WO2021244587A1 - ANTI-PD-L1/TGF-β FUSION PROTEIN - Google Patents

Abstract

The present invention belongs to the technical field of fusion proteins, and provides an anti-PD-L1/TGF-β fusion protein, which contains an anti-PD-L1 antibody, a peptide linker, and a TGF-β R II extracellular domain, wherein the TGF-β R II extracellular domain is connected to a C terminal of a heavy chain of the anti-PD-L1 antibody by means of the peptide linker. The fusion protein of the present invention has potential for treating a disease related to PD-L1 and TGF-β activity.

Description

An anti-PD-L1/TGF-β fusion protein Technical field

The present invention relates to the technical field of fusion proteins, and more specifically, to an anti-PD-L1/TGF-β fusion protein.

Background technique

Human Programmed Cell Death Receptor-1 (PD-1) is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in the body, but also the main mechanism and reason that causes PD-L1 positive tumor cells to evade immune surveillance. By blocking the negative regulation of the PD-1/PD-L1 signaling pathway by cancer cells and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thereby opening the door to a new tumor treatment method-tumor Immunotherapy.

PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction. The structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al. Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus. Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505) (2003)). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs. .

Transforming growth factor-β (transforming growth factor-β, TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β transmits signals through a heterotetrameric receptor complex, which is composed of two type I and two type II transmembrane serine/threonine kinase receptors. TGF-β is a multifunctional cytokine, which exerts a tumor suppressor or tumor promotion effect in a cell- or background-dependent manner. The tumor suppressor effect of TGF-β signaling stems from its ability to induce the expression of multiple genes. When mutations or epigenetic modifications are introduced during tumor development, cancer cells gradually tolerate the suppressive effect of TGF-β signaling, which ultimately leads to tumor development. developing. Studies have found that blocking the TGF-β signaling pathway can reduce tumor metastasis. The art is eager to develop an anti-tumor fusion protein with good specificity, good curative effect and easy preparation.

Summary of the invention

The purpose of the present invention is to provide a new anti-PD-L1/TGF-β fusion protein that can simultaneously block PD-L1 and TGF-β signal pathways. The purpose of the present invention is also to provide a polynucleotide molecule encoding the fusion protein; to provide an expression vector containing the polynucleotide molecule; to provide a host cell containing the expression vector; to provide a method for preparing the fusion protein; to provide A pharmaceutical composition comprising the fusion protein; an application of the fusion protein or the pharmaceutical composition in the preparation of a medicine for treating cancer; and a method for the fusion protein or the pharmaceutical composition for the treatment of cancer are provided.

In order to achieve the above objective, the present invention provides the following technical solutions:

The first aspect of the present invention provides an anti-PD-L1/TGF-β fusion protein, which has the following general formula:

Ab-L-TGFβRII ECD (I)

Wherein Ab is the anti-PD-L1 antibody, L is the peptide linker, and TGFβRII ECD is the extracellular domain of TGFβRII; the N-terminus of the TGFβRII extracellular domain is connected to the C-terminus of the heavy chain of the anti-PD-L1 antibody through a peptide linker; The heavy chain of the anti-PD-L1 antibody includes the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12; the light chain of the anti-PD-L1 antibody includes the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 13, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14. As shown, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 15.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 17 is shown.

In a preferred embodiment, the heavy chain amino acid sequence of the anti-PD-L1 antibody is selected from SEQ ID NO: 1-SEQ ID NO: 3, and the light chain amino acid sequence of the anti-PD-L1 antibody is as SEQ ID NO : Shown in 4.

In a preferred embodiment, the anti-PD-L1 antibody is a monoclonal antibody.

In a preferred embodiment, the anti-PD-L1 antibody is a humanized antibody.

In a preferred embodiment, the anti-PD-L1 antibody is an IgG antibody.

In a preferred embodiment, the TGFβRII extracellular domain is selected from one or a combination of the following groups:

1) Full length TGFβRⅡ extracellular domain;

2) C-terminal truncated TGFβRII extracellular domain of 6-10 amino acids, more preferably, C-terminal truncated TGFβRII extracellular domain of 8 amino acids;

3) N-terminal truncated TGFβRII extracellular domain of 18-22 amino acids, more preferably, N-terminal truncated TGFβRII extracellular domain of 20 amino acids;

4) A full-length or truncated TGFβRII extracellular domain comprising at least one glycosylation site mutation, the mutation selected from S8P, T16P, T16V, and N71Q.

In a preferred embodiment, the amino acid sequence of the extracellular domain of TGFβRII is selected from SEQ ID NO: 5-SEQ ID NO: 9.

In a preferred embodiment, the peptide linker is selected from (G ₄ S) ₃ T or (G ₄ S) ₃ XDYTHTP, wherein X is G or S, and Y is K or A.

