RU2776204C1 - Fused protein containing tgf-beta receptor, and its pharmaceutical use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to fused proteins based on TGF-beta receptor; it can be used in medicine for the treatment of PD-L1-mediated tumor. Bifunctional fused protein is proposed, containing a targeting part – an antibody against PD-L1 and extracellular domain TGF-beta RII.

EFFECT: invention provides for obtaining fused proteins that are specific relatively to binding to human TGF-beta1 and PD-L1, block binding to PD-1 and inhibit TGF-bets-induced pSMAD3 activity, as well as show a higher expression level and a stable structure compared to fused protein of the similar structure based on uncut ECD TGF-beta RII.

19 cl, 8 dwg, 10 tbl, 12 ex

Description

The field of technology to which the invention belongs

The present invention relates to the field of drugs for antitumor immunotherapy. In particular, the invention relates to fusion proteins for cancer treatment, including a fusion protein containing a targeting molecule and an immunomodulatory factor (such as TGF-βRII (transforming growth factor beta receptor - transforming growth factor beta receptor)). More specifically, the present invention relates to a fusion protein formed by a targeting molecule - an antibody against PD-L1 (from the English programmed cell death ligand 1 - programmed cell death ligand 1) and an immunomodulatory factor (TGF-βRII), containing its pharmaceutical composition and its use as an anticancer drug.

Prior Art

In the treatment of cancer, people have recognized the high toxicity caused by chemotherapy and the negative effects that can lead to the formation of drug-resistant cancer cells. Even if the treatment targeted overexpressed or activated proteins that are associated with tumor survival, cancer cells would still rely on mutations to reduce or avoid dependence on the pathways targeted by the targeted therapy and would also survive through other pathways. Antitumor immunotherapy has received a lot of attention in recent years and is at the center of cancer treatment. It is difficult to develop resistance to drugs, and this is an outstanding advantage of this therapy. Based on the immunological theory and method, antitumor immunotherapy mainly enhances the immunogenicity of tumor cells and killing sensitivity on the basis of effector cells, stimulates and enhances the antitumor immune response, and introduces immune cells and effector molecules into the host, coordinating with the immune system to kill tumor cells and inhibition of tumor growth.

Programmed cell death 1 (PD-1) protein is a member of the CD28 superfamily. PD-1 is expressed on activated T cells, B cells, and myeloid cells, which have two ligands, PD-L1 (Programmed Cell Death Ligand 1) and PD-L2. PD-L1 interacts with PD-1 on T cells and plays an important role in the downregulation of immune responses. Expression of the PD-L1 protein can be detected in many human tumor tissues. The tumor microenvironment can induce PD-L1 expression on tumor cells. Expression of PD-L1 beneficially influences the spread and growth of tumors, and induces apoptosis of the antitumor T cell. A PD-1/PD-L1 pathway inhibitor can block the binding of PD-1 to PD-L1 and block this negative regulatory signal, and restore T cell activity with enhanced immune response. Thus, immunoregulation targeting PD-1/PD-L1 is important for tumor suppression.

Transforming growth factor beta (TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β signal through a heterotetrameric receptor complex, which consists of two type I transmembrane receptor serine-threonine kinases and two type II transmembrane receptor serine-threonine kinases.

TGF-β is a multifunctional cytokine that exerts a tumor-suppressing action or a tumor-stimulating action in a cell-dependent or lineage-dependent manner. The tumor-suppressing effect of the TGF-β signaling pathway is due to the ability to induce the expression of multiple genes. When a mutation or epigenetic modification occurs during tumor development, cancer cells gradually become tolerant to the inhibition of the TGF-β signaling pathway, eventually leading to the development of tumors.

During research, it was found that blocking the TGF-β signaling pathway can reduce tumor metastasis. The Smad2/3 truncated dominant negative mutant was used to inhibit the TGF-β signaling pathway of a breast tumor cell line, and it was found that the ability of the tumor cells to metastasize was inhibited. In colon cancer studies on microsatellite instability, inactive TGF-βRII mutations were found to reduce metastasis and increase postoperative survival. However, in general, the use of an inhibitor of the TGF-β signaling pathway alone has little effect in clinical treatment, which may be due to the high level of expression of TGF-β mainly in tumor cells and the bioavailability of inhibitors of the signaling pathways.

Thus, inhibition of the PD-1/PD-L1 pathway based on blocking and neutralizing TGF-β in the tumor microenvironment can restore T cell activity, enhance the immune response, and improve the effect of inhibiting the development of tumor spread. An anti-PD-L1 antibody is proposed by Applicant's prior PCT application PCT/CN2016/104320.

To date, there are merged proteins of antibodies/receptor TGF-β, opened in Wo 2006074451 A2, Wo 2009152610 A1, Wo 2011109789 A2, Wo 2013164694 A1, Wo 2014164427, Wo 20157540 A2, Wo 9309228 A1, Wo 9409815 A1, Wo 20154 WO 2015118175 A2. However, some fusion proteins still have instability or low expression problems. There is still a need to further develop products with better efficacy, both in production and in clinical practice. According to the present invention, a technical solution is proposed that is favorable for production and has more stable performance.

Brief summary of the invention

The present invention provides a TGF-β receptor fusion protein comprising a targeting moiety and a TGF-β receptor moiety, wherein the TGF-β receptor moiety is an N-terminal truncated form of the TGF-βRII extracellular domain.

In a preferred embodiment of the present invention, wherein the N-terminal truncated form of the TGF-βRII extracellular domain comprises a deletion of 26 or fewer contiguous amino acids at the N-terminus of the TGF-βRII extracellular domain, preferably a deletion of 14-26 contiguous amino acids, more preferably a deletion of 14-21 adjacent amino acids, most preferably a deletion of 14-21 adjacent amino acids; As non-limiting examples, the N-terminal truncated form of the extracellular domain of TGF-βRII contains the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

In a preferred embodiment of the present invention, the sequence of the extracellular domain of TGF-βRII is shown as SEQ ID NO: 13.

In a preferred embodiment of the present invention, the targeting moiety is a cell-specific targeting moiety; preferably, the targeting moiety is a cancer cell-specific targeting moiety.

In a preferred embodiment of the present invention, the cancer cell-specific targeting moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a receptor, and a cytokine.

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of full length antibody, chimeric antibody, Fab', Fab, F(ab')2, single domain antibody (DAB), Fv, scFv, small antibody, bispecific antibody, and trispecific antibodies or mixtures thereof.

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof binds to one or more of the following polypeptides or proteins selected from the group consisting of HER2 (human epidermal growth factor receptor 2), HER3 ( human epidermal growth factor receptor 3), immune checkpoint molecules, CD33, VEGF (vascular endothelial growth factor), VEGFR (growth factor receptor vascular endothelial growth factor receptor), VEGFR-2 (vascular endothelial growth factor receptor 2), CD152, TNF (tumor necrosis factor, from English . "tumour necrosis factor"), IL-1, IL-5, IL-17, IL-6R, IL-1, IL-2R, BLYS, PCSK9 (proprotein convertase subtilisin/kexin type 9, from English "proprotein convertase subtilisin/kexin type 9"), EG FR (epidermal growth factor receptor) "epidermal growth factor receptor"), c-Met (hepatocyte growth factor receptor), CD2, CD3, CD11a, CD19, CD30, CD38, CD20, CD52, CD60, CD80, CD86, TNF-α (tumor necrosis factor alpha, from "tumour necrosis factor alpha"), IL-12, IL-17, IL-23, IL-6, IL-1β, RSVF, IgE, RANK (nuclear factor kappa-B activator receptor, from English "receptor activator of nuclear factor-kappa B"), BLyS (B-lymphocyte stimulator, from English. "B Lymphocyte Stimulator"), α4β7, PD-1CCR4 (CC chemokine receptor 4, from English. "CC chemokine receptor 4"), SLAMF7 (7th member of the family of signaling lymphocyte activation molecules, from the English. "Signaling Lymphocytic Activation Molecule Family member 7"), GD2, CD21, CD79b, IL20Rα, CD22, CD79a, CD72, IGF-1 R (insulin-like growth factor receptor 1, from "insulin-like growth factor 1 receptor") and RANKL (receptor activator of nuclear factor kappa-B ligand); preferably wherein the antibody, or antigen-binding fragment thereof, binds to an immune checkpoint molecule.

