Anti-cd3 antibodies and uses thereof

Abstract

The present application relates to an anti-CD3 antibody and its application. The present application develops a specific antibody molecule targeting CD3 epsilon chain, which can activate T cells by combining with CD3 epsilon on T cells. A T cell binding protein containing the CD3 antibody is further designed, which can specifically target and bind to CD3 epsilon on the surface of T cells in vivo and in vitro, so as to achieve moderate activation of T cells. The CAR lentivirus containing the T cell binding protein of the present application can induce the generation of CAR-T cells in vitro and in vivo, and the CAR-T cells have good tumor cell killing efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an anti-CD3 antibody and its applications. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy, as a novel immunotherapy approach, uses genetic engineering to modify a patient's T cells, enabling them to specifically recognize and kill tumor cells, thus revolutionizing the treatment landscape for patients with hematologic malignancies. However, its application is still constrained by labor-intensive preparation processes, limited production capacity, and unstable clinical efficacy, requiring urgent improvement and optimization.

[0003] In vivo CAR-T technology utilizes targeted delivery systems such as lentiviral vectors and lipid nanoparticles to introduce CAR-encoding genetic material into endogenous T cells, directly generating CAR-T cells within the patient's body, eliminating the need for in vitro CAR-T cell processing and complex logistics. Unlike the personalized nature of traditional CAR-T therapy, in vivo CAR-T therapy is similar to off-the-shelf treatment, simplifying the production and treatment process, potentially offering better efficacy and fewer side effects. Because it involves direct genetic modification within the patient's body, it better mimics the in vivo physiological environment, allowing the modified T cells to better adapt to and attack tumor cells, resulting in faster, better, and more affordable treatment. Simultaneously, in vivo CAR-T generation avoids the cell contamination and mutation risks associated with in vitro production, eliminating the need for lymphoma chemotherapy and maintaining a healthy immune system. Early clinical studies have demonstrated its high transduction efficiency, sustained CAR expression, and anti-tumor activity, making it a promising innovative CAR-T technology solution.

[0004] T cell receptors (TCRs) are specifically expressed on the surface of T cell membranes and mediate T cell immune responses by recognizing antigen peptides / major histocompatibility complex (MHC). The TCRs of most mature T cells consist of two heterodimeric peptide chains, α and β. Their variable region is responsible for recognizing antigen signals. TCRα / β and the co-receptor CD3 form the TCR-CD3 receptor complex. This complex and downstream signaling pathways determine T cell development, activation, and immune responses. Antibodies targeting CD3 can directly activate TCR signaling and downstream pathways without TCR-MHC interaction. Theoretically, the greater the affinity of the CD3 antibody, the higher the TCR activation level. However, excessive T cell activation can lead to neurotoxicity and cytokine release syndrome (CRS). Therefore, developing antibodies with moderate activation potency and good safety is crucial for CD3-targeting molecule screening and drug development. Summary of the Invention

[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides an anti-CD3 antibody and its application, developing an anti-CD3 antibody with good specificity and affinity, which can be effectively used in the preparation of CD3-targeting formulations.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-CD3 antibody, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain of the anti-CD3 antibody respectively include the sequences shown in SEQ ID NO.5-SEQ ID NO.7, and the complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain include the sequences shown in SEQ ID NO.8, KAS and SEQ ID NO.9.

[0007] This invention develops an antibody that specifically binds to the ε chain of CD3 (CD3ε), which has good specificity and affinity and can be effectively used to prepare CD3-targeting formulations. It can specifically recognize and activate T cells by binding to CD3ε on T cells.

[0008] Optionally, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.2.

[0009] Secondly, the present invention provides the use of the anti-CD3 antibody described in the first aspect in the preparation of formulations targeting CD3.

[0010] Thirdly, the present invention provides a T-cell binding protein, the T-cell binding protein comprising a signal peptide, the anti-CD3 antibody described in the first aspect, a hinge region, a transmembrane region, and an intracellular domain connected in sequence.

[0011] The present invention further designs a T-cell binding protein that can specifically target T cells and promote T-cell proliferation.

[0012] Optionally, the signal peptide includes the CD8α signal peptide.

[0013] Optionally, the hinge region includes an IgG4 hinge region.

[0014] Optionally, the transmembrane region and intracellular domain include the PDGFR transmembrane region and intracellular domain.

[0015] Optionally, the amino acid sequence of the CD8α signal peptide includes the sequence shown in SEQ ID NO.15.