In a more preferred embodiment, the fusion protein is selected from the following group: 869, 869F, 869J15, 869J16, 869J17, 869M1, 869M3.

In a more preferred embodiment, the fusion protein is selected from the following group:

1) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 18, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

2) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 19, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

3) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 20, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

4) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 21, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

5) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 22, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

6) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 23, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

7) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 24, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4.

The second aspect of the present invention provides a polynucleotide molecule encoding the fusion protein.

Those skilled in the art know that the polynucleotide molecule encoding the amino acid sequence of the above-mentioned fusion protein can be replaced, deleted, changed, inserted or added as appropriate to provide a homolog of the polynucleotide.

The third aspect of the present invention provides an expression vector containing the above-mentioned polynucleotide molecule.

The fourth aspect of the present invention provides a host cell containing the above-mentioned expression vector.

The fifth aspect of the present invention provides a method for preparing a fusion protein, which includes the following steps:

a) Under expression conditions, culture the host cell as described above to express the anti-PD-L1/TGF-β fusion protein;

b) Isolation and purification of the fusion protein described in step a).

The sixth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the above-mentioned fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients.

In another preferred embodiment, the pharmaceutical composition also contains an anti-tumor agent.

In another preferred embodiment, the pharmaceutical composition is in a unit dosage form.

In another preferred embodiment, the anti-tumor agent and the fusion protein may be separately present in a separate package, or the anti-tumor agent may be coupled with the fusion protein.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

In another preferred embodiment, the parenteral administration dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

The seventh aspect of the present invention provides the use of the above-mentioned fusion protein and pharmaceutical composition in the preparation of drugs for the treatment of cancer.

According to the present invention, the cancer is selected from: colorectal cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, Head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma, etc.

The eighth aspect of the present invention provides a method for treating cancer, which comprises administering the above-mentioned fusion protein, or an immunoconjugate thereof, or a pharmaceutical composition to a subject in need.

According to the present invention, the cancer is selected from: colorectal cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, kidney cancer, bladder cancer, Head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma, etc.

The ninth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:

(a) The fusion protein according to the first aspect of the invention; and

(b) A coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

In another preferred embodiment, the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Detect enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) of the product.

In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.

The positive effect of the present invention is that the fusion protein of the present invention has improved charge heterogeneity, can bind to PD-L1 and TGF-β with high affinity, and its affinity with PD-L1 is comparable to the anti-PD-L1 monoclonal antibody positive control M8 It is equivalent, and its affinity with TGF-β is equivalent to that of the fusion protein positive control M7824. The fusion protein of the present invention can effectively block the combination of PD-1 and PD-L1, its blocking ability is equivalent to that of the anti-PD-L1 monoclonal antibody positive control M8, and can inhibit the pSMAD3 report induced by TGF-β in a dose-dependent manner Its inhibitory activity is equivalent to that of the positive control M7824 of the fusion protein. The fusion protein of the present invention exhibits a significant effect of inhibiting tumor growth in a human melanoma transplantation tumor model. The fusion protein of the present invention has the potential to treat diseases related to PD-L1 and TGF-β activity.

Description of the drawings

Figure 1A: Schematic diagram of the fusion protein structure

Figure 1B: Detection diagram of charge heterogeneity of fusion protein 869

Figure 1C: Detection diagram of charge heterogeneity of fusion protein 869F

Figure 1D: Detection diagram of charge heterogeneity of fusion protein 869M1

Figure 1E: Detection diagram of charge heterogeneity of fusion protein 869J15

Figure 2A: HPLC detection image of fusion protein 869M1

Figure 2B: HPLC detection chart of fusion protein 869J15

Figure 3A: ELISA detection diagram of the affinity of fusion proteins 869M1, 869M3, 869J15 and PD-L1

Figure 3B: ELISA detection diagram of affinity between fusion protein 869J15, 869J16, 869J17 and TGF-β1

Figure 3C: ELISA detection diagram of affinity between fusion protein 869M1, 869M3 and TGF-β1

Figure 3D: ELISA detection image of fusion protein 869J15, 869J16, 869J17 simultaneously binding PD-L1 and TGF-β1

Figure 4: FACS detection diagram of the affinity of fusion proteins 869J15 and 869J16 to the surface antigen PD-L1 of target cells

Figure 5A: Cellular experimental detection diagram of fusion proteins 869J15 and 869J16 blocking the binding of PD-1 and PD-L1

Figure 5B: Cellular experimental detection diagram of the fusion protein 869M1 and 869M3 blocking the binding of PD-1 and PD-L1

Figure 6: Detection of SMAD3 reporter gene inhibition by fusion proteins 869F and 869M1

