Anti-CD3 nano antibody capable of activating T cell activity as well as preparation method and application of anti-CD3 nano antibody

Abstract

The invention belongs to the field of molecular biology and immunology, and particularly relates to an anti-CD3 nano antibody capable of activating the activity of T cells as well as a preparation method and application of the anti-CD3 nano antibody. The invention provides a preparation method of the anti-CD3 nano antibody. The preparation method comprises the following steps: constructing a CD3 plasmid and an overexpression cell line, and immunizing alpaca to obtain peripheral blood mononuclear cells (PBMC) of the alpaca; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription to obtain cDNA; carrying out PCR amplification on the antibody gene segment and cloning the antibody gene segment to a phage display vector; and electrically transforming the vector into SS320 competent cells to obtain the nano antibody library. After two rounds of panning, detecting and screening an antibody clone capable of being combined with the CD3 protein through ELISA (Enzyme-Linked Immunosorbent Assay); an antibody sequence is selected through sequencing, and an antibody protein is expressed by using a mammalian cell high-throughput expression system; and then detecting and screening the antibody which is only specifically combined with the CD3. The method disclosed by the invention is simple, convenient and rapid to operate, and can be used for preparing active antibodies for treating potential tumors and proliferative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and immunology, and specifically relates to an anti-CD3 nano antibody capable of activating T cell activity, and a preparation method and application thereof. Background Art

[0002] Vertebrates have developed a highly complex defense mechanism to defend against foreign invaders (such as exogenous macromolecules or pathogenic microorganisms), known as the acquired immune system. In mammals, the core of this system is two types of lymphocytes: T cells from the thymus and B cells from the bone marrow. T cells play a key role in immune responses, and their functions involve specific antigen recognition and cell-mediated immune responses.

[0003] The activation process of T cells is very complex and depends on the synergistic action of multiple cell surface molecules. Unlike antibodies that recognize foreign proteins or small molecule antigens, T cells specifically recognize small peptides presented by antigens through T cell receptors (TCRs). These peptides must bind to major histocompatibility complex (MHC) molecules before they can be recognized by TCRs and trigger immune responses.

[0004] The TCR complex usually exists in the form of α chain and β chain, or γ chain and δ chain, connected by disulfide bonds to form heterodimers, and non-covalently bound to a group of low molecular weight proteins, which constitute the CD3 complex. CD3 is an auxiliary molecule responsible for transmitting the signal after TCR recognizes the antigen from the outside of the cell to the inside of the cell, thereby initiating the activation of T cells.

[0005] In mammals, the CD3 complex is composed of four different subunits: a γ chain, a δ chain, two ε chains, and two ζ chains. These chains together with the TCR form the TCR-CD3 complex, which lays the structural foundation for the activation of T cells. The intracellular tail of CD3 plays an important role in the activation of T cells. This tail region contains the immunoreceptor tyrosine-based activation motif (ITAM), whose phosphorylation is a key step in signal transduction. The activation of ITAM enables CD3 to recruit and bind to the ZAP70 kinase, thereby amplifying the activation signal cascade and triggering a subsequent immune response.

[0006] CD3 molecules play an irreplaceable role in T cell activation and have therefore become an important target in immunotherapy and drug development. For example, in cancer immunotherapy, the use of anti-CD3 antibodies to regulate T cell function and enhance their ability to kill tumors is a potential therapeutic strategy. However, traditional antibodies have certain limitations in therapeutic applications due to their large molecular weight, structural complexity and immunogenicity.

[0007] Nanobodies, as a new type of antibody molecule, have smaller molecular weight, better stability and lower immunogenicity. Nanobodies are derived from single-domain antibody fragments of camelids and have high specificity and high affinity similar to conventional antibodies. Due to their small molecular weight, nanobodies are better able to penetrate tissues and contact target areas, so they show unique advantages in immunotherapy. The development of nanobodies targeting CD3 molecules can not only enhance the activation effect of T cells, but also reduce the adverse immune responses caused by conventional antibodies, providing an effective tool for precise immune regulation. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an anti-CD3 nanobody capable of activating T cell activity and a preparation method and application thereof.

