RU2791683C2 - Pharmaceutical composition of the fusion protein of the transforming growth factor beta receptor and its application - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceuticals and medicine. The inventions are a pharmaceutical composition for the treatment of a tumor containing from 0.5 mg/ml to 100 mg/ml of the fused protein of the transforming growth factor beta receptor (TGF-β), from 5 mM to 30 mM citrate buffer, from 50 mg/ml to 100 mg/ml sucrose or trehalose and from 0.1 mg/ml to 0.8 mg/ml of polysorbate 80; where the fused protein of the TGF-β receptor contains a fused peptide formed by the heavy chain of the anti-PD-L1 antibody and the extracellular domain (ECD) TGF-βRII, with the sequence SEQ ID NO: 23, and the light chain of the anti-PD-L1 antibody with the sequence SEQ ID NO: 13, and the structure of the fused protein of the TGF-β receptor is shown in dwg. 1; the pH of the pharmaceutical composition is from 6.0 to 6.5; and the method for its preparation. The inventions also represent a lyophilized preparation for the treatment of a tumor, which is obtained by lyophilization of a pharmaceutical composition; a reconstituted solution, which is obtained by restoring a lyophilized drug; a product for the treatment of a tumor and the use of a pharmaceutical composition to obtain a drug.

EFFECT: physical and chemical stability of the composition for the treatment of a tumor.

23 cl, 8 dwg, 18 tbl, 21 ex

Description

This application claims priority of patent application 201811328326.1, filed November 9, 2018, which is incorporated herein by reference in its entirety.

Field of invention

The present disclosure belongs to the field of a pharmaceutical preparation and, in particular, relates to a pharmaceutical composition containing an antibody fusion protein against PD-L1 (programmed cell death ligand 1)/extracellular region of TGF-βRII (transforming growth factor receptor II beta), and to its use as a medicinal product.

Prior Art

The statements in this document only provide background information relevant to the present disclosure and do not necessarily constitute prior art.

People have realized that during the treatment of tumors, the high toxicity associated with chemotherapy and chemotherapy can lead to the formation of drug-resistant cancer cells. Even with targeted therapies that target overexpressed or overactivated proteins associated with tumor survival and growth, there will still be cancer cells that mutate to decrease or evade the pathways targeted by the targeted therapies, and these cancer cells would survive by other ways.

Tumor immunotherapy has received considerable attention in recent years and is a priority in the field of tumor treatment. An outstanding advantage of such therapy is the increased difficulty in developing drug resistance. Tumor immunotherapy mainly uses immunological principles and methods to improve the immunogenicity of tumor cells and the sensitivity to killing by effector cells, and to stimulate and enhance the anti-tumor immune response in the body. Tumor immunotherapy involves the infusion of immune cells and effector molecules into the host, and these two components work with the immune system to kill tumors and inhibit tumor growth in the body.

Programmed death receptor 1 (PD-1) is a member of the CD28 superfamily. PD-1 is expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands: programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 interacts with the PD-1 receptor on T cells and plays an important role in downregulating the immune response. Expression of the PD-L1 protein can be detected in many human tumor tissues. The microenvironment at the tumor site can induce PD-L1 expression on tumor cells, and expressed PD-L1 in turn promotes tumorigenesis and growth, and induces T cell apoptosis against the tumor. PD-1/PD-L1 pathway inhibitors block the binding of PD-1 to PD-L1, block negative regulatory signals, restore T cell activity, and enhance the immune response. Therefore, PD-1/PD-L1 immunomodulation as a target is very important for tumor suppression.

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

TGF-β is a multifunctional cytokine that exerts a tumor-suppressing or tumor-stimulating effect in a cell dependent or background dependent manner. The tumor-suppressing effect of TGF-β depends on the ability to induce the expression of many genes. By introducing mutations or epigenetic modifications during tumor development, cancer cells gradually become tolerant to the inhibitory effect of TGF-β, which ultimately leads to tumor development.

Studies have found that blocking the TGF-β signaling pathway can reduce tumor metastasis. It was found that the metastatic ability of tumor cells was inhibited when the TGF-β signaling pathway of breast tumor cell lines was inhibited by the Smad2/3 negative mutant with truncation. In a colon cancer microsatellite instability study, it was found that an inactivating TGF-βRII mutation reduced metastases and increased the postoperative survival rate of patients. However, in general, this effect is weak when TGF-β signaling pathway inhibitor alone is administered in clinical treatment, possibly because TGF-β is mainly abnormally expressed in tumor cells, while TGF-β signaling pathway inhibitor alone It is difficult for TGF-β signaling to be targeted to the tumor, resulting in low efficacy or low bioavailability of this inhibitor.

Therefore, based on the targeting and neutralization of TGF-β in the tumor microenvironment, inhibition of the PD-1/PD-L1 pathway can restore T cell activity, enhance the immune response, and more effectively improve the inhibitory effect on tumorigenesis and development.

According to the previous PCT application of this applicant - PCT/CN2016/104320 (publication number WO 2017084495) proposed an antibody against PD-L1. An antibody/TGF-β receptor fusion protein has now been published, such as, for example, in WO 2006074451 A2, WO 2009152610 A1, WO 2011109789 A2, WO 2013164694 A1, WO 2014164427 A1, WO 20150797540 A2, WO 20150797540 A2, WO 81815 A91,2 , WO 2015077540 A2, WO 2015118175 A2, etc. Among them, Merck discloses a PD-L1/TGF-β bifunctional fusion protein, bintrafusp alpha (WO 2015118175, also known as M7824, FP17022). Currently, bintrafusp alfa is in the phase of clinical research of tumor diseases such as gastric cancer, lung cancer, esophageal cancer, NSCLC (non-small cell lung cancer), gallbladder cancer. However, the antibody-based drugs of the prior art become unstable due to large molecular weights, complex structures, and are susceptible to degradation, polymerization, or unwanted chemical modifications. In order to make an antibody suitable for administration, to maintain stability during storage and subsequent use, and to have a better effect, it is especially important to study stable antibody drug formulations.

Brief summary of the invention

The present disclosure provides a pharmaceutical composition comprising a PD-L1/TGF-βRII fusion protein that is more conducive to manufacture and administration, and more stable in efficacy; this pharmaceutical composition includes:

- TGF-β receptor fusion protein and

- buffer,

wherein the buffer is selected from the group consisting of a histidine salt buffer, a succinate buffer, a phosphate buffer, and a citrate buffer.

In some embodiments, this buffer is a citrate buffer. In some embodiments, the histidine salt buffer is a histidine-hydrochloric acid buffer; and the succinate buffer is succinic acid-sodium succinate buffer; citrate buffer is a citric acid-sodium citrate buffer. In some embodiments, this buffer is a citric acid-sodium citrate buffer.

In an alternative embodiment, the concentration of the TGF-β receptor fusion protein in the pharmaceutical composition described above is from about 0.5 mg/mL to about 100 mg/mL, preferably from about 30 mg/mL to about 70 mg/mL.

In some embodiments, the concentration of the TGF-β receptor fusion protein in the pharmaceutical composition is 0.5 mg/mL to 100 mg/mL, preferably 30 mg/mL to 70 mg/mL. Non-limiting examples of TGF-β receptor fusion protein concentration include: about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg /ml, about 65 mg/ml, about 70 mg/ml, preferably about 50 mg/ml.

In some embodiments, the concentration of the TGF-β receptor fusion protein in the pharmaceutical composition is 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml, 65 mg/ml, 70 mg/ml, more preferably 50 mg/ml.

In an alternative embodiment, the pH of the buffer in the pharmaceutical composition described above is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5, and optionally about 6.0, about 6.1, about 6, 2, about 6.3, about 6.4, about 6.5, more preferably about 6.2.

In some embodiments, the buffer pH is 5.0 to 7.5 or 6.0 to 6.5, preferably 6.0; 6.1; 6.2; 6.3; 6.4 or 6.5, more preferably 6.2.

In an alternative embodiment, the buffer concentration is from about 5 mM to about 30 mM, preferably from about 5 mM to about 20 mM; its non-limiting examples include 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, more preferably 10 mM.

In some embodiments, the buffer concentration is from 5 mm to 30 mm, preferably from 5 mm to 20 mm; and in some embodiments, the buffer concentration is about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, and more preferably about 10 mM.

In an alternative embodiment, the pharmaceutical composition described above also contains a saccharide. "Saccharide" in the present disclosure includes conventional (CH ₂ O) _n compounds/compositions or derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing saccharides, non-reducing saccharides, and the like. In some embodiments, the saccharide is selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melesitose, melitriose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose and so on. The preferred saccharide is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.

In an alternative embodiment, the concentration of saccharide in the pharmaceutical composition described above is from about 50 mg/ml to about 100 mg/ml, preferably from about 60 mg/ml to about 90 mg/ml; non-limiting examples include 60 mg/ml, 65 mg/ml, 70 mg/ml, 75 mg/ml, 80 mg/ml, 85 mg/ml, 90 mg/ml, most preferably 80 mg/ml.

In some embodiments, the saccharide concentration is from 50 mg/ml to 100 mg/ml, preferably from 60 mg/ml to 90 mg/ml; and in some embodiments, the saccharide concentration is about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, or about 90 mg/ml.

In an alternative embodiment, the pharmaceutical composition described above further comprises a surfactant which may be selected from the group consisting of polysorbate 20, polysorbate 80, polyhydroxyalkylene, triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl sulfobetaine, myristyl- sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, laurelamidopropyl betaine, cocaamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearylamidopropyl betaine, myristamidopropyl dimethylamine, palmamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, ethylene propylene glycol copolymer, etc. The preferred surfactant is Polysorbate 80 or Polysorbate 20, more preferably Polysorbate 80.

In another alternative embodiment, the concentration of surfactant in the pharmaceutical composition described above is from about 0.1 mg/mL to about 0.8 mg/mL, more preferably from about 0.4 mg/mL to about 0.8 mg/mL. . In some embodiments, the concentration of the surfactant is from 0.1 mg/ml to 0.8 mg/ml, preferably from 0.4 mg/ml to 0.8 mg/ml, more preferably about 0.4 mg/ml, about 0.45 mg/ml, about 0.5 mg/ml, about 0.55 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml.

In some embodiments, the concentration of the surfactant is 0.4 mg/ml, 0.45 mg/ml, 0.5 mg/ml, 0.55 mg/ml, 0.6 mg/ml, 0.7 mg/ml or 0.8 mg/ml, more specifically 0.4 mg/ml.

In an alternative embodiment, the pharmaceutical composition described above contains:

(a) about 0.5 mg/mL to about 100 mg/mL TGF-β receptor fusion protein, (b) about 5 mM to about 30 mM citrate buffer, (c) about 50 mg/mL to about 100 mg/ml sucrose and (o!) about 0.1 mg/ml to about 0.8 mg/ml polysorbate 80, preferably the pH of the pharmaceutical composition is from about 5.0 to about 7.5, more preferably from about 6 .0 to about 6.5.