[0016] Optionally, the amino acid sequence of the IgG4 hinge region includes the sequence shown in SEQ ID NO.16.

[0017] Optionally, the amino acid sequences of the transmembrane region and intracellular domain of the PDGFR include the sequence shown in SEQ ID NO.17.

[0018] Fourthly, the present invention provides a nucleic acid molecule that encodes the anti-CD3 antibody described in the first aspect or the T-cell binding protein described in the third aspect.

[0019] Fifthly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the fourth aspect.

[0020] Optionally, the recombinant vector includes a lentiviral vector.

[0021] In a sixth aspect, the present invention provides a recombinant cell containing the nucleic acid molecule described in the fourth aspect or the recombinant vector described in the fifth aspect.

[0022] In a seventh aspect, the present invention provides a preparation method, the preparation method comprising: The recombinant vector described in the sixth aspect is introduced into host cells and cultured.

[0023] Eighthly, the present invention provides the use of the anti-CD3 antibody described in the first aspect or the T-cell binding protein described in the third aspect in the in vivo modification of CAR-T or in the preparation of formulations for in vivo modification of CAR-T.

[0024] In this invention, the T cell binding protein vector and the CAR vector can be co-prepared into a lentivirus, which can induce the generation of CAR-T cells in vivo and in vitro. Moreover, the CAR-T cells have good tumor cell killing ability and can be effectively applied to the field of in vivo modified CAR-T technology.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects: This invention develops a specific antibody molecule targeting the CD3ε chain, which can activate T cells by binding to CD3ε on T cells. Furthermore, a T cell binding protein containing the CD3 antibody is designed, which can specifically target and bind to CD3ε on the surface of T cells in vitro and in vivo, thereby achieving moderate activation of T cells. The CAR lentivirus containing the T cell binding protein of this invention can induce the generation of CAR-T cells in vivo and in vitro, and the CAR-T cells have good tumor cell killing ability. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of T cell-binding proteins.

[0027] Figure 2 Map of T-cell binding protein expression vectors.

[0028] Figure 3 This is a flow cytometry result of protein expression after 293T cells transiently transfected into T cells and binding to protein particles.

[0029] Figure 4 This is a curve showing the expansion of T cells after lentivirus infection of human PBMC cells.

[0030] Figure 5 The graph shows the results of flow cytometry analysis of the CD19-CAR positivity rate of T cells after lentiviral infection of PBMCs (Day 4).

[0031] Figure 6 The graph shows the results of flow cytometry analysis of the CD19-CAR positivity rate of T cells after PBMC infection with low concentration of CD3-LVV (Day 4).

[0032] Figure 7 The image shows the results of CD19 CAR-T cell killing function assay prepared from T cell-targeting specific lentiviruses.

[0033] Figure 8 The graph shows the detection results of the targeted infectivity of different lentiviruses.

[0034] Figure 9 The results of verifying CD3-LVV specific infection ability in mice are shown in Figure A, where flow cytometry results are shown and CAR positivity rate results are shown in Figure B.

[0035] Figure 10 The images show the results of CD3-LVV inducing CAR-T cells in tumor-bearing mice. Image A is an in vivo imaging image, and image B is the tumor fluorescence intensity. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0037] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0038] This invention develops an antibody (CD3 antibody) that specifically binds to the ε chain of CD3 (CD3ε). This antibody can specifically recognize and activate T cells by binding to CD3ε on T cells. This invention also provides a binding protein containing a CD3 antibody structure that can specifically target T cells. The structure includes an N-terminal CD3 antibody, a hinge region, a C-terminal transmembrane domain, and an intracellular domain, and can be used in vitro for direct conjugation of small molecules or direct expression on biomembranes.

[0039] Example 1 This embodiment describes the preparation and screening of CD3 antibodies.

[0040] (1) Immunization with New Zealand white rabbit antigens: CD3ε antigen protein (100-200 μg) was thoroughly mixed with Freund's complete adjuvant CFA (Sigma-Aldrich) and then injected subcutaneously multiple times (4-6 times). Blood samples were collected weekly to detect antibody titers using enzyme-linked immunosorbent assay (ELISA).

[0041] (2) Sorting and enrichment of antigen-specific B cells: Lymphocytes were isolated from rabbit spleens and antigen-specific B cells were sorted to the single-cell level by flow cytometry (FACS) using fluorescently labeled CD3ε antigen protein (Acro Biosystems) and distributed into 96-well plates.

