Antibody fusion protein and application

Abstract

The invention discloses an antibody fusion protein which simultaneously targets CD24, IL4R and an integrin receptor. In-vitro determination shows that the antibody fusion protein disclosed by the invention remarkably inhibits proliferation, migration, invasion, colony formation and adhesion of kidney cancer cells, shows a comprehensive and remarkable anti-tumor effect on gastric cancer, colon cancer and breast cancer, and has an effect of inhibiting angiogenesis. The result of an MC38-hPDL1C57BL6 / J mouse subcutaneous transplantation tumor model shows that the antibody fusion protein disclosed by the invention is combined with a PDL1 antagonist azolizumab, so that the growth of tumors can be remarkably inhibited. The antibody fusion protein can be used as a potential candidate drug for treating metastatic renal cell carcinoma, PDL1 overexpression cancer, ophthalmic diseases and inflammation or autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and more specifically, relates to an antibody fusion protein, a polynucleotide encoding the antibody fusion protein, a vector, a cell, a composition, and their use in preparing anti-tumor and ophthalmic disease treatment drugs. Background Art

[0002] Cancer remains one of the top ten diseases that affect the lives of people all over the world. Tumor recurrence, metastasis and drug resistance occur commonly, and there is still a lack of effective therapeutic drugs that can control and solve the problems of cancer recurrence and metastasis. The tumor microenvironment (TME) is a very complex system that contains many suppressed immune cells, such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells, suppressive NK cells and regulatory B cells, and the mechanisms that promote tumor progression are still unclear. Due to the complex heterogeneity of solid tumors and the immunosuppressive tumor microenvironment (TME), the efficacy of cell therapy products is also limited. Therefore, researchers still urgently need to develop more targeted and effective anti-cancer drugs with the ability to regulate TME.

[0003] Cancer cells are highly similar to normal human cells, many of which express immunosuppressive ligands or receptors and secrete inhibitory factors that affect the infiltration, polarization, and phenotype of immune cells. At the same time, the EMT-MET transformation and angiogenesis of cancer cells provide adaptive conditions for tumor growth and metastasis. Developing a multispecific drug that significantly inhibits tumor growth and angiogenesis and regulates the immune system may be a potentially effective therapeutic strategy.

[0004] Jialiang Hu et al. reported that HM-3 and sunitinib regulate integrin α v β3, α5β1, VEGFR2, and α v The β3-VEGFR2 complex is distributed inside and outside the lipid raft region to regulate endothelial cell migration and intracellular RhoGTPase activity (J ExpClin Cancer Res. 2019; 38: 381). VEGF-targeted drugs can inhibit the growth of primary tumors, but ultimately shorten the survival of mice by promoting tumor invasion and metastasis, which may be caused by increased levels of cytokines that stimulate metastasis and angiogenesis (Cancer Cell. 2009 Mar 3; 15 (3): 220-31, and Cancer Cell. 2009 Mar 3; 15 (3): 232-9). The peptide HM-3 was constructed by connecting RGD to the C-terminus of the ES-2 peptide using a glycine linker. RGD is a tripeptide sequence that is regulated by integrin α vβ3 has high affinity. ES-2 corresponds to amino acids 60-70 of endostatin and inhibits endothelial cell migration and differentiation by binding to integrin α5β1 (J Exp Clin Cancer Res. 2019; 38:381). TingLi et al. reported a HM-3-HSA fusion protein with good stability, which inhibited the growth and tumor angiogenesis of H22 and SMMC-7721 tumor xenografts in vivo; at the same time, it was proved that HM-3-HSA can inhibit the growth of H22 and SMMC-7721 tumor xenografts and tumor angiogenesis through integrin α v β3 and α5β1 bind to HUVEC and inhibit the phosphorylation of downstream protein kinases including FAK, Src and PI3K.

[0005] Literature reports that CD24, IL4R, and integrin receptors are highly expressed in malignant tumors such as breast cancer, gastric cancer, colon cancer, kidney cancer, and head and neck cancer. At the same time, CD24, IL4R, and integrin receptors are closely related to the proliferation, migration, invasion, adhesion, and stemness of tumor cells. Bioinformatics analysis shows that CD24, IL4R, and integrin receptors are significantly upregulated in many malignant tumors compared with normal tissues, and their expression levels are highly correlated with the degree of tumor malignancy and clinical prognosis.

[0006] The differential expression of CD24, IL4R, and integrins in tumor cells and normal tissues suggests that these targets could serve as potential specific targets for anti-tumor therapy. Summary of the invention

[0007] The present invention found that integrin receptor α / β is co-expressed in HUVECs and is closely related to integrin-mediated vascular proliferation, migration, invasion and angiogenesis. Based on this, the present invention provides an antibody fusion protein that simultaneously targets CD24, IL4R and integrin receptors, and has significant angiogenesis inhibition and anti-tumor cell effects in vivo and in vitro, including inhibition of proliferation and metastasis of mouse xenografts. The present invention further provides the use of the antibody fusion protein in the preparation of anti-tumor, ophthalmic disease, inflammatory or autoimmune disease treatment drugs.

[0008] [Antibody fusion protein]

[0009] The first aspect of the present invention provides an antibody fusion protein, comprising fused angiostatic polypeptide HM-3, interleukin 4 variant (ILDM) and anti-CD24 monoclonal antibody domain.

[0010] As a preferred embodiment of any technical solution of the first aspect of the present invention, in the antibody fusion protein, angiostatic polypeptide HM-3 or interleukin 4 variant (ILDM) is fused to the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain.

[0011] [Scheme for C-terminal fusion of interleukin-4 variant (ILDM) with the heavy chain domain of anti-CD24 monoclonal antibody]

[0012] As a preferred embodiment of any technical solution of the first aspect of the present invention, in the antibody fusion protein, the interleukin 4 variant (ILDM) is fused to the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain, for example, via a linker.

[0013] As a preferred embodiment of any technical solution of the first aspect of the present invention, in the antibody fusion protein, the interleukin 4 variant (ILDM) and the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain are linked by a first linker. Preferably, in the antibody fusion protein, the angiostatic polypeptide HM-3 and the interleukin 4 variant (ILDM) are linked by a second linker, and the first linker and the second linker are the same or different.

[0014] As a preferred embodiment of any technical solution of the first aspect of the present invention, the antibody fusion protein comprises the amino acid sequence SEQ ID NO.1. (57103-H amino acid sequence)

[0015] As a preferred embodiment of any technical solution of the first aspect of the present invention, the antibody fusion protein comprises the amino acid sequence SEQ ID NO.3. (57103-L amino acid sequence)

[0016] [Scheme for fusion of angiostatic peptide HM-3 with the C-terminus of the heavy chain domain of anti-CD24 monoclonal antibody]

[0017] As a preferred embodiment of any technical solution of the first aspect of the present invention, in the antibody fusion protein, the angiostatic polypeptide HM-3 is fused to the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain, for example, via a linker.

[0018] As a preferred embodiment of any technical solution of the first aspect of the present invention, in the antibody fusion protein, the angiostatic polypeptide HM-3 is linked to the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain through a first linker. Preferably, in the antibody fusion protein, the interleukin 4 variant (ILDM) is linked to the angiostatic polypeptide HM-3 through a second linker, and the first linker is the same as or different from the second linker.

[0019] A pharmaceutically acceptable salt of the antibody fusion protein described in any technical solution of the first aspect of the present invention.

[0020] [Polynucleotide]

[0021] The second aspect of the present invention provides a polynucleotide comprising a nucleotide sequence encoding an antibody fusion protein, wherein the antibody fusion protein comprises an angiostatic polypeptide HM-3, an interleukin 4 variant (ILDM) and an anti-CD24 monoclonal antibody domain linked by a linker.

[0022] As a preferred embodiment of any technical solution of the second aspect of the present invention, the polynucleotide sequence includes the nucleotide sequence of SEQ ID NO.2 encoding the antibody fusion protein. (57103-H nucleotide sequence)

[0023] As a preferred embodiment of any technical solution of the second aspect of the present invention, the polynucleotide sequence further comprises a nucleotide sequence of SEQ ID NO.4 encoding the antibody fusion protein. (57103-L nucleotide sequence)

[0024] [Carrier]

[0025] The third aspect of the present invention provides a vector, wherein the vector comprises the polynucleotide described in any scheme of the second aspect.

[0026] [cell]

[0027] The fourth aspect of the present invention provides a cell, wherein the cell comprises the vector described in any one of the schemes of the third aspect.

[0028] [Composition]

[0029] The fifth aspect of the present invention provides a composition comprising the antibody fusion protein, pharmaceutically acceptable salt of the antibody fusion protein, polynucleotide, vector or cell described in any one of the first to fourth aspects. For example, it may also comprise a pharmaceutically acceptable carrier or excipient.

[0030] As a preferred embodiment of any technical solution of the fifth aspect of the present invention, the composition further comprises a PDL1 antagonist.

[0031] As a preferred embodiment of any technical solution of the fifth aspect of the present invention, the PDL1 antagonist includes atezolizumab or durvalumab.

[0032] [Application in the preparation of medicines]

[0033] The sixth aspect of the present invention provides the use of the antibody fusion protein, or a pharmaceutically acceptable salt of the antibody fusion protein, or a polynucleotide, or a vector, or a cell, or a composition described in any one of the first to fourth aspects in the preparation of anti-tumor drugs, drugs for treating ophthalmic diseases, inflammation or autoimmune diseases.

[0034] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the tumor includes primary cancers, melanomas and sarcomas originating from the human head and neck, brain, thyroid, esophagus, pancreas, lungs, liver, stomach, breast, kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, bladder, and testicles.

[0035] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the tumor includes one or more of renal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, colon cancer, glioma, melanoma or cervical cancer.

[0036] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the ophthalmic disease includes one or more of iris neovascular eye disease, choroidal neovascular eye disease, retinal neovascular eye disease or corneal neovascular eye disease.

[0037] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the iris neovascular eye disease includes one or more of neovascular glaucoma, diabetic retinopathy or iris neovascular eye disease caused by central retinal vein thrombosis;

[0038] The choroidal neovascularization eye disease includes one or more of age-related macular degeneration, central exudative retinopathy, ocular histoplasmosis syndrome or choroidal neovascularization eye disease caused by glaucoma;

[0039] The retinal neovascular eye disease includes one or more of retinal neovascular eye diseases associated with diabetes, tumors, retinal detachment, central retinal vein occlusion, retinal periphlebitis, systemic lupus erythematosus, Eales disease or Coat disease;

[0040] The corneal neovascular eye diseases include corneal neovascular diseases caused by corneal contact lenses, and one or more of the corneal neovascular eye diseases caused by alkali and other chemical burns, corneal surgery, bacterial infection, chlamydia infection, viral infection or protozoan infection.

[0041] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the inflammation includes one or more of rheumatoid arthritis, osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, infectious arthritis and traumatic arthritis.

[0042] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the autoimmune disease includes one or more of lupus erythematosus and psoriasis.

[0043] The sixth aspect of the present invention also provides the use of the antibody fusion protein, or a pharmaceutically acceptable salt of the antibody fusion protein, or a polynucleotide, or a vector, or a cell, or a composition described in any one of the first to fifth aspects in the preparation of a drug for inhibiting FGF2 or VEGFA-induced renal cancer RCCs or other angiogenesis-driven cancers or diseases.

[0044] As a preferred embodiment of any technical solution of the sixth aspect of the present invention, the dosage form of the drug includes one or more of capsules, tablets, pills, water injections, powder injections, injections, eye drops, nasal sprays or aerosols.

[0045] [Method for preparing antibody fusion protein]

[0046] The seventh aspect of the present invention provides a method for preparing the antibody fusion protein described in any scheme of the first aspect, using one or more of a mammalian cell expression system, a yeast expression system or a prokaryotic expression system.

[0047] Preferably, the mammalian cell expression system includes human embryonic kidney cells HEK293 and Chinese hamster ovary cells CHO; the yeast expression system includes one or more of Pichia pastoris, Saccharomyces cerevisiae or Hansenula; the prokaryotic expression system includes one or more of Escherichia coli or Bacillus subtilis.

[0048] [Treatment Method]

[0049] The eighth aspect of the present invention provides a method for treating an individual suffering from a tumor, an ophthalmic disease, an inflammation or an autoimmune disease using the antibody fusion protein, a pharmaceutically acceptable salt of the antibody fusion protein, a polynucleotide, a vector, a cell or a composition as described in any one of the first to fifth aspects, the method comprising administering to the individual an effective amount of the antibody fusion protein, polynucleotide, vector, cell or composition.

[0050] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the tumor includes primary cancers, melanomas and sarcomas originating from the human head and neck, brain, thyroid, esophagus, pancreas, lungs, liver, stomach, breast, kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, bladder, and testicles.

[0051] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the tumor includes one or more of renal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, colon cancer, glioma, melanoma or cervical cancer.

[0052] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the ophthalmic disease includes one or more of iris neovascular eye disease, choroidal neovascular eye disease, retinal neovascular eye disease or corneal neovascular eye disease.

