WO2022063193A1

WO2022063193A1 - BIFUNCTIONAL MOLECULE SIMULTANEOUSLY TARGETING PD-L1 AND TGFβ AND MEDICAL USE THEREOF

Info

Publication number: WO2022063193A1
Application number: PCT/CN2021/120006
Authority: WO
Inventors: 陈奇; 蒋家骅; 周传初; 顾津明
Original assignee: 上海齐鲁制药研究中心有限公司
Priority date: 2020-09-24
Filing date: 2021-09-23
Publication date: 2022-03-31
Also published as: CN116323657B; CN116323657A

Abstract

Provided are a bifunctional molecule simultaneously targeting PD-L1 and TGFβ and a pharmaceutical composition comprising the bifunctional molecule. Further provided is the use of the bifunctional molecule simultaneously targeting PD-L1 and TGFβ in treating and preventing cancers.

Description

A bifunctional molecule targeting PD-L1 and TGFβ simultaneously and its medicinal use

technical field

The invention belongs to the field of biomedicine, in particular to a bifunctional molecule that simultaneously targets PD-L1 and TGFβ and its medical application.

Background technique

Following chemotherapy, radiotherapy and targeted therapy, immunotherapy has become an increasingly important new type of tumor treatment, representing a transformation of thinking in this field. Programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) are the core proteins of the immune check signaling pathway, which can protect their own tissues against normal conditions. Attacked by T cells, but also used by tumor cells to evade clearance by the immune system. Antibody drugs targeting PD-1 or PD-L1 can significantly inhibit tumor growth, and patients with multiple cancer types who respond to PD-1/PD-L1 therapy have significant survival benefits. However, the response rate of tumor patients to PD-1/PD-L1 therapy is not high, one of the reasons is that the complex microenvironment of the tumor has multiple immunosuppressive signaling pathways. Therefore, bispecific antibodies targeting PD-1/PD-L1 and another immune checkpoint at the same time have more therapeutic potential.

Studies have shown that tumor tissues that do not respond to PD-1/PD-L1 therapy are often accompanied by high expression of transforming growth factor beta (TGFβ). Under normal physiological conditions, TGFβ acts as an immunoregulatory factor to protect its own tissues from the attack of the immune system, but in the tumor microenvironment, TGFβ acts on the immune system to help tumor cells achieve immune escape and accelerate tumor progression. TGFβ has three subtypes, namely TGFβ1, TGFβ2 and TGFβ3, which are highly expressed in various tumor tissues, and their high expression in serum is also associated with poor prognosis. At the same time, TGFβ1 can directly prevent the differentiation of T cells and inhibit the function of T cells and NK cells to kill cancer cells.

Therefore, drugs that block both the PD-1/PD-L1 signaling pathway and the TGFβ signaling pathway are expected to further improve the response rate of immunotherapy compared with drugs targeting a single signaling pathway.

At present, bifunctional molecules composed of PD-L1 antibody and TGFβRII fusion protein have been disclosed, such as WO2015118175A2, WO2018205985A1 and the like. However, there are still problems such as short half-life, and the present invention provides a technical solution with better stability in vivo and in vitro.

SUMMARY OF THE INVENTION

The present invention provides a bifunctional molecule targeting PD-L1 and TGFβ at the same time, comprising a PD-L1-targeting part and a TGF-β receptor part, and the PD-L1-targeting part is a PD-L1 antibody, The TGF-β receptor part is the N-terminal truncated form of the extracellular region of TGFβRII (TGF-beta receptor type-2), and the C-terminus of each heavy chain of the PD-L1 antibody is connected to a TGFβRII extracellular region The N-terminal truncated form of the PD-L1 antibody, the light chain and heavy chain variable region CDR sequences of the PD-L1 antibody are as follows:

The sequence of LCDR1 is shown in SEQ ID NO: 9;

The sequence of LCDR2 is shown in SEQ ID NO: 10;

The sequence of LCDR3 is shown in SEQ ID NO: 11;

The sequence of HCDR1 is shown in SEQ ID NO: 12;

The sequence of HCDR2 is shown in SEQ ID NO: 13;

The sequence of HCDR3 is shown in SEQ ID NO:14.

Preferably, the full-length sequence of the extracellular region of TGFβRII is shown in SEQ ID NO: 1, and the N-terminal truncated form is a truncation of 17-27 amino acids.

Preferably, the sequence of the TGF-β receptor part is shown in SEQ ID NO: 2.

Preferably, the light chain variable region sequence of the PD-L1 antibody is shown in SEQ ID NO: 15; the heavy chain variable region sequence is shown in SEQ ID NO: 16.

Preferably, the light chain sequence of the PD-L1 antibody is shown in SEQ ID NO: 5; the heavy chain sequence is shown in SEQ ID NO: 6, and the K at the last position of the C-terminal of the heavy chain is mutated into A.

Preferably, the C-terminal of the heavy chain of the PD-L1 antibody is connected to the N-terminal truncated form of the extracellular region of TGFβRII through a linking peptide.

Preferably, the connecting peptide is (G ₄ S) _X G, and the x is 3-6, preferably 4-5.

Preferably, the light chain sequence of the PD-L1 antibody is shown in SEQ ID NO: 7, and the overall sequence of the heavy chain and the N-terminal truncated form of the extracellular region of TGFβRII is shown in SEQ ID NO: 8.

