Anti-b7-h3 single-chain antibody

Abstract

Provided is an anti-B7-H3 single-chain antibody, comprising: in a light chain variable region, CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 11, CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 12, and CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 13; and in a heavy chain variable region, CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 14, CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 15, and CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 16. The single-chain antibody specifically binds to B7-H3.

Description

Anti-B7-H3 single-chain antibody

[0001] This invention claims priority date 2023-08-15 and priority number 2023110224794. Technical Field

[0002] The present invention relates to the field of antibodies, and in particular to a single-chain antibody against B7-H3. Background Art

[0003] B7-H3 is a key member of the immune checkpoint protein family, primarily expressed on tumor cells, while expression is low in normal tissues. B7-H3 is widely expressed on tumor tissue cells (91.8% in liver cancer, 88.3% in ovarian cancer, and 58% in gastric cancer). It can suppress the immune response in tumor tissues, contributing to tumor growth and spread, and is associated with metastasis and poor prognosis. B7-H3 negatively regulates the activity of key transcription factors such as NFAT, AP-1, and NF-κB, thereby reducing IL-2 production by T cells. This inhibits the proliferation and activation of CD4 and CD8 T cells and suppresses IFN production by Th1 cells. Data indicate that patients with B7-H3 expression in tumor tissues are associated with poor clinical prognosis. Despite the crucial role of B7-H3 in tumor immune responses, its corresponding receptor on T cells is unknown. This makes it difficult to develop therapeutic antibodies to block the B7-H3 signaling pathway, as is the case with PD-L1 / PD-1. Because the immunosuppressive signaling pathways underlying B7-H3 are unclear, developing antibodies to block B7-H3's T cell suppression presents technical challenges.

[0004] Given this situation, most researchers and companies choose to develop and apply B7-H3 solely as a target. For example, in CAR-T cell therapy, B7-H3 is used to attack tumor cells, rather than blocking B7-H3's inhibitory effect on immune T cells. This is because the receptor on T cells is unknown, making it impossible to prevent its inhibitory effect on immune T cells. Similarly, some use B7-H3 as a target for antibody-antibody conjugates (ADCs) to treat tumors. Of course, antibody-dependent cellular cytotoxicity (ADCC) can also be used for immunotherapy. Some also develop bispecific antibodies, one targeting B7-H3 and the other targeting CD3. This mechanism is similar to the therapeutic mechanism of BiTEs, which closes the distance between T cells and tumor cells to kill cancer cells. Some also use B7-H3 as a target to kill tumors through ADCC, while also using PD-1 monoclonal antibodies to block B7-H1's immunosuppression of T cells.

[0005] The role of B7-H3 in the tumor microenvironment is quite complex. However, one thing is clear: B7-H3 has receptors on immune T or NK cells. This allows for the identification of therapeutic antibodies targeting B7-H3 through animal model experiments. Such antibodies may exert their therapeutic effects by blocking the contact of B7-H3 with MHC-II, or they may act through unknown receptors. Literature suggests that B7-H3 can bind to the TLT-2 receptor. The B7-H3 target offers numerous advantages, including immunosuppressive effects in regulating both T and NK cells. It is overexpressed in various tumors, but expressed only minimally in normal cells. This constitutes an important option for tumor immunotherapy and forms the basis for further research into B7-H3 immunotherapy.

[0006] In summary, the role of B7-H3 in the tumor microenvironment is now largely understood. With dozens of preclinical studies and early clinical trials underway, multiple clinical trials are evaluating the therapeutic efficacy of B7-H3-targeted strategies, either alone or in combination with other checkpoint inhibitors. Immunotherapies targeting B7-H3 have demonstrated a high safety profile and relatively low toxicity in clinical practice. Although the receptor for B7-H3 is still unknown, screening for effective antibodies using animal models is an effective approach. Preclinical studies can identify B7-H3 antibodies with tumor-killing effects. Although the mechanism of action of such antibodies is not fully understood, their ability to kill tumors through ADCC or by blocking their interaction with unknown receptors on immune cells remains a sensible and practical option.