In an alternative embodiment, the pharmaceutical composition described above contains:

0.5 mg/mL to 100 mg/mL TGF-β receptor fusion protein

5 mM to 30 mM citrate buffer

50 mg/ml to 100 mg/ml sucrose and

0.1 mg/ml to 0.8 mg/ml polysorbate 80;

preferably the pH of this pharmaceutical composition is from 5.0 to 7.5, more preferably from 6.0 to 6.5.

In an alternative embodiment, the pharmaceutical composition described above contains:

(a) from about 30 mg/mL to about 70 mg/mL of TGF-β receptor fusion protein, (b) from about 5 mM to about 20 mM citric acid-sodium citrate buffer, (c) from about 60 mg/mL to about 90 mg/ml sucrose; and (d) about 0.4 mg/ml to about 0.8 mg/ml polysorbate 80, preferably the pH of the pharmaceutical composition is from about 6.0 to about 6.5.

In an alternative embodiment, the pharmaceutical composition described above contains:

30 mg/mL to 70 mg/mL TGF-β receptor fusion protein

5 mM to 20 mM citric acid-sodium citrate buffer

60 mg/ml to 90 mg/ml sucrose and

0.4 mg/ml to 0.8 mg/ml polysorbate 80;

The pH of this pharmaceutical composition is from about 6.0 to about 6.5.

In an alternative embodiment, this pharmaceutical composition contains:

(a) about 50 mg/ml TGF-β receptor fusion protein, (b) about 10 mM citric acid-sodium citrate buffer, (c) about 80 mg/ml sucrose, and (d) about 0.4 mg/ml polysorbate 80 , the pH of this pharmaceutical composition is preferably about 6.2.

In an alternative embodiment, this pharmaceutical composition contains:

50 mg/mL TGF-β receptor fusion protein

10 mM citric acid-sodium citrate buffer

80 mg/ml sucrose and

0.4 mg/ml polysorbate 80;

preferably the pH of the pharmaceutical composition is about 6.2.

In an alternative embodiment, the TGF-β receptor fusion protein in the pharmaceutical composition described above is shown as the general formula (I):

Ab-L-TGF-βRII ECD (I)

where the extracellular domain (ECD) of TGF-βRII is a truncated form of the extracellular region of TGF-βRII; Ab is an anti-PD-L1 antibody or antigen-binding fragment thereof;

L is a linker sequence.

In an alternative embodiment, the linker sequence in the pharmaceutical composition described above is (G ₄ S) _x G, where x is an integer of 3-6. In an alternative embodiment x is 3, 4, 5 or 6, preferably 4.

In an alternative embodiment, the truncated form of the TGF-βRII extracellular region is the TGF-βRII extracellular domain sequence (shown as SEQ ID NO: 14) with a deletion of at most 26 consecutive amino acid residues at the amino terminus (also referred to as the N terminus). In some embodiments, the truncated form of the extracellular region of TGF-βRII is the sequence of the extracellular domain of TGF-βRII with a deletion of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous amino acid residues per N end. In some embodiments, the ECD sequence of TGF-βRII in the pharmaceutical composition described above is shown as SEQ ID NO: 14, 15, 16 or 17; preferably this sequence is shown as SEQ ID NO: 15.

In an alternative embodiment, the anti-PD-L1 antibody, or antigen-binding fragment thereof, in the pharmaceutical composition described above comprises:

heavy chain complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; And

light chain complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In an alternative embodiment, the anti-PD-L1 antibody, or antigen-binding fragment thereof, in the pharmaceutical composition described above comprises:

HCDR1, HCDR2 and HCDR3, shown as SEQ ID NO: 1, SEQ ID NO: 10 and SEQ ID NO: 3, respectively; And

LCDR1, LCDR2 and LCDR3, shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

In an alternative embodiment, the anti-PD-L1 antibody, or antigen-binding fragment thereof, in the pharmaceutical composition described above comprises:

the heavy chain variable region shown as SEQ ID NO: 7, and

light chain variable region shown as SEQ ID NO: 8;

or contains:

the heavy chain variable region shown as SEQ ID NO: 9, and

light chain variable region shown as SEQ ID NO: 11.

In an alternative embodiment, the amino acid sequence of the heavy chain of the anti-PD-L1 antibody in the pharmaceutical composition described above is shown as SEQ ID NO: 12 or has at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity with the amino acid sequence shown as SEQ ID NO: 12; the amino acid sequence of the light chain of the anti-PD-L1 antibody and is shown as SEQ ID NO: 13 or has at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% a clear sequence identity with the amino acid sequence shown as SEQ ID NO: 13.

In an alternative embodiment of the pharmaceutical composition described above, in the TGF-β ECD receptor fusion protein, TGF-βRII is fused to the carboxyl terminus of the anti-PD-L1 antibody heavy chain via a linker sequence.

In some embodiments, the TGF-β receptor fusion protein comprises:

• a fusion peptide formed by a heavy chain of an anti-PD-L1 antibody fused to a TGF-βRII ECD, the sequence of which is shown as SEQ ID NO: 23 or has at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity with the sequence shown as SEQ ID NO: 23, and

• a light chain of an anti-PD-L1 antibody whose sequence is shown as SEQ ID NO: 13 or has at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% -sequence identity with the sequence shown as SEQ ID NO: 13.

In other embodiments, the TGF-β receptor fusion protein comprises:

• a fusion peptide formed by an anti-PD-L1 antibody heavy chain fused to a TGF-βRII ECD, the sequence of which is shown as SEQ ID NO: 24 or has at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity with the sequence shown as SEQ ID NO: 24, and

• an anti-PD-L1 antibody light chain whose sequence is shown as SEQ ID NO: 13 or has at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% -sequence identity with the sequence shown as SEQ ID NO: 13.

The present disclosure also provides a method for preparing a pharmaceutical composition as described above, which comprises the step of contacting a TGF-β receptor fusion protein with a buffer, for example by performing buffer exchange in a stock solution of the TGF-β receptor fusion protein, and this buffer is preferably a citrate buffer. ; more preferably a buffer citric acid-sodium citrate, and the concentration of this buffer is preferably from about 5 mm to about 20 mm; non-limiting examples include 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, more preferably 10 mM; The pH of this buffer is from about 6.0 to about 6.5, non-limiting examples include 6.0; 6.1; 6.2; 6.3; 6.4; 6.5; preferably 6.2. In an alternative embodiment, the buffer concentration is from 5 mM to 20 mM, non-limiting examples include about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, more preferably about 10 mM; The pH of this buffer is from 6.0 to 6.5, non-limiting examples include about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, preferably about 6, 2.

The present disclosure also provides a method for preparing the pharmaceutical composition described above, further comprising the following steps after contacting the TGF-β receptor fusion protein with a buffer: adding sucrose and polysorbate 80 to the resulting solution (there is no precedence order between them), then adjusting the volume using buffer, where the concentration of the buffer solution is preferably from about 5 mm to about 20 mm, more preferably from 5 mm to 20 mm, non-limiting examples include 5 mm, 8 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, 20 mM; The pH of the buffer is from about 6.0 to about 6.5, non-limiting examples include 6.0; 6.1; 6.2; 6.3; 6.4; 6.5.

The present disclosure also provides a method for preparing a lyophilized preparation containing a TGF-β receptor fusion protein, which includes the step of lyophilizing the pharmaceutical composition described above.

In an alternative embodiment, a method is provided for preparing a lyophilized preparation as described above containing a TGF-β receptor fusion protein, wherein the lyophilization is carried out according to a method known in the art, such as, for example, steps including, but not limited to, pre-freezing, primary drying, and secondary drying. Those of skill in the art will appreciate that any method for removing water from a pharmaceutical composition in the present disclosure is applicable to this disclosure.

The present disclosure also provides a lyfilized preparation containing a TGF-β receptor fusion protein, which is obtained by the method of preparing the lyfilized preparation described above.

The present disclosure also provides a lyfilized formulation containing a TGF-β receptor fusion protein that can be reconstituted to form the pharmaceutical composition described above.

In some embodiments, the lyophilized formulation may be stable at 2°C-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation may be stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

The present disclosure also provides a reconstituted solution containing a TGF-β receptor fusion protein, which is obtained by reconstituting a lyophilized preparation containing the TGF-β receptor fusion protein described above.

The present disclosure also provides a method for preparing a reconstituted solution containing the above-described TGF-β receptor fusion protein, which includes the step of reconstituting the above-described lyophilized preparation, wherein the solution used for reconstitution contains water for injection, saline, or glucose solution, but not limited to them, preferably water for injection.

The present disclosure further provides an article or kit comprising: a pharmaceutical composition of the present disclosure and container(s).

In some embodiments, the container is a glass bottle, such as, but not limited to, an injection bottle made from neutral borosilicate glass.

The present disclosure also provides an article of manufacture comprising container(s) which contain(s) the pharmaceutical composition described above, or a lyophilized preparation thereof, or a reconstituted solution of a lyophilized preparation.

The present disclosure also provides for the use of any of the following in the preparation of a medicament:

a pharmaceutical composition as described above, or a lyophilized preparation, or a reconstituted solution of this lyophilized preparation, or an article of manufacture; wherein the drug is used to treat or inhibit disease(s) or disorder(s) associated with tumor cell proliferation or metastasis.

In some embodiments, the disease(s) or disorder(s) is(are) a tumor.

In some embodiments, the disease(s) or disorder(s) is(are) selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer carcinoma, squamous cell skin carcinoma, elevated dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

The present disclosure also provides a method for treating or inhibiting a disease(s) or disorder(s) related to cancer cell proliferation or metastasis, comprising providing a therapeutically effective amount of a pharmaceutical composition as described above, or a lyophilized preparation, or a reconstituted solution, or article of manufacture to a subject. who needs it. In some embodiments, the method comprises administering to the subject a unit dose of a composition comprising 0.1 mg to 3000 mg of a TGF-β receptor fusion protein as described above, a pharmaceutical composition, or a lyophilized preparation, or a reconstituted solution, or article of manufacture. In some embodiments, the given disease(s) or disorders om (you and) is(are) a tumor. In some embodiments, the disease(s) or disorder(s) is(are) selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer. uterus, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, lung cancer, small intestine cancer, non-small cell lung cancer, melanoma, basal cell skin carcinoma, squamous cell skin carcinoma, elevated dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

The present invention also provides a TGF-β receptor fusion protein, pharmaceutical composition or lyophilized preparation or reconstituted solution or article of manufacture as described above for treating or inhibiting a disease(s) or disorder(s) related to cancer cell proliferation or metastasis. . In some embodiments, the disease(s) or disorder(s) is(are) a tumor. In some embodiments, the disease(s) or disorder(s) is(are) selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma. , lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, lung cancer, small intestine cancer, non-small cell lung cancer, melanoma, basal cell carcinoma cutaneous carcinoma, squamous cell cutaneous carcinoma, dermatofibrosarcoma eminentus, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

As is well known to those skilled in the art, one, some, or all of the characteristics of the various embodiments described in this disclosure may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by the combination are further illustrated in the following detailed description.

Description of graphic materials

Fig. 1: A schematic diagram showing the structure of a fusion protein.

Fig. 2: Results showing in vitro binding of fusion proteins to human TGF-β1.

Fig. 3: Results showing in vitro binding of fusion proteins to human TGF-β1.