In a preferred embodiment of the present invention, the antibody is an anti-PD-L1 antibody; preferably the anti-PD-L1 antibody is selected from the group consisting of: MSB0010718C, MEDI4736, BMS-936559 and MPDL3280A; or the anti-PD-L1 antibody contains one or more CDRs (complementarity determining region - hypervariable region) selected from the group consisting of the following CDRs or their mutants:

where X ₁ represents H or G, preferably G; X ₂ is G or F, preferably F.

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is a chimeric antibody or functional fragment thereof, a humanized antibody or functional fragment thereof, or a human antibody or functional fragment thereof.

In a preferred embodiment of the present invention, the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 7, preferably comprises the heavy chain variable region of SEQ ID NO: 9.

In a preferred embodiment of the present invention, the humanized antibody further comprises the heavy chain of SEQ ID NO: 11.

In a preferred embodiment of the present invention, the humanized antibody comprises the light chain variable region of SEQ ID NO: 8 or 10, or a mutant thereof.

In a preferred embodiment of the present invention, the humanized antibody comprises the light chain of SEQ ID NO: 12.

In a preferred embodiment of the present invention, wherein the TGF-β receptor-containing fusion protein is as shown in general formula (I):

where TGF-βRII ECD is a truncated form of the extracellular domain of TGF-βRII;

Ab is an antibody;

L is a linker.

In a preferred embodiment of the present invention, the linker is (G ₄ S) _x G, where x is 3-6, preferably 4-5.

The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a TGF-β receptor-containing fusion protein as described above and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention provides a DNA molecule encoding a fusion protein containing the TGF-β receptor as described above.

The present invention further provides an expression vector containing a DNA molecule as described above.

The present invention further provides a host cell transformed with this expression vector as described above, wherein the host cell is selected from the group consisting of bacterial, yeast and mammalian cells; preferably mammalian cells.

The present invention further provides the use of a TGF-β receptor-containing fusion protein as described above, or a pharmaceutical composition thereof, for the preparation of a medicament for the treatment of tumors; preferably for the preparation of a medicament for the treatment of a PD-L1-mediated tumor; more preferably a cancer in which PD-L1 is expressed.

The present invention further provides a method for treating or preventing a tumor, comprising administering to a patient in need thereof a therapeutically effective amount of a TGF-β receptor-containing fusion protein as described above.

The present invention further provides a truncated extracellular domain of TGF-βRII, wherein the truncated extracellular domain of TGF-βRII comprises a deletion of 26 or fewer contiguous amino acids at the N-terminus of SEQ ID NO: 13, preferably a deletion of 14-26 contiguous amino acids at the N-terminus, greater than preferably deletion of 14-21 adjacent amino acids at the N-terminus; non-limiting examples of the truncated extracellular domain of TGF-βRII include the sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15.

The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the truncated extracellular domain of TGF-βRII of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention further provides the use of the truncated extracellular domain of TGF-βRII of the present invention or a pharmaceutically acceptable composition thereof for the manufacture of a medicament for the treatment or inhibition of diseases or disorders associated with cancer cell proliferation or metastasis.

The present invention further provides a method for treating or preventing a tumor, comprising administering to a patient in need thereof a therapeutically effective amount of the truncated extracellular domain of TGF-βRII of the present invention or a pharmaceutical composition thereof.

The tumor or cancer described in this disclosure is selected from the group consisting of colorectal, breast, ovarian, pancreatic, gastric, prostate, kidney, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine cancer , bladder, neuroendocrine cancer, head and neck cancer, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma bulging, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

Description of graphic materials

Fig. 1. Schematic representation of the structure of the fusion protein.

Fig. 2. Results showing in vitro binding of the fusion protein to human TGF-β1.

Fig. 3. Results showing in vitro binding of the fusion protein to human TGF-β1.

Fig. 4. Results showing in vitro binding of the fusion protein to human PD-L1.

Fig. 5. Results showing blocking of the PD-1/PD-L1 pathway by the fusion protein in vitro.

Fig. 6. The fusion protein inhibits TGFβ-induced pSMAD3 reporter activity in a dose-dependent manner.

Fig. 7. All fusion protein samples enhance the secretion of the cytokine IFN-γ (from the English interferon gamma - interferon gamma) by activated T-lymphocytes.

Fig. 8. Effect of fusion protein on tumor mass in tumor-bearing mice.

Detailed description of the invention

Terms

For an easier understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning generally understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the one-letter code and the three-letter code for amino acids is as described in J. Biol. Chem, 243, (1968) p. 3558.

As used herein, the term "antibody" refers to an immunoglobulin, a four peptide chain structure formed by two identical heavy chains and two identical light chains linked by an interchain disulfide bond. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and sequences, hence representing different antigenicity. Accordingly, immunoglobulins can be subdivided into five categories, also referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE; their respective heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. According to the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, immunoglobulins can be subdivided into different subcategories, for example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. The light chain can be subdivided into a κ or λ chain, considering the different constant region. Each category Ig of these five categories includes a chain κ or λ.

In the present invention, the antibody light chain referred to herein further comprises a light chain constant region that contains a human or mouse κ, λ chain, or a variant thereof.

In the present invention, the heavy chain of the antibody referred to herein further comprises a heavy chain constant region that contains human or mouse IgG1, IgG2, IgG3, IgG4, or a variant thereof.

At the N-terminus of the heavy and light chains of an antibody, about 110 amino acids are highly variable, which is known as the variable region (the F _V region); the amino acid sequence at the C-terminus is relatively stable, which is known as the constant region. The variable region contains three hypervariable regions (HVR - from the English hypervariable region) and four FR regions (FR - from the English framework region - constant region) with a relatively conservative sequence. Three hypervariable regions determine the specificity of an antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR - light chain variable region) and each heavy chain variable region (HCVR - heavy chain variable region) consists of three CDR regions and four FR regions, located from the N-terminus to C - ends in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDR regions are LCDR1, LCDR2 and LCDR3; the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and position of the amino acid residues of the CDR region in the LCVR and HCVR regions of the antibody or antigen-binding fragment herein follow the known Kabat numbering criteria (LCDR1-3, HCDE2-3) or follow the Kabat and Chothia numbering criteria (HCDR1).

The antibody of the present invention includes a full length antibody selected from the group consisting of a mouse antibody, a chimeric antibody and a humanized antibody, preferably a humanized antibody.

The term "mouse antibody" in the present invention refers to an anti-human PD-L1 monoclonal antibody produced in accordance with the knowledge and skill of the art. During preparation, the test subject was injected with the PD-L1 antigen, and then a hybridoma expressing an antibody that has the desired sequence or functional characteristics was isolated. In a preferred embodiment of the present invention, the mouse anti-PD-L1 antibody, or antigen-binding fragment thereof, further comprises a mouse κ, λ light chain constant region, or a variant thereof, or further comprises a mouse IgG1, IgG2, IgG3 heavy chain constant region, or a variant thereof.

The term "chimeric antibody" is an antibody that is formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, so as to facilitate an immune response elicited by the mouse antibody. To create a chimeric antibody, a hybridoma secreting a specific mouse monoclonal antibody is first created, then the variable region genes are cloned from mouse hybridoma cells, and then the human antibody constant region genes are cloned if desired, the mouse variable region genes are ligated with human constant region genes to form a chimeric gene, which can be inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present invention, the light chain of the chimeric anti-PD-L1 antibody further comprises light chain constant regions derived from the human κ, λ chain, or a variant thereof. The heavy chain of the chimeric anti-PD-L1 antibody further comprises heavy chain constant region(s) derived from human IgG1, IgG2, IgG3, IgG4, or a variant thereof. The constant region(s) of a human antibody may be selected from the constant region(s) of the heavy chain derived(s) from human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably contains a heavy chain constant region derived from human IgG2 or IgG4, or IgG4 without ADCC (antibody-dependent cell-mediated cytotoxicity) after an amino acid mutation.

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody created by grafting murine CDR sequences into a human antibody variable region framework, namely an antibody formed from different types of human germline antibody framework sequences. The humanized antibody overcomes the adversely strong antibody response elicited by a chimeric antibody that carries a large amount of murine components. Such framework sequences can be obtained from a publicly available DNA database covering germline antibody gene sequences, or from published reference sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available at www.mrccpe.com.ac.uk/vbase) and can also be found in Kabat, EA, et al, 1991 Sequences of Proteins of Immunological Interest, 5th Ed. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the framework of the human antibody variable region is subjected to minimal backmutation to maintain activity. The humanized antibody of the present invention also includes a humanized antibody, which is further obtained by phage display, for the purpose of CDR affinity maturation.

