Antibody fusion protein and use

Abstract

Provided is an antibody fusion protein, which simultaneously targets CD24, IL4R and an integrin receptor. An in-vitro assay shows that the antibody fusion protein significantly inhibits the proliferation, migration, invasion, colony formation and adhesion of renal cancer cells, also has a comprehensive and significant anti-tumor effect on gastric cancer, colon cancer and breast cancer, and has the effect of inhibiting angiogenesis. The results of a mouse tumor model of subcutaneous transplantation of MC38-hPDL1C57BL6 / J show that the antibody fusion protein combined with the PDL1 antagonist atezolizumab can significantly inhibit tumor growth. The antibody fusion protein can be used as a potential candidate drug for treating metastatic renal cell carcinoma, PDL1-overexpressing cancer, ophthalmic diseases and inflammation or autoimmune diseases.

Description

Antibody fusion proteins and their applications 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 are common, 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, inhibitory NK cells and regulatory B cells. The mechanism that promotes tumor progression is 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 influence the infiltration, polarization, and phenotype of immune cells. Furthermore, the EMT-MET transition and angiogenesis of cancer cells provide adaptive conditions for tumor growth and metastasis. Developing a multispecific drug that significantly inhibits tumor growth and angiogenesis while modulating 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 Exp Clin 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 due to 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 linking RGD to the C-terminus of the ES-2 peptide using a glycine linker. RGD is a tripeptide sequence that is regulated by integrin αv β3 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). Ting Li et al. reported a HM-3-HSA fusion protein with good stability that inhibited the growth and angiogenesis of H22 and SMMC-7721 tumor xenografts in vivo; they also demonstrated that HM-3-HSA can inhibit the growth of H22 and SMMC-7721 tumor xenografts and angiogenesis through integrin α5β1. 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. Furthermore, CD24, IL4R, and integrin receptors are closely associated with tumor cell proliferation, migration, invasion, adhesion, and stemness. Bioinformatics analysis has shown that CD24, IL4R, and integrin receptors are significantly upregulated in many malignant tumors compared to normal tissues, and their expression levels are highly correlated with 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.

[0007] Summary of the Invention

[0008] The present invention discovered that integrin receptors α / β are co-expressed in HUVECs and are closely associated with integrin-mediated vascular proliferation, migration, invasion, and angiogenesis. Based on this finding, the present invention provides an antibody fusion protein that simultaneously targets CD24, IL4R, and integrin receptors, exhibiting significant anti-angiogenic 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 this antibody fusion protein in the preparation of anti-tumor drugs, drugs for treating ophthalmic diseases, inflammation, or autoimmune diseases.

[0009] [Antibody fusion protein]

[0010] The first aspect of the present invention provides an antibody fusion protein comprising a fused angiostatic polypeptide HM-3, an interleukin-4 variant (ILDM), and an anti-CD24 monoclonal antibody domain.

[0011] 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 domain of the anti-CD24 monoclonal antibody.

[0012] [Scheme for C-terminal fusion of interleukin-4 variant (ILDM) with the heavy chain domain of anti-CD24 monoclonal antibody]

[0013] As a preferred embodiment of any technical solution of the first aspect of the present invention, 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, for example, via a linker.

[0014] 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 linked to the C-terminus of the heavy chain domain of the anti-CD24 monoclonal antibody via a first linker. Preferably, in the antibody fusion protein, the angiostatic polypeptide HM-3 is linked to the interleukin-4 variant (ILDM) via a second linker, and the first linker and the second linker are the same or different.

[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.1. (57103-H amino acid sequence)

[0016] 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)

[0017] [Scheme for fusion of angiostatic peptide HM-3 with the C-terminus of the heavy chain domain of anti-CD24 monoclonal antibody]

[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 fused to the C-terminus of the heavy chain domain of the anti-CD24 monoclonal antibody, for example, via a linker.

[0019] 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 domain of the anti-CD24 monoclonal antibody via a first linker. Preferably, in the antibody fusion protein, the interleukin-4 variant (ILDM) is linked to the angiostatic polypeptide HM-3 via a second linker, and the first linker and the second linker are the same or different.

[0020] A pharmaceutically acceptable salt of the antibody fusion protein described in any technical solution of the first aspect of the present invention.

[0021] [Polynucleotide]

[0022] 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.

[0023] 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)

[0024] As a preferred embodiment of any technical solution of the second aspect of the present invention, the polynucleotide sequence further comprises the nucleotide sequence of SEQ ID NO. 4 encoding the antibody fusion protein. (57103-L nucleotide sequence)

[0025] [Carrier]

[0026] The third aspect of the present invention provides a vector comprising the polynucleotide described in any one of the schemes of the second aspect.

[0027] [cell]

[0028] 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.

[0029] [Composition]

[0030] 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 according to any one of the first to fourth aspects. For example, the composition may further comprise a pharmaceutically acceptable carrier or excipient.

[0031] As a preferred embodiment of any technical solution of the fifth aspect of the present invention, the composition further comprises a PDL1 antagonist.

[0032] As a preferred embodiment of any technical solution of the fifth aspect of the present invention, the PDL1 antagonist includes atezolizumab or durvalumab.

[0033] [Application in drug preparation]

[0034] 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 drugs for treating anti-tumor, ophthalmic diseases, inflammation or autoimmune diseases.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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;

[0039] The choroidal neovascular eye disease includes one or more of age-related macular degeneration, central exudative retinitis, ocular histoplasmosis syndrome or glucocorticoid choroidopathy;

[0040] 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;

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 cell carcinoma RCCs or other angiogenesis-driven cancers or diseases.

[0045] 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.

[0046] [Method for preparing antibody fusion protein]

[0047] 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.

[0048] 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; and the prokaryotic expression system includes one or more of Escherichia coli or Bacillus subtilis.

[0049] [Treatment Method]

[0050] In an eighth aspect, 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 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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;

[0055] The choroidal neovascular eye disease includes one or more of age-related macular degeneration, central exudative retinitis, ocular histoplasmosis syndrome or glucocorticoid choroidopathy;

[0056] 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;

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The antibody fusion protein of the present invention has a novel molecular design and contains HM-3, ILDM and anti-CD24 monoclonal antibody domains. The data in the examples show that the 57103 antibody fusion protein has been shown to be a potential multispecific antibody fusion protein with good anti-tumor effects.

[0062] 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 a specific regulatory effect on the tumor microenvironment.

[0063] The 57103 antibody fusion protein has a more pronounced CD24 / α binding than HM-3, IL4DM, or the monoclonal antibody targeting 571C1. v β3 anti-angiogenic effect.

[0064] The 57103 antibody fusion protein promotes the internalization of CD24 in tumor cells and improves immune cells that highly express CD24 / α through ADCC and ADCP effects. v Disruption of β3 / IL4R in cell lines.

[0065] 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 for tumor metastasis and recurrence.

[0066] The results of the MC38-hPDL1 C57B6 / J mouse subcutaneous transplant tumor model showed that the efficacy of the 57103 antibody fusion protein combined with the PDL1 antagonist was superior to the anti-PDL1 monoclonal antibody Atezolizumab treatment and the anti-PDL1 monoclonal antibody Atezolizumab and Endostar combination treatment. The results were statistically significant, indicating that the 57103 antibody fusion protein and the PDL1 antagonist, especially Atezolizumab, have possible additive or synergistic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 shows paired and unpaired differential expression analysis of pan-cancer target genes. (A, B) Paired analysis of CD24 (A) and IL4R (B). (C) Unpaired analysis of IL4R (C) and CD24 (D). (E) Venn diagram showing the intersection of cancer types with CD24 and IL4R overexpression. Mann-Whitney U statistical analysis was used; *p < 0.05, **p < 0.01, ***p < 0.001.

[0068] Figure 2 shows paired and unpaired target gene analysis of differential gene expression between 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 was performed using Mann-Whitney U. *p < 0.05, **p < 0.01, ***p < 0.001.

[0069] Figure 3 shows the gene correlation analysis between KIRC and KIRP. (A) Correlation analysis of KIRC using ggplot2 [3.3.6] and R (4.2.1); (B) Correlation analysis of KIRP 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 analyzed using the Spearman correlation coefficient. The results showed that most target genes were positively correlated with other target genes, with statistically significant differences (p < 0.05). Red indicates positive correlation; blue indicates negative correlation.

[0070] Figure 4 shows the expression levels of target genes determined by qRTPCR, flow cytometry, and Western Blot. Renal cancer cell lines were subcultured at 37°C, 5% CO2. Cells were then harvested, pretreated, and assayed according to standard qRTPCR, flow cytometry, and Western Blot protocols. (A) qRTPCR results for renal cancer cell lines. CD24, IL4R, and integrin α were analyzed in renal cancer cell lines. v β3, α5β1 and CD274 expression levels. (B) Flow cytometry results of renal cancer cell targets. SN3, LM609, HuM200 and Atezolizumab biosimilars were used to detect CD24, IL4R, integrin α v Primary antibodies were used for β3, α5β1, and CD274, and were conjugated to Alexa-fluor 647-conjugated secondary antibodies according to an indirect cell surface staining protocol. For IL4R detection, FITC-conjugated IL4 and FITC-conjugated IL4DM were used. The table summarizes the relative percentage of positive expression for each target in renal cancer cells as determined by FCM. (C) Western blot analysis of the relative expression levels of target genes in each renal cancer cell line.

