A method for screening tumor-targeting bcr and its application in constructing car-t cells

Abstract

The application provides a method for screening tumor-targeting BCR and application thereof in constructing CAR-T cells, and relates to the technical field of biological pharmacy.The application firstly provides a method for screening tumor-targeting BCR from samples derived from individuals and a BCR sequence of the tumor-targeting BCR targeting tumor antigens, and firstly proposes a method for constructing CAR-T cells for tumor treatment by using the tumor-targeting BCR as an antigen recognition domain of CAR-T and a method for constructing the CAR-T cells.It is verified that the BCR-CAR-T cells obtained by using the method have excellent anti-tumor effects on different tumor types in vivo and in vitro, can effectively inhibit tumor growth, secrete high levels of effector factors IFN gamma and IL-2, realize immune cell activation, enhance the anti-tumor ability, and significantly prolong the survival period of the body.The application provides a new perspective and precise treatment direction for the clinical application of the tumor-targeting BCR, and provides a new strategy for the research and development of engineered immune cells targeting tumor cells and the treatment of solid tumors.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a method for screening tumor-targeting BCRs and its application in constructing CAR-T cells. Background Technology

[0002] The antigen-recognition domain (ARD) of CAR-T cells plays a central role in determining tumor-killing specificity and clinical efficacy. Currently, most CAR-T products in clinical practice use single-chain variable fragments (scFv) derived from animals such as mice and alpacas, or de novo-designed artificial binders as antigen recognition domains. Although these strategies have achieved significant success in hematologic malignancies (such as CD19 CAR-T), they still face the following key limitations: (1) Strong immunogenicity: Non-human antibodies can easily induce antibody or cell-mediated immune responses in vivo, leading to suppression of CAR-T cell function or serious adverse reactions. (2) Vague recognition specificity: Traditional scFvs often target highly expressed universal antigens (such as CD19), lacking sufficient tumor specificity in solid tumors and easily causing off-target toxicity. (3) Poor tumor specificity of target antigens: Many animal-derived antibodies have insufficient affinity and specificity for human tumor antigens, making it difficult to cover antigenic heterogeneity among patients, thus limiting the therapeutic effect of CAR-T cells. Therefore, innovative methods for discovering antigen recognition regions are needed to improve the efficacy of CAR-T cell therapy.

[0003] B-cell receptors (BCRs) are specific antigen recognition molecules expressed on the surface of B cell membranes. During B cell development, the V, D, and J gene segments encoding the variable region of the BCR undergo random combination, linkage, and mutation, resulting in a vast number of different BCR molecules. Each B cell clone expresses only one type of BCR, and therefore can only recognize and bind to a specific antigenic epitope (a small fragment on the antigen). This highly specific one-to-one binding is the basis of an effective immune response. Antibodies are secretory homologs of BCRs, retaining the same antigen-binding site (CDR), but losing the transmembrane structure and becoming freely circulating effector molecules. Therefore, antibodies are essentially BCRs. High-throughput sequencing shows that the V(D)J rearrangement patterns of tumor-infiltrating B cells differ from those of peripheral blood B cells, indicating that they undergo specific clonal expansion and somatic mutations in the tumor microenvironment. Furthermore, these BCRs can specifically recognize tumor cells. The high variability of the BCR CDR3 region determines its specific binding ability to tumor antigens and has been proven to be a key structure for tumor-specific immune recognition.

[0004] Since BCRs derived from individuals have undergone somatic mutation and affinity maturation processes in vivo, they can recognize tumor antigens with low expression or mutation with higher affinity. Based on this, the present invention provides a method for screening tumor-targeting B cells and tumor-targeting BCRs using single-cell sequencing data and BCR immune repertoire information, and further explores the application of tumor-targeting BCRs in the construction of CAR-T cells. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for screening tumor-targeting BCRs and provides an application of using the tumor-targeting BCRs screened by this method as antigen recognition domains of chimeric antigen receptors for constructing CAR-T cells.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] First, this invention provides a method for screening tumor-targeting BCRs, the method comprising the following steps:

[0010] (1) Extract B cells from in vitro samples from individuals, analyze them using high-throughput single-cell sequencing and BCR immune repertoire information, and screen out B cells with complete BCR variable region sequences;

[0011] Specifically, the assembly and consensus sequence assembly and annotation methods for the immune repertoire are as follows: When performing data analysis using SeekOne® Tools software, clean reads are first compared with known V(D)J fragments. Paired reads that align to at least 15 bp of the fragments are retained. Then, contig assembly and annotation screening are performed for each cell. The selected and retained single-cell contigs are used for subsequent consensus sequence assembly in samples. For high-quality contigs in a sample, consensus sequence assembly is further performed by combining contigs from all effective cells in that sample. The results of the consensus sequence assembly and annotation are then used for subsequent BCR genotyping.

[0012] Specifically, the B cell with a complete BCR sequence refers to a B cell that simultaneously possesses complete heavy chain variable region (VH) and light chain variable region (VL) sequences, wherein the variable region sequence includes a complete complementarity determining region (CDR) sequence and a framework region (FR) sequence.

[0013] It should be noted here that the complete BCR variable region sequence includes the CDR sequence and the backbone region sequence. Since the CDR sequence region forms a spatial conformation complementary to the antigen epitope, the six CDRs (VH and VL) together constitute the antigen-binding site of the antibody, which determines the specificity of the antibody. The backbone region mainly plays the role of stabilizing the spatial conformation of the CDR region, and the amino acid composition and sequence do not change much. Therefore, this invention mainly uses the six CDR regions of BCR for screening. However, it should be noted that the complete BCR variable region sequence, including the CDR and its corresponding backbone region, should be used to synthesize the CAR nucleic acid molecule.

[0014] (2) Further analysis and screening of BCR recognition sequences obtained from peripheral blood-derived B cells and tumor tissue-infiltrating B cells were performed. The specific method was as follows:

[0015] All CDR sequences from the light chain variable region (VL) and heavy chain variable region (VH) of each B cell were extracted individually and uniformly defined as "one BCR recognition sequence" for subsequent clonal frequency calculation, comparison, and screening. Based on the clonal frequency of each BCR recognition sequence, BCR recognition sequences that appear only in tumor tissue infiltrating B cells but are not detected in peripheral blood B cells were first selected. These BCR recognition sequences meeting the above requirements were used as the first screening results, with n sequences as the first screening results. When n < 3, the sequences were then sorted according to the clonal frequency of the heavy chain, and the top m sequences by heavy chain frequency were selected and their corresponding light chain variable regions were compared with the m sequences. The chains are combined to form BCR recognition sequences as the second screening result; where n+m=3, n=0,1,2,3, m=0,1,2,3; that is, when the number of BCR recognition sequences in the first screening result is 3 or more, the top three BCR recognition sequences are selected according to their cloning frequency from high to low; when the number of BCR recognition sequences in the first screening result is less than 3, they are sorted according to the cloning frequency of the heavy chain, and the top 3-m sequences of the heavy chain frequency are selected and combined with their corresponding light chains to form BCR recognition sequences as the second screening result. A total of 3 BCR recognition sequences are obtained from the first and second screenings.

[0016] Specifically, the BCR identification sequence includes three CDR sequences corresponding to the light chain variable region (VL) and the heavy chain variable region (VH), namely VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3.

[0017] (3) The three BCR recognition sequences obtained according to the method in step (2) are supplemented with their corresponding skeleton region sequences to form the tumor-targeting BCR sequences.

[0018] Secondly, the present invention provides a CAR-T cell based on tumor-targeting BCR, wherein the CAR-T cell is obtained by modifying T cells using genetic engineering technology to use tumor-targeting BCR derived from a single individual as the antigen recognition domain of chimeric antigen receptor (CAR).