The terms "human antibody" and "antibody of human origin" are used interchangeably to mean that one or more variable and constant regions are derived from a human immunoglobulin sequence. In a preferred embodiment, all variable and constant regions are derived from human immunoglobulin sequences, ie "fully human-derived antibodies" or "fully human antibodies". These antibodies can be obtained in various ways, including phage display technology; isolation of B cells from human PBMCs (from the English. peripheral blood mononuclear cells - mononuclear cells of peripheral blood), spleen or lymph nodes; constructing a library of phage native single chain human antibodies; or by immunizing transgenic mice that express human antibody light and heavy chains; and screening for antibodies thus obtained.

As used herein, the term "antigen binding fragment" or "functional fragment" refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment with antigen binding activity, and a scFv fragment of an Fv fragment, binding to human PD-L1. The Fv fragment is the minimum fragment of an antibody that includes all antigen binding sites, the Fv fragment contains a heavy chain variable region and a light chain variable region, but no constant region. Typically, the Fv antibody further comprises a polypeptide linker between the VH and VL domains to form the structure required for antigen binding. Also, different linkers can be used to join the variable regions of two antibodies to form a polypeptide called a single chain antibody or single chain Fv (scFv). As used herein, the term "PD-L1 binding" means the ability to interact with human PD-L1. As used herein, the term "antigen-binding site" of the present invention refers to discrete, three-dimensional sites on an antigen recognized by an antibody or antigen-binding fragment of the present invention.

As used herein, the term "ADCC", namely antibody-dependent cellular cytotoxicity, refers to cells expressing Fc receptors that directly kill antibody-coated target cells by recognizing the Fc segment of an antibody. The ADCC effector function of an antibody can be attenuated or eliminated by modifying the Fc segment in IgG. Modification refers to mutations in the constant region of the heavy chain of the antibody, such as mutations selected from N297A, L234A, L235A in IgG1; IgG2/4 chimera; or F234A/L235A mutations in IgG4.

"Mutation" in the "mutant sequence" of the present invention includes, but is not limited to, "back mutation", "conservative modification", or "conservative substitution or substitution". "Conservative modification" or "conservative substitution or substitution" in the present disclosure refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) , so that these changes can often be made without changing the biological activity of a given protein. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-replaceable region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., p.224 (4th edition)). In addition, substitutions of structurally or functionally similar amino acids are unlikely to impair biological activity.

The term "mutant sequence", as used in the present invention, means that the nucleotide sequence and amino acid sequence of the present invention undergoes a substitution, insertion, or deletion, such that the resulting nucleotide sequence and amino acid sequence share a varying percentage of identity with nucleotide sequence and amino acid sequence of the present invention.

As used herein, the term "identity" indicates the degree of similarity between two nucleic acids or two amino acid sequences. The sequence identity in the present invention is at least 85%, 90% or 95%, preferably at least 95%. Representative examples include, but are not limited to 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, 100%. Sequence comparison and determination of percent identity between two sequences can be performed using the default settings of the BLASTN/BLASTP algorithm available from the National Center for Biotechnology Institute website.

The phrase "anti-PD-L1 antibody or antigen-binding protein thereof" of the present invention could include any of the anti-PD-L1 antibodies or antigen-binding fragments thereof described in the art. An anti-PD-L1 antibody may be an anti-PD-L1 antibody that is commercially available or has been disclosed in the literature, including but not limited to anti-PD-L1 antibody BMS-936559, MPDL3280A, MEDI4736, MSB0010718C (see US 2014341917, US 20130034559, US 8779108), etc. The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. An antibody fragment includes a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment having antigen-binding activity, and an Fv fragment and a scFv fragment that binds to an antigen.

As an exemplary method for producing an anti-PD-L1 antibody of the present invention, see PCT/CN2016/104320, an anti-PD-L1 antibody contains heavy chain variable region CDRs as described below:

In an alternative embodiment, X ₁ is selected from H or G; and X ₂ is selected from G or F.

In another embodiment, an exemplary anti-PD-L1 antibody of the present invention further comprises light chain variable region CDR sequences as described below:

In another embodiment, the above CDR regions are humanized by CDR grafting and the humanized light chain template FRs are IGKV7-3*01 and hjk2.1, the humanized heavy chain template FRs are IGHV1-46*01 and hjh6.1, and the sequences humanized variable regions look like this:

Humanized Heavy Chain Variable Region:

Humanized Light Chain Variable Region:

Note. The order is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the fragment in italics represents the FR sequence, and the underlined fragment represents the CDR sequence.

In another embodiment, a reverse mutation scheme was implemented on a humanized antibody of the present invention, see table below:

A new humanized antibody can be generated by a different combination of heavy chain and light chain mutations shown in the table above.

In another aspect of the invention, an embodiment for constructing a humanized clone is provided, as follows:

Primers were designed and the VH/VK gene fragments of each humanized antibody were constructed by PCR and then inserted into a pHr expression vector (with signal peptide and constant region gene fragment (CH1-Fc/CL)) to perform homologous recombination to construct a full length antibody expression vector: VH-CH1-Fc-pHr/VL-CL-pHr.

1. Primer design

Online DNAWorks software (v3.2.2)

(http: //helixweb.nih.gov/dnaworks/) was used to design a set of primers for the synthesis of VH/VK containing gene fragments required for recombination: 5'-30 p. (bp) signal peptide plus VH/VK plus 30 bp CH1/CL-3'.

2. Fragment splicing

According to the operating instructions for DNA polymerase Primer STAR GXL from TaKaRa, using the primers designed above, VH/VK containing gene fragments required for recombination were obtained by two-step PCR amplification.

3. Construction of pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)) and enzymatic digestion

The pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)) was designed and constructed by using a specific restriction endonuclease such as BsmBI that recognizes the distinguishing feature between sequence and restriction site. BsmBI cleaved the vector and then the cleaved fragments were isolated by gel and stored for use.

4. Recombinant construction of the VH-CH1-Fc-pHr/VK-CL-pHr expression vector

VH/VK containing gene fragments required for recombination and pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)), which was digested with BsmBI, were added to competent DH5H cells in a ratio of 3:1, incubated at 0°C on ice for 30 min, subjected to heat shock at 42°C for 90 s, combined with 5 volumes of LB medium, incubated at 37°C for 45 min, placed on a plate with LB-Amp and cultured at 37°C during the night. A single clone was selected for sequencing and a test clone was obtained.

5. The plasmid was constructed in accordance with the scheme of this example, then the protein was purified, and the affinity of the obtained protein was measured by the detection described in Example SPR (surface plasmon resonance).

6. Finally, the affinity of the humanized reverse mutant or hybridoma antibodies for human PD-L1-His was measured by BIACORE, the resulting humanized reverse mutation sites and combination of sequences by screening are as follows:

heavy chain variable region:

where CDR2 is the sequence in which X ₁ SEQ ID NO: 7 is G and X ₂ is F.

light chain variable region:

Note. The order is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the fragment in italics represents the FR sequence, and the underlined fragment represents the CDR sequence.

In another aspect, the present invention provides an embodiment for constructing and expressing an anti-PD-L1 antibody of the human IgG4 type, and further provides an anti-PD-L1 antibody used to construct a fusion protein. An anti-PD-L1 antibody can also be used as a control molecule in the assay examples of the present invention.

Since PD-L1 is also expressed in activated T cells, therefore, the use of wild-type IgG1 constant regions can cause Fc-mediated effects such as ADCC and CDC, which could lead to a decrease in activated T cells. According to the present invention, mutated IgG4 is selected to produce antibodies without ADCC and CDC. The clone resulting from affinity maturation was converted to the IgG4 type and the IgG4 core hinge contained the S228P mutation, and the F234A and L235A mutations were additionally introduced (mAb 4:3, 310-318; May/June 2012).