Figure 7: Anti-tumor effect of fusion protein 869M1 on human melanoma A375 xenograft tumor model

detailed description

Through extensive and in-depth research, the inventors constructed an anti-PD-L1/TGF-β fusion protein for the first time. Specifically, on the basis of the anti-PD-L1 antibody (see PCT/CN2020/090442) independently developed by the applicant, the C-terminus of the heavy chain of the monoclonal antibody was connected to the extracellular domain of TGFβRII with a flexible peptide linker to obtain binding PD -Dual target fusion protein of L1 and TGF-β molecule. In addition, the present inventors modified the glycosylation site of the extracellular domain of TGFβRII in the fusion protein to obtain a fusion protein with significantly improved charge heterogeneity, thereby increasing and improving the stability and immunogenicity of the fusion protein. The fusion protein of the present invention has significant anti-tumor activity in mice, so it is expected to be developed as an anti-tumor drug with superior curative effect. On this basis, the inventor completed the present invention.

the term

In the present invention, the term "fusion protein" refers to a new polypeptide sequence obtained by the fusion of two or more identical or different polypeptide sequences. The term "fusion" refers to direct connection by peptide bonds or operative connection via one or more connecting peptides (peptide linkers). The term "connecting peptide (peptide linker)" refers to a short peptide that can connect two polypeptide sequences, generally a peptide of 2-30 amino acids in length.

As used herein, the term "peptide linker (linker)" refers to an immunoglobulin that is inserted into an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold into one or more immunoglobulins that exchange dual variable regions. Multiple amino acid residues. In the present invention, the preferred peptide linker refers to peptide linker L, where L connects the N-terminus of the extracellular domain of TGFβRII to the C-terminus of the heavy chain of the anti-PD-L1 antibody.

Examples of suitable peptide linkers include single glycine (Gly) or serine (Ser) residues. The identity and sequence of amino acid residues in the peptide linker can vary with the type of secondary structural elements that need to be implemented in the peptide linker. In the present invention, the peptide linker is selected from (G4S) ₃ T or (G4S) ₃ XDYTHTP, wherein X is G or S, and Y is K or A.

In the present invention, the term "antibody (Ab)" is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC) and so on. The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.

In the present invention, the term "monoclonal antibody (monoclonal antibody)" refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins.

In the present invention, the term "humanized" means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, framework residues can be changed to maintain binding affinity.

As used herein, the term "framework region" (FR) refers to the portion of the immunoglobulin variable region that has relatively small changes in amino acid composition and sequence outside the hypervariable region. The light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.

Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.

As used herein, the term "human framework region" is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .

Bifunctional fusion protein

The bifunctional fusion protein of the present invention is an anti-PD-L1×TGF-β bifunctional fusion protein, including an anti-PD-L1 antibody part and a TGFβRII extracellular domain part responsible for capturing TGF-β

Preferably, the sequence of the anti-PD-L1 antibody of the present invention is as described in the patent application PCT/CN2020/090442. Those skilled in the art can also modify or transform the anti-PD-L1 antibody of the present invention through techniques well known in the art, such as adding , Deletion and/or substitution of one or several amino acid residues, thereby further increasing the affinity or structural stability of anti-PD-L1, and obtaining modified or modified results through conventional measurement methods.

In the present invention, the bifunctional fusion protein of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the bifunctional fusion protein of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5 At best, at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitutions.

Table A

Initial residues	Representative substitution	Preferred substitution
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln; His; Lys; Arg	Gln
Asp(D)	Glu	Glu
Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
Glu(E)	Asp	Asp
Gly(G)	Pro; Ala	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe	Leu
Leu(L)	Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
Met(M)	Leu; Phe; Ile	Leu
Phe(F)	Leu; Val; Ile; Ala; Tyr	Leu
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

In the present invention, the terms "anti" and "binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. Generally, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of ^{less than about 10 -7} M, for example, less than about 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^{-11 M or less.} The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, Surface Plasmon Resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or an ELISA is used to measure the relative binding affinity of an antibody to the antigen.

The bifunctional fusion protein of the present invention can be used alone, or can be combined or coupled with a detectable label (for diagnostic purposes), a therapeutic agent, or any combination of these substances.

Coding nucleic acid and expression vector

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. In the present invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substances, such as a plasmid, a viral vector (such as adenovirus, retrovirus), a phage, a yeast plasmid or other vectors. For example, conventional expression vectors in the art that include appropriate regulatory sequences, such as promoters, terminators, enhancers, etc., the expression vectors include, but are not limited to: viral vectors (such as adenovirus, retrovirus), plasmids, bacteriophages , Yeast plasmid or other vectors. The expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR or pTT5.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

The present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

In the present invention, the term "host cell" refers to various conventional host cells in the art, as long as the vector can replicate itself stably and the polynucleotide molecule carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO, NS0, sf9, sf21, DH5α, BL21 (DE3), TG1, BL21 (DE3), 293F or 293E cells.

