Double-target CD19 / CD79b CAR structure and application

Abstract

The invention relates to the technical field of adoptive immunotherapy, in particular to a double-target CD19 / CD79b CAR structure and application thereof. The structure comprises a Loop CAR structure or a tan CAR structure, and the Loop CAR structure or the tan CAR structure comprises a signal peptide sequence, an antigen binding structural domain, a connecting peptide, an antigen binding structural domain, a hinge region, a transmembrane structural domain, a costimulatory signal molecule and a cytoplasm signal transduction sequence which are connected in sequence; the antigen binding structural domain is an independent scFV structure aiming at specific targets, and the specific targets comprise CD19 and CD79b. The structure can be applied to immune cells, nucleic acid molecules and carriers. The structure can improve the killing effect.

Description

Technical Field

[0001] This invention relates to the field of adoptive immunotherapy technology, and in particular to the structure and application of dual-target CD19 / CD79b CARs. Background Technology

[0002] In recent years, chimeric antigen receptor T-cell (CAR-T) therapy, as a powerful new adoptive immunotherapy technology, has shown very effective therapeutic effects and has been used to treat a variety of solid and hematological cancers. In particular, CD19-CAR-T therapy for B-cell lymphoblastic leukemia and lymphoma has shown very good clinical therapeutic effects. In addition, CD79b is also an ideal target for B-cell tumors.

[0003] Currently, CAR-T cell therapy has made significant progress in treating B-cell malignancies; however, nearly half of patients with R / RB cell lymphoma still experience treatment failure, with poor prognosis. Studies have shown that the main reasons for treatment failure include: antigen loss and tumor heterogeneity: tumor cells may lack expression of target antigens (such as CD19), leading to the failure of single-target therapy. Rapid depletion of T cells in vivo: CAR-T cells are rapidly depleted due to continuous activation during treatment, reducing their anti-tumor efficacy.

[0004] The drawback of single-target therapy is that relapse is easily triggered when tumor cells downregulate antigen expression. Therefore, constructing bispecific CAR-T cells that can simultaneously target two targets can reduce tumor cell escape and lower the relapse rate. Although multi-antigen-targeted CAR-T cells hold promise for reversing the aforementioned resistance mechanisms, no effective multi-antigen-targeted regimens for R / RB cell lymphoma have yet been found clinically. Existing commercially available CD19 and CD79b svFv antibodies suffer from insufficient antigen affinity and low binding accessibility, which may lead to unsatisfactory treatment outcomes.

[0005] Therefore, there is an urgent need in this field to develop a novel dual-target CD19 / CD79b CAR-T cell based on CD19 and CD79b antibodies. This invention, starting from a practical clinical problem, not only possesses academic innovation but also represents a technological breakthrough over existing treatment methods, demonstrating excellent clinical translational potential and application prospects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a dual-target CD19 / CD79b CAR structure and its applications.

[0007] The technical solution of the present invention is as follows: Firstly, the dual-target CD19 / CD79b CAR structure, including the LoopCAR structure or the tandem CAR structure. Both the Loop CAR and Tandem CAR structures include a signal peptide sequence, an antigen-binding domain, a linker peptide, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence, all connected in sequence. The antigen-binding domain is an independent scFV structure targeting specific targets, including CD19 and CD79b.

[0008] Furthermore, the Loop CAR structure includes a signal peptide sequence, a first scFV structure targeting CD19, a first scFV structure targeting CD79b, a linker peptide, a second scFV structure targeting CD79b, a second scFV structure targeting CD19, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence, connected in sequence. Furthermore, the tandem CAR structure includes, in sequence, a signal peptide sequence, a first scFV structure targeting CD19, a second scFV structure targeting CD19, a linker peptide, a second scFV structure targeting CD79b, a first scFV structure targeting CD79b, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence.

[0009] Preferably, the first scFV structure targeting CD19 includes the antibody light chain variable region as shown in SEQ ID NO.1-SEQ ID NO.5.

[0010] Preferably, the second scFV structure targeting CD19 includes the antibody heavy chain variable region as shown in SEQ ID NO.7-SEQ ID NO.11.

[0011] Preferably, the first scFV structure targeting CD79b includes a light chain variable region as shown in SEQ ID NO.12-SEQ ID NO.17.

[0012] Preferably, the second scFV structure targeting CD79b includes a heavy chain variable region as shown in SEQ ID NO.18-SEQ ID NO.23.