[0019] Specifically, the tumor-targeting BCR is obtained by analyzing and screening in vitro samples from a single individual using high-throughput single-cell sequencing and BCR immune repertoire information to obtain the BCR sequences of tumor-targeting B cells and their targeting tumor antigens, and an antigen recognition domain is constructed using the tumor-targeting BCR sequence.

[0020] Specifically, the chimeric antigen receptor further includes a transmembrane region, a co-stimulatory domain, and an activation domain.

[0021] Specifically, the in vitro samples from a single individual are peripheral blood B cells and tumor-infiltrating B cells.

[0022] Secondly, this invention provides a method for constructing CAR-T cells based on tumor-targeting BCR, the method comprising the following steps:

[0023] I. Tumor-targeting BCR sequences were obtained by screening using the above method.

[0024] II. Construction of chimeric antigen receptor CAR

[0025] The CAR nucleic acid molecule is synthesized from the whole genome. The CAR contains, from the N-terminus to the C-terminus, an antigen recognition domain targeting tumor antigens, a transmembrane domain, a co-stimulatory domain, and an activation domain.

[0026] III. Construction of CAR-T cells based on tumor-targeting BCR

[0027] The synthesized CAR was cloned into a viral vector to obtain a plasmid expressing a CAR targeting the tumor antigen. After extracting the CAR plasmid, 293T17 cells were transduced using calcium phosphate transfection reagent to produce the virus. The viral supernatant was collected after 48 hours. T cells were isolated and then activated using anti-CD3 / CD28 activation magnetic beads. After 50 hours of activation, the viral supernatant was added to the T cells for centrifugation infection with an infection MOI of 10-30. After the infection was completed, the medium was replaced with fresh 10% RIPM1640 medium and placed in an incubator for normal cell culture and expansion, thus obtaining CAR-T cells based on tumor-targeting BCR.

[0028] Specifically, the antigen recognition domain targeting the tumor antigen in step (ii) is constructed from the three BCR recognition sequences obtained in step (i);

[0029] Specifically, the transmembrane domain in step (ii) can be derived from a natural polypeptide or can be artificially designed; the artificially designed transmembrane domain is a polypeptide that mainly includes hydrophobic residues such as leucine and valine; preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthesized transmembrane domain; optionally, a short oligopeptide linker or polypeptide linker, such as a linker with a length of 2 to 10 amino acids, can be provided between the transmembrane domain and the intracellular domain; in one embodiment, a linker sequence having a glycine-serine continuous sequence can be used, optionally (G4S)3 or (G4S)4.

[0030] Specifically, the co-stimulatory domain in step (ii) is one or more of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10; preferably, the co-stimulatory domain is 4-1BB.

[0031] Specifically, the activation domains in step (ii) include one or more of CD3ζ, CD3γ, CD3δε, or CD3ε.

[0032] Furthermore, this invention also provides the application of CAR-T cells constructed based on tumor-targeting BCR in the preparation of immunotherapy drugs.

[0033] (III) Beneficial Effects

[0034] This invention first provides a method for screening tumor-targeting BCRs. The method involves analyzing and screening in vitro samples from individuals using high-throughput single-cell sequencing and BCR immune repertoire information to obtain the BCR sequences of tumor-targeting B cells and their targeting tumor antigens. This method provides conditions for in-depth exploration of tumor-targeting BCRs.

[0035] Furthermore, this invention is the first to propose a method for constructing CAR-T cells for tumor therapy using tumor-targeting BCR derived from individuals as the antigen recognition domain of CAR-T cells. This provides a new perspective and direction for precision treatment in the clinical application of tumor-targeting BCR. Experimental verification shows that BCR-CAR-T cells obtained using this method exhibit excellent anti-tumor effects against different tumor types, both in vivo and in vitro. They can effectively inhibit tumor growth, secrete high levels of effector factors IFNγ and IL-2, activate immune cells, enhance anti-tumor capabilities, and significantly prolong the body's survival.