[0071] Figure 5 shows the detection of target expression levels using flow cytometry and confocal laser fluorescence microscopy. (A) Renal cancer cells were collected, washed, and stained using target-specific antibodies based on cell surface staining. (B) Confocal laser fluorescence microscopy analysis of 786O and 769P. The results showed that both 786O and 769P expressed high levels of CD24, IL4R, and integrin α. v β3.

[0072] Figure 6 shows the construction and quality characterization of antibody fusion proteins 571, 571C1, and 57103. (A) Schematic diagram of gene fusion strategy and protein structure. (B) Molecular dynamics simulation and docking, as well as minimum free energy values. The monomer models were predicted by LOMETS software from @zhanglab. Docking and optimization results based on haddock software from @BonvinLab. (C) Map of the antibody fusion protein expression vector 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.

[0073] Figure 7 shows the physicochemical properties of the antibody fusion proteins. (A) Sample hydrophobicity analysis using HPLC-IHC. The ASBCs of samples 571, 571C1, and 57103 were 1.13, 0.99, and 0.99, respectively, exceeding the ASBCs of the reference products bevacizumab (0.71) and trastuzumab (0.92), indicating good hydrophilicity. (ASBC concentration of ammonium sulfate when bound to a hydrophobic column (relative value under specific conditions); a higher ASBC indicates stronger hydrophilicity.) (B) Sample thermal stability analysis using PR Panta (Nano Temper). Sample thermal stability and DLS particle uniformity. (C) Accurate molecular weight determination results for 57103 by LC-MS. (D) Accurate molecular weight determination results for the sample.

[0074] Figure 8 shows the results of MST affinity determination and ELISA binding and functional verification of the antibody fusion protein. (A) Analysis of the interaction between Cy5-labeled 57103 and CD24 by microfluidic thermophoresis (MST) detection (Kd = 1.97 nM) (B) Analysis of the interaction between Cy5-labeled 57103 and IL4R by MST detection (Kd = 1.1 nM). (C) Analysis of the interaction between Cy5-labeled 57103 and α v The MST affinity assay results for 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, R 2=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 of 57103 and α5β1. (H) Competitive ELISA test of 57103 inhibiting the binding of IL4 to IL4R, EC50 was 538.1ng / ml (R 2 =0.9809). (I) Schematic diagram of the binding and basic functions of the 57103 fusion protein.

[0075] Figure 9 shows the detection of HUVEC target expression levels and drug binding analysis. (A) qRT-PCR analysis 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 drug target expression levels 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 performed in triplicate. Quantitative analysis data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0076] Figure 10 shows that 57103 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 images of HUVECs wound healing. (E) Representative images of HUVECs Transwell chamber migration. (F) Microscopic photos 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 cells migrating in the Transwell chamber, and (iv) number of HUVECs cells invading in the 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 were measured for different drugs. Migration and invasion inhibition rate (%) = [(control - experimental) / control] × 100. (H) Fluorescence imaging of HUVEC tube formation assay. (I) Fluorescence imaging of rat aortic rings. (J) Quantitative analysis of HUVEC tube formation assay ((i) sprouting area and (ii) inhibition rate) and rat aortic ring assay ((iii) total tube length (mm) and (iv) number of branch points). (K) Target expression levels in HUVEC samples decreased with increasing 57103 concentration. HUVECs treated with 5.12 μM 57103 showed significant differences compared to the control (NC). Data are expressed as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0077] Figure 11 shows the 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 diagram 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 by BECKMAN COULTER (CytoFLEX) flow cytometry. The affinities of 57103 targeting ACHN, A498, and 786O were 9.64 nM (R-Reduced, reduced sample; NR-Non-reduced, non-reduced sample), respectively. 2 =0.9664),1.45nM(R 2 =0.9931) and 1.63 nM (R2 =0.9931). (D) Fluorescence imaging results of Cy7-labeled 571, 571C1, and 57103 in a subcutaneous renal cell carcinoma cell line xenograft model. The results show that the fluorescently labeled antibody fusion protein molecules can be targeted and enriched in subcutaneously transplanted tumor tissue.

[0078] Figure 12 shows that 57103 significantly inhibited the proliferation, metastasis, and adhesion of RCCs. (A) CCK8 cell viability assays showed that 57103 significantly inhibited the growth of three RCC cell lines. 57103 at 5.12 μM exhibited the highest inhibitory rate against renal cancer cell proliferation, exceeding that of 571C1 or 571 (P < 0.001). The inhibitory rates of 5.12 μM 57103 against 786O, A498, and ACHN were 79.27% ​​(P < 0.001), 58.30% (P < 0.001), and 72.02% (P < 0.001), respectively. (B-C) Representative micrographs (B) and quantitative analysis results (C) of RCC colony formation assays under different drug treatment conditions. (D) Quantitative analysis of RCC migration inhibition in (i) wound healing assays, (ii) Transwell chamber migration assays, and (iii) Transwell chamber invasion assays. (EG) Micrographs of RCCs (E) Wound healing, (F) Transwell migration assay, (G) Transwell invasion assay. (H) Adhesion assay to evaluate the ability of 57103 to inhibit vitronectin-driven or fibronectin-driven adhesion interactions. 2 × 10 cells per well 4 Cells were cultured overnight in 96-well plates coated with either vitronectin or fibronectin. Drugs were added and cultured for an additional hour. (I) Platelet adhesion assay was used to determine the inhibitory effect, reducing the number of platelets specifically attached to renal cancer cells. Quantitative analysis data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0079] Figure 13 shows the analysis of ADCP and ADCC internalization mediated by 571C1 and 57103. (A) Microscopic imaging analysis of phagocytosis. TM (Red 600, SE) labeled, in the presence or absence of drugs, after three washes, and co-cultured with THP1-derived M0. The number of red dots reflects the intensity of phagocytosis. (B) Representative images of Luciferase-positive GFP-positive RCC cells (LUC+GFP+RCC) and hPBMC co-cultured under different drug treatment conditions. (C) Expression of different CD24 and integrin α vThree human renal carcinoma cells with high β3 levels 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 hours in the presence or absence of 5.12 μM 57103 or 571C1. Luciferase reporter assay was used to measure the expression of CD24-positive α3 in NK-92MI-CD16a and renal carcinoma cells at an E:T = 10:1 ratio. v β3-positive ACHN, A498, and 786O renal carcinoma 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) Fluorescence microscopy (FM) was used to observe the internalization of pH-sensitive fluorescent dye-labeled 57103-mediated targets in different cancer cell types. The green fluorescence intensity represents the internalization level. (F) (i) Flow cytometric analysis of cell samples treated with 571C1-Cy5-labeled antibodies for 48 hours after 786O was added at a concentration of 5.12 μM (red line); (ii) Flow cytometric analysis of cell samples treated with 571C1-Cy5-labeled antibodies for 48 hours after 786O was added at a concentration of 5.12 μM (red line); (iii) Flow cytometric analysis of cell samples treated with 571C1-Cy5-labeled antibodies for 48 hours after 786O was added at a concentration of 5.12 μM (red line); (iv) Flow cytometric analysis of cell samples treated for 48 hours with 571C1-Cy5 antibody staining (red line); (iv) Flow cytometric analysis of cell samples treated with 57103 at a concentration of 5.12 μM for 48 hours with 571C1-Cy5 antibody staining (red line); the blue line represents the control group, which refers to the flow cytometric analysis of cell samples treated with the corresponding vehicle for 48 hours with 571C1-Cy5 antibody staining. Flow cytometry was used to determine the internalization of Cy5-labeled 571C1 (571C1-Cy5) on the cell surface after staining. Quantitative data are mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0080] Figure 14 shows that 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 every three days with 57103 (30 mg / kg), 571C1 (30 mg / kg), 571 (30 mg / kg), or Avastin (10 mg / kg), and tumor volume was monitored for 48 days. (B) Tumor growth curves (i) and quantitative analysis of tumor volume inhibition rates (ii) in different groups. Tumor volume data are presented as mean ± standard deviation (Mean ± SD, n = 6). (C) IHC scoring analysis of Ki67, TUNEL, and CD31 tumor proliferation, apoptosis, and angiogenesis. (D) Infiltration of ACHN cancer cells in lung tissue was identified by HE staining. (E) IHC and HE data were quantified using IHC Profiler (ImageJ) and Image-Pro Plus, respectively. (F) MC38-hPDL1 tumors were harvested after animal sacrifice and washed with DPBS. (G) Average tumor growth curves of different treatments in the MC38-hPDL1 mouse tumor xenograft model. (H) Tumor inhibition rates of monotherapy and combination therapy in the MC38-hPDL1 subcutaneously transplanted C57B6 / J model were superior to those in the PBS control group. Quantitative analysis data are mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

[0081] Figure 15 shows the anti-tumor effect of 57103 in subcutaneous MGC-803 transplanted tumors. (A) Four-week-old female C57BL / 6J mice (n=10 / group) were injected with 4×10 6 The cells were treated for 21 days and tumor volume was monitored. (B) Tumor growth was observed in mice after dissection at the end of the experiment. (C) Tumor volume growth curve.