[0013] Furthermore, the signal peptide sequence includes the sequence shown in SEQ ID NO.24.

[0014] Furthermore, the hinge region includes a hinge region derived from CD8α, the transmembrane domain includes a transmembrane domain derived from CD8α, the co-stimulatory signaling molecule includes 41BB, and the cytoplasmic signal transduction sequence includes CD3 Zeta.

[0015] Secondly, immune cells, including CAR-T cells, express the aforementioned dual-target CD19 / CD79b CAR structure.

[0016] Thirdly, the nucleic acid molecule encodes the aforementioned dual-target CD19 / CD79b CAR structure.

[0017] Fourthly, the vector contains the aforementioned nucleic acid molecules.

[0018] The beneficial effects of this invention are: (1) This invention utilizes scFv containing anti-CD19 and anti-CD79b antibodies to design a bispecific CAR molecule. After introducing the CAR molecule into T cells, bispecific CAR-T cells, namely CD19 / CD79b CAR-T cells, were successfully constructed. Clinically, these cells can simultaneously target B-cell malignancies expressing CD19 antigen and CD79b antigen, such as B-cell leukemia and lymphoma.

[0019] (2) The dual-specific CD19 / CD79b CAR-T cells constructed in this invention can simultaneously target two targets, thus expanding the scope of treatment and eliminating tumor recurrence caused by single-target seroconversion, laying the foundation for better targeted therapy of B-cell-derived leukemia and lymphoma.

[0020] (3) The dual-specific CD19 / CD79b CAR-T cells constructed using the present invention exhibited very good in vitro killing effects. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 These are structural diagrams of the single-target CD19 CAR-T, single-target CD79b, and two dual-target CD19 / CD79b CAR-T vectors of the present invention. Figure 2 This is a flow cytometry graph showing the transfection efficiency of MOCK-T cells according to the present invention; Figure 3 This is a flow cytometry graph showing the transfection efficiency of single-target CD19 CAR-T cells according to the present invention. Figure 4 This is a flow cytometry graph showing the transfection efficiency of single-target CD79b CAR-T cells according to the present invention. Figure 5 This is a flow cytometry graph showing the transfection efficiency of loop CAR-T cells in this invention; Figure 6 This is a flow cytometry graph showing the transfection efficiency of tandem CAR-T cells according to the present invention. Figure 7 This is a comparison diagram of the cytotoxicity of different CAR-T cells against LY8 B-cell lymphoma cells that naturally express CD20 and CD19. Figure 8 This is a comparison diagram of the cytotoxicity of different CAR-T cells of the present invention against U2932 B lymphocyte lymphoma cells that naturally express CD20 and CD19; Figure 9 This is a comparison of the cytotoxicity of different CAR-T cells against SU-DHL4 B-cell lymphoma cells that naturally express CD20 and CD19. Figure 10 This is a comparison diagram of the secretion of IL-2 by different CAR-T cells according to the present invention; Figure 11 This is a comparison diagram of the secretion of different CAR-T cells and the cytokine IFN-γ in this invention; Figure 12 This is a comparison diagram of the secretion of different CAR-T cells and the cytokine TNF-α in this invention; Figure 13 This describes the antitumor effects of the single-target CD19 CAR-T, single-target CD79b, and two dual-target CD19 / CD79b CAR-T cells of the present invention in mice. Figure 14 This is a statistical in vivo imaging of tumors in mice on day 21 of the animal experiment of this invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the equipment and materials used in the embodiments can be easily obtained from commercial companies. In the following description, the manufacturer / model has been noted after the first appearance of key features, and will not be repeated multiple times.

[0023] The dual-target CD19 / CD79b CAR structure and its application provided by this invention address the shortcomings of existing single-target CAR-T cells in the treatment of R / RB cell lymphoma, such as antigen loss, tumor heterogeneity, and rapid T cell depletion. Through a series of rigorous experimental procedures including multiple rounds of animal immunization, antibody screening, CAR molecular design and construction, T cell gene modification, and functional verification, a dual-target CAR-T cell with highly effective anti-tumor activity and good safety was finally obtained. The specific implementation steps of this invention will be described in detail below, covering experimental materials, reagent preparation, antibody screening, CAR construction, T cell isolation and transduction, in vitro functional testing, in vivo animal model experiments, data statistics, and discussion.

[0024] Where specific techniques or conditions are not described in detail in the embodiments, operations should be performed according to general techniques or conditions in the art, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products commonly used in the art.