Fig. 4: Results showing in vitro binding of fusion proteins to human PD-L1.

Fig. 5: result showing detection of blocking of the PD-1/PD-L1 pathway by fusion proteins in vitro.

Fig. 6: Fusion proteins inhibit TGFβ-induced pSMAD3 reporter activity in a dose-dependent manner.

Fig. 7. All fusion protein samples enhance the secretion of the cytokine IFN-γ by activated T-lymphocytes.

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

Detailed description of the invention

Terminology

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

The term "buffer" refers to a solution that is resistant to pH change through the action of acid-base conjugate components. Examples of buffers that can control the pH within a suitable range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, and glycylglycine.

"Histidine salt buffer" is a buffer containing histidine radical ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like; preferably histidine hydrochloride buffer. Histidine hydrochloride buffer is prepared from histidine and hydrochloric acid.

"Citrate buffer" is a buffer that contains citrate radical ions. Examples of citrate buffers include citric acid-sodium citrate, potassium citrate-citrate, calcium citrate-citrate, magnesium citrate-citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

"Succinate buffer" is a buffer that contains succinate radical ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate.

"Phosphate buffer" is a buffer that contains phosphate radical ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

"Acetate buffer" is a buffer containing acetate radical ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or its physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carrier(s) or excipient(s). The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient as an antibody, promote administration to the body, and facilitate absorption of the active ingredient so that it exhibits biological activity. Used in this document, the terms "pharmaceutical composition" and "preparation" are not mutually exclusive.

Unless otherwise specified, when referring to a solution form of a pharmaceutical composition described in this disclosure, the solvent used therein is water.

The term "lyophilized preparation" refers to a preparation or pharmaceutical composition obtained after a freeze-drying step (e.g., a vacuum freeze-drying step) of a pharmaceutical composition in its liquid or solution form, or lyophilization of a drug in its liquid or solution form.

The term "about" or "approximately" as used in this disclosure means that a given value is within the acceptable error range of a particular value as determined by those of ordinary skill in the art, and this value depends in part on whether how it is measured or defined (i.e. the limits of a measuring system). For example, the term "about" or "approximately" refers in the art to a standard deviation of less than one or greater than one. Alternatively, the term "about" or "approximately" or "substantially comprising" refers to a range of up to 20%. Also, in particular for biological systems or processes, the term means an order of magnitude of up to one or up to 5 times the given value. Unless otherwise specified, "about XX" or "about XX" or "substantially containing XX" as used in this disclosure refers to a value within the acceptable error range of a particular value of "XX" (including the value of "XX" itself, as well as values within an acceptable error range for this value as determined by those of ordinary skill in the art).

The pharmaceutical composition described in the present disclosure is capable of achieving a stable effect: the TGF-β receptor fusion protein or pharmaceutical composition thereof substantially retains physical stability and/or chemical stability and/or biological stability after storage; preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is usually determined based on the predetermined shelf life of the pharmaceutical composition. Currently, there are many analytical methods for measuring the stability of active ingredients, which can measure the stability after storage at a given temperature for a given period of time.

A stable pharmaceutical preparation of an antibody or protein is one for which no significant changes are observed under the following conditions: storage at a refrigerated temperature (2-8°C) for at least 3 months, preferably for 6 months, more preferably for 1 year and even more preferably up to 2 years. In addition, stable liquid formulations include liquid formulations that exhibit desirable characteristics after storage at a temperature (including 25°C) for 1 month, 3 months, 6 months, or after storage at 40°C for a period of 28 days.

Typical acceptable standards for stability are as follows: HF-HPLC (High Performance Liquid Chromatography Gel Filtration) measurement typically degrades no more than about 10%, preferably no more than about 5% of the active ingredients (such as proteins, antibodies). By visual inspection, this pharmaceutical preparation is a pale yellow almost colorless, clear or colorless liquid, or a clear to slightly milky white or pale yellow almost colorless transparent liquid. The change in concentration, pH and osmolality of the drug is no more than plus / minus 10%. Typically, a decrease of no more than about 10%, preferably no more than about 5%, is observed. Typically no more than about 10%, preferably no more than about 5% aggregates are formed.

An active ingredient in a pharmaceutical product is considered to "retain its physical stability" if the antibody does not show any significant increase in aggregation, precipitation and/or denaturation by visual inspection of color and/or clarity, or UV (ultraviolet light) scattering. , gel filtration (GF) and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) or by FTIR (Fourier transform infrared spectroscopy) spectroscopy (which determines the secondary structure of the protein).

An active ingredient (such as a protein or antibody) in a pharmaceutical product is said to "retain its chemical stability" if that active ingredient (such as a protein or antibody) does not exhibit any significant chemical change. By identifying and quantifying chemically altered forms of proteins or antibodies, chemical stability can be assessed. Degradation processes that often result in a change in the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as gel filtration or SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combined with mass spectrometry or MALDI/TOF/MS (time-of-flight mass spectrometry with matrix-assisted laser desorption/ionization), etc.), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, measurement of isoaspartic acids, etc.) and isomerization (as measured by isoaspartic acid measurement, peptide mapping, etc.).

An active ingredient (e.g., a protein or antibody) "retains its biological stability" in a pharmaceutical product if that active ingredient (e.g., protein or antibody) exhibits, for a given period of time, biological activity within a given interval when preparing a pharmaceutical product. The biological activity of an active ingredient (such as a protein or antibody) can be determined, for example, by antigen binding assay.

The three letter code and one letter code for amino acids as used in this disclosure are as described in J. Biol. Chem, 243, p 3558 (1968).

The term "antibody" as used in this disclosure refers to an immunoglobulin, a four peptide chain structure formed by two identical heavy chains and two identical light chains linked by an interchain disulfide bond(s) .

In the present disclosure, the antibody light chain described in the present disclosure further comprises light chain constant region(s) that contains the human or mouse κ, λ chain, or variant(s) thereof.

In the present disclosure, the heavy chain of an antibody described in this disclosure further comprises a heavy chain constant region(s) that human or murine IgG1, IgG2, IgG3, IgG4, or variant(s) thereof contain(s). .

At the N-terminus of the heavy chain and light chain of an antibody, a region of about 110 amino acids varies greatly, which is known as the variable region (Fv region); the amino acid sequence at the C-terminus, which is known as the constant region, is relatively stable. The variable region contains three hypervariable regions (HVR) and four FR regions (FR) with a relatively conserved sequence. Three hypervariable regions determine the specificity of an antibody and are also known as the complementarity determining region (CDR). Each light chain variable region (LCVR or VL) and each heavy chain variable region (HCVR or VH) consists of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDR regions are LCDR1, LCDR2 and LCDR3; the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and arrangement of amino acid residues of the CDR region of the LCVR and HCVR regions of the antibody or antigen-binding fragment in this document corresponds to the known Kabat numbering criteria (LCDR1-3, HCDR1-3) or according to the Kabat and Chothia numbering criteria; the Kabat numbering criteria (see Kabat et al (1991), Sequences of Proteins of Immunological Interest, the ^5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), and the Chothia numbering criteria (see Al-Lazikani et al (1997) JMB 273: 927-948).

An antibody of the present disclosure includes a mouse antibody, a chimeric antibody, and a humanized antibody, preferably a humanized antibody.

The term "antibody or its binding fragment" or "functional fragment" as used in the present disclosure refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment having antigen-binding activity, as well as a fragment Fv, an scFv fragment that binds to an antigen. The Fv fragment is the minimum antibody fragment that contains all antigen binding sites, the Fv fragment contains the heavy chain variable region and the light chain variable region, but without the constant region(s). Typically, the Fv antibody further comprises a polypeptide linker between the VH and VL domains to form the structure required for antigen binding. Different linkers can also be used to join the variable regions of two antibodies to form a polypeptide chain, referred to as a single chain antibody or single chain Fv (sFv). The term "binding to PD-L1" as used in this disclosure means the ability to interact with human PD-L1. The term "antigen-binding site" as used in this disclosure refers to non-sequential or consecutive three-dimensional sites on an antibody or antigen-binding fragment that recognizes a target antigen and binds specifically to that antigen.

The term "mouse antibody" in the present disclosure refers to a monoclonal antibody against human PD-L1, obtained according to the knowledge and skill in this field. During preparation, the analyzed subject is injected with the PD-L1 antigen, and then a hybridoma is isolated expressing an antibody that has the desired sequence or functional characteristics.

A "chimeric antibody" is an antibody that is formed by fusing the variable region of a non-human (such as a mouse) antibody with the constant region of a human antibody in such a way as to facilitate an immune response induced by the non-human (such as a mouse) antibody. To create a chimeric antibody, a hybridoma secreting a specific monoclonal antibody is first created, then the variable region genes are cloned from the hybridoma cells, and then, if desired, the constant region genes of a human antibody, the variable region genes of a non-human (such as mouse) antibody, are cloned. are ligated to human constant region genes to form a chimeric gene that can be inserted into a human vector, and the chimeric antibody molecule is finally expressed in a eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present disclosure, the light chain of the chimeric anti-PD-L1 antibody further comprises constant region(s) derived from human chain k, X, or variant(s) thereof. The heavy chain of the chimeric anti-PD-L1 antibody further comprises heavy chain constant region(s) derived from human IgG1, IgG2, IgG3, IgG4 or variant(s) thereof. The constant region(s) of the human antibody may be selected from the constant region(s) of the heavy chain derived(s) from human IgG1, IgG2, IgG3, IgG4 or variant(s) thereof, preferably contains a heavy chain constant region(s). a chain derived from human lgG2 or lgG4 or lgG4 without ADCC (antibody dependent cellular cytotoxicity) due to an amino acid mutation.

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody obtained by grafting non-human (such as murine) CDR sequences onto a human antibody variable region framework, i.e. an antibody formed from different types of human germline antibody backbone sequences. The humanized antibody overcomes the strong anti-antibody response induced by a chimeric antibody that carries a large number of non-human (such as murine) components. Such framework sequences can be obtained from a public DNA database or published references covering germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available online at www.mrccpe.com.ac.uk/vbase) and are also found in Kabat , EA et al. 1991, Sequences of Proteins of Immunological Interest, the 5th Ed. In order to avoid a decrease in activity caused by reduced immunogenicity, the human antibody variable region framework undergoes minimal backmutation to maintain activity. A humanized antibody of the present disclosure also refers to a humanized antibody that is further produced by phage display for the purpose of CDR affinity maturation.

The term "ADCC" (from the English antibody-dependent cell-mediated cytotoxicity), namely, antibody-dependent cellular cytotoxicity, as used in this disclosure, refers to cells expressing Fc receptors that directly kill target cells coated with antibody, by recognizing the Fc segment of the antibody. The effector function of an ADCC antibody can be reduced or eliminated by modifying the IgG Fc segment. This modification refers to mutations in the constant region of the heavy chain of the antibody, such as mutations selected from the group consisting of N297A, L234A, L235A in lgG1; chimera lgG2/4; or F234A/L235A mutations in lgG4.

