WO2019096137A1

WO2019096137A1 - Hybridoma comprising immune checkpoint inhibitor, preparation method for same, and applications thereof

Info

Publication number: WO2019096137A1
Application number: PCT/CN2018/115286
Authority: WO
Inventors: 韩化敏; 张叔人; 高荣凯; 褚丽新; 徐义; 金瑾; 魏晓莉
Original assignee: 拜西欧斯（北京）生物技术有限公司
Priority date: 2017-11-14
Filing date: 2018-11-13
Publication date: 2019-05-23
Also published as: CN109503718B; CN109503718A

Abstract

Provided in the present application are a hybridoma comprising an immune checkpoint inhibitor and cytokines, nucleic acid molecules encoding the hybridoma, a corresponding expression vector, a host cell, and a method comprising the preparation of the hybridoma. In addition, also provided in the present application are a method for detecting the activity of the hybridoma and a method for using the hybridoma in treating a disease and improving on or relieving discomfort.

Description

Fusion containing immunological checkpoint inhibitor and preparation method and application thereof

Technical field

The present application belongs to the field of immunotherapy, and relates to an immunotherapeutic drug for cancer, and particularly relates to a fusion structure of an immunological checkpoint and a cytokine for immunotherapy.

background

In recent years, the clinical application of cancer immunotherapy products and technologies has made significant progress, and has become another effective cancer treatment method after surgery, radiotherapy, chemotherapy and targeted therapy.

Cytokines are important immunotherapeutic products in the field of cancer immunotherapy. Cytokines act as molecular messengers, allowing cells of the immune system to communicate with each other to produce coordination of target antigens to exert regulatory and effector functions in many diseases. As an immunomodulator, cytokines can be used to activate immunotherapy, immunosuppressive therapy, and the like. Clinical application of cytokines for the treatment of cancer and other diseases has been more than 20 years. Cytokines such as interferon (IFN) and interleukin (IL) have been widely used in the treatment of hairy cell leukemia. The treatment of cytokines is summarized as follows: (1) There is no simple dose-response relationship: cytokines mainly act through immune regulation, and overdosing may inhibit the expected immune response, while the dose is too low. It can not effectively cause an immune response; (2) long-term daily medication can induce immune tolerance; (3) the effect shows slow but long-lasting: biological immunotherapy is different from radiotherapy and chemotherapy, even tumors that are sensitive to cytokines, It disappears completely after treatment, and the treatment effect may be displayed after a few months. The condition will continue to improve after the cytokine is stopped. (4) Combination therapy is better than monotherapy: various cytokine combinations Application is more effective than treatment with only one cytokine, because multiple cytokines can make up for each other's shortcomings, and exert their own advantages, so that the effect is better; (5) can prolong the life of patients: cytokines can inhibit tumor cell growth And the toxic side effects are small, so the life of the patient can be significantly prolonged.

However, although cytokines are highly efficient, they still have the disadvantages of single component, targeting and killing.

Therefore, there is still a need in the art to develop a fusion that effectively enhances the anti-tumor effect of cytokines as an improved therapeutic agent to address the drawbacks of the prior art described above.

Summary of invention

The object of the present application is to provide a novel disease treatment product which can combine the high anti-tumor effect of anti-tumor cytokines and the characteristics of immunological checkpoint inhibition to provide a new choice for tumor treatment.

Illustratively, embodiments of the present application that achieve the above-described objectives include the following:

WHAT IS CLAIMED IS: 1. A fusion for immunotherapy comprising an immunological checkpoint inhibitor and a cytokine.

2. The fusion of embodiment 1, wherein the immunological checkpoint inhibitor comprises, but is not limited to, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a guanamine 2,3-double Oxygenase (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator (BTLA) inhibitor, T cell immunoglobulin Protein inhibitors, TIM-3 inhibitors, and Killer-cell Immunoglobulin-like Receptor (KIR) inhibitors expressed on the surface of NK cells and part of T cells.

3. The fusion according to embodiment 1, wherein the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a guanamine 2,3-dual oxygenation Enzyme (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator, T cell immunoglobulin, TIM-3, and expressed in NK cells and partial T Killer cell immunoglobulin-like receptor on the cell surface.

4. The fusion of embodiment 1, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

5. The fusion of any of embodiments 1-4, wherein the immune checkpoint inhibitor is an anti-immunological checkpoint antibody.

6. The fusion of embodiment 5, wherein the anti-immunization checkpoint antibody is an anti-PD-1 antibody.

7. The fusion according to embodiment 6, which comprises a domain that specifically recognizes and binds to the immune cell surface antigen PD-1 and a constant region from an immunoglobulin constant region (Fc), The domain that specifically recognizes and binds to the immune cell surface antigen PD-1 includes a light chain variable region having three CDRs (anti-PD-1 VL) and a heavy chain variable region having three CDRs (anti-PD-1) VH), wherein the light chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOs: 1-16; and the heavy chain variable region (Anti-PD-1 VH) comprises a heavy chain CDR (HCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 17-31.

8. The antibody or functional fragment thereof of embodiment 7, wherein the light chain variable region comprises:

LCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 1, 2, 3, 4 and 5,

LCDR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 6, 7, 8, 9 and 10, and

LCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 11, 12, 13, 14, 15 and 16;

The heavy chain variable region comprises:

HCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,

HCDR2, the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and

HCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.

The antibody or functional fragment thereof according to embodiment 7 or 8, which comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;

b) a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;

c) a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;

d) a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;

e) a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;

f) Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.

The antibody or functional fragment thereof according to embodiment 7 or 8, which comprises a heavy chain variable region selected from the group consisting of:

g) a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;

h) a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;

i) a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;

j) a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;

k) Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.

The antibody or functional fragment thereof according to embodiment 7 or 8, which comprises a light chain variable region selected from any one of VL1, VL2, VL3, VL4, VL5 and VL6 and selected from VH1, VH2 Heavy chain variable region of any of VH3, VH4 and VH5.

12. The antibody or functional fragment thereof of embodiment 7, wherein the antibody or functional fragment thereof comprises:

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 1, 7, and 13, respectively, and the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 17, 22, and 27, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 2, 6 and 12, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 18, 23 and 28, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 5, 10 and 16, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 19, 24 and 29, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 21, 26 and 31, respectively;

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 3, 7 and 13, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 21, 26 and 31, respectively, and

Wherein the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.

13. The fusion of embodiment 7, the amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 47, 48, 49, 50, 51, 58.

The fusion according to embodiment 7, which comprises the nucleotide sequence of SEQ ID NO: 53, 54, 55, 56, 57, 59.

The fusion of any of embodiments 1-14, wherein the cytokine is capable of inhibiting tumor growth.

The fusion according to embodiment 15, wherein the cytokine is selected from the group consisting of: interleukin (IL), tumor necrosis factor (TNF), interferon (IFN), colony stim μlating factor (CSF) , transforming growth factor, growth factor (GF) and chemokine family.

The fusion according to embodiment 16, wherein the colony stim μlating factor (CSF) comprises G (granulocyte)-CSF, M (macrophage)-CSF, GM (granulocyte, macrophage) Cell)-CSF, Multi(multiplex)-CSF (IL-3), stem cell factor (SCF), erythropoietin (EPO);

The tumor necrosis factor (TNF) is selected from the group consisting of TNF-α and TNF-β;

The transforming growth factor is a transforming growth factor-βfamily (TGF-βfamily) TGF-β1, TGF-β2, TGF-β3, TGFβ1β2 or bone morphogenetic protein (BMP);

The growth factor (GF) includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I. (IGF-I), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF) -α), vascular endothelial growth factor (VEGF);

The chemokine family is selected from the group consisting of CXC/α subfamily, CC/β subfamily, C-type subfamily, and CX3C subfamily; wherein the CXC/α subfamily includes IL-8, a growth-regulated oncogene / melanoma growth stimulating factor (GRO/MGSA), platelet factor-4 (PF-4), platelet basic protein (PBP/CXCL7), proteolytic-derived product CTAP-III and β-platelet globulin (β- Thromboglobulin, β-TG), interferon-inducible protein-10, Epithelial Neutrophil-Activating Protein 78 (ENA-78); Phage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES (regulated upon activation normal T-cell expressed and secreted (CCL5), monocyte chemotactic protein-1 (MCP-1/MCAF), MCP-2 MCP-3 and I-309; the C-type subfamily comprises a lymphocyte chemotactic protein; the CX3C subfamily comprises a chemokine fractal (Fractalkine).

The fusion according to embodiment 16, wherein the cytokine is selected from any one of IFN-α, IFN-β and IFN-γ, preferably IFN-γ.

19. The fusion of claim 18, wherein the cytokine is IFN-[gamma], preferably an IFN-[gamma] dimer.

The fusion according to claim 18 or 19, wherein the antibody or a functional fragment thereof comprises: the light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 3, 7 and 13, respectively; The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 21, 26 and 31, respectively; or the light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 21, 26 and 31, respectively.

21. A method of making the fusion of any of embodiments 1-20, comprising the steps of:

(1) preparing an immunological checkpoint inhibitor;

(2) determining the amino acid sequence of the immunological checkpoint inhibitor obtained in the step (1);

(3) determining a nucleotide sequence encoding the immunological checkpoint inhibitor according to the amino acid sequence of the immunological checkpoint inhibitor obtained in the step (2);

(4) using the nucleotide sequence encoding the immunological checkpoint inhibitor obtained by the step (3) and the nucleotide sequence encoding the cytokine as a template, respectively designing primers and amplifications for amplifying the immunological checkpoint inhibitor Adding primers for the cytokine;

(5) Recombinant construction of an immunological checkpoint inhibitor-cytokine fusion protein expression vector;

(6) transforming an immunological checkpoint inhibitor-cytokine fusion protein expression vector into a recipient strain;

(7) screening an immunological checkpoint inhibitor-expression strain of a cytokine fusion protein;

(8) expressing an immunological checkpoint inhibitor-cytokine fusion protein and sequencing;

(9) Purification of an immunological checkpoint inhibitor-cytokine fusion protein.

