CN117088981A - Single chain antibody against B7-H3 - Google Patents

Abstract

The invention discloses a single-chain antibody for resisting B7-H3. Comprising: the light chain variable region CDR1 comprises SEQ ID NO:1 or SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no; CDR2 comprises SEQ ID NO:2 or SEQ ID NO:12, an amino acid sequence shown in seq id no; CDR3 comprises SEQ ID NO:3 or SEQ ID NO:13, an amino acid sequence shown in seq id no; the heavy chain variable region CDR1 comprises SEQ ID NO:4 or SEQ ID NO:14, an amino acid sequence shown in seq id no; CDR2 comprises SEQ ID NO:5 or SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no; CDR3 comprises SEQ ID NO:6 or SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no; wherein the single chain antibody specifically binds to B7-H3.

Description

Anti-B7-H3 single chain antibody

Technical field

The present invention relates to the field of antibodies, in particular to anti-B7-H3 single-chain antibodies.

Background technique

B7-H3 is an important member of the immune checkpoint protein family and is mainly expressed on tumor cells, with lower expression in normal tissues. B7-H3 is widely expressed on tumor tissue cells (expression rate of liver cancer is 91.8%, ovarian cancer is 88.3%, and gastric cancer is 58%). It can inhibit the immune response of tumor tissue, cause tumor growth and spread, and is related to tumor metastasis and poor prognosis. . B7-H3 reduces the production of IL-2 by T cells by negatively regulating the activity of major transcription factors such as NFAT, AP-1, and NF-B, thereby inhibiting the proliferation and activation of CD4 and CD8 T cells and inhibiting the IFN production of Th1 cells. Some data indicate that patients with B7-H3 expression in tumor tissues have poor clinical prognosis. Despite the important role of B7-H3 in tumor immune responses, its corresponding receptor on T cells is not known. This makes it difficult to develop therapeutic antibodies like PD-L1/PD-1 in blocking the B7-H3 signaling pathway. Because the immunosuppressive signaling pathway of B7-H3 is unclear, there are technical difficulties in using what kind of antibodies to block the inhibition of T cells by B7-H3.

Based on this situation, most researchers or companies only use B7-H3 as a target when they choose to develop and apply it. For example, when using CAR-T cell therapy, B7-H3 is used as a target to attack tumor cells. , instead of blocking the suppression of immune T cells by B7-H3, because its receptor on T cells is not known, so it cannot block its suppression of immune T cells. Similarly, some people use B7-H3 as a target to use antibody conjugates (ADC) to treat tumors. Of course, antibody-dependent cellular cytotoxicity (ADCC) can also be used for immunotherapy. Some people also make bispecific antibodies, one of which targets B7-H3 and the other target targets CD3. This is similar to the therapeutic mechanism of BiTE, which closes the distance between T cells and tumor cells to kill cancer cells. Some people also use B7-H3 as a target to kill tumors through ADCC, and also use PD-1 monoclonal antibodies to block the immunosuppression of T cells by B7-H1.

The role of B7-H3 in the tumor microenvironment is quite complex. One thing is clear, that is, B7-H3 has its receptor on immune T or NK cells. This can be used to screen out B7-H3 treatments through animal model experiments. Sexual antibodies, this antibody may have a therapeutic effect on tumors by blocking the contact between B7-H3 and MHC-II, and may also work through an unknown receptor. Literature shows that B7-H3 can bind to the TLT-2 receptor. The B7-H3 target has many advantages. It exhibits immunosuppressive effects when regulating T cells and NK cells. It is overexpressed in a variety of tumors and has little expression in normal cells. This constitutes an important option for tumor immunotherapy and also becomes the basis for further research on B7-H3 immunotherapy in the future.