[0082] Figure 16 shows the anti-tumor effect of 57103 in subcutaneous HT29-Luc transplanted tumors. (A) Female nude mice aged 4 weeks (n=6 / group) were injected with 1×10 7 The cells were treated for 21 days and tumor volume was monitored. (B) Tumor growth was observed in mice after dissection at the end of the experiment. (C) Tumor volume growth curve.

[0083] Figure 17 shows the anti-tumor effect of 57103 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 administered for 21 days, and tumor volume was monitored. (B) Tumor growth was observed in mice after dissection at the end of the experiment. (C) Tumor volume growth curve shows that the high-dose 57103 group showed a better effect.

[0084] Figure 18 shows the construction of MC38-hCD24 cell line to investigate the anti-tumor effect of 57103 in subcutaneous transplanted tumors. (A) Female nude mice aged 4 weeks (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 observed after dissection at the end of the experiment.

[0085] Figure 19 is an investigation of the anti-tumor effect of 57103 in subcutaneously transplanted renal cancer cell line 786-O. (A) Four-week-old female C57BL / 6J mice were injected with 1×10 7 The cells were treated for 21 days, and tumor volume was monitored. (B) Tumor growth was observed in mice dissected at the end of the experiment. (C) Tumor volume growth curve shows that 57103 was more effective and outperformed the positive control drugs.

[0086] Figure 20 is an investigation of the anti-tumor effect of 57103 in subcutaneous transplanted AGS tumors. (A) 4-week-old female C57BL / 6J mice were injected with 8×10 6 The cells were treated for 21 days, and tumor volume was monitored. (B) Tumor growth was observed in mice dissected at the end of the experiment. (C) Tumor volume growth curve shows that 57103 was more effective and outperformed the positive control drugs.

[0087] Figure 21 is an investigation of the anti-tumor effect of 57103 in the subcutaneously transplanted renal cancer cell line ACHN. (A) Overall experimental process and experimental endpoint: tumor growth in dissected mice; (B) (i) tumor volume growth curve; (ii) tumor inhibition rate.

[0088] Figure 22 shows that 57103 prolongs the survival of mice with MGC-803-Luc metastases after tail vein injection. (A) Overall experimental process; (B) Survival curve.

[0089] Figure 23 shows that 57103 prolongs the survival of mice with HT29-Luc metastases after tail vein injection. (A) Overall experimental process; (B) Survival curve; (C) Fluorescence imaging display.

[0090] Figure 24 shows the mechanism of 57103 antibody fusion protein.

[0091] In the picture:

[0092] AbQ15 refers to: Atezolizumab;

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

[0094] AT refers to: Atezolizumab;

[0095] ED refers to: Endostar.

[0096] Figures 25 to 29 are data related to the anti-rheumatoid arthritis experiment:

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

[0098] Figure 26 Changes in HE staining and IHC indicators of ankle joint.

[0099] Figure 27 shows the biochemical indicators of anticoagulated whole blood.

[0100] Figure 28 shows 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] FIG29 shows the inhibition of inflammatory factor expression in serum.

[0102] Figures 30-31 are data related to in vitro anti-inflammatory experiments on human synovial cells:

[0103] FIG30 shows the effects of drugs on the proliferation of human synoviocytes.

[0104] FIG31 shows the detection of relevant inflammatory indicators by ELISA.

[0105] Figures 32 and 33 show the data related to the in vitro anti-inflammatory experiment of RAW264.7:

[0106] Figure 32 shows the safety evaluation of RAW264.7 at different drug concentrations.

[0107] FIG33 shows the ELISA detection of relevant inflammatory indicators.

[0108] Figures 34 and 35 show the data related to the in vitro anti-inflammatory experiment of THP1 cells:

[0109] FIG34 is the safety evaluation of different drug concentrations on THP-1 cells.

[0110] FIG35 shows ELISA detection of relevant inflammatory indicators.

[0111] Figures 36 and 37 show data related to in vitro anti-inflammatory experiments on spleen cells:

[0112] FIG36 is the safety evaluation of different drug concentrations on spleen cells.

[0113] FIG37 shows the ELISA detection of relevant inflammatory indicators. DETAILED DESCRIPTION

[0114] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; 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 those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0116] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[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-mentioned aspects.

[0120] As used herein, the term "effective amount" 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, an effective amount is an amount sufficient to delay progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An 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; (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 neovascular disorders, an effective amount includes an amount sufficient to treat, inhibit, delay and / or prevent the neovascular 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 choroidal neovascularization. (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, cows, 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, so 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 in that it is derived from a substantially homogeneous antibody population and should not be construed as requiring antibody production by any ad hoc method.

[0124] The present invention will be further described below with reference to 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 the 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 (DMEM, HyClone, Palo Alto, CA). MDA-MB-231 cells were cultured in Leibovitz's L-15 medium 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 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 a previously published method (reference: Gene-modified NK-92MI cells expressing a chimeric CD16-BB-ζor CD64-BB-ζreceptor exhibit enhanced cancer-killing ability in combination with therapeutic antibodies).

[0128] 1.2 Data download and analysis

[0129] Transcriptome data of 33 cancers were downloaded from TCGA Pan-Cancer (TCGA). Differential expression analysis of target genes was performed using the DESeq2 method. The threshold parameters for differential expression analysis were FDR < 0.05 and |Log2FC| > 2. Then, 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 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 shows that the Spearman's correlation coefficient (ρ) values ​​ranged from -1.0 (negative correlation, green panel) to 1.0 (positive correlation, red panel).

[0130] 1.3 Target expression level detection

[0131] All qPCR experiments were performed in triplicate for each biological sample and are presented as mean ± SD. For all qPCR results, two-tailed Student's t-tests were used to calculate P values ​​using GraphPad. Cells were harvested and lysed using RIPA lysis buffer (Beyotime, China). Qualified samples were loaded onto SDS-PAGE, transferred to PVDF membranes (Millipore), and stained with anti-ITGAV (1:500–1:1,000, Cell Signaling Technology). β-actin was detected by Western blotting using an anti-β-actin antibody, as a loading control for cell lysates (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA). Finally, Western blotting was performed using a Tanon 5200 Chemiluminescence Imaging System (Tianneng, China), and grayscale analysis was performed using β-actin as an internal control. All flow cytometric analyses were performed using a Beckman Coulter CytoFLEX instrument.

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

[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 and 57103 utilize the human IgG1 (L234A, L235A) antibody backbone sequence, and IL4DM linked to HM-3 is fused to the C-terminus of the antibody heavy chain via a GGGGSGGGGSGGGGS linker. Figure 6 shows a schematic diagram of the fusion strategy and structure. Plasmids encoding the heavy and light chains were transfected into CHO-K1 suspension-adapted cells and selected under MSX conditions in serum-free medium. Ultimately, CHO-K1 cells stably expressing the antibody or antibody fusion protein were obtained. The fermentation supernatant was collected on day 15, centrifuged, and filtered through a 0.22 μm membrane. The filtered supernatants were purified on Protein A HP columns (GE Healthcare) and 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 purification steps were performed by FPLC (AKTA PURE, GE Healthcare Life Sciences). The antibody fusion protein stock solution was subjected to a series of tests to verify its 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, accurate molecular weight mass spectrometry data were obtained using a Waters XEVO G2-XS QTOF and an Acquity H-Class HPLC. The thermal stability of the protein sample was analyzed using a Prometheus NT.48 (NanoTemper Technologies).

[0138] 1.5 Antibody fusion protein structure simulation

[0139] The monomer models of this invention were predicted using LOMETS software from @zhanglab. The final model was obtained through protein-protein docking and manual optimization using haddock software from @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 using β3 ECD-6×His and α5β1 ECD-6×His with ligand proteins such as vitronectin, fibronectin, interleukin-4 (IL-4), and interleukin-13 (IL-13). Horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated streptavidin were purchased. Biotin-labeled proteins or antibodies were also purchased or prepared as needed. ELISA binding assays were performed between specific targets and ligands. An appropriate amount of target protein was coated overnight at 4°C, and the primary antibody was added the next day. After three washes with PBST or TBST, the primary antibody was added and incubated at 37°C for 1–3 hours, and the secondary antibody was added and incubated for 1 hour at 37°C. After washing with PBST or TBST, TMB substrate was added for color development and detection. Competitive ELISAs were performed as follows: the target protein was coated with the drug, followed by the addition of the drug and incubation for 1–3 hours. 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 plated 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 48-72h (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). Cell proliferation was determined by CCK-8. For the luciferase reporter assay, cell viability was detected after adding luciferase detection reagent. The inhibition rate (%) was calculated as equation (1): Inhibiton rate (%) = (1-(OD experiment -OD blank ) / (OD control -OD blank ))×100(1)

[0144] 1.8 Plate colony formation experiment

[0145] Human renal cancer cell line was cultured at 1×10 3 Cells were plated in six-well culture plates and cultured in a 5% CO2, 37°C incubator. Treatment with various drug concentrations was performed for 1-2 weeks, and colonies were counted after staining with 0.1% crystal violet solution. Colonies of 50 or more cells were counted and recorded. Experiments were performed in triplicate.

[0146] 1.9 Cell migration and invasion

[0147] The effects of drugs on the migration and invasion of tumor cells and vascular endothelial cells were evaluated using wound healing assays and Transwell chambers. The invasive potential of cells was determined using the Transwell chamber invasion assay. Cells were serum starved overnight and 2×10 4 -1×10 5 Cells were seeded in serum-free or 1% FBS-containing culture medium onto the upper chamber of an 8-μm pore chamber 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.