The present invention also provides a pharmaceutical composition comprising the above-mentioned bifunctional molecule and a pharmaceutically acceptable carrier.

The present invention also provides a nucleic acid molecule encoding the above-mentioned bifunctional molecule.

The present invention also provides an expression vector, which contains the above-mentioned nucleic acid molecule.

The present invention also provides a host cell comprising the above-mentioned expression vector, the host cell is selected from bacteria, yeast and mammalian cells; preferably mammalian cells; more preferably HEK293E cells, expi293 cells or CHO cells.

The present invention also provides the use of the above-mentioned bifunctional molecule in the preparation of a medicament for treating cancer.

Preferably, the cancer is a PD-L1 positive tumor.

Preferably, the cancer is selected from the group consisting of lung cancer, gastric cancer, melanoma, kidney cancer, breast cancer, bowel cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer and head and neck cancer.

The present invention also provides a method for treating and preventing tumors, comprising administering to a patient in need a therapeutically effective amount of the above-mentioned bifunctional molecule or the above-mentioned pharmaceutical composition.

Description of drawings

Figure 1: Schematic diagram of the bifunctional molecular structure.

Figure 2: Results of in vitro binding of bifunctional molecules to human PD-L1.

Figure 3: Results of in vitro binding of bifunctional molecules to human TGFβ1.

Figure 4: Detection of bifunctional molecule 6 binding to human PD-L1 and human TGFβ1 using SPR technology.

Figure 5: The ability of bifunctional molecules to block the PD-1/PD-L1 signaling pathway was tested at the cellular level.

Figure 6: The ability of bifunctional molecules to block the TGFβ/SMAD signaling pathway was tested at the cellular level.

Figure 7: Effect of bifunctional molecules on tumor weight in the MDA-MB-231 model.

Figure 8: Effect of bifunctional molecules on tumor volume and tumor weight in a humanized MC-38 model.

detailed description

the term

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. into this article.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Certain embodiments disclosed herein encompass numerical ranges, and certain aspects of the invention may be described in terms of ranges. Unless otherwise indicated, it should be understood that numerical ranges or descriptions of ranges are presented for brevity and convenience only and should not be construed as limiting the scope of the invention. Accordingly, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within that range, as if such subranges and numerical points were expressly written herein. The above principles apply equally regardless of the breadth of the stated numerical values. When a range is used, the range includes the endpoints of the range.

When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include ±20% of the specified value, or in some cases ±10%, or in some cases ±5%, or within ±1% in some cases, or ±0.1% in some cases.

The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody", as used herein, typically refers to a Y-type tetramer comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions protein. Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and constant region.

Five main classes of antibodies are known in the art: IgA, IgD, IgE, IgG and IgM, the corresponding heavy chain constant domains are called α, δ, ε, γ and μ, respectively, IgG and IgA can be further divided into different The subclasses, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

In the case of IgG, IgA and IgD antibodies, the constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length proline and cysteine rich segment. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.

The terms "variable region" or "variable domain" exhibit significant changes in amino acid composition from one antibody to another, and are primarily responsible for antigen recognition and binding. The variable regions of each light/heavy chain pair form the antibody binding site such that an intact IgG antibody has two binding sites (ie it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) domain of the light chain each contain three regions of extreme variability known as hypervariable regions (HVRs), or more commonly, known as Complementarity determining regions (CDRs), VH and VL each have four framework regions FR, which are represented by FR1, FR2, FR3, and FR4, respectively. Thus, CDR and FR sequences typically occur in the following sequences of the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)- FR4.

The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

As used herein, types of "antibodies" in a broad sense may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and primatized antibodies, CDR-grafted antibodies (CDR- grafted antibodies), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies ( including mutant proteins and their variants) and so on.

The term "monoclonal antibody" (or "monoclonal antibody") refers to a substantially homogeneous antibody produced by a single cell clone directed against only a particular epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, synthetic techniques, or a combination of the foregoing, and the like.

It should be noted that the division of CDRs and FRs in the variable region of the monoclonal antibody of the present invention is determined according to the Kabat definition. While other nomenclature and numbering systems, such as Chothia, IMGT or AHo, etc., are also known to those skilled in the art. Accordingly, humanized antibodies comprising one or more CDRs derived from any nomenclature system based on the sequences of the mAbs of the invention are expressly within the scope of the invention.

The term "antibody fragment" encompasses at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to an immunoglobulin or antibody that specifically binds a selected antigen or an immunogenic determinant thereof Or reacted polypeptide fragments, or fusion protein products further derived from such fragments, such as single-chain antibodies, extracellular binding domains in chimeric antigen receptors, and the like. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, single domain antibodies, linear Antibodies, single chain antibodies (scFv) and bispecific or multispecific antibodies formed from antibody fragments, etc.

The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including mono-epitopes, poly-epitopes, mono-structures domain, multi-domain, intact extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, parts thereof, and combinations thereof can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, and the like. "Antigen" can also refer to a molecule that elicits an immune response. Any form of the antigen or cells or preparations containing the antigen can be used to generate antibodies specific for an antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (eg, immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (eg, immunized with only the ECD portion of the protein), or a protein Constructs (eg, Fc antigens). The antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (eg, cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Cells in which the antigen is expressed can be transformed using any vector including, but not limited to, adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

The term "epitope" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically exist in unique spatial conformations and include at least 3-15 amino acids. Methods for determining the epitope to which a given antibody binds are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). The term " _KD " refers to the dissociation constant for a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultrafast Centrifugation and flow cytometry, etc.