In summary, the role of B7-H3 in the tumor microenvironment has been largely clarified. With dozens of preclinical studies and early clinical trials ongoing, multiple clinical trials are evaluating the therapeutic efficacy of B7-H3 targeting strategies alone or in combination with other checkpoint inhibitors. Immunotherapy targeting B7-H3 has shown high clinical safety and low toxicity. Although the receptor of B7-H3 has not yet been clarified, it is an effective method to screen effective antibodies through animal models, that is, to screen out B7-H3 antibodies that have a killing effect on tumors through preclinical experiments. The mechanism of action is not very clear. It exerts a tumoricidal effect through ADCC or blocking its interaction with unknown receptors on immune cells. It is a wise and practical choice under the current circumstances.

Contents of the invention

The object of the present invention is to provide an anti-B7-H3 single-chain antibody.

In order to achieve the above object, the present invention provides an anti-B7-H3 single-chain antibody, which is characterized in that it contains:

Light chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11;

Light chain variable region CDR2, which includes the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12;

Light chain variable region CDR3, which includes the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13;

Heavy chain variable region CDR1, which includes 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

Heavy chain variable region CDR3, which includes the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 16;

The single chain antibody specifically binds to B7-H3.

The invention also protects the polynucleotide encoding the anti-B7-H3 single-chain antibody.

The invention also protects a vector, which is characterized in that it contains the polynucleotide.

The invention also protects a recombinant cell, which is characterized in that it contains the vector.

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

Specifically, embodiments of the present invention provide anti-B7-H3 monoclonal antibodies:

The light chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GASENIYGALN , SEQ ID NO: 1) sequence; the double underline Represents CDR2(/> SEQ ID NO:2) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:3) sequence.

The heavy chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GFNIKDT , SEQ ID NO:4) sequence; the double underline Represents CDR2(/> SEQ ID NO:5) sequence; wavy line/> Represents CDR3/> SEQ ID NO:6) sequence.

The embodiment of the present invention provides another anti-B7-H3 monoclonal antibody:

The light chain sequence is as follows:

or

The single underline "_" represents the CDR1 ( SASSSVSYMH , SEQ ID NO:11) sequence; the double underline Represents CDR2(/> SEQ ID NO:12) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:13) sequence.

The heavy chain sequence is as follows:

or:

The single underline "_" represents the CDR1 ( GYTFTEY , SEQ ID NO:12) sequence; the double underline Represents CDR2(/> SEQ ID NO:13) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:14) sequence.

Description of the drawings

Figure 1 is a graph showing the binding ability of mouse B7-H3 antibody measured by Octet instrument.

Figure 2. FD3-34-scFv affinity chromatography results chart.

Figure 3. Data graph showing that FD3-34-scFv single chain antibody specifically binds to cancer tissues and cells. (A) Data on the binding of FD3-34-scFv single chain antibody to lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, colorectal cancer and melanoma. The staining method is immunohistochemical staining. (B) FD3-34-scFv single chain antibody binds 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, ovarian cancer cell line OVCAR- 8), the detection method is flow cytometry.

Figure 4 is a picture of the protein electrophoresis results of humanized antibodies.

Figure 5 is a diagram showing the effect of detecting and eliminating tumors in lung cancer model mice.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1: Preparation of single-chain antibodies against B7-H3

1. Production of anti-B7-H3 monoclonal antibody (mouse source)

1.1 Immunization of mice

Take 8-week-old female Balb/c mice and use human B7-H3 recombinant antigen protein (purchased from Shanghai YEASEN Company, product number 93111ES60, 100ug, RMB3185, Uniport No.Q5ZPR3.1) and Freund's complete adjuvant (CFA , Sigma) mixed emulsion was subcutaneously inoculated at multiple points, with 80-100 μg protein per mouse; 3 weeks later, protein and incomplete adjuvant (IFA, Sigma) emulsion was used for booster immunization, with a total of 3 immunizations, each mouse Mouse 80-100μg protein. The serum was taken to measure the antibody titer about 2 weeks after each mouse immunization. The serum titer was measured using the ELISA method with a microplate reader SpectraMax i3. When the serum titer reaches a high enough level, 100 μg of human B7-H3 recombinant antigen protein is dissolved in PBS, and intraperitoneal injection is given to boost the immunity once. After 3 days, the spleen is removed for fusion.