[0009] The present invention aims to develop an anti-CD3 nanobody, which has a simple and efficient preparation method and has a wide range of application potential in immunotherapy and disease prevention and control. This nanobody can be used as a single drug or in combination with other therapeutic molecules to enhance the immune response of T cells and is widely used in the treatment of autoimmune diseases, infectious diseases and cancer.

[0010] The technical solution of the present invention to solve the technical problem is as follows:

[0011] In the first aspect of the present invention, an anti-CD3 nanobody capable of activating T cell activity is provided, wherein the VHH chain of the nanobody comprises a framework region and a complementary determining region; the complementary determining region comprises CDR1, CDR2 and CDR3, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 are selected from group 1) or group 2):

[0012] 1) The complementary determining region CDR1 sequence is shown as SEQ ID NO.1, the complementary determining region CDR2 sequence is shown as SEQ ID NO.2, and the complementary determining region CDR3 sequence is shown as SEQ ID NO.3;

[0013] 2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 are at least 90% identical to the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 described in group 1).

[0014] Preferably, the amino acid sequence of the VHH chain of the Nanobody is as shown in SEQ ID NO.4.

[0015] In the second aspect of the present invention, a nucleic acid encoding the anti-CD3 Nanobody as described in the first aspect is provided.

[0016] Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.5.

[0017] In the third aspect of the present invention, a vector containing the nucleic acid described in the second aspect is provided. Further, the vector is pComb3XSS.

[0018] In the fourth aspect of the present invention, a host cell containing the vector described in the third aspect is provided. Further, the host cell is TG1 or SS320, and SS320 host cell is preferably selected.

[0019] In the fifth aspect of the present invention, a method for preparing an anti-CD3 nanobody as described in the first aspect is provided, comprising the following steps: constructing a CD3 plasmid and an overexpression cell line and immunizing an alpaca to obtain its peripheral blood mononuclear cells (PBMC); extracting its RNA for reverse transcription to obtain cDNA; PCR amplifying the antibody gene fragment and cloning it into a phage display vector; electrotransforming the vector into SS320 competent cells to obtain a nanobody library. After panning, ELISA is used to detect and screen antibody clones that can bind to CD3 protein; antibody sequences are selected by sequencing, and antibody proteins are expressed using a mammalian cell high-throughput expression system; and then a series of methods are used to detect, screen and select antibody molecules that specifically bind to and activate CD3.

[0020] In the present invention, the primers used in the step of alpaca immunization, antibody gene fragment amplification, panning method and construction of CD3 high-expressing cell line are all obtained after optimization in conventional operations.

[0021] In the sixth aspect of the present invention, a detection reagent or kit is provided containing the Nanobody as described in the first aspect or the biomaterial as described in the second aspect, the third aspect or the fourth aspect.

[0022] In the seventh aspect of the present invention, provided is the use of the anti-CD3 nanobody as described in the first aspect in the preparation of a medicament for treating diabetes and obesity, and in the preparation of an agent or medicament for diagnosing, preventing or treating a CD3-related disease or a hypoglycemia-related disease.

[0023] In the eighth aspect of the present invention, there is provided the use of the anti-CD3 nanobody as described in the first aspect in the preparation of a product for detecting CD3.

[0024] In the ninth aspect of the present invention, there is provided the use of the anti-CD3 nanobody as described in the first aspect in the preparation of a product binding to CD3.

[0025] The present invention has the following technical effects:

[0026] 1) The present invention provides a novel anti-CD3 nanobody, which is obtained by screening through phage display technology and has high affinity and specificity. Compared with traditional CD3 antibodies, the CD3 nanobody of the present invention is smaller in size and can better penetrate tissues, and is particularly suitable for targeted treatment of tumor tissues and complex lesions. At the same time, it has better thermal stability and chemical stability, and the production process is simple, which reduces the production cost and is more suitable for large-scale production.

[0027] 2) The nano-antibodies of the present invention have low immunogenicity, and can reduce side effects and immune rejection reactions when used for a long time, thereby improving the tolerance of patients.