The method according to embodiment 21, wherein the immunological checkpoint inhibitor is an anti-immunization checkpoint antibody, which is prepared by a phage library screening method, a hybridoma method, preferably by a phage library screening method. preparation.

The method of embodiment 22, wherein the anti-immunization checkpoint antibody is an anti-PD-1 antibody.

The method of any one of embodiments 21 to 23, wherein the cytokine is IFN-γ, preferably an IFN-γ dimer.

25. A nucleic acid molecule encoding the fusion of any of embodiments 1-20.

26. An expression vector comprising the nucleic acid molecule of embodiment 25, which is selected from the group consisting of a eukaryotic expression vector and a prokaryotic expression vector.

27. A host cell comprising the expression vector of embodiment 26.

28. A protein or polypeptide comprising the fusion of any of embodiments 1-20.

29. A method of detecting the activity of an immune checkpoint inhibitor-cytokine fusion according to any of embodiments 1-20.

30. Use of an immunological checkpoint inhibitor-cytokine fusion according to any one of embodiments 1 to 20 for the manufacture of a medicament for treating a disease, ameliorating or ameliorating discomfort.

31. A method for treating a disease, ameliorating or ameliorating discomfort, the method comprising administering the immune checkpoint inhibitor-cytokine fusion of any one of embodiments 1-20.

32. The use of embodiment 30 or the method of claim 31, wherein the disease and discomfort are PD-1 mediated diseases and discomforts.

The method of embodiment 30 or the method of claim 31, wherein the disease and discomfort comprise cancer, an inflammatory disease, and an infectious disease.

34. The use or method of embodiment 33, wherein the cancer, inflammatory disease, and infectious disease are PD-1 mediated.

The use or method of embodiment 33, wherein the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, Parathyroid carcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer , rectal cancer, adrenal cancer, anal cancer, vulvar cancer, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodkin Lymphoma, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, T cell Lymphoma, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), childhood solid tumors and lymphocytic leukemia One or more tumors.

The use or method of embodiment 33, wherein the infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infectious disease caused by the following viruses: Hepatitis virus (eg hepatitis A, B or C), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia Virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus , rotavirus, measles virus, rubella virus, parvovirus, JC virus or arbovirus viral encephalitis virus, or infectious diseases caused by bacteria: pneumococcal, mycobacteria, staphylococcus, streptococcus, meningococcus , conococci, Serratia, Klebsiella, Proteobacteria, Pseudomonas, Salmonella, Vibrio cholerae, Diphtheria, Botox, Bacillus anthracis, Clostridium tetani, Legionella, Plague Infectious diseases caused by bacteria, leptospirosis or Lyme disease, or fungi: Aspergillus (eg Aspergillus fumigatus, Aspergillus niger, etc.), Candida (eg Candida albicans), Candida krusei , Candida glabrata, Candida tropicalis, Cryptococcus neoformans, dermatitis bud, Mucor genus (such as Mucor, Absidia or Rhizopus), S. cerevisiae, Brazilian subsphere Sporozoites, Coccidioides or granulosa histoplasma, or infectious diseases caused by parasites: amoeba in dysentery, colonic guinea worm, flucler, amoeba, vivax malaria, voles Babesia, sucking Giardia, Cryptosporidium, Pneumocystis carinii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania polygala, Rat toxoplasma or Brassica elegans.

The use or method of embodiment 33, wherein the disease is cancer; the cancer is selected from the group consisting of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glial Tumor, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, intestinal cancer, and non-small cell lung cancer; the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection disease, the chronic virus For HIV, HBV or HCV.

BRIEF abstract

Figure 1 is a DNA electrophoresis pattern of colony PCR identification of the ID1 fusion protein gene. DNA molecular weight standard - (M): DL5000 (TAKARA, 3428A)

Figure 2 is a SDS-PAGE electropherogram of recombinant expression of the ID1 fusion protein. Lane 1: Ultrasound Broken Wall Supernatant of ID1 Fusion Protein Expression Strain; Lane 2: Ultrasound Broken Wall Supernatant of Negative Control BL21 Strain. Protein molecular weight standard (M): Rainbow 245 broad-spectrum protein Marker (Solebao, PR1920)

Figure 3 is a SDS-PAGE electropherogram of the ID1 fusion protein purified by affinity chromatography. Lane 1: unpurified sample; Lane 2: unbound protein, ie, protein eluted in the effluent; Lane 3: eluted ID1 fusion protein. Protein molecular weight standard (M: Rainbow 245 broad-spectrum protein Marker (Solebao, PR1920)

Figure 4 shows the lysate supernatant of E. coli BL21 (DE3) cell culture transfected with pGEX-6p-1-ID1 and control E. coli BL21 (DE3) strain (BL21) treated with different dilution factors to treat HepG2- Luc's mean fluorescence intensity (MFI).

Figure 5 shows that purified ID1 fusion protein promotes killing of HepG2-Luc cells by CTL cells.

Figure 6 shows the effect of a target-to-target ratio of 50 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells. ** indicates p < 0.01; *** indicates p < 0.001.

Figure 7 shows the effect of in vivo imaging on the efficiency of CTL killing HepG2-Luc cells compared with 50 to 1 and different concentrations of ID1 fusion protein. The higher the fluorescence intensity, the more viable cells represent, the lower the killing efficiency. .

Figure 8 shows the effect of a target-to-target ratio of 10 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells. * indicates p<0.05

Figure 9 shows the effect of in vivo imaging on the efficiency of CTL killing HepG2-Luc cells compared with 10 to 1 and different concentrations of ID1 fusion protein. The higher the fluorescence intensity, the more viable cells represent, the lower the killing efficiency. .

Figure 10 shows the effect of a target-to-target ratio of 50 to 1 and different concentrations of ID1 fusion protein on the efficiency of CTL killing THP-1 cells. **** indicates p<0.0001

Figure 11 shows the purification of the tID1 fusion protein using a chromatography column Superdex 200 10/30.

Figure 12 shows the results of SDS-PAGE detection of the tID1 fusion protein. M: Rainbow 245 broad-spectrum protein Marker (Solebao, PR-1920); Lane 1 is the second tube eluate shown in Figure 11, Lane 2 is the third tube collection solution shown in Figure 11, Lane 3 is Figure 11. The fourth tube collection liquid is shown, and the lane 4 is the collected liquid after the 2-4 tubes are combined.

Figure 13 shows the ELISA binding curve of the tID1 fusion protein to the PD-1 protein.

Figure 14 shows the ELISA binding curve of tID1 fusion protein to IFNGR. Panel A shows the binding curve of tID1 fusion protein to IFNGR, and Panel B shows the binding curve of IFN-γ and IFNGR.

Figure 15 shows the SPR assay for detecting the binding of the tID1 fusion protein to the PD1 antigen and IFNGR.

Figure 16 shows the ELISA binding curves of tID1 fusion protein to different species PD-1.

Figure 17 shows an ELISA profile of the tID1 fusion protein blocking PD-1 binding to PD-L1.

Figure 18 shows the binding curves of tID1 fusion protein to 293T-PD-1 cells.

Figure 19 shows that the tID1 fusion protein blocks the binding curve of 293T-EGFP/PD-1 to PD-L2.

Figure 20 shows by flow cytometry (FACS) that the tID1 fusion protein promotes tumor cell expression of PD-L1.

Figure 21 and Figure 22 show the inhibition of proliferation of hepG2 cells and Hela cells by tID1 fusion protein, respectively.

Figure 23 shows the binding of tID1 fusion protein to CD3 induced CD4+ T cells.

Figure 24 shows that the tID1 fusion protein promotes the killing effect of PBMC on hepG2 cells.

Figure 25 shows that tID1 promotes the killing effect of PBMC on human bladder cancer cell line T24.

Detailed

This application relates to the following definitions:

A fusion refers to a structure obtained by biological genetic manipulation such as recombination or by chemical synthesis of functional structures having different mechanisms of action and targeted targets. In the present application is a fusion of an anti-tumor cytokine with an immunological checkpoint inhibitor, more specifically, a fusion protein of IFN-γ and an anti-PD-1 antibody.

Immunotherapy refers to the immune state of the body that is low or hyperactive, artificially enhancing or inhibiting the body's immune function to achieve treatment for disease purposes. In the present application, it specifically refers to immunotherapy for tumors and cancer diseases using fusions of cytokines and immunological checkpoint inhibitors, particularly tumor-inhibiting therapies using fusion proteins of IFN-γ and PD-1 antibodies.

Tumor inhibition refers to the inhibition of the occurrence or progression of a tumor by manipulating an immunological pathway at a molecular level by a drug that specifically binds to a tumor-associated gene or a regulatory factor associated with tumor production. In the present application, it specifically means that the PD-1 antibody binds to the PD-1 antigen, and the PD-1 inhibits the weakening of the T cell activity, and provides the anti-tumor activity of the T cell, thereby suppressing the growth, reproduction and slowing down of the tumor cell. The purpose of tumor growth rate.

Cytokine is a tumor-associated substance produced by immune cells such as lymphocytes, macrophages, monocytes, and related cells. The cytokines related to tumor biological therapy include: tumor necrosis factor TNF, granulocyte-macrophage colony-stimulating factor GM-CSF, interleukin, interferon and the like. In a preferred embodiment of the present application, the cytokine is an interferon.

Interferon, a cytokine produced by monocytes and lymphocytes, is also a lymphokine with extensive immunomodulatory effects. It is a group of active proteins with multiple functions (mainly glycoproteins). Efficient antiviral biologically active substance. Interferon does not directly kill or inhibit the virus, but mainly causes the cells to produce antiviral proteins through the action of cell surface receptors, thereby inhibiting the replication of the virus. The types are classified into three types, α-(white blood cell) type and β-(成Fibroblast type, γ-(lymphocyte) type; at the same time, it can enhance the vitality of natural killer cells (NK cells), macrophages and T lymphocytes, thereby playing an immunomodulatory role and enhancing antiviral ability.

Lymphocyte-type interferon, IFN-γ, is produced by human T cells and NK cells, and its related genes are located on human chromosome 12. Human IFN-γ belongs to type II interferon, also known as γ-IFN or immunointerferon. It is a secreted protein. It is composed of 143 amino acids after removing the signal peptide. It exists in the form of homodimer or tetramer. The molecular weight is 40 kDa.