The term "identity" as used in the present disclosure indicates the degree of similarity between the sequences of two polynucleotides or two polypeptides. The sequence identity in the present disclosure is at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% , but are not limited to them. Comparison and determination of percent identity between two sequences can be performed using the default settings of the BLASTN/BLASTP algorithm available from the National Center for Biotechnology Information website.

The term "TGF-β receptor II" or "TGFβRII" or "transforming growth factor β receptor II" refers to the binding of ligands (including, but not limited to, TGFβ1, TGFβ2, and TGFβ3) by which these cell surface receptors trigger the intracellular transduction pathway. alarms.

The term "PD-L1" refers to programmed death ligand 1, also known as CD274 and B7H1. PD-L1 is a 290 amino acid protein having an extracellular IgV-like and IgC-like domain (amino acids 19-239 of full-length PD-L1), a transmembrane domain, and an intracellular domain of about 30 amino acids. PD-L1 is constitutively expressed on many cells, such as antigen-presenting cells (such as dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (such as vascular endothelial cells, islets of Langerhans, and an immunologically privileged site). PD-L1 is also expressed on a variety of tumors and virus-infected cells and is an element in an immunosuppressive environment (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of two T cell co-inhibitors (PD-1 and B7-1).

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

As an exemplary preparation method for an anti-PD-L1 antibody of the present disclosure, it has been published in PCT Application PCT/CN 2016/104320 (Publication No. WO 2017084495), wherein the anti-PD-L1 antibody contains CDR sequences in the heavy chain variable regions as described below:

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

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

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

Heavy chain variable region of a humanized anti-PD-L1 antibody:

SEQ ID NO: 7, where X ₁ is selected from H or G; and X ₂ is selected from G or F.

Light chain variable region of a humanized anti-PD-L1 antibody:

NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the part in italics is the FR sequence, and the underlined part is the CDR sequence (CDR amino acid residues are defined and designated based on Kabat numbering criteria).

In another embodiment, a backmutation(s) plan is performed on a humanized antibody of the present disclosure, and the planned backmutations are shown in Table 1 below:

Note: For example, Y91F indicates a backmutation from Y to F at position 91 according to natural numbering. "Grafted" indicates that the mouse antibody CDR is implanted on human germline FR sequences.

New humanized antibodies can be generated by different combinations of the heavy chain and light chain mutations shown in Table 1.

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

Primers were designed, and the VH/VK gene fragments of each humanized antibody were constructed by PCR, and then inserted into a pH expression vector (having a signal peptide and a constant region gene fragment (CH1-Fc/CL)) to effect homologous recombination in order to construct an expression vector. vector for the full length antibody VH-CH1-Fc-pHr/VK-CL-pHr.

1. Designing primers:

The online DNAWorks (v3.2.2) program (http://helixweb.nih.gov/dnaworks/) was used to design numerous primers for the synthesis of gene fragments containing VH/VK required for recombination: a 30 bp 5'-signal peptide .n. plus VH/VK plus 30 bp CH1/CL-3'.

2. Splicing fragments:

According to the guidelines for DNA polymerase Primer STAR GXL from TaKaRa, using the primers designed above, two-step PCR (polymerase chain reaction) amplification generated gene fragments containing VH/VK required for recombination.

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

The pHr expression vector (having a signal peptide and a constant region gene fragment (CH1-FC/CL)) was designed and constructed by using some specific restriction endonuclease such as BsmBl that recognizes a distinguishable element between the sequence and the restriction site. This vector was digested with BsmBI and then the digested fragments were gel extracted and stored for use.

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

Gene fragments containing VH/VK required for recombination and the pHr expression vector (having a signal peptide and a constant region gene fragment (CH1-FC/CL)) that had been cleaved with BsmBl were added to competent DH5H cells in a 3:1 ratio, incubated at 0°C on ice for 30 min, subjected to heat shock at 42°C for 90 s, added 5 volumes of LB medium, then incubated at 37°C for 45 minutes, then planted on a LB-Amp dish, cultured at 37°C overnight. A single clone was selected for sequencing and the clone of interest was obtained.

5. A plasmid was constructed according to the construct in the present example, then the purified protein was expressed, and the affinity of the resulting protein was measured by the detection described in the SPR example.

6. Finally, the affinity of the humanized reverse mutant(s) or hybridoma antibodies for human PD-L1-his was measured by BIACORE, with the humanized reverse mutation sites and sequence combinations obtained from the screening being as follows:

Heavy chain variable region of anti-PD-L1 antibody:

where HCDR2 is as shown in RIGPNSGFTSYNEKFKN SEQ ID NO: 10, i.e., X ₁ in SEQ ID NO: 7 is G and X ₂ in SEQ ID NO: 7 is F;

Anti-PD-L1 antibody light chain variable region:

SEQ ID NO: 11;

NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the part in italics represents the FR sequence, and the underlined part represents the CDR sequence (CDR amino acid residues are defined and designated based on Kabat numbering criteria).

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

Since PD-L1 is also expressed in activated T cells, therefore, the use of wild-type IgG1 constant regions can cause Fc-mediated effects (such as ADCC and CDC), which could lead to a decrease in the level of activated T cells. In the present disclosure, mutated lgG4 is selected to generate antibodies without ADCC and CDC. The clone obtained by affinity maturation was converted to type lgG4, and the central hinge region of lgG4 contains the mutation S228P (corresponding to position 227 in the natural sequence of SEQ ID NO: 12). Additionally, the mutation F234A (corresponding to position 233 in the natural sequence of SEQ ID NO: 12) and L235A (corresponding to position 234 in the natural sequence of SEQ ID NO: 12) was introduced (mAb 4:3, 310-318; May/June 2012). At the same time, in order to avoid the breakage that occurs at the C-terminus of the heavy chain of the antibody when the linker peptide (which is used to bind the extracellular domain of TGF-βRII) is introduced, the K at the final position of the heavy chain of the anti-PD-L1 antibody was further mutated to A (corresponding to the last position in the natural sequence of SEQ ID NO: 12) so as to increase the stability of the fusion protein. The sequence of the anti-PD-L1 antibody of the present disclosure used to construct the fusion protein is as follows:

anti-PD-L1 antibody heavy chain: lgG4 (AA) (S228P)

NOTE: The underlined part is the heavy chain variable region sequence and the ununderlined part is the heavy chain constant region sequence (part in italics is the mutation site);

Anti-PD-L1 antibody light chain:

SEQ ID NO: 13;

NOTE: The underlined part is the light chain variable region sequence and the ununderlined part is the light chain constant region sequence.

As used in the present disclosure, the fusion protein described in the present disclosure is a protein product obtained by co-expression of two genes through a genetic engineering technique. Methods for making and purifying antibodies and antigen-binding fragments are well known in the art and can be found (eg, Antibodies, A Laboratory Manual, Cold Spring Harbor, chapters 5-8 and 15). For example, mice can be immunized with human PD-L1 or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced for amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be obtained by conventional methods. The antibody or antigen-binding fragments of the present disclosure are designed to graft CDRs derived from a non-human antibody into one or more human FRs. By aligning against a human antibody variable region germline IMGT database using the MOE program, human framework germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351.

The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using known methods. For example, cDNA sequences encoding the heavy chain and light chain can be cloned and constructed in the GS expression vector. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. As a more preferred method known in the art, a mammalian expression system will result in glycosylation of the antibody, typically at highly conserved N-terminal sites in the Fc region. Stable clones can be obtained by expression of an antibody that specifically binds to human PD-L1. Positive clones can be expanded in serum-free culture medium to produce antibodies in bioreactors. The culture medium into which the antibody has been secreted can be purified by conventional techniques. For example, this medium can be loaded onto a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer. This column is washed to remove non-specific binding components. Bound antibody is eluted with a pH gradient, antibody fractions are detected by SDS-PAGE and then collected. This antibody can be filtered and concentrated using conventional techniques. Soluble aggregate and multimers can be effectively removed by conventional techniques, including gel filtration or ion exchange chromatography. The product may be frozen immediately, for example at -70°, or may be lyophilized.

The "immunomodulatory molecule" of the present disclosure can be used to reduce the immunotolerance of cancer cells. The present disclosure uses a truncated form of the extracellular domain of TGF-βRII as an immunomodulatory molecule in a fusion protein. "TGF-β receptor II (TGF-βRII)" binds to TGF-β1 and TGF-β3 ligands with high affinity. The TGF-βRII/TGF-β complex recruits TGF-βRI to form a signal transduction complex (Won et al, Cancer Res. 1999; 59: 1273-7). The extracellular domain of TGF-βRII is a 136 amino acid residue peptide from the extracellular N-terminus of TGF-βRII, a typical example of which is shown in SEQ ID NO: 14. Other variants are about 136 amino acids in length and derived from the extracellular domain of human TGF-βRII, which methods to bind to TGF-β1 and TGF-p3 also belong to the extracellular domain of TGF-βRII of this disclosure. In the present disclosure, it has been found that the structure and function of the N-terminally truncated form of the extracellular domain of TGF-βRII is more stable than that of the untruncated molecule. The fusion protein containing the N-terminally untruncated form of the TGF-βRII extracellular domain (polypeptide shown as aa (amino acids) 1-136 of SEQ ID NO: 14) is susceptible to degradation. In particular, the extracellular domain of TGF-βRII, which is truncated by less than 26 consecutive amino acids from the N-terminus, is more stable; preferably the extracellular domain of TGF-βRII, which is truncated at 14-26 and more preferably truncated at 14-21 consecutive amino acids from the N-terminus, has a higher level of expression; and most preferably truncated by 19 or 21 consecutive amino acids.

A "TGF-β receptor fusion protein" is a fusion protein containing the TGF-β receptor. In some embodiments, the TGF-β receptor fusion protein of the present disclosure is a TGF-β receptor fusion protein as described in International Patent Application PCT/CN 2018/086451 (WO 2018205985 A1). The entire content of WO 2018205985 A1 is incorporated into this disclosure in its entirety. In some embodiments, the TGF-β receptor fusion protein is an anti-PD-L1 antibody/TGF-βRII extracellular domain (PD-LI/TGF-β decoy) fusion protein with the TGF-βRII extracellular domain serving as the immunomodulatory portion of the fusion protein molecule. , the anti-PD-L1 antibody serves as the targeting portion of this fusion protein, the extracellular domain of TGF-βRII (e.g. shown as SEQ ID NO: 14, 15, 16, or 17) is fused to the C-terminus (also known as the carboxyl terminus) of the heavy chain of an anti-PD-L1 antibody via a linker sequence (e.g., (G ₄ S) _x G, x is 3-6) to form a fusion sequence, and this fusion sequence is connected to the light chain of an anti-PD-L1 antibody via an interchain disulfide(s) link(s) finally to form a PD-LI/TGF-β trap fusion protein, this structure is shown in FIG. 1. In some embodiments, the TGF-β receptor fusion protein is the fusion protein described in Table 2 of Example 1 of this disclosure.

The term "linker" or "linker sequence" refers to a connecting peptide sequence used to connect domains of a protein, usually with some degree of flexibility, and the use of linkers will not result in loss of the original function of the protein domain. In some embodiments of the present disclosure, the linker sequence is (G ₄ S) _x G, where x is 3-6, for example, this linker sequence is a polypepid such as: (G ₄ S) ₃ G, (G ₄ S) ₄ G, (G ₄ S) ₅ G or (G ₄ S) ₆ G.