[0053] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the iris neovascular eye disease includes one or more of neovascular glaucoma, diabetic retinopathy or iris neovascular eye disease caused by central retinal vein thrombosis;

[0054] The choroidal neovascularization eye disease includes one or more of age-related macular degeneration, central exudative retinopathy, ocular histoplasmosis syndrome or choroidal neovascularization eye disease caused by glaucoma;

[0055] The retinal neovascular eye disease includes one or more of retinal neovascular eye diseases associated with diabetes, tumors, retinal detachment, central retinal vein occlusion, retinal periphlebitis, systemic lupus erythematosus, Eales disease or Coat disease;

[0056] The corneal neovascular eye diseases include corneal neovascular diseases caused by corneal contact lenses, and one or more of the corneal neovascular eye diseases caused by alkali and other chemical burns, corneal surgery, bacterial infection, chlamydia infection, viral infection or protozoan infection.

[0057] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the inflammation includes one or more of rheumatoid arthritis, osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, infectious arthritis and traumatic arthritis.

[0058] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the autoimmune disease includes one or more of lupus erythematosus and psoriasis.

[0059] As a preferred embodiment of any technical solution of the eighth aspect of the present invention, the administration method of the treatment method includes intravenous injection, intraperitoneal injection or subcutaneous injection.

[0060] The antibody fusion protein molecule of the present invention is novel in design and contains HM-3, ILDM and anti-CD24 monoclonal antibody domains. The data in the examples show that the 57103 antibody fusion protein is proven to be a potential multi-specific antibody fusion protein with good anti-tumor effect.

[0061] The 57103 antibody fusion protein can target tumor tissues that highly express CD24 and integrins, reduce the systemic effects of HM-3 and ILDM, improve specific killing efficacy, and exhibit specific regulatory effects on the tumor microenvironment.

[0062] The 57103 antibody fusion protein has a more significant CD24 / α than HM-3, IL4DM or the monoclonal antibody targeting 571C1 v β3 anti-angiogenic effect.

[0063] 57103 antibody fusion protein promotes internalization of CD24 tumor cells and improves immune cells that highly express CD24 / α through ADCC and ADCP effects v Disruption of β3 / IL4R in cell lines.

[0064] The 57103 antibody fusion protein showed good anti-tumor metastasis ability both in vivo and in vitro, and can be used as a potential therapeutic agent against tumor metastasis and recurrence.

[0065] The results of the MC38-hPDL1 C57B6 / J mouse subcutaneous transplantation tumor model showed that the efficacy of 57103 antibody fusion protein combined with PDL1 antagonist was better than that of Anti-PDL1 monoclonal antibody Atezolizumab treatment and Anti-PDL1 monoclonal antibody Atezolizumab and Endostar combined treatment. The results were statistically significant, indicating that 57103 antibody fusion protein and PDL1 antagonists, especially Atezolizumab, have possible additive or synergistic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Paired and unpaired differential expression analysis of pan-cancer target genes. (AB) Paired analysis of CD24 (A) and IL4R (B). (CD) Unpaired analysis of IL4R (C) and CD24 (D). (E) Venn diagram showing the intersection of CD24 and IL4R overexpressing cancer types. Mann-Whitney U statistical analysis was used, *p < 0.05, **p < 0.01, ***p < 0.001.

[0067] Figure 2 Paired and unpaired target gene analysis of differential gene expression of KIRC and KIRP. (A) Paired and unpaired analysis of CD24 and IL4R. (B) Paired and unpaired analysis of KIRC integrin receptor genes; (C) Paired and unpaired analysis of KIRP integrin receptor genes; Statistical analysis method was Mann-Whitney U. *p<0.05, **p<0.01, ***p<0.001.

[0068] Figure 3Gene correlation analysis of KIRC and KIRP. (A) Correlation analysis of KIRC was performed using ggplot2[3.3.6] and R(4.2.1); (B) Correlation analysis of KIRP was performed using ggplot2[3.3.6] and R(4.2.1); the statistical method was Spearman(log2(value+1)). (C) Target correlation network diagram of KIRC. (D) Target correlation network diagram of KIRP. The target correlation network diagram was constructed using the circlize[v0.4.1] R package, and the Spearman correlation coefficient was used for analysis. The results showed that most target genes were positively correlated with other target genes, with statistical significance (p<0.05). Red, positive correlation; blue, negative correlation.

[0069] Figure 4 To determine the expression level of target genes using qRTPCR, flow cytometry, and Western Blot, renal cell carcinoma cell lines were subcultured at 37°C, 5% CO2. Cells were then harvested, pretreated, and tested according to standard qRTPCR, flow cytometry, and Western Blot protocols. (A) qRTPCR results for renal cell carcinoma cell lines. CD24, IL4R, and integrin α were analyzed in renal cell carcinoma cell lines. v SN3, LM609, HuM200, and Atezolizumab biosimilars were used to detect CD24, IL4R, integrin α v Primary antibodies for β3, α5β1, and CD274 were used and combined with Alexa-fluor 647-labeled secondary antibodies according to the indirect cell surface staining protocol. For IL4R detection, FITC-labeled IL4 and FITC-labeled IL4DM were used. The relative positive percentage data for each target in renal cancer cells determined by FCM are summarized in the table. (C) Western Blot results of relative expression levels of target genes in each renal cancer cell line.

[0070] Figure 5 Flow cytometry and confocal laser fluorescence microscopy were used to detect target expression levels. (A) Renal cancer cells were collected, washed, and stained using target-specific antibodies based on cell surface staining. (B) Confocal laser fluorescence microscopy of 786O and 769P. The results showed that both 786O and 769P expressed high levels of CD24, IL4R, and integrin α v β3.

[0071] Figure 6 shows the construction and quality characterization of 571, 571C1, and 57103 antibody fusion proteins. (A) Schematic diagram of gene fusion strategy and protein structure. (B) Molecular dynamics simulation and docking and minimum free energy value. The monomer models predicted by LOMETS software from @zhanglab. Docking and optimization results based on haddock software from @BonvinLab. (C) Antibody fusion protein expression vector map and restriction enzyme digestion identification of transfection-grade plasmid samples. The samples were also sequenced and confirmed. The arrows in the figure indicate the target genes. (D) Results of HPLC and Coomassie Brilliant Blue detection of antibody fusion proteins.

[0072] Figure 7 shows the physicochemical properties of the antibody fusion protein. (A) The hydrophobicity of the samples was analyzed by HPLC-IHC. The ASBC of samples 571, 571C1, and 57103 were 1.13, 0.99, and 0.99, respectively, which were greater than the reference ASBC of bevacizumab (0.71) and trastuzumab ASBC (0.92), indicating good hydrophilicity. (ASBC concentration of ammonium sulfate when combined with a hydrophobic column (relative value under specific conditions), the larger the ASBC, the stronger the hydrophilicity.) (B) The thermal stability of the samples was analyzed by PR Panta (Nano Temper). The thermal stability of the samples and the uniformity of the DLS particles. (C) The precise molecular weight determination results of 57103 by LC-MS. (D) The precise molecular weight determination results of the samples.

[0073] Figure 8 The results of MST affinity determination and ELISA binding and functional verification of antibody fusion proteins. (A) Analysis of the interaction between Cy5-labeled 57103 and CD24 detected by microscale thermophoresis (MST) (Kd = 1.97 nM) (B) Analysis of the interaction between Cy5-labeled 57103 and IL4R detected by MST (Kd = 1.1 nM). (C) Analysis of the interaction between Cy5-labeled 57103 and α v The MST affinity assay result of β3 showed an affinity of 92 nM. (D) 571 (no binding), 571C1 (EC50 = 18.03 ng / mL, R 2 =0.9938) and 57103 (EC50 = 34.03 ng / mL, R 2 =0.9963) and CD24 ELISA binding test results. (E) 571 (EC50 = 11.83 ng / mL, R 2 =0.9700), 571C1 (no binding) and 57103 (EC50 = 5.10 ng / mL, R 2 =0.9895) and IL4R ELISA binding test results. (F) 571 (EC50 = 741.90 ng / mL, R2 =0.9680), 571C1 (EC50 = 4290.00ng / mL, R 2 =0.9863) and 57103 (EC50 = 520.50 ng / mL, R 2 =0.9843) and α v ELISA binding results between β3. (G) 571 (EC50 = 1.03 × 10 4 ng / mL, R 2 =0.9832), 571C1 (no binding) and 57103 (EC50 = 1.37 × 10 4 ng / mL, R 2 =0.9891) ELISA binding test results with α5β1. (H) Competitive ELISA test of 57103 inhibiting the binding of IL4 to IL4R, EC50 is 538.1ng / ml (R 2 =0.9809). (I) Schematic diagram of the binding and basic functions of the 57103 fusion protein.

[0074] Figure 9 shows the detection of HUVEC target expression levels and drug binding analysis. (A) qRT-PCR of HUVEC markers VWF and CD31. (B) Flow cytometric analysis of the binding capacity of 57103 (EC50 = 1.86 μg / ml, R 2 =0.9865). (C) Analysis of the expression levels of drug targets in HUVEC cells, CD31 (90.6%), α v β3 (85%), α5β1 (92.9%), αv (85.3%), KDR (19.4%), IL4RA (38.2%), CD24A (53.0%), β1 (95.2%). Each experiment was tested in triplicate. Quantitative analysis data are expressed as mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0075] Fig.1057103 has a significant anti-angiogenic effect. (AC) HUVECs were subjected to drug proliferation assays using CCK8 under different treatments (no growth factor addition (A), 50ng / mL VEGF165 stimulation (B) and 50ng / mL FGF2 stimulation (C). (D) Representative image of HUVECs wound healing. (E) Representative image of HUVECs Transwell chamber migration. (F) Micrograph of HUVECs Transwell chamber invasion. (G) HUVECs wound healing assay ((i) wound healing migration distance and (ii) wound healing migration inhibition rate, (iii) number of HUVECs cell migration in Transwell chamber, (iv) number of HUVECs cell invasion in Transwell chamber. The results showed that 57103 could significantly inhibit the migration and invasion of HUVECs. Image-ProPlus 6.0 The migration distance, number of migrating cells and number of invasive cells of different drugs were determined. Migration and invasion inhibition rate (%) = [(control - experimental) / control] × 100. (H) Fluorescence imaging photos of HUVECs tube formation assay. (I) Fluorescence imaging photos of rat aortic rings. (J) Quantitative analysis of HUVECs tube formation assay ((i) sprouting area and (ii) inhibition rate), rat aortic ring assay ((iii) total tube length (mm) and (iv) number of branch points). (K) The target expression level of HUVECs samples decreased with the increase of 57103 concentration. HUVECs treated with 5.12μM 57103 were significantly different from the control (NC). Data are expressed as mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0076] Fig.11 In vitro and in vivo targeting studies of 571, 571C1, and 57103. (A) Schematic diagram of the structures of 571, 571C1, and 57103 molecules after Cy7 labeling. (B) SDS-PAGE Coomassie Brilliant Blue staining results and fluorescence detection diagrams of Cy7-labeled 571, 571C1, and 57103 molecules (R-Reduced, reduced sample; NR-Non-reduced, non-reduced sample). (C) Affinity test results of ACHN, A498, and 786O renal cancer cells and Cy7-labeled 57103 (57103-Cy7); ACHN, A498, and 786O cells were washed and stained, and antibody binding was analyzed using BECKMAN COULTER (CytoFLEX) flow cytometry. The affinities of 57103 targeting ACHN, A498, and 786O were 9.64 nM (R- 2 =0.9664),1.45nM(R 2 =0.9931) and 1.63nM (R2 =0.9931). (D) Fluorescence imaging results of 571, 571C1 and 57103 labeled with Cy7 in a subcutaneous renal cancer cell line xenograft model. The results show that the fluorescently labeled antibody fusion protein molecules can be targeted and enriched in subcutaneously transplanted tumor tissues.

