma, and the activity is obviously higher than that of a PD-1 monoclonal antibody and TGF beta Trap combined group and a PD-1 monoclonal antibody group (figure 9 b).

Example 10: inhibition of human melanoma A375 tumor growth in mice by bifunctional protein JS-TZO3

1. Purpose of testing

The anti-tumor effect of the bifunctional protein JS-TZO3 in a human melanoma A375 Mixeno subcutaneous transplantation model is evaluated.

2. Test procedure

18 animals are taken for 6-7 weeks of ageFemale NCG mice (Jiangsu Ji Yikang Biotechnology Co., ltd.) were inoculated subcutaneously on the right back with premixed 4.5X 10 ⁶ (individual/0.1 mL) A375 cells (ATCC CRL-1619) and 1X 10 ⁶ (0.1 mL) activated T cells (# 250PBMC donor, medium Meorostachian Biotechnology (Tai House) Co., ltd.) in suspension (0.2 mL/min.). Administration was started 1 hour after cell inoculation. The day of tumor cell inoculation was defined as day 0. The weight of the animal was equally divided into 3 groups of 6 animals:

TGF beta Trap control group (G4 Fc TGF beta R2 ecd), 10mg/kg;

PD-1 mab (Torapalimab) control group, 10mg/kg;

the treatment group of the bifunctional protein (JS-TZO 3) is 10mg/kg.

All groups were given by intraperitoneal injection, 2 times per week, 6 weeks continuously, and the experiment was ended 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mice body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition TGI% (tgi=1-T/C (%). T/C% is calculated as tumor proliferation rate, i.e. the percentage value of tumor volume or tumor weight in the treated and control groups at a certain time point. T and C are the Tumor Volumes (TV) of the treated and control groups, respectively, at a certain time point.

As shown in fig. 10, at day 31 after the start of administration, the tgfβtrap control group (G4 Fc tgfβr2 ecd) had an average tumor volume of 2009±217mm ³ . Average tumor volumes of PD-1 mab (Torilimab) control group and bifunctional protein (JS-TZO 3) group were 579+ -217 mm, respectively ³ And 404+ -123 mm ³ Tumor inhibition rates were 71.17% and 79.89%, respectively, compared to tgfβtrap control. The humanized antibody JS-TZO3 of the double-targeting PD-1 and TGF beta R2ecd can obviously inhibit the growth of human melanoma A375 cell subcutaneous transplantation tumor at the dosage level of 10mg/kg, and is superior to PD-1 monoclonal antibody (Torapalimab) and TGF beta Trap control (G4 Fc TGF beta R2 ecd).

Example 11: the stability evaluation of the bifunctional protein JS-TZO3

11.1: thermal stability of the bifunctional protein JS-TZO3

1. Purpose of testing

The heat stability of the bifunctional protein JS-TZO3 of the invention is detected. The stability of the bifunctional proteins was examined at different temperatures using QPCR-7500 in a buffer system at pH 5.2 (20 mM acetic acid-sodium acetate/50 mM sodium chloride/150 mM mannose).

2. Test procedure and results

The sample was replaced with the above buffer solution, and the concentration of the sample was controlled to about 10mg/ml, and the sample was detected by QPCR-7500. The results are shown in Table 3, and the bifunctional protein JS-TZO3 of the present invention shows good heat stability in the above buffer.

Table 3: thermal stability of bifunctional protein JS-TZO3

Sample of	Tm1(℃)	Tm2(℃)
Dual-function protein JS-TZO3	65.7	76.8

Note that: tm: thermal transition temperature.

11.2: high temperature stability of the bifunctional protein JS-TZO3

1. Purpose of testing

And (3) researching stability of the bifunctional protein JS-TZO3 under a high-temperature condition.

2. Test procedure and results

The bifunctional protein JS-TZO3 is replaced to a buffer system with pH of 5.2 (20 mM acetic acid-sodium acetate/50 mM sodium chloride/150 mM mannitol), the concentration of a sample is controlled to be about 10mg/ml, a certain sample volume (200 mu l/tube) is subpackaged, and the solution is placed in a constant temperature oven with the temperature of 40 ℃, so that the stability of 0W, 1W, 2W and 4W is inspected, and the sample is sent according to a time point for detection.