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

The term "possible" or "possible" means that the event or situation that follows may, but does not necessarily occur, and this description includes instances in which the event or circumstance actually occurs or does not occur. For example, "possibly containing 1-3 heavy chain variable regions" means that an antibody heavy chain variable region may be present, but not necessarily present.

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

"Treat" means to administer a therapeutic agent, such as a composition of the present disclosure, internally or externally to a subject having one or more symptoms of a disease for which the agent has a known therapeutic activity. Typically, the agent is administered in an effective amount to alleviate one or more symptoms of the disease in the subject or population being treated, to induce regression or prevent the progression of such symptom(s) to a clinically measurable extent. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary according to such factors as the state of the disease, the age and weight of the subject, and the ability of the agent to elicit the desired response in the subject. Whether a symptom of a disease improves can be assessed by any clinical measure commonly used by physicians or other healthcare professionals to assess the severity or state of progression of a symptom. While one embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target symptom(s) of the disease in each subject, it should alleviate the target symptom(s) of the disease in a statistically significant number of subjects. as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann and Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test.

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

"Tm value" refers to the temperature at which thermal denaturation of the protein occurs, that is, the temperature at which half of the protein is unfolded. At this time, the spatial structure of the protein is disturbed. Therefore, the higher the Tm value, the higher the thermal stability of a given protein.

The term "replacement" refers to the replacement of a solvent system that dissolves the antibody protein. For example, a high salt or hypertonic solvent system containing an antibody protein is replaced using a physical operation on a buffer system to obtain a stable preparation such that the antibody protein can be present in a stable preparation. This physical operation includes, but is not limited to, ultrafiltration, dialysis, or recovery from centrifugation.

Detailed description of the invention

Below, the present invention is further described with reference to examples, analysis examples, or production examples. However, these examples, analysis examples or production examples are for the purpose of illustration only, the scope of the present disclosure is not limited to them.

In the examples, assays, or preparations of the present disclosure, where specific conditions are not described, they are generally carried out under conventional conditions or under conditions suggested by the substance or product manufacturers. When the source of reagents is not specifically indicated, these reagents are conventional commercially available reagents.

Examples

Example 1 Cloning and Expression of PD-L1 Fusion Protein/TGF-β Trap

The extracellular domain of TGF-βRII (full-length or truncated form of SEQ ID NO: 14) was used as part of the immunomodulatory molecule in the fusion protein, and the anti-PD-L1 antibody was used as the targeting part of the fusion protein to form the anti-PD-L1 antibody/extracellular protein. TGF-βRII domain (PD-L1/TGF-β decoy).

Surprisingly, the truncated form of the extracellular domain of TGF-βRII was found to be relatively stable, especially more stable after truncation of less than 26 amino acids from its N-terminus, preferably a higher level of expression and a more stable structure is obtained after truncation of 14-26 amino acids, more preferably truncating 14-21 consecutive amino acids from the N-terminus, and more preferably truncating 14, 19 or 21 consecutive amino acids from the N-terminus.

The sequences of non-limiting examples of the extracellular domain of TGF-βRII and its truncated form in the present disclosure are as follows:

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

SEQ ID NO: 14;

TGF-βRII extracellular domain sequence with 19 amino acid truncation or deletion at the N-terminus: ECD (20-136)

SEQ ID NO: 15;

TGF-βRII extracellular domain sequence with 21 amino acid truncation or deletion at the N-terminus: ECD (22-136)

SEQ ID NO: 16;

TGF-βRII extracellular domain sequence with 14 amino acid truncation or deletion at the N-terminus: ECD (15-136)

SEQ ID NO: 17.

As an example, the amino acid of the C-terminus of the heavy chain of an anti-PD-L1 antibody of the present disclosure (an anti-PD-L1 antibody in which the heavy chain is shown as SEQ ID NO: 12 and the light chain is shown as SEQ ID NO: 13) was ligated to the extracellular domain of TGF-βRII with a variable linker length (G ₄ S) _x G (x is 3-6) by homologous recombination technique and conveniently expressed in the 293 expression system, along with the anti-PD-L1 antibody light chain, and the resulting fusion proteins shown in Table 2.

Note: Ab represents an anti-PD-L1 antibody of the present disclosure (heavy chain shown as SEQ ID NO: 12 and light chain shown as SEQ ID NO: 13); ECD (n-136) in the sequence description is the full-length or truncated form of the extracellular domain of TGF-βRII; n represents the original number of amino acids after truncation of the extracellular domain of TGF-βRII. The structure of the fusion protein of the present disclosure is shown in FIG. 1; N19A indicates that the amino acid at position 19 of the full length extracellular domain of TGF-βRII (SEQ ID NO: 14) is mutated from N to A.

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

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

The cell culture medium was centrifuged at high speed, the supernatant was collected, and the first purification step was carried out by affinity chromatography. The chromatographic medium is protein A or a derived excipient that interacts with an Fc such as Mabselect from GE. The balancing buffer was 1xPBS (137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na ₂ HPO ₄ , 2 mmol/l KH ₂ PO ₄ , pH 7.4). After equilibrating with 5× column volumes, the cell supernatant was loaded for binding, and the flow rate was controlled such that the sample was allowed to remain in the column for 1 min or more. After loading the sample, the column was washed with 1×PBS (pH 7.4) until the absorption in UV (ultraviolet region) A280 (at 280 nm) decreased to its original level. The column was then washed with 0.1 M glycine (pH 3.0) elution buffer, the eluted peak was taken according to the absorption peak in UV A280, and the eluted sample was neutralized with 1 M Tris (pH 8.5).

The neutralized eluted sample was concentrated by ultrafiltration and then subjected to gel filtration, the buffer was 1×PBS and the column was XK26/60 Superdex 200 (GE). The flow rate was controlled at 4 ml/min, the loading volume was less than 5 ml, and the target protein peak was pooled according to UV A280 absorbance. The purity of the selected protein was greater than 95% when identified by GP-HPLC and was confirmed by LC-MS. The confirmed sample was aliquoted for use. Received PD-L1/trap TGF-β.

Analysis example (biological assessment in vivo, in vitro)

Assay Example 1: ELISA detection of PD-L1 binding/trap TGF-β cTGF-β1 in vitro

The detection process is described as follows:

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

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

c. Washing 3 times with 250 μl 1×PBST, gradient dilutions of PD-L1/TGF-β trap were added, and the TGF-β trap was used as a positive control, and incubated for 1 hour at 37°C.

d. Washing 3 times with 250 µl 1×PBST.

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

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

g. Absorbance at 450 nm was measured on a microplate reader and the data was analyzed with Graphpad Prism 5.

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

Experimental Example 2: PD-LI Binding ELISA/TGF-β Trap to PD-L1 in vitro

Antigen used for detection: PD-L1-His

SEQ ID NO: 18.

The detection method is described as follows:

a. 96-well plates were coated with 100 μl/well of human PD-L1-His (SEQ ID NO: 18) at a concentration of 5 μg/ml at 4° C. overnight.

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

c. Washing 3 times with 250 μl 1×PBST, gradient dilutions of PD-LI/TGF-β trap, and anti-PD-L1 antibody as a positive control were added and incubated for 1 hour at 37°C.

d. Washing 3 times with 250 µl 1×PBST.

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

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

e. Absorbance at 450 nm was measured on a microplate reader and the data was analyzed with Graphpad Prism 5.

The results of in vitro binding of the fusion proteins of the present disclosure to human PD-L1 are shown in FIG. 4. ELISA showed that all fusion proteins retained binding activity to human PD-L1.

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

1. Purpose of the analysis:

In order to investigate the blocking effect of PD-LI/TGF-β decoy on the PD-1/PD-L1 signaling pathway, a cell-based antibody blocking experiment was performed on cells bearing human PD-1 and PD-L1 receptor molecules that were engineered Promaga respectively.

2. Prototypes

1) Anti-PD-L1 antibody with heavy chain shown as EEO. ID NO: 12 and the light chain shown as EEO ID NO: 13;

2) Control 1 (20T-Fc): ECD(20-136)-Fc, a fusion protein containing the missing TGF-βRII extracellular domain fragment - ECD(20-136) and Fc, and the sequence is as follows:

3) Control 2 (22T-Fc): ECD(22-136)-Fc - TGF-βRII extracellular domain truncated protein fusion protein - ECD(22-136) and Fc and the sequence is as follows:

4) TGF-β receptor fusion protein obtained in Example 1 of the present disclosure: fusion protein 9, fusion protein 15:

In fusion protein 9, the sequence of the fusion peptide - heavy chain of anti-PD-L1-(G ₄ S) ₄ G-ECD TGF-β R11 antibody (20-136) is as follows:

NOTE: Regular script is the anti-PD-L1 antibody heavy chain sequence, italics is the linker sequence, and underline is the sequence of the truncated ECD fragment (20-136) of the TGF-βRII extracellular region.

The light chain sequence of the anti-PD-L1 antibody in fusion protein 9 is as follows:

SEQ ID NO: 13;

The sequence of the anti-PD-L1-(G ₄ S) ₅ G-ECD TGF-β R11 (22-136) heavy chain fusion peptide in fusion protein 15 is as follows:

NOTE: Regular script is the anti-PD-L1 antibody heavy chain sequence, italics is the linker sequence, and underline is the sequence of the truncated ECD fragment (22-136) of the TGF-βRII extracellular region.

The light chain sequence of the anti-PD-L1 antibody in fusion protein 15 is as follows:

5) human IgG: blank control, human immunoglobulin obtained from mixed normal human serum by purification using a conventional affinity chromatography method, such as for protein A;

6) Positive control (FP17022): anti-PD-I antibody 2 fusion protein/TGF-βRII extracellular domain;

Amino acid sequence of the anti-PD-L1 antibody 2 light chain in the FP17022 fusion protein:

SEQ ID NO:21;

The amino acid sequence of the fusion peptide - anti-PD-L1 antibody 2 heavy chain/TGF-βRII extracellular domain (1-136) in the FP17022 fusion protein:

3. Method of analysis

CHO/PD-L1 cells (CS187108, Promega) were digested and resuspended in F-12 nutrient medium complete medium (Ham). Cell density was adjusted to 4×10 ⁵ /ml using complete medium according to cell count results. The cell suspension was transferred to a loading container, added to a 96-well plate at a volume of 100 μl/well using a multichannel pipette and incubated in an incubator at 37°C, 5% CO ₂ for 20-24 h; Jurkat/PD-1 cell suspension (CS187102, Promega) was prepared the next day and cells were resuspended according to the results of cell count using assay medium and cell density was adjusted to 1.25×10 ⁶ /ml; cell culture plates containing CHO/PD-L1 cells were removed from the incubator, 95 μl of culture solution per well was withdrawn using a multichannel pipette, and a gradient diluted fusion protein, anti-PD-L1 antibody, and positive control (FP17022), respectively, were added, in a volume of 40 µl/well. Then, the Jurkat/PD-1 cell suspension was transferred to a loading container, added to a cell culture plate in a volume of 40 μl/well, and incubated at 37°C, 5% CO ₂ for 5-6 hours. Bio-Glo™ and allowed to cool to room temperature. Cell culture plates were taken out and placed at room temperature for 5-10 min. Then the Bio-Glo™ reagent was added to each well, incubated in a laminar for 5-10 min, and the value of the chemiluminescence signal was read using a multifunctional microplate reader.