IFN-γ is commonly used clinically for antiviral and antitumor. IFN-γ inhibits the proliferation of a variety of viruses and is widely used in the treatment of viral infections. IFN-γ can inhibit tumor cell growth and prevent tumor metastasis and recurrence in vivo. Clinical application shows that IFN-γ has good effects on hairy cell leukemia, melanoma, skin tumor, chronic myeloid leukemia, glioma, lymphoma and myeloma. IFN-γ can directly kill tumor cells by inducing apoptosis of tumor cells and interfering with the growth cycle of tumor cells. It can also inhibit tumor growth by inhibiting the growth of blood vessels in tumor tissues, and can also enhance the body by regulating the body's immune system. The ability to remove tumor cells.

IFN-γ activates the immune system through the following pathways: (1) activates the function of dendritic cells (DC cells), promotes the differentiation of hematopoietic cells into DC cells and the maturation of DC cells, and enhances the infiltration of DC cells into tumor sites. Enhance the antigen presentation of DC cells to T cells; (2) Activate T cells to produce specific immunity to tumor cells; at the same time, through the NF-κB pathway, T cells are protected from apoptosis and maintain long-term survival of T cells; 3) It can also be converted into macrophages by activating monocytes, which produce tumor killing effects by releasing tumor necrosis factor (such as TNF-α). The anti-angiogenic effect of IFN-γ is mainly to inhibit the formation of tumor blood vessels by down-regulating the expression of pro-angiogenic factors.

However, at present, in the field of tumor treatment, the treatment of cytokines such as IFN-γ alone has a defect of poor targeting, and the effect of cytokines in tumor treatment is not satisfactory.

As an important cellular immune function enhancer, cytokines have been used to prepare a variety of conjugate and fusion protein drugs. However, it has not been contemplated in the art to fuse cytokines with immunological checkpoint inhibitors that have important applications in anti-tumor research. Unexpectedly, the inventors of the present application found that the fusion of the two drugs not only makes them better maintain their respective functions, but also has a synergistic effect. Therefore, fusion proteins of cytokines and immunological checkpoint inhibitors have good development and application prospects for tumor therapy.

Immune checkpoints are a class of immunosuppressive molecules that modulate the intensity and breadth of immune responses and prevent normal tissue from being damaged and destroyed. These "checkpoints" inhibit the immune regulatory signaling pathway, which normally inhibits the function of T cells, and may be used by tumor cells to form immune escape in tumor tissues. Therefore, in the process of tumor development and development, immune checkpoints become one of the main causes of immune tolerance.

Immunological checkpoint-based therapies are treatments that enhance T cell activity by co-suppressing or co-stimulating the corresponding signals to enhance anti-tumor immune responses. Drugs that act on immune checkpoints have completely different characteristics in terms of anti-cancer compared to previous drugs. First, they do not directly act on tumor cells, but indirectly kill tumor cells by acting on T cells; in addition, they are not specific to the specific surface of the tumor surface, but systematically enhance the systemic anti-tumor immunity. reaction.

Programmed cell death protein 1 (PD-1) and CTLA-4, BTLA, cellular immunoglobulin, and TIM-3, T cell immunoglobulin are all "immunoassay" molecules in tumors. Tissues may be used by tumor cells to form immune escapes, which control cell cycle progression by controlling extracellular and intracellular signals.

The strategy of blocking immune checkpoints such as the PD-1 pathway mainly enhances the killing effect on tumor cells from the following aspects: (1) promoting the aggregation of effector T cells at the tumor site by enhancing homing ability; (2) reducing tumor micro The number of regulatory T cells in the environment reduces its activity; (3) increases the number of effector T cells; (4) increases the cytotoxic effect of tumor-specific T cells, and the tumor-specific cytotoxic T cells reach the tumor site and pass TCR recognizes tumor cells, releases IFN-γ and T cell particles to kill tumor cells to enhance killing of tumor cells; (5) enhances the production of pro-inflammatory cytokines; and (6) down-regulates potential inhibitory cytokines such as IL- 10.

An immunological checkpoint inhibitor of the present application refers to a structure that can bind to an immunological checkpoint and inactivate an immunological checkpoint. The inhibitor may be either an organic compound that non-specifically binds to an immunological checkpoint or a ligand or antibody that specifically binds to an immunological checkpoint. In the present application, an immunological checkpoint inhibitor is preferably an antibody.

In some exemplary embodiments, the immunological checkpoint antibody is an immunoglobulin or a functional fragment thereof, such as an antigen-binding fragment, that specifically binds to an immunological checkpoint. In some exemplary embodiments, the antibody of the present application may be a whole antibody or a single chain antibody. In some exemplary embodiments, the immunological checkpoint inhibitor of the present application may be a functional fragment of an antibody, an antigen binding fragment, such as Fab, F(ab')2, Fv, scFv, Fd or dAb.

In some exemplary embodiments of the present application, the immunological checkpoint inhibitor is an anti-PD-1 antibody. PD-1 is expressed on activated T cells, B cells, macrophages, and monocytes. The ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC). The interaction of PD-1 with its ligand can down-regulate the central and peripheral immune responses and inhibit the anti-tumor activity of T cells.

It has not been contemplated in the art that immunological checkpoint inhibitors, which have important application prospects in anti-tumor research, are fused with cytokines to treat diseases. Unexpectedly, the inventors of the present application have found that blocking the immunological checkpoint pathway can effectively perform anticancer treatment by suppressing an immune response in a tumor environment. The fusion of immunological checkpoint inhibitors with cytokines not only allows them to maintain their respective functions well, but also has a synergistic effect.

Based on the comprehensive consideration of the anti-tumor effect of cytokines such as IFN-γ and the mechanism of action of immunological checkpoints in the tumor microenvironment, the present application uses an prokaryotic expression system to immunological checkpoint inhibitors and IFN- in one embodiment. Gamma fusion expression achieves the effect of targeting to tumor cells, such that IFN-γ and immunological checkpoint inhibitors, such as anti-immunoassay antibodies, synergistically enhance anti-tumor effects; this application uses eukaryotic expression in another embodiment The expression system fuses the dimeric structure of IFN-γ with an immunological checkpoint inhibitor, and also achieves a synergistic anti-tumor effect.

Accordingly, the present application provides new products and uses with highly potent anti-tumor activity. In particular, the present application will have an anti-immunization checkpoint antibody (in some exemplary embodiments, an anti-PD-1 antibody) with a specific sequence and a cytokine (in some exemplary embodiments, an IFN-γ immunomodulatory factor) The fusion forms a fusion protein as a new drug with high antitumor activity.

In the immunological checkpoint inhibitor and cytokine fusion of the present application, IFN-γ can activate antigen presenting cells, and promote differentiation of type I helper T cells (ThI cells) by up-regulating the transcription factor T-bet. An important immunopotentiator with antiviral, immunomodulatory and antitumor properties. It can directly inhibit tumor proliferation, promote its apoptosis, and can promote tumor tissue necrosis through anti-angiogenesis, and can also promote the expression of MHC molecules in tumor cells, which is conducive to T cell recognition and killing. However, IFN-γ can also stimulate tumor cells to enhance the expression level of PD-L1, and the PD-1/PD-L1 signal leads to inhibition of immune cell killing by tumor cells. PD-1 monoclonal antibody inhibits the inhibition of T cell immune killing by blocking PD-1/PD-L1 signaling, and is suitable for tumor types with high PD-L1 expression. Therefore, the ID1 fusion protein of the present application organically integrates the two, and can directly exert the positive tumor killing effect of IFN-W and PD-1 monoclonal antibody, and the PD-1 monoclonal antibody can effectively block the induction by IFN- Tumor cells express PD-L1-induced immunosuppression, which enhances the killing effect on tumors and compensates for their unfavorable characteristics compared with IFN-I and PD-1 monoclonal antibodies alone, further expanding the anti-tumor application. The scope is expected to become a kind of artificial immunotherapy drug with stronger tumor letting ability and wider application range.

The embodiments of the present application are described below by way of examples, and those skilled in the art should understand that the specific embodiments are merely illustrative of the embodiments of the invention. Improvements to the technical solutions of the present application are apparent in light of the teachings of the present application, and are within the scope of the present disclosure.

The drugs and reagents used in the following examples are not generally described, and are all commercially available products.

Example 1: Preparation of PD-1 scFv antibody with high PD-1 binding activity

(1) A plurality of monoclonal candidate phage containing anti-PD-1 scFv were selected by a human phage antibody library screening method, and the phage obtained by screening was tested for binding ability to PD-1 by an ELISA method.

The method was as follows: ELISA plate was coated with human PD-1 recombinant protein (ACRO Biosystems, PD1-H5221) at 50 μL/well 2 μg/mL, overnight at 4 ° C; ELISA plate was blocked with 200 μL of 3% MPBS, 37 ° C for 3 h; 50 μL The selected phage antibody was added to the ELISA plate well, incubated at 37 ° C for 1.5 h; PBST was washed 3 times, and anti-M13 monoclonal antibody-HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05-50) was added, and incubated at 37 ° C for 1 h. ; TMB color developer color, A450 value after 2M sulfuric acid termination. At the same time, M13KO7 helper phage was used as a negative control.

The results are shown in Table 1 below.

Table 1

The ELISA assay of Table 1 demonstrates the human anti-PD-1 scFv obtained by the phage screening method (the amino acid sequence is shown in SEQ ID NO. 32-36, and the nucleotide sequence is shown in SEQ ID NO. 42-46) It is capable of binding to PD-1 with high affinity.

(2) The sequences of the heavy and light chains of the PD-1 scFv antibody having high PD-1 binding activity and the CDR regions are exemplified as follows:

A plurality of specific light chains are screened using a human phage antibody library screening method, comprising any of the CDRs of SEQ NO. 1-16, wherein

LCDR1: selected from any one of SEQ ID NOS. 1 to 5.

LCDR2: selected from any one of SEQ ID NOS. 6 to 10.