Table 4: high temperature stability of bifunctional protein JS-TZO3

Table 5: high temperature stability of bifunctional protein JS-TZO3

Note that: NA indicates undetected.

Example 12 preparation of bifunctional protein JS-TZO4 targeting PD-L1 and TGF-beta

12.1 construction of bifunctional protein expression vectors

The human antibody heavy chain IgG4 constant region gene fragment was synthesized and the IgG4 constant region gene fragment was ligated to pcdna3.1 vector by EcoRI and NotI restriction enzymes to obtain vector HXT4s. The nucleotide sequence encoding the anti-human PD-L1 antibody is synthesized by the Nanjing Jinsri, and the gene synthesis fragment 9B1HC2 is connected to the vector HXT4s by BSPQI restriction enzyme to obtain the vector HXT4s-9B1HC2. The nucleotide sequence encoding the extracellular domain (ECD) of TGF-beta RII was delegated to Nanjing Jinsri. Connecting peptide ((G) ₄ S) ₄ G) The nucleotide sequence of (2) is obtained by means of a technique in the art. Nucleotide sequence encoding anti-human PD-L1 antibody, nucleotide sequence encoding TGF-beta RII extracellular region, and adaptor protein fragment ((G) ₄ S) ₄ G) The nucleotide sequence of (2) is obtained by means of a technique in the art.

The primers TGF beta R2-G4SBSPQIFV2 and TGF beta R2-BSPQIRV2 are synthesized, the human TGF beta R2 is used as a template to carry out PCR amplification to obtain a human TGF-beta RII ECD gene fragment, and the TGF-beta RII ECD gene fragment is connected to a vector HXT4s-9B1HC2 through BSPQI restriction endonuclease to obtain an expression vector HXT4s-9B1HC 2-TGF beta RII.

Wherein,

the nucleotide sequence of primer TGF-beta R2-G4SBSPQIFV2 (SEQ ID NO: 14) in 5'-3' order is as follows:

the nucleotide sequence of primer TGF-beta R2-BSPQIRV2 in 5'-3' order (SEQ ID NO: 15) is as follows:

the amino acid sequences of the anti-human PD-L1 antibody constructed by the bifunctional protein JS-TZO4 are shown as SEQ ID NO. 31 (heavy chain) and SEQ ID NO. 27 (light chain); the amino acid sequence of the extracellular region of TGF-beta RII is shown as SEQ ID NO. 10; the expression vector HXT4s-1A6H2-TGF beta RII expresses a bifunctional protein JS-TZO4, the second subunit amino acid sequence of the bifunctional protein JS-TZO4 is shown as SEQ ID NO. 27, and the encoding nucleic acid is shown as SEQ ID NO. 30; the first subunit amino acid sequence is shown as SEQ ID NO. 28, and the encoding nucleic acid is shown as SEQ ID NO. 29.

12.2 expression and purification of bifunctional proteins

Liquid nitrogen stored 293F cells (FreeStyle) ^TM 293-F Cells, thermosipher) were resuscitated and added to expression medium (Freestyle ^TM 293 expression medium, gibco). After several generations of culture, 293F cells were diluted to (1.5-2.0). Times.10 the day before transfection ⁶ During transfection, 1/10 transfection volume of the culture medium is firstly used for incubating an expression vector HXT4s-9B1HC2-TGF beta RII and a transfection reagent (PEI), the dosage of the expression vector is 0.5-2 mug/mL, and the dosage of the transfection reagent is 3-14 mug/mL. Incubation time is 20-30min, and the transfection mixture is added after incubation is completed and treated one day in advanceIn the 293F cells described above, shaking was performed while adding the medium. Placing the transfected shake flask into a carbon dioxide shaking table for culturing at 36.5deg.C, 120rpm and 7% CO ₂ . After 7 days, the supernatant was collected by centrifugation and purified. Purification was performed using AKTA AVANT instrument using protein a affinity packing (MabSelect SuRe ^TM GE Healthcare) preloaded purification column, centrifuging the supernatant, filtering the supernatant by a 0.22 mu m filter, loading the supernatant, washing impurities by PBS, eluting by a citric acid buffer solution, and neutralizing by a Tris buffer solution after the elution is completed to obtain the bifunctional protein JS-TZO4. The prepared bifunctional protein JS-TZO4 can be used in each of the following examples.