4. Results

As shown in FIG. 5, similar to the positive control molecule, fusion protein 9 of the present disclosure could effectively block the binding of PD-1 expressing Jurkat cells to CHO/PD-L1 cells, and there was a drug concentration and dose-dependent effect. Fusion protein 15 has the same blocking ability as the blocking ability of the fusion protein 9.

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

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

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

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

Assay Example 5: SMAD3 Reporter Gene Inhibition Assay

1. Purpose of the analysis:

In this experiment, a Smad3 binding element (SBE) with a luciferase reporter gene was expressed in HepG2 cells to investigate the inhibitory effect of PD-L1/TGF-β Trap on TGF-β1-induced Smad3 activation and PD-L1/TGF-β Trap activity in vitro. was evaluated according to the IC50 value (half maximum inhibitory concentration).

2. Sample for analysis: fusion protein 9, positive control (FP17022).

3. Method of analysis

HepG2 cells were cultured in complete MEM medium (GE, SH30243.01) containing 10% FBS (fetal calf serum) and subcultured every 3 days. On the first day of the experiment, 25,000 cells per well were inoculated into 96-well plates (Corning, 3903) and cultured at 37° C., 5% CO ₂ for 24 hours. The following day, the medium in the cell culture plates was discarded and 100 ng of 3TP-Lux plasmid was transfected per well. These cells were additionally cultured at 37°C, 5% CO ₂ for 24 hours. Six hours prior to adding the assay sample, the complete medium in the 96-well plate was discarded and 80 μl of incomplete medium (MEM plus 0.5% FBS) was added to each well. After 6 hours, 10 µl of human TGF-β1 solution (R&D, 240-B-010) prepared in incomplete medium (final concentration 2 ng/ml) and 10 µl of assay solution (final concentration 500; 50; 5; 0) were added. .5, 0.05, 0.005, 0.0005, and 0 nM), TGF-β1 human solvent was used as a control, and cells were cultured at 37°, 5% CO ₂ for an additional 18 h. Then 100 µl of prepared luciferase substrate of the ONE-Glo™ Luciferase Assay System (Promega, E6110) and incubated at room temperature for 10 minutes in the dark, and then the luminescent signal value was read using a Victor 3 microplate reader (Perkin Elmer). The IC50 value of the sample for analysis was obtained by calculation using the data analysis program Graphpad Prism 5.0.

On FIG. 6 shows that fusion protein 9 dose-dependently inhibited TGF-β-induced pSMAD3 reporter activity and had comparable potency and IC50 (concentration required to inhibit 50% maximal activity) with that of the positive control FP17022. The results of the analysis of the anti-PD-L1 antibody showed that it had no inhibitory effect (IC50 greater than 500 nM).

Assay Example 6: In Vitro Detection of IFNγ (Interferon-gamma) PBMC (Peripheral Blood Mononuclear Cells) Secretion Due to Tuberculin (TB) Stimulation

1. Purpose of the analysis

To study T-lymphocyte activation by PD-L1/TGF-β trap, human peripheral blood mononuclear cells (PBMCs) were selected, purified and stimulated in vitro with tuberculin (TB) for 5 days to detect the level of secretion of the cytokine IFNγ.

2. Sample for analysis

1) Human IgG;

2) Antibody against PD-L1;

3) Fusion protein 9;

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

5) Anti-PD-L1 plus control 1 (20T-Fc).

3. Method of analysis

20 μl tuberculin was added to freshly isolated and purified PBMC, 15 ml, approximately 3×10 ⁷ and cultured in an incubator for 5 days at 37°C, 5% CO ₂ . On day 6, cultured cells were collected and centrifuged, washed once with PBS and resuspended in fresh medium at a density adjusted to 1×10 ⁶ cells/ml, 90 μl of resuspended cells were added to a 96-well plate. In the respective wells of the above 96-well cell culture plate, 10 μl/well of different concentrations of antibodies were individually added, 10 μl of PBS were added to the control and blank groups, respectively. The cell culture plate was then incubated in an incubator for 3 days at 37° C., 5% CO ₂ . The cell culture plate was taken out and the supernatant was taken from each well after centrifugation (4000 rpm, 10 min). After a 10-fold dilution, IFN-γ secretion was detected by ELISA (human IFN-γ detection kit, NEOBIOSCIENCE, EHC 102d.96) according to the reagent instructions for specific operations. As shown in Table 4, all PD-LI/TGF-β trap samples could increase IFN-γ cytokine secretion by activated T lymphocytes and there was a dose concentration effect of the drug.

4. Result

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

Assay Example 7: Pharmacokinetic Evaluation

Three female SD rats were purchased from Jie Si Jie Laboratory Animal Co., Ltd. and maintained on a 12/12 hour light-dark cycle (temperature was 24 plus/minus 3°C, relative humidity was 50-60%), the rats had free access to water and food. On the day of the experiment, SD rats were injected with the fusion protein in the tail vein at a dose of 6 mg/kg and with an injection volume of 5 ml/kg.

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

The measurement method is described as follows:

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

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

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

d. Wash 5 times with 250 µl 1×PBST.

e. 100 μl/well of biotinylated anti-human TGF-βRII antibody (R&D) was added and incubated for 1 hour at 37°C.

f. Wash 5 times with 250 µl 1×PBST.

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

h. The absorbance at 450 nm was measured on a microplate reader and the data was analyzed by Graphpad Prism 5.

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

Assay Example 8: Effect of PD-L1/TGF-β Trap on MDA-MB-231 Mouse Subcutaneous Human Breast Cancer Xenograft

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

1) Empty control: PBS;

2) Fusion protein 9: 4.8 mg/kg;

3) Fusion protein 9: 24 mg/kg;

4) Anti-PD-L1 antibody: 4 mg/kg;

5) Anti-PD-L1 antibody: 20 mg/kg;

6) Anti-PD-L1 4 mg/kg plus control 1 (20T-Fc) 2.14 mg/kg;

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

MDA-MB-231 cells were resuspended in serum-free RPMI-1640 medium and mixed with an equal volume of Matrigel, 100 μl (2.3×10 ⁶ ) were inoculated subcutaneously into the right flank of NOD/SCID mice. After 11 days, animals with tumors of excessive or insufficient size were excluded, mice were randomized into groups with 9 animals in each group. 5x10 ⁵ stimulated PBMCs (60 μl) were injected into the tumor tissues and the remaining PBMCs were further cultured without stimulation. One week later, 5×10 ⁶ PBMCs (100 μl) were intraperitoneally injected into tumor bearing mice as the first injection cycle. Throughout the experimental period, 2 and a half cycles were provided: a total of 5 injections of PBMC. On the day of the first intratumoral injection, intraperitoneal administration was carried out, three times a week, a total of 14 injections. The administration schedule was shown in Table 6. Tumor volume and body weight were measured twice a week. The experimental results are shown in Table 7. At the end of the experiment, the tumor bearing mice were sacrificed and the tumor was removed and weighed.

The results are shown in FIG. 8, antibody fusion protein 9 (4.8 mg/kg, 24 mg/kg) can significantly inhibit the growth of the mouse subcutaneous human breast cancer xenograft MDA-MB-231. There was a dose-dependent relationship between high and low doses and was superior to the anti-PD-L1 antibody control (4 mg/kg, 20 mg/kg), the TGF-βRII control molecule 20T-FC (2.14 mg/kg) and the combined group (anti-PD-L1 antibody - 4 mg/kg plus 20T-FC - 2.14 mg/kg) at the equivalent molar dose, respectively. Each dose of fusion protein 9 maintained the desired antitumor effect from day 14 post-administration; compared to anti-PD-L1 antibody - 20 mg/kg, fusion protein 9 at a higher dose had a clear advantage (p less than 0.05). On the 25th day after administration, the antitumor effect of each antibody reached the optimal level. The antitumor rate of the low and high dose of fusion protein 9 and anti-PD-L1 antibody and the combination group was 37.24%, 52.38%, 30.24%, 28.01% and 31.38%, respectively. On the 32nd day after administration, the antitumor effect of fusion protein 9 was still very significant. The %TGI of the low and high dose groups was 36.68% and 50.76%, respectively, and the tumor volume was statistically different when compared to the control group (p<0.05).

Assay Example 9: Physical Stability of PD-LI/TGF-β Traps

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

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

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

Periodic stability at a certain concentration was examined by monitoring purity by GP-HPLC, typical conditions, for example, sample concentration was controlled at about 50 mg/ml in 10 mM acetate/135 mM NaCl (pH 5.5), and stability was compared under conditions such as 5 cycles of freeze and thaw at -80°C relative to the state after storage at 40°C for one month. An HPLC column for protein Xbridge BEN SEC 200A (Waters) was used for detection. The results are shown in Table 9 as follows: these two fusion proteins showed good stability.

Assay Example 10: Chemical Stability of the Fusion Protein Deamidation is a common chemical modification that will affect late stage stability of an antibody, in particular it is usually chosen to avoid or reduce highly deamidated modification of certain amino acids in the CDR regions as much as possible. through mutation. 1600 μg of the antibody to be analyzed was dissolved in 200 μl of 10 mM acetate/135 mM NaCl (pH 5.5) and placed in an incubator at 40°C. Samples were taken on days 0, 14 and 28 for enzymatic hydrolysis analysis. 100 μg of each sample taken at different time points was dissolved in 100 μl of 0.2 M His-HCl, 8 M guanidine-HCl, pH 6.0; 3 μl of 0.1 g/ml DTT (dithiothreitol) was added, and then the sample was incubated in a water bath at 50° C. for 1 hour. This sample was then ultrafiltered twice with 0.02 M His-HCl (pH 6.0) and digested overnight at 37° C. in a water bath by adding 3 μl of 0.25 mg/ml trypsin. The deamidation modification was verified using an Agilent 6530 Q-TOF (quadrupole-time-of-flight) GC-MS (gas chromatography-mass spectrometry) and the results are shown in Table 10 below.

Note: N is a detectable modified asparagine and the number represents the position in the light chain or heavy chain from the N-terminus. Percentage is the ratio of deamidation modification detected by GC-MS to the signal of all peptides at a given site.

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

Example receiving

Exemplary Methods for the Preparation of a Fusion Protein for a Pharmaceutical Composition (Preparation)

First step: a certain amount of stock solution of the purified TGF-β receptor fusion protein was withdrawn and solvent exchanged (preferably by ultrafiltration) using a protein-free buffer (such as 10 mM citric acid-sodium citrate buffer, pH 6.2), by passing through an ultrafiltration membrane for at least 6-fold volume, then this protein was concentrated to about 70 mg/ml. A certain volume of sucrose stock solution was added and mixed to achieve a final sucrose concentration of 80 mg/mL. A certain volume of Tween-80 stock solution was added and mixed to achieve a final Tween-80 concentration of 0.4 mg/mL. Added 10 mm citrate buffer, pH 6.2, to achieve a precisely defined volume so as to obtain a concentration of 50 mg/ml protein (other preparations to be analyzed, or stable preparations were obtained according to similar stages).