Pharmaceutical composition and application

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 4-8, preferably about 5-7, although the pH can be The nature of the formulated substance and the condition to be treated vary. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection.

In the present invention, the term "pharmaceutical composition" means that the bifunctional fusion protein of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more stably exert the curative effect. These preparations can ensure the bifunctionality disclosed in the present invention. The conformational integrity of the amino acid core sequence of the fusion protein also protects the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bifunctional fusion protein (or conjugate thereof) of the present invention, and A pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the bifunctional fusion protein of the present invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the bifunctional fusion protein or its immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are all within the skill range of a skilled physician.

In the present invention, the term "effective amount" refers to the amount or dose that produces the expected effect in the treated individual after the pharmaceutical composition of the present invention is administered to the subject, and the expected effect includes the improvement of the individual's condition. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats, mice, and the like.

The sequence information involved in the following examples is summarized in Table 1 of the Sequence Listing.

Table 1 Sequence Listing

The anti-human PD-L1 antibody positive control M8 used in the following examples is derived from PCT/CN2020/090442, and its heavy chain and light chain amino acid sequences are SEQ ID NO: 1 and SEQ ID NO: 4 in the Sequence Listing Table 1, respectively .

The anti-PD-L1/TGF-β fusion protein positive control M7824 used in the following examples is derived from US20150225483A1, and its heavy chain and light chain amino acid sequences are SEQ ID NO: 25 and SEQ ID NO: 26 in the Sequence Listing Table 1, respectively. .

Unless otherwise specified, the reagents and raw materials used in the following examples can be purchased from commercial sources.

The following experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods or conventional methods in the art, such as methods for preparing polynucleotide molecules, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or Methods for introducing plasmids into host cells, methods for culturing host cells, etc., such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Example 1. Construction of anti-PD-L1/TGF-β bifunctional fusion protein

The extracellular domain of TGFβRⅡ is used as the immunomodulatory molecule part of the fusion protein, and the anti-PD-L1 antibody M8 is used as the targeting part of the fusion protein to form an anti-PD-L1/TGFβ bifunctional fusion protein (the schematic diagram is shown in Figure 1A). The general formula is as follows:

Ab-L-TGFβRII ECD (I)

Among them, TGFβRII ECD is the extracellular domain of TGFβRII, including full-length, truncated or mutant forms of TGFβRII extracellular domain. Wherein Ab is an M8 antibody or a mutant of M8 antibody. Where L is a peptide linker, including (G ₄ S) ₃ T or (G ₄ S) ₃ XDYTHTP, where X is G or S, and Y is K or A.

The inventors have carried out continuous research and improvement on the structure of the fusion protein. First, using homologous recombination technology, the C-terminal amino acid of the heavy chain of the anti-PD-L1 antibody M8 is connected through a peptide linker (G ₄ S) ₃ T to the N-terminal end of the extracellular domain of TGFβRⅡ Linked and expressed together with the light chain of M8 to obtain fusion protein 869. Due to the serious charge heterogeneity of 869, the inventors optimized it. The study found that there are 3 N-glycosylation sites in the extracellular domain of TGFβRII, namely N47, N71, N131 (underlined), and The N-terminal O-glycosylation sites S8 and T16 (shown underlined). Glycosylation leads to a very complicated charge heterogeneity in the anti-PD-L1/TGF-β bifunctional fusion protein. In order to reduce the complexity of glycosylation modification, the glycosylation site was mutated with N71Q and the C-terminal was truncated by 8 amino acids to obtain the sequence 869F; the glycosylation site was mutated with S8P, T16P, N71Q, and the C-terminal was truncated 8 amino acids, the sequence 869M1 is obtained; the N-terminal glycosylation site is mutated with S8P, T16V, N71Q, and the C-terminal is truncated by 8 amino acids to obtain the sequence 869M3; in addition, the N-terminal 20 amino acids are truncated, and the C-terminal 8 amino acids are truncated , And add different peptide linkers to get 869J15-17 series protein. Add peptide linker (G ₄ S) ₃ GDATHTP to get 869J15, add peptide linker (G ₄ S) ₃ GDATHTP to get 869J16, add peptide linker (G ₄ S) ₃ SDKTHTP to get 869J17 (see Table 2 for the description of the fusion protein sequence constructed above) . The charge heterogeneity test results (Figure 1B-Figure 1E) found that the charge heterogeneity of the fusion protein 869F was significantly improved compared to the fusion protein 869, while the charge heterogeneity of the 869M1, 869M3, and 869J15-17 series of fusion proteins was improved. It is even more significant. Charge heterogeneity is an important quality attribute of recombinant protein drugs, and the improved charge heterogeneity is conducive to improving the stability of the fusion protein and improving immunogenicity.