1.2 Preparation and screening of hybridomas

Hybridoma cells are produced by fusion of spleen cells from immunized mice and mouse myeloma cell line (SP2/0-Ag14) under the action of polyethylene glycol (PEG1450, SIGMA) fusion agent. The fused cells are suspended in a suspension containing HAT. (SigmaAldrich) selected culture medium was added to a 96-well culture plate and cultured for about 11 days, and the culture supernatant was identified by SpectraMax i3 using ELISA method. The selected positive clones were then subjected to subclone selection by the limiting dilution method more than three times to ensure that they were positive clones from a single source.

1.3 Purification of anti-B7-H3 monoclonal antibody (mouse source)

1.2 After the subclone is cultured with conventional hybridoma cells, the culture supernatant is captured by HiTrap Protein G HP (purchased from GE Healthcare, product number 29048581), and then eluted with 0.1M glycine (pH 3.5), and the elution peaks are collected. Then use HiTrap Desalting (purchased from GE Healthcare, product number: 29048684) column to desalt, and replace the buffer with 1×PBS (pH7.4). Store at -20 degrees for later use.

2. Characteristics (specific binding ability) of anti-B7-H3 monoclonal antibody (mouse source)

SPC-A1, A549, and CA-9-22 (human oral epithelial cancer cell lines) cells that highly express B7-H3 were used, and B7-H3/ was labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (purchased from Thermo, product number: A30006). Fc of human IgG1 (sequence derived from the Fc segment of human IgG1, sequence number: AAB2181.1, as shown in SEQ ID NO:19, a total of 456 amino acids, 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 is purified using a protein A column and thoroughly mixed with the anti-B7-H3 monoclonal antibody (mouse source) purified according to 1.3 above, incubated at 4°C for 20 to 30 minutes, and washed. , tested on FACSVerse, and the results are shown in Figure 1. Figure 1 shows the binding ability of mouse B7-H3 antibody measured by Octet instrument (molecular interaction instrument detection method, using biolayer interference technology), where the horizontal axis represents the antibody The time course of binding the B7-H3 antigen on cells (in seconds). The rising curve in the first half represents the time it takes for the antibody to bind to the antigen (Association). The gentle curve in the second half represents the separation after the antibody binds to the antigen (Dissociation). . The shorter the time required for binding (the steeper the curve), the stronger the binding force of the antibody. The flat curve (not decreasing) after binding means that the antibody does not separate very much after binding to the antigen. This experiment shows that the single-chain antibody FD3-34-scFv binds to B7- The ability of H3 antigen is good, specific data are shown in (Figure 1 and Table 1).

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

Note: KD represents the binding force 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.

QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHTAAPFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGKABATGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO:19.

3. Sequence determination of anti-B7-H3 antibody (mouse source)

According to hybridoma preparation technology (Kohler and Milstein, 1975) and "Molecular Cloning Experimental Guide" (3rd edition) [Molecular Cloning, written by J. Sambrook, D.W. Russell, translated by Huang Peitang et al.] cDNA library preparation and its Chapter operations such as genetic identification and DNA sequencing. Total RNA was extracted from UM1-51, UM1-56 and UM1-69 hybridoma cells, and cDNA was synthesized and sequenced.

