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] This invention belongs to the field of molecular biology and immunology, specifically relating to an anti-CD3 nanobody capable of activating T cell activity, its preparation method, and its application. Background Technology

[0002] Vertebrates have developed a highly complex defense mechanism called the adaptive immune system to protect themselves from foreign invaders, such as exogenous macromolecules or pathogenic microorganisms. In mammals, the core of this system consists of two types of lymphocytes: T cells from the thymus and B cells from the bone marrow. T cells play a crucial role in the immune response, their functions involving specific antigen recognition and cell-mediated immune responses.

[0003] The activation process of T cells is highly complex, relying 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 via the T cell receptor (TCR). These peptides must bind to the major histocompatibility complex (MHC) molecule in order to be recognized by the TCR and trigger an immune response.

[0004] The TCR complex typically exists as an α-chain and β-chain, or a γ-chain and δ-chain, linked by disulfide bonds to form a heterodimer. It then non-covalently binds to a group of low-molecular-weight proteins that constitute the CD3 complex. CD3 is a helper molecule responsible for transmitting the signal from the extracellular space to the intracellular space after the TCR recognizes the antigen, thereby initiating T cell activation.

[0005] In mammals, the CD3 complex consists of four distinct subunits: one γ chain, one δ chain, two ε chains, and two ζ chains. These chains, together with the TCR, form the TCR-CD3 complex, providing the structural basis for T cell activation. The intracellular tail of CD3 plays a crucial role in T cell activation. This tail region contains the immune receptor tyrosine activation motif (ITAM), whose phosphorylation is a key step in signal transduction. Activation of the ITAM enables CD3 to recruit and bind to ZAP70 kinase, thereby amplifying the activation signal cascade and triggering subsequent immune responses.

[0006] CD3 molecules play an irreplaceable role in T cell activation, thus becoming an important target in immunotherapy and drug development. For example, in cancer immunotherapy, using anti-CD3 antibodies to regulate T cell function and enhance their tumor-killing ability is a promising therapeutic strategy. However, traditional antibodies have certain limitations in therapeutic applications due to their large molecular weight, structural complexity, and immunogenicity issues.

[0007] Nanobodies, as a novel type of antibody molecule, possess smaller molecular weight, better stability, and lower immunogenicity. Derived from single-domain antibody fragments from camelids, nanobodies exhibit high specificity and affinity similar to conventional antibodies. Due to their small molecular weight, nanobodies can better penetrate tissues and reach target regions, thus demonstrating unique advantages in immunotherapy. The development of nanobodies targeting the CD3 molecule can not only enhance T cell activation but also reduce adverse immune responses induced by conventional antibodies, providing an effective tool for precise immune regulation. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-CD3 nanobody that can activate T cell activity, its preparation method, and its application.

[0009] This invention aims to develop an anti-CD3 nanobody with a simple and efficient preparation method and broad application potential in immunotherapy and disease control. This nanobody can be used as a single drug or in combination with other therapeutic molecules to enhance T-cell immune responses, and can be widely used in the treatment of autoimmune diseases, infectious diseases, and cancer.

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

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

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

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

[0014] Preferably, the VHH chain of the nanobody has the amino acid sequence shown in SEQ ID NO.4.

[0015] In a second aspect of the invention, a nucleic acid encoding an 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 a third aspect of the invention, a vector containing the nucleic acid as described in the second aspect is provided. Further, the vector is -pComb3XSS.

[0018] In a fourth aspect of the invention, a host cell containing the vector as described in the third aspect is provided. Further, the host cell is TG1 or SS320; preferably, SS320 host cells are used.

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

[0020] In this invention, the primers, selection methods, and CD3-overexpressing cell lines used in steps such as alpaca immunization and antibody gene fragment amplification are all obtained through optimization of conventional operations.

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

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

[0023] In an eighth aspect of the invention, the application of the anti-CD3 nanobody as described in the first aspect in the preparation of products for detecting CD3 is provided.