The term "biological activity" refers to the ability of an antibody to bind an antigen and cause a measurable biological response, which can be measured in vitro or in vivo.

The term "pharmaceutical formulation" or "formulation" or "formulation formulation" means a product that exists in a form that allows the biological activity of the active ingredient to be effective and that does not contain other components that are toxic to the subject to which the formulation is to be administered .

The term "solution formulation" means a formulation that is liquid at a temperature of at least about 2°C to about 8°C at atmospheric pressure.

The term "deamidation" means that one or more asparagine residues in an antibody have been derivatized, eg, aspartic acid or iso-aspartic acid.

The term "aggregated" antibody is an antibody that has been found to aggregate with other antibody molecules, especially after freezing and/or agitation.

The term "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, after storage. The shelf life is generally selected based on the shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected time. Stability can be assessed qualitatively and/or quantitatively in many different ways, including assessing aggregate formation (eg, by size exclusion chromatography, by measuring turbidity, and/or by visual observation); by use of cation exchange chromatography or capillary partition electrophoresis to assess charge heterogeneity; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping analysis; assessment of biological activity or antigen-binding function of antibodies; etc. Instability may include any one or more of the following: aggregation, deamidation (eg, Asn deamidation), oxidation (eg, Met oxidation), isomerization (eg, Asp isomerization), shearing Cleavage/hydrolysis/fragmentation (eg hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, differential glycosylation, etc.

The term "buffer" or "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to: histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer, or the like mixture, etc. The pH of the buffer is adjusted with acids or bases known in the art, the pH can be adjusted to a value in the range of 4.5-6.0, in particular to a value in the range of 4.5-5.5, most particularly to pH 5.5. 2.

The term "stabilizer" means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application. Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, and the like.

The term "surfactant" refers to a pharmaceutically acceptable excipient used to protect a protein formulation against physical stress such as stirring and shearing. Pharmaceutically acceptable surfactants include: polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (such as those sold under the trademark Brij ^™ ) and polyoxyethylene-polyoxypropylene Copolymer (Poloxamer, Pluronic). Polyoxyethylene sorbitan-fatty acid esters include polysorbate 20 (sold under the trademark Tween 20 ^™ ) and polysorbate 80 (sold under the trademark Tween 80 ^™ ).

The term "combination drug" refers to a combination comprising two or more pharmaceutical formulations, each having an active ingredient, which is required to be used in combination when administered to a subject. The active ingredients can be mixed together to form a single administration unit, or they can be separately formed into administration units and used separately.

The term "effective amount" refers to the dose of a pharmaceutical formulation of an antibody or fragment of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in the treated patient. An effective amount can be readily determined by the attending physician, who is skilled in the art, by taking into account a variety of factors such as ethnic differences; weight, age, and health; the specific disease involved; the severity of the disease; the individual patient's response; The particular antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapy.

The term "kit" includes an effective amount of one or more pharmaceutical formulations or combinations of the invention in unit dosage form. In some embodiments, the kit may contain a sterile container of the therapeutic or prophylactic composition; such container may be a box, ampule, bottle, vial, tube, bag, blister pack, or other suitable known in the art container form. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding drugs. In addition, the kit also includes instructions for administering the pharmaceutical formulation or combination of the present invention to an individual. A method of treating or preventing a disease using the pharmaceutical formulation or combination of the present invention is generally included in the specification.

Methods of producing and purifying antibodies and antigen-binding fragments are well known and available in the art, eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15.

The engineered antibodies or antigen-binding fragments thereof of the present invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian-like expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.

The term "individual" or "subject" as used herein refers to any animal, such as a mammal or a marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (eg, cynomolgus or rhesus monkeys or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and any species of poultry.

The term "tumor" as used herein refers to a disease characterized by the pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). The tumors described in the present invention may include, but are not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, Acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, severe Chain disease, solid tumors such as sarcomas and cancers (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, Bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, neuroma Schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, the "tumor" includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

As used herein, the terms "disease" or "condition" or "disorder" and the like refer to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, "diseases" include, but are not limited to, tumors, pathogen infections, autoimmune diseases, T cell dysfunctional diseases, or defective immune tolerance (e.g., transplant rejection).

The term "treatment", as used herein, refers to clinical intervention in an attempt to alter an individual or to manipulate a cell-induced disease process, either prophylactically or in a clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, improving or relieving the condition, relieving or improving the prognosis, etc.

The term "pharmaceutical composition" as used herein refers to a mixture comprising one or more of the compounds described herein, or a physiological/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiological/pharmaceutically acceptable salts or prodrugs. Pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

Example

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Example 1: Cloning and expression of PD-L1/TGFβ bifunctional molecules

The structure of the bifunctional molecule is to connect the extracellular domain of TGFβRII protein at the C-terminus of the heavy chain of the PD-L1 antibody, as shown in Figure 1. The full length of the extracellular domain of TGFβRII protein is composed of 136 amino acids. It has been found that truncation of the N-terminal less than 27 amino acids will not affect the binding ability of TGFβ cytokines, nor its function, and can improve the dual function. The stability of the molecule, therefore, is preferably 17-27 amino acid truncation, more preferably the N-terminal truncated 22 amino acid form, wherein the full-length sequence ECD (1-136) of the extracellular region of the TGFβRII protein is as SEQ ID NO: 1 As shown, the N-terminal truncated 22 amino acid sequence ECD (23-136) of the extracellular region of the TGFβRII protein is shown in SEQ ID NO: 2.