[0007] Summary of the Invention

[0008] The object of the present invention is to provide a single-chain antibody against B7-H3.

[0009] To achieve the above objectives, the present invention provides an anti-B7-H3 single-chain antibody, characterized in that it comprises:

[0010] a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11;

[0011] a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12;

[0012] a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13;

[0013] a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14;

[0014] a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 15; and

[0015] a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 16;

[0016] The single-chain antibody specifically binds to B7-H3.

[0017] The present invention also protects the polynucleotide encoding the anti-B7-H3 single-chain antibody.

[0018] The present invention also protects a vector, characterized in that it contains the polynucleotide.

[0019] The present invention also protects a recombinant cell, characterized in that it contains the vector.

[0020] The present invention also protects a pharmaceutical composition, characterized in that it contains the anti-B7-H3 single-chain antibody and a pharmaceutically acceptable carrier.

[0021] Specifically, the present invention provides an anti-B7-H3 monoclonal antibody:

[0022] The light chain sequence is as follows:

[0023] Among them, the single underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; the double underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; Represents CDR2( SEQ ID NO: 2) sequence; lower wavy line Represents CDR3( SEQ ID NO:3) sequence.

[0024] The heavy chain sequence is as follows:

[0025] Among them, the single underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; the double underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; Represents CDR2( SEQ ID NO: 5) sequence; lower wavy line Represents CDR3( SEQ ID NO:6) sequence.

[0026] The present invention provides another anti-B7-H3 monoclonal antibody:

[0027] The light chain sequence is as follows:

[0028] or

[0029] The single underline "__" represents the CDR1 (SASSSVSYMH, SEQ ID NO: 11) sequence; the double underline "__" represents the CDR1 (SASSSVSYMH, SEQ ID NO: 11) sequence; Represents CDR2( SEQ ID NO: 12) sequence; lower wavy line Represents CDR3( SEQ ID NO:13) sequence.

[0030] The heavy chain sequence is as follows:

[0031] or:

[0032] The single underline "__" represents the CDR1 (GYTFTEY, SEQ ID NO: 12) sequence; the double underline "__" represents the CDR1 (GYTFTEY, SEQ ID NO: 12) sequence; Represents CDR2( SEQ ID NO: 13) sequence; lower wavy line Represents CDR3( SEQ ID NO:14) sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a graph showing the binding capacity of mouse B7-H3 antibody measured by an Octet instrument.

[0034] Figure 2. FD3-34-scFv affinity chromatography results.

[0035] Figure 3. Data showing specific binding of FD3-34-scFv single-chain antibody to cancer tissues and cells. (A) Data showing binding of FD3-34-scFv single-chain antibody to lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and melanoma, using immunohistochemical staining. (B) Data showing binding of FD3-34-scFv single-chain antibody to cancer cells (lung cancer stem cell line SPC-A1, non-small cell lung cancer cell line A549, head and neck squamous cell carcinoma cell line CA-9-22, and ovarian cancer cell line OVCAR-8), using flow cytometry.

[0036] FIG4 is a graph showing the protein electrophoresis results of humanized antibodies.

[0037] FIG5 is a diagram showing the effect of eliminating tumors in lung cancer model mice. DETAILED DESCRIPTION

[0038] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0039] Example 1: Preparation of anti-B7-H3 single-chain antibody

[0040] 1. Production of anti-B7-H3 monoclonal antibody (mouse origin)

[0041] 1.1 Immunization of mice

[0042] Eight-week-old female Balb / c mice were immunized subcutaneously at multiple sites with a mixture of human B7-H3 recombinant antigen protein (purchased from Shanghai YEASEN, product number 93111ES60, 100 μg, RMB 3185, Uniport No. Q5ZPR3.1) and complete Freund's adjuvant (CFA, Sigma) emulsion, at a dose of 80-100 μg protein per mouse. Three weeks later, booster immunizations were performed with an emulsion of protein and incomplete adjuvant (IFA, Sigma) for a total of three immunizations, at a dose of 80-100 μg protein per mouse. Sera were collected approximately two weeks after each immunization for antibody titer determination using ELISA on a SpectraMax i3 microplate reader. When serum titers reached a sufficiently high level, a booster immunization was performed intraperitoneally with 100 μg of human B7-H3 recombinant antigen protein dissolved in PBS. Three days later, spleens were removed and fused.