[0077] Fig.12 57103 significantly inhibited the proliferation, metastasis and adhesion of RCCs. (A) CCK8 cell viability analysis showed that 57103 significantly inhibited the growth of three RCCs cells, and 57103 5.12μM had the highest inhibition rate on renal cancer cell proliferation, which was higher than 571C1 or 571 (P<0.001). The inhibition rates of 5.12μM 57103 on 786O, A498, and ACHN were 79.27% ​​p<0.001, 58.30% (p<0.001), and 72.02% (p<0.001), respectively. (BC) Representative micrographs of RCC colony formation assay under different drug treatment conditions (B) and quantitative analysis results (C). (D) Quantitative analysis of RCCs (i) wound healing migration inhibition rate, (ii) Transwell chamber migration assay, and (iii) Transwell chamber invasion assay. (EG) Micrographs of RCCs (E) Wound healing, (F) Transwell chamber migration assay, (G) Transwell chamber invasion assay. (H) Adhesion assay to evaluate the ability of 57103 to inhibit vitronectin-driven or fibronectin-driven adhesion interactions. 2×10 4 Cells were cultured in 96-well plates coated with glass protein or fibronectin overnight, and the culture was continued for 1 h after adding drugs. (I) Platelet adhesion assay to determine the inhibitory effect to reduce the number of platelets specifically attached to renal cancer cells. Quantitative analysis data are mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0078] Fig.13 Figure 5 shows the analysis of ADCP and ADCC internalization mediated by 571C1 and 57103. (A) Microscopic imaging analysis of phagocytosis. Renal cancer cell lines were stained with pH-sensitive fluorescent dye (Protonex TM (B) Representative images of Luciferase-positive GFP-positive RCC cells (LUC+GFP+RCC) and hPBMC co-cultured under different drug administration conditions. (C) Expression of different CD24 and integrin α vThree human renal cancer cells with high levels of β3 were incubated as target cells overnight and then co-cultured with (i) THP1-derived M0 (E:T = 4:1) and (ii) IL2-stimulated human PBMC (E:T = 30:1) for 72 h with or without 5.12 μM 57103 or 571C1, and the expression of luciferase reporter assay was used. (D) Expression of CD24-positive α3 in NK-92MI-CD16a and renal cancer cells with or without 57103 at an E:T = 10:1 ratio was detected. v β3-positive ACHN, A498, and 786O renal cancer cell cytotoxicity. Luciferase assay showed that NK-92MI-CD16a cells had a concentration-dependent enhanced cytotoxic effect on RCCs in the presence of 57103. (E) The internalization of 57103-labeled with pH-sensitive fluorescent dye in different types of cancer cells was observed by fluorescence microscopy (FM). The green fluorescence intensity represents the internalization level. (F) (i) Flow cytometry results of 786O cell samples treated with 571C1 at a concentration of 5.12 μM for 48 hours after staining with 571C1-Cy5 labeled antibody (red line); (ii) Flow cytometry results of 786O cell samples treated with 57103 at a concentration of 5.12 μM for 48 hours after staining with 571C1-Cy5 labeled antibody (red line); (iii) Flow cytometry results of 769P cell samples treated with 571C1 at a concentration of 5.12 μM for 48 hours after staining with 571C1-Cy5 labeled antibody (red line); Flow cytometry results of cell samples treated for 48 hours after staining with 571C1-Cy5 labeled antibody (red line); (iv) Flow cytometry results of cell samples treated with 769P for 48 hours after adding 57103 at a concentration of 5.12 μM after staining with 571C1-Cy5 labeled antibody (red line); the blue line represents the control group, which refers to the flow cytometry results of cell samples treated with the corresponding solvent for 48 hours after staining with 571C1-Cy5 labeled antibody. Flow cytometry was used to determine the internalization effect of Cy5-labeled 571C1 (571C1-Cy5) on the cell surface after staining. Quantitative analysis data are mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0079] Fig.14 57103 has a high anti-tumor efficacy and regulates the tumor immune microenvironment in nude mice containing ACHN. (A) Female nude mice aged 4 weeks (n = 6 / group) were injected with 5×10 6ACHN cells were injected with 57103 (30 mg / kg), 571C1 (30 mg / kg), 571 (30 mg / kg) or Avastin (10 mg / kg) every three days, and the tumor volume was monitored for 48 days. (B) Quantitative analysis of tumor growth curves (i) and tumor volume inhibition rates (ii) in different groups. The data of tumor volume are presented as mean ± standard deviation (Mean ± SD, n = 6). (C) IHC staining scoring analysis of Ki67, TUNEL and CD31 tumor proliferation, apoptosis and angiogenesis. (D) HE staining was used to identify the infiltration of ACHN cancer cells in lung tissue. (E) IHC and HE data were quantified using IHCProfiler (ImageJ) and Image-Pro Plus, respectively. (F) MC38-hPDL1 tumors were collected after animal sacrifice and washed with DPBS. (G) Average tumor growth curves of different treatments for the mouse tumor transplantation model MC38-hPDL1. (H) The tumor inhibition rate of the single therapy and combined therapy in the MC38-hPDL1 subcutaneous transplantation C57B6 / J model was better than that of the PBS control group. Quantitative analysis data are mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0080] Fig.15 57103 has antitumor effects in subcutaneous MGC-803 xenografts (A) Female C57BL / 6J mice aged 4 weeks (n = 10 / group) were injected intra-axillary with 4×10 6 The cells were treated for 21 days and the tumor volume was monitored. (B) The tumor growth of mice was analyzed at the end of the experiment.

[0081] (C) Tumor volume growth curve.

[0082] Fig.16 57103 has antitumor effect in subcutaneous HT29-Luc xenografts (A) Female nude mice aged 4 weeks (n=6 / group) were injected intra-axillary with 1×10 7 cells, and the tumor volume was monitored for 21 days. (B) Tumor growth of mice dissected at the end of the experiment. (C) Tumor volume growth curve.

[0083] Fig.17 57103 has antitumor effect in the subcutaneous transplantation model of breast cancer cell line MDA-MB-231. (A) Female nude mice aged 4 weeks (n=5 / group) were injected with 5×10 6 The cells were treated for 21 days and the tumor volume was monitored. (B) The tumor growth of mice was dissected at the end of the experiment. (C) Tumor volume growth curve, the high-dose 57103 group had a better effect.

[0084] Fig.18 To construct the MC38-hCD24 cell line and investigate the antitumor effect of 57103 in subcutaneous xenografts (A), 4-week-old female nude mice (n=6 / group) were injected with 5×10 6 The cells were treated for 21 days and the tumor volume was monitored. (B) The tumor growth of mice was analyzed at the end of the experiment.

[0085] Fig.19 To investigate the antitumor effect of 57103 in subcutaneously transplanted renal cancer cell line 786-O, (A) 4-week-old female C57BL / 6J mice were injected intra-axillary with 1×10 7 cells, and the drug was administered for 21 days, and the tumor volume was monitored. (B) The tumor growth of the mice was dissected at the end of the experiment. (C) Tumor volume growth curve, 57103 has a better effect and is superior to positive drugs.

[0086] Fig. 20 To investigate the antitumor effect of 57103 in subcutaneous AGS tumors (A), 4-week-old female C57BL / 6J mice were injected intra-axillary with 8×10 6 cells, and the drug was administered for 21 days, and the tumor volume was monitored. (B) The tumor growth of the mice was dissected at the end of the experiment. (C) Tumor volume growth curve, 57103 has a better effect and is superior to positive drugs.

[0087] Fig.21 To investigate the anti-tumor effect of 57103 in subcutaneously transplanted renal cancer cell line ACHN, (A) overall experimental process and experimental endpoints, tumor growth of dissected mice; (B) (i) tumor volume growth curve; (ii) tumor inhibition rate.

[0088] Fig. 22 57103 prolongs the survival of mice with MGC-803-Luc metastasis injected into the tail vein. (A) Overall experimental process; (B) Survival curve.

[0089] Fig.23 57103 prolongs the survival of mice with HT29-Luc metastatic tumors injected into the tail vein. (A) Overall experimental process; (B) Survival curve; (C) Fluorescence imaging display.

[0090] Fig.24 The mechanism of the 57103 antibody fusion protein.

[0091] In the figure:

[0092] AbQ15 refers to: Atezolizumab;

[0093] AbQ16 refers to: hIgG1-κIsotype isotype control;

[0094] AT stands for: Atezolizumab;

[0095] ED refers to: Endostar.

[0096] Figures 25 to 29 Related data for anti-rheumatoid arthritis experiments:

[0097] Fig.25 Anti-arthritis effect of the drug on RA rats; A: Experimental process; B: Photos of ankle joints of AA rats in different groups; C: Effects of the drug on changes in toe thickness (upper left), arthritis score (upper right), periodic body weight (lower left), and inhibition rate (lower right).

[0098] Fig.26 Changes of ankle joint HE staining and IHC indexes.

[0099] Fig. 27 It is a biochemical indicator of anticoagulation in whole blood.

[0100] Fig.28 The effects of different drugs on serum inflammatory factors in RA rats. All data are expressed as mean±SD. Compared with the control group, ####p<0.0001, compared with the model group, ****p<0.0001, ***p<0.001, *p<0.05.

[0101] Fig.29 It is the inhibition of inflammatory factor expression in serum.

[0102] Figures 30-31 Related data for in vitro anti-inflammatory experiments on human synovial cells:

[0103] Fig.30 Effects of drugs on proliferation of human synoviocytes.

[0104] Fig.31 To detect relevant inflammatory indicators by ELISA.

[0105] Figures 32-33 Related data of RAW264.7 in vitro anti-inflammatory experiment:

[0106] Fig.32 To evaluate the safety of RAW264.7 at different drug concentrations.

[0107] Fig.33 ELISA was used to detect relevant inflammatory indicators.

[0108] Figures 34-35 Related data for in vitro anti-inflammatory experiment of THP1 cells:

[0109] Fig.34 To evaluate the safety of different drug concentrations on THP-1 cells.

[0110] Fig.35 ELISA was used to detect relevant inflammatory indicators.

[0111] Figures 36-37 Related data for spleen cell in vitro anti-inflammatory experiment:

[0112] Fig.36 To evaluate the safety of different drug concentrations on spleen cells.

[0113] Fig.37 ELISA was used to detect relevant inflammatory indicators. DETAILED DESCRIPTION

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0115] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0116] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0117] [definition]

[0118] As used herein, "angiogenesis" or "neovascularization" refers to the formation, growth and / or development of new blood vessels.

[0119] As used herein, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, favorable or desired clinical results include, but are not limited to, one or more of the following results: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread (e.g., cancer cell metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, providing disease relief (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life and / or prolonging life. "Treatment" also encompasses reducing the pathological consequences of cancer or ocular neovascularization disorders. The method of the present invention treats any one or more of the above aspects.

[0120] The term "effective amount" as used herein refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition or disease, such as to improve, alleviate, mitigate and / or delay one or more of its symptoms. For cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (such as inhibiting tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, the effective amount is an amount sufficient to delay development. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. The effective amount can be administered in one or more administrations. The effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, slow down, slow down and preferably prevent cancer cells from infiltrating into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably prevent) tumor cancer cell metastasis to a certain extent; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. For ophthalmic diseases, such as ocular neovascularization disorders, an effective amount includes an amount sufficient to treat, inhibit, delay and / or prevent the neovascularization disorder or its symptoms, such as an amount sufficient to achieve one or more of the following: (i) inhibit or prevent drusen formation; (ii) cause a decrease in the number and / or size of drusen (drusen regression); (iii) cause a reduction or prevention of lipofuscin deposition; (iv) inhibit or prevent vision loss or slow the rate of vision loss; (v) inhibit choroidal neovascularization or slow the rate of choroidal neovascularization; (vi) cause a decrease in choroidal neovascularization characterized by (vii) inhibit choroidal neovascularization or slow the rate of retinal neovascularization; (viii) cause a reduction in the size and / or number of ablation lesions characterized by retinal neovascularization; (ix) improve visual acuity and / or contrast sensitivity; (x) reduce macular edema and / or reduce abnormal macular thickness; (xi) inhibit or prevent photoreceptor or RPE cell atrophy or apoptosis, or reduce the rate of photoreceptor or RPE cell atrophy or apoptosis; (xii) inhibit or prevent the progression of non-exudative macular degeneration to exudative macular degeneration.

[0121] As used herein, "individual" or "subject" or "patient" refers to a mammal, including but not limited to humans, cattle, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0122] As used herein, the term "antibody" or "antibody portion" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.

[0123] As used herein, the term "monoclonal antibody" refers to an antibody derived from substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being derived from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any particular method.

[0124] The present invention is further described below in conjunction with specific embodiments.

[0125] 1. Materials and Methods

[0126] 1.1 Cells and Animals

[0127] Cell lines 769P (SCSP-5060), ACHN (SCSP-5063), and Caki-1 (SCSP-5064) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). 786O, A498, OSRC2, and Caki-2 were provided by Procell Life Science & Technology Co., Ltd. 769-P, 786O, and OSRC2 were cultured in RPMI-1640 complete medium (PM150110, Procell, Wuhan, China). ACHN cells were cultured in MEM complete medium (MEM, PM150410, Procell, Wuhan, China). Caki-1 and Caki-2 were cultured in McCoy's 5A complete medium. 293T cells were cultured in Dulbecco's modified Eagle's medium complete medium (DMEM, HyClone, Palo Alto, CA). MDA-MB-231 was cultured in Leibovitz's L-15 containing 10% FBS (Procell, China). Cells were maintained in an incubator at 37°C, 80-90% humidity, and 5% carbon dioxide. All methods were performed in accordance with the relevant guidelines and regulations of the institutional biosafety committee. HUVECs were cultured in ECM medium (ScienCell) containing 1% ECGS, 5% FBS, and 1% P / S. NK92mi-CD16a was prepared according to the method in previously published literature (reference: Gene-modifiedNK-92MI cells expressing a chimeric CD16-BB-ζor CD64-BB-ζreceptor exhibitenhanced cancer-killing ability in combination with therapeutic antibody).

[0128] 1.2 Data download and analysis

[0129] Transcriptome data of 33 cancers were downloaded from TCGAPan-Cancer (TCGA). The DESeq2 method was used to perform differential expression analysis of target genes. The threshold parameters for differential expression analysis were FDR < 0.05 and |Log2FC| > 2. Then the RNAseq data of TCGA-KIRP (renal papillary cell carcinoma) and TCGA-KIRC (renal clear cell carcinoma) were downloaded from the TCGA database (https: / / portal.gdc.cancer.gov), and the data were extracted in TPM format and clinical data (log2(value+1)). The correlation between target genes was studied by Pearson's correlation analysis. Figure 3 Heat map showing Spearman's correlation coefficient (ρ) values ​​ranging from -1.0 (negative correlation, green panels) to 1.0 (positive correlation, red panels).