[0025] Example 1 Construction of CAR molecular vectors This embodiment constructed a chimeric antigen receptor CD19 CAR targeting CD19, a chimeric antigen receptor CD79b CAR targeting CD79b, and a dual-target chimeric antigen receptor CD19 / CD79b CAR capable of simultaneously targeting both CD19 and CD79b. The structural schematic diagram is shown below. Figure 1 As shown, the corresponding CAR sequence was synthesized by a gene sequencing company, inserted into a lentiviral vector (GV401 by Jikai Gene), and after transformation and sequencing, the plasmid and bacterial culture were sent back.

[0026] The structure of the CD19 CAR is shown in Equation I below: singal peptide-CD19VL-G4S Linker-CD19VH-CD8α Hinge-CD8α TM-41BB-CD3Zeta (I) In the formula, each "-" represents a linking peptide or peptide bond; The signal peptide sequence is called the signal peptide sequence. CD19VL is the variable region of the light chain of the CD19 antibody; G4S Linker is a G4S linker peptide that connects VL and VH. CD19VH is the variable region of the CD19 antibody heavy chain; CD8α Hinge is the hinge region originating from CD8α; CD8α™ is a transmembrane domain derived from CD8α; 41BB is used as a co-stimulatory signaling molecule; CD3 Zeta is a cytoplasmic signal transduction sequence derived from CD3 Zeta; Specifically, The antigen-binding domain (scFv) of CD19 CAR is VL-Linker-VH, where "-" indicates the absence of the preceding and following elements and simply signifies that the elements are connected.

[0027] More specifically, 1) The antigen-binding domain (scFv) of the CD19 CAR contains the antibody light chain variable region (VL) as shown in SEQ ID NO.1-SEQ ID NO.5, see Table 1.

[0028] 2) The antigen-binding domain (scFv) of the CD19 CAR contains a linker as shown in SEQ ID NO. 6: SEQ ID NO.6: GGGGSGGGGSGGGGS Among them, glycine (Gly, G) and serine (Ser, S).

[0029] 3) The antigen-binding domain (scFv) of the CD19 CAR contains the antibody heavy chain variable region (VH) as shown in SEQ ID NO.7-SEQ ID NO.11, see Table 2.

[0030] For better illustration, taking 2343A1 as an example, its ScFv sequence is SEQ ID NO.1-Linker-SEQ ID NO.7, as follows: DIKMTQSPSSMYASLGERVTITCKASQDIKSYLRWYQQKPGKSPKTLIYETSNLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGSGTKVEIKGGGGSGGGGSGG GGSQVQLQQPGAEVLRPGSSVKLSCKASGYSFTNYWMHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCSSLSYSFDYWGQGTTLTVSS The other ScFv sequences are also similar to the examples above.

[0031] Table 1

[0032] Table 2

[0033] CD79b CAR, its structure is shown in Tables 3 and 4. Figure 1 .

[0034] The structure of the CD79b CAR is shown in Formula II below: singal peptide-CD79bVL-G4S Linker-CD79bVH-CD8α Hinge-CD8α TM-41BB-CD3Zeta (Ⅱ) The difference between formulas I and II is that... CD79bVL is the variable region of the light chain of the CD79b antibody; CD79bVH is the variable region of the CD79b antibody heavy chain; The rest is the same as Equation I.

[0035] More specifically, 1) The antigen-binding domain (scFv) of CD79b CAR contains the antibody light chain variable region (VL) as shown in SEQ ID NO.12-SEQ ID NO.22, see Table 3.

[0036] 2) The antigen-binding domain (scFv) of the CD79b CAR contains a linker as shown in SEQ ID NO. 6 above: SEQ ID NO.6: GGGGSGGGGSGGGGS Among them, glycine (Gly, G) and serine (Ser, S).

[0037] 3) The antigen-binding domain (scFv) of CD79b CAR contains the antibody heavy chain variable region (VH) as shown in SEQ ID NO.23-SEQ ID NO.28, see Table 4.

[0038] Table 3

[0039] Table 4

[0040] Loop CAR and tandem CAR were also constructed, referencing Figure 1 .

[0041] The structure of the Loop CAR is shown in Equation III below: singal peptide-CD19VL-CD79bVH-G4S Linker-CD79bVL-CD19VH-CD8α Hinge-CD8α TM-41BB-CD3 Zeta(Ⅲ) The structure of the tandem CAR is shown in Equation IV below: singal peptide-CD19VL-CD19VH-G4S Linker-CD79bVL-CD79VH-CD8α Hinge-CD8α TM-41BB-CD3 Zeta(Ⅳ) It should be noted that the specific sequences of CD19VL, CD19VH, CD79bVL, and CD79bVH have been given above.