After filtration, samples of this product were taken for sterility analysis in connection with the control of the environment. The mother liquor was passed through a 0.22 μm PVDF (polyvinylidene fluoride) filter and the filtrate was collected.

Second stage: the fill volume was adjusted to 6.3 ml, the filtrate was loaded into a 6 ml vial, which was then stoppered, and samples were taken at the beginning, in the middle and at the end of the fill in order to detect the difference in the fill volume in order to control the medium.

The third stage: the work of the capper was started, covered with aluminum caps.

Fourth stage: a visual inspection was carried out to confirm that the product had no deficiencies such as inaccurate loading. Printed labels and applied to vials; cardboard packaging was printed, cartons were folded, bottles were loaded into them and labeled.

Preparation Example 1 pH Screening of the Buffer System of a TGF-β Receptor Fusion Protein Preparation

TGF-β receptor fusion protein preparations (fusion protein 9) were prepared using the following buffers at a protein concentration of 50 mg/mL:

1) 10 mM histidine-acetic acid, pH 5.0;

2) 10 mM histidine-acetic acid, pH 6.0;

3) 10 mM histidine-acetic acid, pH 6.5;

4) 10 mM sodium dihydrophosphate-disodium hydrogen phosphate, pH 7.0;

5) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.5.

Each preparation was filtered and added at a volume of 1.2 ml/vial to a 2 ml injection vial made of neutral borosilicate glass. The injection vial was fitted with a stopper, cap and sealed. Samples were taken and subjected to high temperature 40° C. and shaking experiments. Experimental results are shown in Table 11. These results show that TGF-β receptor fusion proteins have better stability at pH 6.0-6.5.

Preparation Example 2 Screening of Buffer System for TGF-β Receptor Fusion Protein Preparations

TGF-β receptor fusion protein preparations (fusion protein 9) were prepared using the following buffers at a protein concentration of 50 mg/mL:

1) 10 mM succinic acid-sodium succinate, pH 6.0;

2) 10 mM citric acid-sodium citrate, pH 6.0;

3) 10 mM citric acid-sodium citrate, pH 6.5;

4) 10 mM sodium dihydrophosphate-disodium hydrogen phosphate, pH 6.5;

5) 10 mM histidine hydrochloride, pH 6.5.

Each preparation was filtered and added in a volume of 1.2 ml/vial to a 2 ml neutral borosilicate glass injection vial. The injection vial was stoppered, capped and sealed. Samples were taken for a shaking experiment (at 25° C., 300 rpm). The experimental results are shown in Table 12. These results show that in the sodium dihydrogen phosphate-disodium hydrogen phosphate group on the 6th day, a large number of small particles were observed with shaking, and aggregates reached 1.8% when detected by HF. However, only tiny particles were occasionally observed in other groups. It can be seen that the stability of the TGF-β receptor fusion protein in buffer systems based on citric acid, histidine and succinate is better than that in phosphate buffer systems.

Preparation Example 3 Additional Buffer Screening for TGF-β Receptor Fusion Protein Preparation

A pH 6.2 buffer containing 10 mM histidine hydrochloride or 10 mM citric acid-sodium citrate was used to prepare a formulation containing 80 mg/mL sucrose, 0.4 mg/mL polysorbate 80, a TGF-β receptor fusion protein ( fusion protein 9) at a concentration of 50 mg/ml.

Each preparation was filtered and added at a volume of 1.2 ml/vial to a 2 ml injection vial made of neutral borosilicate glass. The injection vial was stoppered, capped and sealed. Samples were stored at 25°C for stability analysis for 6 months with the detection of GF or CE-SDS (sodium dodecyl sulfate capillary electrophoresis) under non-reducing conditions.

The experimental results are shown in Table 13. These results demonstrate that the citric acid-sodium citrate system is better than the histidine-hydrochloride system (Mb HF aggregate: 1.8% vs. 2.2%; CE-SDS under non-reducing conditions: 94.5 % relative to 92.2%); thus, the citric acid system can be chosen as a buffer system for the TGF-β receptor fusion protein.

Preparation Example 4 Screening for Stabilizers for TGF-β Receptor Fusion Protein Preparations

TGF-β receptor fusion protein preparations (fusion protein 9) were prepared using the following buffers or different saccharides at a protein concentration of 50 mg/mL:

1) 10 mM citric acid-sodium citrate, 80 mg/ml sucrose, pH 6.2;

2) 10 mM citric acid-sodium citrate, 80 mg/ml α,α-trehalose dihydrate, pH 6.2.

Each preparation was filtered and added at a volume of 1.2 ml/vial to a 2 ml injection vial made of neutral borosilicate glass. The injection vial was stoppered, capped and sealed. Samples were taken for long-term storage experiments at room temperature 25°C and low temperature 2-8°C.

The experimental results are shown in Table 14. These results show that sucrose and trehalose have similar effects on the stability of the TGF-β receptor fusion protein (fusion protein 9). Sucrose was chosen as the stabilizer for the TGF-β receptor fusion protein (fusion protein 9). At a sucrose concentration of 80 mg/mL, the osmotic pressure is about 300 mosm/kg, which is close to isotonic, so the sucrose concentration can be 80 mg/mL.

Production Example 5 Surfactant Screening for TGF-β Receptor Fusion Protein Preparations

TGF-β receptor fusion protein preparations (fusion protein 9) were prepared using the following buffers and different types of surfactants at different concentrations with a protein concentration of 50 mg/ml.

1) 10 mM histidine hydrochloride, 0.1 mg/ml polysorbate 20, pH 6.2;

2) 10 mM histidine hydrochloride, 0.2 mg/ml polysorbate 20, pH 6.2;

3) 10 mM histidine hydrochloride, 0.4 mg/ml polysorbate 20, pH 6.2;

4) 10 mM histidine hydrochloride, 0.6 mg/ml polysorbate 20, pH 6.2;

5) 10 mM histidine hydrochloride, 0.8 mg/ml polysorbate 20, pH 6.2;

6) 10 mM histidine hydrochloride, 0.1 mg/ml polysorbate 80, pH 6.2;

7) 10 mM histidine hydrochloride, 0.2 mg/ml polysorbate 80, pH 6.2;

8) 10 mM histidine hydrochloride, 0.4 mg/ml polysorbate 80, pH 6.2;

9) 10 mM histidine hydrochloride, 0.6 mg/ml polysorbate 80, pH 6.2;

10) 10 mM histidine hydrochloride, 0.8 mg/ml polysorbate 80, pH 6.2.

Each preparation was filtered, 0.5 ml of the preparation was injected into 50 ml saline injection or 5% glucose injection to achieve a protein concentration of 0.5 mg/ml after dilution. Sample stability was observed after dilution. The results of this experiment are shown in Table 15. The results show that when the concentration of Polysorbate 20 reached more than 0.2 mg/ml in this preparation, the level of insoluble particles was significantly reduced after dilution; as for polysorbate 80, the level of insoluble particles produced due to dilution with sodium chloride decreased along with an increase in the concentration of polysorbate 80. When the concentration of polysorbate 80 reached 0.4 mg/ml or more, the level of particles larger than 10 μm was reduced to less than 10 particles/ml.

Preparation Example 6 Additional Surfactant Screening for TGF-β Receptor Fusion Protein Preparations

TGF-β receptor fusion protein preparations (fusion protein 9) were prepared using the following buffers with different types of surfactants at a protein concentration of 50 mg/ml:

1) 10 mM citric acid-sodium citrate, 0.4 mg/ml polysorbate 80, pH 6.2;

2) 10 mM citric acid-sodium citrate, 0.6 mg/ml polysorbate 20, pH 6.2. Each preparation was filtered and added at a volume of 1.2 ml/vial to a 2 ml injection vial made of neutral borosilicate glass. The injection vial was stoppered, capped and sealed.

Samples were taken for experiments on long-term storage at a low temperature of 2-8°C.

The experimental results are shown in Table 16. These results show that polysorbate 80 has a better effect on the stability of the TGF-β receptor fusion protein (fusion protein 9). Therefore, polysorbate 80 was chosen as the surfactant for the TGF-β receptor fusion protein (fusion protein 9).

Preparation Example 7 Filter Membrane Compatibility Assay for TGF-β Receptor Fusion Protein Preparations

TGF-β receptor fusion protein (fusion protein 9) was prepared at a concentration of 50 mg/ml in 10 mM citric acid-sodium citrate buffer, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, pH 6.2. These preparations were passed through a 0.22 μm PES (polyethersulfone) filter membrane and a PVDF filter membrane, respectively, and samples were taken at the beginning, in the middle and at the end of the analysis.

Experimental results are shown in Table 17. Analysis of protein content, appearance and purity shows that the TGF-β receptor fusion protein (fusion protein 9) was stable during contact with the filter membrane and the formulation was compatible with both PES and PVDF.

Preparation Example 8 Lyophilization of TGF-β Receptor Fusion Protein Preparation

A TGF-β receptor fusion protein preparation (fusion protein 9) containing a concentration of 50 mg/ml TGF-β receptor fusion protein (fusion protein 9), 80 mg/ml sucrose, and 0.4 mg/ml polysorbate 80 was prepared with buffer c pH 6.2 containing 10 mM citric acid-sodium citrate. The antibody was added at a volume of 6.3 mg/vial to a 20 ml vial and placed in a low temperature freezer for lyophilization.

Lyophilization procedures include pre-freezing, primary drying and secondary drying. Once the lyophilization process was completed, the vial was sealed under vacuum. The samples were reconstituted and a comparison was made of the state before and after lyophilization. The results show that the reconstituted solution can maintain a beneficial efficacy similar to that of the solution formulation.