First, total RNA was extracted and purified from hybridoma cells using an RNA purification kit (RAeasy Mini Kit, Qiagen 74104, QIAshredder, Qiagen 79654). Then use a kit (SMARTer RACE cDNAAmplification Kit, Clontech) and follow the company's reagent manual to reverse-transcribe RNA to synthesize the first strand of cDNA. Utilize 5'RACE technology, use the universal primer UPM (universal primer) provided in the kit as the upstream primer, and the gene specific primer GSP (gene specific primer) designed based on the mouse IgG1 heavy chain variable region and light chain kappa chain gene sequences. As a downstream primer, PCR amplification was performed using SMART first-strand cDNA as a template. The PCR products were then ligated into T vectors (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10) respectively. Select clones for sequence determination and analysis to obtain the light chain and heavy chain variable region sequences of the antibody, and then design primers based on the heavy chain variable region sequence and mouse IgG1 sequence, obtain the full-length heavy chain sequence through PCR, and use IGBLAST analysis The tool (KABAT) analyzes the sequence and determines the light and heavy chains and their CDR regions, as shown below.

The light chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GASENIYGALN , SEQ ID NO: 1) sequence; the double underline Represents CDR2(/> SEQ ID NO:2) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:3) sequence.

The heavy chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GFNIKDT , SEQ ID NO:4) sequence; the double underline Represents CDR2(/> SEQ ID NO:5) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:6) sequence.

4. Humanization of anti-B7-H3 antibody (mouse source)

In order for antibodies to be better used in the treatment of clinical patients, the antibody sequence needs to be humanized to retain its high affinity for the antigen and other beneficial biological properties (such as reducing its immunogenicity in the human body) . To this end, humanized antibodies are prepared according to preferred methods by analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Almost all murine antibodies can be humanized via CDR grafting to maintain 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. Immunol., 151: 2623; Carter et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4285; Lo Benny K. C. et al., in Antibody Engineering: Methods and Protocols, Volume 248, Humana Press, New Jersey , 2004.

Based on the aforementioned work, it is preferred to humanize FD3-34-scFv. First, the antibody heavy chain constant region is replaced with a human IgG4 constant region sequence, and the antibody light chain constant region is replaced with a human kappa chain constant region to obtain a human-mouse chimeric parent antibody (Chimeric Ab). After confirming the functional activity of the parent antibody, humanize the heavy chain variable region and light chain variable region sequences, and carry out reverse mutation of key amino acids in the FR region based on CDR transplantation. A series of IgG mutants were constructed by degenerate synthesis of amino acids between FRs that could not be determined to affect the binding of antigens and antibodies. After screening based on antibody affinity and expression level, 1 heavy chain and 1 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 the Kozak sequence. And human IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 22), its corresponding nucleotide sequence is:

The light chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GASENIYGALN , SEQ ID NO: 1) sequence; the double underline Represents CDR2(/> SEQ ID NO:2) sequence; wavy line/> Represents CDR3/> SEQ ID NO:3) sequence.

The heavy chain sequence is as follows:

Among them, the single underline "_" represents the CDR1 ( GFNIKDT , SEQ ID NO:4) sequence; the double underline Represents CDR2(/> SEQ ID NO:5) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:6) sequence.

The above light chain and heavy chain expression vectors are transiently transfected into ExpiCHO-S (Invitrogen) cells to obtain a humanized antibody (FD3-34-scFv). The purified antibody is screened by ELISA and then combined with the molecular interaction method (BLI). optimal humanized antibody (Figure 1).

FD3-34-scfv humanized amino acid sequence

DIQMTQSPSSSLSASVGDRVTITTCGASENIYGALNWFQRKPGKSPQFLIYGATNLADGVPSRFSGSGSGRQYSLTISSLQPEDFATYYCQNVLTSPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYMHWVKERPGQGLEWIGRIDPANGNTKYDPRFQGKATITADTSSNTAYME LSSLSEDTAVYYCTRREVYWGQGTLVTVSS.

SEQ ID NO:23.

Same as above, humanized transformation of FD3-23 scFv, the following sequence is the modified one:

The light chain sequence is as follows:

or

The single underline "_" represents the CDR1 ( SASSSVSYMH , SEQ ID NO:11) sequence; the double underline Represents CDR2(/> SEQ ID NO:12) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:13) sequence.