LCDR3: selected from any one of SEQ ID NOS. 11 to 16.

Heavy chain sequence: comprises any of the HCDRs of SEQ NO. 17-31.

Heavy chain CDR sequences:

HCDR1: any one of SEQ ID NOS. 17-21

HCDR2: any of SEQ ID NOS. 22-26

HCDR3: any of SEQ ID NOS. 27-31

The above antibody is a fully human antibody or a murine antibody, preferably a fully human antibody.

Example 2. Construction and expression of a fusion protein comprising IFN-γ-PD-1 scFv antibody (ID1)

I. Construction of an expression vector containing the ID1 gene

1. Template: human anti-PD-1 scFv sequence (the amino acid sequence of which is shown in SEQ ID NO. 32-36, the nucleotide sequence is shown in SEQ ID NO. 42-46) and truncated human IFN- The γ sequence (the amino acid sequence of which is shown in SEQ ID NO. 41 and the nucleotide sequence is shown in SEQ ID NO. 52) was synthesized by Jin Weizhi Biotechnology Co., Ltd.

2. Primers:

IFN-γ amplification primers:

pIFN-F: 5'-gcggatccCAGGACCCATATGTTAAAG-3' (SEQ ID No. 37)

pIFN-R: 5'-CTCCACCAGCTGCACCTG-3' (SEQ ID No. 38)

Anti-PD-1ScFv Amplification Primer:

pNFV-F: 5'-CAGGTGCAGCTGGTGGAG-3' (SEQ ID No. 39)

pNFV-R: 5'-GCCTCGAGttaACGTTTGATCTCCACGTTG-3' (SEQ ID No. 40).

3. Expression vector: pGEX-6p-1.

4. Method:

(1) IFN-γ and anti-PD-1ScFv were amplified by Primestar polymerase mix (TAKARA, R045A), respectively.

Amplification system: Primer F: 1 μl, primer R: 1 μl, template 1 μl, Primestar polymerase mix 25 μl, ddH2O to 50 μl.

Amplification conditions: 94 ° C 90 s, (94 ° C 15 s, 50 ° C 5 s, 72 ° C 10 s) × 30 cycles, 72 ° C 5 min.

(2) The amplified fragment was subjected to a gel recovery kit (QIAGEN, 28704) and subjected to overlap PCR.

Amplification system: IFN-γ recovery fragment: 100 ng, anti-PD-1 scFv recovery fragment: 100 ng, polymerase 12.5 μl, supplement ddH2O to 25 μl.

Amplification conditions: 94 ° C 90 s, (94 ° C 15 s, 50 ° C 5 s, 72 ° C 5 s) × 10 cycles, 72 ° C 5 min.

(3) Taking 5 μl of the previous product as a template, PCR amplification was continued with the IFN-γ upstream primer pIFN-F and the anti-PD-1 ScFv downstream primer pNFV-R.

Reaction system: pINF-F: 1 μl, pNFV-R: 1 μl, template 5 μl, Primestar polymerase mix 25 μl, supplement ddH2O to 50 μl.

Amplification conditions: 94 ° C 90 s, (94 ° C 15 s, 55 ° C 5 s, 72 ° C 5 s) × 30 cycles, 72 ° C 5 min.

(4) pGEX-6P-1 and fusion target fragment were digested with restriction endonuclease BamHI (NEB, R3136S) and XhoI (NEB, R0146S), respectively, and digested at 37 °C for 2 h, and DNA was carried out. Recycling.

(5) The vector and the fragment were ligated with T4 ligase (NEB, M0202L), and the ligation system was: pGEX-6P-1 vector fragment: 1 μl, fusion target fragment: 4 μl, buffer: 1 μl, T4 ligase: 1 μl, Supplement ddH ₂ O to 10 μl. After 4 h at room temperature, it was transfected into E. coli Trans-T1 competent cells (TransGen Biotech Co., Ltd., CD501). Plates were plated on 2YT medium agar plates containing a final concentration of 100 μg/ml ampicillin.

(6) Picking clones for colony PCR verification and extracting plasmids for sequencing verification.

(7) The correctly sequenced recombinant vector was transformed into E. coli BL21 (DE3) competent cells.

5. Results: The full-length sequence of the amplified ID1 gene is shown in SEQ ID Nos. 53 to 57 (which respectively encode the amino acid sequences shown in SEQ ID No. 47 to 51), and colony PCR verification and sequencing results showed recombination. The vector pGEX-6p-1-ID1 was successfully constructed.

Example 3. Prokaryotic expression and purification of ID1 fusion protein

1. Medium:

(1) Seed medium (g·L ^-1 ): Tryptone 16, yeast powder 10, NaCl 5, pH 7.0-7.4.

(2) Expression medium (g·L ^-1 ): Tryptone 12, yeast powder 24, glycerol 4, KH ₂ PO ₄ 2.31, K ₂ HPO ₄ 12.54, pH 7.0.

2. Method:

(1) The recombinant strain pGEX-6p-1-ID1/E.coli BL21 (DE3) was cultured overnight, and transferred to 100 ml of the expression medium the next day, and cultured at 37 ° C and 200 rpm. The negative control group was set to pGEX-6p-1/E.coli BL21 (DE3).

(2) When OD ₆₀₀ = 0.6-1.0, isopropyl thiogalactoside (IPTG) having a final concentration of 0.2 mM was added, and the mixture was transferred to 16 ° C, 200 rpm for induction, and cultured overnight.

(3) The next day, the cells were collected and centrifuged, the supernatant was removed, 30 ml of 50 mM Tris-HCl was added, and phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) at a final concentration of 1 mM were added for sonication. The cells were broken, 200w power, 3/6s, 15min. After centrifugation at 10,000 rpm for 15 min, the supernatant was taken and centrifuged at 10,000 rpm for 15 min. The supernatant was collected and filtered through a 0.45 μm filter.

(4) GSTrap HP purification column purification:

Binding solution: 50 mM Tris-HCl pH 7.5-8.0;

Eluent: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0.

The column was equilibrated with 5 column volumes of binding solution; 0.5 ml/min was loaded; 5 column volumes, 1 ml/min flow rate washing column; 3-5 column volume eluent, 1 ml/min flow rate eluted to collect proteins.

3. Results: As can be seen from Fig. 2, compared with the control group, a target band of about 70 kD (including the GST tag of 26 kD) was found, indicating that a soluble protein component was present. The purification results are shown in Figure 3.

Example 4. Detection of ID1 fusion protein activity by enzyme-linked immunosorbent assay (ELISA)

(1) Detection of anti-PD-1 activity

Experimental design

A fusion protein having the amino acid sequence of SEQ ID No. 50 (anti-PD-1 scFv and IFN-γ comprising SEQ ID NO: 35) was tested for its anti-PD-1 activity. Positive control 1 was set to anti-PD-1 scFv (obtained by phage expression as in Example 1), positive control 2 - human anti-PD-1 antibody (crobiosystems, anti-PD-1 mAb, Human IgG4) (concentration 2.5 Μg/ml), negative control: BL21 (DE3) bacterial cell wall supernatant, sample 1 is purified ID1 protein.

2. The solution used

1 coating solution: 0.05mol / L carbonate buffer (pH 9.6)

0.75 g of sodium carbonate, 1.46 g of sodium hydrogencarbonate, and deionized water to a volume of 500 ml.

20.02 mol/L phosphate buffer (pH 7.4)

0.2 g of potassium dihydrogen phosphate, 2.90 g of disodium hydrogen phosphate, 8 g of sodium chloride, and deionized water to a volume of 1000 ml.

3 antibody dilution: 0.02mol / L PBS (pH 7.4) + 0.2% BSA

4 blocking solution: 0.05mol / L carbonate buffer (pH 9.6) + 2.0% BSA

5 washing solution: 0.02mol / L PBS (pH 7.4) +0.05% Tween-20

6 color solution: TMB-hydrogen peroxide urea solution

Liquid A (3,3',5,5'-tetramethylbenzidine, TMB): 20 mg of TMB was weighed and dissolved in 10 ml of absolute ethanol, and after completely dissolved, double distilled water was added to 100 ml.

Solution B (0.1mol/L citric acid-0.2mol/L disodium hydrogen phosphate buffer, pH 5.0-5.4): Weigh out 14.6g of Na ₂ HPO ₄ , 9.33g of citric acid dissolved in 180ml of double distilled water, add 0.75 % hydrogen peroxide urea 6.4ml, to a volume of 1000ml, adjust the pH to 5.0-5.4.

Mixing liquid A and liquid B in a ratio of 1:1 to form a TMB-hydrogen peroxide urea application liquid.

7 stop solution: 2mol / L H ₂ SO ₄ solution

3. Method

(1) The sample was added to an ELISA plate coated with PD-1 antigen, and incubated at 37 ° C for 1.5 h;

(2) The ELISA plate was washed 5 times with a 100-fold dilution of 10% Tween-20 stock solution (TPBS), and a 1:1000 diluted primary antibody was added to the sample group: mouse anti-GST antibody (Conway Reagent Biotechnology Co., Ltd.) , CW0084), incubate for 40 min; the positive control group did not add;

(3) After washing 5 times with TPBS, the sample group was added with 1:3000 diluted secondary antibody: goat anti-mouse antibody (Kangwei Century Biotechnology Co., Ltd., CW0102S), and incubated for 40 min (positive control 1 added anti-M13 antibody (Beijing) Yiqiao Shenzhou Biotechnology Co., Ltd., 11973-MM05-50)), positive control 2 added goat anti-human antibody (Abeam, ab6858);

(4) TMB color development for 5 min, 2 mol/L H ₂ SO _{4 was added to} terminate the reaction, and the absorbance at A450 nm was detected by a microplate reader.

2. Results: The results showed that the ID1 fusion protein has anti-PD-1 activity compared with the control group.

Table 1: Anti-PD-1 activity of ID1 fusion protein

Description: Based on space considerations, the inventors only displayed the A450 value-centered scFv sequence obtained by phage screening. SEQ ID NO: 35 was prepared to obtain the ID1 fusion protein as an example display. Other scFv sequences obtained by phage screening also have good when prepared as ID1 fusion protein. effect.