Table 2 Description of fusion protein sequence

Examples of Fusion Proteins	Fusion protein sequence description
869	M8-LSPGK(G 4 S) 3 T-ECD(1-136)
869F	M8-LSPGK(G 4 S) 3 T-ECD(1-128)/N71Q
869J15	M8-LSPGG(G 4 S) 3 GDATHTP-ECD(21-128)/N71Q
869J16	M8-LSPG(G 4 S) 3 GDATHTP-ECD(21-128)/N71Q
869J17	M8-LSPG(G 4 S) 3 SDKTHTP-ECD(21-128)/N71Q
869M1	M8-LSPGK(G 4 S) 3 T-ECD(1-128)/S8P, T16P, N71Q
869M3	M8-LSPGK(G 4 S) 3 T-ECD(1-128)/S8P, T16V, N71Q

Note: M8-LSPGK represents the heavy chain of M8, M8-LSPGG represents the mutation of amino acid residue K at position 447 of the heavy chain of M8 to G, and M8-LSPG represents the deletion of amino acid residue K at position 447 of the heavy chain of M8.

ECD (1-136) represents the extracellular domain of TGFβRII with a full length of 136 amino acids.

ECD (21-128) represents the extracellular domain of TGFβRII truncated by 20 amino acids at the N-terminus and 8 amino acids at the C-terminus.

ECD (1-128) represents the extracellular domain of TGFβRII with a C-terminal truncation of 8 amino acids.

Example 2. Expression and purification of anti-PD-L1/TGF-β bifunctional fusion protein

The DNA fragments of the heavy and light chains of the anti-PD-L1/TGF-β bifunctional fusion protein constructed above were respectively subcloned into pTT5 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells and/or 293F cells. After the cells were cultured for 7 days, the culture solution was subjected to high-speed centrifugation, vacuum filtration with a microporous membrane, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with 100 mM citric acid, pH 3.5 eluent, and the target sample was recovered and dialyzed Change the medium to PBS. The purified protein was detected by HPLC. The detection results of Figures 2A-2B showed that the fusion protein molecule was in a uniform state, and the monomer purity reached more than 97%.

Example 3. Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PD-L1/TGF-β bifunctional fusion protein to antigen

3.1 ELISA to detect the affinity of anti-PD-L1/TGF-β bifunctional fusion protein and PD-L1

Prepare recombinant PD-L1-ECD-Fc protein (for the preparation method refer to WO2018/137576A1), plate with 100ng/well at 4°C overnight. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the antibody to 100 nM with the diluent, dilute by 4 times to form 12 concentration gradients, and add them to the blocked microtiter plate successively, 100 μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled goat anti-human Fab antibody (purchased from abcam, Cat.#ab87422), and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and place at room temperature (20±5°C) in the dark for 5 minutes; add stop solution to each well to stop the substrate reaction, enzyme label instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50.

The experimental results are shown in Figure 3A. The anti-PD-L1/TGFβ bifunctional fusion protein 869M1, 869M3, 869J15 and the positive control M8 monoclonal antibody can effectively bind PD-L1-ECD, and the EC50 (nM) values are 0.2374, 0.3278, 0.399 and 0.3335 have the same affinity.

3.2 ELISA to detect the affinity of anti-PD-L1/TGF-β bifunctional fusion protein and TGF-β1

In order to detect the binding ability of anti-PD-L1/TGF-β bifunctional fusion protein and TGF-β1, TGF-β1 protein (purchased from acrobiosystems, Cat.#TG1-H4212) was plated at 20ng/well at 4°C overnight. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the anti-PD-L1/TGF-β bifunctional fusion protein with diluent to the initial concentration of 327nM (869J15), 418nM (869J16), 582nM (869J17) and 200nM (869M1, 869M3 and M7824), 3 times ratio ( Fig. 3B) or 4-fold dilution (Fig. 3C) to form 12 concentration gradients, which were added to the blocked microtiter plate successively, 100 μl/well, and placed at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place it at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and keep it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50.

The experimental results are shown in Figures 3B and 3C. The anti-PD-L1/TGF-β bifunctional fusion protein can specifically bind to TGF-β1, and the anti-PD-L1/TGF-β bifunctional fusion protein 869J15, 869J16, 869J17, 869M1 The EC50 (nM) values of 869M3 and the positive control M7824 are 1.466, 1.508, 1.552, 0.8196, 0.8983 and 1.475, respectively, and their affinity is comparable to that of the positive control M7824.