[0042] A mock car was also set up as a control, for reference. Figure 1 .

[0043] The structure of a Mock CAR is shown in Equation V below: singal peptide-G4S Linker-CD8α Hinge-CD8α TM-41BB-CD3 Zeta(Ⅴ) Furthermore, the specific sequence of the signal peptide (SEQ ID NO.24) is given below: ATGGCTCTGCCTGTGACCGCACTGCTGCTGCCCCTGGCTCTGCTGCTGCACGCCGCAAGACCT Example 2 Plasmid amplification The corresponding plasmid was transformed into competent bacteria. 5 ml of bacterial culture was added to autoclaved LB medium and placed in a shaker at 37°C and 235 r / min for 16 h. Plasmid was then extracted using an endotoxin-free plasmid macromolecular kit.

[0044] Example 3 Lentiviral Packaging In this embodiment, the plasmid constructed in Example 2 was used for lentiviral packaging. A three-plasmid system was used. The helper plasmid pMD2.G (Jiangyuan Biotechnology, JY03029) and the lentiviral vector psPAX2 (Jiangyuan Biotechnology, JY03027) were mixed according to the product instructions and added to serum-free DMEM. At the same time, PEI (polyethyleneimine) with a total plasmid mass of 2 times was added. The mixture was mixed and left to stand for 20 min to obtain the mixture. The above mixture was added to a cell culture flask containing HEK 293T cells, gently mixed, and cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. After completion, the culture medium was replaced with fresh medium and cultured for another 48 hours and 72 hours. The lentivirus culture supernatant was collected twice for concentration, filtration, and purification.

[0045] Example 4 Isolation and activation of T cells Peripheral blood was collected from healthy volunteers or ethically approved donors, and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation. CD3-positive T cells were then isolated using magnetic bead sorting (MACS) technology. Strict aseptic conditions were maintained throughout the isolation process to ensure cell viability and function.

[0046] CD / CD28 magnetic beads were added to the previously isolated T cells to activate them, and the growth factor IL-2 was added to obtain activated T cells. Activation conditions were generally controlled within 24–48 hours. The activated T cells exhibited good proliferative behavior, preparing them for gene transduction.

[0047] Example 5 T-cell lentiviral infection The lentivirus from Example 3 was used to infect the activated T cells from Example 4. Polycationic acid (polyethyleneimine) could be added during the infection process to improve transduction efficiency. After 24 hours, the T cells were resuspended in fresh culture medium containing 200 IU / mL IL-2, maintaining the CAR-T cell density at 1 × 10⁻⁶ cells / mL. 6 The volume of solution should be around 10 cells / mL, and half of the solution should be replaced every 2-3 days.

[0048] Following infection, the cells are cultured in a medium containing cytokines such as IL-2 for 7–10 days to allow for sufficient expansion of the transduced T cells. The expression of CAR molecules is then detected by flow cytometry. A GFP marker is designed onto the CAR molecules to facilitate quantitative analysis of transduction efficiency.

[0049] Results reference Figures 2-6 . Figures 2-6 This is a flow cytometry graph showing the transfection efficiency of single-target CD19 CAR-T, single-target CD79b, and two dual-target CD19 / CD79b CAR-T cells according to the present invention.

[0050] Example 6 In vitro detection of the killing effect of CAR-T cells on B-cell lymphoma cells To verify the cytotoxic effect of dual-target CAR-T cells on B-cell lymphoma cells, B-cell lymphoma cells (LY8, U2932, and SU-DHL-4) were selected as target cells in an in vitro co-culture experiment. Different ratios of effector cells to target cells (E:T ratios of 1:1, 1:5, 1:10, and 1:20) were set and co-cultured at 37°C for 24 hours. Cell death rate was then detected using flow cytometry. The results showed that dual-target CAR-T cells significantly improved the tumor cell killing rate even at a lower E:T ratio, and their cytotoxicity was significantly higher than that of single-target and control T cell groups.

[0051] Results reference Figures 7-9 . Figures 7-9 This reflects the cytotoxicity of the single-target CD19 CAR-T, single-target CD79b, and two dual-target CD19 / CD79b CAR-T cells of the present invention against B-cell lymphoma cells (LY8, U2932, SU-DHL4) that naturally express CD20 and CD19.