The CDR sequences of the light chain and heavy chain variable regions of the PD-L1 antibody are as follows:

The sequence of LCDR1 is shown in SEQ ID NO: 9;

The sequence of LCDR2 is shown in SEQ ID NO: 10;

The sequence of LCDR3 is shown in SEQ ID NO: 11;

The sequence of HCDR1 is shown in SEQ ID NO: 12;

The sequence of HCDR2 is shown in SEQ ID NO: 13;

The sequence of HCDR3 is shown in SEQ ID NO:14.

The light chain variable region sequence of the PD-L1 antibody is shown in SEQ ID NO: 15; the heavy chain variable region sequence is shown in SEQ ID NO: 16.

The light chain sequence of the PD-L1 antibody is shown in SEQ ID NO: 5; the heavy chain sequence is shown in SEQ ID NO: 6.

The extracellular domain of different truncated forms of TGFβRII protein is linked to the C-terminus of the heavy chain of the PD-L1 antibody through (G ₄ S) _x G, and the K at the last position of the C-terminus of the heavy chain of the original antibody is mutated to A, and the light Chain together, expressed by HEK293E or expi293 cell system, to obtain bifunctional molecules as shown in Table 1 below:

Table 1 Structure description of each bifunctional molecule

双功能分子例Examples of bifunctional molecules	序列描述sequence description	N端截短氨基酸数Number of N-terminal truncated amino acids
双功能分子1 bifunctional molecule 1	anti-PDL1-(G ₄S) ₄G-ECD(18-136,N19S) anti-PDL1-(G ₄ S) ₄ G-ECD(18-136,N19S)	1717
双功能分子2bifunctional molecule 2	anti-PDL1-(G ₄S) ₄G-ECD(19-136,N19S) anti-PDL1-(G ₄ S) ₄ G-ECD(19-136,N19S)	1818
双功能分子3bifunctional molecule 3	anti-PDL1-(G ₄S) ₄G-ECD(20-136) anti-PDL1-(G ₄ S) ₄ G-ECD(20-136)	1919
双功能分子4bifunctional molecule 4	anti-PDL1-(G ₄S) ₄G-ECD(21-136) anti-PDL1-(G ₄ S) ₄ G-ECD(21-136)	2020
双功能分子5 bifunctional molecule 5	anti-PDL1-(G ₄S) ₅G-ECD(21-136) anti-PDL1-(G ₄ S) ₅ G-ECD(21-136)	2020
双功能分子6bifunctional molecule 6	anti-PDL1-(G ₄S) ₅G-ECD(23-136) anti-PDL1-(G ₄ S) ₅ G-ECD(23-136)	22twenty two
双功能分子7 bifunctional molecule 7	anti-PDL1-(G ₄S) ₅G-ECD(26-136) anti-PDL1-(G ₄ S) ₅ G-ECD(26-136)	2525
双功能分子8bifunctional molecule 8	anti-PDL1-(G ₄S) ₅G-ECD(28-136) anti-PDL1-(G ₄ S) ₅ G-ECD(28-136)	2727

Note: ECD(n-136) in the sequence is the truncated form of the extracellular domain of TGFβRII protein, n is the sequence number of the truncated starting amino acid; N19S means that the 19th amino acid is mutated to S.

The light chain sequence of the above-mentioned bifunctional molecule 6 is shown in SEQ ID NO: 7, and the overall sequence of the heavy chain and the N-terminal truncated form of the extracellular region of TGFβRII is shown in SEQ ID NO: 8.

The bifunctional molecules expressed in cells were purified by Example 2, and the resulting proteins were used in the experiments described below.

Example 2: Purification of PD-L1/TGFβ bifunctional molecules

The cell culture medium was centrifuged at 4500 g for 30 min to collect the supernatant, and filtered with a 0.22 μm filter. The supernatant was purified using MabSelect SuRe Protein A column (GE Healthcare); the equilibration buffer was 1×PBS, equilibrated for 10 column volumes, and the cell supernatant was loaded with Protein A combined. and 0.1% Triton X114 in 1×PBS for 10 column volumes, then with 1×PBS for 10 column volumes, then with 100mM sodium acetate (pH3.5) elution buffer, according to A280 UV The eluted sample was collected from the absorption peak, and the collected eluted sample was neutralized with 1 M Tris-HCl (pH 9.0).

The neutralized eluted samples were filtered with a 0.22 μm filter membrane, concentrated by ultrafiltration, and then subjected to molecular sieve chromatography with HiLoadTM26/600SuperdexTM200pg (GE Healthcare), the buffer was 1×PBS, and the target protein peaks were merged according to A280 ultraviolet absorption. The collected protein samples were identified by SEC-HPLC with a purity greater than 95%; the collected protein samples were detected by LAL (Endosafe nexgen-PTS) method for endotoxin, and the result was less than 1EU/mg.