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

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

[0026] 1) This invention provides a novel anti-CD3 nanobody, obtained through phage display technology, which exhibits high affinity and specificity. Compared with traditional CD3 antibodies, the CD3 nanobody of this invention is smaller in size, enabling better tissue penetration, and is particularly suitable for targeted therapy of tumor tissue and complex lesions. It also possesses better thermal and chemical stability, and the production process is simple, reducing production costs and making it more suitable for large-scale production.

[0027] 2) The nanobody of the present invention has low immunogenicity, which can reduce side effects and immune rejection reactions and improve patient tolerance during long-term use.

[0028] 3) The anti-CD3 nanobody of the present invention can effectively activate T cells and enhance the 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 effect of immunotherapy and shows a strong advantage in tumor immunotherapy. Attached Figure Description

[0029] Figure 1 Results of immune serum titer determination by cell flow cytometry. Figure 1 A is associated with Jurkat cell flow cytometry binding. Figure 1 B is associated with Jurkat KO CD3 cell flow cytometry binding.

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

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

[0032] Figure 4 This represents the results of antigen-binding activity assays and non-specific binding assays for CD3 antibodies. From... Figure 4 A is the protein that binds to the human CD3-αβTCR complex in ELISA. Figure 4 B represents the ELISA results for the recombinant human αβTCR protein, showing that Anb1359-3A11 does not specifically bind to the recombinant human αβTCR protein.

[0033] Figure 5 The results are from the FACS binding experiment.

[0034] Figure 6 This is the result of the CD3 antibody activation activity assay. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Reagents or instruments used, unless otherwise specified, are all conventional products that can be purchased through legitimate channels.

[0036] Example 1

[0037] In this embodiment, the method for preparing CD3 nanobodies includes the following steps:

[0038] 1) Based on the gene and protein sequences of CD3 and TCR, CD3 overexpression plasmids and cell lines were constructed. Overexpression cell lines, including CHOK1-hCD3 / αβTCR and HEK-hCD3 / αβTCR, were constructed using the following plasmid sequences. These were used for subsequent alpaca immunization, serum titer assays, CD3 phage antibody library screening, and clone ELISA analysis. First, lentiviral plasmids and the target gene were transfected into packaging cell lines (e.g., HEK293T). After a certain period, the cell culture supernatant, containing the generated lentiviral particles, was collected, and the viral titer was concentrated using PEG precipitation. Next, the target cells were co-cultured with the collected viral supernatant. Approximately 48 hours post-infection, successfully transfected cells were screened using appropriate antibiotics. Cells successfully integrating the lentivirus were typically selected within 1-2 weeks. Finally, FACS detection was primarily used to ensure successful cell line construction.

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

[0040] 2) Use the plasmids or cells obtained in step 1) to immunize alpacas 6 times. The first and second immunizations use Human TCR and Human CD3 plasmids, and the last four immunizations use CHOK1-hCD3 / αβTCR cells. Each cycle is 7 days to obtain alpaca PBMC cells. One week after each immunization, blood is collected to detect the serum titer of Anti-CD3. One week after the sixth immunization, 50 mL of blood is collected for screening and library construction.

[0041] 3) Anti-CD3 serum titer was detected by FACS. The detection procedure was as follows: 1E was seeded into each 96-well plate. 5Cells were collected by serially diluting alpaca serum 1 / 100 in the first well and 1 / 1000 in the second well, followed by 2-fold serial dilutions in each well. 50 μL of cell suspension was added to each well of a 96-well plate, mixed thoroughly, and incubated at 4°C for 1 hour. 100 μL of LACS buffer was added to each well, and the plate was centrifuged at 300g for 5 minutes, discarding the supernatant. Cells were resuspended in 200 μL of LACS buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. This process was repeated twice. Finally, 100 μL of Alexa serum was added to each well. 488 anti-alpaca IgG (H+L) was incubated at 4°C for 0.5 hours. 100 μL of MACS buffer was added to each well, and the cells were centrifuged at 300g for 5 minutes, discarding the supernatant. The cells were then resuspended in 200 μL of MACS buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. This process was repeated twice. Finally, the fluorescence intensity was detected by flow cytometry. A positive antiserum titer was defined as a result of an MFI value more than three times that of the negative control. The results are as follows: Figure 1 As shown. Figure 1 Figure A shows the flow cytometry binding with Jurkat cells, and Figure B shows the flow cytometry binding with Jurkat KO CD3 cells. Comparing Figures A and B, it can be seen that the fifth and sixth immunized sera showed significant binding with human CD3.