[0043] 1.2 Preparation and screening of hybridomas

[0044] Hybridoma cells were generated by fusing spleen cells from immunized mice with a mouse myeloma cell line (SP2 / 0-Ag14) using polyethylene glycol (PEG1450, SIGMA) as a fusion agent. The fused cells were suspended in HAT selection medium (Sigma Aldrich) and cultured in 96-well plates for approximately 11 days. The culture supernatant was then identified using ELISA on a SpectraMax i3. Positive clones were then subcloned using limiting dilution methods three or more times to ensure they were single-origin positive clones.

[0045] 1.3 Purification of anti-B7-H3 monoclonal antibody (mouse origin)

[0046] The subclones described in 1.2 were cultured with conventional hybridoma cells, and the culture supernatant was captured using HiTrap Protein G HP (purchased from GE Healthcare, Catalog No. 29048581). The supernatant was eluted with 0.1 M glycine (pH 3.5), and the eluted peak was collected and desalted using a HiTrap Desalting column (purchased from GE Healthcare, Catalog No. 29048684) with 1× PBS (pH 7.4) as the replacement buffer. The supernatant was stored at -20°C until use.

[0047] II. Characteristics of Anti-B7-H3 Monoclonal Antibody (Mouse) (Specific Binding Ability)

[0048] SPC-A1, A549, and CA-9-22 (human oral epithelial cancer cell lines) that highly express B7-H3 were used, and B7-H3 / Fc of human IgG1 (sequence derived from the Fc segment of human IgG1, sequence number: AAB2181.1, as shown in SEQ ID As shown in NO:19, a total of 456 amino acids were cloned into the vector according to standard methods, and then transferred to 293T cells to express B7-H3 / IgG1-Fc. The expressed B7-H3 / IgG1-Fc was purified using a protein A column and thoroughly mixed with the anti-B7-H3 monoclonal antibody (mouse source) purified according to 1.3 above. The mixture was incubated at 4°C for 20-30 minutes, washed, and detected on a FACSVerse. The results are shown in Figure 1. Figure 1 shows the binding capacity of the mouse B7-H3 antibody measured by the Octet instrument (molecular interaction instrument detection method, using bio-layer interferometry). The horizontal axis represents the time course (in seconds) of antibody binding to the B7-H3 antigen on cells. The first half of the rising curve represents the time required for the antibody to bind to the antigen (Association), while the second half of the flat curve represents the dissociation of the antibody after binding (Dissociation). A shorter binding time (a steeper curve) indicates stronger antibody binding, while a flat curve (no dip) after binding indicates minimal dissociation of the antibody after binding. This experiment demonstrates that the single-chain antibody FD3-34-scFv binds well to the B7-H3 antigen. See Figure 1 and Table 1 for detailed data.

[0049] Table 1 Binding parameters of FD3-34-scfv single-chain antibody to B7-H3 antigen

[0050] Note: KD represents the binding affinity of the antibody; Kon represents the binding constant between the antibody and the antigen; Kdis represents the separation constant between the antibody and the antigen.

[0051] 3. Sequence Determination of Anti-B7-H3 Antibody (Mouse)

[0052] According to the hybridoma preparation technique (Kohler and Milstein, 1975) and the sections on cDNA library preparation, gene identification, and DNA sequencing in Molecular Cloning (3rd edition, by J. Sambrook and D.W. Russell, translated by Huang Peitang et al.), total RNA was extracted from UM1-51, UM1-56, and UM1-69 hybridoma cells, and cDNA was synthesized and sequenced.