The heavy chain sequence is as follows:

or:

The single underline "_" represents the CDR1 ( GYTFTEY , SEQ ID NO:14) sequence; the double underline Represents CDR2(/> SEQ ID NO:15) sequence; wavy line/> Represents CDR3(/> SEQ ID NO:16) sequence.

FD3-23-1-scfv amino acid sequence (VL6-link-VH5):

Question LTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS. SEQ ID NO:24.

FD3-23-2-scfv amino acid sequence (VL6-link-VH8):

QIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVKQSHGKSLEWIGGFNPNNNGGTTYNQKFKGRATMTGDRSSSTAYM ELRSLTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS. SEQ ID NO:25.

FD3-23-3-scfv amino acid sequence (VL9-Link-VH5):

Question LTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS. SEQ ID NO:26.

Example 2 Characteristics and functional experiments of humanized antibodies

1. Expression and purification of humanized antibodies

The pcDNA3.1 plasmid of the antibody light chain and heavy chain DNA sequences prepared in Example 1 was transformed into E. coli DH5α. After sequencing and verification, the glycerol bacteria were stored at -80 degrees. Take a glycerol bacteria plate and streak it. After overnight culture, pick a single colony and inoculate it into 3 mL of LB medium containing 100 μg/mL ampicillin. Cultivate overnight at 37°C; use the Tiangen endotoxin-free plasmid maximal extraction kit to extract the antibody light and heavy chain plasmids respectively (see the instructions for specific operation procedures). After measuring the plasmid concentration, store it at -20°C until transfection.

ExpiCHO-S cells were cultured with 30 mL of ExpiCHO expression medium (containing 0.5% Anti-clumping Agent) at 37°C, 135 rpm, and 8% CO _2. When the cell density was 4-6X10 ⁶ cells/mL, 5X10 ⁵ cells/mL was used. mL seeding density for passage; after 2 to 3 consecutive passages, some passage cells can be taken, counted by trypan blue staining, and transfected. Use ExpiFectamine CHO Transfection Kit for transient transfection; centrifuge 1.5X10 ⁸ cells, discard the supernatant, add 25.5 mL of fresh ExpiCHO expression medium to resuspend the cells; take 20 μg of antibody plasmid (10 μg of light chain plasmid, 10 μg of heavy chain plasmid) , dissolve in 1 mL of pre-cooled OptiPRO SFM serum-free medium, and mix gently; take 80 μL of transfection reagent, add 920 μL of pre-cooled OptiPRO SFM serum-free medium, and mix gently; mix the transfection reagent solution with the plasmid Mix the solution and let it stand at room temperature for 1-5 minutes before adding it to the ExpiCHO-S cell culture medium. Use Standard culture mode. 18-22 hours after transfection, add 150 μL Enhancer and 6 mL Feed. When the cell viability drops to 75%, harvest the cells. ; Filter the cell supernatant obtained after centrifugation with a 0.22 μm filter, load the cell culture supernatant on AKTAavant, and pass it through a ProA prepacked column (GE Hitrap ProteinA HP 1ml). Separation conditions: After loading, wash the column with PBS buffer, elute with 0.1M glycine (pH3.5), and regenerate with 0.1M glycine (pH2.0) (Figure 2, Figure 2 is FD3-34-scFv affinity chromatography Result picture. It can be seen from the picture that the B7-H3 scFv single-chain antibody peak is clearly visible.

Pour the purified protein FD3-34-scFv (about 2 ml) into an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kD, add about 15 ml of PBS buffer, and centrifuge at 4000×g for 5 min*2 times, and centrifuge at 4000×g for 10 min*3 times. Aspirate the collected protein with a 2ml syringe, filter with 0.22μm, aliquot, and store at -80°C. According to the DNA sequencing and protein N-terminal sequencing results, 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.

