Tumor specific glycoprotein antibody D8 and application thereof

Abstract

The invention discloses a tumor specific glycoprotein antibody D8 and application thereof. The amino acid sequence of the CDR1 of the tumor specific glycoprotein antibody D8 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the CDR2 of the tumor specific glycoprotein antibody D8 comprises a sequence as shown in SEQ ID NO. 2; the amino acid sequence of the CDR3 of the tumor specific glycoprotein antibody D8 comprises a sequence as shown in SEQ ID NO. 3. The tumor specific glycoprotein antibody D8 has affinity to GPC3 and is smaller in molecular weight, and antibody drugs developed on the basis of the tumor specific glycoprotein antibody D8 are better in effectiveness, higher in tissue penetrating power, better in in-vivo stability and better in drug effect.

Description

Technical Field

[0001] The present invention belongs to the field of antibody biotechnology and relates to a tumor-specific glycoprotein antibody D8 and an application thereof. Background Art

[0002] Glypican-3 (GPC3) protein is a liver cancer-specific antigen protein located on the cell membrane surface. It is usually highly expressed on the surface of embryonic and hepatocellular carcinoma (HCC) patients' tumor cells, and lowly expressed in normal adult tissues, and even in liver pathological tissues such as fatty liver, cirrhosis, hepatitis, or liver damage. GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, contains a 580-amino acid core protein and two HS chains at the C-terminus. The GPC3 core protein is anchored to the cell membrane surface by glycosylphosphatidylinositol (GPI) and can be cleaved into an approximately 40KDa N-terminal soluble protein (sGPC3) and a 30KDa C-terminal membrane protein. The cleaved sGPC3 can be released from cancer cells into the microenvironment and enter the body fluid / blood circulation. Studies have shown that GPC3 acts through the Wnt signaling pathway, activating the Wnt signaling pathway to highly express the Wnt gene, thereby promoting tumor growth.

[0003] GPC3 is expressed in tumor cells of liver cancer, lung cancer, ovarian cancer, and melanoma, and has been considered to be a therapeutic target with great application potential for hepatocellular carcinoma. Therapeutic monoclonal antibodies, antibody-durg conjugates (ADC), bispecific antibodies (BsAb), and chimeric antigen receptor T (CAR-T) cells targeting GPC3 have been developed, and some drugs have been put into clinical research. However, since the GPC3 protein can be cleaved into an approximately 40KDa N-terminal soluble protein (sGPC3) and a 30KDa C-terminal membrane protein, the cleaved sGPC3 can be released from cancer cells into the microenvironment. Therefore, in the antibody screening process, a specific antibody that binds to the 30KDa C-terminal membrane protein is required. This also makes factors such as steric hindrance affect the efficiency of existing ScFv antibodies binding to GPC3. The above-mentioned existing technologies are all developed based on antibodies targeting GPC3. Therefore, there is still room for improvement in the therapeutic effect of related drugs. In addition, the GPC3 antibodies in the prior art are all mouse / humanized antibodies. When applied to antibody drugs, their larger molecular weight is not conducive to improving the drug's penetration rate into solid tumor lesions; when applied to cell drugs, they are usually expressed in the form of single-chain antibodies (scFv), and their longer coding sequences are not conducive to the construction and modification of expression vectors, and may also cause non-specific activation of cell drugs due to cross-pairing of VH and VL of adjacent scFv molecules.

[0004] Therefore, there is an urgent need to provide a GPC3 antibody that has both high affinity and high specificity and a small molecular weight. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a tumor-specific glycoprotein antibody D8 and its application, aiming to solve the problem that the existing GPC3 antibodies cannot have both affinity and specificity, and to obtain a GPC3 antibody with a smaller molecular weight than the existing GPC3 antibody, so as to improve the drugability of related antibodies and cell drugs.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a tumor-specific glycoprotein antibody D8, wherein the amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.2; and the amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.3.

[0008] SEQ ID NO. 1: GSIFSINV.

[0009] SEQ ID NO. 2: STDGGST.

[0010] SEQ ID NO.3:NAHIWSTPRKYDNY.