[0052] Example 7 Cytokine release detection After the co-culture experiment, the supernatant was collected and the secretion levels of key cytokines (IFN-γ, TNF-α, and IL-2) were detected by flow cytometry. The data showed that dual-target CAR-T cells, while killing target cells, could induce full activation of T cells and produce higher levels of cytokines, thereby further enhancing the anti-tumor immune effect, demonstrating that the dual-target design has advantages in prolonging the activated state of T cells.

[0053] Results reference Figures 10-12 . Figures 10-12 This reflects the cytokine secretion of the single-target CD19 CAR-T, single-target CD79b, and two dual-target CD19 / CD79b CAR-T cells of the present invention after incubation with LY8, U2932, and SU-DHL4 cells.

[0054] Example 8 In vivo detection of the killing effect of CAR-T cells on B-cell lymphoma cells An R / RB cell lymphoma xenograft model was established using immunodeficient mice. First, a certain number (approximately 1 × 10^6) of U2932 cells were subcutaneously injected into the mice to establish the lymphoma model. Tumor progression was monitored using in vivo imaging techniques during tumor growth.

[0055] After the model was established, mice were randomly assigned to groups for treatment trials. The experimental group received intravenous injections of dual-target CAR-T cells, while the control group received either single-target CAR-T cells or untransduced T cells. Mice were housed in a standard environment, and tumor changes were continuously observed using in vivo imaging techniques to evaluate the anti-tumor effect of CAR-T cell therapy. Data showed that tumor growth was significantly inhibited in mice treated with dual-target CAR-T cells.

[0056] Results reference Figure 13 and Figure 14 .

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dual-target CD19 / CD79b CAR structure, characterized in that, Dual-target CD19 / CD79b CAR structures include LoopCAR or tandem CAR structures. Both the Loop CAR and Tandem CAR structures include a signal peptide sequence, an antigen-binding domain, a linker peptide, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence, all connected in sequence. The antigen-binding domain is an independent scFV structure targeting specific targets, including CD19 and CD79b.

2. The dual-target CD19 / CD79b CAR structure according to claim 1, characterized in that, The Loop CAR structure includes, in sequence, a signal peptide sequence, a first scFV structure targeting CD19, a first scFV structure targeting CD79b, a linker peptide, a second scFV structure targeting CD79b, a second scFV structure targeting CD19, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence. The tandem CAR structure includes, in sequence, a signal peptide sequence, a first scFV structure targeting CD19, a second scFV structure targeting CD19, a linker peptide, a second scFV structure targeting CD79b, a first scFV structure targeting CD79b, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and a cytoplasmic signal transduction sequence.

3. The dual-target CD19 / CD79b CAR structure according to claim 2, characterized in that, The first scFV structure targeting CD19 includes the antibody light chain variable region as shown in SEQ ID NO.1-SEQ ID NO.

5.

4. The dual-target CD19 / CD79b CAR structure according to claim 2, characterized in that, The second scFV structure targeting CD19 includes the antibody heavy chain variable region as shown in SEQ ID NO.7-SEQ ID NO.

11.

5. The dual-target CD19 / CD79b CAR structure according to claim 2, characterized in that, The first scFV structure targeting CD79b includes a light chain variable region as shown in SEQ ID NO.12-SEQ ID NO.

17.

6. The dual-target CD19 / CD79b CAR structure according to claim 2, characterized in that, The second scFV structure targeting CD79b includes the heavy chain variable region as shown in SEQ ID NO.18-SEQ ID NO.

23.

7. The dual-target CD19 / CD79b CAR structure according to claim 1, characterized in that, The signal peptide sequence includes the sequence shown in SEQ ID NO.

24.

8. The dual-target CD19 / CD79b CAR structure according to claim 1, characterized in that, The hinge region includes the hinge region derived from CD8α, the transmembrane domain includes the transmembrane domain derived from CD8α, the co-stimulatory signaling molecule includes 41BB, and the cytoplasmic signal transduction sequence includes CD3 Zeta.

9. Immune cells, characterized in that, Immune cells include CAR-T cells that express the dual-target CD19 / CD79b CAR structure according to any one of claims 1-8.

10. A carrier, characterized in that, The vector contains a nucleic acid molecule that encodes the dual-target CD19 / CD79b CAR structure according to any one of claims 1-8.

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