(II) Detection of IFN-γ activity of ID1 fusion protein

The IFN-γ activity was verified by verifying that the ID1 fusion protein acts on the PD-L1 expression level of HepG2-Luc cells for a certain period of time.

Experimental program:

1. Hepatoma cells HepG2-Luc were cultured in 24-well plates at 2 x 10 ⁴ cells per well, 500 μl/well.

2. HepG2-Luc cells were lysed for 6-8 hours, and lysate supernatant and IFN of different concentrations of pGEX-6p-1-ID1/E.coli BL21(DE3) strain and control E.coli BL21(DE3) strain were added. - γ, 3 replicate wells per concentration.

Preparation of lysate supernatant (BL21 supernatant) of different concentrations of pGEX-6p-1-ID1/E.coli BL21(DE3) strain (ID1 supernatant) and control E.coli BL21(DE3) strain: The ID1 bacterial supernatant and the BL21 (DE3) supernatant were freeze-dried (about 24 h), and the dried sample was reconstituted with an equal volume of DMEM complete medium according to the sample volume before drying, and diluted as follows:

A: 5 μl ID1 supernatant + 495 μl DMEM

B: 50 μl ID1 supernatant + 495 μl DMEM

C: 500μl ID1 bacterial supernatant + 0μl DMEM

D: 5 μl BL21 supernatant + 495 μl DMEM

E: 50 μl BL21 supernatant + 495 μl DMEM

F: 500μl BL21 supernatant +0μl DMEM

IFN-γ: mother liquor concentration 50ng/mL, working concentration 25ng/mL

3. Take 0.5 mL of the diluted sample and the control, add to the well-attached 24-well culture plate of HepG2-Luc cells, and add 0.5 mL of DMEM complete medium to a final volume of 1 mL/well.

4. Incubate for 24 h in a 37 ° C 5% CO ₂ incubator.

5, CCK8 detection of cell proliferation inhibition: CCK8 was added at a volume of 1/20 of the final volume of each well, placed in a 37 ° C 5% CO ₂ incubator for 1 h, and the corresponding well supernatant was taken up to 100 μL to a new 96-well plate, and the detection was performed at a wavelength of 450 nm. The OD of the ID1 fusion protein and the positive control and the negative control solution; the cells of each well were digested and collected in a 1.5 mL centrifuge tube, and the PD-L1 expression level of HepG2-Luc cells was detected by flow cytometry.

6, the results

The effect of the lysate supernatant of the recombinant strain expressing ID1 and the supernatant of the bacterial BL21(DE3) supernatant on the expression of HepG2-Luc expressing PD-L1 is shown in Fig. 4.

The results showed that 1) compared with the negative control, the supernatant of supernatant of BL21(DE3) was not affected by the expression of PD-L1 in HepG2-Luc cells, and the supernatant of BL21(DE3) containing the ID1 fusion protein was lysed by ultrasound. The supernatant has an effect on the expression of PD-L1 in HepG2-Luc cells, and there is a dose-effect relationship; 2) It is proved that the IFN-γ component of the ID1 fusion protein has an active effect.

Example 5. ID1 fusion protein promotes CTL killing of tumor cells

Experimental operation:

1. Peripheral blood mononuclear cells (PBMC) separation

(1) Blood collection: 50 mL of peripheral blood of the donor was taken under aseptic conditions, heparin was added at 30 U/mL, and dispensed into two sterile 50 mL centrifuge tubes.

(2) The upper layer plasma was aspirated at 3000 rpm (acceleration 7, deceleration 6) for 10 min, and the lower layer cells were diluted with an equal volume of physiological saline.

(3) Take a 50mL centrifuge tube, first add the same volume of human peripheral blood lymphocyte separation solution (Ficoll, Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1077) with the diluted cells of step (2), and then use a straw Carefully pipette the cells onto the Ficoll level.

(4) Centrifugation at 1800 rpm for 10 min, after centrifugation, the centrifuge tube was divided into four layers from top to bottom, and the second layer of the ring-shaped milky white substance was lymphocytes.

(5) Carefully pipette the second layer of lymphocytes with a pipette and transfer it to a new 15 mL centrifuge tube, and add 10 mL of physiological saline to the obtained centrifuge tube and mix.

(6) At 1800 rpm, centrifuge for 10 min, discard the supernatant, add 1 mL of red blood cell lysate to each 15 mL centrifuge tube, mix gently by pipetting and let stand for 5 min at room temperature.

(7) 9 mL of physiological saline was added, mixed, centrifuged at 1800 rpm for 10 min, the supernatant was discarded, and the cells were resuspended by adding GT-T551 complete medium, and counted.

2. Mixed culture of HepG2-Luc, THP-1 and PBMC cells

(1) Mitomycin C treatment of HepG2-Luc and THP-1 cells: Resolving mitomycin C in serum-free medium (sigma, M0503) at a working concentration of 10 μg/mL at 37 ° C 5% CO ₂ Incubate in the incubator, and incubate for 1.5 h every 10 min; collect the stimulated HepG2 and THP-1 cells, and wash them with PBS three times to remove residual mitomycin C as a stimulating cell.

(2) Inoculated in a T75 flask according to the ratio of PBMC cells to HepG2-Luc or THP-1 tumor cells in a ratio of 10:1, cultured with GT-T551 medium containing 10% autologous plasma and supplemented with IL-2 (100 u/ml). (Tongli Haiyuan, article number: TL-104) and CD3 (50u/ml) (Tongli Haiyuan, article number: TL-101), incubated for 3 days in a 37 ° C 5% CO2 incubator.

3. Specific killing experiments of CTL on HepG2-Luc and THP-1

(1) The cells co-cultured in the step 2 were collected, that is, specific CTL cells, washed twice with PBS, counted, and the cell concentration was adjusted.

(2) In a 96-well plate, HepG2-Luc and THP-1 cells (1×10 ⁴ /well) were mixed with specific CTL in a ratio of 50:1 or 10:1, and grouped according to Table 2, Purified ID1 fusion protein samples and controls, total volume 200 μL, 3 replicate wells in each group.

Table 2

(3) After mixing the groups, incubate for 18 h in a 37 ° C 5% CO ₂ incubator, carefully pipet 100 μL of the supernatant, and place in a new 96-well plate, after which they are treated as follows:

The target cells were THP-1 using a lactate dehydrogenase (LDH) cytotoxicity test kit (Biyuntian, C0016), and the absorbance was measured at 490 nm, and the killing efficiency of each group of samples and the control substance against the cells was statistically analyzed;

The target cells were HepG2-Luc group, and 100 μL of D-luciferin (0.075 mg/mL) (MCE, HY-12591A) was added to the wells of the remaining 100 μL medium for 5 min in the dark, and then detected by a living imager (spectral Amix). HepG2-Luc (with Fg-Luc fluorescent gene) was used to measure the fluorescence intensity of cells, and the cell killing efficiency of each group was statistically analyzed.

The purified ID1 fusion protein promotes the killing effect of CTL cells on HepG2-Luc cells as shown in FIG. The effect of different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells was determined by detecting the effective target ratios of 50:1 and 10:1, respectively. The results are shown in Fig. 6 (** indicates p<0.01; *** indicates p< 0.001) and Figure 8 (* indicates p < 0.05). In vivo imaging was used to detect the effect of different concentrations of ID1 fusion protein on the efficiency of CTL killing HepG2-Luc cells at 50:1 and 10:1. The results are shown in Figure 7 and Figure 9, where the fluorescence intensity is higher. High, representing more living cells, the lower the killing efficiency. The effect of the ID1 fusion protein on the efficiency of CTL killing of THP-1 cells at 50:1 was shown in Figure 10 (**** indicates p < 0.0001).

The results showed that the ID1 fusion protein of the present application can promote the killing effect of specific CTL on HepG2-Luc and THP-1, and the experimental group added with the ID1 fusion protein has significant difference compared with the positive control.

Example 6. Construction of eukaryotic expression vector pBudCE4.1 containing the tID1 fusion protein gene of (IFN-γ) ₂ -PD-1 scFv2

We selected the anti-PD-1 scFv2 sequence of SEQ ID No: 36 (which comprises the light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 3, 7 and 13, respectively, and SEQ ID NO: 21, respectively. The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in 26 and 31;) fused with two IFN-γ molecules to form a (IFN-γ) ₂ -PD-1 scFv2 structure, further examining two IFN-γ The nature and function of the molecular PD-1 scFv fusion protein. Based on this, we synthesized the sequence SEQ ID NO: 60 containing the (IFN-γ)2-PD-1 scFv2 gene.

The specific construction process is as follows:

(1) Gene synthesis The sequence containing the (IFN-γ) ₂ -PD-1 scFv2 gene SEQ ID NO: 60, which is provided at the 5' end of the sequence of the sequence (IFN-γ) ₂ -PD-1 scFv2 gene HindIII cleavage site, and XbaI restriction site is set at the 3'end;

(2) The synthesized sequences of SEQ ID NO: 60 and pBudCE4.1 were treated with HindIII and XbaI endonuclease, and digested at 37 ° C for 2 h, and DNA recovery was carried out;

(3) The vector and the fragment were ligated with T4 ligase (NEB, M0202L), and the ligation system was: pBudCE4.1 vector fragment: 1 μL, fusion target fragment: 4 μL, buffer: 1 μL, T4 ligase: 1 μL, supplement ddH ₂ O to 10 μL. After 4 h at room temperature, it was transfected into E. coli Trans-T1 competent cells (TransGen Biotech Co., Ltd., CD501). Plates were plated on 2YT medium agar plates containing a final concentration of 100 μg/ml ampicillin.

(4) Picking clones for sequencing verification.

RESULTS: The sequencing result showed that the recombinant vector pBudCE4.1-tID1 fusion protein was successfully constructed, and the full-length DNA sequence of the amplified tID1 fusion protein gene is shown in SEQ ID No. 59, which encodes the amino acid shown as SEQ ID No. 58. a sequence comprising the light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 3, 7 and 13, respectively, and the heavy chain CDR1, CDR2 of the amino acid sequences set forth in SEQ ID NOs: 21, 26 and 31, respectively. And CDR3.