2. SDS-PAGE detects the molecular weight of antibodies

1), prepare 6% or 12% separating gel and 5% stacking gel;

2) Sample processing: Take 6ul of sample, add 1.5ul 5*reducing loading buffer and 1.5ul 5*non-reducing loading buffer respectively, and boil in 95℃ metal bath for 3 minutes;

3). Sample loading: Add 5ul of processed sample to each well. Run the stacking gel for electrophoresis at 60V and the separation gel at 120V until the samples are separated. Coomassie brilliant blue staining for 40 minutes. Use destaining solution to destain until the bands are clear, take pictures with a gel imager and save them. The results are shown in Figure 4. Figure 4 is a picture of the protein electrophoresis results of humanized antibodies. A is the band of FD3-34 under reducing conditions. The heavy chain is about 50KD, the light chain is about 25KD, and there are no other hybrid bands. B shows that the molecular weight of the main band is around 150kD under non-reducing conditions. Positive control: PD-1mAb; negative control PBS is Ctrl.

3. UPLC-SEC detects the purity of antibodies

Prepare 100mM sodium phosphate buffer solution to adjust the pH to 6.80, equilibrate the column (Waters, BEHSEC 200 4.6*150mm) at 0.1mL/min until the pressure fluctuation is <10psi, and then adjust the flow rate to 0.4mL/min. The sample is filtered through a 0.22um filter membrane, and then added to the corresponding sample tray position. Each sample is at least 20uL. After the baseline is stable, select the corresponding instrument sodium phosphate 100mM, injection volume 2uL, time 7min, click run. After the injection is completed, flush the chromatographic column with a low flow rate (less than 0.2 mL/min) for at least 10 column volumes and store it in a 20% methanol solution.

The purification of FD3-34-scFv antibody is shown in Figure 2, with the main peak accounting for 96.70%.

4.Octet affinity determination

Prepare the antibody to be tested into 6 concentrations of 50nM half-diluted, and set up a Buffer blank hole and a 50nM antigen (Human B7-H1 Protein His-Tagged) blank hole; fix the 5ug/ml antigen on the SA sensor, and set the solidification height About 0.6nm; follow the set procedures; (Baseline: 120s; Loading: 60s; Baseline: 120s; Association: 300s; Dissociation: 1800s) to measure the affinity of the antibody to be tested on the Octet Red96 instrument (Fortebio Company). Octet Red96 supporting software was used to fit and calculate the affinity data (Figure 1 and Table 1).

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

5. Immunohistochemical staining shows the binding activity to cancer tissue antigens and flow cytometry determines the binding activity.

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 conventional graded alcohol, embedded in paraffin, cut into 5-micron slices, and spread on a glass slide on the chip, then incubate with FD3-34-scFv primary antibody, rinse with PBS, incubate with ligase secondary antibody, add enzyme substrate and incubate for 15 minutes, rinse with PBS, HE stain, dehydrate, seal, observe and take photos under a microscope. The immunohistochemical staining results are shown in Figure 3, A. A is a case of FD3-34-scFv antibody binding to B7-H3 antigen in various human cancer tissues (immunohistochemical staining method).

Adherently culture the 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 OVCAR-8 expressing B7-H3 to the appropriate cell density. Aspirate the culture medium and use PBS Wash twice, add 0.2-0.5ml 0.25% trypsin-EDTA and place it in a cell culture incubator for digestion for 1-2 minutes. If the indigestible time is too long, use RPMI-1640 (10% FBS) to terminate the digestion. Count the cells, 1 per tube. ~5*10^5 cells. Centrifuge at 2000 rpm for 3 minutes. Aspirate the upper culture medium, add an appropriate amount of 1*PBS to resuspend the cells, and centrifuge again at 2000 rpm for 3 minutes. Repeat twice. Add different concentrations of the antibody to be tested and fluorescently labeled B7-H3 mixture; and set up isotype and positive controls. Add 100ng of B7-H3/hFc-FITC and different concentrations of the anti-B7-H3 antibody to be tested (100ng) into each tube, and mix Mix well and place one tube of isotype control at 4 degrees Celsius for 20-30 minutes. Wash 1 to 3 times with 1*PBS, add an appropriate amount of 1*PBS to adjust the cell concentration, and test on the machine (the results are shown in Figure 4, B).