The schematic molecular structure of JS-TZO4 bifunctional protein is shown in FIG. 11.

Example 13: ELISA detection of binding of bifunctional protein JS-TZO4 and humanized TGF-beta

1. Purpose of testing

ELISA method is used for detecting the combination of the bifunctional protein JS-TZO4 and human TGF-beta 1, TGF-beta 2 and TGF-beta 3.

2. Test procedure and results

300 μl 1 XPBST was washed 4 times, and the gradient diluted bifunctional protein JS-TZO4 and negative control antibody (Anti-KLH IgG 4) (initial concentration 100 μg/ml, 2.5-fold gradient diluted 12 concentration spots) were added, respectively, and incubated at 37℃for 1 hour.

d.300. Mu.l 1 XPBST were washed 4 times.

As shown in FIGS. 12, 13 and 14, the bifunctional protein JS-TZO4 of the invention has stronger binding with TGF-beta 1 and TGF-beta 3 and relatively weaker binding with TGF-beta 2.

Example 14: ELISA (enzyme-Linked immuno sorbent assay) for detecting combination of bifunctional protein JS-TZO4 and PD-L1

1. Test purpose:

ELISA detects the binding of the bifunctional protein JS-TZO4 to PD-L1.

2. Test procedure

a. Human PD-L1 (0.3. Mu.g/ml) was used as antigen to coat 96-well plates at 100. Mu.l/well and incubated at 37℃for 1.5 hours.

300 μl1 XPBST was washed 4 times, and was incubated with a gradient of diluted bifunctional protein JS-TZO4, positive control Anti-PD-L1 mAb and negative control antibody (Anti-KLH IgG 4) (initial concentration 1 μg/ml, 2.5-fold gradient diluted 12 concentration points) for 1 hour at 37 ℃.

d.300. Mu.l 1 XPBST were washed 4 times.

Positive control anti-PD-L1 mab: from Jundi organisms (patent application No. PCT/CN 2018/076669), the heavy chain is shown as SEQ ID NO. 26, and the light chain is shown as SEQ ID NO. 27.

As shown in FIG. 15, the bifunctional protein JS-TZO4 of the invention has stronger combination with PD-L1, and is equivalent to a positive control antibody.

Example 15: reporter gene test for detecting blocking of bifunctional protein JS-TZO4 to PD-1/PD-L1 passage

1. Test purpose:

in order to study the blocking effect of the bifunctional protein JS-TZO4 on the PD-1/PD-L1 signal pathway, a PD-1/PD-L1 reporter gene system constructed by Promega corporation is adopted to detect the cell biological activity of the PD-L1 end in the bifunctional protein.

2. Test procedure

CHO/PD-L1 cells (Promega) were taken, digested and resuspended in complete medium of F1-2 nutrient mix (F-12 Nutrient Mixture), and the cell density was adjusted to 5X 10 based on the result of cell counting using complete medium ⁵ Cell suspensions were transferred to a loading well and 100. Mu.l/well into 96-well plates using a multichannel pipette and placed at 37℃in 5% CO ₂ Culturing in an incubator for 16-20 h; the next day a Jurkat/PD-1 (Promega) cell suspension was prepared, the cells were resuspended using analytical medium according to the cell count results, and the cell density was adjusted to 2.5X10 ⁶ Individual cells/ml; the cell culture plate to which CHO/PD-L1 cells were added was removed from the incubator, 100. Mu.l of the culture solution was removed per well using a multi-channel pipette, and a gradient of bifunctional protein JS-TZO4 (initial concentration 16.5nM, 3-fold gradient 10 concentration points), positive control PD-L1 mab (initial concentration 18.33nM, 3-fold gradient 10 concentration points) and negative control antibody (Anti-KLH IgG4; initial concentration 18.33nM, 3-fold gradient 10 concentration points) were added to each well, respectively, and then the Jurkat/PD-1 cell suspension was transferred to the addition tank, and 40. Mu.l/well was added to the cell culture plate, and the mixture was placed at 37℃with 5% CO ₂ Culturing in an incubator for 5-6 h. During protein incubation, one-Glo reagent (Promega, E6130) was removed to bring its temperature back to room temperature. The cell culture plate was removed, left at room temperature for 5-10 min, and then 40. Mu.l of luciferase substrate ONE-Glo was added to each well ^TM The reagent (promega, E6130) was placed on a mixer and mixed for 5-10 min, and then the luminescence signal value was read using an M1000 pro multifunctional enzyme-labeled instrument (Tecan).