In addition, the light chain sequence of the PD-L1 monoclonal antibody in the following experiments is shown in SEQ ID NO: 5; the heavy chain sequence is shown in SEQ ID NO: 6. In the following experiments, the positive control molecule is SHR-1701, which is a bifunctional molecule targeting PD-L1 and TGFβ of Hengrui Medicine. The sequence is shown in Patent WO2018205985A1. Specifically, its light chain sequence is as shown in SEQ ID NO:3. As shown, the overall sequence of the heavy chain and the N-terminal truncated form of the extracellular region of TGFβRII is shown in SEQ ID NO: 4. Expression purification was also performed as described above.

The following Examples 3-5 are binding activity evaluation experiments, Examples 6-7 are cell function evaluation experiments, Example 8 is a pharmacokinetic evaluation experiment, and Examples 9-10 are in vivo pharmacodynamic evaluation experiments.

Example 3: ELISA detection of PD-L1/TGFβ bifunctional molecule combined with human PD-L1 experiment

The experimental procedure is described as follows:

a) Coat a 96-well plate with human PD-L1 protein (purchased from Sino biological, Cat. No. 10084-H08H) at a concentration of 1 μg/mL, 100 μL per well, and place overnight at 4°C;

b) Wash the plate 3 times with 200 μL PBST per well, add 200 μL blocking reagent 1% BSA, and incubate at 37°C for 1.5 hours;

c) Wash the plate 3 times with 200 μL of PBST per well, add 100 μL of progressively diluted PD-L1/TGFβ bifunctional molecule, positive control SHR-1701 and PD-L1 mAb, and incubate at room temperature for 2 hours;

d) Wash the plate 3 times with 300 μL PBST per well, add 100 μL goat anti-human Fc (HRP) secondary antibody (purchased from abcam, product number ab98624), dilute 1:20000, and incubate at room temperature for 1 hour;

e) Wash the plate 6 times with 300 μL of PBST per well, add 100 μL of TMB, and place it in the dark for 6 minutes, then add 100 μL of stop solution to stop the color reaction;

f) The M5 plate reader of MD Company detects the absorbance value at the wavelength of 450 nm, and uses the softmax software to process the data.

The experimental results of ELISA binding are shown in Figure 2. The bifunctional molecule 6 retains the binding activity to human PD-L1, and the binding ability is comparable to that of the positive control molecule.

Example 4: ELISA detection of PD-L1/TGFβ bifunctional molecule combined with human TGFβ1 experiment

The experimental procedure is described as follows:

a) Coat a 96-well plate with human TGFβ1 protein (purchased from CST, catalog number #8915) at a concentration of 1 μg/mL, 100 μL per well, and place overnight at 4°C;

The experimental results of ELISA binding are shown in Figure 3. The ability of bifunctional molecule 6 to bind human TGFβ1 protein is comparable to that of the positive control molecule.

Example 5: Biacore detects the affinity and kinetic properties of PD-L1/TGFβ bifunctional molecule and antigen

The affinity and kinetic properties of the bifunctional molecule to human PD-L1 and human TGFβ1 were analyzed using a Biacore 8K instrument. The CM5 chip was first activated with EDC and NHS, then anti-human Fc mouse mAb was immobilized, and then blocked with ethanolamine.

To determine the affinity and kinetic properties of human PD-L1, the bifunctional molecule was diluted to 5 μg/mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer at 10 μL/mL The flow rate of min was captured for 30s. Human PD-L1 was diluted to serial concentrations (5nM-0.078nM) two-fold stepwise, and was bound for 180s at a flow rate of 30μL/min, and dissociated for 300s.

To determine the affinity and kinetic properties with human TGFβ1, the bifunctional molecule was diluted to 1 μg/mL with HBS-EP+ buffer and captured at a flow rate of 10 μL/min for 60 s. Human TGFβ1 was two-fold serially diluted to serial concentrations (2nM-0.00156nM), bound for 120s at a flow rate of 30μL/min, and dissociated for 1200s.

After each round of experiments, rinse with 3M MgCl ₂ solution at a flow rate of 30 μL/min for 30 s to remove the captured antibody together with the antigen to complete the regeneration of the chip. The original data was analyzed using Biacore Insight Evaluation Software (version 2.0.15.12933), and the fitting model was 1:1. The experimental data are shown in Figure 4, and the obtained affinity and kinetic properties are shown in Table 2 below. The results show that the bifunctional Molecules 6 have high affinity for human PD-L1 and human TGFβ1.

Table 2 Affinity data of 6 bifunctional molecules

Ligand(配体)Ligand (ligand)	Analyte(分析物)Analyte	ka(1/Ms)ka(1/Ms)	kd(1/s)kd(1/s)	KD(M)KD(M)
双功能分子6bifunctional molecule 6	人源PD-L1Human PD-L1	1.41E+061.41E+06	7.44E-047.44E-04	5.26E-105.26E-10
双功能分子6bifunctional molecule 6	人源TGFβ1Human TGFβ1	7.44E+087.44E+08	3.65E-043.65E-04	4.90E-134.90E-13

Example 6: Experiment to detect bifunctional molecules blocking PD-1/PD-L1 signaling pathway

The biological activity of the molecule was detected by reporter gene method, and CHO cells transfected with PD-L1 and anti-CD3-single-chain antibody fragment (scFv) were used as target cells to transfect PD-1 and NFAT element-regulated CHO cells. Jurkat cells with the luciferase gene were used as effector cells. After the anti-CD3-scFv on the CHO cell membrane binds to CD3 on the surface of Jurkat cells, it will present an activation signal to Jurkat cells, thereby expressing luciferase; PD-L1 on the surface of CHO cells binds to PD-1 on the surface of Jurkat cells. The inhibitory signal is delivered to Jurkat cells to inhibit the expression of luciferase; while the bifunctional molecule can block the binding of PD-1 and PD-L1, thereby releasing the delivery of the inhibitory signal, restoring the expression of luciferase, and generating a fluorescent signal.