[0011] The tumor-specific glycoprotein antibody D8 of the present invention has affinity for GPC3 and a smaller molecular weight. Antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy; engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower transformation difficulty; detection reagents developed based on it have higher sensitivity and lower false positives.

[0012] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, namely heavy chain antibodies. Compared with ordinary antibodies, it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single domain antibody (also known as nanobody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity.

[0013] This project used CHO-S cells overexpressing GPC3 to immunize alpacas. After determining the titer of GPC3-specific antibodies in alpaca serum by ELISA and flow cytometry, alpaca peripheral blood mononuclear cells (PBMCs) were isolated, RNA was extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers were used to amplify the VHH sequence and cloned into a phage expression plasmid to construct a phage display library. CHO-S cells were used to incubate the phage display library to eliminate antibody clones that only bound to CHO-S cells. Then, virus-like particles (VLPs) containing GPC3 were used to enrich and pan the phage display library for 3-4 rounds. Monoclones were picked from the enriched products, and the specific binding of candidate antibodies to GPC3-VLPs was detected by ELISA. After the positive antibodies were expressed and purified, their affinity to CHO-S cells expressing GPC3 was tested respectively. We selected antibody clones with strong affinity for GPC3, cloned the GPC3 single-domain antibody sequence into the CAR expression vector, constructed CAR-T cells with GPC3 single-domain antibody as the antigen binding domain, and tested its ability to kill GPC3-positive Huh7 cells in vivo and in vitro. The results showed that compared with the existing technology, the GPC3 single-domain antibody obtained in this project has a killing effect on the cell line expressing GPC3 and is specific.

[0014] Preferably, the amino acid sequence of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] AVQLVESGGGLVQAGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAVSTDGGSTS YEDSVKGRFTLSRDNTKNTVYLQMNSLKPEDTAVYYCNAHIWSTPRKYDNYWGQGTQVT VSS.

[0017] In a second aspect, the present invention provides a single domain antibody, which comprises the variable region sequence of the tumor-specific glycoprotein antibody D8 described in the first aspect and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin.

[0018] Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc fragment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0019] Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.5.

[0020] Preferably, the amino acid sequence of the single-domain antibody is the sequence shown in SEQ ID NO.6.

[0021] SEQ ID NO.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] SEQ ID NO.6:

[0024] AVQLVESGGGLVQAGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAVSTDGGSTSYEDSVKGRFTLSRDNTKNTVYLQMNSLKPEDTAVYYCNAHIWSTPRKYDNYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] In a third aspect, the present invention provides a composition for detecting tumor-specific glycoproteins, wherein the composition comprises the tumor-specific glycoprotein antibody D8 described in the first aspect or the single domain antibody described in the second aspect.

[0026] In a fourth aspect, the present invention provides a chimeric antigen receptor, comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; the antigen binding domain comprises the tumor-specific glycoprotein antibody D8 described in the first aspect.

[0027] Preferably, the signal peptide comprises any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ.

[0028] Preferably, the hinge region comprises any one of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ, or a combination of at least two thereof.

[0029] Preferably, the transmembrane region includes any one of CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314, or a combination of at least two of them.

[0030] Preferably, the signal transduction domain comprises a co-stimulatory domain and a human CD3 intracellular signal transduction domain.

[0031] Preferably, the co-stimulatory domain comprises any one of CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR or OX40L, or a combination of at least two of them.

[0032] Preferably, the human CD3 intracellular signal transduction domain includes any one of CD3ζ, CD3ε, CD3γ or CD3δ, or a combination of at least two of them.

[0033] Preferably, the signal peptide comprises a human CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO.7.

[0034] Preferably, the hinge region comprises the human CD8 hinge region as shown in SEQ ID NO.8 or the human IgG4 hinge region as shown in SEQ ID NO.9.

[0035] Preferably, the transmembrane region comprises the human CD8 transmembrane region as shown in SEQ ID NO.10.

[0036] Preferably, the signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.11 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.12.

[0037] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0038] SEQ ID NO.8:

[0039] TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA.

[0040] SEQ ID NO.9:

[0041] K.