At the same time, in order to avoid that a break occurs at the C-terminus of the heavy chain of the antibody when the linker peptide (which is used to bind the extracellular domain of TGF-βRII) is introduced, the last amino acid K of the heavy chain of the anti-PD-L1 antibody was further mutated. to obtain A, thus increasing the stability of the fusion protein. The sequence of the anti-PD-L1 antibody of the present invention used to construct the fusion protein is as follows:

Anti-PD-L1 heavy chain: IgG4(AA) (S228P)

Anti-PD-L1 antibody light chain:

Note. The underlined fragment represents the sequence of the variable region of the heavy or light chain of the antibody or its coding nucleotide sequence. The rest of the fragment is the sequence of the constant region of the antibody and its coding nucleotide sequence.

As used herein, the fusion protein described in the present invention is a protein product resulting from the co-expression of two genes through recombinant DNA technology. Methods for making and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in the Cold Spring Harbor Antibody Experimental Technologies Manual, chapters 5-8 and 15. For example, mice can be immunized with human PD-L1 or fragments thereof, and the resulting antibodies can then be annealed, purified and sequenced for amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be obtained by conventional methods. The antibody or antigen-binding fragments of the present invention are constructed by grafting CDRs derived from a non-human antibody into one or more human FRs. By alignment against the IMGT human germline antibody variable region database using the MOE software, human germline framework sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified using known methods. For example, cDNA sequences encoding the heavy chain and light chain can be cloned and constructed into a GS expression vector. The engineered immunoglobulin expression vector can then be stably transfected with CHO cells (Chinese hamster ovary cell). As a more preferred method known in the art, a mammalian expression system will result in glycosylation of the antibody, typically at highly conserved N-terminal sites in the Fc region. Stable clones can be obtained by expression of an antibody that specifically binds to human PD-L1. Positive clones can be expanded in serum-free culture media to generate antibodies in bioreactors.

The culture medium into which the antibody has been secreted can be purified by conventional techniques. For example, this medium can be loaded onto a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer. The column is washed to remove non-specific binding components. Bound antibody is eluted with a pH gradient and antibody fragments are detected by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and then harvested. The antibody can be filtered and concentrated using conventional techniques. Soluble aggregate and multimers can be effectively removed by conventional techniques, including gel filtration or ion exchange. The product may be immediately frozen, for example at -70°C, or may be lyophilized.

The "immunomodulatory molecule" of the present invention can be used to reduce the immune tolerance of cancer cells. The present invention uses a truncated form of the extracellular domain of TGF-βRII as an immunomodulatory molecule in a fusion protein. "TGF-β receptor II (TGF-βRII)" binds to TGF-β1 and 3 ligands with high affinity. The TGF-βRII/TGF-β complex enhances TGF-βRI to form a signal transduction complex (Won et al, Cancer Res. 1999; 59: 1273-7). The TGF-βRII extracellular domain is a 136 amino acid residue peptide from the N-terminus of TGF-βRII, an illustrative example of which is shown in SEQ ID NO: 13. bind to TGF-β1 and 3 also belong to the extracellular domain of TGF-βRII of the invention. The present invention has found that the structure and function of the N-terminal contiguous truncated form of the TGF-βRII extracellular domain is more stable than the structure and function of the untruncated molecule. A fusion protein containing the N-terminal untruncated form of the extracellular domain of TGF-βRII (polypeptide shown as a.k. 1-136 of SEQ ID NO: 13) is susceptible to cleavage. In particular, a truncated form containing a maximum 26 amino acid deletion at its N-terminus is more stable, preferably a 14-26 amino acid truncation, more preferably a 14-21 amino acid truncation at the N-terminus with a higher level of expression, most preferably a 19 or 21 adjacent amino acids at the N-terminus.

The term "administration" and "treatment", as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to bringing an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition into contact with an animal. , human, subject, cell, tissue, organ, or biological fluid. The terms "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Processing a cell encompasses bringing a reagent into contact with a cell, as well as bringing the reagent into contact with a fluid, where the fluid is in contact with the cell. The terms "administration" and "treatment" also mean in vitro or ex vivo treatments of, for example, cells with a reagent, diagnostic, binding compound, or other cell. The term "treatment", as applied to a human subject, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

The term "treat" means to administer a therapeutic agent, such as a composition containing any of the binding compounds of the present invention, orally or externally to a patient who has one or more symptoms of a disease for which the agent has known therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, causing regression or preventing the development of such symptom(s) to any clinically measurable extent. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary according to factors such as the course of the disease, the age and weight of the patient, and the ability of the drug to elicit the desired response in patient. Whether a symptom of a disease has been alleviated can be assessed by any clinical measurement commonly used by physicians or other qualified healthcare professionals to assess the severity or progression status of a given symptom. While an embodiment of the present invention (e.g., method of treatment or article of manufacture) may not be effective in alleviating the target symptom(s) of the disease in each patient, it should alleviate the target symptom(s) of the disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann and Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test .

"Conservative modifications" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that these changes can often be made without changing the biological activity of the protein. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-replaceable region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., p.224 (4.sup.th Ed.)) In addition, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity.

The term "effective amount" encompasses an amount sufficient to alleviate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing protocol that avoids significant side effects or toxic effects.

The term "exogenous" refers to substances that are formed outside the body, cell, or human body, depending on the context. The term "endogenous" refers to substances that are formed within a cell, organism, or human body, depending on the context.

The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. Molecules are considered homologous at one position when that position in both sequences to be compared is occupied by the same base or amino acid monomeric subunit, for example, when the position in each of two DNA molecules is occupied by adenine. The percent homology of two sequences is a function of the number of matching or homologous positions that the two sequences share, divided by the number of all positions compared, and then multiplied by 100. For example, in a possible alignment, if 6 out of 10 positions in two sequences match or are homologous, then these two sequences are 60% homologous. In general, comparison is made by aligning two sequences to obtain the maximum percentage of homology.

The term "immune checkpoint molecules" includes a stimulatory immune checkpoint molecule and an inhibitory immune checkpoint molecule, and exemplary molecules include CD27, CD28, CD40, CD40L, CD122, OX40, OX40L, GITR, ICOS, A2AR, B7-H3, B7- H4, BTLA, CTLA-4, IDO, KIR (from the English Killer-cell Immunoglobulin-like Receptor - a receptor containing immunoglobulin-like domains), LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, etc.

As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include the primary test cells and cultures derived from them, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in DNA content due to targeted or random mutations. Progeny with mutations that have the same function or biological activity as the function and biological activity of the originally transformed cells are obtained by screening and will be included in the invention. In the case where distinct designations are intended, this will be clear from the context.

As used herein, the phrase "polymerase chain reaction" or "PCR" refers to a process or procedure in which a small amount of specific nucleic acid, RNA and/or DNA fragments are amplified as described, for example, in US Pat. No. 4,683,195. Typically, there is a need for sequence information at the ends or outside of the region of interest so that oligonucleotide primers can be designed; the sequence of these primers will be identical or similar to the reverse strand of the template to be amplified. The 5'-terminal nucleotides of these two primers coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cell RNA, bacteriophage or plasmid sequences, and so on. Generally see Mullis et al. (1987) Cold Spring Harbor Symp. Ouant Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used in the present invention is considered to be one, but not the only, example of a method for amplifying a nucleic acid sample to be analyzed based on a polymerase reaction. The method involves using known nucleic acids as primers and a nucleic acid polymerase to amplify or produce a particular nucleic acid fragment.

The term "possible" or "possible" means that the event or situation described hereinafter may occur (but does not necessarily occur), and this description includes instances in which the event or situation occurs or does not occur. For example, the phrase "possibly contains 1-3 antibody heavy chain variable region(s)" means that an antibody heavy chain variable region of a particular sequence may be present, but need not be present.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds according to the present invention or their physiologically/pharmaceutically acceptable salt or prodrug and other chemical components, and these chemical components are such as physiologically/pharmaceutically acceptable(s) carrier(s) ) and excipient(s) substance(s). The purpose of the pharmaceutical composition is to promote administration to the body, to facilitate the absorption of the active substance and thus to produce a biological effect.

Examples and Sample Assays

Hereinafter, the present invention is further described with reference to examples. However, the scope of the present invention is not limited to them.

In examples of the present invention that do not describe specific conditions, experiments are usually carried out under traditional conditions or under conditions suggested by the manufacturers of the material or product. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. When the source of the reagents is not specifically indicated, the reagents are commercially available conventional reagents.