[0148] 1.10 Tubule formation assay

[0149] The anti-angiogenic effects of drugs were evaluated using a HUVEC tubule formation assay. HUVECs were seeded at 30,000 cells per well in 96-well plates coated with 50 μL of Matrigel. After incubation at 37°C for 4 hours in the presence or absence of drug, tubule formation was observed under a microscope, and the cumulative length and number of branch points of the tubules were measured.

[0150] 1.11 Rat arterial ring experiment

[0151] The aortic ring assay was performed according to a published protocol. The mouse thoracic aorta was isolated, cut into rings of 0.5 mm width, 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 5% FBS (1%) supplemented with 1% ECGS to stimulate vascular sprouting, with or without drugs. After incubation for 8-12 days, the rings were treated with calcein. AM Staining and imaging using an inverted fluorescence microscope.

[0152] 1.12 Antibody-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC) assays

[0153] Cytotoxicity was determined by luciferase reporter assay using IL2-stimulated human PBMCs or NK92mi-CD16a as effector cells and renal cancer cells as target cells. Briefly, target cells (5×10 3 ~1×10 4 Cells) were seeded into 96-well U-bottom plates and pre-incubated at 37°C, 5% CO2 for 1 hour. Then, effector cells were added at the specified dose and incubated for 24-72 hours. Three replicates were set up 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 TM Detection of cell viability of luciferase-positive renal cell carcinoma (LUC+GFP+RCC). Inhibition rate (%) = (1-(RLU experiment -RLU blank ) / (RLU control -RLU blank ))×100(2)

[0154] 1.13 Antibody internalization

[0155] pH-sensitive dyes for antibodies (Protonex TM Antibody internalization and localization in cultured cells were assessed using fluorescence microscopy after labeling with 571C1 (Green 500, SE-AAT, Bioquest). Antibody-target complex interactions were quantified using FACS analysis. After incubation with 571C1 or 57103 for 48 hours, cells were harvested and stained using an indirect method. The primary antibody was 57103 (10 μg / mL), and the secondary antibody was goat anti-human IgG Fc labeled with AlexaFluor 647.

[0156] 1.14 Animal Experimentation

[0157] 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. 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 within 7-14 days. Mice were randomly divided according to tumor size, and when the tumor reached 150-200mm 3 Each 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.

[0158] 2) MC38-hPDL1 C57B6 / J tumor transplantation model

[0159] Female C57B6 / J mice (4 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. MC38 cells overexpressing human PDL1 were screened and verified by qRT-PCR and western blot. 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. Different treatments were then administered for 21 days. Tumor volume and mouse weight were assessed three times a week using digital caliper measurements and calculated using the formula (length × width × height) × 0.5. Tumor inhibition rate % = (1-experimental group volume / control group volume) × 100%. The ratio of the change in treated tumor volume (ΔT) to the average change in control tumor volume (%T / C = ΔT / AverageΔC × 100%) was calculated. The relative tumor proliferation index (TGI) represents antitumor activity, TGI = TRTV / CRTV × 100%.

[0160] 1.15 Quantitative and statistical analysis

[0161] All data are expressed as mean ± standard deviation (SD), with significant values ​​of p ≤ 0.05 as *, p ≤ 0.01 as **, and p ≤ 0.001 as ***. Statistical differences were analyzed using t-tests, one-way or two-way ANOVA multiple comparison tests, and data were analyzed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA).

[0162] 2. Results

[0163] 2.1 Differential expression of target genes and correlation analysis

[0164] We collected gene expression and copy number data from TCGA samples from UCSC Xena. Paired and unpaired differential gene expression analysis compared gene expression changes across the pan-cancer transcriptome. Results showed that CD24 and IL4R were highly expressed in CHOL, KIRC, and KIRP (Figures 1A-1B). Further DEQ analysis of KIRP and KIRC revealed elevated expression levels of all target genes identified above, including many integrin receptor subtypes such as ITGAV, ITGB3, ITGA5, and ITGB1 (Figures 2A-2C).

[0165] 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. IL4R was positively correlated with ITGA5, ITGB1, and ITGB3, with correlation coefficients of 0.65, 0.61, and 0.50, respectively (Figures 3A and 3C). CD24 was positively correlated with IL4R and ITGB1 in KIRP, with correlation coefficients of 0.53 and 0.47, respectively (Figures 3B and 3D). In summary, integrin genes were positively and significantly correlated with other genes. Compared with paraneoplastic or normal tissues, KIRC and KIRP contained more immune-related genes, including CD24, IL4R, and CD274 (Figures 3A-3D).

[0166] 2.2 Overexpression of CD24, IL4R, and Integrin Receptors in Renal Cancer Cell Lines

[0167] 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 verified by QRT-PCR (Figure 4A), flow cytometry (Figure 4B, Figure 5A), western blot (Figure 4C), and laser confocal microscopy (Figure 5B). Flow cytometric 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, and 786O also have relatively high expression levels in renal cancer cell lines (Figure 4B).

[0168] 2.3 Molecular structure design and docking simulation of 57103 antibody fusion protein

[0169] Previous laboratory studies have shown that HM-3 targets integrin α vAntagonist 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, and its schematic diagram is shown in Figure 6A. Figure 6B shows the results of molecular docking simulation. The binding free energies of 57103 targeting targets AF-P25063, 1hik, and 1u8c were -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).

[0170] 2.4 Production and Quality Characterization of 57103, 571, and 571C1

[0171] The target gene was amplified and cloned into the laboratory platform expression vector pRT2101 (Figure 6C), transformed into Escherichia coli DH5α, screened for positive strains using aminocillin resistance plates, and verified by colony PCR and sequencing. The correct transfection-grade recombinant expression plasmid was identified by restriction enzyme digestion and confirmed by sequencing (Figure 6C). The target gene expression plasmid was screened by CHO-K1 MSX and verified by ELISA, then transfected into 25 μM host cells to generate a cell line with high expression levels. Fermentation broth was obtained after 15 days of fed-batch culture. Protein A affinity chromatography, low pH inactivation, cation exchange chromatography, viral nanofiltration, and stepwise endotoxin removal were used to obtain a raw material solution with a purity greater than 95% and an endotoxin content less than 1 EU / mg (Figure 6D). Physicochemical properties further demonstrated that the fusion protein met the requirements for subsequent in vitro and in vivo assays (Figure 7).

[0172] 2.5 57103 can bind to CD24, IL4R and integrin targets and inhibit their function

[0173] 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 vβ3 binding with affinities of 1.97nM, 1.1nM, and 92nM, respectively (Figures 8A-8C). ELISA results showed that the EC50 of 57103CD24 was 34.03ng / ml, and the EC50 of 571C1 monoclonal antibody was 18.03ng / ml, with affinity comparable to 571C1 (Figure 8D). The EC50 of 57103 to IL4R was 5.098ng / ml, and the EC50 of 571 monoclonal antibody was 11.83ng / ml, with affinity comparable to 571. v The EC50 of β3 was 520.5 ng / ml, which was superior to the EC50 of 571 (741.9 ng / mL) and the EC50 of 571C1 (4290 ng / mL). The results of competitive ELISA showed that 57103 could inhibit the binding of IL4 and IL4R with an EC50 of 538.1 ng / mL (Figure 8H). The combined results showed that 57103 could inhibit the binding of IL4 and IL4R with an EC50 of 538.1 ng / mL (Figure 8H). 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.

[0174] 2.6 571, 571C1, and 57103 showed good anti-angiogenic effects

[0175] 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).

[0176] Flow cytometry was used to detect the expression level of 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) (Figure 9B).

[0177] 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 ( Figure 10 ).

[0178] The antiproliferative potential of the drugs was tested in an in vitro HUVEC model stimulated with no growth factor or with 50 ng / mL VEGF165 or 50 ng / mL FGF2 (Figures 10A-10C). The results showed that 57103 exhibited a stronger antiproliferative effect on HUVEC compared to control molecules (571, 571C1, HM-3, and IL4DM) (Figures 10A-10C). Figure 10B shows that both 57103 and 571C1 exhibited significant inhibitory effects in the VEGF165-induced HUVEC proliferation model, with inhibition rates of 48.06% and 48.74%, respectively, higher than 571 (26.95%) (p < 0.001). In the FGF2-stimulated HUVEC proliferation model, the inhibition rates of 57103, 571C1, and 571 reached 64.54%, 57.21%, and 44.31%, respectively (p < 0.001) (Figure 10C). Microscopic images (Figures 10D-10F) and quantitative analysis (Figure 10G) showed that 57103 exhibited a superior inhibitory effect on wound healing compared to 571 and 571C1. 57103 exhibited an inhibition rate of 64.25% (p<0.001) on HUVEC wound wound healing, 86.85% (p<0.001) on HUVEC transwell migration, and 68.11% (p<0.001) on HUVEC transwell invasion.

[0179] Microscopic images (Figure 10H) and quantitative analysis (Figure 10I) further demonstrated that 57103 exhibited significant anti-angiogenic activity. At a concentration of 5.12 μM, the optimal inhibition rates for total tubule length, branch point number, and sprouting area were 86.12% (p < 0.001), 88.81% (p < 0.001), and 90.67% (p < 0.001), respectively.