Example 7: Eukaryotic expression and purification of tID1 fusion protein

(1) The pBudCE4.1-tID1 construct was electroporated into 293T/17 cells, and after 7 days, the cell culture medium was collected to obtain a supernatant expressing the tID1 fusion protein (with a His purification tag).

(2) Ni column affinity chromatography purification:

Equilibrate: 20 mM PB buffer (16.8 mM Na ₂ HPO4·12H ₂ O + 3.2 mM NaH ₂ PO 4 · 2H ₂ O, pH = 7.4), 150 mM NaCl;

Washing solution: 20 mM PB buffer (16.8 mM Na ₂ HPO ₄ · 12H ₂ O + 3.2 mM NaH ₂ PO 4 · 2H ₂ O, pH = 7.4), 150 mM NaCl, 10 mM imidazole;

Eluent: 20 mM PB buffer (16.8 mM Na ₂ HPO ₄ · 12H ₂ O + 3.2 mM NaH ₂ PO 4 · 2H ₂ O, pH = 7.4), 150 mM NaCl, 500 mM imidazole;

Steps:

The Ni column (GE Healthcare Ni Sepharose ^TM excel, GE Cat. No. 45-003-011) was equilibrated with an equilibration solution of 20 column volumes;

The collected fusion protein expression supernatant was applied at 0.5 ml/min; the Ni column was washed at a flow rate of 1 ml/min using 5 column volumes of the washing solution;

The Ni column was eluted at a flow rate of 1 ml/min using 3-5 column volumes of eluate to collect the tID1 fusion protein;

The eluate was concentrated using an ultrafiltration centrifuge tube to obtain a tID1 fusion protein at a concentration of 8.9 mg/mL.

(3) Molecular sieve chromatography purification:

Balanced molecular sieve column Superdex 20010/30 (GE) using an equilibration solution, ie 20 mM PB buffer (16.8 mM Na ₂ HPO4·12H ₂ O + 3.2 mM NaH ₂ PO 4 ·2H ₂ O, pH=7.4), 150 mM NaCl) Then, the 8.9 mg/mL tID1 fusion protein obtained by eluting 0.5 mL of the above was applied to the chromatography, and the flow rate was 0.3 ml/min. According to the A280 absorption value, 1 ml of the No. 2 tube, 1 mL of the No. 3 tube, and 0.68 mL of the No. 4 tube were collected (Fig. 11). 20 μL of each tube was taken for SDS-PAGE electrophoresis.

3. Results: As can be seen from Fig. 12, compared with the control group, a target band of about 63 kD was found, indicating the presence of the soluble target protein tID1 fusion protein component. The 72 kD band above the band of interest indicates the glycosylated form of the tID1 fusion protein.

Example 8: tID1 fusion protein fusion protein blocks PD-1 binding to PD-L1

(1) Determination of the binding of tID1 fusion protein to PD1 by ELISA

method:

1. ELISA plate coated antigen PD1 (His-tag): 2ug/mL 100μL/well; 2. TID1 fusion protein (0.257mg/mL, purity 98%), diluted according to the following table 2 series, to 100μL/well Add ELISA plate and incubate at 37 ° C for 1.5h

3, add primary antibody: APC-anti human IFNG Ab (1:400) 100 μL / well, incubate at 37 ° C for 1 h;

4. Add secondary antibody: Goat-anti-mouse IgG (H+L) (1:2000) 100 μL/well, incubate for 1 h at 37 °C.

5. The detection OD value was detected at a wavelength of 450 nm, and for each tID1 fusion protein concentration, three duplicate wells were set.

Table 2

See Figure 13 for the binding curve.

(B) Determination of the binding of tID1 fusion protein to IFNGR by ELISA

method:

1. The antigen IFNGR (Fc-tag) was coated on the ELISA plate at 2.5 ug/mL and 100 μL/pack;

2. The tID1 fusion protein (0.257 mg/mL purity 98%) was diluted according to the following 3A series; the positive control IFN-γ was serially diluted according to the following Table 3B;

3. The tID1 fusion protein and the positive control were added to the corresponding wells of the ELISA plate at 100 μL/well, and incubated at 37 ° C for 1.5 h;

4, add primary antibody: APC-anti human IFNG Ab (1:400), 100 μL / well, incubate at 37 ° C for 1 h;

5, add secondary antibody: Goat-anti-mouse IgG (H + L) (1: 2000), 100 μL / well, incubate at 37 ° C for 1 h;

6. The detection OD value was detected at a wavelength of 450 nm, and for each tID1 fusion protein concentration, three duplicate wells were set.

See Table 3A below for the results.

Table 3A ELISA for detection of binding of tID1 fusion protein to IFNGR

The binding of IFNG to IFNGR was examined in the same manner. See Table 3B for the results.

Table 3B ELISA for detection of binding of IFNG to IFNGR

The binding curve is shown in Figure 14, in which Figure A is the binding curve of tID1 fusion protein to IFNGR, and Figure B is the binding curve of IFN-γ and IFNGR.

The results showed that the EC50 of tID1 fusion protein binding to IFN receptor was _more than 10 times higher than that of positive control IFN-γ, indicating that tID1 fusion protein has good binding activity to IFNGR.

(C) Detection of the binding of tID1 fusion protein to PD1 antigen and IFNGR by SPR method

As a result, as seen from Fig. 15, the ka (1/(M*s)) of the tID1 fusion protein bound to the PD-1 antigen was 8.17e+05, Kd(1/s) was 5.17e-03, and KD(M) was 6.32. E-09, positive control anti-PD-1 antibody (Pdab) binding to PD-1 antigen ka (1/(M*s)) was 3.88e+05, Kd(1/s) was 1.21e-03, KD (M) is 3.11e-09; ka(1/(M*s)) of tID1 fusion protein binding to IFN-γ receptor is 2.04e+06, Kd(1/s) is 1.64e-03, KD (M) is 8.05e-10. The above data indicate that the tID1 fusion protein has a good affinity with the PD-1 antigen and the IFN-γ receptor, and can meet the therapeutic needs.

(4) Determination of the binding of tID1 fusion protein to PD-1 of different animals by ELISA

Methods: ELISA was used to detect the binding of tID1 fusion protein to human, mouse, dog and monkey PD-1.

1. The antigen human PD-1 mouse PD-1, canine PD-1, and monkey PD-1 were coated with ELISA plate at a concentration of 2 ug/mL, respectively.

2. Sample dilution: The tID1 fusion protein (initial concentration: 0.257 mg/mL, purity 98%) was diluted to different concentrations according to the following Table 4, and added to the ELISA plate at 100 μL/well, and incubated at 37 ° C for 1.5 h.

3. Add primary antibody: APC-anti IFN-gamma Ab (1:400), 100 μL/well, incubate for 1 h at 37 °C.

4. Add secondary antibody: Goat anti mouse IgG HRP (1:2000), 100 μL/well, incubate for 1 h at 37 °C.

5. Detection and detection of OD at a wavelength of 450 nm, see Table 4

Table 4

See Figure 16 for the binding curve. According to the binding curve, the tID1 fusion protein binds to human PD-1 and monkey PD-1, and does not bind to both mouse PD-1 and canine PD-1, thereby showing that the fusion tID1 fusion protein obtained by the present invention is human PD- 1 specific.

(5) Detection of the binding effect of tID1 fusion protein fusion protein on PD-1 and PD-L1 binding by ELISA at the molecular level

Methods: The blocking effect of tID1 fusion protein on PD-1/PD-L1 binding was detected by biotinylated PD-1:PD-L1 inhibitor screening kit (Acro Biosystems, EP-101). Refer to the product manual. Briefly, ELISA plate was coated with 100 μL/well 2 μg/mL of PD-L1 at 4 ° C overnight; 200 μL/well 3% MPBS was added, blocked at 37 ° C for 3 h; 0.6 μL of organism was added to the centrifuge tube. 100 μL of the sampled test solution of PD-1 (100 μg/mL) and different dilutions of tID1 fusion protein were incubated at 37 ° C for 1.5 h; the test sample and the control sample were added to the ELISA plate, incubated at 37 ° C for 1.5 h; PBST was washed 4 times. 100 μL/well of HRP-labeled streptavidin (0.4 μg/mL) was added and incubated at 37 ° C for 1 h; PBST was washed 4 times to develop color; A450 value was measured.

The OD values at a wavelength of 450 nm are shown in Table 5 below:

table 5

logC(nM)logC(nM)	nMnM	ug/mlUg/ml	A450A450
3.22916973.2291697	16951695	100100	0.09240.0924
2.92813972.9281397	847.5847.5	5050	0.10580.1058
2.62710972.6271097	423.75423.75	2525	0.16180.1618
2.32607972.3260797	211.875211.875	12.512.5	0.21910.2191
2.02504972.0250497	105.9375105.9375	6.256.25	0.39430.3943
1.72401971.7240197	52.9687552.96875	3.1253.125	0.68260.6826
1.42298971.4229897	26.48437526.484375	1.56251.5625	1.02071.0207
1.12195971.1219597	13.24218813.242188	0.781250.78125	1.10971.1097
0.82092970.8209297	6.62109386.6210938	0.3906250.390625	1.37921.3792
0.51989970.5198997	3.31054693.3105469	0.19531250.1953125	1.41291.4129
0.21886970.2188697	1.65527341.6552734	0.09765630.0976563	1.47831.4783
-0.08216-0.08216	0.82763670.8276367	0.04882810.0488281	1.42451.4245
	00	00	1.44121.4412

See Figure 17 for the blocking curve

The results showed that the tID1 fusion protein has a blocking effect on the binding of PD-1 to PD-L1.

(6) Determination of binding of tID1 fusion protein to PD-1 at the cellular level by flow cytometry

The 293T-PD-1 cell line is a cell line constructed by the applicant to stably express the PD-1 antigen, and the 293T cell line used for construction is ATCC product number CRL-11268.

Table 6: tID1 fusion protein to bind EC 293T-PD-1 cells in ₅₀

The results of detecting the binding of the tID1 fusion protein to 293T-PD-1 cells by flow cytometry are shown in the binding curve of FIG.