[0130] 1.3 Target expression level detection

[0131] Data for each independent biological sample in all qPCR experiments were obtained by repeating the experiment at least three times, and the results are shown as mean ± SD. For all qPCR results, the two-tailed Student's t test of GraphPad was used to calculate the P value. Cells were collected and lysed with RIPA lysis buffer (Beyotime, China), and qualified samples were loaded into SDS PAGE, transferred to PVDF membrane (Millipore), and stained with anti-ITGAV (1:500-1:1,000, Cell Signaling Technology). WB detection of β-actin was performed using anti-β-actin antibody to control the loading of cell lysates (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). Finally, the results of WB were detected by Tanon 5200 chemiluminescence imaging system (Tianneng, China), and grayscale analysis was performed using β-actin as the internal reference standard. All flow cytometry assays were analyzed using Beckman Coulter (CytoFLEX).

[0132] 1.4 Construction, expression and purification of antibody fusion protein

[0133] In the embodiment of the present invention, three antibody fusion proteins are constructed, namely:

[0134] 571: amino acid sequence: SEQ ID NO.5; nucleotide sequence: SEQ ID NO.6;

[0135] 571C1: heavy chain amino acid sequence: SEQ ID NO.7, light chain amino acid sequence: SEQ ID NO.8; heavy chain nucleotide sequence: SEQ ID NO.9, light chain nucleotide sequence: SEQ ID NO.10;

[0136] 57103: Heavy chain amino acid sequence: SEQ ID NO.1, light chain amino acid sequence: SEQ ID NO.3; heavy chain nucleotide sequence: SEQ ID NO.2; light chain nucleotide sequence: SEQ ID NO.4.

[0137] 571, 57103 used the Human IgG1 (L234A, L235A) antibody backbone sequence, and IL4DM connected to HM-3 was fused to the C-terminus of the antibody heavy chain through a GGGGSGGGGSGGGGS linker. Figure 6 shows a schematic diagram of the fusion strategy and structure. The plasmids encoding the heavy and light chains were transfected into CHO-K1 suspension adaptive cells and selected under MSX conditions containing a serum-free medium. Finally, CHO-K1 cells that stably expressed antibodies or antibody fusion proteins were obtained. The fermentation supernatant was collected on the 15th day and filtered through a 0.22 μm membrane after centrifugation. The filtered supernatants were purified on protein A HP columns (GE Healthcare) and then further purified by cation exchange chromatography using HiTrap SP HP cation exchange columns (GE Healthcare Life Sciences). Endotoxins were subsequently removed using a high-load endotoxin removal kit (88275; Thermo Fisher Scientific), and all steps were purified by FPLC (AKTAPURE, GE Healthcare Life Sciences). The antibody fusion protein stock solution was tested through a series of tests to confirm the molecular physicochemical properties and stability. Specifically, the hydrophobicity of the molecule was analyzed using Arc (Waters) and a chromatographic column (HIC; TSKgel Butyl-NPR; Tosoh, Tokyo, Japan). High-resolution and accurate molecular weight mass spectrometry data were obtained using Waters XEVO G2-XS QTOF and Acquity H-Class (HPLC). The thermal stability of the protein sample was analyzed using Prometheus NT.48 (NanoTemper Technologies).

[0138] 1.5 Antibody fusion protein structure simulation

[0139] The present invention is based on the monomer models predicted by LOMETS software of @zhanglab. The final model is obtained by protein-protein docking and manual optimization based on haddock software of @BonvinLab.

[0140] 1.6 Antibody fusion protein binding and functional assay

[0141] Purchase or purify the extracellular domain (ECD) or secreted form of target proteins CD24 ECD, IL4R ECD, α v Antibody fusion proteins were prepared with β3ECD-6×His, α5β1ECD-6×His and ligand proteins such as vitronectin, fibronectin, interleukin-4 (IL-4), interleukin-13 (IL-13), etc., and horseradish peroxidase (HRP)-labeled goat anti-human IgG Fc and HRP-labeled streptavidin were purchased. Biotin-labeled proteins or antibodies were also purchased or prepared according to experimental needs. ELISA binding assays were performed step by step between specific targets and ligands. Appropriate amounts of target proteins were coated overnight at 4°C, and primary antibodies were added the next day. After three PBST or TBST washes, primary antibodies were added and incubated at 37°C for 1-3h, and secondary antibodies were added and incubated at 37°C for 1h. Then, TMB substrate was added after PBST or TBST washes for color development and detection. Competitive ELISA was performed according to the following steps: first, the target protein of the drug was coated, then the drug was added and incubated for 1-3h. Then, biotinylated protein and HRP-Strepvidin were mixed together and incubated for another 1 h. The color change of each well was recorded on a SpectraMax M3 plate reader at 450 nm (Molecular Devices).

[0142] 1.7 Cell proliferation

[0143] The cells were coated on 96-well plates, with 2 to 3 × 10 3 cells. After overnight starvation and treatment with various concentrations, the cells were incubated at 37°C for 48-72 h (the concentrations of the tested drugs were 5.12, 1.28, 0.32, and 0.08 μM, and the positive control Docetaxel was 20 μg / mL. The drug solvent was used as a blank control), and cell proliferation was determined by CCK-8. For the luciferase reporter method, cell viability was detected after adding luciferase detection reagent. The inhibition rate (%) was calculated as equation (1):

[0144] Inhibition rate (%) = (1-(OD experiment -OD blank ) / (OD control -OD blank ))×100(1)

[0145] 1.8 Plate colony formation experiment

[0146] Human renal cancer cell line was cultured at 1×10 3 The cells were plated in six-well culture plates and cultured in a 5% CO2, 37°C incubator. The cells were treated with different concentrations of drugs for 1-2 weeks and the number of clones was counted after staining with 0.1% crystal violet solution. Colonies formed by more than 50 cells were counted and recorded. The experiment was repeated three times independently.

[0147] 1.9 Cell migration and invasion

[0148] The effects of drugs on the migration and invasion of tumor cells and vascular endothelial cells were evaluated by wound healing assay and Transwell chamber. The invasion potential of cells was determined by Transwell chamber invasion assay. Cells were serum starved overnight and 2×10 4 -1×10 5 Cells were seeded in the upper chamber of 8-μm pore chambers with serum-free or 1% FBS-containing culture medium for migration assays. For cell invasion assays, the upper chamber was first coated with matrigel (BD Biosciences) before adding cell suspension and drugs for invasion assays.

[0149] 1.10 Tubule formation assay

[0150] The anti-angiogenic effect of drugs was evaluated by HUVEC tubule formation method. HUVECs were seeded at 30,000 cells per well in a 96-well plate coated with 50 μL Matrigel. After incubation at 37°C for 4 h in the presence or absence of drugs, tubule formation was observed under a microscope, and the cumulative length and number of branch points of the tubules were measured.

[0151] 1.11 Rat arterial ring experiment

[0152] Aortic ring assay was performed according to published protocols. The mouse thoracic aorta was isolated, cut into ~0.5 mm wide rings, and incubated in ECM complete medium (1% P / S). Each aortic ring was then added to a 96-well plate coated with 50 μL of Matrigel and cultured in 150 μL of ECM containing FBS (5%) supplemented with 1% ECGS to stimulate vascular sprouting in the presence or absence of drugs. After incubation for 8-12 days, calcein was used to stimulate the growth of blood vessels. AM Staining and imaging using a fluorescent inverted microscope.

[0153] 1.12 Antibody-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC) assays

[0154] IL2-stimulated human PBMCs or NK92mi-CD16a were used as effector cells and renal cancer cells were used as target cells. Cytotoxicity was determined by luciferase reporter assay. 3 ~1×10 4 Cells) were inoculated into 96-well U-bottom plates and pre-incubated for 1 h at 37°C and 5% CO2. Then, effector cells were added at the specified dose and incubated for 24-72 h. Three replicate wells were set for each condition, including target cell group, target cell plus effector cell group, and target cell plus effector cell and antibody drug group. Luciferase reporter assay kit Beyotime was used on M3 (Molecular Devices) TM Detect the cell viability of luciferase-positive renal cancer cell RCC (LUC+GFP+RCC).

[0155] Inhibiton rate (%) = (1-(RLU experiment -RLU blank ) / (RLU control -RLU blank ))×100(2)

[0156] 1.13 Antibody internalization

[0157] pH-sensitive dyes for antibodies (Protonex TM The antibody was labeled with 571C1 or 57103 for 48 hours and the cells were collected and stained by indirect method. The primary antibody was 57103 (10 μg / mL) and the secondary antibody was goat anti-human IgG Fc labeled with AlexaFluor 647.

[0158] 1.14 Animal experiments

[0159] 1) ACHN xenograft Balb / c nude mouse model. Balb / c nude mice (female, 4-6 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The animal experiments were approved by the Animal Research Committee of China Pharmaceutical University and performed in accordance with the ARRIVE guidelines. ACHN tumor cells (5.0×10 6 cells) to the left axilla, and tumors formed in 7-14 days. Mice were randomly divided according to tumor size, and when the tumor reached 150-200mm 3Each group was treated 16 times with PBS, 57103 (30 mg / kg), 571C1 (30 mg / kg), and 571 (30 mg / kg), and the tumor size and animal survival rate were monitored.

[0160] 2) MC38-hPDL1 C57B6 / J tumor transplantation model

[0161] Female mice C57B6 / J (4 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. MC38 cells overexpressing human PDL1 were screened and verified by qRTPCR and WB. The xenograft model was established by subcutaneous injection of MC38-hPDL1 tumor cells (1.0×10 6 Cells) were injected into the left axilla for 5-7 days. Then different treatments were carried out for 21 days. Tumor volume and mouse weight were evaluated three times a week using digital caliper measurements, and the calculation formula was (length × width × height) × 0.5. Tumor inhibition rate % = (1-experimental group volume / control group volume) × 100%. The ratio of the change in treatment tumor volume (ΔT) to the average change in control tumor volume (%T / C = ΔT / AverageΔC × 100%) was calculated. The relative tumor proliferation rate (TGI) represents the antitumor activity, TGI = TRTV / CRTV × 100%.

[0162] 1.15 Quantitative and statistical analysis

[0163] All data are expressed as mean ± standard deviation (SD), and significant values ​​are * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001. Statistical differences were analyzed using t-test, one-way or two-way ANOVA multiple comparison test, and data were analyzed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA).

[0164] 2. Results

[0165] 2.1 Differential expression of target genes and correlation analysis

[0166] We collected gene expression and copy number data of TCGA samples from UCSC Xena. Paired and unpaired differential gene expression analysis compared gene expression changes of pan-cancer data transcriptomes and showed that CD24 and IL4R were highly expressed in CHOL, KIRC, and KIRP ( Figure 1 A-1B). Further DEQ analysis of KIRP and KIRC showed that the target genes analyzed above, including many subtypes of integrin receptors such as ITGAV, ITGB3, ITGA5, and ITGB1, had high expression levels ( Figure 2 A-2C).

[0167] The results showed that CD24 in KIRC was positively correlated with ITGAV, ITGB1 and ITGB3, with correlation coefficients of 0.46, 0.37 and 0.31, respectively, and IL4R was positively correlated with ITGA5, ITGB1 and ITGB3, with correlation coefficients of 0.65, 0.61 and 0.50, respectively. Figure 3 A and 3C). CD24 was positively correlated with IL4R and ITGB1 of KIRP, with correlation coefficients of 0.53 and 0.47, respectively ( Figure 3 B and Figure 3 D). In summary, integrin genes were positively and significantly correlated with other genes. Compared with paraneoplastic or normal tissues, KIRC and KIRP contained immune-related genes CD24, IL4R, CD274 ( Figure 3 A- Figure 3 D).

[0168] 2.2CD24, IL4R, and integrin receptors are overexpressed in renal cancer cell lines

[0169] Bioinformatics analysis showed that CD24, IL4R, integrin, and checkpoint CD274 were overexpressed in RCC tissues. Data downloaded from the CCLE database also provided evidence that RCC had high expression levels of these target genes. This result was confirmed by QRT-PCR ( Figure 4 A) Flow cytometry ( Figure 4 B. Figure 5 A) western blot Figure 4 C) was verified by laser confocal microscopy ( Figure 5 B) Flow cytometry analysis showed that both KIRC and KIRP renal cancer cell lines highly expressed integrin α v β3, α5β1, especially Caki-2, 786O, ACHN, 769P and A498. In addition, the CD274 gene is also upregulated in renal cancer, and Caki1, Caki2, A498, ACHN, 786O also have relatively high expression levels in renal cancer cell lines ( Figure 4 B).

[0170] 2.3 Molecular structure design and docking simulation of 57103 antibody fusion protein

[0171] Previous laboratory studies have reported that HM-3 targets integrin α vIL4DM is an antagonist of β3 and α5β1 integrins. IL4DM is an IL4 mutant that can bind to the IL4 receptor (IL4R) with high affinity to competitively inhibit the binding of IL4 and IL4R. 571C1 is a high-affinity humanized monoclonal antibody targeting CD24. In this study, an antibody fusion protein 57103 containing HM-3, IL4DM and 571C1 monoclonal antibodies was designed. The schematic diagram is shown in Fig. 6A . Figure 6B The molecular docking simulation results show that the binding free energies of 57103 targeting targets AF-P25063, 1hik, and 1u8c are -197.44 kcal / mol, -193.87 kcal / mol, and -428.72 kcal / mol, respectively. The results predict that the antibody fusion protein molecule 57103 can stably bind to each target ( Figure 6B ).