[0180] Furthermore, qRT-PCR results of target expression levels in HUVECs treated with variable concentrations of 57103 (Figure 10K) 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.

[0181] 2.7 Targeted delivery capability of fusion protein 57103

[0182] To verify that fusion protein 57103 targets CD24, IL4R, integrin α vFor tumors with high expression of the β3 target, we purified 571, 571C1, and 57103 fusion proteins, labeled the fusion proteins with Cy7 antibodies, and performed SDS-PAGE-Coomassie Brilliant Blue staining and fluorescence detection (Figure 11B). Figure 11A 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 to detect the binding ability of 571, 571C1, and 57103 to ACHN, A498, and 786O cells using indirect flow cytometry staining (Figure 11C). The results showed that the affinity of 57103 targeting ACHN, A498, and 786O was 9.64nM (R 2 =0.9664),1.45nM(R 2 =0.9931) and 1.63 nM (R 2 =0.9931). We subcutaneously inoculated ACHN, A498, and 786O renal cancer cells, which highly express the target, into Balb / c nude mice. Following subcutaneous injection of Cy7-labeled 571, 571C1, and 57103, we assessed tumor targeting using in vivo imaging. Figure 11D shows fluorescence imaging of Cy7-labeled 571, 571C1, and 57103 in a subcutaneous renal cancer cell line xenograft model. These results demonstrate that all fluorescently labeled antibody fusion proteins are capable of targeted accumulation in subcutaneously transplanted tumor tissue.

[0183] 2.8 57103 significantly inhibits the proliferation, metastasis and adhesion of renal cancer cells

[0184] The effect of 571033 on renal cell carcinoma (RCCs) was evaluated by in vitro proliferation, migration, invasion, and adhesion assays of renal cancer cells. The results of the CCK8 proliferation assay showed that 57103 had a significant inhibitory effect on the proliferation of A498, ACHN, and 786O cells. 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-proliferative effects of 571 or 571C1 (Figure 12A). The cell colony formation assay also showed that 57103 had a high cytotoxicity against RCC. In addition, the scratch assay, Transwell chamber migration assay, and chamber invasion assay showed that 57103 had significant anti-metastatic ability (Figures 12D-12G). At 5.12 μM, 57103 inhibited wound healing by 36.85%, 68.11%, and 90.93% for renal cancer cells 786O, ACHN, and A498, respectively (p < 0.001). At 5.12 μM, 57103 inhibited transwell migration by 67.48%, 61.29%, and 71.76% for renal cancer cells 786O, ACHN, and A498, respectively (p < 0.001). Invasion inhibition by 57103 was 87.40%, 77.39%, and 80.40% for renal cancer cells 786O, ACHN, and A4978, respectively, with p < 0.001 for the positive control. Adhesion assays demonstrated that 57103 could inhibit vitronectin- or fibronectin-driven adhesion (Figure 12H). Platelet adhesion assays also demonstrated that 57103 has the potential to inhibit platelet adhesion in renal cancer cells (Figure 12I). In summary, the in vitro experimental results indicate that 57103 may be a potential drug for the treatment of renal cancer.

[0185] 2.9 57103 can mediate ADCP, ADCC and antibody internalization effects to exert effective anti-tumor activity

[0186] The ADCP, ADCC and antibody internalization effects mediated by the antibody fusion protein were determined by co-culture of THP1-polarized M0 or human PBMC isolated from whole blood with tumor cells (Figure 13). TMRenal cancer cell lines were labeled with 57103 (Red 600, SE) and co-cultured with THP1-polarized M0 macrophages. Macrophage phagocytosis (ADCC) of tumor cells was observed under a fluorescence microscope in the presence or absence of 57103 or 571C1. The results showed that M0 macrophages promoted tumor cell phagocytosis in the presence of 57103 or 571C1. The number of red dots reflects the degree of macrophage phagocytosis of tumor cells (Figure 13A). 57103 achieved optimal inhibition at a concentration of 5.12 μM against 786O, ACHN, and A498, reaching 69.26% (p < 0.001), 59.71% (p < 0.001), and 95.30% (p < 0.001), respectively. Furthermore, the ADCC effect plays a crucial role in the anti-tumor effects of antibody drugs. Figure 13BLUC-labeled tumor cells were cultured overnight and then co-cultured with PBMC (E:T=30:1) or NK92mi-CD16a cells (E:T=10:1) stimulated with 1000 IU / mL IL2 overnight, and then 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 demonstrated the expression of CD16a CAR. Microscopic imaging (Figure 13B and Figure 13D (i)) and quantitative analysis (Figure 13C and Figure 13D (ii)) showed that 57103 could mediate immune cell responses to CD24, IL4R, and α v β3-overexpressing renal cancer cells activated ADCC, thereby killing tumor cells. The inhibition rates of hPBMC against 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 (Figure 13C(ii)). At 571035.12 μM, NK92mi-CD16a inhibited the proliferation of 786O-Luc, ACHN-Luc, and A498-Luc by 51.83% (p < 0.001), 82.86% (p < 0.001), and 85.37% (p < 0.001), respectively (Figure 13D). All marketed antibodies can specifically bind to their targets with high affinity, leading to internalization and degradation of the antibody-antigen complex. In this study, fluorescence microscopy (Figure 13E) and flow cytometry (Figure 13F) were used to qualitatively and quantitatively analyze the internalization of antibodies 57103 and 571C1. TMWhen antibodies containing 571C1 (Green500, SE) bind to their targets, they are internalized within different experimental cells and emit green fluorescence in a low-pH environment, indicating this process. Flow cytometry results showed that treatment of cells with 571C1 and 57103 significantly reduced the binding of 571C1 to target cells. For 786O treated with 571C1, the binding rate decreased from 97.1% to 64.0%, and for 786O treated with 57103, the binding rate decreased from 96.9% to 64.0%. For 769P treated with 571C1, the binding rate decreased from 95.6% to 7.66%, and for 769P treated with 57103, the binding rate decreased from 95.8% to 24.9%.

[0187] 2.10 In the ACHN xenograft model, 57103 inhibited tumor growth and affected the tumor microenvironment

[0188] To evaluate the efficacy of 571, 571C1, and 57103 in inhibiting the growth of ACHN renal tumors, 5×10 6 ACHN cells were implanted subcutaneously in the flanks of female nude mice. Xenograft tumor models were treated with different dosing regimens (including a saline control group, 57103 30 mg / kg, 571C1 30 mg / kg, 571 30 mg / kg, or Avastin 10 mg / kg). At the end of treatment, the 57103 group had the strongest inhibitory effect on the growth of subcutaneous ACHN xenograft tumors in nude mice, with tumor volume and weight inhibition rates reaching 68.30% and 70.00%, respectively, comparable to the inhibitory effect of the positive control drug Avastin (tumor volume inhibition rates of 62.74% and 68.40%). Tumor volume inhibition rates for 571C1 and 571 were 55.86% and 42.36%, respectively, and tumor weight inhibition rates for 571C1 and 571 were 65.40% and 36.60%, respectively. Growth inhibition varied among the treatment groups, but the greatest growth inhibition occurred in the 57103 group (Figure 14B and Table 1). The pro-apoptotic, anti-proliferative, and anti-angiogenic activities of 57103 were quantified using TUNEL, Ki67, and CD31 IHC staining scores, respectively (Figures 14C and 14D). HE staining analysis of lung metastases in ACHN showed that 57103 had a significant anti-metastatic effect compared to the saline control group (Figure 14E). In summary, 57103 may be a potential effective anti-tumor candidate with good anti-proliferative, anti-angiogenic, and anti-metastatic properties.

[0189] Table 1 Effects of drugs on tumor growth of xenografted renal cancer cells ACHN

[0190] 2.11 57103 enhances the inhibitory activity of the PDL1 antagonist atezolizumab against MC38-hPDL1 xenografts

[0191] First, bioinformatics data analysis revealed that CD24, CD124, and integrin are associated with CD274 (PD-L1). To explore whether 57103 can synergistically promote the therapeutic effect of PDL1 antagonists, we established an 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 50-100 mm 3 The mice were randomly divided into PBS negative control, atezolizumab 5 mg / kg positive control, 571 5 mg / kg, 571 5 mg / kg + atezolizumab 5 mg / kg, Endostar 5 mg / kg, Endostar 5 mg / kg + atezolizumab, 57103 5 mg / kg, and 57103 5 mg / kg + atezolizumab 5 mg / kg. Tumor volume and mouse body weight were monitored, and mice were sacrificed after 21 days of treatment. Data from dissected tumor tissue, tumor growth, and schematic diagrams of inhibitory effects indicate that the combination of 57103 and atezolizumab is more effective than the combination of atezolizumab with 571 or Endostar, and also more effective than treatment with 57103 alone (Figures 14F-14H). The data showed that the combination of atezolizumab and 571 or atezolizumab and Endostar had no significant effect 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 superior to 57103 monotherapy (38.3%, p < 0.001) and atezolizumab monotherapy (40.9%, p < 0.001) (Figures 14F and 14H).