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

The human lung cancer cell line SPC-A1, which highly expresses B7-H3, was cultured in RPMI 1640 medium containing 10% FBS. It was passaged once in about 2-3 days to the appropriate cell number, and healthy adult PBMCs were extracted by density gradient centrifugation. , wash, count and set aside after extraction. 4-5 week old female immunodeficient NCG mice (purchased from Nanjing University-Nanjing Institute of Biomedicine, raised with independent water and gas supply systems on the IVC rack) were used. The SPC-A1 cells were digested and mixed with PBMC in an appropriate proportion and then pressed The inoculation density of 3x10^7 cells/ml was inoculated on the mice, and each mouse was inoculated 1 under the right wing. Generally, tumors will form within 3-5 days after inoculation, and the tumors will reach ^100-250mm3 within 7-9 days. The body weight and tumor volume of the mice will be measured when entering the group, and mice with tumors smaller than 80mm3 or larger than 250mm3 or with other abnormal conditions will be eliminated. Mice that meet the requirements are then randomly assigned to different experimental groups.

After enrollment, the experimental antibody (FD3-34), positive control antibody (PD-1mAb) and negative control antibody (human IgG4, recorded as hIgG4 or Ctrl) were given according to the group. The antibody concentration was 1mg/ml, intraperitoneal injection, first The dose is increased. The initial dose of FD3-34 antibody is 250ug/animal, and then 200ug/animal/time, once every 3 days, until 6 times, that is, q3dx6 times; measure the tumor volume before each administration. Test 2-3 times a week, usually before administration.

After 6 times of antibody treatment, as shown in Figure 5A, the tumor volume growth in the FD3-34 group was more significantly inhibited compared with the PD-1mAb group and hIgG4 group (i.e., Ctrl). From 2 to 6 times of administration, the ability of FD3-34 to inhibit tumor proliferation was lower than that of PD-1 mAb. From the survival curve, as shown in Figure 5B, the survival rate of the FD3-34 group is similar to that of PD-1mAb. Among them, A is the weight of the tumor after NCG treatment in mice (unit is grams, the number of mice is 6. Compared with the control group, the FD3-34 antibody and the positive control group PD-1mAb have a significant reduction in tumor weight. , *P<0.05); B is the survival curve of NCG mice after treatment with FD3-34 and PD-1mAb (number of animals: 6 in each group).

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art will not deviate from the principles and purposes of the present invention. Under the circumstances, the above-described embodiments can be changed, modified, replaced and modified within the scope of the present invention.

Claims (5)

1. A single chain antibody against B7-H3 comprising:

a light chain variable region CDR1 comprising SEQ ID NO:1 or SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no;

a light chain variable region CDR2 comprising SEQ ID NO:2 or SEQ ID NO:12, an amino acid sequence shown in seq id no;

a light chain variable region CDR3 comprising SEQ ID NO:3 or SEQ ID NO:13, an amino acid sequence shown in seq id no;

a heavy chain variable region CDR1 comprising SEQ ID NO:4 or SEQ ID NO:14, an amino acid sequence shown in seq id no;

a heavy chain variable region CDR2 comprising SEQ ID NO:5 or SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no; and

a heavy chain variable region CDR3 comprising SEQ ID NO:6 or SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no;

wherein the single chain antibody specifically binds to B7-H3.

2. A polynucleotide encoding the single chain antibody of claim 1 against B7-H3.

3. A vector comprising the polynucleotide of claim 2.

4. A recombinant cell comprising the vector of claim 3.

5. A pharmaceutical composition comprising the anti-B7-H3 single chain antibody of claim 1 and a pharmaceutically acceptable carrier.