[0053] First, total RNA was extracted and purified from hybridoma cells using RNA purification kits (RAeasy Mini Kit, Qiagen 74104, QIAshredder, Qiagen 79654). The RNA was then reverse transcribed into first-strand cDNA using the SMARter RACE cDNA Amplification Kit (Clontech) according to the company's reagent manual. PCR amplification was performed using the 5' RACE technique, using the universal primer UPM (provided by the kit) as the upstream primer and the gene-specific primer GSP (designed based on the mouse IgG1 heavy chain variable region and light chain kappa chain gene sequences) as the downstream primer, using the SMARter first-strand cDNA as the template. The PCR products were then ligated into T-vectors (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10). Select clones for sequencing and analysis to obtain the light and heavy chain variable region sequences of the antibody, respectively. Primers were then designed based on the heavy chain variable region sequence and the mouse IgG1 sequence. The full-length heavy chain sequence was obtained by PCR. The sequence was analyzed using the IGBLAST analysis tool (KABAT) to determine the light and heavy chains and their CDR regions, as shown below.

[0054] The light chain sequence is as follows:

[0055] Among them, the single underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; the double underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; Represents CDR2( SEQ ID NO: 2) sequence; lower wavy line Represents CDR3( SEQ ID NO:3) sequence.

[0056] The heavy chain sequence is as follows:

[0057] Among them, the single underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; the double underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; Represents CDR2( SEQ ID NO: 5) sequence; lower wavy line Represents CDR3( SEQ ID NO:6) sequence.

[0058] IV. Humanization of anti-B7-H3 antibody (mouse origin)

[0059] In order to make antibodies better applicable to clinical patient treatment, it is necessary to carry out humanization transformation to the antibody sequence so that it retains high affinity to antigen and other favorable biological properties (such as reducing its immunogenicity in the human body). To achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Almost all mouse antibodies can be humanized by CDR grafting, thereby maintaining antigen binding. For relevant technical literature, please refer to Sims et al., 1987, J. Immunol., 151: 2296; Chothia et al., 1987, J. Mol. Biol., 196: 901; Presta et al., 1993, J. Immnol., 151: 2623; Carter et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4285; Lo Benny KC et al., in Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004.

[0060] Based on the above work, FD3-34-scFv is preferably humanized. First, the antibody heavy chain constant region is replaced with the human IgG4 constant region sequence, and the antibody light chain constant region is replaced with the human κ chain constant region to obtain a human-mouse chimeric parent antibody (Chimeric Ab). After confirming the functional activity of the parent antibody, the heavy chain variable region and light chain variable region sequences are humanized, and the key amino acids in the FR region are reversely mutated on the basis of CDR transplantation. For amino acids between FRs that are not sure whether they affect the binding of antigen and antibody, merger synthesis is performed to construct a series of IgG mutants. After screening based on antibody affinity and expression level, one heavy chain and one light chain sequence were screened and cloned into the pcDNA3.1 expression vector using conventional molecular cloning technology. The 5' end of the light chain or heavy chain sequence carries a Kozak sequence and a human IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 22), and its corresponding nucleotide sequence is:

[0061] The light chain sequence is as follows:

[0062] Among them, the single underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; the double underline "__" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; Represents CDR2( SEQ ID NO: 2) sequence; lower wavy line Represents CDR3( SEQ ID NO:3) sequence.

[0063] The heavy chain sequence is as follows:

[0064] Among them, the single underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; the double underline "__" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; Represents CDR2( SEQ ID NO: 5) sequence; lower wavy line Represents CDR3( SEQ ID NO:6) sequence.

[0065] Transient transfection of the above light and heavy chain expression vectors into ExpiCHO-S (Invitrogen) cells yielded a humanized antibody (FD3-34-scFv). The purified antibody was screened by ELISA and then biochemically analyzed by biochemical interaction assay (BLI), identifying the humanized antibody with the best binding affinity (Figure 1).

[0066] FD3-34-scfv humanized amino acid sequence

[0067] As above, FD3-23scFv was humanized, and the following sequence is the humanized sequence:

[0068] The light chain sequence is as follows:

[0069] or

[0070] The single underline "__" represents the CDR1 (SASSSVSYMH, SEQ ID NO: 11) sequence; the double underline "__" represents the CDR1 (SASSSVSYMH, SEQ ID NO: 11) sequence; Represents CDR2( SEQ ID NO: 12) sequence; lower wavy line Represents CDR3( SEQ ID NO:13) sequence.