[0192] Table 2 Effects of drugs on the growth of transplanted colon cancer cell MC38-hPDL1 tumors

[0193] 2.12 Inhibitory activity of 57103 against MGC803 xenograft tumors

[0194] 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. 3Mice were randomly divided into PBS, Avastin, oxaliplatin, 571, 571C1, and 57103 groups. Each group received 57103 via tail vein injection. Three days later, the same dose was administered once more. Mouse body weight and tumor volume were monitored every two to three days. Deaths were observed and recorded, and survival rates were calculated. Results showed that 57103 exhibited a significant dose-dependent inhibitory effect on tumor growth (Figure 15 and Table 3).

[0195] Table 3 Effects of drugs on tumor growth of xenografted gastric cancer cells MGC803

[0196] 2.13 Inhibitory activity of 57103 against HT29-Luc xenograft tumors

[0197] 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, or 57103 via the tail vein every three days. Oxaliplatin was injected intraperitoneally every seven days. Mouse body weight and tumor volume were monitored every two to three days. Deaths were recorded and survival rates were calculated. Results showed that 57103 exhibited a significant tumor growth inhibitory effect, surpassing the positive drug group (Figure 16 and Table 4).

[0198] Table 4 Effects of drugs on tumor growth of xenografted colon cancer cells HT29-Luc

[0199] 2.14 Inhibitory activity of 57103 against MDA-MB-231 xenograft tumors

[0200] 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, or 57103 via the tail vein every three days. Body weight and tumor volume were monitored every two to three days, and mortality was recorded and analyzed. The results showed that 57103 significantly inhibited tumor growth, surpassing the positive drug group, with the high-dose group showing even greater efficacy (Figure 17 and Table 5).

[0201] Table 5 Effects of drugs on tumor growth of xenograft breast cancer cells MDA-MB-231

[0202] 2.15 Inhibitory activity of 57103 against MC38-hCD24 xenograft tumors

[0203] 5×10 6 MC38-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, or 57103 via the tail vein every three days. Body weight and tumor volume were monitored every two to three days. Deaths were observed and recorded, and survival rates were calculated. The results showed that 57103 exhibited a significant inhibitory effect on tumor growth (Figure 18 and Table 6).

[0204] Table 6 Effects of drugs on tumor growth of xenografted colon cancer cells MC38-hCD24

[0205] 2.16 Inhibitory activity of 57103 against 786-O xenograft tumors

[0206] 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, or atezolizumab plus 57103 every three days. Mouse body weight and tumor volume were monitored every two to three days, and mortality was recorded and analyzed. The results showed that 57103 exhibited a significant tumor growth inhibitory effect, and the effect was even greater when used in combination with atezolizumab (Figure 19 and Table 7).

[0207] Table 7 Effects of drugs on tumor growth of xenografted renal cancer cell 786-O

[0208] 2.17 Inhibitory activity of 57103 against AGS xenograft tumors

[0209] 8×10 6 AGS cells were inoculated subcutaneously in the left armpit of NXG mice. About 7 days after inoculation, the tumor size of the mice was about 50-100 mm. 3The mice were randomly divided into groups and intraperitoneally injected with PBS, Avastin, 571, 571C1, or 57103 every three days. Body weight and tumor volume were monitored every two to three days. Deaths were recorded and survival rates were calculated. The results showed that 57103 exhibited a significant inhibitory effect on tumor growth (Figure 20 and Table 8).

[0210] Table 8 Effects of drugs on tumor growth of xenografted gastric cancer cells AGS

[0211] 2.18 Inhibition of tumor growth-ACHN

[0212] 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, or 57103 via the tail vein every three days. Body weight and tumor volume were monitored every two to three days. The dosing schedule is shown in Table 9. Deaths were recorded and survival rates were calculated. As shown in Table 9 and Figure 21, 57103 demonstrated a significant inhibitory effect on tumor growth.

[0213] Table 9 Inhibitory effects of drugs on xenograft tumor ACHN

[0214] 2.19 Life Extension Experiment-MGC-803-Luc

[0215] 5×10 6 Balb / c nude mice were injected with MGC-803-Luc cells via the tail vein, with eight mice per group. Approximately seven days after inoculation, the mice were randomly divided into groups and given the drug regimen shown in Table 10. Deaths were recorded over time, and survival rates were calculated. As shown in Figure 22, the results show that 57103 significantly prolonged the lifespan of the experimental mice.

[0216] Table 10 Drug administration regimen for extending the life cycle of MGC-803-Luc tail vein metastases

[0217] 2.20 Tumor metastasis inhibition experiment-HT29-Luc

[0218] 5×10 6 HT29-Luc cells were injected into the tail vein of Balb / c nude mice, with 8 mice per group. Approximately 7 days after inoculation, the mice were randomly divided into groups and the dosing schedule was shown in Table 11. Deaths were recorded over time, and survival rates were calculated. As shown in Figure 23, the results show that 57103 can prolong the lifespan of the experimental mice.

[0219] Table 11 Drug administration regimen for prolonging the life cycle of HT29-Luc tail vein metastases

[0220] The above results indicate that 57103 can be used as a potential multispecific antibody fusion protein with good anti-tumor effects by regulating immune cells in the tumor microenvironment, inhibiting angiogenesis, and inhibiting tumor cell proliferation, migration, and invasion. Mechanistic 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 macrophage, PBMC and NK92mi-CD16a co-culture models also confirmed the ADCC and ADCP of 57103. The results of the ACHN nude mouse subcutaneous xenograft tumor experiment showed that 57103 had comparable anti-tumor effects compared with Avastin (no significant difference between the two). The results of the MC38-hPDL C57B6 / J mouse subcutaneous xenograft 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 the combination of atezolizumab 5mg / kg + endostatin (5mg / kg). In summary, the present invention has developed a new multispecific and multifunctional antibody fusion protein that has anti-angiogenesis, anti-proliferation, and anti-metastasis activities and the possibility of combined immunotherapy, which can be used as a new strategy for the treatment of refractory renal cancer, metastatic renal cancer and other tumors ( Figure 24 ).

[0221] Renal cell carcinoma (RCC) consists of different subtypes with significant molecular and histological tumor heterogeneity and contains a complex immune response in the tumor microenvironment (TME). Alterations in the immune balance may lead to severe side effects unless the dose of combination therapy is reduced. Inhibition of VEGF signaling or targeting T cells with immune checkpoint blockade (ICB) are commonly used 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 the immune microenvironment.

[0222] 2.21 Anti-rheumatoid arthritis experiment

[0223] Experimental methods:

[0224] 1. Experimental Animals

[0225] 42 female SD rats, 200±20 g; certificate number: 320730240100010000, Changzhou Cavens.

[0226] 2. Experimental consumables and instruments

[0227] 3. Modeling and Experimental Grouping

[0228] After acclimation for one week in a laboratory at 22-25°C, 55-65% relative humidity, and a 12h / 12h light cycle, SD rats were randomly divided into a control group and an adjuvant arthritis group. Control rats received a subcutaneous injection of 150 μL of normal saline into the plantar surface of the right hind paw, while arthritis rats received a 150 μL injection of Freund's complete adjuvant (CFA, Bio-Tech). A second injection was given on day 7, and a final injection was given on day 21. Subsequently, adjuvant arthritis rats were randomly divided into six groups and given 57103 (2.4 mg / kg, 4.8 mg / kg, and 9.6 mg / kg), Humira M03C2 (5 mg / kg), or methotrexate (MTX, 1 mg / kg), respectively.

[0229] IV. Method of administration

[0230] 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.

[0231] 5. Weight and foot thickness measurement

[0232] Body weight measurement: The body weights of the 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 the rats in the model group were compared with those in the control group.

[0233] The thickness of the rat's right paw was measured using 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.

[0234] 6. Arthritis Index (AI) Evaluation:

[0235] The degree of joint inflammation was evaluated using the arthritis index, which ranged from 0 to 4, and the cumulative score was the arthritis index of each rat.

[0236] 0 points: no erythema and swelling of the paw;

[0237] 1 point: redness or swelling of the toes;

[0238] 2 points: redness or swelling on the dorsum of the foot, without involving the ankle joint;

[0239] 3 points: erythema and mild swelling from the ankle joint;

[0240] 4 points: erythema and severe swelling involving the ankles, feet, and toes;

[0241] 7. Specimen Collection

[0242] Twenty-four hours after the last administration, rats were anesthetized and blood was collected from the abdominal aorta. The blood was allowed to stand at room temperature for 1 hour, then centrifuged at 4500 rpm for 10 minutes at 4°C. The supernatant was aspirated and frozen at -80°C. Synovial tissue from both knee joints was collected and frozen at -80°C until homogenization. The surrounding muscle and connective tissue from both ankle joints were isolated and fixed in 4% paraformaldehyde.

[0243] 8. Hematoxylin and Eosin Staining

[0244] After paraformaldehyde fixation, the ankle joint was rinsed in tap water for 1 hour, then immersed in pure water for 20 minutes, repeated three times, and then decalcified in EDTA decalcification solution. Decalcification was performed at 37°C for 6 weeks. Decalcification was considered complete when a thumbtack inserted into the bone felt free of obstruction. The joint was then dehydrated, transparentized, wax-impregnated, embedded, and sectioned to a thickness of 5 μm. Slides were baked overnight at 60°C before pathological staining to prevent detachment.