[0028] 3) The anti-CD3 nanoantibody of the present invention can effectively activate T cells and enhance immune response, providing a new and effective tool for immunotherapy. It can also be used in combination with immune cells in vitro to improve the immunotherapy effect, showing a strong advantage in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of immune serum titer were determined by flow cytometry. Figure 1 A is combined with Jurkat cell flow cytometry, Figure 1 B is a flow cytometry analysis of Jurkat KO CD3 cells.

[0030] Figure 2 This is an SDS-PAGE gel image of the first-round PCR products.

[0031] Figure 3 This is the SDS-PAGE gel image of the second-round PCR products.

[0032] Figure 4 The results of the antigen binding activity test and non-specific binding test of CD3 antibody are shown in Figure 2. Figure 4 A is binding to human CD3-αβTCR complex protein ELISA, Figure 4 B is the ELISA result with human αβTCR recombinant protein, which shows that Anb1359-3A11 does not non-specifically bind to human αβTCR recombinant protein.

[0033] Figure 5 The figure shows the results of FACS binding experiment.

[0034] Figure 6 This is the result of the activation activity test of CD3 antibody. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0036] Example 1

[0037] In this embodiment, the method for preparing CD3 nanobody comprises the following steps:

[0038] 1) According to the gene sequence and protein sequence information of CD3 and TCR, construct CD3 overexpression plasmid and cell line. Overexpression cell lines were constructed using the following plasmid sequences, including CHOK1-hCD3 / αβTCR and HEK-hCD3 / αβTCR. They are used for subsequent alpaca immunization, serum titer determination, CD3 phage antibody library panning, clone ELISA determination and analysis, etc. First, the lentiviral plasmid and target gene are transfected into the packaging cell line (such as HEK293T). After a certain period of time, the cell culture supernatant containing the produced lentiviral particles is collected, and the virus titer is concentrated by PEG precipitation method. Then the target cells are co-cultured with the collected viral supernatant, and about 48 hours after infection, the corresponding antibiotics are used to screen the successfully transfected cells. Cells that have successfully integrated the lentivirus are usually screened out within 1-2 weeks. Finally, FACS detection is mainly used to ensure the successful construction of the cell line.

[0039] Name ReferenceSequence humanCD3E Uniprotkb_P07766 humanCD3G Uniprotkb_P09693 humanCD3D Uniprotkb_P04234 humanCD3Z Uniprotkb_P20963 humanTCRαβchain NCBI_XGT88490.11G4hi_TCR

[0040] 2) The alpaca was immunized 6 times with the plasmid or cell obtained in step 1), wherein the first and second immunizations were immunized with Human TCR and Human CD3 plasmids, and the last four immunizations were immunized with CHOK1-hCD3 / αβTCR cells, with a cycle of 7 days each, to obtain alpaca PBMC cells: one week after each immunization, blood was collected to detect the Anti-CD3 serum titer; one week after the sixth immunization, 50 mL of blood was collected for screening and library construction.

[0041] 3) Anti-CD3 serum titer was detected by FACS. The detection process was as follows: 1E 5cells, dilute the alpaca serum 1 / 100 in the first well and 1 / 1000 in the second well, then perform two-fold gradient dilutions in each well, add 50 μL of cell suspension to each well of the 96-well plate, mix thoroughly, and incubate at 4°C for 1 hour; add 100 μL MACS buffer to the well, centrifuge at 300 g for 5 minutes, and discard the supernatant; add 200 μL MACS buffer to the well to resuspend the cells, centrifuge at 300 g for 5 minutes, discard the supernatant, and repeat twice; then add 100 μL Alexa Fluor 488 anti-alpaca IgG (H+L), incubate at 4°C for 0.5 hours; add 100 μL MACS buffer to the wells, centrifuge at 300 g for 5 minutes, and discard the supernatant; add 200 μL MACS buffer to the wells to resuspend the cells, centrifuge at 300 g for 5 minutes, discard the supernatant, repeat twice; finally detect the fluorescence intensity by flow cytometry. When the MFI value of the sample to be tested is more than 3 times that of the negative control, the antiserum titer is judged to be positive. The results are as follows: Figure 1 shown. Figure 1 Figure A is the flow cytometry binding with Jurkat cells, and Figure B is the flow cytometry binding with Jurkat KO CD3 cells. Comparing Figure A and Figure B, it can be seen that the fifth immune serum and the sixth immune serum have significant binding with human CD3.