[0042] SEQ ID NO. 10: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0043] SEQ ID NO.11:

[0044] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0045] SEQ ID NO.12:

[0046] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0047] Preferably, the chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the first aspect, the human CD8 hinge region shown in SEQ ID NO.8, or the human IgG4 hinge region shown in SEQ ID NO.9, the human CD8 transmembrane region shown in SEQ ID NO.10, the human 4-1BB intracellular region shown in SEQ ID NO.11 and the human CD3ζ intracellular region shown in SEQ ID NO.12.

[0048] Preferably, the amino acid sequence of the chimeric antigen receptor comprises a sequence as shown in SEQ ID NO.13 (using the CD8 hinge region to link the single domain antibody and the CD8 transmembrane region) or SEQ ID NO.14 (using the IgG hinge region to link the single domain antibody and the CD8 transmembrane region).

[0049] SEQ ID NO.13:

[0050] MALPVTALLLPLALLLHAARPSAVQLVESGGGLVQAGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAVSTDGGSTSYEDSVKGRFTLSRDNTKNTVYLQMNSLKPEDTAVYYCNAHIWSTPRKYDNYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0051] SEQ ID NO.14:

[0052] MALPVTALLLPLALLLHAARPSAVQLVESGGGLVQAGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAVSTDGGSTSYEDSVKGRFTLSRDNTKNTVYLQMNSLKPED TAVYYCNAHIWSTPRKYDNYWGQGTQVTVSSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0053] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the tumor-specific glycoprotein antibody D8 described in the first aspect, the single domain antibody described in the second aspect, or the chimeric antigen receptor described in the fourth aspect.

[0054] In a sixth aspect, the present invention provides an expression vector, comprising a gene encoding the chimeric antigen receptor according to the fourth aspect.

[0055] Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor described in the fourth aspect, preferably a lentiviral vector.

[0056] In a seventh aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the sixth aspect.

[0057] In an eighth aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor described in the fourth aspect.

[0058] Preferably, the chimeric antigen receptor immune cell comprises the expression vector described in the sixth aspect.

[0059] Preferably, the chimeric antigen receptor immune cell comprises any one or a combination of at least two of T cells, TIL cells, B cells, NK cells, mast cells or macrophages.

[0060] In the ninth aspect, the present invention provides the use of the tumor-specific glycoprotein antibody D8 described in the first aspect, the chimeric antigen receptor described in the fourth aspect, the expression vector described in the sixth aspect, or the chimeric antigen receptor immune cell described in the eighth aspect in the preparation of a product that can prevent and / or treat cancer or tumors.

[0061] Preferably, the tumor comprises a GPC3-positive solid tumor; and the cancer comprises any one of liver cancer, melanoma, ovarian cancer or lung cancer.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) The tumor-specific glycoprotein antibody D8 of the present invention has affinity for GPC3 and has a smaller molecular weight;

[0064] (2) Antibody drugs developed based on the tumor-specific glycoprotein antibody D8 of the present invention have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy;

[0065] (3) The engineered immune cells developed based on the tumor-specific glycoprotein antibody D8 of the present invention have higher exogenous gene expression levels, better effectiveness, better drug efficacy, and lower modification difficulty;

[0066] (4) The detection reagent developed based on the tumor-specific glycoprotein antibody D8 of the present invention has higher sensitivity and lower false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1A This is the first round PCR amplification diagram of the VHH region of the alpaca antibody;

[0068] Figure 1B This is the second round PCR amplification diagram of the VHH region of the alpaca antibody;

[0069] Figure 2 The specificity diagram of recombinant single domain antibodies was detected by flow cytometry;

[0070] Figure 3 The concentration-effect curve of the antibody tested in CHO-S-GPC3 cells;

[0071] Figure 4A Schematic diagram of the molecular structure of CAR;

[0072] Figure 4B This is a graph showing the expression results of BVHGC3-004 and BVHGC3-005 in T cells detected by flow cytometry;

[0073] Figure 5 Figure 3 IncuCyte real-time quantitative live cell imaging analysis of the killing function and specificity of CAR-T cells targeting GPC3. DETAILED DESCRIPTION