Examples

Example 1 Cloning and expression of PD-L1 fusion protein/TGF-β trap

The extracellular domain of TGF-βRII (full length or truncated form of SEQ ID NO: 13) was used as a fragment for an immunomodulatory molecule in a fusion protein, and an anti-PD-L1 antibody was used as a targeting fragment of the fusion protein to form an anti-PD-L1 antibody/ fusion protein. extracellular domain of TGF-βRII (PD-L1/TGF-β decoy). Studies have found that the truncated form of the extracellular domain of TGF-βRII is relatively stable, in particular the truncated form includes a deletion of less than 26 amino acids at its N-terminus, preferably a deletion of 14-26 amino acids, more preferably a deletion of 14-21 adjacent amino acids, which shows higher expression level and stable structure; more preferably a deletion of 14, 19 or 21 adjacent amino acids. The sequences of non-limiting examples of the extracellular domain of TGF-βRII and its truncated form according to the invention are as follows:

TGF-βRII extracellular domain sequence: ECD (1-136)

The sequence of the truncated extracellular domain of TGF-βRII, which includes a deletion of 19 adjacent amino acids at the N-terminus: ECD (20-136)

The sequence of the truncated extracellular domain of TGF-βRII, which includes a deletion of 21 adjacent amino acids at the N-terminus: ECD (22-136):

The sequence of the truncated extracellular domain of TGF-βRII, which includes a deletion of 14 adjacent amino acids at the N-terminus: ECD (15-136):

The C-terminal amino acid of the heavy chain of the anti-PD-L1 antibody of the present invention was ligated with a (G ₄ S) _x G linker to the extracellular domain of TGF-βRII with varying lengths by a homologous recombination technique, and expressed by the 293 expression system together with the light chain in a conventional manner. , and the resulting fusion proteins are shown in Table 2:

The nucleotide sequence encoding the anti-PD-L1 antibody, the nucleotide sequence encoding the extracellular domain of TGF-βRII, and the nucleotide sequence of the linker protein fragment ((G ₄ S) _x G) were obtained by conventional techniques in the art. The C-terminal nucleotide of the anti-PD-L1 antibody was ligated with a linker protein to the N-terminal nucleotide of the TGF-βRII extracellular domain of various lengths by homologous recombination technique, and then cloned into the Phr-BsmbI vector. The recombinant PD-L1 fusion protein/TGF-β decoy was expressed in 293 cells and purified as described in Example 2. The purified protein can be used in the experiments of the following examples.

Example 2 Purification of PD-L1 Fusion Protein/TGF-β Trap

The cell culture medium was centrifuged at high speed, and the supernatant was collected, and the first purification step was carried out by affinity chromatography. The chromatographic medium is Protein A or a derived excipient that reacts with Fc, such as Mabselect GE. The balancing buffer was 1×PBS (from the English. Phosphate buffered saline - phosphate-buffered saline solution) (137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na ₂ HPO ₄ , 2 mmol/l KH ₂ PO ₄ , pH 7.4). After 5 column volumes were equilibrated, the cell supernatant was loaded for binding and the flow rate was controlled to allow the sample to remain on the column for more than 1 min or for 1 min. After sample loading, the column was washed with 1×PBS (pH 7.4) until the A280 reading decreased to baseline. Then, the column was washed with 0.1 M containing glycine (pH 3.0) elution buffer, and the eluting peak was collected in accordance with the absorbance peak in the ultraviolet A280 region, and the collected eluting sample was neutralized with 1 M Tris (pH 8.5).

The neutralized eluted sample was concentrated by ultrafiltration and then subjected to size exclusion chromatography, the buffer was 1×PBS and the column was XK26/60 Superdex 200 (GE). The flow rate was controlled at 4 ml/min, the loading volume was less than 5 ml, and the target protein peak was pooled according to A280 ultraviolet absorbance. The purity of the assembled protein was greater than 95% as identified by size exclusion HPLC (High Performance Liquid Chromatography) and was verified by LC-MS (liquid chromatography/mass spectrometry). The tested sample was divided into aliquots for use. Received fusion protein PD-L1/trap TGF-β.

The efficacy and benefits of the present invention were tested by biochemical analytical methods as follows.

Assessment of biological activity in vitro

Assay Example 1 In Vitro Detection of PD-L1 Fusion Protein/TGF-β Trap Binding to TGF-β1 by ELISA

The detection method is described below:

a. 96-well plates were coated with 100 μl/well of human TGF-β1 (8915LC, CST) at 1 μg/ml at 4° C. overnight.

b. Washing, 3 times, 250 μl 1×PBST, 250 μl 5% milk in PBS was added to block at 37° C. for 2 hours.

in. Wash, 3 times, 250 μl 1xPBST, gradient dilution of PD-L1 fusion protein/TGF-β trap, TGF-p trap and positive control were added and incubated for 1 hour at 37°C.

d. Wash, 3 times, 250 µl 1×PBST.

e. 100 μl of anti-human Fc antibody conjugated with HRP (from the English horseradish peroxidase - horseradish peroxidase) (1:4000) was added to each well and incubated for 40 minutes at 37°C.

e. 100 μl TMB (from the English. tetramethylbenzidine - tetramethylbenzidine) was added to each well, incubated for 10 minutes at room temperature, and the reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ .

and. Absorbance at 450 nm was measured on a microplate reader and the data was analyzed with Graphpad Prism5.

The results of in vitro binding of the fusion protein to human TGF-β1 are shown in FIG. 2 and 3. ELISA showed that fusion protein 1 in Table 2 did not retain binding activity to human TGF-β1. Mass spectrometric analysis showed that fusion protein 1 (i.e., the non-truncated form of the TGF-βRII extracellular domain (1-136)) was unstable, and it was easily degraded in the TGF-βRII heavy chain, and the positive control had the same defect. Fusion proteins containing an N-terminal truncated form of the TGFβRII extracellular domain, such as fusion proteins 7, 9, 10, 12-15, are specific for binding to human TGF-β1.

Assay Example 2 In Vitro Detection of PD-L1 Fusion Protein/TGF-β Trap Binding to PD-L1 by ELISA

Antigen used for detection: PD-L1-His

The detection method is described below:

a. 96-well plates were coated with 100 μl/well of human PD-L1-His (SEQ ID NO: 17) at a concentration of 5 μg/ml at 4° C. overnight.

b. Washing, 3 times, 250 μl 1×PBST, 250 μl 5% milk in PBS was added to block at 37°C for 2 hours.

in. Wash, 3 times, 250 μl 1×PBST, gradient dilution of PD-L1 fusion protein/TGF-β trap, anti-PD-L1 antibody as a positive control was added and incubated for 1 hour at 37°C.

d. Wash, 3 times, 250 µl 1×PBST.

e. 100 μl of anti-human Fc antibody conjugated with HRP (1:4000) was added to each well and incubated for 40 minutes at 37°C.

e. 100 μl of TMB was added to each well, incubated for 10 minutes at room temperature, and the reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ .

and. Absorbance at 450 nm was measured on a microplate reader and the data was analyzed with Graphpad Prism5.

The results of in vitro binding of the fusion protein of the present invention to human PD-L1 are shown in FIG. ELISA showed that all fusion proteins retained binding activity to human PD-L1.

Assay Example 3 In Vitro Detection of PD-1/PD-L1 Blocking

1. Purpose of the analysis

To investigate the blocking effect of the PD-L1 fusion protein/TGF-β decoy on the PD-1/PD-L1 signaling pathway, an antibody blocking experiment on cells was performed on cells bearing human PD-1 and PD-L1 receptor molecules, which were engineered by Promaga, respectively.