Positive control PD-L1 mab: from Jundi organisms (patent application No. PCT/CN 2018/076669), the heavy chain is shown as SEQ ID NO. 26, and the light chain is shown as SEQ ID NO. 27.

3. Results

As shown in FIG. 16, the bifunctional protein JS-TZO4 and the anti-PD-L1 monoclonal antibody can effectively block the interaction of PD-1/PD-L1, promote the enhancement of NFAT signals, have obvious drug concentration dose-dependent effect and have equivalent activity levels.

Example 16: test for detecting blocking of TGF beta channel by bifunctional protein JS-TZO4 through reporter gene method

1. Purpose of testing

Inhibition of TGF- β1-induced Smad3 activation by bifunctional protein JS-TZO4 was studied by 4T1 cells expressing Smad3 binding original (SBE) with luciferase reporter.

2. Test procedure

4T1-SBE cells (Junychia) were cultured using 1640 complete medium (Gibco, 11875-093) containing 10% FBS, once every 2 days. Experiment first day at a density of 100,000 cells per well, seeded in 96-well plates (Costar, 3917), at 37 ℃, 5% co ₂ Culturing for 16-20 hours under the condition. The following day, the medium in the cell culture plates was discarded and a gradient dilution (initial concentration of 1nM, 4-fold gradient of 9 concentration points) of bifunctional protein JS-TZO4 and positive control G4Fc TGF beta R2ecd (TGF beta Trap, jun. RTM.) was added at 50. Mu.l/well. Cells were incubated at 37℃with 5% CO ₂ Culturing for 0.5-1 hr. 50 μl of recombinant humanized TGF-beta 1 was added per well. Cells were incubated at 37℃with 5% CO ₂ Culturing for 5-6 hr. Then 50 μl of luciferase substrate ONE-Glo was added per well ^TM The reagent (promega, E6130) was placed on a mixer and mixed for 5-10 min, and then the luminescence signal value was read using an M1000 pro multifunctional enzyme-labeled instrument (Tecan). IC50 values of the samples to be tested were calculated using the data processing software Graphpad prism 7.0.

3. Results

As shown in FIG. 17, the bifunctional proteins JS-TZO4 and G4Fc TGF beta R2ecd (TGF beta Trap) of the present invention are both capable of inhibiting TGF beta 1-induced activation of pSMAD3 in a dose-dependent manner.

Example 17: endocytosis test of bifunctional protein JS-TZO4

1 purpose of test

Endocytic activity of bifunctional protein JS-TZO4 was detected at the in vitro level using an antibody endocytic assay.

2. Test procedure

The CypHer 5Mono NHS Ester dye labels the sample to be tested: using Label buffer (labelling buffer, PBS: naCO) ₃ The concentrations of the samples to be tested (bifunctional protein JS-TZO4, anti-PD-L1 mab and negative control anti-KLH IgG 4) were adjusted to 1.0mg/ml, respectively, =9:1, ph: 8.3). And calculating the mass of a sample to be detected, adding CypHer 5Mono NHS Ester dye (GE, PA 15401), and uniformly mixing for 1h at room temperature in a dark place. Transferring the reacted sample to be tested and the dye mixed solution into a 0.5ml ultrafiltration tube (30 KD), centrifuging for 10min, discarding the waste liquid, supplementing Label buffer into the ultrafiltration tube, centrifuging for 4 times, and washing away the unbound excessive dye.

CHO/PD-L1 cells (Promega) were taken, digested and resuspended in F-12 Nutrient Mixture complete medium, 293F JS1E 4 cells were taken, digested and resuspended in 1640 complete medium, and cell density was adjusted to 2X 10 using 1640 complete medium based on cell count results ⁶ Individual cells/ml.