3.3 ELISA to detect the ability of anti-PD-L1/TGF-β bifunctional fusion protein to simultaneously bind PD-L1 and TGF-β1

The steric hindrance may affect the ability of the anti-PD-L1/TGF-β bifunctional fusion protein to simultaneously bind to two antigens. In order to test the ability of the anti-PD-L1/TGF-β bifunctional fusion protein to simultaneously bind PD-L1 and TGF-β1, TGF-β1 was plated at 20ng/well at 4°C overnight. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate with PBST once for later use. Dilute the anti-PD-L1/TGF-β bifunctional fusion protein with diluent to the initial concentration of 130nM (869J15), 167nM (869J16), 233nM (869J17) and 139nM (M8), 4 times the dilution to form 10 concentration gradients , Add the blocked microtiter plate successively, 100μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add PD-L1-ECD-Fc-biotin at 150ng/well, and place at 37°C for 1 hour. After washing the plate 3 times with PBST, HRP-labeled Streptavidin was added and placed at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and keep it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50.

The experimental results are shown in Figure 3D. Anti-PD-L1/TGF-β bifunctional fusion proteins 869J15, 869J16, and 869J17 can simultaneously bind PD-L1 and TGF-β1, with EC50 (nM) values of 1.556, 1.303, 0.9251, respectively.

Example 4. Determination of the binding affinity of anti-PD-L1/TGF-β bifunctional fusion protein to the surface antigen PD-L1 of target cells by FACS method

In this experiment, cell surface PD-L1 expression in PD-L1aAPC / CHO-K1 cells (available from promega, Cat. # J1252) as target cells, the target cells according to 2 × 10 ⁵ / well were seeded in 96-well plates, containing Wash three times with 0.5% BSA in PBS, centrifuge at 300g for 5 minutes each time, and discard the supernatant. Add 100μl of antibodies with a 3-fold gradient from the initial concentration of 65nM (869J15), 84nM (869J16), and 69nM (M8) serially diluted 11 gradients as the primary antibody to a 96-well plate, suspend the cells and incubate at 4°C for 1h . The cells were washed twice with PBS containing 0.5% BSA to remove unbound antibodies, and then the cells were incubated with 100 μl of 1 μg/ml goat anti-human IgG-FITC (purchased from sigma, Cat. #F9512) at 4° C. for 30 minutes. Centrifuge at 300g for 5 minutes. Wash the cells twice with PBS containing 0.5% BSA to remove unbound secondary antibodies. Finally, the cells are resuspended in 200μl PBS. The binding affinity of L1. The data obtained was fitted and analyzed by GraphPad Prism6 software.

The experimental results are shown in Figure 4. The anti-PD-L1/TGF-β bifunctional fusion protein 869J15, 869J16 and the positive control M8 can specifically bind to PD-L1 expressed on the cell surface, with EC50 (nM) of 0.4600 and 0.5763, respectively It has the same affinity as 0.7123.

Example 5. Cellular experiment of anti-PD-L1/TGF-β bifunctional fusion protein blocking the binding of PD-1 and PD-L1

Take the PD-L1aAPC/CHO-K1 cells (purchased from promega, Cat.#J1252) grown in the logarithmic phase, trypsinize them into single cells and transfer them to a white bottom permeable 96-well plate, 100μl/well, 40,000 cells/well, Place at 37°C, 5% CO ₂ , and incubate overnight. The anti-PD-L1/TGF-β bifunctional fusion protein, positive control M8 and negative control NC (isotype-negative control antibody IgG1) according to the initial concentration of 32nM (869J15), 42nM (869J16), 34nM (M8) triple gradient Diluted into 2× working solution, a total of 8 concentration gradients (Figure 5A), and the initial concentration of 200nM (869M1, 869M3 and M8) was diluted three times into 2× working solution, a total of 10 concentration gradients (Figure 5B). At the same time, the PD-1 effector cells (purchased from promega, Cat.#J1252) with a ^{density of 1.4-2×10 6 /ml and a cell viability above 95% were trypsinized to 1.25×10 6} cells/ml. Cell suspension. Take the PD-L1aAPC/CHO-K1 cells plated the day before, discard the supernatant, add 40 μl of the antibody working solution of gradient dilution, and then add an equal volume of PD-1 effector cells. Place at 37°C, 5% CO ₂ , and incubate for 6 hours. After the cells were incubated at 37°C for 6 hours, 80 μl of detection reagent Bio-Glo (purchased from promega, Cat. #G7940) was added to each well. After incubating for 10 minutes at room temperature, read the luminescence with SpectraMax i3. All data are double-replicated holes, and the obtained signal values are averaged and then fitted with the 4-parameter method, and the data is analyzed with GraphPad Prism6.