[0042] 4) Dilute the 50 ml blood collected in step 2) twice with PBS, separate the PBMC cells with lymphocyte separation solution (Ficoll reagent), and then add Trizol to dissolve the cells to obtain a cell concentration of 10 7 / ml PBMC cells, stored at -80 degrees.

[0043] 5) RNA was extracted from PBMC using Trizol method, and reverse transcribed into cDNA using oligo(dT) (the reverse transcription kit was TaKaRa-SMARTcribe Reverse Transcript), and PCR was performed twice using nested PCR to obtain nanobody gene fragments. Figure 2 This is the result of the first round of PCR. The target fragment is about 750 bp. Figure 3 The second round of PCR results, the target band is about 400bp. The obtained nano antibody gene fragment is inserted into the phage expression vector, the phage vector with the antibody fragment is obtained and electrotransferred to the SS320 electrotransfer competent state, the bacteria are collected, and the CD3 immune phage antibody library is obtained.

[0044] 6) CD3 phage antibody library panning: First, the obtained phage library is packaged and its titer is measured and recorded as input phage. Then, an appropriate amount of input phage is incubated with negative screening cells, and then incubated with CD3 overexpressing cells. Subsequently, the phage is eluted with trypsin, infects SS320 competent cells, and plated for culture. After two rounds of panning, a single clone is selected for culture and clone ELISA detection is performed.

[0045] 7) Clone ELISA: CD3 overexpressing cell lines were inoculated into 96-well plates at 37°C and cultured overnight. The next day, cells were fixed with 4% paraformaldehyde for 30 minutes and then blocked with 1% BSA for 1 hour at room temperature. Next, the cultured monoclonal phage solution was centrifuged, diluted to a certain ratio and added to the assay plate, incubated at room temperature for 2 hours, and then anti-M13-HRP secondary antibody was added and incubated at room temperature for 30 minutes. Finally, TMB colorimetric substrate was added, and the OD450 value was detected with an enzyme reader. The clones with an OD value greater than three times that of the negative control were selected as positive clones. All washing buffers were 0.05% PBST (pH 7.4). After sequencing, the screened positive clones were selected, and antibodies with a single heterosexual gene sequence were expressed through a mammalian cell expression system. After purification, they were used in the following in vitro functional detection experiments to further screen specific antibodies.

[0046] 8) Antigen-antibody binding detection: Perform ELISA binding experiment. Use human CD3-αβTCR complex protein and humanαβTCR recombinant protein to perform binding analysis with the expressed VHH antibody. In a 96-well ELISA plate, the antigen protein was coated at a concentration of 1μg / mL at 4°C overnight, and blocked with 1% BSA at room temperature for 2 hours the next day. Subsequently, add gradient diluted antibodies and incubate at room temperature for 1 hour, then add anti-human IgG Fc HRP (Sigma, Cat#A0170, 1:1000) and incubate at room temperature for 30 minutes. Finally, add TMB colorimetric substrate and detect OD450 value with an enzyme-linked microplate reader. Antibody molecules that strongly bind to the human CD3-αβTCR complex protein and do not non-specifically bind to the humanαβTCR recombinant protein are selected for subsequent evaluation. The results are as follows. Figure 4 As shown. Figure 4 A shows that the antibody Anb1359-3A11 strongly binds to the human CD3-αβTCR complex protein. Figure 4 B shows that Anb1359-3A11 does not non-specifically bind to human αβTCR recombinant protein.

[0047] 9) Cell binding detection: Use flow cytometry (FACS) for cell binding detection. Prepare the cultured Jurkat cells to 2×10^6 cells / ml with MACS buffer, and add 50μL of cell suspension to a 96-well plate. Dilute the prepared antibody in a 6-fold gradient with MACS buffer, and add 50μL of antibody dilution to the designated wells, and add positive and negative controls at the same time. Incubate the assay plate in the dark at 4°C for 1 hour and wash 3 times with MACS buffer. Subsequently, add the fluorescent secondary antibody to resuspend the cells, incubate in the dark at 4°C for 30 minutes, wash 3 times with MACS buffer, and finally add 200μL MACS buffer to resuspend the cells, use a flow cytometer for measurement, and use FlowJo software for data analysis. The results are as follows Figure 5 As shown. Figure 5 It can be seen that the antibody Anb1359-3A11 has a significant binding to Jurkat cells and is concentration gradient dependent.