Endocytosis test (co-incubation of sample to be tested and target cells) (temperature is required to be noted in this step, endocytosis of antibody is required to be carried out at 37 ℃ and can be inhibited at 4 ℃) A96-well ultra-low adsorption U bottom plate is adopted, cells are spread in a 96-well plate, 50 mu.l of cell suspension is added per well by a multichannel pipetting gun (the number of cells per well is 1×10) ⁵ Individual cells/wells); then the samples to be tested (bifunctional protein JS-TZO4: initial concentration of 55.56nM, 3-fold gradient dilution of 12 concentration points; anti-PD-L1 monoclonal antibody and Anti-KLH IgG4: initial concentration of 66.7nM, 3-fold gradient dilution of 12 concentration points) with the cells at different concentrations are mixed with CO ₂ Incubate for 4h. Taking out the 96-well cell plate after incubation, centrifuging at 1000rpm for 5 minutes, and discarding the supernatant; cells were then resuspended in staining buffer (PBS+1% BSA) and centrifuged at 1000rpm for 5 min, the supernatant discarded, and unbound excess sample washed away by repeating the procedure twice, 150 μl of staining buffer was added to each well of a 96 well cell plate, examined on-press FACS and analyzed by FlowJo software.

Positive control PD-L1 mab: jundian organism (patent application No. PCT/CN 2018/076669), the heavy chain is shown as SEQ ID NO. 26, and the light chain is shown as SEQ ID NO. 27.

3. Results

As shown in FIG. 18, the bifunctional protein JS-TZO4 and the anti-PD-L1 monoclonal antibody have strong endocytic activity and have dose-dependent effects. The negative control Anti-KLH IgG4 had no endocytosis.

Example 18: in vitro mixed lymphocyte reaction of bifunctional protein JS-TZO4

1. Purpose of testing

The invention researches the release level of IL2 and IFN gamma cytokines after T cell activation by the bifunctional protein JS-TZO4 by using a mixed lymphocyte reaction experiment at an in vitro level.

2. Test procedure

The adherent cells were stimulated with commercial human PBMC (SAILY BIO, cat#SLB-HP 050B) with serum-containing medium at 37℃for 2h, after which the suspension cells were washed out, and then with 10ng/ml GM-CSF (Sino Biological, cat#GMP-11846-HNAE) and 10ng/ml IL-4 (Sino Biological, cat#GMP-10015-HNAH), respectively, for 5 days, followed by 5ng/ml IL-1β (Sino Biological, cat#10139-HNAE), 10ng/ml IL-6 (Sino Biological, cat#GMP-10395-HNAE), 10ng/ml TNF- α (Sino Biological, cat#10602-HNAE) and 1. Mu. Mol/L PGE2, respectively, for 2 days to induce dendritic cells (mDC). Commercial Human PBMC (SAILY BIO, cat#SLB-HP050B, lot# 190100) were purified with CD4T microblades, human (Miltenyi Biotec, cat#130-045-101) to give CD4T cells.

Samples to be tested (blank control, bifunctional protein JS-TZO4, negative control antibody (anti-KLH IgG 4) +TGF-beta Trap (monad)) control, PD-L1 mab and TGF-beta Trap combination (PD-L1 antibody+TGF-beta Trap control) were mixed with constant concentrations (100 ng/ml) of TGF-beta 1 recombinant protein (novoprotein, cat#CA 59) and mDC cells (10000 cells per well) and purified CD4T cells (100000 cells per well) in 96-well round bottom plates at 37℃CO ₂ Incubate for 5 days. Part of the reaction supernatant was collected from each well of a 96-well plate on day 3 of co-incubation and assayed for IL-2 release using BD CBA human IL2Flex set (BD, cat# 558270) and BD C on day 5 of co-incubationThe BA human IFN gamma Flex set (BD, cat# 558269) detects the IFN gamma content in the supernatant.

PD-L1 mab: jundian organism (patent application No. PCT/CN 2018/076669), the heavy chain is shown as SEQ ID NO. 26, and the light chain is shown as SEQ ID NO. 27.