[0074] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0075] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0076] Reagents used in the following examples:

[0077] Huh7 cells: Cell Bank of Chinese Academy of Sciences, SCSP-526;

[0078] SK-Hep-1 cells: ATCC, HTB-52;

[0079] CHO-S cells: Gibco, R80007;

[0080] PE-labeled anti-human IgG antibody: Biolegend, 410708;

[0081] FITC-labeled Rabbit anti-Llama IgG (H+L) antibody: Invitrogen, A16155;

[0082] Anti-M13-HRP antibody: SinoBiological, 11973-MM05T-H;

[0083] AF647-labeled Anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170;

[0084] AF647-labeled anti-human GPC3 antibody: R&D system, FAB2119A.

[0085] Anti-VHH antibody: GenScript, A02017;

[0086] Example 1

[0087] Alpaca immunization and antibody titer determination.

[0088] (1) Construction of GPC3 overexpressing cells

[0089] Based on the amino acid sequence information of human GPC3 (UniProtAccession: P51654), GPC3 was overexpressed in CHO-S cells to obtain CHO-S-GPC3 cells, which were used for alpaca immunization, serum titer detection and antibody affinity verification.

[0090] (2) Alpaca immunization and serum titer testing

[0091] Three alpacas (73#, 74# and 194#) were immunized using the CHO-S-GPC3 cells constructed as described above. 5 mL of peripheral blood was collected two weeks before immunization, the second, third and fourth immunizations, and the upper serum was collected from the blood samples. The GPC3-His recombinant protein was diluted with PBS to a final concentration of 1 μg / mL, and 100 μL / well was added to a 96-well ELISA plate and coated overnight at 4°C. The antigen coating solution was shaken off and washed 5 times with PBST (containing 0.05% Tween 20). The blocking solution was added at 200 μL / well and blocked at 37°C for 2h; after shaking off the blocking buffer, the plate was washed 5 times with PBST. The serum collected above was gradiently diluted with PBS, and 100 μL of the gradient diluted serum was added to the 96-well ELISA plate, incubated at room temperature for 1h, and the control well was PBS. The liquid in the well was shaken off and washed 5 times with PBST. Add 100 μL HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour. After shaking off the liquid in the wells, wash the plate 5 times with PBST. Add TMB colorimetric solution at 100 μL / well; incubate at room temperature in the dark for 10 minutes. Add stop solution at 50 μL / well; read the OD in the wells using an ELISA reader. 450 The results are shown in Tables 1, 2 and 3. The binding titers of the serum of the three alpacas before immunization with the GPC3 protein were all lower than 1:2000 (the antibody titer is expressed in the form of 1:X, where X is the maximum dilution multiple at which the antibody can be detected), indicating that the anti-GPC3 antibody content was extremely low. On the contrary, the anti-GPC3 antibody titers in the serum of the 73# and 74# alpacas after the fourth immunization were both higher than 1:32K, and the anti-GPC3 antibody titer in the serum of the 194# alpacas after the fourth immunization was higher than 1:8K, indicating that the content of anti-GPC3 specific antibodies in the serum of the three alpacas was significantly increased after multiple rounds of immunization.

[0092] Table 1

[0093]

[0094] Table 2

[0095]

[0096] Table 3

[0097]

[0098] Example 2

[0099] Construction of single domain antibody phage display library

[0100] (1) VHH antibody fragment cloning

[0101] 100 mL of alpaca peripheral blood was collected, PBMC was separated, and RNA was extracted. TM II 1st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers were used to PCR amplify the alpaca heavy chain antibody sequence. Agarose gel electrophoresis was used to analyze the PCR product, and the target fragment of about 750 bp was recovered ( Figure 1A ). Then, using the first round PCR product as a template, specific primers (the upstream primer binds to the antibody FR1 region, and the 5' end contains an SfiI restriction site; the downstream primer binds to the antibody Hinge and FR4 regions, and the 5' end contains an SfiI restriction site) are used to amplify the heavy chain antibody VHH fragment. The PCR product is analyzed by agarose electrophoresis, and the target fragment of about 400 bp is recovered ( Figure 1B ). The target fragment was stored at -80℃.