The results indicate that the tID1 fusion protein of the present application is capable of binding to the 293T-PD-1 cell line.

(VII) The binding of tID1 fusion protein to 293T-EGFP/PD-1 and PD-L2

The PD-L2 used in the method was HumanB7-DC/CD273protain-His (Cat: 10292-H08H, SinoBiological). Detection was performed by flow cytometry.

The OD450 detection results of tID1 fusion protein blocking the binding of 293T-EGFP/PD-1 to PD-L2 are shown in Table 7 below.

Table 7

The binding curve of the tID1 fusion protein blocking 293T-EGFP/PD-1 to PD-L2 is shown in FIG.

The results indicate that the tID1 fusion protein is able to block the binding of 293T-EGFP/PD-1 to PD-L2.

Example 9 tID1 fusion protein promotes tumor cell expression of PD-L1

The effect of tID1 fusion protein on the expression level of PD-L1 in hepatoma cells hepG2 and cervical cancer cell hela was tested. Methods as below:

1. Hepatoma cells hepG2 and cervical cancer hela were seeded in 24-well culture plates at appropriate densities, respectively.

2. Prepare 2× diluted tID1 fusion protein sample (tID1 fusion protein concentration 4.3 μM, purity 98%. Dilution method: dilute to 272nM (2×136) with complete medium, then double dilution, take 500μL/ The pores have a final concentration of 136 nM).

3 Replace the cells in the 24-well plate of step 1 with a new complete medium, 500 μL/well.

4 Take 500 μL of the prepared sample and the positive control IFN-γ, add to the corresponding cell wells, and incubate for 24 h in 37 ° C 5% CO ₂ culture.

5. Collect the cells of each well in a 1.5 ml centrifuge tube and wash twice with PBS.

6. After resuspending the cells with 100 μL of PBS, 1 μL of APC-CD274 antibody (APC anti-human CD274 (PDL1, B7H1) Antibody APC, eBioscience) was added and incubated on ice for 30 min in the dark.

7. Wash twice with PBS buffer and analyze the expression level of PD-L1 using flow cytometry.

The results are shown in Figure 20.

The results showed that there was a dose-effect relationship between the concentration of tID1 fusion protein and the expression level of PD-L1 in tumor cell lines.

For hepG2 cells, PD-L1 expression increased with increasing concentration of tID1 fusion protein, but when the concentration of tID1 fusion protein increased above 2.72 nM, PD-L1 expression level reached saturation and no longer increased (Fig. A); For hela cells, the tID1 fusion protein at a lower concentration of 0.34 nM resulted in a higher level of hela cells than the positive control IFN-γ PD-L1 (see Figure B), but did not increase PD- as the concentration continued to increase. The expression level of L1.

Therefore, for the tID1 fusion protein, the hepatoma cell hepG2 and the cervical cancer cell hela can be more effectively promoted to express PD-L1 when it is lower than the same molar IFN-γ, which indicates that the tID1 fusion protein has better targeting specificity.

On the other hand, the data in Figure 1 also showed that the tID1 fusion protein promoted the different concentrations of hepatoma cell hepG2 and cervical cancer cell hela expression in PD-L1 equivalent to the IFN-γ positive control, the former being 2.72 nM, the latter being lower than the latter 0.34 nM, which also provides a basis for subsequent screening of drug concentrations in different tumor cell lines.

Example 9: Effect of tID1 fusion protein on tumor cell proliferation

The effect of tID1 fusion protein on tumor cell proliferation was tested using hepatoma cell hepG2 and cervical cancer cell hela. Methods as below:

1. Hepatoma cells hepG2 and cervical cancer cells hela were inoculated separately into 96-well culture plates at appropriate densities.

2. A 2× diluted sample of the tID1 fusion protein was prepared the next day (original concentration: 6.3 μM purity 98%).

3. Replace the cells in the 96-well plate with a new complete medium, 50 μL/well.

4. Take 50 μL of the prepared sample and add it to the corresponding cell well.

Incubate in a 37 ° C 5% CO ₂ incubator.

5. After 24h and 48h, the cell proliferation assay was performed using the enhanced CCK8 assay kit (Biyuntian Biotechnology Co., Ltd., Cat. No. C0041).

The results are shown in Fig. 21 and Fig. 22.

As shown in Fig. 2 and Fig. 3, when the concentrations were 84.7 nM, 169.4 nM and 338.8 nM, respectively, the inhibition effect of tID1 fusion protein on the proliferation of hepG2 cells for 48 h was gradually enhanced, but the effect of 24 h increased cell proliferation; for hela cells With the increase of the concentration of tID1 fusion protein, the inhibition of cell proliferation is gradually enhanced, and the inhibition of proliferation is also enhanced with the prolongation of the action time.

Example 10: Binding of tID1 fusion protein to CD3 induced CD4+ T cells

Binding of the tID1 fusion protein to CD3-induced CD4+ T cells was detected by flow cytometry. Methods as below:

1. Isolation of PBMC: lymphocyte separation solution Ficoll separation of human peripheral blood mononuclear cells PBMC.

2. Induction of CD4+ T cells: CD4+ T cells were induced and cultured by adding 50 ng/mL of CD3 and 100 U/mL of IL-2 to PBMC cells.

3. Two-step CD4+ T cells were seeded at 3 x 10 ⁵ /50 μL into V-type 96-well plates.

4. According to the experimental group, add different concentrations of tID1 fusion protein, and set up adjustment and compensation control wells, the final volume of each well was 100 μL, and incubate on a horizontal shaker for 90 min.

5. Wash twice with PBS and centrifuge for 5 min at 500 g.

6. Add the flow detection antibody His-tag, antiCD4 and antiCD8 to the tID1 fusion protein group, and add 1 μL of antiCD4 and antiCD8. The corresponding adjustment antibody was also added to these flow detection antibodies, and incubated on a horizontal shaker for 30 min.

7. Wash twice with PBS and centrifuge for 5 min at 500 g.

8. The cells of each well were resuspended in 100 μL of PBS, transferred to a 1.5 mL centrifuge tube, and the binding of the tID1 fusion protein to CD3-induced CD4+ T cells was detected on a flow cytometer.

The results are shown in Table 8 below and Figure 23. The results indicate that the tID1 fusion protein is capable of binding to CD3-induced CD4+ T cells.

Table 8

Example 11. tID1 fusion protein promotes killing of target cells by PBMC

(1) To examine the effect of tID1 fusion protein on PBMC killing HepG2-luc cells. Methods as below:

1) separation of PBMC: separation of human peripheral blood mononuclear cells PBMC using lymphocyte separation solution Ficoll;

2) Cell count: Count HepG2-luc cells, adjust the concentration to 2 × 10 ⁵ /mL; count PBMC, adjust the concentration to 10 ⁷ /mL;

3) PBMC cells were added to a round-bottom 96-well plate at 50 μL/well, and different concentrations of tID1 fusion protein were added according to the experiment, and 50 U/mL of IL-2 was added for stimulation, and the final volume of each well was adjusted to 100 μL;

4) HepG2-luc cells were added to a round-bottom 96-well plate at 50 μL/well, and different concentrations of tID1 fusion protein samples were also added according to the experiment, and the final volume of each well was adjusted to 100 μL;

5) The cells of steps 3) and 4) were each placed in a 37 ° C incubator for 10 min;

6) Move the HepG2-luc cells of step 4) to the PBMC of step 3), mix well, prepare a killing hole with a target ratio of 50:1, and a final volume of 200 μL per well, and incubate in a 37 ° C incubator;

7) 96-well plates were taken at 16 h and 24 h, respectively, and 100 μL of D-luc substrate was added to each well, and the fluorescence intensity of bioluminescence was measured using a small animal imager (AMIX, model: SI-Image AMIX).

The result is shown in Fig. 24.

The tID1 fusion protein was incubated with PBMC and hepatocellular carcinoma cell hepG2 at three concentrations of 1.02 nM, 10.2 nM and 102 nM for 16 h and 24 h. The results showed that: with anti-PD1 antibody: Anti-PD1 mAb, Human (IgG4) Lot No. B52- Compared with the 63NS1-AS, ACRO Biosystems group, these three concentrations showed significant activity in promoting PBMC killing of hepG2 cells, with the highest killing effect at 10.2nM; 10.2nM tID1 fusion protein compared with the same molar anti-PD1 antibody It has a stronger promotion of the killing activity of PBMC on hepG2 and is statistically significant.

(II) Detection of the effect of tID1 fusion protein on PBMC killing human bladder cancer cell line T24 cells

The method is as described in the experiment (I) of the present embodiment. The human bladder cancer cell line T24 cell line was purchased from ATCC article number HTB-4. See Figure 25 for the results.

The tID1 fusion protein was incubated with PBMC and human bladder cancer cell line T24 for 24 h at both concentrations of 10.2 nM and 102 nM. The results showed that the 10.2 nM tID1 fusion protein was stronger than the same molar positive control (anti-PD1 antibody, sourced from above). The PBMC promoted the killing activity of human bladder cancer cell line T24, and it was statistically significant.

The above experimental data of the present application proves that the immunotherapeutic product provided by the present application has both the tumor killing effect against tumor cytokines and the precise targeting advantage of immunological checkpoint inhibitors. Therefore, the fusion of cytokines and immunological checkpoint inhibitors not only makes them better maintain their respective functions, but also has a synergistic effect, so that the fusion of the present application is a new tumor treatment plan with important development prospects in the field of tumor treatment.

The embodiments disclosed in the present application are as described above, but the description is only for the purpose of understanding the present application, and is not intended to limit the present application. Any modifications and changes in the form and details of the embodiments may be made by those skilled in the art without departing from the spirit and scope of the disclosure. The scope defined by the appended claims shall prevail.

Industrial applicability

The fusion comprising the immunological checkpoint inhibitor and the cytokine or the protein or polypeptide comprising the same provided by the embodiments of the present application can effectively treat the disease, improve or alleviate the discomfort, and is suitable for the development and industrial production of the new drug.