[0172] 2.4 Production and quality characterization of 57103, 571 and 571C1

[0173] The target gene was amplified and cloned into the laboratory platform expression vector pRT2101 ( Figure 6C The expression plasmid was transformed into E. coli DH5α, and positive strains were screened by aminocillin resistance plate, and identified by colony PCR and sequencing. The correct transfection-grade recombinant expression plasmid was determined by restriction endonuclease digestion and confirmed by sequencing ( Figure 6C ). The target gene expression plasmid was screened by CHO-K1 MSX and identified by ELISA, and then transfected into 25μM host cells to obtain a cell line with high expression level. The fermentation broth was obtained after 15 days of fed-batch culture. Protein A affinity chromatography, low pH inactivation, cation exchange chromatography, virus nanofiltration, and step-by-step endotoxin removal were used to obtain a cell line with a purity greater than 95% and endotoxin less than 1EU / mg ( Fig.6D The results of physicochemical properties further demonstrated that the fusion protein met the requirements of subsequent in vitro and in vivo assays ( FIG. 7 ).

[0174] 2.5 57103 can bind to CD24, IL4R and integrin targets and inhibit their functions

[0175] The results of the binding assay showed that the antibody fusion protein 57103 could specifically bind to the target and inhibit the function of the target. The MST results showed that 57103 could bind to CD24, IL4R and α v β3 binding, with affinities of 1.97nM, 1.1nM and 92nM respectively ( Figure 8A-8C). The ELISA results showed that the EC50 of 57103CD24 was 34.03 ng / ml, and the EC50 of 571C1 monoclonal antibody was 18.03 ng / ml, with similar affinity to 571C1 ( Figure 8 D) Equivalent. The EC 50 of 57103 to IL4R is 5.098 ng / ml, and the EC 50 of 571 monoclonal antibody is 11.83 ng / ml, and the affinity is equivalent to 571. 57103 binds to α v The EC 50 of β3 was 520.5 ng / ml, which was better than the EC50 of 571 (741.9 ng / mL) and the EC50 of 571C1 (4290 ng / mL). The results of competitive ELISA assay showed that 57103 could inhibit the binding of IL4 and IL4R with an EC 50 of 538.1 ng / mL ( Figure 8 H). The comprehensive results show that 57103 can be compared with α v β3 and α5β1, inhibiting the binding of IL4 to IL4R. These results demonstrate that 57103 can bind to these target genes and may exhibit anti-tumor and angiogenic activities.

[0176] 2.6 571, 571C1, and 57103 showed good anti-angiogenic effects

[0177] HUVECs were used for in vitro angiogenesis assays. qRT-PCR results showed that the purchased primary HUVEC cells highly expressed VWF and CD31, which was verified by flow cytometry (FCM) results (Figure 9).

[0178] Flow cytometry was used to detect the expression level and KDR in HUVECs. Binding assays showed that 57103 had a high affinity for HUVECs, with an EC 50 of 1.86 μg / ml (R 2 =0.9865)( Fig. 9B ).

[0179] The in vitro anti-angiogenic effects of antibody fusion proteins 57103, 571C1, and 571 were evaluated using CCK8 proliferation assay, wound healing assay, Transwell chamber migration assay, Transwell chamber invasion assay, rat aortic ring assay, and qRT-PCR assay. Fig.10 ).

[0180] The HUVEC in vitro model without growth factors or stimulated with 50ng / mL VEGF165 or 50ng / mL FGF2 was used to test the antiproliferative ability of the drugs. Fig.10A-10C). The results showed that 57103 had a stronger antiproliferative effect on HUVEC compared with the control molecules (571, 571C1, HM-3, IL4DM) ( Fig.10 A-10C). Fig.10 B shows that 57103 and 571C1 have significant inhibitory effects in the HUVEC proliferation model induced by VEGF165, with inhibition rates of 48.06% and 48.74%, respectively, which are higher than 571 (26.95%) (p < 0.001). When the HUVEC proliferation model was stimulated by FGF2, the inhibition rates of 57103, 571C1, and 571 reached 64.54%, 57.21%, and 44.31%, respectively (p < 0.001) ( Fig.10 C). Microscope photo ( Fig.10 D-10F) and quantitative analysis ( Fig.10 G) The results showed that 57103 had a better inhibitory effect on wound healing than 571 and 571C1. The inhibition rate of 57103 on HUVEC scratch was 64.25% (p < 0.001), the inhibition rate on HUVEC Transwell chamber migration was 86.85% (p < 0.001), and the inhibition rate on HUVEC Transwell chamber invasion assay was 68.11% (p < 0.001).

[0181] Microscope image ( Fig.10 H) and quantitative analysis results ( Fig.10 I) further showed that 57103 has significant anti-angiogenic ability. At a concentration of 5.12 μM, the optimal inhibition rates of the total length, branch point number and sprouting area of ​​tubule formation were 86.12% (p < 0.001), 88.81% (p < 0.001) and 90.67% (p < 0.001), respectively.

[0182] In addition, the qRT-PCR results of target expression levels in HUVECs samples treated with variable concentrations of 57103 ( Fig.10 K) showed that 57103 significantly downregulated the mRNA levels of CD24, IL4R, CD274, ITGAV, ITGB3, ITGA5, and ITGB1. Therefore, we propose that these findings may provide a new anti-angiogenic therapeutic strategy for inhibiting FGF2- or VEGFA-induced RCCs or other angiogenesis-driven cancers or diseases.

[0183] 2.7 Targeted delivery capability of fusion protein 57103

[0184] To verify that fusion protein 57103 targets CD24, IL4R, and integrin α vFor tumors with high expression of β3 target, we purified 571, 571C1 and 57103 fusion proteins, labeled the fusion proteins with Cy7 antibody, and performed SDS-PAGE-Coomassie Brilliant Blue staining and fluorescence detection ( Fig.11 B). Fig.11 A is a schematic diagram of the structure of 571, 571C1, and 57103 molecules after Cy7 labeling. In addition, we used a BECKMAN COULTER (CytoFLEX) flow cytometer and indirect flow cytometric staining to detect the binding ability of 571, 571C1, and 57103 to ACHN, A498, and 786O cells ( Fig.11 C). The results showed that 57103 targeted ACHN, A498 and 786O with an affinity of 9.64 nM (R 2 =0.9664),1.45nM(R 2 =0.9931) and 1.63nM (R 2 =0.9931). We inoculated ACHN, A498, and 786O renal cancer cells with high target expression into Balb / c nude mice subcutaneously, injected Cy7-labeled 571, 571C1, and 57103 subcutaneously, and then used an in vivo imaging device to detect the targeting of the antibody to the tumor. Fig.11 D is the fluorescence imaging result of 571, 571C1 and 57103 labeled with Cy7 in the subcutaneous renal cancer cell line xenograft model. The results show that the fluorescently labeled antibody fusion protein molecules can be targeted and enriched in the subcutaneously transplanted tumor tissue.

[0185] 2.8 57103 significantly inhibits the proliferation, metastasis and adhesion of renal cancer cells

[0186] The effect of 571033 on renal cancer cell RCCs was evaluated by in vitro proliferation, migration, invasion and adhesion assays of renal cancer cells. The results of CCK8 proliferation assay showed that 57103 had a significant inhibitory effect on the proliferation of A498, ACHN and 786O. The inhibition levels reached 79.27% ​​(p < 0.001), 58.30% (p < 0.001) and 72.02% (p < 0.001), respectively, which were superior to the anti-proliferation effects of 571 or 571C1 ( Fig.12 A). Cell colony formation assay also showed that 57103 had high cytotoxicity against RCC. In addition, scratch assay, Transwell chamber migration assay and chamber invasion assay showed that 57103 had significant anti-metastatic ability ( Fig.12D-12G). The wound healing inhibition rates of 57103 5.12μM on renal cancer 786O, ACHN, and A498 were 36.85%, 68.11%, and 90.93%, respectively (p < 0.001). The Transwell migration inhibition rates of 57103 5.12μM on renal cancer 786O, ACHN, and A498 were 67.48%, 61.29%, and 71.76%, respectively (p < 0.001). The invasion inhibition rates of renal cancer 786O, ACHN, and A4978 were 87.40%, 77.39%, and 80.40%, respectively, and the positive control was p < 0.001. The adhesion test showed that 57103 could inhibit the adhesion effect driven by vitronectin or fibronectin ( Fig.12 H). Platelet adhesion analysis also showed that 57103 has the potential to inhibit renal cancer cells from adhering to platelets ( Fig.12 I). In summary, the in vitro experimental results indicate that 57103 may be a potential drug for the treatment of renal cancer.

[0187] 2.9 57103 can mediate ADCP, ADCC and antibody internalization effects to exert effective anti-tumor activity

[0188] The ADCP, ADCC and antibody internalization effects mediated by antibody fusion proteins were determined by co-culture of THP1-polarized M0 or whole blood-isolated human PBMCs with tumor cells ( Fig.13 ). Using pH-sensitive fluorescent dye (Protonex TM Renal cancer cell lines were labeled with 57103 Red 600, SE) and co-cultured with THP1 polarized M0 macrophages. The phagocytosis (ADCP) of tumor cells by macrophages was observed under a fluorescence microscope in the presence or absence of 57103 or 571C1. The results showed that M0 macrophages promoted the phagocytosis of tumor cells in the presence of 57103 or 571C1. The number of red dots reflects the degree of macrophage phagocytosis of tumor cells ( Fig.13 A), 57103 achieved the best inhibition rate against 786O, ACHN, and A498 at a concentration of 5.12 μM, with inhibition rates reaching 69.26% (p < 0.001), 59.71% (p < 0.001), and 95.30% (p < 0.001), respectively. In addition, the ADCC effect plays an important role in the anti-tumor effect of antibody drugs. Fig.13After overnight culture, B LUC-labeled tumor cells were co-cultured with PBMC (E:T=30:1) or NK92mi-CD16a cells (E:T=10:1) stimulated with 1000 IU / mL IL2 overnight and cultured for another 24 to 72 hours. The NK92mi-CD16a cell line was constructed using the method reported by Ying Chen et al. pRT-PCR results confirmed the expression of CD16a CAR. Microscope imaging ( Fig.13 B and Fig.13 D(i)) and quantitative analysis ( Fig.13 C and Fig.13 The results of D(ii)) showed that 57103 could mediate the immune cell responses to CD24, IL4R, α v The ADCC effect of β3-high-expressing renal cancer cells kills tumor cells. The inhibition rates of hPBMC on 786O-Luc, ACHN-Luc and A498-Luc were 69.98% (p < 0.001), 57.96% (p < 0.001) and 86.37% (p < 0.001) respectively. Fig.13 C(ii)). Under the condition of 571035.12μM, the proliferation inhibition rate of NK92mi-CD16a on 786O-Luc, ACHN-Luc and A498-Luc reached 51.83% (p<0.001), 82.86% (p<0.001), 85.37% (p<0.001) ( Fig.13 D). All marketed antibodies can bind to the target with high affinity and specificity, causing the antibody-antigen complex to be internalized and degraded. This study used fluorescence microscopy imaging ( Fig.13 E) and flow cytometry ( Fig.13 F) Qualitative and quantitative analysis of antibody internalization of 57103 and 571C1. Once labeled with a pH-sensitive dye (Protonex TM Green500, SE) antibodies bind to the target, they will be internalized in different experimental cells and emit green fluorescence in a low pH environment to show this progress. Flow cytometry results showed that cells treated with 571C1 and 57103 significantly reduced the binding of 571C1 to target cells. The binding rate of 786O treated with 571C1 decreased from 97.1% to 64.0%, the binding rate of 786O treated with 57103 decreased from 96.9% to 64.0%, the binding rate of 769P treated with 571C1 decreased from 95.6% to 7.66%, and the binding rate of 769P treated with 57103 decreased from 95.8% to 24.9%.

[0189] 2.10 In the ACHN xenograft model, 57103 inhibits tumor growth and affects the tumor microenvironment

[0190] To evaluate the efficacy of 571, 571C1, and 57103 in inhibiting the growth of ACHN renal tumors, 5×10 6 ACHN cells were implanted into the subcutaneous flanks of female nude mice. The xenograft tumor models were treated with different drugs (dosing groups included saline control group, 57103 30 mg / kg, 571C1 30 mg / kg, 571 30 mg / kg or Avastin 10 mg / kg). At the end of the treatment, the 57103 group had the strongest inhibitory effect on the growth of subcutaneous transplanted tumors in ACHN nude mice, with the inhibition rates of tumor volume and tumor weight reaching 68.30% and 70.00%, respectively, which was comparable to the inhibitory effect of the positive control drug Avastin (tumor volume inhibition rates of 62.74% and 68.40%). The tumor volume inhibition rates of 571C1 and 571 were 55.86% and 42.36%, respectively, and the tumor weight inhibition rates of 571C1 and 571 were 65.40% and 36.60%, respectively. The growth inhibition effects of different treatment groups were different, but the maximum growth inhibition occurred in the 57103 group ( Fig.14 B and Table 1). The pro-apoptotic, anti-proliferative and anti-angiogenic activities of 57103 were quantified by IHC staining scores of TUNEL, Ki67 and CD31, respectively ( Fig.14 C and 14D). HE staining analysis of ACHN lung metastasis showed that 57103 had a significant anti-metastatic effect compared with the saline control group ( Fig.14 E). In summary, 57103 may be a potential effective antitumor candidate with good antiproliferative, antiangiogenic and anti-metastatic properties.