[0102] (2) Electroporation of library vectors and detection of library capacity and diversity

[0103] The VHH fragment library obtained above was cloned into the SfiI site of the phage surface display vector pComf (the linearized pComf vector and the VHH fragment library were connected overnight at 16°C using T4ligase), and the connection product was stored at -80°C. The connection product was electrotransformed into E. coli SS320 competent cells, SOC medium was added to resuspend the cells, and the shaking table was used for recovery culture. In addition, the bacterial solution was diluted and spread on an LB plate containing ampicillin and cultured overnight. The number of clones that can be produced by each connection reaction was calculated to obtain the capacity of the single domain antibody phage surface display library.

[0104] The results showed that the library capacity of the single domain antibody phage display library obtained from 73#, 74# and 194# alpaca PBMC was 1.60×10 9 , 1.32×10 9 and 1.15×10 9 At the same time, Sanger sequencing results showed that the phage library sequences were highly different, with no repeated sequences and good diversity.

[0105] (3) Enrichment of phage display libraries

[0106] Take the bacterial solution after electrotransformation and recovery and adjust it to OD 600 When OD is 0.25, ampicillin is placed in a constant temperature shaker for cultivation. 600When the pH was 0.6, M13KO7 helper phage was added, shaken and allowed to stand at 37°C for 30 min, and cultured on a shaker at 37°C for 1 h. Afterwards, the supernatant was discarded by centrifugation, and the suspension was resuspended in 2YT-AK medium and cultured on a shaker overnight. The bacterial solution was then centrifuged, the phage precipitate was collected and resuspended 3 times to obtain the original library of single-domain antibody phage surface display.

[0107] Take 10 μL of the precipitate and perform 10-fold gradient dilutions. Add 200 μL of OD 600 ER2738 Escherichia coli was added at 0.5, mixed well, placed in a 37°C water bath, and allowed to stand for 10 minutes before being spread on an LB plate. After overnight culture at 37°C, the plaques were counted to obtain the titer of the phage surface display library.

[0108] Example 3

[0109] Target antibody library screening.

[0110] (1) First round of panning and product amplification

[0111] Add 50 μg / mL of GPC3 protein to a 96-well solid phase plate and coat overnight. 7 CHO-S-GPC3 cells were washed 3 times with PBS. Resuspended in 3% PBSA, and blocked by rotation and mixing at 37°C for 1h. At the same time, the precipitated product of the original library displayed on the surface of single-domain antibody phage was taken, PBSA was added, and the mixture was blocked by rotation and mixing at 4°C for 1h as a premix. The blocked CHO-S cells were centrifuged to remove the supernatant, and the phage premix was added and incubated for 1h to remove the phage clones that non-specifically bound to CHO-S. The GPC3 protein in the 96-well plate was discarded, and MPBS buffer was added and allowed to stand at room temperature. After blocking, the CHO-S-GPC3 cells were centrifuged to remove the supernatant, and added to the GPC3 protein wells for incubation for 1h. The phage supernatant in the GPC3 protein wells was discarded, washed, eluted, incubated, and the specifically bound phages were repeatedly eluted. The eluted products were stored in pre-blocked centrifuge tubes at 4°C.

[0112] Escherichia coli ER2738 was cultured in 2YT medium (containing tetracycline) until OD 600 Add the eluted phage product to the ER2738 bacterial solution, mix well, and incubate until the bacterial solution OD is 600 When the pH is 0.5, add M13KO7 helper phage, shake well and let stand. Add ampicillin to the bacterial solution, culture, centrifuge, discard the supernatant and collect the bacteria. Resuspend with 2YT-AK medium and culture overnight. Centrifuge the phage suspension that has been infected and amplified overnight and divide the supernatant. After standing on ice to precipitate the phage, centrifuge, discard the supernatant and resuspend the phage. This is the phage product obtained in the first round of panning amplification.

[0113] (2) Second to fourth rounds of panning and product amplification

[0114] The GPC3 antigen was diluted to 10 μg / mL using CBS solution, added to a 96-well solid phase plate, and coated overnight at 4° C. The phage products amplified in the first round of panning were used for the second to fourth rounds of panning in the same manner as above.