[0245] Dewax paraffin sections to dehydrate: Place sections in xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and pure water for 5 minutes. Hematoxylin staining: Stain sections in hematoxylin solution for 3-5 minutes, rinse with tap water, differentiate with differentiation solution, rinse with tap water, blue with bluing solution, and rinse with running water. Eosin staining: Dehydrate sections in 85% and 95% graded alcohol for 4 minutes each, then stain in eosin solution for 10 seconds. Dehydrate and mount sections: Place sections in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to clear them. Mount sections with mounting medium. Microscopic examination and image acquisition and analysis.

[0246] IX. Immunofluorescence Staining

[0247] 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.

[0248] Antigen retrieval: Place the tissue sections in a microwave oven filled with antigen retrieval buffer and microwave on medium heat for 8 minutes until boiling. Reduce heat to medium-low heat for 8 minutes, then keep warm for 7 minutes. During this process, prevent excessive evaporation of the buffer and avoid drying the slides. After cooling naturally, wash the slides three times in PBS (pH 7.4) on a decolorizing shaker, shaking for 5 minutes each time. (The retrieval solution and conditions will depend on the tissue.)

[0249] 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 30 minutes, wash thoroughly, and place in 4% paraformaldehyde solution for 10 minutes to prevent DNA renaturation.

[0250] Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9) and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

[0251] Serum blocking: Add 3% BSA to the tissue circle to evenly cover the tissue and block at room temperature for 30 minutes. (If the primary antibody is goat-derived, use rabbit serum; if the primary antibody is from other sources, use BSA)

[0252] Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in a certain ratio of PBS to the slice, and incubate the slice flat in a humidified box at 4°C overnight. (Add a small amount of water to the humidified box to prevent the antibody from evaporating)

[0253] Add secondary antibody: Wash slides three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each time. After drying the sections slightly, add a secondary antibody (HRP-labeled) of the same species as the primary antibody to the circle, covering the tissue and incubating at room temperature for 50 minutes.

[0254] DAB staining: Wash slides three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each time. After the sections are slightly dried, add freshly prepared DAB staining solution to the circle. Monitor the color development time under a microscope. A positive color is brownish-yellow. Rinse the sections with tap water to terminate the color development.

[0255] Counterstaining of cell nuclei: counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, and rinse with tap water to return to blue.

[0256] Dehydration and sealing: Place the slices in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and xylene I for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0257] Microscopy, image acquisition and analysis.

[0258] 10. ELISA test

[0259] Rat serum was collected and assayed for IL-6, IL-1β, TNF-α, and IL-17 levels using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. OD values ​​were measured at 450 nm using an automated microplate reader, and cytokine concentrations were determined by reference to a standard curve.

[0260] 11. Statistical Analysis

[0261] GraphPad Prism 8.00 software was used to perform statistical analysis of the experimental data. The experimental data were expressed as X±SD, and the differences between the groups were compared using t-test or ANOVA; P<0.05 was considered statistically significant, and P<0.01 was considered statistically significant.

[0262] Experimental results:

[0263] Figure 25A shows the experimental cycle of rheumatoid arthritis, and Figure 25B shows photographs of the hind paws of normal rats and RA rats after drug treatment. It can be seen that compared with the model group, the symptoms improved after drug treatment, and the effect of 57103 was dose-dependent, with the high-dose group achieving the best results and surpassing the positive drugs MO3C2 and MTX. Figure 25C shows consistent results in terms of toe thickness, i.e., swelling. Furthermore, from a safety perspective, 57103 had no effect on rat body weight and was non-toxic. The inhibition rate of 57103 reached 84.6%.

[0264] Figure 26 shows the results of HE staining of the ankle joints of rats and immunohistochemistry of related indicators. HE pathological results showed that the joint tissue structure of the normal group (Control) was normal, with no obvious swelling, normal number of articular chondrocytes, and no obvious degeneration; the joint cavity was normal, with osteoblasts and osteoclasts in the bone marrow cavity in a dynamic balance, and no obvious dilation or bleeding of blood vessels in the bone marrow cavity; no obvious infiltration of lymphocytes and neutrophils; the joint tissue structure of the model group was abnormal, with joint swelling, gradual repair of articular cartilage, gradual appearance of chondrocytes, cartilage degeneration, exposure of the subchondral bone plate, and dense and hard bone; osteoblasts and osteoclasts gradually began to appear in the bone marrow cavity, infiltration of fat tissue and lymphocytes; dilation of blood vessels in the bone marrow cavity, and bleeding filling the bone marrow cavity; the 57103 treatment group showed significant improvement, the high-dose group basically returned to normal, the low-dose group showed little change, and the positive drug group also showed some improvement, but not as significant as the high-dose 57103 group.

[0265] Figure 27 shows the anticoagulation-induced whole blood biochemical parameters. Compared with the normal group, the model group showed a significant increase in white blood cell count and a significant decrease in lymphocyte count. After drug administration, 57103 significantly reduced white blood cell count, with the highest effect observed at the high dose. 57103 also increased lymphocyte count, with the highest effect observed at the high dose. These results demonstrate that 57103 is superior to the active drugs M03C2 and MTX.

[0266] Figure 28 shows the expression of inflammatory factors in serum. Compared with the normal group, the model group showed a significant increase in inflammatory factors in serum. After drug intervention, 57103 significantly downregulated the expression levels of various inflammatory factors, with the highest effect being achieved at the high dose. In these downregulation effects, 57103 was superior to the positive drugs M03C2 and MTX.

[0267] Figure 29 shows the inhibition of inflammatory cytokine expression in serum. Compared with the model group, after drug intervention, the high-dose 57103 molecule inhibited IL-6 upregulation by 89.77%. The inhibition rates for TNF-α and IL-17 were 73.30% and 86.12%, respectively, achieving the best effect and surpassing the positive drugs M03C2 and MTX.

[0268] 2.22 Human synovial cell anti-inflammatory experiment

[0269] Experimental methods:

[0270] 1. Cell Proliferation Assay

[0271] The CCK8 assay was used to detect the inhibitory effect of drugs on MH7A cell proliferation. The following steps were performed: cells in the logarithmic growth phase were collected and plated at 5×10 3Cells were seeded in 96-well plates and incubated in an incubator for 24 hours. After the cells recovered, they were treated with TNF-α (10 ng / mL) to establish an in vitro injury model. The cells were treated with different drugs for 24, 48, and 72 hours. After the cell incubation period, 10 μL of CCK8 solution was added to each well and the absorbance of each well was measured using a microplate reader. All experiments were repeated three times.

[0272] Drug concentration: M03C2: 5.12μM; MTX: 5.12μM; 57103: low-dose group: 1.28μM, medium-dose group: 2.56μM; high-dose group: 5.12μM.

[0273] 2. Detection of Inflammatory Factors in Cell Supernatants

[0274] MH7A cells in the logarithmic growth phase were taken and 1×10 4 Cells were seeded in 96-well plates and cultured for 24 hours. The cells were then stimulated with TNF-α (10 ng / mL) and treated with various drugs for 48 hours. The supernatant from each well was collected. IFN-γ, IL-4, IL-6, and IL-10 levels were measured according to the inflammatory cytokine kit instructions. The absorbance values ​​were analyzed using GraphPad software.

[0275] Drug concentration: M03C2: 5.12μM; MTX: 5.12μM; 57103: low-dose group: 1.28μM, medium-dose group: 2.56μM; high-dose group: 5.12μM.

[0276] Experimental results:

[0277] Figure 30A shows the effects of drugs on human synovial cell proliferation over a 72-hour period. After drug administration, 57103 inhibited cell proliferation in a dose-dependent manner, with the high-dose group showing superior effects compared to the positive drugs M03C2 and MTX.

[0278] Figure 30B shows 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 in the high-dose group reached 80%, which was superior to the positive drugs M03C2 and MTX.

[0279] Figure 31 shows the detection of inflammatory indicators related to human synovial cells by ELISA. The results show that compared with the normal group (Control group), the levels of pro-inflammatory factors IL-6, IL-1β, and IFN-γ in the model group were significantly increased, and the levels of anti-inflammatory factors IL-4 and IL-10 were significantly decreased. After drug intervention, 57103 significantly downregulated the levels of IL-6, IL-1β, and IFN-γ, with the high-dose group having the best effect. 57103 also upregulated the levels of IL-4 and IL-10, with the high-dose group having the best effect. In these up-regulation and down-regulation effects, 57103 is superior to the positive drugs M03C2 and MTX.

[0280] 2.23 RAW264.7 in vitro anti-inflammatory assay

[0281] Experimental methods:

[0282] 1. Detection of RAW264.7 cell viability using CCK-8 assay

[0283] 1×10 4 Individual cells were pipetted into a 96-well plate (200 μL / well) 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.

[0284] Figure 32 shows the safety evaluation of different drug concentrations on RAW264.7 cells. The results show that 57103 inhibits the proliferation of RAW264.7 cells at a concentration of 20.48 μM, while other drug concentrations have no effect on RAW264.7 cells.

[0285] 2. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 levels in cell supernatants

[0286] 1. M1 polarization: 2.5 ng / mL IFN-γ + 200 ng / mL LPS (1640 + 10% FBS inactivated + 1% triple antibody) were co-acted for 12 hours to induce polarization;

[0287] 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;

[0288] Polarized cells were seeded in 24-well plates. A blank control group (0.2 mL of RPMI-1640 per well) was added, followed by an 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 incubation for 48 hours in a 37°C, 5% CO2 incubator, the cells were centrifuged at 1000 g for 15 minutes, and the supernatant was collected. The OD values ​​were measured at a wavelength of 450 nm using a microplate reader according to the ELISA kit instructions. A standard curve was plotted, and the levels of IL-6, TNF-α, IL-1β, and IL-10 in the corresponding test samples were calculated.