[0042] (3) Single B cell PCR and gene cloning: The sorted single B cells were lysed, and the variable region genes of the rabbit antibody heavy chain (VH) and light chain (VL) were amplified by nested RT-PCR (Life Technologies); then the obtained VH and VL gene fragments were cloned into an expression vector containing the constant region of rabbit IgG to construct a complete antibody expression plasmid.

[0043] (4) Antibody expression and screening: The correctly sequenced heavy chain and light chain expression plasmids were co-transfected into 293T cells. After several days of culture, the cell supernatant was collected, and clones that could bind CD3ε antigen in the supernatant were detected by ELISA. Positive clones with strong binding signals and low background were screened out.

[0044] A CD3 antibody clone (named 5G3) was obtained through screening. Sequencing analysis revealed the following variable region sequence of the CD3 antibody: The amino acid sequence of the 5G3 heavy chain variable region (5G3-VH) (SEQ ID NO.1): QSVKESEGGLFKPTDTLTLTCTASGFSLTTYDMSWVRQAPGKGLEWIGWSGTDSRAWYATWAKSRSTITRNTNLNTVTLKMTSLTGADTATYFCARGAAGDIWGPGTLVTVSS。

[0045] Amino acid sequence of the variable region of the 5G3 light chain (5G3-VL) (SEQ ID NO.2): ADIVMTQTPASVSGAVGGTVTINCQASESIRSWLAWYQQKPGQRPKLLIYKASTLESGVPSRFKGSRSGTQFTLTISDLECADAATYYCQSYYGSNSNYDNNFGGGTEVVVK。

[0046] Nucleotide sequence of 5G3-VH (SEQ ID NO.3): CAGTCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCACCACCTACGACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGATGGTCTGGTACAGATAGTAGAGCATGGTACGCGACCTGGGCGAAAAGTCGATCCACCATCACCAGAAACACCAACCTGAATACGGTGACTCTGAAAATGACCAGTCTGACCGGCGCGGACACGGCCACCTATTTCTGTGCGAGAGGTGCGGCTGGTGACATCTGGGGCCCAGGCACTCTGGTCACCGTCTCCTCA。

[0047] Nucleotide sequence of 5G3-VL (SEQ ID NO.4): GCCGACATCGTGATGACCCAGACTCCAGCCTCCGTGTCTGGAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAGCATTAGGAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATAAGGCATCCACTCTGGAA TCTGGGGTCCCATCGCGGTTCAAAGGCAGTCGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTATTGTCAAAGCTATTATGGTAGTAATAGTAATTATGATAATAATTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0048] The heavy chain and light chain CDR sequences are shown in Table 1.

[0049] Table 1 (5) CD3 antibody antigen binding activity test: CD3 positive T cells were co-incubated with different concentrations of 5G3 antibody (30 min at room temperature), and then APC fluorescently labeled goat anti-rabbit secondary antibody (Abcam) was added for a second incubation. The antibody binding activity was detected by flow cytometry. The results of the CD3 antibody antigen binding activity test are shown in Table 2. The results show that 5G3 showed a relatively moderate antigen binding ability at both high and low concentrations. OKT3 is a commercial CD3 antibody and was used as a positive reference.

[0050] (6) CD3 antibody activation ability test: 5G3 antibody at different dilution ratios (six concentration gradients between 0.0001 μg / mL and 10 μg / mL) was co-incubated with Jurkat-NFAT-luc cells (37℃, 24 h), followed by the addition of luciferase substrate. The ability of CD3 antibody to activate T cells was tested by detecting bioluminescence values. The results of the CD3 antibody activation ability test are shown in Table 3. The results showed that the half-maximal effective concentration (EC50) of 5G3 was 0.435, indicating that the tested antibodies could activate CD3-positive Jurkat cells at low concentrations and express luciferase by activating the NFAT signaling pathway, thereby generating a fluorescent signal in the presence of luciferase substrate. Among them, OKT3 is a commercially available CD3 antibody with strong T cell activation activity and was used as a positive control in this experiment; G07 is an irrelevant antibody and was used as an irrelevant control.

[0051] Table 2 Table 3 Example 2 This embodiment describes the design of T-cell binding proteins.