[0115] (3) ELISA detection and sequencing of monoclonal phage

[0116] Infect ER2738 E. coli with the selected single-domain antibody phage display library, mix well and place in a 37℃ water bath for 10 minutes. Spread on LB plate and incubate at 37℃ overnight. Add 2YT-A medium to a 96-well deep-well plate and pick a single clone on the plate and culture overnight. Culture until OD 600 The concentration of M13KO7 helper phage was 0.5, and the cells were cultured statically. After centrifugation and discarding the supernatant, each well was resuspended in 2YT-AK medium and cultured overnight. After centrifugation, the supernatant obtained was the monoclonal phage particles.

[0117] While amplifying the phage monoclonal, coat the GPC3 antigen protein onto the ELISA plate with CBS at pH 9.6 and coat overnight at 4°C. Then discard the antigen, wash, and block overnight. After discarding the blocking solution, wash each well and add the above monoclonal phage supernatant. After incubation for 1 hour, wash with PBST. Add 100 μL anti M13-HRP antibody to each well, incubate at 4°C for 45 minutes, color develop at room temperature, and then stop. Read OD on the ELISA plate 450 The sample / negative control value was calculated, and clones with a ratio significantly greater than that of the positive serum control group were selected for sequencing to obtain antibody 73-G3-1-D8.

[0118] Example 4

[0119] Expression of recombinant single domain antibodies and detection of binding to target proteins.

[0120] The 73-G3-1-D8 sequence was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, and its C-terminus was fused with the human IgG1 Fc fragment for expression. The obtained expression plasmid was transiently transfected into HEK293 cells, and the harvested cell culture supernatant contained the 73-G3-1-D8 antibody. In the same manner, the GPC3-specific antibody hYP7 was expressed as a positive control (YP7 light chain variable region amino acid sequence (SEQ ID NO.15): DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQK NYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQY YNYPLTFGAGTKLELK. YP7 heavy chain variable region amino acid sequence (SEQ ID NO.16): EVQLVETGGGMVQPEGSLKLSCAAS GFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQ MNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA).

[0121] The binding specificity of 73-G3-1-D8 antibody was tested. The culture supernatant containing 73-G3-1-D8 antibody was mixed with 3×10 5 Huh7 (GPC3 positive), SK-Hep-1 overexpressing GPC3, 293T (GPC3 negative) cells were incubated at room temperature for 1 hour. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. 100μL PE-labeled anti-human IgG antibody was added and incubated at room temperature in the dark for 45 minutes. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500μL PBS for flow cytometry analysis.

[0122] The results are as follows Figure 2 As shown, the expression supernatant of the negative control group had no significant binding to Huh7, SK-Hep-1 overexpressing GPC3, and 293T; however, the 73-G3-1-D8 recombinant single domain antibody had a high binding efficiency to Huh7 and SK-Hep-1 cells overexpressing GPC3, but almost no binding to 293T, indicating that the 73-G3-1-D8 recombinant single domain antibody can specifically bind to GPC3 on the surface of tumor cells.

[0123] Example 5

[0124] Purification and half effective concentration (EC 50 ) determination.

[0125] To determine the EC of 110-1-E02 antibody 50 , transiently transfect the relevant expression plasmid into 293F cells, and culture them in a shaking flask. Since the target recombinant antibody contains human IgG fragments, ProteinA magnetic beads can be used for affinity purification. Wash the ProteinA magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of ProteinA magnetic beads (20mg IgG / mL ProteinA magnetic beads) to the 293F cell shaking flask. Incubate at room temperature for 4h at 120rpm in a shaking incubator. Collect the ProteinA magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and H2O, resuspend them with 1mL elution buffer. After incubation at room temperature for 5min, collect the magnetic beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a centrifuge tube. After eluting the ProteinA magnetic beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. The eluted sample is dialyzed with at least 100 times the volume of the sample with PBS. First, dialyze at 18-25℃ for 2h. After changing the solution once, dialyze at 2-8℃ for 14-16h. Finally, determine the protein concentration and filter the sample with a 0.22μm sterile filter membrane and divide it into aliquots and store it in a -80℃ refrigerator for later use.