[0071] The heavy chain sequence is as follows:

[0072] or:

[0073] The single underline "__" represents the CDR1 (GYTFTEY, SEQ ID NO: 14) sequence; the double underline "__" represents the CDR1 (GYTFTEY, SEQ ID NO: 14) sequence; Represents CDR2( SEQ ID NO: 15) sequence; lower wavy line Represents CDR3( SEQ ID NO:16) sequence.

[0074] FD3-23-1-scfv amino acid sequence (VL6-linked-VH5):

[0075] FD3-23-2-scfv amino acid sequence (VL6-linked-VH8):

[0076] FD3-23-3-scfv amino acid sequence (VL9-linked-VH5):

[0077] Example 2 Characteristics and Functional Experiments of Humanized Antibodies

[0078] 1. Expression and Purification of Humanized Antibodies

[0079] The pcDNA3.1 plasmids of the antibody light chain and heavy chain DNA sequences prepared in Example 1 were transformed into Escherichia coli DH5α, and after sequencing verification, the glycerol bacteria were stored at -80 degrees. Streaks were taken from the glycerol bacteria plate, and after overnight culture, single colonies were picked and inoculated into 3 mL of LB medium containing 100 μg / mL ampicillin and cultured overnight. The culture was then inoculated into 150 mL of LB liquid medium (with a final concentration of 100 μg / mL ampicillin) and cultured at 37°C overnight. The antibody light and heavy chain plasmids were extracted separately using the Tiangen endotoxin-free plasmid extraction kit (see the instructions for the specific operation process). After measuring the plasmid concentration, the plasmids were stored at -20°C for transfection.

[0080] ExpiCHO-S cells were cultured in 30 mL of ExpiCHO expression medium (containing 0.5% Anti-clumping Agent) at 37°C, 135 rpm, and 8% CO2 to a cell density of 4-6 × 10 6 cells / mL, 5X10 5 After 2 to 3 consecutive passages, a portion of the passaged cells can be taken and counted by trypan blue staining for transfection. Use the ExpiFectamine CHO Transfection Kit for transient transfection; take 1.5X10 8 The cells were centrifuged, the supernatant was discarded, and 25.5 mL of fresh ExpiCHO expression medium was added to resuspend the cells; 20 μg of antibody plasmid (10 μg of light chain plasmid, 10 μg of heavy chain plasmid) was dissolved in 1 mL of pre-cooled OptiPRO SFM serum-free medium and gently mixed; 80 μL of transfection reagent was added to 920 μL of pre-cooled OptiPRO SFM serum-free medium and gently mixed; the transfection reagent solution and plasmid solution were mixed, and the mixture was allowed to stand at room temperature for 1-5 minutes before being added to the ExpiCHO-S cell culture medium; the Standard culture mode was used, and 18-22 hours after transfection, 150 μL of Enhancer and 6 mL of Feed were added; the cells were harvested when the cell viability dropped to 75%; the cell supernatant obtained after centrifugation was filtered through a 0.22 μm filter and the cell culture supernatant was loaded on an AKTA avant and passed through a ProA pre-packed column (GE Hitrap ProteinA HP 1 ml). Separation conditions: After loading, the column was washed with PBS buffer, eluted with 0.1 M glycine (pH 3.5), and regenerated with 0.1 M glycine (pH 2.0) (Figure 2, Figure 2 is the result of FD3-34-scFv affinity chromatography. As can be seen in the figure, the B7-H3scFv single-chain antibody peak is clearly visible.

[0081] The purified protein FD3-34-scFv (approximately 2 ml) was transferred to a 30 kD molecular weight cutoff ultrafiltration centrifuge tube. Approximately 15 ml of PBS buffer was added and centrifuged at 4000 × g for 5 min twice and at 4000 × g for 10 min three times. The collected protein was aspirated with a 2 ml syringe, filtered through 0.22 μm, aliquoted, and stored at -80°C. Based on DNA sequencing and protein N-terminal sequencing, the light chain amino acid sequence of the purified antibody FD3-34-scFv is shown in Seq ID No: 7, and the heavy chain amino acid sequence is shown in Seq ID No: 8.