The experimental procedure is described as follows:

a) CHO-PDL1-CD3L cells were harvested, washed and resuspended to 4E5 cells/mL;

b) Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight in a 37°C, 5% CO ₂ cell incubator;

c) Dilute the molecule to be tested twice from 200nM to 10 concentration points;

d) Jurkat-PD1-NFAT cells were harvested, washed and resuspended to 1E6 cells/mL;

e) Take out the 96-well plate containing CHO-PDL1-CD3L cells from the incubator, remove 95 μL of supernatant from each well, and add 50 μL of the molecule solution to be tested;

f) Add 50 μL of Jurkat-PD1-NFAT cells and incubate for 6 h at 37°C, 5% CO ₂ cell incubator;

g) Add 50 μL of Bio-Glo Luciferase staining solution to each well, and read the chemiluminescence signal.

The results are shown in Figure 5. The blocking ability of the bifunctional molecule 6 to PD-1/PD-L1 is comparable to that of the positive control molecule SHR-1701, and also to that of the PD-L1 mAb.

Example 7: Experiment to detect bifunctional molecules blocking TGFβ/SMAD signaling pathway

The SBE Reporter-HEK293 cell line purchased from BPS Bioscience was used to monitor the activity of TGFβ/SMAD signaling pathway. TGFβ protein binds to cell surface receptors and initiates a signaling cascade leading to phosphorylation and activation of SMAD2 and SMAD3, which then form a complex with SMAD4. The SMAD complex then translocates to the nucleus and binds to the SMAD-binding element (SBE) in the nucleus, resulting in the transcription and expression of TGFβ/SMAD-responsive genes, including the luciferase gene transfected into the cell, resulting in a fluorescent signal. The bifunctional molecule contains TGFβRII fusion protein, which can prevent the binding of TGFβ protein to cell surface receptors, thereby inhibiting the expression of fluorescent signals.

The experimental procedure is described as follows:

a) 25,000 SBE Reporter-HEK293 cells were added to each well of a 96-well plate;

b) Incubate for 24h in a 37°C, 5% _CO2 cell incubator;

c) adding serially diluted antibody molecules of different concentrations;

d) TGFβ protein was added after 4 hours, the final concentration was 10ng/mL;

e) Incubate overnight in a 37°C, 5% _CO2 cell incubator

f) Add 100 μL of ONE-Step ^™ Luciferase reagent to each well, shake at room temperature for 15-30 min, and read the fluorescent signal.

The results are shown in Fig. 6. The blocking ability of multiple bifunctional molecules on the TGFβ/SMAD signaling pathway is similar and comparable to that of the positive control molecule SHR-1701.

Example 8: Pharmacokinetic experiment in cynomolgus monkeys

Two cynomolgus monkeys, one female and one male, were used for the pharmacokinetic experiments of each molecule, and they were fasted for more than 12 hours before administration and 4 hours after administration, with free access to water. The dosage of bifunctional molecule and positive control was 10mg/kg, and the intravenous infusion was completed in 30min. Blood collection time points were Pre-dose, 30min, 1hr, 6hr, 24hr, 2d, 4d, 7d, 10d, 14d, 21d, 28d, 35d, 42d. After the whole blood was collected, it was left to stand at room temperature for half an hour, and the supernatant was collected by centrifugation (6000 rpm, 8 minutes, 4°C) to collect serum.

The concentration of drug molecules in serum was detected by ELISA, and the detection process was described as follows:

a) Coat a 96-well plate with human PD-L1 protein at a concentration of 1 μg/mL, 100 μL per well, and place overnight at 4°C;

b) Wash the plate 3 times with 200 μL PBST per well, add 300 μL blocking reagent 5% milk powder, and incubate at 37°C for 1 hour;

c) Wash the plate 3 times with 200 μL of PBST per well, add 100 μL of standard, QC and samples to be tested, and incubate at 37°C for 1 hour;

d) Wash the plate 6 times with 300 μL PBST per well, add 100 μL anti-human TGFβRII biotinylated antibody (purchased from R&D, product number BAF241), dilute 1:5000, and incubate at 37°C for 1 hour;

e) Wash the plate 6 times with 300 μL PBST per well, add 100 μL Streptavidin HRP (purchased from BD, Cat. No. 554066), dilute 1:10000, and incubate at 37°C for 1 hour;

f) Wash the plate 6 times with 300 μL of PBST per well, add 100 μL of TMB, and place in the dark for 6 minutes, then add 100 μL of stop solution to stop the color reaction;

g) The M5 plate reader of MD Company detects the absorbance value at the wavelength of 450 nm, and uses the softmax software to process the data.