[0052] A T-cell binding protein containing the CD3 antibody obtained in Example 1 was constructed, and its structural diagram is shown below. Figure 1 As shown, the binding protein expressed under the drive of the CMV promoter includes the CD8α signal peptide sequence, the single-chain antibody sequence of CD3 antibody (heavy chain variable region and light chain variable region), the hinge region of IgG4, and the transmembrane and intracellular sequences of PDGFR, as shown in Table 4.

[0053] Table 4 Example 3 This embodiment constructs the expression vector for the T-cell binding protein designed in Example 2.

[0054] (1) Based on the theoretical sequence of the T cell binding protein, the gene was optimized so that it could be efficiently expressed in human cells. The gene encoding the T cell binding protein was prepared by codon optimization and whole gene synthesis method, and the whole gene was synthesized at Guangzhou Aiji Biotechnology Co., Ltd. (2) The T cell binding protein gene synthesized by double digestion with Xba I and Xho I and the empty vector pMD2-CMV-MCS were digested in a water bath at 37℃ for 30 min. Then, DNA electrophoresis was performed using 1.5% agarose gel, and then purified and recovered using Tiangen's agarose gel kit. (3) Ligation of the pMD2-CMV-MCS vector with the T cell binding protein gene fragment; the ligation system is shown in Table 5: Table 5 Ligation was performed at 22℃ for 1 h. The ligation product was directly transformed into Stbl3 competent E. coli cells. 200 μL of the transformation product was plated onto kanamycin-resistant LB agar plates and incubated upside down overnight at 37℃. The next morning, three single clones were randomly selected for colony PCR identification, and positive clones were sent for sequencing.

[0055] (4) The map of the constructed T cell-binding protein expression vector is as follows: Figure 2 As shown.

[0056] Example 4 This embodiment uses flow cytometry to detect the protein expression of T cells constructed in Example 3 after transient transduction of 293T cells and binding to protein particles.

[0057] First, 293T cells were prepared and cultured to an appropriate density. Then, the expression plasmid was mixed with the transfection reagent PEI (Polysciences) according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently agitated to ensure even distribution. Next, the cells were placed in an incubator and cultured under appropriate conditions to promote transfection efficiency. 24 h post-transfection, cells were collected and washed with PBS. Cells were then labeled with GS-linker flow cytometry antibody and incubated. Subsequently, flow cytometry was used to detect the labeled cells and analyze the binding of the expression plasmid-transfected 293T cells to the GS-linker flow cytometry antibody, which recognizes the linker sequence between the heavy and light chains of the 5G3 antibody. Finally, flow cytometry data were collected and analyzed to assess transfection efficiency and the expression level of T cell-binding proteins. The results are shown below. Figure 3 As shown, flow cytometry results indicated that after transient transfection with T cell binding protein particles containing the 5G3 antibody sequence, 293T cells were able to correctly express T cell binding protein, with a positive expression rate of 25.28%.

[0058] Example 5 This embodiment involves lentiviral packaging targeting T cells.

[0059] The five-plasmid system was used, and the specific steps are as follows: (1) The five plasmid system includes gag / pol, Rev, VSV-G, CD19-CAR expression vector (the scFv sequence of which is derived from clone FMC63, a mouse antibody that recognizes human CD19 protein) and the T cell binding protein expression vector constructed in Example 3: the five plasmids were transiently transfected into 293T cells with a DNA content of 2 μg / mL. Non-targeting CD19-CAR lentiviruses were prepared by transfection with gag / pol, Rev, VSV-G, and CD19-CAR expression vectors; (2) Mix the above plasmid with PEI transfection reagent, add it to a certain volume of serum-free DMEM, mix well and let stand for 15 minutes, add the above mixture to a T75 culture flask containing 293T cells, mix gently, and incubate at 37℃ and 5% CO2 for 6 h. After 6 hours, the culture medium was replaced with fresh medium and cultured for another 72 hours. The culture supernatant of the lentivirus was collected for purification and detection.

[0060] Example 6 This embodiment uses the lentivirus packaged in Example 5 to infect human PBMC cells.