[0082] 2. SDS-PAGE detection of antibody molecular weight

[0083] 1) Prepare 6% or 12% separation gel and 5% stacking gel;

[0084] 2) Sample treatment: Take 6 μl of sample, add 1.5 μl 5* reducing loading buffer and 1.5 μl 5* non-reducing loading buffer respectively, and boil in a 95°C metal bath for 3 min;

[0085] 3) Sample loading: Add 5 μl of treated sample to each well. Run the stacking gel at 60V and the separating gel at 120V until the sample is separated. Stain with Coomassie Brilliant Blue for 40 minutes. Destain with destaining solution until the bands are clear, then photograph and save using a gel imager. The results are shown in Figure 4. Figure 4 shows the protein electrophoresis results of the humanized antibody. A shows the FD3-34 protein under reducing conditions, with a heavy chain band of approximately 50 kD and a light chain band of approximately 25 kD, with no other contaminants. B shows the main band under non-reducing conditions, with a molecular weight of approximately 150 kD. Positive control: PD-1 mAb; negative control: PBS (Ctrl).

[0086] 3. UPLC-SEC detection of antibody purity

[0087] Prepare 100mM sodium phosphate buffer, adjust the pH to 6.80, and equilibrate a chromatography column (Waters, BEH SEC 200 4.6*150mm) at 0.1mL / min until the pressure fluctuation is <10psi. Then adjust the flow rate to 0.4mL / min. Filter the sample through a 0.22µm filter and add at least 20µL of each sample to the appropriate sample tray. Once the baseline stabilizes, select the instrument's sodium phosphate buffer (100mM), inject 2µL of the sample, and run for 7 minutes. After the injection is complete, flush the column at a low flow rate (less than 0.2mL / min) for at least 10 column volumes and store in 20% methanol.

[0088] The purification of FD3-34-scFv antibody is shown in FIG2 , and the main peak accounts for 96.70%.

[0089] 4. Octet affinity determination

[0090] The antibody to be tested was prepared at six 50 nM half-dilution concentrations. A buffer blank well and a 50 nM antigen (Human B7-H1 Protein His-Tagged) blank well were also set up. 5 μg / ml of antigen was immobilized on the SA sensor to a solidification height of approximately 0.6 nm. The affinity of the test antibody was measured on an Octet Red 96 instrument (Fortebio) according to the pre-set protocol (Baseline: 120 s; Loading: 60 s; Baseline: 120 s; Association: 300 s; Dissociation: 1800 s). Affinity data were calculated using the Octet Red 96 software (Figure 1 and Table 1).

[0091] The affinity KD (M) of the B7-H3 antibody (FD3-34-scFv) is 8.37E -10 ±1.10E -9 , the binding coefficient is 2.96E+05(1 / Ms), the dissociation coefficient is 1.17E-04(1 / s), and the Full R 2 The value is 0.9991. See Figure 1 and Table 1 for details.

[0092] 5. Immunohistochemical staining to show the activity of binding to cancer tissue antigens and flow cytometry to measure the binding activity

[0093] Various cancer tissue sections were prepared according to routine pathology procedures. Cancer tissues (lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, colorectal cancer, malignant melanoma, etc.) were fixed with 4% formalin, dehydrated with a standard graded alcohol solution, embedded in paraffin, cut into 5-micron slices, and mounted on glass slides. The sections were then incubated with the primary antibody FD3-34-scFv, rinsed with PBS, incubated with the secondary antibody linked to the enzyme, and incubated with the enzyme substrate for 15 minutes. The sections were then rinsed with PBS, stained with hematoxylin and eosin, dehydrated, mounted, and observed and photographed under a microscope. The immunohistochemical staining results are shown in Figure 3A, which shows an example of FD3-34-scFv binding to the B7-H3 antigen in various human cancer tissues (immunohistochemical staining method).