Claims

A fusion for immunotherapy comprising an immunological checkpoint inhibitor and a cytokine.
The fusion according to claim 1 wherein said immunological checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, indoleamine 2,3-dual oxygenation Enzyme (IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator (BTLA) inhibitor, T cell immunoglobulin inhibition Agent, TIM-3 inhibitor, and Killer-cell Immunoglobulin-like Receptor (KIR) inhibitor expressed on the surface of NK cells and part of T cells.
The fusion according to claim 1, wherein the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a guanamine 2,3-dioxygenase ( IDO-1) inhibitor, 4-1BB (CD137) inhibitor, OX40 (CD134) inhibitor, B cell and T cell attenuator, T cell immunoglobulin, TIM-3, and expressed on NK cells and part of T cell surface Killer cell immunoglobulin-like receptor.
The fusion according to claim 1, wherein the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.
The fusion according to any one of claims 1 to 4, wherein the immunological checkpoint inhibitor is an anti-immunization checkpoint antibody.
The fusion according to claim 5, wherein the anti-immunization checkpoint antibody is an anti-PD-1 antibody.
The fusion according to claim 6, wherein said anti-PD-1 antibody comprises a domain that specifically recognizes and binds to immune cell surface antigen PD-1 and a constant region derived from an immunoglobulin constant region (Fc), said specificity The domain that recognizes and binds to the immune cell surface antigen PD-1 includes a light chain variable region (anti-PD-1 VL) having three CDRs and a heavy chain variable region (anti-PD-1 VH) having three CDRs. Wherein the light chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 1-16; and the heavy chain variable region ( The anti-PD-1 VH) comprises a heavy chain CDR (HCDR) selected from the amino acid sequences set forth in SEQ ID NOs: 17-31.
The antibody or functional fragment thereof according to claim 7, wherein the light chain variable region comprises:

LCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 1, 2, 3, 4 and 5,

LCDR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 6, 7, 8, 9 and 10, and

LCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 11, 12, 13, 14, 15 and 16;

The heavy chain variable region comprises:

HCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,

HCDR2, the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and

HCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.
The antibody or functional fragment thereof according to claim 7 or 8, which comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;

b) a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;

c) a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;

d) a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;

e) a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;

f) Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.
The antibody or functional fragment thereof according to claim 7 or 8, which comprises a heavy chain variable region selected from the group consisting of:

g) a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;

h) a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;

i) a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;

j) a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;

k) Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.
The antibody or functional fragment thereof according to claim 7 or 8, which comprises a light chain variable region selected from any one of VL1, VL2, VL3, VL4, VL5 and VL6 and selected from the group consisting of VH1, VH2, VH3, Heavy chain variable region of any of VH4 and VH5.
The antibody or functional fragment thereof according to claim 7, wherein the antibody or functional fragment thereof comprises:

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 1, 7, and 13, respectively, and the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 17, 22, and 27, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 2, 6 and 12, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 18, 23 and 28, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 5, 10 and 16, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 19, 24 and 29, respectively; or

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOs: 21, 26 and 31, respectively;

The light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 3, 7 and 13, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 21, 26 and 31, respectively, and

Wherein the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.
The fusion according to claim 7, which comprises the amino acid sequence of SEQ ID NO: 47, 48, 49, 50, 51, 58.
The fusion according to claim 7, which comprises the nucleotide sequence of SEQ ID NO: 53, 54, 55, 56, 57, 59.
The fusion according to any one of claims 1 to 14, wherein the cytokine is capable of inhibiting tumor growth.
The fusion according to claim 15, wherein the cytokine is selected from the group consisting of: interleukin (IL), tumor necrosis factor (TNF), interferon (IFN), colony stim μlating factor (CSF), transformation Growth factors, growth factors (GF) and the chemokine family.
The fusion according to claim 16, wherein the colony stimμlating factor (CSF) comprises G (granulocyte)-CSF, M (macrophage)-CSF, GM (granulocyte, macrophage) -CSF, Multi (multiple)-CSF (IL-3), stem cell factor (SCF), erythropoietin (EPO);

The tumor necrosis factor (TNF) is selected from the group consisting of TNF-α and TNF-β;

The transforming growth factor is a transforming growth factor-βfamily (TGF-βfamily) TGF-β1, TGF-β2, TGF-β3, TGFβ1β2 or bone morphogenetic protein (BMP);

The growth factor (GF) includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I. (IGF-I), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF) -α), vascular endothelial growth factor (VEGF);

The chemokine family is selected from the group consisting of CXC/α subfamily, CC/β subfamily, C-type subfamily, and CX3C subfamily; wherein the CXC/α subfamily includes IL-8, a growth-regulated oncogene / melanoma growth stimulating factor (GRO/MGSA), platelet factor-4 (PF-4), platelet basic protein (PBP/CXCL7), proteolytic-derived product CTAP-III and β-platelet globulin (β- Thromboglobulin, β-TG), interferon-inducible protein-10, Epithelial Neutrophil-Activating Protein 78 (ENA-78); Phage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES (regulated upon activation normal T-cell expressed and secreted (CCL5), monocyte chemotactic protein-1 (MCP-1/MCAF), MCP-2 MCP-3 and I-309; the C-type subfamily comprises a lymphocyte chemotactic protein; the CX3C subfamily comprises a chemokine fractal (Fractalkine).
The fusion according to claim 16, wherein the cytokine is selected from any one of IFN-α, IFN-β and IFN-γ, preferably IFN-γ.
The fusion according to claim 18, wherein said cytokine is IFN-γ, preferably IFN-γ dimer.
The fusion according to claim 18 or 19, wherein the antibody or a functional fragment thereof comprises: the light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOS: 3, 7 and 13, respectively, and The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence set forth in SEQ ID NOS: 21, 26 and 31; or the light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; The heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 21, 26 and 31.
A method of preparing the fusion of any of claims 1-20, comprising the steps of:

(1) preparing an immunological checkpoint inhibitor;

(2) determining the amino acid sequence of the immunological checkpoint inhibitor obtained in the step (1);

(3) determining a nucleotide sequence encoding the immunological checkpoint inhibitor according to the amino acid sequence of the immunological checkpoint inhibitor obtained in the step (2);

(4) using the nucleotide sequence encoding the immunological checkpoint inhibitor obtained by the step (3) and the nucleotide sequence encoding the cytokine as a template, respectively designing primers and amplifications for amplifying the immunological checkpoint inhibitor Adding primers for the cytokine;

(5) Recombinant construction of an immunological checkpoint inhibitor-cytokine fusion protein expression vector;

(6) transforming an immunological checkpoint inhibitor-cytokine fusion protein expression vector into a recipient strain;

(7) screening an immunological checkpoint inhibitor-expression strain of a cytokine fusion protein;

(8) expressing an immunological checkpoint inhibitor-cytokine fusion protein and sequencing;

(9) Purification of an immunological checkpoint inhibitor-cytokine fusion protein.
The method according to claim 21, wherein said immunological checkpoint inhibitor is an anti-immunization checkpoint antibody, which is prepared by a phage library screening method, a hybridoma method, preferably by a phage library screening method.
The method of claim 22, wherein the anti-immunization checkpoint antibody is an anti-PD-1 antibody.
The method according to any one of claims 21 to 23, wherein the cytokine is IFN-γ, preferably an IFN-γ dimer.
A nucleic acid molecule encoding a fusion according to any one of claims 1-20.
An expression vector comprising the nucleic acid molecule of claim 25, the expression vector being selected from the group consisting of a eukaryotic expression vector and a prokaryotic expression vector.
A host cell comprising the expression vector of claim 26.
A protein or polypeptide comprising the fusion of any of claims 1-20.
A method of detecting the activity of an immune checkpoint inhibitor-cytokine fusion according to any one of claims 1-20.
Use of the immunological checkpoint inhibitor-cytokine fusion according to any one of claims 1 to 20 for the preparation of a medicament for treating a disease, ameliorating or ameliorating discomfort.
A method for treating a disease, ameliorating or ameliorating discomfort, the method comprising administering the immune checkpoint inhibitor-cytokine fusion of any one of claims 1 to 20.
The use according to claim 30 or the method of claim 31, wherein the disease and discomfort are PD-1 mediated diseases and discomforts.
The invention according to claim 30 or the method of claim 31, wherein the diseases and discomforts include cancer, inflammatory diseases, and infectious diseases.
The use or method of claim 33, wherein the cancer, inflammatory disease, and infectious disease are PD-1 mediated.
The use or method according to claim 33, wherein the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, and thyroid Adenocarcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectum Cancer, adrenal cancer, anal cancer, vulvar cancer, urinary tract cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodgkin's lymph Tumor, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, T-cell lymphoma , chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), childhood solid tumors and lymphocytic lymphoid One or more of these.
The use or method according to claim 33, wherein the infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infectious disease caused by a virus: hepatitis virus (eg hepatitis A, B or C), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, round Virus, measles virus, rubella virus, parvovirus, JC virus or arbovirus viral encephalitis virus, or infectious diseases caused by bacteria: pneumococcal, mycobacteria, staphylococcus, streptococcus, meningococcus, conococci , Serratia, Klebsiella, Proteobacteria, Pseudomonas, Salmonella, Vibrio cholerae, Diphtheria, Botox, Bacillus anthracis, Clostridium tetani, Legionella, Yersinia , leptospirosis or Lyme disease bacteria, or infectious diseases caused by the following fungi: Aspergillus (eg Aspergillus fumigatus, Aspergillus niger, etc.), Candida (eg Candida albicans), Candida krusei, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, dermatitis bud, Mucor genus (such as Mucor, Absidia or Rhizopus), S. skrjab, Brazil paraspora Infectious diseases caused by bacteria, Coccidioides or granulosa histoplasma, or the following parasites: amoeba in dysentery, colonic guinea worm, flucuff, amoeba, vivax malaria, voles Beetle, sucking Giardia, Cryptosporidium, Pneumocystis carinii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania polygala, Rat toxoplasma gondii or Nematode.
The use or method according to claim 33, wherein the disease is cancer; the cancer is selected from the group consisting of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, Kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, intestinal cancer, and non-small cell lung cancer; the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection, the chronic virus is HIV , HBV or HCV.