[0191] Table 1 Effects of drugs on tumor growth of xenografted renal cancer cell ACHN

[0192]

[0193] 2.11 57103 enhances the inhibitory activity of PDL1 antagonist atezolizumab against MC38-hPDL1 xenografts

[0194] First, bioinformatics data analysis revealed that CD24, CD124, and integrin were associated with CD274 (PD-L1). To explore whether 57103 could synergistically promote the therapeutic effect of PDL1 antagonists, we constructed a MC38-hPDL1-C57B6 / J subcutaneous transplantation model. MC38 cells overexpressing human PDL1 were screened and verified by qRT-PCR and WB. Then, we used 1×10 6 C57B6 / J cells were seeded at a density of 10 cells (4 weeks). The tumor volume reached 50-100mm 3, randomly divided into PBS negative control, atezolizumab 5 mg / kg positive control, 571 5mg / kg, 571 5mg / kg + atezolizumab 5mg / kg, Endostar 5mg / kg, Endostar 5mg / kg + atezolizumab, 57103 5mg / kg, 57103 5mg / kg + atezolizumab 5mg / kg. Tumor volume and mouse body weight were monitored, and mice were killed after 21 days of treatment. The data of dissecting tumor tissue, tumor growth and inhibitory effect diagram showed that the combination of 57103 and atezolizumab was more effective than the combination of atezolizumab with 571 or Endostar, and was also more effective than 57103 monoclonal antibody treatment ( Fig.14 F-14H) is more effective. The data showed that the combination of atezolizumab and 571 or atezolizumab and Endostar was not significant compared with the atezolizumab positive control as shown in Table 2. In these studies, the combination of 57103 and PDL1 atezolizumab achieved the best inhibition rate (78.6%, p < 0.001), which was better than 57103 monotherapy (38.3%, p < 0.001) and atezolizumab monotherapy (40.9%, p < 0.001) ( Fig.14 F and 14H).

[0195] Table 2 Effects of drugs on the growth of transplanted colon cancer cell MC38-hPDL1 tumors

[0196]

[0197] 2.12 Inhibitory activity of 57103 against MGC803 xenograft tumors

[0198] 4×10 6 MGC-803 cells were inoculated subcutaneously in the left axilla of C57BL / 6J mice. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm. 3 The mice were randomly divided into PBS, Avastin, Oxaliplatin, 571, 571C1, and 57103 groups, and the drugs were injected into the tail vein. The same dose was given to the mice once 3 days later. The weight and tumor volume of the mice were monitored every 2 to 3 days, and the death of the mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth, and it was dose-dependent ( Fig.15 and Table 3).

[0199] Table 3 Effects of drugs on tumor growth of xenografted gastric cancer cell MGC803

[0200]

[0201] 2.13 Inhibitory activity of 57103 against HT29-Luc xenograft tumors

[0202] 1×10 7 HT29-Luc cells were inoculated subcutaneously in the left axilla of Balb / c nude mice, with 10 mice in each group. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm 3 The mice were randomly divided into groups and injected with PBS, Avastin, 571, 571C1, and 57103 by tail vein every 3 days, and oxaliplatin was injected intraperitoneally every 7 days. The weight and tumor volume of mice were monitored every 2 to 3 days, and the death of mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth, and the effect was better than that of the positive drug group ( Fig.16 and Table 4).

[0203] Table 4 Effects of drugs on tumor growth of xenografted colon cancer cells HT29-Luc

[0204]

[0205] 2.14 Inhibitory activity of 57103 against MDA-MB-231 xenograft tumors

[0206] 5×10 6 MDA-MB-231 cells were inoculated subcutaneously in the left axilla of Balb / c nude mice, with 10 mice in each group. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm 3 The mice were randomly divided into groups and injected with PBS, Avastin, 571, and 57103 through the tail vein every 3 days. The weight and tumor volume of the mice were monitored every 2 to 3 days. The death of the mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth, and the effect was better than that of the positive drug group, and the high-dose group had a better effect ( Fig.17 and Table 5).

[0207] Table 5 Effects of drugs on tumor growth of xenograft breast cancer cells MDA-MB-231

[0208]

[0209] 2.15 Inhibitory activity of 57103 against MC38-hCD24 xenograft tumors

[0210] 5×10 6MC38-hCD24 cells were inoculated subcutaneously in the left axilla of C57BL / 6J mice, with 10 mice in each group. About 7 days after inoculation, the tumor volume of the mice was about 50-100 mm 3 The mice were randomly divided into groups and injected with PBS, hIgG1 isotype control, 571, 571C1, and 57103 through the tail vein every 3 days. The weight and tumor volume of the mice were monitored every 2 to 3 days. The death of the mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth ( Fig.18 and Table 6).

[0211] Table 6 Effects of drugs on tumor growth of xenografted colon cancer cells MC38-hCD24

[0212]

[0213] 2.16 Inhibitory activity of 57103 against 786-O xenograft tumors

[0214] 1×10 7 786-O cells were inoculated subcutaneously in the left axilla of NXG mice. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm. 3 The mice were randomly divided into groups and intraperitoneally injected with PBS, Sunitinib, Atezolizumab, 57103, and Atezolizumab + 57103. The mice were given the drug once every 3 days. The weight and tumor volume of the mice were monitored every 2 to 3 days. The death of the mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth, and the effect was better when used in combination with Atezolizumab ( Fig.19 and Table 7).

[0215] Table 7 Effects of drugs on tumor growth of xenografted renal cancer cell 786-O

[0216]

[0217] 2.17 Inhibitory activity of 57103 against AGS xenograft tumors

[0218] 8×10 6 AGS cells were inoculated subcutaneously in the left axilla of NXG mice. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm. 3 The mice were randomly divided into groups and intraperitoneally injected with PBS, Avastin, 571, 571C1, and 57103 every 3 days. The weight and tumor volume of the mice were monitored every 2 to 3 days. The death of the mice was observed and recorded at any time, and the survival rate was calculated. The results showed that 57103 showed a good effect in inhibiting tumor growth ( Fig. 20and Table 8).

[0219] Table 8 Effects of drugs on tumor growth of xenografted gastric cancer cells AGS

[0220]

[0221] 2.18 Inhibition of tumor growth-ACHN

[0222] 5×10 6 ACHN cells were inoculated subcutaneously in the left axilla of Balb / c nude mice, with 10 mice in each group. About 14 days after inoculation, the tumor size of the mice was about 50-100 mm 3 The mice were randomly divided into groups and injected with PBS, Avastin, and 57103 through the tail vein every 3 days. The body weight and tumor volume of the mice were monitored every 2 to 3 days. The dosing schedule is shown in Table 9. The death of the mice was observed and recorded at any time, and the survival rate was calculated. Fig.21 The results showed that 57103 showed a good effect in inhibiting tumor growth.

[0223] Table 9 Inhibitory effect of drugs on xenograft tumor ACHN

[0224]

[0225] 2.19 Life Extension Experiment-MGC-803-Luc

[0226] 5×10 6 MGC-803-Luc cells were injected into the tail vein of Balb / c nude mice, with 8 mice in each group. About 7 days after inoculation, the mice were randomly divided into groups and given medication. The medication schedule is shown in Table 10. The death of mice was observed and recorded at any time, and the survival rate was calculated. Fig. 22 The results showed that 57103 significantly prolonged the life of experimental mice.

[0227] Table 10 Drug administration regimen for extending the life cycle of MGC-803-Luc tail vein metastasis

[0228]

[0229] 2.20 Tumor metastasis inhibition experiment-HT29-Luc

[0230] 5×10 6 HT29-Luc cells were injected into the tail vein of Balb / c nude mice, with 8 mice in each group. About 7 days after inoculation, the mice were randomly divided into groups. The dosing schedule was shown in Table 11. The death of the mice was observed and recorded at any time, and the survival rate was calculated. Fig.23 The results showed that 57103 was shown to prolong the life of experimental mice.

[0231] Table 11 Drug administration regimen for prolonging the life cycle of HT29-Luc tail vein metastasis

[0232]

[0233] The above results indicate that 57103 can be used as a potential multi-specific antibody fusion protein with good anti-tumor effect by regulating immune cells in the tumor microenvironment, inhibiting angiogenesis, and inhibiting tumor cell proliferation, migration and invasion. Mechanism studies have shown that IL4 or IL4DM inhibits the proliferation, migration and invasion of HUVEC and RCC through the IL4 / IL4R / STAT6 pathway. ELISA results show that VH / VL can simultaneously target CD24 and integrin α v β3 exerts a synergistic effect. 57103 can be rapidly enriched on the surface of HUVEC and cancer cells, thereby enhancing the anti-tumor and angiogenic effects of HM-3 and IL4 or IL4DM. THP1-derived macrophages, PBMC and NK92mi-CD16a co-culture models also confirmed the ADCC and ADCP of 57103. The results of the ACHN nude mouse subcutaneous transplantation experiment showed that 57103 had a comparable anti-tumor effect compared with Avastin (no significant difference between the two). The results of the MC38-hPDL C57B6 / J mouse subcutaneous transplantation tumor showed that the combination of 57103 5mg / kg + atezolizumab 5mg / kg had better tumor inhibitory activity than the anti-PDL1 antagonist atezolizumab alone (5mg / kg) and atezolizumab 5mg / kg + endostatin (5mg / kg) combined treatment. In summary, the present invention has developed a new multi-specific multifunctional antibody fusion protein, which has anti-angiogenesis, anti-proliferation, anti-metastasis activity and the possibility of combined immunotherapy, and can be used as a new strategy for the treatment of refractory renal cancer and metastatic renal cancer and other tumors ( Fig.24 ).

[0234] Renal cell carcinoma (RCC) consists of different subtypes with significant molecular and histological tumor heterogeneity, containing complex immune responses in the tumor microenvironment (TME). Alterations in immune balance may lead to severe side effects unless the dose of combination therapy is reduced. Inhibition of VEGF signaling or targeting T cells by immune checkpoint blockade (ICB) are common strategies for the treatment of ccRCC. However, the above treatment strategies often lead to drug resistance and are prone to recurrence and metastasis, which can be partly attributed to other tumor-protective effects in the microenvironment. Hanahan et al. reviewed that the development of cancer is often accompanied by cumulative changes in gene mutations, cell proliferation, migration, invasion, vascular neovascularization, and immune microenvironment.

[0235] 2.21 Anti-rheumatoid arthritis experiment

[0236] Experimental methods:

[0237] 1. Experimental Animals

[0238] 42 SD rats, female, 200±20g; certificate number: 320730240100010000, Changzhou Cavens.

[0239] 2. Experimental consumables and instruments

[0240]

[0241]

[0242] 3. Modeling and Experimental Grouping

[0243] SD rats were acclimated to the laboratory at room temperature of 22-25°C, relative humidity of 55-65%, and 12h / 12h light cycle for 1 week, and then randomly divided into a control group and an adjuvant arthritis group. The rats in the control group were injected with 150μL of normal saline subcutaneously at the plantar of the right hind foot, and the rats in the arthritis group were injected with 150μL of Freund's complete adjuvant (CFA, Biyuntian). The second injection was performed on the 7th day, and the last injection was performed on the 21st day. After that, the rats with adjuvant arthritis were randomly divided into 6 groups, which were given 57103 (2.4mg / kg, 4.8mg / kg, 9.6mg / kg), Humira M03C2 (5mg / kg), and methotrexate (Methotrexate MTX, 1mg / kg), respectively.

[0244] IV. Method of administration

[0245] The drugs were prepared and packaged, and 57103 was injected intraperitoneally once every 3 days, and M03C2 and MTX were injected intraperitoneally once every 7 days.

[0246] 5. Measurement of body weight and sole thickness

[0247] Body weight measurement: The body weights of rats were recorded on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 and 42, and the changes in body weights of rats in the model group were compared with those in the control group.

[0248] The thickness of the rat's right paw was measured with a vernier caliper before modeling (day 0) and every 3 days after successful modeling (days 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 and 42). The data were recorded when the two sides of the vernier caliper just touched the surface of the foot.

[0249] 6. Arthritis Index (AI) Evaluation:

[0250] The degree of joint inflammation was evaluated by the arthritis index, which ranged from 0 to 4 points, and the cumulative score was the arthritis index of each rat.

[0251] 0 points: no erythema and swelling of the paw;

[0252] 1 point: redness or swelling of the toes;

[0253] 2 points: redness or swelling on the dorsum of the foot, without involving the ankle joint;

[0254] 3 points: erythema and mild swelling from the ankle joint;

[0255] 4 points: erythema and severe swelling involving the ankles, feet, and toes;

[0256] 7. Specimen Collection

[0257] 24 hours after the last administration, the rats were anesthetized and blood was collected from the abdominal aorta. After blood collection, the blood was allowed to stand at room temperature for 1 hour, centrifuged at 4500rpm for 10 minutes at 4℃, and the supernatant was aspirated and frozen at -80℃. The synovial tissue of the knee joints on both sides was collected and frozen in a -80℃ refrigerator for homogenization. The surrounding muscles and connective tissues of the ankle joints on both sides were separated and fixed in 4% paraformaldehyde.

[0258] 8. Hematoxylin and eosin staining

[0259] The ankle joint fixed with paraformaldehyde was rinsed in tap water for 1 hour, soaked in pure water for 20 minutes, repeated 3 times, and then decalcified in EDTA decalcification solution. Decalcification was performed at 37℃ for 6 weeks. The decalcification was completed when the thumbtack was inserted into the bone without any obstruction. Normal dehydration, transparency, wax immersion, embedding, and sectioning were performed. The section thickness was 5μm. Before pathological staining, the slides were baked at 60℃ overnight to prevent detachment.