[0126] Serial dilutions of the target antibody were added to 3×10 5 Incubate CHO-S-GPC3 and CHO-S at room temperature for 1 hour. Centrifuge at 800×g for 5 minutes at room temperature. Discard the supernatant containing antibodies and wash the cells 3 times with PBS. Add 100μL PE-labeled Anti-human IgG antibody, mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant containing antibodies, wash the cells 3 times with PBS, resuspend the cells with 500μL PBS, and perform flow cytometry analysis.

[0127] The results are as follows Figure 3 As shown in Table 4, Table 4 is the EC of recombinant single domain antibodies detected by flow cytometry 50 Results. EC of positive control antibody HYP7 against CHO-S-GPC3 50 The value is 0.04346μg / mL, and the EC value of 73-G3-1-D8 for CHO-S-GPC3 50 The value was 0.0946 μg / mL, which was similar to the EC 50 This result indicates that the affinity of 73-G3-1-D8 to GPC3 is comparable to that of HYP7.

[0128] Table 4

[0129]

[0130] Example 6

[0131] Construction of CAR-T cells based on target antibodies.

[0132] (1) Design of CAR molecules

[0133] The target gene structure of the lentiviral vector involved in this example is as follows Figure 4A shown.

[0134] BVHGC3-004 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human GPC3 single-domain antibody 73-1-D8 [VHH (73-1-D8) for short], human CD8 hinge region (CD8 hinge for short), human CD8 transmembrane domain (CD8TM for short), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD for short), and human CD3ζ intracellular signal transduction domain (CD3ζICD for short).

[0135] BVHGC3-005 is composed of the following structures in series: human CD8 signal peptide, anti-human GPC3 single-domain antibody 73-1-D8, human IgG4 hinge region, human CD8 transmembrane domain, human 4-1BB intracellular co-stimulatory domain, and human CD3ζ intracellular signal transduction domain.

[0136] (2) Lentivirus preparation

[0137] The above CAR molecule expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it was expressed under the regulation of the human EF-1α promoter and Kozak sequence. The above lentiviral vector expression plasmids were co-transfected with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000. The viral supernatant was collected 48 hours after transfection. Centrifuged at 3000rpm for 15min at 4°C, then filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm for 3h at 4°C. After resuspending with physiological saline, the obtained virus concentrate was stored at -80°C and named BVHGC3-004 and BVHGC3-005 respectively. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0138] (3) CAR-T cell preparation

[0139] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were added and placed in a cell culture incubator for 24 h. Lentivirus was added at an MOI of 5 TU / cell to transduce T cells, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were supplemented and placed in a cell culture incubator for culture. After 24 h, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300IU / mL hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for another 5 days, during which the cell density was maintained at 1×10 6 / mL. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0140] (4) Detection of CAR molecule expression

[0141] The BVHGC3-004 and BVHGC3-005 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer. FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 1 hour. The supernatant was then removed by centrifugation, washed twice with FACS buffer, and resuspended. Using Ctrl T cells as negative control, the CAR molecule expression rate of BVHGC3-004 and BVHGC3-005 cells was detected by flow cytometry.

[0142] The results are as follows Figure 4B The expression rates of BVHGC3-004 and BVHGC3-005 CARs were shown to be 20.2% and 25.3%, respectively.

[0143] Example 7

[0144] Study on the functions of CAR-T cells in vivo and in vitro.