Production Example 9 Other Possible Formulation Compositions

In addition, according to the present disclosure, other

pharmaceutical formulations of TGF-β receptor fusion protein (fusion protein 9):

(1) 70 mg/ml fusion protein 9, 75 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 20 mM citric acid-sodium citrate buffer, final pH 6.4;

(2) 80 mg/ml fusion protein 9, 85 mg/ml sucrose, 0.5 mg/ml polysorbate 80, and 15 mM citric acid-sodium citrate buffer, final pH 6.2;

(3) 60 mg/ml fusion protein 9, 90 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 5 mM citric acid-sodium citrate buffer, final pH 6.2;

(4) 30 mg/ml fusion protein 9, 60 mg/ml sucrose, 0.3 mg/ml polysorbate 80, and 30 mM citric acid-sodium citrate buffer, final pH 6.3;

(5) 90 mg/ml fusion protein 9, 95 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 6.0;

(6) 100 mg/ml fusion protein 9, 70 mg/ml sucrose, 0.1 mg/ml polysorbate 80, and 25 mM citric acid-sodium citrate buffer, final pH 6.5;

(7) 50 mg/ml fusion protein 9, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 7.0;

(8) 50 mg/ml fusion protein 9, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 7.5;

(9) 50 mg/ml fusion protein 9, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 5.0;

(10) 60 mg/ml fusion protein 9, 70 mg/ml sucrose, 0.5 mg/ml polysorbate 80, and 15 mM citric acid-sodium citrate buffer, final pH 5.5;

(11) 40 mg/ml fusion protein 9, 80 mg/ml sucrose, 0.5 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 6.2;

(12) 55 mg/ml fusion protein 9, 75 mg/ml sucrose, 0.3 mg/ml polysorbate 80, and 5 mM citric acid-sodium citrate buffer, final pH 6.0;

(13) 65 mg/ml fusion protein 9, 90 mg/ml sucrose, 0.7 mg/ml polysorbate 80, and 30 mM citric acid-sodium citrate buffer, final pH 7.5;

(14) 70 mg/ml fusion protein 9, 75 mg/ml sucrose, 0.8 mg/ml polysorbate 80, and 30 mM citric acid-sodium citrate buffer, final pH 7.0;

(15) 50 mg/ml fusion protein 9, 80 mg/ml sucrose, 0.8 mg/ml polysorbate 80, and 10 mM citric acid-sodium citrate buffer, final pH 7.0.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO., LTD.;

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> RECEPTOR FUNCTION PROTEIN PHARMACEUTICAL COMPOSITION

TRANSFORMING GROWTH FACTOR BETA AND ITS APPLICATIONS

<130> 719079CPCT

<150> 201811328326.1

<151> 2019-11-09

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> Heavy chain complementarity determining region 1 (HCDR1)

<400> 1

Ser Tyr Trp Met His

15

<210> 2

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> HCDR2

<220>

<221> Domain

<222> (3)..(3)

<223> Xaa selected from His or Gly

<220>

<221> Domain

<222>(8)..(8)

<223> Xaa selected from Gly or Phe

<400> 2

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

1 5 10 15

Asn

<210> 3

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> HCDR3

<400> 3

Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> Light chain complementarity determining region 1 (LCDR1)

<400> 4

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

1 5 10 15

<210> 5

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> LCDR2

<400> 5

Ala Ala Ser Asn Leu Glu Ser

15

<210> 6

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> LCDR3

<400> 6

Gln Gln Ser Phe Glu Asp Pro Leu Thr

15

<210> 7

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> Heavy chain variable region of a humanized antibody against

programmed cell death ligand-1 (PD-L1)

<220>

<221> Domain

<222> (52)..(52)

<223> Xaa selected from His or Gly

<220>

<221> Domain

<222> (57)..(57)

<223> Xaa selected from Gly or Phe

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 8

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

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

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 9

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> Anti-PD-L1 antibody heavy chain variable region

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 10

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

<223> HCDR2

<400> 10

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

1 5 10 15

Asn

<210> 11

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<221> Domain

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

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 12

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<221> Chain

<223> Anti-PD-L1 antibody heavy chain sequence: Ig G4(AA) (S228P)

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 13

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<221> Chain

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

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 14

<211> 136

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

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

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 15

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> Truncated TGF-beta RII extracellular domain sequence or

lack of 19 amino acids at the N-terminus: ECD (20-136)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ser Asn Pro Asp

115

<210> 16

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> Truncated TGF-beta RII extracellular domain sequence or

lack of 21 amino acids at the N-terminus: ECD (22-136)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asn Pro Asp

115

<210> 17

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> Truncated TGF-βRII extracellular domain sequence or

lack of 14 amino acids at the N-terminus: ECD (15-136)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

115 120

<210> 18

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

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

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235

<210> 19

<211> 346

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

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

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

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

340 345

<210> 20

<211> 344

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

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

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

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Lys Ser Leu Ser Leu Ser Leu Gly

340

<210> 21

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<221> Chain

<223> In the fusion protein FP17022, the amino acid sequence of the light chain

antibodies 2 against PD-L1

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 22

<211> 607

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> In the FP17022 fusion protein, the fused peptide sequence is heavy chain

antibodies 2 against PD-L1/extracellular domain of TGF-beta RII (1-136)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

<210> 23

<211> 584

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> In fusion protein 9, the fusion peptide sequence is heavy chain

antibodies against PD-L1-(G4S)4G-extracellular domain of TGF-beta RII (20-136)

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Glu Tyr Asn Thr Ser Asn Pro Asp

580

<210> 24

<211> 587

<212> Protein

<213> Artificial sequence

<220>

<221> Peptide

<223> In fusion protein 15, the fusion peptide sequence is heavy chain

antibodies against PD-L1-(G4S)5G-extracellular domain of TGF-beta RII (22-136)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Val Lys Phe Pro Gln Leu Cys Lys

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

580 585

<---

Claims (60)

1. Pharmaceutical composition for the treatment of a tumor, containing: 0.5 mg/ml to 100 mg/ml of transforming growth factor receptor beta (TGF-β) fusion protein, 5 mM to 30 mM citrate buffer, 50 mg/ml to 100 mg/ml saccharide and 0.1 mg/ml to 0.8 mg/ml polysorbate 80; Where the TGF-β receptor fusion protein comprises a fusion peptide formed between the heavy chain of an anti-PD-L1 antibody and the extracellular domain (ECD) of TGF-βRII, the sequence of which is shown as SEQ ID NO: 23, and the light chain of an anti-PD-L1 antibody, the sequence of which is shown as SEQ ID NO: 13, and the structure of the TGF-β receptor fusion protein is shown in Fig. 1; the saccharide is sucrose or trehalose; And The pH of the pharmaceutical composition is from 6.0 to 6.5. 2. Pharmaceutical composition according to claim 1, in which: citrate buffer is a citric acid-sodium citrate buffer. 3. Pharmaceutical composition according to claim. 1 or 2, in which the concentration of citrate buffer is from 5 mm to 20 mm. 4. Pharmaceutical composition according to claim 3, wherein the concentration of citrate buffer is 10 mM. 5. Pharmaceutical composition according to any one of paragraphs. 1-4, in which the concentration of the TGF-β receptor fusion protein is from 30 mg/ml to 70 mg/ml. 6. The pharmaceutical composition of claim 5 wherein the concentration of the TGF-β receptor fusion protein is 50 mg/mL. 7. Pharmaceutical composition according to any one of paragraphs. 1-6, where the pH of the pharmaceutical composition is 6.2. 8. Pharmaceutical composition according to any one of paragraphs. 1-7, in which the concentration of saccharide is from 60 mg/ml to 90 mg/ml. 9. Pharmaceutical composition according to claim 8, wherein the saccharide concentration is 80 mg/ml. 10. Pharmaceutical composition according to any one of paragraphs. 1-9, where the saccharide is sucrose. 11. Pharmaceutical composition according to any one of paragraphs. 1-10, in which the concentration of polysorbate 80 is from 0.4 mg/ml to 0.8 mg/ml. 12. Pharmaceutical composition according to claim 11, in which the concentration of polysorbate 80 is 0.4 mg/ml. 13. Pharmaceutical composition according to claim 1, containing: 30 mg/ml to 70 mg/ml TGF-β receptor fusion protein, 5 mM to 20 mM citric acid-sodium citrate buffer, 60 mg/ml to 90 mg/ml sucrose and 0.4 mg/ml to 0.8 mg/ml polysorbate 80; The pH of this pharmaceutical composition is from 6.0 to 6.5. 14. Pharmaceutical composition according to claim 13, containing: 50 mg/ml TGF-β receptor fusion protein, 10 mM citric acid-sodium citrate buffer, 80 mg/ml sucrose and 0.4 mg/ml polysorbate 80. 15. The pharmaceutical composition according to claim 14, wherein the pH of the pharmaceutical composition is 6.2. 16. A method for preparing a pharmaceutical composition according to claim 1, the method comprising: the step of contacting the TGF-β receptor fusion protein with a citrate buffer, the pharmaceutical composition comprising: 0.5 mg/ml to 100 mg/ml TGF-β receptor fusion protein, 5 mM to 30 mM citrate buffer, 50 mg/ml to 100 mg/ml saccharide and 0.1 mg/ml to 0.8 mg/ml polysorbate 80; Where the TGF-β receptor fusion protein comprises a fusion peptide formed between the heavy chain of an anti-PD-L1 antibody and the extracellular domain (ECD) of TGF-βRII, the sequence of which is shown as SEQ ID NO: 23, and the light chain of an anti-PD-L1 antibody, the sequence of which is shown as SEQ ID NO: 13, and the structure of the TGF-β receptor fusion protein is shown in FIG. 1; the saccharide is sucrose or trehalose; And The pH of the pharmaceutical composition is from 6.0 to 6.5. 17. The method of claim 16 wherein the citrate buffer is citric acid-sodium citrate buffer. 18. The method of claim 16 wherein the concentration of the citrate buffer is 5 mM to 20 mM and the pH of the citrate buffer is 6.0 to 6.5. 19. A lyophilized preparation containing a TGF-β receptor fusion protein for treating a tumor, which is obtained by lyophilization of a pharmaceutical composition according to any one of paragraphs. 1-15, wherein the TGF-β receptor fusion protein comprises a fusion peptide formed between the heavy chain of an anti-PD-L1 antibody and the extracellular domain (ECD) of TGF-βRII, the sequence of which is shown as SEQ ID NO: 23, and a light chain of an anti-PD-L1 antibody. L1, the sequence of which is shown as SEQ ID NO: 13, and the structure of the TGF-β receptor fusion protein is shown in FIG. 1. 20. A reconstituted solution containing a TGF-β receptor fusion protein for treating a tumor, which is obtained by reconstituting a lyophilized preparation according to claim 19, wherein the reconstituted solution contains: 0.5 mg/mL to 100 mg/mL TGF-β receptor fusion protein, 5 mM to 30 mM citrate buffer, 50 mg/ml to 100 mg/ml saccharide and 0.1 mg/ml to 0.8 mg/ml polysorbate 80; where the saccharide is sucrose or trehalose; and the pH of the pharmaceutical composition is from 6.0 to 6.5, wherein the TGF-β receptor fusion protein comprises a fusion peptide formed between the heavy chain of an anti-PD-L1 antibody and the extracellular domain (ECD) of TGF-βRII, the sequence of which is shown as SEQ ID NO: 23, and the light chain of an anti-PD-L1 antibody, the sequence of which is is shown as SEQ ID NO: 13 and the structure of the TGF-β receptor fusion protein is shown in FIG. 1. 21. Product for the treatment of tumors, containing one or more than one container containing: pharmaceutical composition according to any one of paragraphs. 1-15. 22. The use of a pharmaceutical composition according to any one of paragraphs. 1-15 to obtain a medicinal product: wherein the drug is used to treat or inhibit the disease(s) or disorder(s), wherein the disease(s) or disorder(s) is(are) a tumor. 23. Use according to claim 22, wherein the tumor is selected from the group consisting of head and neck cancer, glioblastoma, glioma, nasopharyngeal carcinoma, thyroid cancer, lung cancer, myeloma cancer, myelodysplastic syndrome, neuroendocrine cancer, lymphoma, leukemia, melanoma, basal cell skin carcinoma, squamous cell skin carcinoma, elevated dermatofibrosarcoma, Merkel cell carcinoma, sarcoma, mesothelioma, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, colorectal cancer, breast cancer, endometrial cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer and testicular cancer; the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.

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