The measured monkey serum concentration was calculated by Phoenix Winnolin software to obtain pharmacokinetic parameters, as shown in Table 3 below.

Table 3 Monkey serum concentrations and PK parameters of bifunctional molecule 6

The units of the concentrations in the table are all μg/mL, and BLQ is below the detection limit.

Table 4 Monkey serum concentrations and PK parameters of positive controls

The units of concentrations in the above table are all μg/mL, and BLQ is below the detection limit.

The results show that the half-life of bifunctional molecules in monkeys is about 164 hours. Since PD-L1 is a target with a relatively fast internalization rate, bifunctional molecules have target-mediated drug clearance in vivo, and there are certain immunogens. Therefore, the bifunctional molecule has good pharmacokinetic properties, stable properties in cynomolgus monkeys, and no obvious off-target binding.

At the same time, we found that the half-life of bifunctional molecule 6 in monkeys was twice that of the positive control molecule SHR-1701 (see Table 4). The reason may be that the bifunctional molecule 6 is of the IgG1 subtype and the positive control is of the IgG4 subtype, and antibodies of the IgG1 subtype are generally considered to be more stable in vivo than those of the IgG4 subtype. Therefore, bifunctional molecule 6 is expected to achieve a longer half-life than the positive control in subsequent clinical experiments, further reducing the frequency of administration and the cost of medication.

Example 9: Evaluation of the tumor inhibition rate of bifunctional molecules in the subcutaneous xenograft model of human breast cancer MDA-MB-231 mice

This experiment evaluates the antitumor effect of the test drugs in the subcutaneous transplantation of human breast cancer cells MDA-MB-231 mixed PBMC in NCG mice.

MDA-MB-231 cells were cultured in L-15 medium containing 10% fetal bovine serum (FBS). MDA-MB-231 cells in logarithmic growth phase were collected and resuspended in HBSS to an appropriate concentration for subcutaneous tumor inoculation of NCG mice. Normal human peripheral blood was taken, and human PBMCs were separated by density gradient centrifugation. The separated PBMCs were added to MDA-MB-231 cells treated with Mitomycin C, and PBMCs and MDA-MB-231 cells were co-cultured for 6 days. IL-2 and 10% FBS in RPMI 1640 medium. After culturing for 6 days, the cultured PBMCs were collected, and the PBMCs were mixed with freshly digested MDA-MB-231 cells and inoculated into the right subcutaneous of 64 NCG mice (Jiangsu JiCui Yaokang Biotechnology Co., Ltd.), with 8 mice in each group. 8 groups. After inoculation, the mice were randomly administered into groups according to their body weight. The detailed administration method, dose and route of administration were shown in Table 5. The day of group administration was Day 0.

Table 5 Dosing schedule of each group

The tumor volume was measured twice a week using a vernier caliper, and the tumor volume was calculated with the formula V=0.5a×b ² , where a and b represent the long and short diameters of the tumor, respectively. Tumor growth inhibition rate TGI (%)=[1-(T _i -T ₀ )/( _V _i -V ₀ )]×100, where Ti is the average tumor volume after the compound group started to be administered, and T ₀ is the compound The mean tumor volume of the group at the first administration, V ₀ is the mean tumor volume of the vehicle control group at the first administration, and _Vi is the mean tumor volume of the vehicle control group after the start of administration. Body weights of all tumor-bearing mice were measured twice a week. At the same time, the change rate of the body weight of the mice after administration was calculated: the weight change used the formula RCBW%=(BW _i -BW ₀ )/BW ₀ ×100%, BW _i was the current body weight of the mice, and BW ₀ was the body weight of the mice on the day of the grouping . After the experiment, the tumor mass was weighed and photographed. The results are shown in Table 6 below:

Table 6 Experimental results of each group

The results of tumor weighing are shown in Figure 7. It can be seen that the bifunctional molecule 6 has obvious tumor inhibitory effect. At the doses of 5mg/kg, 10mg/kg and 20mg/kg, the tumor inhibition rates were 59.92%, 62.78% and 71.48%, respectively. The tumor inhibition rates increased with the dose. increase with the increase. At the level of equimolar dose, the efficacy of bifunctional molecule 6 is better than that of PD-L1 monoclonal antibody and the combination of PD-L1 monoclonal antibody and TGFbRII fusion protein. At the same time, the mice in the bifunctional molecule 6 administration group did not die, and the weight of the mice increased steadily, indicating that the bifunctional molecule 6 is safe in mice.

Example 10: Evaluation of the tumor inhibition rate of bifunctional molecules in the human colon cancer MC-38 mouse subcutaneously transplanted tumor model

In this experiment, the tumor inhibitory effect of bifunctional molecules on PD-L1 humanized mouse MC38 (humanized PD-L1) colon cancer model was evaluated and compared with the positive control antibody SHR-1701.

MC38 (humanized PDL1) cells were subcutaneously inoculated into the right axilla of PD-L1 humanized mice at 1×10 ⁶ cells/0.1 mL, totaling 60 mice. When the average tumor volume reached 60-120 mm ³ , 40 mice with moderate tumor volume were selected into the group and randomly divided into 5 groups (8 mice in each group) according to the tumor volume: the first group was IgG1 isotype antibody (20 mg/kg) , The second group is bifunctional molecule 6 (5mg/kg), the third group is bifunctional molecule 6 (10mg/kg), the fourth group is bifunctional molecule 6 (20mg/kg), and the fifth group is positive control SHR -1701 (20mg/kg). The animals were grouped on the day of administration, and the administration volume was 10 mL/kg, and the administration method was intraperitoneal injection (ip). 2 times a week for a total of 6 doses.