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

[0043] 5) RNA was extracted from PBMCs using the Trizol method. The RNA was then reverse transcribed into cDNA using oligo(dT) (using the TaKaRa-SMARTcribe Reverse Transcript kit). Nested PCR was performed twice to obtain the nanobody gene fragment. Figure 2 These are the results of the first round of PCR; the target fragment is around 750bp. Figure 3 The results of the second round of PCR show that the target band is approximately 400 bp. The obtained nanobody gene fragment was inserted into a phage expression vector to obtain a phage vector containing the antibody fragment. This vector was then electroporated into SS320 electroporation competent cells, and the cells were collected to obtain a CD3-immunized phage antibody library.

[0044] 6) CD3 phage antibody library panning: First, the obtained phage library was packaged and its titer was determined, recorded as input phage. Next, an appropriate amount of input phage was incubated with negative screening cells, followed by co-incubation with CD3-overexpressing cells. Subsequently, the phage was eluted with trypsin, infecting SS320 competent cells, and plate-cultured. After two rounds of panning, single clones were selected for culture and clone ELISA detection.

[0045] 7) Cloning ELISA: CD3-overexpressing cell lines were seeded into 96-well plates at 37°C and cultured overnight. The next day, cells were fixed with 4% paraformaldehyde for 30 minutes, followed by blocking with 1% BSA at room temperature for 1 hour. Next, the cultured monoclonal phage solution was centrifuged, diluted to a specific ratio, and added to the assay plate. The plate was incubated at room temperature for 2 hours, followed by incubation with anti-M13-HRP secondary antibody for 30 minutes at room temperature. Finally, TMB chromogenic substrate was added, and the OD450 value was measured using a microplate reader. Clones with OD values ​​more than three times greater than the negative control were selected as positive clones. All washing buffers were 0.05% PBST (pH 7.4). After sequencing, antibodies with a single heterologous gene sequence were selected and expressed using a mammalian cell expression system. After purification, these antibodies were used for the following in vitro functional assays to further screen for specific antibodies.

[0046] 8) Antigen-antibody binding detection: ELISA binding assays were performed. Human CD3-αβTCR complex protein and recombinant human αβTCR protein were used to analyze the binding with expressed VHH antibody. In 96-well microplates, the antigen protein was coated overnight at 4°C with a concentration of 1 μg / mL. The next day, the plates were blocked with 1% BSA at room temperature for 2 hours. Then, serially diluted antibodies were added and incubated at room temperature for 1 hour. Next, anti-human IgG Fc HRP (Sigma, Cat#A0170, 1:1000) was added and incubated at room temperature for 30 minutes. Finally, TMB substrate was added, and the OD450 value was measured using a microplate reader. Antibody molecules that strongly bind to the human CD3-αβTCR complex protein and do not non-specifically bind to the recombinant human αβTCR protein were selected for further evaluation. The results are as follows: Figure 4 As shown. From Figure 4 As can be seen from A, antibody Anb1359-3A11 binds strongly to the human CD3-αβTCR complex protein. Figure 4 As can be seen from B, Anb1359-3A11 does not bind nonspecifically to the human αβTCR recombinant protein.