[0145] In vitro killing experiment of BVHGC3-004 and BVHGC3-005 cells

[0146] Use culture medium at 1×10 5Huh7 cells with mCherry reporter gene were resuspended at a density of 100 μL / mL and inoculated into 96-well plates at a volume of 100 μL per well. After being cultured overnight in the IncuCyte live cell imaging analysis system, CAR-T cells were added for co-culture at an effect-target ratio of 4.5:1, and the killing effect of CAR-T cells on tumor cells was recorded in real time. After the co-culture, the changes in the mCherry fluorescence signal of the target cells in each group were calculated using the IncuCyte software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Figure 2. Figure 5 As shown, for GPC3-positive Huh7 cells, the killing effect of BVHGC3-004 and BVHGC3-005 groups was significantly improved compared with the Mock group (P<0.05). In addition, compared with BVHGC3-004 (the relative fluorescence area at the experimental endpoint was 1.48±0.03), BVHGC3-005 (the relative fluorescence area at the experimental endpoint was 1.35±0.02) had a stronger killing effect on target cells (P<0.05), indicating that the construction of CAR molecules using different hinge regions will affect the killing effect of the final CAR-T cells on target cells. The above results show that compared with existing antibodies, CAR-T cells constructed based on 73-G3-1-D8 antibodies have significant specific killing function on GPC3-positive tumor cells.

[0147] In summary, the tumor-specific glycoprotein antibody D8 of the present invention has affinity for GPC3 and a smaller molecular weight. Antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy; engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower transformation difficulty; detection reagents developed based on it have higher sensitivity and lower false positives.

[0148] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A tumor-specific glycoprotein antibody D8, characterized in that: The amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.1; The amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.2; The amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.

3.

2. The tumor-specific glycoprotein antibody D8 according to claim 1, characterized in that: The amino acid sequence of the tumor-specific glycoprotein antibody D8 includes the sequence shown in SEQ ID NO.

4.

3. A single domain antibody, characterized in that The single-domain antibody comprises the variable region sequence of the tumor-specific glycoprotein antibody D8 according to claim 1 or 2 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin; The Ig Fc comprises the full length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof; The Fc amino acid sequence of the IgG1 includes the sequence shown in SEQ ID NO.5; The amino acid sequence of the single domain antibody is the sequence shown in SEQ ID NO.

6.

4. A composition for detecting tumor-specific glycoprotein, characterized in that: The composition comprises the tumor-specific glycoprotein antibody D8 according to claim 1 or 2 or the single domain antibody according to claim 3.

5. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; The antigen binding domain comprises the tumor-specific glycoprotein antibody D8 according to claim 1 or 2; The signal peptide includes any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ; The hinge region includes any one or a combination of at least two of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ; The transmembrane region includes any one or a combination of at least two of CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314; The signal transduction domain includes a co-stimulatory domain and a human CD3 intracellular signal transduction domain; The co-stimulatory domain includes any one or a combination of at least two of CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR or OX40L; The human CD3 intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ; The signal peptide includes a human CD8 signal peptide whose amino acid sequence is shown in SEQ ID NO.7; The hinge region includes the human CD8 hinge region shown in SEQ ID NO.8 or the human IgG4 hinge region shown in SEQ ID NO.9; The transmembrane region includes the human CD8 transmembrane region shown in SEQ ID NO.10; The signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.11 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.12; The chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the tumor-specific glycoprotein antibody D8 according to claim 1 or 2, the human CD8 hinge region shown in SEQ ID NO.8 or the human IgG4 hinge region shown in SEQ ID NO.9, the human CD8 transmembrane region shown in SEQ ID NO.10, the human 4-1BB intracellular region shown in SEQ ID NO.11, and the human CD3ζ intracellular region shown in SEQ ID NO.12; The amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.13 or SEQ ID NO.

14.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the tumor-specific glycoprotein antibody D8 according to claim 1 or 2, the single domain antibody according to claim 3, or the chimeric antigen receptor according to claim 5.

7. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 6; The expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to claim 5.

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 5; The chimeric antigen receptor immune cell comprises the expression vector of claim 7; The chimeric antigen receptor immune cells include any one or a combination of at least two of T cells, TIL cells, B cells, NK cells, mast cells or macrophages.

9. Use of the tumor-specific glycoprotein antibody D8 according to claim 1 or 2, the chimeric antigen receptor according to claim 5, the expression vector according to claim 7 or the chimeric antigen receptor immune cell according to claim 8 in the preparation of a product for preventing and / or treating cancer or tumors.

10. The use according to claim 9, characterized in that: The tumor includes a GPC3-positive solid tumor; the cancer includes any one of liver cancer, melanoma, ovarian cancer or lung cancer.

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