[0260] Paraffin sections were dewaxed to water: the sections were placed in xylene I for 10 min-xylene II for 10 min-xylene III for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-85% alcohol for 5 min-75% alcohol for 5 min-pure water and allowed to stand for 5 min. Hematoxylin staining: the sections were dyed in hematoxylin solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. Eosin staining: the sections were dehydrated in 85% and 95% gradient alcohol for 4 min each, and then dyed in eosin solution for 10 s. Dehydration and sealing: the sections were placed in anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-xylene I for 5 min-xylene II for 5 min to make them transparent, and sealed with sealing agent. Microscope examination, image acquisition and analysis.

[0261] IX. Immunofluorescence staining

[0262] Dewax the paraffin sections to water: sequentially place the sections in xylene I for 15 min - xylene II for 15 min - xylene III for 15 min - anhydrous ethanol for 5 min - anhydrous ethanol for 5 min - 85% alcohol for 5 min - 75% alcohol for 5 min - and wash with distilled water.

[0263] Antigen repair: Place the tissue sections in a repair box filled with antigen repair buffer in a microwave oven for antigen repair, boil for 8 minutes on medium heat, keep warm for 8 minutes, and then turn to medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the slides. After natural cooling, place the slides in PBS (PH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue)

[0264] DNA denaturation: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the reagent from flowing away), add 1N (1mol / L) hydrochloric acid in the histochemical circle, treat at 37℃ for 30min, wash thoroughly, and place in 4% paraformaldehyde solution for 10min to prevent DNA renaturation.

[0265] Blocking of endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9), incubated at room temperature in the dark for 25 min, and the slides were placed in PBS (PH 7.4) and washed 3 times on a decolorizing shaker, each time for 5 min.

[0266] Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes. (If the primary antibody is from goat, use rabbit serum to block, and other sources use BSA to block)

[0267] Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain proportion of PBS on the slice, and incubate the slice flat in a humidified box at 4°C overnight. (Add a small amount of water in the humidified box to prevent the antibody from evaporating)

[0268] Add secondary antibody: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the same species as the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0269] DAB color development: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

[0270] Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water to return to blue.

[0271] Dehydration and sealing: Put the slices into 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0272] Microscope examination, image acquisition and analysis.

[0273] 10. ELISA test

[0274] The serum of rats was collected and the levels of cytokines IL-6, IL-1β, TNF-α and IL-17 in the serum of experimental rats were detected according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit. The OD value was measured at a wavelength of 450 nm using an automatic microplate reader, and the cytokine concentration in the sample was determined by referring to the standard curve.

[0275] 11. Statistical Analysis

[0276] GraphPad prism 8.00 software was used to perform statistical analysis on the experimental data. The experimental data were expressed as X±SD, and t-test or ANOVA was used to compare the differences among the groups; P<0.05 was statistically significant, and P<0.01 was statistically significant.

[0277] Experimental results:

[0278] Fig.25 A is the experimental cycle diagram of anti-rheumatoid arthritis. Fig.25 B is a photo of the hind feet of normal rats and RA rats after drug intervention. It can be seen intuitively that compared with the model group, the symptoms improved after drug intervention, and 57103 was dose-dependent. The high-dose group of 57103 was the best and better than the positive drugs M03C2 and MTX. Fig.25 The thickness of C toe, i.e. the degree of swelling, was consistent with the above results. From the perspective of safety, 57103 had no effect on the body weight of rats and no toxicity. The inhibition rate of 57103 reached 84.6%.

[0279] Fig.26The figure shows the results of HE staining of the ankle joint of rats and the immunohistochemistry of related indicators. The HE pathological results show that the joint tissue structure of the normal group (Control) is normal, the joints are not obviously swollen, the number of articular chondrocytes is normal, and no obvious degeneration is observed; the joint cavity is normal, the osteoblasts and osteoclasts in the bone marrow cavity reach a dynamic balance, and the blood vessels in the bone marrow cavity do not show obvious expansion and bleeding; there is no obvious infiltration of lymphocytes and neutrophils; the joint tissue structure of the model group is abnormal, the joints are swollen, the articular cartilage gradually begins to repair, chondrocytes gradually begin to appear, the cartilage degenerates, the subchondral bone plate is exposed, and the bones become dense and hard; osteoblasts and osteoclasts gradually begin to appear in the bone marrow cavity, and adipose tissue and lymphocytes infiltrate; the blood vessels in the bone marrow cavity dilate and hemorrhage fills the bone marrow cavity; the drug group 57103 has improved significantly, the high-dose group has basically returned to normal, the low-dose group has not changed much, and the positive drug group has also improved but not as significantly as the high-dose group of 57103.

[0280] Fig. 27 It is a biochemical index of anticoagulation whole blood. Compared with the normal group, the number of white blood cells in the model group increased significantly, and the number of lymphocytes decreased significantly; after drug intervention, 57103 significantly reduced the number of white blood cells, and the high dose had the best effect, and 57103 increased the number of lymphocytes, and the high dose had the best effect. The above results show that 57103 is superior to positive drugs M03C2 and MTX.

[0281] Fig.28 The expression of inflammatory factors in serum. Compared with the normal group, the inflammatory factors in the serum of the model group were significantly upregulated. After drug intervention, 57103 significantly downregulated the expression levels of various inflammatory factors, and the high dose had the best effect. In the above downregulation effect, 57103 was superior to the positive drugs M03C2 and MTX.

[0282] Fig.29 The inhibition of inflammatory factor expression in serum. Compared with the model group, after drug intervention, the high-dose 57103 molecule inhibited the upregulation of IL-6 by 89.77%, and the inhibition rates of TNF-α and IL-17 were 73.30% and 86.12%, respectively. The effect was the best, better than the positive drugs M03C2 and MTX.

[0283] 2.22 Anti-inflammatory experiment of human synovial cells

[0284] Experimental methods:

[0285] 1. Cell proliferation assay

[0286] The CCK8 method was used to detect the inhibitory effect of drugs on MH7A cell proliferation. The following steps were followed: cells in the logarithmic growth phase were collected and plated at 5 × 10 3The cells were inoculated in a 96-well plate and placed in an incubator for 24 hours. After the cells recovered, TNF-α (10 ng / mL) was used to induce the cells to construct an in vitro injury model. Different drugs were used for 24 hours, 48 ​​hours, and 72 hours. After the cell culture treatment time was over, 10 μL of CCK8 solution was added to each well and the absorbance value of each well was detected using an ELISA reader. All experiments were repeated 3 times.

[0287] Drug concentration: M03C2: 5.12μM; MTX: 5.12μM; 57103: low-dose group: 1.28μM, medium-dose group: 2.56μM; high-dose group: 5.12μM.

[0288] 2. Detection of inflammatory factors in cell supernatant

[0289] MH7A cells in the logarithmic growth phase were taken and 1×10 4 The cells were inoculated in 96-well plates and cultured for 24 hours. The cells were stimulated with TNF-α (10 ng / mL) and then treated with different drugs for 48 hours. The supernatant of each well was collected. The contents of IFN-γ, IL-4, IL-6 and IL-10 were detected according to the instructions of the inflammatory factor kit. The absorbance values ​​obtained by the detection were analyzed using GraphPad software.

[0290] Drug concentration: M03C2: 5.12μM; MTX: 5.12μM; 57103: low-dose group: 1.28μM, medium-dose group: 2.56μM; high-dose group: 5.12μM.

[0291] Experimental results:

[0292] Fig.30 A is the effect of drugs on the proliferation of human synovial cells for 72 hours. After drug intervention, 57103 inhibited cell proliferation in a dose-dependent manner, and the effect of the high-dose group was better than that of positive drugs M03C2 and MTX.

[0293] Fig.30 B is the inhibition rate of drug on human synovial cell proliferation. After 72 hours of drug intervention, 57103 inhibited cell proliferation in a dose-dependent manner, and the inhibition rate of the high-dose group reached 80%, which was better than the positive drugs M03C2 and MTX.

[0294] Fig.31In order to detect the inflammatory indexes related to human synovial cells by ELISA, the results showed that compared with the normal group (Control group), the content of pro-inflammatory factors IL-6, IL-1β, and IFN-γ in the model group increased significantly, and the content of anti-inflammatory factors IL-4 and IL-10 decreased significantly; after drug intervention, 57103 significantly down-regulated the amount of IL-6, IL-1β, and IFN-γ, and the high-dose group had the best effect; 57103 up-regulated the content of IL-4 and IL-10, and the high-dose group had the best effect. The above up-regulation and down-regulation effects of 57103 are better than those of positive drugs M03C2 and MTX.

[0295] 2.23 RAW264.7 in vitro anti-inflammatory experiment

[0296] Experimental methods:

[0297] 1. Detection of RAW264.7 cell activity by CCK-8 assay

[0298] 1×10 4 The cells were added to a 96-well plate (200 μL / well) using a pipette and cultured at a constant temperature. When the confluence increased to 80%, the complete medium was replaced with an incomplete medium and cultured at 200 μL / well for 3 h. The cells were then treated with 57103 at concentrations of 20.48, 5.12, 1.28, 0.32, 0.08, 0.02, and 0 μM and incubated for 4 h. 20 μL of CCK-8 was added and the cells were cultured in an incubator for 1 h. The absorbance A at 450 nm was measured using a microplate reader.

[0299] Fig.32 The results showed that 57103 at a concentration of 20.48 μM inhibited the proliferation of RAW264.7 cells, while other drug concentrations had no effect on RAW264.7 cells.

[0300] 2. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 content in cell supernatant

[0301] 1. M1 polarization: 2.5ng / mL IFN-γ + 200ng / mL LPS (1640 + 10% FBS inactivated + 1% triple antibody) acted together for 12h to induce polarization;

[0302] 2. M2 polarization: Add 10 ng / mL IL-4 (1640+10% FBS inactivated+1% triple antibody) to M1 cells for 12 hours to induce polarization;

[0303] Polarized cells were inoculated in 24-well plates. They were divided into a blank control group (0.2 mL RPMI-1640 was added to each well), a M03C2 group (5.12 μM), a MTX group (5.12 μM), and a 57103 group with final concentrations of 1.28, 2.56, and 5.12 μM, respectively. After culturing in a 37°C 5% CO2 incubator for 48 h, the cells were centrifuged at 1000 g for 15 min, and the supernatant was collected and analyzed by ELISA. TM The OD value was measured by microplate reader at a wavelength of 450 nm according to the kit instructions, and the standard curve was drawn to calculate the content of IL-6, TNF-α, IL-1β and IL-10 in the corresponding test samples.

[0304] Experimental results:

[0305] like Fig.33 As shown in the figure, ELISA was used to detect the inflammatory indexes of RAW264.7 cells. The results showed that compared with the normal group (Control group), the content of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group was significantly increased, and the content of anti-inflammatory factor IL-10 was significantly reduced; after drug intervention, 57103 significantly downregulated the amount of IL-6, IL-1β, and TNF-α, and the high-dose group had the best effect; 57103 upregulated the content of IL-10, and the high-dose group had the best effect. The above up-regulation and down-regulation effects of 57103 high-dose group are better than positive drugs M03C2 and MTX.

[0306] 2.24 THP1 anti-inflammatory assay in vitro

[0307] Experimental methods:

[0308] 1. Detection of THP-1 cell activity by CCK-8 method

[0309] 1×10 4 The cells were added to a 96-well plate (200 μL / well) using a pipette and cultured at a constant temperature. When the confluence increased to 80%, the complete medium was replaced with an incomplete medium and cultured at 200 μL / well for 3 h. The cells were then treated with 57103 at concentrations of 20.48, 5.12, 1.28, 0.32, 0.08, 0.02, and 0 μM and incubated for 4 h. 20 μL of CCK-8 was added and the cells were cultured in an incubator for 1 h. The absorbance A at 450 nm was measured using a microplate reader.

[0310] Fig.34 The results showed that 57103 at a concentration of 20.48 μM would inhibit the proliferation of THP-1 cells, while other drug concentrations had no effect on THP-1 cells.

[0311] 2. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 content in cell supernatant

[0312] Polarization steps of THP1 anti-inflammatory model:

[0313] 1. M0 polarization: 150nM PMA (1640 + 10% FBS inactivation + 1% triple antibody)

[0314] 2. M1 polarization: 20ng / mL IFN-γ + 20ng / mL LPS (1640 + 10% FBS inactivated + 1% triple antibody)

[0315] 3. M2 polarization: 20ng / mL IL-4 + 20ng / mL IL-13 (1640 + 10% FBS inactivated + 1% triple antibody)

[0316] 4. Pancreatic enzyme digestion solution: lidocaine injection: pancreatic enzyme (containing EDTA) = 1:4

[0317] Polarized THP-1 cells were inoculated in 24-well plates. They were divided into a blank control group (0.2 mL RPMI-1640 was added to each well), M03C2 group (5.12 μM), MTX group (5.12 μM), and 57103 with final concentrations of 1.28, 2.56, and 5.12 μM, respectively. After culturing in a 37°C 5% CO2 incubator for 48 hours, the cells were centrifuged at 1000 g for 15 minutes, and the supernatant was collected. The ELISA kit instructions were followed, and the OD value was measured at a wavelength of 450 nm using an enzyme marker. A standard curve was drawn to calculate the contents of IL-6, TNF-α, IL-1β, and IL-10 in the corresponding test samples.