The experimental results are shown in Figure 5. In Figure 5A, the anti-PD-L1/TGF-β bifunctional fusion protein 869J15, 869J16 and the positive control M8 can effectively block the interaction between PD-1 and PD-L1. IC50(nM) are 0.1977, 0.1592 and 0.154, respectively, and the blocking ability of the three is equivalent. In Figure 5B, the anti-PD-L1/TGF-β bifunctional fusion protein 869M1, 869M3 and the positive control M8 can effectively block the interaction between PD-1 and PD-L1, with IC50 (nM) values of 1.559 , 1.846 and 1.169, the blocking ability of the three is equivalent.

Example 6. SMAD3 reporter gene suppression experiment

SBE Reporter HEK293 Cell (purchased from BPS bioscience, Cat.#60653) expresses the Smad3 binding element (SEB) with a luciferase reporter gene. By studying the inhibitory effect of the antibody protein on the activation of Smad3 induced by TGF-β1 in the cell, it can be Evaluate the in vitro activity of the antibody. Take SBE293 cells grown in logarithmic phase of adherent culture, wash once with DPBS, digest with trypsin to neutralize trypsin. Trypan blue cell count, 300g centrifugation for 5min. Resuspend in MEM (10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate) medium (all purchased from Gibico, Cat.#10095-098, 10091-148, 11140-050, 11360-070) and count Pave the plate, adjust the density to 35000/well, 100μl/well, place at 37°C, 5% CO ₂ , and incubate overnight, about 24h. Dilute TGF-β1 (10ng/ml) with MEM (0.5% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate) medium, and dilute the fusion protein to 200 nM, 4-fold dilution, 10 gradients, and place at room temperature for 1 hour. Replace the original medium of the cell plate and place it in a 37°C incubator overnight. Add 100μL/well of the detection reagent Bio-Glo (put in a 25°C water bath 30 minutes in advance to thaw the equilibrium temperature). After incubating for 10 minutes at room temperature, read the luminescence with SpectraMax i3.

The experimental results are shown in Figure 6. The fusion protein inhibits the activity of the pSMAD3 reporter induced by TGF-β in a dose-dependent manner. The IC50 (nM) values of the fusion protein 869F, 869M1 and the positive control M7824 are 0.236, 0.1324 and 0.1404, respectively. The activity is comparable.

Example 7 The anti-tumor effect of human PBMC to rebuild the immune system and inoculate human melanoma A375 transplantation tumor model

The human melanoma A375 cell line was purchased from ATCC, and the NPG mouse was purchased from Beijing Weitongda Biotechnology Co., Ltd. The dose of the test sample 869M1 is 25mg/kg, the dose of the control drug IMFINZI (Durvalumab, anti-PD-L1 monoclonal antibody, purchased from AstraZeneca) is 20.5mg/kg, and the dose of M7824 is 25mg/kg. The blank control group was given the same volume of normal saline. Collect human melanoma A375 cells cultured in vitro, adjust the cell suspension concentration to 2×10 ⁸ /ml, and mix with an equal volume of Matrigel. Resuscitate the frozen human PBMC cells and adjust the cell concentration to 1×10 ⁸ /ml. An equal volume of A375 cell suspension was mixed with PBMC suspension, and under aseptic conditions, 200 μl of cell suspension was inoculated under the skin of the right rib of NPG mice. The diameter of the transplanted tumor was measured with a vernier caliper, and the animals were randomly divided into groups after the ^{average tumor volume grew to about 70mm 3.} IMFINZI, M7824 and 869M1 were administered according to the above-mentioned doses. The control group was given the same amount of normal saline by intraperitoneal injection twice a week for a total of 4 administrations. Throughout the experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time. The calculation formula of tumor volume (TV) is: TV=1/2×a×b ² . Among them, a and b represent length and width respectively. According to the measurement results, the relative tumor volume (RTV) is calculated, and the calculation formula is: RTV=V _t /V ₀ . Where V ₀ is the tumor volume measured at the time of group administration (ie d ₀ ), and V _t is the tumor volume at each measurement. The evaluation index of anti-tumor activity is the relative tumor proliferation rate T/C(%), and the calculation formula is as follows: T/C(%)=(T _RTV /C _RTV )×100(T _RTV : treatment group RTV; C _RTV : negative Control group RTV).

The results of the experiment are shown in Fig. 7. On the human PBMC mouse A375 xenograft tumor model, IMFINZI tumor inhibition rate TGI was 69.79%, M7824 tumor inhibition rate TGI was 96.33%, and 869M1 tumor inhibition rate TGI was 98.52%. The results show that in this transplanted tumor model, 869M1 can significantly inhibit tumor growth, and its efficacy is better than anti-PD-L1 monoclonal antibody.

Example 8. Physical stability of anti-PD-L1/TGF-β bifunctional fusion protein.