[0094] Adherently culture B7-H3-expressing human lung cancer stem cell line SPC-A1, non-small cell lung cancer A549, head and neck squamous cell carcinoma CA-922, and ovarian cancer cell line OVCAR-8 to an appropriate cell density. Aspirate the culture medium, wash twice with PBS, add 0.2-0.5 ml of 0.25% trypsin-EDTA, and incubate in a cell culture incubator for 1-2 minutes. If digestion is too long, terminate the digestion with RPMI-1640 (10% FBS). Count the cells, averaging 1 to 5 x 10^5 cells per tube. Centrifuge at 2000 rpm for 3 minutes, aspirate the supernatant, resuspend the cells in an appropriate amount of 1 x PBS, and centrifuge again at 2000 rpm for 3 minutes. Repeat twice. Add a mixture of the test antibody and fluorescently labeled B7-H3 at varying concentrations. For isotype and positive controls, add 100 ng of B7-H3 / hFc-FITC and varying concentrations of the anti-B7-H3 antibody to each tube. Mix thoroughly. For the isotype control, incubate at 4°C for 20-30 minutes. Wash 1-3 times with 1x PBS, adjust the cell concentration with an appropriate amount of 1x PBS, and analyze the cells (results shown in Figure 4B).

[0095] 6. Detection of tumor elimination effect in lung cancer model mice

[0096] The human lung cancer cell line SPC-A1, which highly expresses B7-H3, was cultured in RPMI 1640 medium containing 10% FBS and passaged once every 2-3 days until the appropriate cell number was reached. PBMCs from healthy adults were extracted by density gradient centrifugation, washed, counted, and set aside. Four- to five-week-old female immunodeficient NCG mice (purchased from Nanjing University-Nanjing Institute of Biomedicine, and raised on an IVC rack with independent water and air supply systems) were used. SPC-A1 cells were digested and mixed with PBMCs in an appropriate ratio. The cells were then inoculated into the mice at a density of 3x10^7 cells / ml, with one inoculation per mouse under the right wing. Tumors generally form 3-5 days after inoculation, and tumors can reach 100-250 mm in 7-9 days. 3 The weight and tumor volume of mice were measured at the time of enrollment, and mice with tumors smaller than 80 mm3 or larger than 250 mm3 or other abnormal mice were eliminated. Then, mice that met the requirements were randomly assigned to different experimental groups.

[0097] After enrollment, the experimental antibody (FD3-34), positive control antibody (PD-1 mAb), and negative control antibody (human IgG4, denoted as hIgG4 or Ctrl) were administered according to the group. The antibody concentration was 1 mg / ml, and it was injected intraperitoneally. The initial dose was increased. The initial dose of FD3-34 antibody was 250 μg / mouse, and then 200 μg / mouse / time, administered once every 3 days for up to 6 times, that is, q3dx6 times; the tumor volume was measured before each dose, and measured 2-3 times a week, generally before administration.

[0098] After six rounds of antibody treatment, as shown in Figure 5A, the FD3-34 group showed a more significant inhibition of tumor volume growth compared to the PD-1 mAb group and the hIgG4 group (i.e., Ctrl). From the second to the sixth dose, FD3-34's ability to inhibit tumor proliferation was lower than that of PD-1 mAb. From the survival curve, as shown in Figure 5B, the FD3-34 group had a survival rate similar to that of PD-1 mAb. A represents the tumor weight of mice after NCG treatment (in grams, 6 mice. Compared with the control group, the FD3-34 antibody and the positive control group PD-1 mAb showed a significant reduction in tumor weight, *P<0.05); B represents the survival curve of NCG mice after FD3-34 and PD-1 mAb treatment (6 animals per group).

[0099] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. An anti-B7-H3 single-chain antibody, characterized in that: Include: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11; a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12; a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13; a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14; A heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 15; and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 16; The single-chain antibody specifically binds to B7-H3.

2. A polynucleotide encoding the anti-B7-H3 single-chain antibody according to claim 1.

3. A carrier, characterized in that Contains the polynucleotide according to claim 2.

4. A recombinant cell, characterized in that Containing the vector according to claim 3.

5. A pharmaceutical composition, characterized in that A composition comprising the anti-B7-H3 single-chain antibody according to claim 1 and a pharmaceutically acceptable carrier.

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