[0047] 9) Cell binding assay: Cell binding was detected using flow cytometry (FACS). Cultured Jurkat cells were prepared to a concentration of 2 × 10^6 cells / ml with MACS buffer, and 50 μL of cell suspension was added to each well of a 96-well plate. The prepared antibody was serially diluted 6-fold with MACS buffer, and 50 μL of antibody dilution was added to each well, along with positive and negative controls. The assay plate was incubated at 4°C in the dark for 1 hour, followed by washing three times with MACS buffer. Subsequently, the cells were resuspended with fluorescent secondary antibody, incubated at 4°C in the dark for 30 minutes, washed three more times with MACS buffer, and finally resuspended in 200 μL of MACS buffer. Flow cytometry was used for assay, and data analysis was performed using FlowJo software. The results are shown below. Figure 5 As shown. From Figure 5 It can be seen that the antibody Anb1359-3A11 binds significantly to Jurkat cells in a concentration gradient-dependent manner.

[0048] 10) Antibody Activation Assay: Reporter gene cell activation bioassay was performed. The antibody was serially diluted and coated overnight at 4°C in 96-well microplates. The liquid was discarded the next day, and the cells were washed three times with PBS. Jurkat-NFAT-Luci cells were centrifuged at 300g for 5 minutes and counted with trypan blue to ensure cell viability exceeded 95%. Cells were resuspended in serum-free medium to 1×10^6 cells / ml, and 100 μL of cell suspension was aliquoted into antibody-coated 96-well plates and incubated at 37°C, 5% CO2 for 6 hours. Bio-Glo Reagent (Vazyme, #DD1201) was equilibrated with the 96-well plates at room temperature for 15 minutes. Then, 100 μL of Bio-Glo reagent was added to each well containing cells using a multichannel pipette, and the plates were shaken at 400 rpm for 10 minutes. Finally, the reaction mixture was transferred to 96-well white plates, and measurements were taken using a microplate reader with a luminescent detection module. Data analysis results are shown below. Figure 6 As shown. From Figure 6 It can be seen that the antibody Anb1359-3A11 can activate Jurkat-NFAT-Luci reporter cells in a concentration gradient-dependent manner. The reporter gene mimics the T cell activation pathway, which shows that the CD3 nanobody can effectively activate T cells and enhance the immune system's recognition and killing of tumor cells.

[0049] The nanobody Anb1359-3A11 has the VHH amino acid sequence shown in SEQ ID NO.4 and the nucleic acid sequence shown in SEQ ID NO.5. The VHH includes a framework region and a complementarity-determining region (CDR). The CDR1 sequence is shown in SEQ ID NO.1, the CDR2 sequence is shown in SEQ ID NO.2, and the CDR3 sequence is shown in SEQ ID NO.3.

[0050] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

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

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

4.

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

5.

4. A vector containing the nucleic acid as described in claim 3.

5. A host cell containing the vector as described in claim 4.

6. The method for preparing the anti-CD3 nanobody as described in claim 2, characterized in that, The process includes the following steps: constructing a CD3 plasmid and establishing an overexpressing cell line; immunizing alpacas to obtain peripheral blood mononuclear cells (PBMCs); extracting RNA and performing reverse transcription to obtain cDNA; PCR amplifying the antibody gene fragment and cloning it into a phage display vector; and electroporating the vector into SS320 competent cells to obtain a nanobody library. After screening, antibody clones that can bind to CD3 protein were selected by ELISA; antibody sequences were selected by sequencing and antibody proteins were expressed using a mammalian cell high-throughput expression system; antibodies that specifically bind to CD3 were screened by ELISA and FACS and their activation ability was tested, finally obtaining antibody molecules that can bind to and activate CD3.

7. Detection reagents or kits containing nanobodies as described in claim 1 or 2 or biomaterials as described in any one of claims 3-5.

8. The use of the anti-CD3 nanobody as described in claim 1 or 2 in the preparation of medicaments for treating diabetes and obesity, and in the preparation of reagents or medicaments for diagnosing, preventing or treating CD3-related diseases or hypoglycemia-related diseases.

9. The application of the anti-CD3 nanobody as described in claim 1 or 2 in the preparation of products for detecting CD3.

10. The use of the anti-CD3 nanobody as described in claim 1 or 2 in the preparation of products that bind to CD3.

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