3. Results

As can be seen from fig. 19, in the mixed lymphocyte reaction, the dual function proteins JS-TZO4, PD-L1 mab and PD-L1 mab combined with tgfβtrap of the present invention all promoted IL-2 release, but the difference was not obvious (fig. 19 a). The bifunctional protein JS-TZO4 can obviously promote release of IFN gamma, and the activity is equivalent to that of a PD-L1 monoclonal antibody and TGF beta Trap combined group and a PD-L1 monoclonal antibody group (figure 19 b).

Example 19: the stability evaluation of the bifunctional protein JS-TZO4

8.1: thermal stability of the bifunctional protein JS-TZO4

1. Purpose of testing

Is used for detecting the thermal stability of the bifunctional protein JS-TZO 4. The thermal stability of the different antibodies was examined using QPCR-7500, and the stability at different temperatures was examined in a buffer at pH 5.2 (20 mM acetate (acetic acid-sodium acetate)/50 mM sodium chloride/150 mM mannose).

2. Test procedure and results

The sample was replaced with the above buffer solution, and the concentration of the sample was controlled to about 10mg/ml, and the sample was detected by QPCR-7500. In particular, as shown in Table 6, the bifunctional protein JS-TZO4 of the present invention exhibits good thermal stability in the above buffer.

Table 6: thermal stability of bifunctional protein JS-TZO4

Sample of	Tm1(℃)	Tm2(℃)
Dual-function protein JS-TZO4	65.4	76.9

Note that: tm: thermal transition temperature.

8.2: high temperature stability of the bifunctional protein JS-TZO4

1. Purpose of testing

And (5) researching stability of the bifunctional protein JS-TZO4 under a high-temperature condition.

2. Test procedure and results

The samples were replaced in a buffer system at pH 5.2 (20 mM acetic acid-sodium acetate/50 mM sodium chloride/150 mM mannitol), the concentration of the samples was controlled at about 10mg/ml, and the samples were dispensed in a volume (200. Mu.l/tube) and placed in a 40℃incubator to examine the stability at 0W, 1W, 2W and 4W until the time point was reached for sample feeding detection. Stability was assessed by the following parameters: (a) SEC-HPLC (size exclusion chromatography) measures the content of antibody monomers, polymers or fragments; (b) Detecting the molecular weight of the antibody by a CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method; (c) ELISA and reporter gene methods detect antibody biological activity.

The results show that the bifunctional protein JS-TZO4 has good thermal stability, and the specific results are shown in a table 7 and a table 8.

Table 7: high temperature stability of bifunctional protein JS-TZO4

Table 8: high temperature stability of bifunctional protein JS-TZO4

Note that: NA indicates undetected.

Claims

Bifunctional proteins targeting PD-1 or PD-L1 and TGF-beta comprising

(i) An antigen binding fragment of an anti-PD-1 or PD-L1 antibody;