[0061] After resuscitating human PBMCs, they were divided into two batches. One batch was activated with X-VIVO medium containing 50 ng / mL OKT3 and 300 IU / mL IL-2 (PBMCs activated), while the other batch was not activated with OKT3 (PBMCs not activated). Two days later, the cell culture medium was completely replaced with X-VIVO medium containing 300 IU / mL IL-2 for expansion culture. RetroNectin (recombinant human fibronectin fragment) was used to enhance the infection efficiency of lentivirus on T cells. 30 μg of RetroNectin was coated into 6-well plates and incubated at 37°C for 2 h. RetroNectin was then used to block the coated 6-well plates with Hank's solution containing 2.5% BSA, and the plates were incubated at 37°C for 0.5 h. The blocking solution was then removed, and the 6-well plates were washed with Hank's solution containing 2% Hepes. X-VIVO medium was added, along with an appropriate amount of lentivirus solution (T cell-targeting lentivirus or non-targeting lentivirus). The plates were centrifuged at 2000×g for 2 h. The supernatant was discarded, and 1×10⁻⁶ ppm of the supernatant was added. 6 Unactivated or activated PBMC cells were centrifuged at 1000×g for 10 min and cultured in a cell culture incubator at 37℃, 5% CO2, and controlled humidity. Cell counts were performed every two days, and the medium was replaced with X-VIVO containing 300 IU / mL IL-2, maintaining a cell concentration of 0.5×10⁻⁶ cells / day. 6 -1×10 6 / mL, cultured continuously for 6 days. CAR-T cell expansion was assessed using a CountStar IC1000 automated cell counter (USA). Results are as follows: Figure 4 As shown, cell counting results indicate that T-cell-targeted lentiviruses effectively stimulated the in vitro expansion of unactivated PBMCs (unactivated PBMCs + T-cell-targeted lentivirus, marked in red), while non-targeted lentiviruses failed to stimulate the in vitro expansion of unactivated PBMCs (unactivated PBMCs + non-targeted lentivirus, marked in purple). Furthermore, T-cell-targeted lentiviruses additionally enhanced the in vitro expansion of activated PBMCs (activated PBMCs + T-cell-targeted lentivirus, marked in blue), while non-targeted lentiviruses did not enhance the in vitro expansion of PBMCs (activated PBMCs + non-targeted lentivirus, marked in green).

[0062] Example 7 This embodiment evaluates the expression and function of CAR in the CAR-T cells prepared in Example 6.

[0063] (1) Flow cytometry was used to detect the expression of CAR molecules on the surface of CAR-T cells and their binding ability to corresponding antigen proteins. T cell populations were labeled with APC-anti-CD3 antibody, and the CAR expression positivity rate was detected using FITC-CD19 protein (ACRO Biosystems). Results are as follows: Figure 5 As shown: Non-targeting CD19-CAR lentivirus (VSVG-LVV) and T-cell-targeting specific CD19-CAR lentivirus (CD3-LVV) were used to infect unactivated PBMCs, respectively. Among them, only the T-cell-targeting specific lentivirus could specifically infect T cells, with a CAR positivity rate of 33.61%. VSVG-LVV and CD3-LVV were used to infect OKT3-activated PBMCs, respectively. The results showed that both lentiviruses could effectively infect cells, with CAR positivity rates of 57.34% and 58.10%, respectively.

[0064] (2) Unactivated PBMCs were infected with different low concentrations (MOI=0.2, MOI=0.5, MOI=1) of CD19-CAR lentivirus (CD3-LVV) with T cell targeting specificity. On the fourth day post-infection, the expression of CD19-CAR on T cells was detected by flow cytometry. Results are as follows: Figure 6 As shown, low concentrations of CD3-LVV can effectively infect unactivated PBMCs, and the CAR positivity rate increases with the increase of MOI.

[0065] (3) Using Raji (CD19 positive) and K562 (CD19 negative) tumor cells as target cells, a killing assay was performed to test the in vitro killing function of CAR-T cells. At an effector-target ratio of 1:1, effector cells CD19 CAR-T and target cells were co-incubated. After 24 h, the culture supernatant was collected, and the secretion of interferon-gamma (IFN-γ) in the supernatant was detected by ELISA. The results showed ( Figure 7 T cell-targeting specific lentivirus (CD3-LVV) can effectively transduce T cells and generate CD19 CAR-T cells. In the presence of CD19-positive target cells Raji, it produces a large amount of effector molecule IFN-γ, which effectively kills the target cells Raji.

[0066] Example 8 This embodiment tests the CD3-LVV specific infectivity.