During the experiment, the body weight and tumor growth status of the experimental animals were continuously observed, the tumor was measured and weighed twice a week, and the tumor volume and tumor growth inhibition rate were calculated. At the end of the test (the 4th day after the last administration), tumor tissue was collected and photographed, the tumor tissue was weighed and the tumor weight inhibition rate was calculated (see Figure 8).

The results showed that the body weight of animals in each group increased steadily during the test period, and no obvious effect of the test drug on the daily activities and body weight of the animals was observed. As shown in Figure 8, the tumor growth curve showed that the bifunctional molecule 6 could significantly inhibit the growth of the MC38 tumor model at doses of 5, 10 and 20 mg/kg (P<0.01), and showed a significant dose-dependent manner. Tumor growth inhibition rates at endpoint were 41.0%, 52.9%, and 60.8%, respectively. At the same time, at the same dose (20 mg/kg), bifunctional molecule 6 and positive control antibody SHR-1701 had comparable tumor inhibitory effects (P>0.05), and their TGIs were 60.8% and 63.2%, respectively.

The tumor tissue weight at the end of the experiment showed that the bifunctional molecule 6 could significantly increase the tumor tissue growth at doses of 5, 10 and 20 mg/kg (P<0.01), and showed a significant dose-dependence, and its tumor weight inhibition rates were respectively were 43.2%, 54.4% and 64.8%. At the same time, the bifunctional molecule 6 and the positive control antibody SHR-1701 showed similar anti-tumor activity (P>0.05) at the same dose (20 mg/kg), and their tumor weight inhibition rates (IR%) were 64.8% and 61.0, respectively. %.

Claims

A bifunctional molecule targeting both PD-L1 and TGFβ, characterized in that it comprises a PD-L1-targeting part and a TGF-β receptor part, and the PD-L1-targeting part is a PD-L1 antibody The TGF-β receptor part is the N-terminal truncated form of the extracellular region of TGF-β receptor 2TGFβRII, and the C-terminus of each heavy chain of the PD-L1 antibody is connected to an N-terminal of the extracellular region of TGFβRII Truncated form, the light chain and heavy chain variable region CDR sequences of the PD-L1 antibody are as follows:

The sequence of LCDR1 is shown in SEQ ID NO: 9;

The sequence of LCDR2 is shown in SEQ ID NO: 10;

The sequence of LCDR3 is shown in SEQ ID NO: 11;

The sequence of HCDR1 is shown in SEQ ID NO: 12;

The sequence of HCDR2 is shown in SEQ ID NO: 13;

The sequence of HCDR3 is shown in SEQ ID NO:14.
The bifunctional molecule of claim 1, wherein the full-length sequence of the extracellular region of TGFβRII is shown in SEQ ID NO: 1, and the N-terminal truncated form is a truncation of 17-27 amino acids.
The bifunctional molecule according to any one of claims 1-2, wherein the sequence of the TGF-β receptor part is shown in SEQ ID NO: 2.
The bifunctional molecule according to any one of claims 1-3, wherein: the light chain variable region sequence of the PD-L1 antibody is shown in SEQ ID NO: 15; the heavy chain variable region sequence is shown in SEQ ID NO: 15 ID NO: 16.
The bifunctional molecule according to any one of claims 1-4, wherein the light chain sequence of the PD-L1 antibody is shown in SEQ ID NO: 5; the heavy chain sequence is shown in SEQ ID NO: 6 , and the last K at the C-terminus of the heavy chain is mutated to A.
The bifunctional molecule according to any one of claims 1-5, wherein the C-terminus of the heavy chain of the PD-L1 antibody is linked to the N-terminal truncated form of the extracellular region of TGFβRII through a linking peptide.
The bifunctional molecule according to claim 6, wherein the connecting peptide is (G 4 S) X G, and the x is 3-6, preferably 4-5.
The bifunctional molecule according to any one of claims 1-7, wherein the light chain sequence of the PD-L1 antibody is shown in SEQ ID NO: 7, and the heavy chain is truncated with the N-terminus of the extracellular region of TGFβRII The sequence of the entire short form is shown in SEQ ID NO:8.
A pharmaceutical composition, characterized in that it comprises the bifunctional molecule according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
A nucleic acid molecule, characterized in that it encodes the bifunctional molecule according to any one of claims 1-8.
An expression vector, characterized in that: it contains the nucleic acid molecule of claim 10.
A host cell is characterized in that: it comprises the described expression vector of claim 11, and the host cell is selected from bacteria, yeast and mammalian cells; preferably mammalian cells; more preferably HEK293E cells, expi293 or CHO cell.
Use of the bifunctional molecule according to any one of claims 1-8 in the preparation of a medicament for treating cancer.
The use of claim 13, wherein the cancer is a PD-L1 positive tumor.
The use of claim 13, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, melanoma, kidney cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, and pancreatic cancer and head and neck tumors.
A method for treating and preventing tumors, comprising administering to a patient in need a therapeutically effective amount of the bifunctional molecule according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9.