2. Analyzed samples

Антитело против PD-L1: SEQ ID NO: 11, SEQ ID NO: 12;

Anti-PD-L1 antibody: SEQ ID NO: 11, SEQ ID NO: 12;

Контроль 1 (20T-Fc): ECD(20-136)-Fc, слитый белок, содержащий фрагмент усеченного внеклеточного домена TGF-βRII ECD (20-136) и Fc

Control 1 (20T-Fc): ECD(20-136)-Fc, fusion protein containing a truncated extracellular domain fragment of TGF-βRII ECD(20-136) and Fc

The sequence looks like this:

Контроль 2 (22T-Fc): ECD(22-136)-Fc, слитый белок, содержащий фрагмент усеченного внеклеточного домена TGF-βRII ECD (22-136) и Fc

Control 2 (22T-Fc): ECD(22-136)-Fc, fusion protein containing a truncated extracellular domain fragment of TGF-βRII ECD(22-136) and Fc

The sequence looks like this:

Слитый белок 9, слитый белок 15;

Fusion protein 9, fusion protein 15;

Человеческий IgG: холостой контроль, человеческий иммуноглобулин, полученный из смешанной нормальной сыворотки человека, посредством очистки, используя традиционный метод аффинной хроматографии, как например Белок А;

Human IgG: blank control, human immunoglobulin derived from mixed normal human serum by purification using a conventional affinity chromatography method such as Protein A;

Положительный контроль (М7824, полученный посредством патента, на который делается ссылка, WO 2015118175): слитый белок антитело против PD-L1/внеклеточный домен TGF-βRII;

Positive control (M7824, obtained through the referenced patent, WO 2015118175): anti-PD-L1 antibody/TGF-βRII extracellular domain fusion protein;

Amino acid sequence of the light chain of an anti-PD-L1 antibody:

Amino acid sequence of the heavy chain of an antibody against PD-L1/extracellular domain of TGF-βRII (1-136):

3. Analysis process

CHO/PD-L1 cells (CS187108, Promega) were digested and resuspended in complete F-12 Nutrient Mixture (Ham). Cell density was adjusted to 4×10 ⁵ /ml using complete medium according to cell count results. The cell suspension was transferred to a loading tank, added to a 96-well plate at 100 μl/well using a multichannel pipette, and incubated at 37° C., in a 5% CO ₂ incubator for 20-24 h; The next day, a Jurkat/PD-1 cell suspension (CS187102, Promega) was prepared and the cells were resuspended according to the results of cell count using assay medium, and the cell density was adjusted to 1.25×10 ⁶ /ml; Cell culture plates containing CHO/PD-L1 cells were removed from the incubator, 95 µl of culture solution was withdrawn per well using a multichannel pipette, and the gradient diluted fusion protein, anti-PD-L1 antibody, and positive control (M7824), respectively, were added in an amount 40 µl/well. Then, the Jurkat/PD-1 cell suspension was transferred to the loading tank, added to the cell culture plate at 40 µl/well, and incubated at 37°C, 5% CO ₂ for 5-6 hours. During the protein incubation, the reagent Bio-Glo™ was taken out and left at room temperature. Cell culture plates were removed and placed at room temperature for 5–10 min. Then 40 µl of Bio-Glo™ reagent was added to each well, incubated in a laminar flow hood for 5-10 min, and the chemiluminescence signal value was read using a multifunctional microplate reader.

4. Results

As shown in FIG. 5, as well as a positive control molecule, the fusion protein 9 of the present invention could effectively block the binding of PD-1 expressing Jurkat cells to CHO/PD-L1 cells and act in a dose-dependent manner along with drug concentration. Fusion protein 15 has the same blocking ability as fusion protein 9.

Assay Example 4: In Vitro Affinity and Binding Kinetics Detection with Biacore

The affinity of the test molecule for human or mouse TGF-β1 or human PD-L1 protein was determined by Biacore T200 (GE). The experimental method is described below:

A certain amount of PD-LI/TGF-β fusion protein/TGF-β trap was captured by the protein A chip, and then human or mouse TGF-β1 (8915LC, CST) or human PD-L1 (Sino Biological) was passed through the surface of this chip. The reaction signal was detected in real time using Biacore to obtain association and dissociation curves. The biochip was then washed and regenerated with glycine hydrochloride (pH 1.5, GE). The buffer solution used in the experiment was HBS-EP (GE) buffer. Experimental data were fitted to a (1:1) Langmuir model using evaluation version 4.1 of the BIA(GE) software, and affinity values were obtained as shown in Table 3.

The binding activity of the fusion proteins is shown in Table 3. The results indicate that fusion proteins 9 and 15 of the present invention have extremely high affinity for human, murine TGF-β1 and human PD-L1.

Assay Example 5 SMAD3 Reporter Gene Inhibition Assay

1. Purpose of the analysis

In this experiment, a Smad3 binding element (SBE) with a luciferase reporter gene was expressed in HepG2 cells to study the inhibitory effect of the PD-L1 fusion protein/TGF-β trap on TGF-β1-induced activation of Smad3, and the activity of the fusion PD-L1 protein/TGF-β trap in virto was evaluated according to the IC50 value.

2. Sample for analysis: fusion protein 9, positive control (M7824)

3. Method of analysis

HepG2 cells were cultured in complete MEM medium (minimal essential medium) (GE, SH30243.01) containing 10% FBS (Fetal Bovine Serum) and subcultured every 3 days. On the first day of the experiment, 25,000 cells per well were inoculated into 96-well plates (Corning, 3903) and cultured at 37° C., 5% CO ₂ for 24 hours. The following day, the medium in the cell culture plates was discarded and 100 ng of 3TP-Lux plasmid per well was transfected. Cells were additionally cultured at 37°C, 5% CO ₂ for 24 hours. Six hours before adding the test sample, the complete medium in the 96-well plate was discarded and 80 μl of incomplete medium (MEM + 0.5% FBS) was added to each well. After 6 hours, 10 µl of human TGF-β1 solution (R&D, 240-B-010) prepared in incomplete medium (final concentration 2 ng/ml) and 10 µl of test sample added (final concentration is 500, 50, 5, 0 .5, 0.05, 0.005, 0.0005, and 0 nM), human TGF-β1 solvent was used as a control, and the cells were cultured at 37°C, 5% CO ₂ for another 18 hours. Then 100 μl of the obtained substrate Luciferases of the ONE-Glo™ Luciferase Assay System (promega, E6110) were added to each well and incubated at room temperature for 10 minutes in the dark, and then the luminescent signal value was read using a Victor3 miniplate reader (Perkin Elmer). The IC50 value of the analyzed sample was obtained by calculation using Graphpad Prism 5.0 data processing software.

On FIG. 6 shows how fusion protein 9 inhibits TGFβ-inducible pSMAD3 activity in a dose-dependent manner and has comparable potency and IC50 to positive control M7824. The results of the analysis of the antibody against PD-L1 showed that it has no inhibitory effect (IC50 greater than 500 nm).

Assay Example 6 In Vitro Detection of PBMC IFNγ Secretion by Tuberculin (TB) Stimulation

1. Purpose of the analysis

To study T-lymphocyte activation by PD-L1 fusion protein/TGF-β decoy, human peripheral blood mononuclear cells (PBMCs) were harvested and purified, and IFNγ secretion was detected after tuberculin (TB) stimulation for 5 days.

2. Sample for analysis

Человеческий IgG;

антитело против PD-L1;

Слитый белок 9

Контроль 1 (20T-Fc): ECD(20-136)-Fc;

антитело против PD-L1 + контроль 1 (20Т-Fc).

Human IgG;

anti-PD-L1 antibody;

Fusion protein 9

Control 1 (20T-Fc): ECD(20-136)-Fc;

anti-PD-L1 antibody + control 1 (20T-Fc).

3. Method of analysis

To the above samples were added 15 ml of purified fresh PBMC, approximately 3×10 ⁷ cells, and 20 μl of tuberculin and cultured in an incubator for 5 days at 37°C, 5% CO ₂ . On day 6, cultured cells were harvested and centrifuged, washed once with PBS and resuspended in fresh medium at a density adjusted to 1×10 ⁶ cells/ml, 90 µl of resuspended cells were added to a 96-well plate. 10 µl/well of different antibody concentrations were separately added to the respective wells of the above 96-well cell culture plate, 10 μl of PBS were added to the control and blank group, respectively. Then, the plate with cell culture was incubated in an incubator for three days at 37°C, 5% CO ₂ . The cell culture plate was taken out and the supernatant was taken from each well after centrifugation (4000 rpm, 10 min). After 10-fold dilution, IFN-γ secretion was detected by ELISA (human IFN-γ detection kit, Xinbosheng, EHC 102g.96) referring to the reagent instructions for specific operations. As shown in the figure, the PD-L1 fusion protein/TGF-β decoy samples were able to enhance the secretion of the cytokine IFN-γ by activated T lymphocytes, and had a dose effect of drug concentration.

4. Results

As shown in FIG. 7 and Table 4, fusion protein 9 could enhance activated T-lymphocyte secretion of cytokine IFN-γ in a dose-dependent manner, and had a stronger activation effect than that of anti-PD-L1 and 20T-FC antibodies.