[0289] Experimental results:

[0290] As shown in Figure 33, ELISA was used to detect RAW264.7 cell-related inflammatory indicators. The results showed that compared with the normal group (Control group), the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group were significantly increased, and the level of anti-inflammatory factor IL-10 was significantly reduced. After drug intervention, 57103 significantly downregulated the levels of IL-6, IL-1β, and TNF-α, with the high-dose group having the best effect. 57103 upregulated the level of IL-10, with the high-dose group having the best effect. The above up-regulation and down-regulation effects of the high-dose group of 57103 were superior to those of the positive drugs M03C2 and MTX.

[0291] 2.24 THP1 in vitro anti-inflammatory assay

[0292] Experimental methods:

[0293] 1. Detection of THP-1 cell activity by CCK-8 assay

[0294] 1×10 4 Individual cells were pipetted into a 96-well plate (200 μL / well) 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.

[0295] FIG34 shows that 57103 inhibits the proliferation of THP-1 cells at a concentration of 20.48 μM, while other drug concentrations have no effect on THP-1 cells.

[0296] 2. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 levels in cell supernatants

[0297] Polarization steps of THP1 anti-inflammatory model:

[0298] 1. M0 polarization: 150nM PMA (1640 + 10% FBS inactivation + 1% tertiary antibody)

[0299] 2. M1 polarization: 20 ng / mL IFN-γ + 20 ng / mL LPS (1640 + 10% FBS inactivated + 1% tertiary antibody)

[0300] 3. M2 polarization: 20 ng / mL IL-4 + 20 ng / mL IL-13 (1640 + 10% FBS inactivated + 1% tertiary antibody)

[0301] 4. Pancreatic enzyme digestion solution: lidocaine injection: pancreatic enzyme (containing EDTA) = 1:4

[0302] Polarized THP-1 cells were seeded in 24-well plates. The cells were divided into a blank control group (0.2 mL of RPMI-1640 was added to each well), an 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 incubation for 48 hours in a 37°C, 5% CO2 incubator, the cells were centrifuged at 1000 g for 15 minutes, and the supernatant was collected. The OD values ​​were measured at a wavelength of 450 nm using a microplate reader according to the ELISA kit instructions. A standard curve was plotted, and the levels of IL-6, TNF-α, IL-1β, and IL-10 in the corresponding test samples were calculated.

[0303] Conclusion: As shown in Figure 35, ELISA was used to detect THP1 cell-related inflammatory indicators. The results showed that compared with the normal group, the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group were significantly increased, and the level of the anti-inflammatory factor IL-10 was significantly decreased. After drug intervention, 57103 significantly downregulated the levels of IL-6, IL-1β, and TNF-α, with the high dose having the best effect. 57103 also upregulated the level of IL-10, with the high dose having the best effect. The high-dose group of 57103 was superior to the positive drugs M03C2 and MTX in these up-regulation and down-regulation effects.

[0304] 2.25 Splenocyte anti-inflammatory assay in vitro

[0305] Experimental methods:

[0306] 1. Preparation of Mouse Splenic Lymphocyte Suspension

[0307] Mice were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. The spleens were aseptically isolated, rinsed with PBS, and the connective tissue and fat components were removed. The spleen tissue was placed on a 200-mesh stainless steel screen and ground with a glass syringe needle. The PBS was rinsed and the collected rinse was added dropwise to a centrifuge tube containing an equal volume of mouse lymphocyte separation medium. The cells were centrifuged at 4°C and 2000 rpm for 15 minutes. The cloudy lymphocyte layer was collected and washed twice with PBS. The cells were suspended in RPMI-1640 medium to prepare a single-cell suspension of a certain concentration. The cells were counted with trypan blue staining. If the viable cell count was greater than 95%, the cell concentration was adjusted to 2 × 10 6 pieces / mL.

[0308] 2. Experimental methods for evaluating the safety of splenocyte drugs:

[0309] Detection of splenocyte activity by CCK-8 assay

[0310] After the cells stabilized, 1×10 4 Cells were pipetted into a 96-well plate (200 μl / well) to ensure uniform cell numbers in each well. The cells were cultured in a constant temperature atmosphere of CO2. When the growth rate reached 80%, the complete medium was aspirated and incomplete medium was added, 200 μl / well, and cultured for 3 hours. 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 hours. 20 μl of CCK-8 was added and the cells were cultured in an incubator for 1 hour. The absorbance A at 450 nm was measured using a microplate reader.

[0311] 3. ELISA determination of TNF-α, IL-6, IL-1β, and IL-10 levels in cell supernatants

[0312] The prepared mouse spleen lymphocyte suspension was inoculated into 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), an 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 48 hours of incubation at 37°C in a 5% CO2 incubator, the cells were centrifuged at 1000 g for 15 minutes, and the supernatant was collected. The OD value was measured at a wavelength of 450 nm using an enzyme reader according to the ELISA kit instructions. A standard curve was drawn to calculate the content of IL-6, TNF-α, IL-1β, and IL-10 in the corresponding test samples.

[0313] Experimental results:

[0314] As shown in FIG36 , 57103 inhibited the proliferation of splenocytes at a concentration of 20.48 μM, while other drug concentrations had no effect on splenocytes.

[0315] As shown in Figure 37, ELISA was used to detect inflammatory indicators related to spleen cells. The results showed that compared with the normal group (Control group), the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the model group were significantly increased, and the level of anti-inflammatory factor IL-10 was significantly reduced. After drug intervention, 57103 significantly downregulated the levels of IL-6, IL-1β, and TNF-α, with the high dose having the best effect. 57103 also upregulated the level of IL-10, with the high dose having the best effect. The above up-regulation and down-regulation effects of the high-dose group of 57103 were superior to those of the positive drugs M03C2 and MTX.

[0316] 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 and it was confirmed that KIRC and KIRP RCCs highly expressed integrin α v β3, α5β1, CD24 and IL4R (Figures 1 and 2). Most of the targets showed a significant positive correlation (Figure 3). Gene expression levels were determined by qRT-PCR, flow cytometry and Western Blot immunoblotting (Figures 4 and 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 generation process (Figures 6 and 7). MST and ELISA demonstrated the binding ability of 571, 571C1, 57103 and the target corresponding to the design. 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 8F). 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 8E and Figure 8H). In vivo imaging method using Cy7 labeling 57103, 571C1, and 571 demonstrated the targeted delivery ability of the fusion protein (Figure 11).

[0317] In the present invention, we also demonstrated that 57103 has a significant anti-angiogenic effect using HUVEC and rat aortic rings in in vitro models (Figures 9 and 10). qRT-PCR of HUVEC cells treated with 57103 and untreated showed that each target was downregulated, which may be related to drug-induced cytotoxicity or apoptosis or target protein degradation due to antibody internalization (Figure 10K). In addition, 57103 significantly inhibited the proliferation and metastasis of renal cancer cells 786O, ACHN and A498 (Figure 12). The THP1-derived M0, IL2-stimulated PBMC and IL2-stimulated NK92mi-CD16a co-culture model further demonstrated that 57103 can induce effective ADCP, ADCC and target internalization (Figure 13). qRT-PCR results also detected a significant downregulation of the target protein.

[0318] Results from the ACHN xenograft model demonstrated that 57103 inhibited tumor volume and weight by 68.30% and 70.00%, respectively, superior to 571 (42.36% and 36.60%) and 571C1 (55.86% and 65.40%), and comparable to the active agent Avastin (62.74% and 68.40%). Immunohistochemistry also demonstrated that 57103 significantly inhibited tumor growth and angiogenesis, as well as induced apoptosis (Figures 14C-14D). HE staining confirmed the ability of 57103 to inhibit the lung metastasis of subcutaneously transplanted ACHN tumors. Furthermore, combination therapy with 57103 and the PDL1 antagonist atezolizumab demonstrated a robust antitumor effect, significantly superior to either monotherapy or the saline negative control group. The 57103 30 mg / kg administration group of the present invention achieved the best tumor volume inhibition rate of 68.30% and the best tumor weight inhibition rate of 70.00%, and the relative tumor proliferation rate T / C% was 14.3% (T / C%=TRT / CRT×100%, calculated based on tumor volume change).

[0319] The anti-tumor activity evaluation index is the relative tumor proliferation rate T / C (%) T / C% = TRT / CRT × 100%

[0320] TRT: treatment group RT; CRT: negative control group RT.

[0321] Therapeutic efficacy evaluation criteria: T / C%>60% was considered ineffective; T / C%≤60% and P<0.05 after statistical analysis was considered effective.

[0322] The above description is merely an illustrative description of the present invention and its embodiments, which is not restrictive. The embodiment shown in the embodiment is only one embodiment of the present invention, and the actual embodiment is not limited thereto. Therefore, if a person skilled in the art is inspired by the above description and, without departing from the purpose of the present invention, designs an embodiment and examples similar to the technical solution without creatively designing, they shall 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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