[0048] 10) Antibody activation experiment: Perform reporter gene cell activation bioassay. The antibody was diluted in a gradient manner and coated in a 96-well ELISA plate at 4°C overnight. The next day, the liquid was discarded and washed three times with PBS. Jurkat-NFAT-Luci cells were centrifuged at 300g for 5 minutes and counted with trypan blue to ensure that the cell viability was over 95%. The cells were resuspended to 1×10^6 cells / ml in serum-free medium, and then 100μL of the cell suspension was distributed to the 96-well plate coated with the antibody and cultured in a 37°C, 5% CO2 incubator for 6 hours. The Bio-Glo Reagent (Vazyme, #DD1201) was equilibrated with the 96-well plate at room temperature for 15 minutes, and then 100μL Bio-Glo reagent was added to each well containing cells using a multichannel pipette and shaken on a shaker at 400 rpm for 10 minutes. Finally, the reaction solution was transferred to a 96-well white bottom plate and measured using an ELISA reader with a luminescence detection module. The data analysis results are as follows. Figure 6 As shown. Figure 6 It can be seen that the antibody Anb1359-3A11 can activate Jurkat-NFAT-Luci reporter gene cells, and there is a concentration gradient dependence. The reporter gene simulates the T cell activation pathway. This shows that CD3 nanoantibodies can effectively activate T cells and enhance the immune system's recognition and killing of tumor cells.

[0049] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-CD3 nanobody capable of activating T cell activity, wherein the VHH chain of the nanobody comprises a framework region and a complementary determining region; the complementary determining region comprises CDR1, CDR2 and CDR3, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 are selected from group 1) or group 2): 1) The complementary determining region CDR1 sequence is shown as SEQ ID NO.1, the complementary determining region CDR2 sequence is shown as SEQ ID NO.2, and the complementary determining region CDR3 sequence is shown as SEQ ID NO.3; 2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 are at least 90% identical to the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 described in group 1).

2. The anti-CD3 nanobody according to claim 1, characterized in that The amino acid sequence of the VHH chain of the nanobody is shown in SEQ ID NO.

4.

3. A nucleic acid encoding an anti-CD3 nanobody according to claim 1 or 2, characterized in that: The nucleic acid sequence of the anti-CD3 nanobody is as shown in SEQ ID NO.

5.

4. A vector containing the nucleic acid according to claim 3.

5. A host cell containing the vector according to claim 4.

6. The method for preparing an anti-CD3 nanobody according to claim 2, characterized in that: The method comprises the following steps: constructing a CD3 plasmid and establishing an overexpression cell line, immunizing alpacas to obtain peripheral blood mononuclear cells (PBMCs); extracting RNA thereof for reverse transcription to obtain cDNA; PCR amplifying antibody gene fragments and cloning them into a phage display vector; and electrotransforming the vector into SS320 competent cells to obtain a nano antibody library. After screening, antibody clones that can bind to CD3 protein are screened out through ELISA detection; antibody sequences are selected through sequencing, and antibody proteins are expressed using a mammalian cell high-throughput expression system; antibodies that only specifically bind to CD3 are screened and selected through ELISA and FACS detection for activation ability testing, and ultimately antibody molecules that can bind to and activate CD3 are obtained.

7. A detection reagent or kit containing the Nanobody according to claim 1 or 2 or the biomaterial according to any one of claims 3 to 5.

8. Use of the anti-CD3 nanobody according to claim 1 or 2 in the preparation of a drug for treating diabetes and obesity, and in the preparation of an agent or drug for diagnosing, preventing or treating a CD3-related disease or a hypoglycemia-related disease.

9. Use of the anti-CD3 nanobody as claimed in claim 1 or 2 in the preparation of a product for detecting CD3.

10. Use of the anti-CD3 nanobody as claimed in claim 1 or 2 in the preparation of a product that binds to CD3.

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