Pharmacokinetic evaluation

Analysis example 7

Three female SD (Sprague Dawley) rats were purchased from Jiesijie Experimental Animal Co., Ltd. and kept under conditions of a 12/12 hour light/dark cycle (temperature is 24 plus/minus 3°C, relative humidity is 50-60%), the rats had free access to water and food. On the day of the experiment, SD rats were injected with the fusion protein via the tail vein at a dose of 6 mg/kg and an injection volume of 5 ml/kg.

Blood sampling was carried out at the time point: 15 min, 7 h (on the first day), 24 h ( ^2nd day), ^3rd day, ^4th day, ^6th day, ^8th day, ^10th day and ^15th day after administration, 200 μl of blood (equivalent to 100 μl of serum) was collected from a rat fundus vein. The blood sample was placed at room temperature for 30 minutes to ensure agglutination and then centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was taken and immediately placed in storage at -80°C. Serum fusion protein concentration was measured by ELISA.

The measurement method is described below:

a. 96-well plates were coated with 100 μl/well of human PD-L1-His at a concentration of 2 μg/ml overnight at 4°C.

b. Washing, 4 times, 250 μl 1×PBST, 250 μl 5% milk in PBS was added to block at 37°C for 3 hours.

in. Washing, 4 times, 250 μl 1×PBST, 100 μl gradient diluted serum sample was added and incubated at 37° C. for 1 hour with fusion protein 9 as a positive control.

d. Wash, 5 times, 250 µl 1×PBST.

d. Added 100 μl/well of biotinylated anti-human TGF-pRII antibody (R&D) and incubated for 1 hour at 37°C.

e. Wash, 5 times, 250 µl 1×PBST.

and. Added 100 μl/well TMB, incubated for 10 minutes at room temperature and the reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ .

h. Absorbance at 450 nm was measured on a microplate reader and the data was analyzed with Graphpad Prism5.

The results of PK analysis showed that the half-life of the fusion protein 9 of the present invention in rats was approximately 236 hours (9.8 days), see Table 5.

Assessment of biological activity in vivo

Assay Example 8: Effect of PD-L1 Fusion Protein/TGF-β Trap on MDA-MB-231 Human Breast Tumor Xenograft Subcutaneously Transplanted in Mouse

The mouse strain used in this experiment was a female NOD/SCID mouse (Cavens). Human peripheral blood mononuclear cells used in the experiment were isolated from freshly collected blood, and the isolation method was as follows: venous blood subjected to anti-clotting heparin treatment was mixed with PBS containing 2% FBS in the same volume, and after mixing 25 ml The diluted blood was slowly added to a centrifuge tube containing 15 ml of lymphocyte separation solution and centrifuged at 1200 g for 10 minutes at room temperature. The layer of lymphocytes was taken with a pipette into another centrifuge tube, the cells were washed with PBS and centrifuged at 300 g for 8 minutes at room temperature. After one repetition, cells were resuspended in RPMI-1640 medium containing 10% FBS and cells were added to a 6-well plate precoated with anti-CD3 antibody (OCT3, 40 ng/ml) at 2×10 ⁶ cells/well (2 ml), and then placed in an incubator at 37°C for 4 days.

Sample being analyzed

холостой контроль: PBS;

слитый белок 9-4,8 мг/кг;

слитый белок 9-24 мг/кг;

антитело против PD-L1-4 мг/кг;

антитело против PD-L1-20 мг/кг;

антитело против PD-L1 -4 мг/кг + контроль 1 (20T-Fc)-2,14 мг/кг;

контроль 1 (20Т-Fc)-2,14 мг/кг.

blank control: PBS;

fusion protein 9-4.8 mg/kg;

fusion protein 9-24 mg/kg;

antibody against PD-L1-4 mg/kg;

antibody against PD-L1-20 mg/kg;

anti-PD-L1 antibody -4 mg/kg + control 1 (20T-Fc) -2.14 mg/kg;

control 1 (20T-Fc) - 2.14 mg/kg.

MDA-MB-231 cells were resuspended in serum-free RPMI-1640 medium and mixed with an equal volume of Matrigel, 100 μg (2.3×10 ⁶ ) were inoculated subcutaneously into the right flank of NOD/SCID mice. 11 Days later, animals bearing too large or insufficiently large tumor were excluded, mice were randomly assigned to groups of 9 animals in each group. 5x10 ⁵ Stimulated PBMCs (60 μl) were injected into the tumor tissues and the remaining PBMCs were further cultured without stimulation. One week later, 5x10 ⁶ PBMCs (100 μl) were intraperitoneally injected into tumor-bearing mice as the first injection round. During the experimental period, for 2 and a half rounds, a total of 5 injections of PBMC were provided. On the day of the first intratumoral injection, an intraperitoneal injection was performed, three times a week, for a total of 14 injections. The administration schedule is shown in Table 6. Tumor volume and weight were measured twice a week. The results of the experiment are shown in Table 7.

The results are shown in FIG. 8, the fusion protein containing antibody 9 (4.8, 24 mg/kg) can significantly inhibit the growth of murine subcutaneous human breast tumor xenograft MDA-MB-231. There was a dose-dependent relationship between high and low doses and was superior to the reference drug anti-PD-L1 antibody (4.20 mg/kg), TGF-pRII 20T-FC control molecule (2.14 mg/kg) and the combined group (anti-PD-L1 antibody-4 mg/kg + 20 T-FC - 2.14 mg/kg) at the equivalent molar dose, respectively. Each dose of fusion protein 9 maintained an ideal ^antitumor effect from day 14 post-administration; when compared to anti-PD-L1-20 mg/kg, high dose fusion protein 9 had a clear advantage (p less than 0.05). On the ^25th day after administration, the antitumor effect of each antibody reached the optimal level. The degrees of inhibition achieved by fusion protein group 9 in combination with anti-PDL-1 antibody (both high and low dose) were 37.24%, 52.38%, 30.24%, 28.01% and 31 .38%, respectively. At ³² days post-administration, the antitumor effect of fusion protein 9 was still very significant. %, TGI (from the English tumor growth inhibition - inhibition of tumor growth) of the low and high dose groups was 36.68% and 50.76%, respectively, and the tumor volume was statistically different compared to the control group (p less than 0.05 ).

Assay Example 9 Physical Stability of PD-L1 Fusion Protein/TGF-β Trap

This assay example was used to determine the stability of fusion protein 9 and fusion protein 15.

DSC (differential scanning colorimetry) was used to determine the thermal stability of different antibodies, and the stability in different buffer systems was compared. Buffer systems include 10 mM acetate/135 mM NaCl (pH 5.5) and 10 mM acetate/9% trehalose (pH 5.5).

The sample was dissolved in appropriate buffers and the concentration was controlled at about 50 mg/ml. Detection was performed using a capillary differential scanning calorimeter (DSC) MicroCal* VP (Malvern). Prior to analysis, each sample and blank buffer was degassed using a vacuum degassing device for 1–2 min. 400 μl of sample or blank buffer was added to each well of the plate (loading amount was 300 μl). Finally, two pairs of well plates were added with 14% Decon 90 and ddH ₂ O, respectively, and were ready for washing. The sample was loaded into the plate, and then the plate was sealed with a plastic cap. Scanning started at 25°C and ended at 100°C, and the scanning speed was 60°C/h. The results are shown in Table 8, indicating that both fusion protein 9 and fusion protein 15 show good thermal stability in these two test systems.

Periodic stability at a certain concentration was examined by monitoring purity by size-exclusion HPLC, illustrative conditions, eg sample concentration was controlled at about 50 mg/mL. The stability of different antibodies in 10 mM acetate/135 mM NaCl (pH 5.5) was compared under conditions such as 5 cycles of freeze and thaw at -80° C. and stored at 40° C. for one month. For detection, a HPLC column with protein Xbridge BEN SEC 200A (Waters) was used. The result is shown in Table 9 below, these two fusion proteins showed good stability.

Analysis Example 10 Chemical Stability of the Fusion Protein

Deamidation is a common chemical modification that will affect the stability of an antibody at a later stage. In particular, overdeamidation of amino acids in CDR regions will be avoided or reduction of mutations in these regions is preferred. 1600 μg of the antibody to be tested was dissolved in 200 μl of 10 mM acetate/135 mM NaCl (pH5.5) and placed in an incubator at 40°C. Samples were taken on days 0, 14 and 28 for analysis based on enzymatic hydrolysis. 100 μg of each sample taken at different time points was dissolved in 100 μl of 0.2 M His-HCl, 8 M Gua-HCl solution, pH 6.0; 3 μl of 0.1 g/ml DTT was added, and then the sample was incubated in a 50° C. water bath for 1 hour. The sample was then ultrafiltered twice with 0.02 M His-HCl (pH 6.0) and digested overnight at 37° C. in a water bath by adding 3 μl of 0.25 mg/ml trypsin. Modification - deamidation was investigated using an Agilent 6530 time-of-flight liquid chromatography-mass spectrometer, and the results are shown below in Table 10.