[0318] Conclusion: If Fig.35 As shown in the figure, ELISA was used to detect THP1 cell-related inflammatory indicators. The results showed that compared with the normal group, the content of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group was significantly increased, and the content of anti-inflammatory factor IL-10 was significantly reduced; after drug intervention, 57103 significantly downregulated the amount of IL-6, IL-1β, and TNF-α, and the high dose had the best effect; 57103 upregulated the content of IL-10, and the high dose had the best effect. The above up-regulation and down-regulation effects of the high-dose group of 57103 were better than those of the positive drugs M03C2 and MTX.

[0319] 2.25 Splenocyte anti-inflammatory assay in vitro

[0320] Experimental methods:

[0321] 1. Preparation of Mouse Splenic Lymphocyte Suspension

[0322] Mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, and the spleen was aseptically separated and rinsed with PBS to remove connective tissue and fat components. The spleen tissue was placed on a 200-mesh stainless steel screen, ground with a glass syringe needle, rinsed with PBS, and the rinse solution was collected and added dropwise to a centrifuge tube containing an equal volume of mouse lymphocyte separation solution. The cells were centrifuged at 4°C and 2000 rpm for 15 minutes, and the cloudy lymphocyte layer was collected and washed twice with PBS. The cells were suspended in RPMI-1640 culture medium to prepare a single cell suspension of a certain concentration, and counted by trypan blue staining. When the number of live cells was greater than 95%, the cell concentration was adjusted to 2×10 6 Pieces / mL.

[0323] 2. Experimental methods for evaluating the safety of splenocyte drugs:

[0324] CCK-8 assay for detecting splenocyte activity

[0325] After the cells stabilized, 1×10 4 Each cell was added to a 96-well plate (200 μl / well) by a pipette to ensure that the cells in each well were uniform and the number was basically the same. The cells were cultured at a constant temperature in CO2. When the cells grew to 80%, the complete medium was aspirated and incomplete medium was added. 200 μL / well was cultured for 3 hours. Then, the cells were treated with 57103 at concentrations of 20.48, 5.12, 1.28, 0.32, 0.08, 0.02, and 0 μM and incubated for 4 hours. After adding 20 μL CCK-8, the cells were placed in an incubator and cultured for 1 hour. The absorbance A at 450 nm was measured by an enzyme marker.

[0326] 3. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 content in cell supernatant

[0327] The prepared mouse spleen lymphocyte suspension was inoculated in a 24-well plate, 0.8 mL per well. ConA (20 μg / mL) was added to stimulate the establishment of an anti-inflammatory model. The experiment was divided into a blank control group (0.2 mL RPMI-1640 was added to each well), M03C2 group (5.12 μM), MTX group (5.12 μM), and 57103 with final concentrations of 1.28, 2.56, and 5.12 μM, respectively. After culturing in a 37°C 5% CO2 incubator for 48 hours, centrifuged at 1000 g for 15 minutes, and the supernatant was collected. According to the instructions of the ELISA kit, the OD value was measured by an enzyme marker at a wavelength of 450 nm, and a standard curve was drawn to calculate the content of IL-6, TNF-α, IL-1β, and IL-10 in the corresponding test samples.

[0328] Experimental results:

[0329] like Fig.36As shown, 57103 at a concentration of 20.48 μM inhibits the proliferation of spleen cells, while other drug concentrations have no effect on spleen cells.

[0330] like Fig.37 , spleen cell related inflammatory indexes were detected by ELISA, and the results showed that compared with the normal group (Control group), the content of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group increased significantly, and the content of anti-inflammatory factor IL-10 decreased significantly; after drug intervention, 57103 significantly downregulated the amount of IL-6, IL-1β, and TNF-α, and the high dose had the best effect; 57103 upregulated the content of IL-10, and the high dose had the best effect. The above up-regulation and down-regulation effects of 57103 high-dose group are better than positive drugs M03C2 and MTX.

[0331] Many cancers overexpress immunosuppressive receptors or ligands on the cell surface, inhibiting the infiltration and function of immune cells. In the present invention, differentially expressed gene analysis was performed, confirming that KIRC and KIRP RCCs highly expressed integrin α v β3, α5β1, CD24 and IL4R ( Figure 1 and Figure 2 Most of the targets showed significant positive correlations ( Figure 3 qRT-PCR, flow cytometry and Western Blot were used to determine gene expression levels ( Figure 4 and Figure 5 Then, an antibody fusion protein 57103 was designed for CD24, IL4R and integration (Figure 6). The fusion proteins were prepared and characterized according to the standard antibody production process (Figures 6 and 7). MST and ELISA demonstrated the binding ability of 571, 571C1, 57103 corresponding to the design and the target. In addition, the present invention found that 57103 can bind to CD24 and α v β3 target, thereby further promoting its binding to tumor targets and improving selectivity ( Figure 8 F). Competitive ELISA also verified that 57103 and 571 could competitively block the binding of IL4 and IL4R, indicating that the IL4 mutant of the fusion protein can exert the designed biological effect ( Figure 8 E and Figure 8 H). The in vivo imaging method using Cy7 labeled 57103, 571C1, and 571 demonstrated the targeted delivery capability of the fusion protein ( Fig.11 ).

[0332] In the present invention, we also demonstrated that 57103 has an in vitro model (Figures 9 and Fig.10) showed significant anti-angiogenic effects using HUVEC and rat aortic rings. qRT-PCR of HUVEC cells treated with 57103 and untreated cells showed that each target was downregulated, which may be related to drug-induced cytotoxicity or apoptosis or target protein degradation due to antibody internalization ( Fig.10 K). In addition, 57103 significantly inhibited the proliferation and metastasis of renal cancer cells 786O, ACHN and A498 ( Fig.12 ). THP1-derived M0, IL2-stimulated PBMC and IL2-stimulated NK92mi-CD16a co-culture models further demonstrated that 57103 could induce effective ADCP, ADCC and target internalization ( Fig.13 ). qRT-PCR results also detected a significant downregulation of the target protein.

[0333] The results of the ACHN xenograft model showed that 57103 had an inhibition rate of 68.30% and 70.00% on tumor volume and tumor weight, respectively, which was better than 571 (42.36% and 36.60%) and 571C1 (55.86% and 65.40%), and comparable to the positive drug Avastin (62.74% and 68.40%). Immunohistochemistry results also showed that 57103 significantly inhibited tumor growth and angiogenesis, and induced cell apoptosis ( Fig.14 C-14D). HE staining provides evidence for the ability of 57103 to inhibit lung metastasis of subcutaneously transplanted ACHN tumors. In addition, the combined treatment of 57103 and PDL1 antagonist atezolizumab monoclonal antibody has a good anti-tumor effect, which is superior to single treatment and the saline negative control group, with significant differences. In the present invention, the 57103 30 mg / kg administration group achieved an optimal tumor volume inhibition rate of 68.30% and an optimal tumor weight inhibition rate of 70.00%, and a relative tumor proliferation rate T / C% of 14.3% (T / C% = TRT / CRT×100%, calculated based on tumor volume changes).

[0334] The anti-tumor activity evaluation index is the relative tumor proliferation rate T / C (%)

[0335] T / C%=TRT / CRT×100%

[0336] TRT: treatment group RT; CRT: negative control group RT.

[0337] Therapeutic effect evaluation criteria: T / C%>60% is ineffective; T / C%≤60% and P<0.05 after statistical analysis is effective.

[0338] The above content is a schematic description of the present invention and its implementation methods, which is not restrictive. What is shown in the embodiment is only one of the implementation methods of the present invention, and the actual implementation method is not limited thereto. Therefore, if a person of ordinary skill in the art is inspired by it and designs implementation methods and embodiments similar to the technical solution without creative design without departing from the purpose of the invention, they should all fall within the scope of protection of the present invention.

Claims

1. An antibody fusion protein, characterized in that: It includes the fusion of angiostatic polypeptide HM-3, interleukin 4 variant (ILDM) and anti-CD24 monoclonal antibody domain.

2. The antibody fusion protein according to claim 1, characterized in that In the antibody fusion protein, angiostatic polypeptide HM-3 or interleukin 4 variant (ILDM) is fused to the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain.

3. The antibody fusion protein according to claim 1, characterized in that: In the antibody fusion protein, interleukin 4 variant (ILDM) is fused to the C-terminus of the heavy chain domain of the anti-CD24 monoclonal antibody.

4. The antibody fusion protein according to claim 3, characterized in that: In the antibody fusion protein, interleukin 4 variant (ILDM) and the C-terminus of the heavy chain of the anti-CD24 monoclonal antibody domain are linked through a first linker.

5. The antibody fusion protein according to claim 4, characterized in that: In the antibody fusion protein, the angiostatic polypeptide HM-3 and the interleukin-4 variant (ILDM) are linked via a second linker, and the first linker and the second linker are the same or different.

6. The antibody fusion protein according to claim 1, characterized in that: The antibody fusion protein comprises the amino acid sequence SEQ ID NO.

1.

7. The antibody fusion protein according to claim 6, characterized in that: The antibody fusion protein comprises the amino acid sequence SEQ ID NO.

3.

8. A pharmaceutically acceptable salt of the antibody fusion protein according to any one of claims 1 to 7.

9. A polynucleotide, characterized in that The invention comprises a nucleotide sequence encoding an antibody fusion protein, wherein the antibody fusion protein comprises an angiostatic polypeptide HM-3, an interleukin 4 variant (ILDM) and an anti-CD24 monoclonal antibody domain connected by a linker.

10. The polynucleotide according to claim 9, characterized in that The polynucleotide sequence includes the nucleotide sequence of SEQ ID NO.2 encoding the antibody fusion protein.

11. The polynucleotide according to claim 10, characterized in that The polynucleotide sequence also includes the nucleotide sequence of SEQ ID NO.4 encoding the antibody fusion protein.

12. A vector comprising the polynucleotide according to claim 10 or 11.

13. A cell comprising the vector of claim 12.

14. A composition comprising the antibody fusion protein according to any one of claims 1 to 7, or a pharmaceutically acceptable salt of the antibody fusion protein according to claim 8, or a polynucleotide according to any one of claims 9 to 11, or a vector according to claim 12, or a cell according to claim 13. The composition according to claim 14 , further comprising a pharmaceutically acceptable carrier or excipient.

16. The composition according to claim 14 or 15, further comprising a PDL1 antagonist.

17. The composition according to claim 16, wherein the PDL1 antagonist comprises Atezolizumab or Durvalumab.

18. Use of the antibody fusion protein according to any one of claims 1 to 7, or a pharmaceutically acceptable salt comprising the antibody fusion protein according to claim 8, or the polynucleotide according to any one of claims 9 to 11, or the vector according to claim 12, or the cell according to claim 13, or the composition according to any one of claims 14 to 17 in the preparation of anti-tumor drugs for treating ophthalmic diseases, inflammation or autoimmune diseases.

19. The use according to claim 18, characterized in that The tumors include primary cancers, melanomas, and sarcomas originating from the head and neck, brain, thyroid, esophagus, pancreas, lungs, liver, stomach, breast, kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, bladder, and testicles of humans; Preferably, the tumor includes one or more of renal cancer, gastric cancer, lung cancer, liver cancer, breast cancer, colon cancer, glioma, melanoma or cervical cancer.

20. The use according to claim 18, characterized in that The ophthalmic diseases include one or more of iris neovascular eye disease, choroidal neovascular eye disease, retinal neovascular eye disease or corneal neovascular eye disease; Preferably, the iris neovascular eye disease includes one or more of neovascular glaucoma, diabetic retinopathy or iris neovascular eye disease caused by central retinal vein thrombosis; The choroidal neovascularization eye disease includes one or more of age-related macular degeneration, central exudative retinopathy, ocular histoplasmosis syndrome or choroidal neovascularization eye disease caused by glaucoma; The retinal neovascular eye disease includes one or more of retinal neovascular eye diseases associated with diabetes, tumors, retinal detachment, central retinal vein occlusion, retinal periphlebitis, systemic lupus erythematosus, Eales disease or Coat disease; The corneal neovascular eye diseases include corneal neovascular diseases caused by corneal contact lenses, and one or more of the corneal neovascular eye diseases caused by alkali and other chemical burns, corneal surgery, bacterial infection, chlamydia infection, viral infection or protozoan infection.

21. The use according to claim 18, characterized in that The inflammation includes one or more of rheumatoid arthritis, osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, infectious arthritis and traumatic arthritis.

22. The use according to claim 18, characterized in that The autoimmune diseases include one or more of lupus erythematosus and psoriasis.

23. The composition according to any one of claims 14 to 17, or the use according to any one of claims 18 to 22, characterized in that: The dosage form of the medicine or composition includes one or more of capsules, tablets, pills, water injections, powder injections, eye drops, nasal sprays or aerosols.

24. The method for preparing an antibody fusion protein according to any one of claims 1 to 7, characterized in that: The protein is expressed using mammalian cell expression system, yeast expression system or prokaryotic expression system.

25. The method for preparing an antibody fusion protein according to claim 24, characterized in that: The mammalian cell expression system includes one or more of human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO; the yeast expression system includes one or more of Pichia pastoris, Saccharomyces cerevisiae or Hansenula; the prokaryotic expression system includes one or more of Escherichia coli or Bacillus subtilis.

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