DSC (Differential scanning calorimetry) was used to detect the thermal stability of 869M1 in the PBS buffer system. Replace the sample with PBS buffer, control the sample concentration at 1 mg/ml, and use MicroCal*Vp-Capillary DSC (Malvern) for detection. Before testing, filter the sample and blank buffer with a 0.22μm filter membrane. Add 400μl sample or blank buffer to each well of the sample plate (set 6 groups of blank buffer pairs), and add ddH ₂ O to the last three pairs of well plates for cleaning. After loading the sample plate, put on the plastic soft cover. The scanning temperature starts at 25°C and ends at 100°C, and the scanning rate is 150°C/h. The specific results are shown in Table 3 below. The 869M1 protein showed good thermal stability.

Table 3. Thermal stability of 869M1 protein

Sample	TmOnset(℃)	Tm1(℃)
869M1	64.59	77.11

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (18)

1. An anti-PD-L1/TGF-β fusion protein, which is characterized in that it has the following general formula:

   Ab-L-TGFβRII ECD (I)

   Wherein Ab is the anti-PD-L1 antibody, L is the peptide linker, and TGFβRII ECD is the extracellular domain of TGFβRII; the N-terminus of the TGFβRII extracellular domain is connected to the C-terminus of the heavy chain of the anti-PD-L1 antibody through a peptide linker; The heavy chain of the anti-PD-L1 antibody includes the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12; the light chain of the anti-PD-L1 antibody includes the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 13, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14. As shown, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 15.
2. The fusion protein of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is shown in SEQ ID NO: 16, and the light chain of the anti-PD-L1 antibody can be The amino acid sequence of the variable region is shown in SEQ ID NO: 17.
3. The fusion protein of claim 2, wherein the heavy chain amino acid sequence of the anti-PD-L1 antibody is selected from SEQ ID NO: 1-SEQ ID NO: 3, and the light chain of the anti-PD-L1 antibody The amino acid sequence is shown in SEQ ID NO: 4.
4. The fusion protein of claim 1, wherein the TGFβRII extracellular domain is selected from one or a combination of the following groups:

   1) Full length TGFβRⅡ extracellular domain;

   2) C-terminal truncated 6-10 amino acid TGFβRⅡ extracellular domain;

   3) TGFβRII extracellular domain with 18-22 amino acids truncated at the N-terminus;

   4) A full-length or truncated TGFβRII extracellular domain comprising at least one glycosylation site mutation, the mutation selected from S8P, T16P, T16V, and N71Q.
5. The fusion protein of claim 4, wherein the amino acid sequence of the extracellular domain of TGFβRII is selected from SEQ ID NO: 5-SEQ ID NO: 9.
6. The fusion protein of claim 1, wherein the peptide linker is selected from (G ₄ S) ₃ T or (G ₄ S) ₃ XDYTHTP, wherein X is G or S, and Y is K or A.
7. The fusion protein according to any one of claims 1-6, wherein the fusion protein is selected from the following group:

   1) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 18, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   2) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 19, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   3) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 20, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   4) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 21, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   5) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 22, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   6) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 23, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4;

   7) The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 24, and the light chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 4.
8. A polynucleotide molecule, characterized in that the polynucleotide molecule encodes the fusion protein according to any one of claims 1-7.
9. An expression vector, characterized in that the expression vector contains the polynucleotide molecule according to claim 8.
10. A host cell, characterized in that it contains the expression vector according to claim 9.
11. A method for preparing a fusion protein according to any one of claims 1-7, characterized in that the preparation method comprises the following steps:

    a) Under expression conditions, culture the host cell according to claim 10 to express the anti-PD-L1/TGF-β fusion protein;

    b) Isolation and purification of the fusion protein described in step a).
12. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises an effective amount of the fusion protein according to any one of claims 1-7 and one or more pharmaceutically acceptable carriers, diluents or excipients. Shape agent.
13. Use of the fusion protein according to any one of claims 1-7 or the pharmaceutical composition according to claim 12 in the preparation of a medicine for the treatment of cancer.
14. The use according to claim 13, wherein the cancer is selected from the group consisting of colorectal cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, Prostate cancer, kidney cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
15. A method for treating cancer, characterized in that the method comprises administering the fusion protein according to any one of claims 1-7, or an immunoconjugate thereof, or according to claim 12 to a subject in need The pharmaceutical composition.
16. The method of claim 15, wherein the cancer is selected from the group consisting of colorectal cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, Prostate cancer, kidney cancer, bladder cancer, head and neck cancer, lymphoma, melanoma, skin cancer, glioma, mesothelioma.
17. An immunoconjugate, characterized in that, the immunoconjugate comprises:

    (a) The fusion protein of any one of claims 1-7; and

    (b) A coupling part selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
18. The use of the immunoconjugate according to claim 17, characterized in that it is used to prepare a pharmaceutical composition for treating tumors.

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