[0067] CD3-negative tumor cells (Raji) were infected with a non-targeting CD19-CAR lentivirus (VSVG-LVV) and a T-cell-targeting specific CD19-CAR lentivirus (CD3-LVV), respectively. Cells were harvested on Day 6 post-infection, and CD19-CAR expression was detected by flow cytometry.Figure 8 As shown, VSVG-LVV can infect Raji tumor cells, but it does not exhibit targeted infection. In contrast, CD3-LVV cannot infect Raji tumor cells, indicating that CD3-LVV has targeted infection capabilities.

[0068] Example 9 This embodiment verifies the CD3-LVV specific infection ability and tumor killing ability in mice.

[0069] Immunodeficient mice (NDG) were injected via the tail vein with 1×10⁻⁶ mmol / L. 6 5 × 10 CD19-positive Raji-luc tumor cells (Day-2), followed by tail vein inoculation. 6 Personal PBMC cells (Day-1), followed by drug administration 24 h later (Day-0), with the test lentivirus CD3-LVV injected into mice via the tail vein. Another identical mouse was injected with 1×10⁻⁶ cells via the tail vein. 6 CD19 CAR-T cells prepared using conventional in vitro methods (Ex vivo CD19 CAR-T) served as the positive control group in this experiment. Additionally, the same mice were injected with an equal volume of PBS as the negative control group. Blood samples were collected at different time points after injection for flow cytometry and in vivo imaging analysis. The experimental results are as follows: Figure 9 As shown, CD19-CAR-positive human CAR-T cells were not detected in mice in the PBS control group, while CD19-CAR-positive human CAR-T cells were detected in mice in the CD3-LVV experimental group. Furthermore, the detected CD19-CAR expression was limited to CD3-positive human T cells, indicating that CD3-LVV can specifically infect CD3-positive human T cells and express CAR molecules. In vivo imaging of mice yielded the following results: Figure 10 As shown, the number of tumor cells in mice gradually decreased after treatment with CD3-LVV, suggesting that CD3-LVV can infect CD3-positive human T cells in mice and generate CD19 CAR-T cells, which can effectively eliminate CD19-positive Raji-luc tumor cells.

[0070] In summary, the CD3ε-targeting specific antibody molecule (CD3 antibody) provided by this invention possesses good specificity and affinity. Furthermore, a T-cell binding protein containing the CD3 antibody is designed. This T-cell binding protein can specifically target and bind to CD3ε on the surface of T cells both in vitro and in vivo, achieving moderate T-cell activation. CAR lentiviruses containing the T-cell binding protein described in this invention can induce the generation of CAR-T cells both in vivo and in vitro, and these CAR-T cells exhibit good tumor cell killing ability. Therefore, the CD3 antibody provided by this invention, and the T-cell binding protein containing it, can be applied to in vivo modified CAR-T technology.

[0071] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-CD3 antibody, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the anti-CD3 antibody include the sequences shown in SEQ ID NO.5-SEQ ID NO.7, respectively, and the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain include the sequence shown in SEQ ID NO.8, KAS, and the sequence shown in SEQ ID NO.

9.

2. The anti-CD3 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-CD3 antibody includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.

2.

3. The use of the anti-CD3 antibody according to claim 1 or 2 in the preparation of formulations targeting CD3.

4. A T-cell binding protein, characterized in that, The T-cell binding protein comprises, in sequence, a signal peptide, the anti-CD3 antibody as described in claim 1 or 2, a hinge region, a transmembrane region, and an intracellular domain.

5. The T-cell binding protein according to claim 4, characterized in that, The signal peptide includes the CD8α signal peptide; Optionally, the hinge region includes an IgG4 hinge region; Optionally, the transmembrane region and intracellular domain include the PDGFR transmembrane region and intracellular domain; Optionally, the amino acid sequence of the CD8α signal peptide includes the sequence shown in SEQ ID NO.15; Optionally, the amino acid sequence of the IgG4 hinge region includes the sequence shown in SEQ ID NO.16; Optionally, the amino acid sequences of the transmembrane region and intracellular domain of the PDGFR include the sequence shown in SEQ ID NO.

17.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD3 antibody as described in claim 1 or 2, or the T-cell binding protein as described in claim 4 or 5.

7. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 6.

8. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 6 or the recombinant vector of claim 7.

9. A preparation method, characterized in that, The preparation method includes: The recombinant vector according to claim 7 is introduced into host cells and cultured.

10. The use of the anti-CD3 antibody of claim 1 or 2 or the T-cell binding protein of claim 4 or 5 in in vivo modification of CAR-T or in the preparation of formulations for in vivo modification of CAR-T.

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