(ii) An immunoglobulin Fc domain; and

(iii) TGF-. Beta.RII extracellular domain (ECD).
The bifunctional protein of claim 1, wherein the antigen-binding fragment of the anti-PD-1 antibody comprises an HCDR1, an HCDR2, and an HCDR3 having amino acid sequences of SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and an LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively; preferably, the antigen binding fragment of the anti-PD-1 antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO. 7 and the light chain variable region set forth in SEQ ID NO. 8.
The bifunctional protein of claim 1, wherein the antigen-binding fragment of the anti-PD-L1 antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID No. 21, SEQ ID No. 22 and SEQ ID No. 23, respectively; preferably, the antigen binding fragment of the anti-PD-L1 antibody comprises a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 24 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 25; further preferably, the antigen binding fragment of the anti-PD-L1 antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 24 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 25.
The bifunctional protein of any one of claims 1-3, wherein the antigen-binding fragment of an anti-PD-1 or PD-L1 antibody is a Fab, fab ', F (ab') 2, fv, scFv, or sdAb.
The bifunctional protein of any one of claims 1-3, wherein said immunoglobulin Fc domain is a human IgG1, igG2, igG3, or IgG4 Fc domain; an Fc domain, preferably human IgG 4; preferably, the Fc domain of IgG4 comprises 1, 2, 3, 4 or 5 amino acid differences, preferably with an S228P amino acid substitution in the Fc domain; it is further preferred to have a C-terminal deletion of both K and G amino acids in the Fc domain.
The bifunctional protein of claim 5, wherein the immunoglobulin Fc domain comprises an Fc domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 9; preferably, the immunoglobulin Fc domain comprises an amino acid sequence as shown in SEQ ID NO. 9.
The bifunctional protein of any one of claims 1-6, wherein the extracellular domain of TGF- βrii has the amino acid sequence shown in SEQ ID No. 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 10.
The bifunctional protein of any one of claims 1-7, wherein said bifunctional protein is in the order of (i), (ii) and (iii) from N-terminus to C-terminus; the order of (iii), (i) and (ii); or (iii), (ii) and (i) in the order of active linkage; preferably, the (i) and (ii) are connected through a hinge region, and the (i) and (iii) and (ii) and (iii) are connected through a connecting peptide, wherein the amino acid sequence of the connecting peptide contains or consists of glycine residues and serine residues, and the length of the connecting peptide is 2-31 amino acid residues; preferably each independently is (GGGGS) nG, wherein n is independently selected from 3, 4, 5 or 6.
The bifunctional protein of any one of claims 1-8, comprising an anti-PD-1 or PD-L1 antibody and one TGF- βrjj ECD operably linked at the C-terminus of each of the two heavy chains of the antibody.
The bifunctional protein of claim 9, wherein the anti-PD-1 antibody has a heavy chain amino acid sequence as set forth in SEQ ID No. 11 and a light chain amino acid sequence as set forth in SEQ ID No. 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 11 and/or SEQ ID No. 12.
The bifunctional protein of claim 9, wherein the anti-PD-L1 antibody comprises a heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 31, and a light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 27. Further preferably, the anti-PD-L1 antibody comprises a heavy chain amino acid sequence as set forth in SEQ ID NO. 31 and a light chain amino acid sequence as set forth in SEQ ID NO. 27.
The bifunctional protein of any one of claims 1-11, comprising a bifunctional protein first subunit having an amino acid sequence shown in SEQ ID NO. 13, and a bifunctional second subunit having an amino acid sequence shown in SEQ ID NO. 12; or comprises a bifunctional protein first subunit with an amino acid sequence shown as SEQ ID NO. 28, and a bifunctional second subunit with an amino acid sequence shown as SEQ ID NO. 27.
A polynucleotide encoding the bifunctional protein of any one of claims 1-12, or an antigen-binding fragment thereof; preferably, the polynucleotide comprises the nucleotide sequence of the first subunit shown as SEQ ID NO. 16 and/or the nucleotide sequence of the second subunit shown as SEQ ID NO. 17; preferably, the polynucleotide comprises the nucleotide sequence of the first subunit as shown in SEQ ID NO. 29 and/or the nucleotide sequence of the second subunit as shown in SEQ ID NO. 30.
An expression vector comprising the polynucleotide of claim 13, preferably the expression vector is a eukaryotic expression vector.
A host cell comprising the polynucleotide of claim 13 or the expression vector of claim 14, preferably the host cell is a mammalian cell.
A method of making the bifunctional protein of any one of claims 1-12, comprising expressing the bifunctional protein in the host cell of claim 15 under conditions suitable for expression of the bifunctional protein, and recovering the expressed bifunctional protein from the host cell.
A pharmaceutical composition comprising the bifunctional protein of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the host cell of claim 15, and a pharmaceutically acceptable carrier or excipient.
Use of a bifunctional protein of any one of claims 1-12, a polynucleotide of claim 13, an expression vector of claim 14, a host cell of claim 15, or a pharmaceutical composition of claim 17 in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder associated with PD-1, PD-L1 or TGF- β activity, preferably the disease or disorder is a cancer, more preferably the cancer is selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, and rectal cancer.
A kit comprising the bifunctional protein of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the host cell of claim 15, or the pharmaceutical composition of claim 17.
A method of detecting the presence of PD-1, PD-L1 or TGF- β in a sample using the bifunctional protein of any one of claims 1-12.