The results of mass spectrometry showed that these two fusion proteins do not have obvious modification-deamidation sites, indicating that these fusion proteins have good chemical stability.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO.,LTD., SHANGHAI HENGRUI PHARMACEUTICAL CO.,

Ltd.

<120> FUSION PROTEIN CONTAINING TGF-b RECEPTOR AND ITS PHARMACEUTICAL

APPLICATION

<130> 780027CPCT

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 5

<212> PRT

<213> Mus musculus

<400> 1

Ser Tyr Trp Met His

fifteen

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody HCDR2 mutant

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa is His or Gly.

<220>

<221> MISC_FEATURE

<222>(8)..(8)

<223> Xaa is Gly or Phe.

<400> 2

Arg Ile Xaa Pro Asn Ser Gly Xaa Thr Ser Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 3

<211> 10

<212> PRT

<213> Mus musculus

<400> 3

Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 15

<212> PRT

<213> Mus musculus

<400> 4

Arg Ala Ser Glu Ser Val Ser Ile His Gly Thr His Leu Met His

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> Mus musculus

<400> 5

Ala Ala Ser Asn Leu Glu Ser

fifteen

<210> 6

<211> 9

<212> PRT

<213> Mus musculus

<400> 6

Gln Gln Ser Phe Glu Asp Pro Leu Thr

fifteen

<210> 7

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region mutant

against PD-L1

<220>

<221> MISC_FEATURE

<222> (52)..(52)

<223> Xaa is His or Gly.

<220>

<221> MISC_FEATURE

<222> (57)..(57)

<223> Xaa is Gly or Phe.

<400> 7

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Xaa Pro Asn Ser Gly Xaa Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 8

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized anti-PD-L1 antibody light chain variable region

<400> 8

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain variable region of anti-PD-L1 antibody with mature

affinity

<400> 9

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 10

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody light chain variable region with mature

affinity

<400> 10

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 11

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody heavy chain

<400> 11

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 12

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody light chain

<400> 12

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 13

<211> 136

<212> PRT

<213> Homo sapiens

<220>

<223> TGF-beta RII extracellular domain sequence: ECD(1-136)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 14

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> The sequence of the truncated extracellular domain of TGF-beta RII, which

includes deletion of 19 adjacent amino acids at the N-terminus: ECD(20-136)

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ser Asn Pro Asp

115

<210> 15

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> The sequence of the truncated extracellular domain of TGF-beta RII, which

includes deletion of 21 adjacent amino acids at the N-terminus: ECD(22-136)

<400> 15

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

1 5 10 15

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

20 25 30

Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asn Pro Asp

115

<210> 16

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> The sequence of the truncated extracellular domain of TGF-beta RII, which

includes deletion of 14 adjacent amino acids at the N-terminus: ECD(15-136)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

115 120

<210> 17

<211> 236

<212> PRT

<213> Artificial sequence

<220>

<223> Antigen used for detection: PD-L1-His

<400> 17

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

1 5 10 15

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

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

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

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Asn Glu Arg Glu Gln Lys Leu

210 215 220

Ile Ser Glu Glu Asp Leu His His His His His His

225 230 235

<210> 18

<211> 346

<212> PRT

<213> Artificial sequence

<220>

<223> Control 1 (20T-Fc): ECD(20-136)-Fc fusion protein containing fragment

truncated extracellular domain TGF-beta RII ECD (20-136) and Fc

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ser Asn Pro Asp Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

115 120 125

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

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

180 185 190

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

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

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

340 345

<210> 19

<211> 344

<212> PRT

<213> Artificial sequence

<220>

<223> Control 2 (22T-Fc): ECD(22-136)-Fc, fusion protein containing fragment

truncated extracellular domain of TGF-beta RII ECD(22-136) and Fc

<400> 19

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

1 5 10 15

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

20 25 30

Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asn Pro Asp Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

115 120 125

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

130 135 140

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

145 150 155 160

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

165 170 175

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

180 185 190

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

195 200 205

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

210 215 220

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

225 230 235 240

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

245 250 255

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

260 265 270

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

275 280 285

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

290 295 300

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

305 310 315 320

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

325 330 335

Lys Ser Leu Ser Leu Ser Leu Gly

340

<210> 20

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 antibody light chain amino acid sequence

<400> 20

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

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

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

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

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

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 21

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> The amino acid sequence of the heavy chain of an anti-PD-

L1/extracellular domain of TGF-beta RII

<400> 21

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

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

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

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

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Ile Pro His Val Gln Lys Ser Val

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

<---

Claims (19)

1. TGF-β receptor-containing fusion protein for the treatment of PD-L1-mediated tumor, where the fusion protein has the general formula (I): Ab-L- TGF-βRII ECD (I), where TGF-βRII ECD is N -terminal truncated form of the extracellular domain of TGF-βRII, having a deletion of 14, 19 or 21 adjacent amino acids at the N-terminus of the extracellular domain of TGF-βRII, where the extracellular domain of TGF-βRII contains the amino acid sequence of SEQ ID NO: 13; Ab is an anti-PD-L1 antibody or antigen-binding fragment containing: HCDR1 sequence of SEQ ID NO: 1; HCDR2 sequence SEQ ID NO: 2; HCDR3 sequence SEQ ID NO: 3; LCDR1 sequence SEQ ID NO: 4; LCDR2 sequence SEQ ID NO: 5; and LCDR3 sequences SEQ ID NO: 6; L is a linker; and the TGF-βRII ECD is linked to the C-terminus of the anti-PD-L1 antibody heavy chain via a linker. 2. A TGF-β receptor fusion protein according to claim 1, wherein the TGF-βRII ECD comprises the sequence of SEQ ID NO: 14, 15 or 16. 3. The TGF-β receptor fusion protein of claim 2, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a chimeric antibody, a bispecific antibody, and a trispecific antibody. 4. The TGF-β receptor fusion protein of claim 1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is a chimeric antibody or functional fragment thereof, a humanized antibody or functional fragment thereof, or a human antibody or functional fragment thereof. 5. The TGF-β receptor fusion protein of claim 4, wherein the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 7. 6. The TGF-β receptor fusion protein of claim 4, wherein the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 9. 7. The TGF-β receptor fusion protein of claim 4, wherein the humanized antibody further comprises the heavy chain of SEQ ID NO: 11. 8. A TGF-β receptor fusion protein according to claim 4, wherein the humanized antibody comprises the light chain variable region of SEQ ID NO: 8 or SEQ ID NO: 10. 9. The TGF-β receptor fusion protein of claim 4, wherein the humanized antibody comprises a light chain of SEQ ID NO: 12. 10. A TGF-β receptor fusion protein according to claim 1, wherein the linker is (G4S)xG, where x is 3-6. 11. A TGF-β receptor fusion protein according to claim 1, wherein the linker is (G4S)xG, where x is 4-5. 12. Pharmaceutical composition for the treatment of PD-L1-mediated tumors, containing a therapeutically effective amount of a fusion protein containing the TGF-β receptor, according to any one of paragraphs. 1-11, and one or more pharmaceutically acceptable carriers, diluents, or excipients. 13. A DNA molecule encoding a fusion protein containing the TGF-β receptor according to any one of paragraphs. 1-11. 14. An expression vector containing a DNA molecule according to claim 13. 15. A host cell comprising the expression vector of claim 14, wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell, and a mammalian cell. 16. The host cell of claim 15, wherein the host cell is a mammalian cell. 17. The use of a fusion protein containing the TGF-β receptor, according to any one of paragraphs. 1-11, or a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment of a PD-L1-mediated tumor. 18. The use of claim 17 wherein the PD-L1 mediated tumor is a cancer in which PD-L1 is expressed. 19. A method of treating or preventing a PD-L1-mediated tumor, comprising administering to a patient in need thereof a therapeutically effective amount of a fusion protein containing the TGF-β receptor, according to any one of paragraphs. 1-11, or a pharmaceutical composition according to claim 12.

Images