[0036] This invention provides BCR-CAR-T cells and their construction method, which construct CAR-T cells by discovering tumor-targeting antibodies in the absence of unknown antigens. This changes the traditional CAR-T cell research and development strategy that focuses on antigen discovery and creatively changes the means of discovering tumor-targeting antibodies. This method is expected to solve the problems of insufficient immunogenicity, specificity and low tumor antigen coverage caused by traditional non-human scFv, so as to improve the efficacy and safety of CAR-T cells.

[0037] This invention provides a new strategy for the development of engineered immune cells that target tumor cells and for the treatment of solid tumors. Attached Figure Description

[0038] Figure 1 Flowchart for collecting mouse B cell sequencing samples.

[0039] Figure 2 UMAP diagram of dimensionality reduction clustering of B cells with complete BCR variable region sequences.

[0040] Figure 3 These are the six complete BCR light and heavy chain variable region sequences that were finally selected.

[0041] Figure 4 To construct the structure maps of plasmids M11-BBz-CAR, M12-BBz-CAR, M13-BBz-CAR, M21-BBz-CAR, M22-BBz-CAR, and M23-BBz-CAR.

[0042] Figure 5 The plasmids constructed using six BCR sequences successfully infected M11-BBz-CAR-T, M12-BBz-CAR-T, M13-BBz-CAR-T, M21-BBz-CAR-T, M22-BBz-CAR-T, and M23-BBz-CAR-T cells (abbreviated as M11, M12, M13, M21, M22, and M23).

[0043] Figure 6 The apoptosis of tumor cells was measured by flow cytometry after co-incubating six types of CAR-T cells with B16-WT cells.

[0044] Figure 7 The specific effector secretion of six BCR CAR-T cells on B16-WT cells; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0045] Figure 8The results show the changes in tumor volume in each group of tumor-bearing mice during the experiment; Note: Compared with the UTD group, *P<0.05, **P<0.01, ***P<0.001.

[0046] Figure 9 To replicate animal experiments and verify the antitumor effects of M12 CAR-T and M21 CAR-T; Note: Compared with the UTD group, *P<0.05, **P<0.01, ***P<0.001.

[0047] Figure 10 The apoptosis of tumor cells was measured by flow cytometry after co-incubating six types of CAR-T cells with YUMM1.7 cells.

[0048] Figure 11 The specific effector secretion of six BCR CAR-T cells in YUMM1.7 cells was evaluated. Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figure 12 To assess the different antitumor effects of six BCR CAR-T cells in the mouse YUMM1.7 tumor model, tumor volume was used as the detection index. Note: Compared with the UTD group, *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0051] Example 1

[0052] A method for screening tumor-targeting BCR sequences, the method comprising the following steps:

[0053] (1) Extraction of in vitro samples from a single individual: Two healthy, immune-functioning female C57BL / 6J mice aged 6-8 weeks and weighing approximately 18-22g were selected. Peripheral blood samples were separated by orbital blood collection. Then, tumor-bearing cells were collected, centrifuged, washed, and resuspended in sterile PBS, adjusted to 1×10⁻⁶. 6 The concentration was 100 µL / cell, and then each mouse was anesthetized and shaved before being given 1 × 10⁻⁶ cells / 100 µL. 6The cell volume was subcutaneously injected into the groin to complete tumor implantation. Fourteen days after tumor implantation, peripheral blood B cells and tumor tissue B cells were extracted from the same mouse. The preparation flowchart is as follows: Figure 1 As shown.

[0054] (2) Peripheral blood B cells and tumor tissue B cells from the same mouse were analyzed using high-throughput single-cell sequencing and BCR immune repertoire information. B cells with complete BCR variable region sequences were screened. The dimensionality reduction clustering UMAP diagram of all effective B cells and the expression of three B cell markers, CD19, CD79a, and CD79b, are shown below. Figure 2 As shown;

[0055] Specifically, the assembly and consensus sequence assembly and annotation methods for the immune repertoire are as follows: When performing data analysis using SeekOne® Tools software, clean reads are first compared with known V(D)J fragments. Paired reads that align to at least 15 bp of the fragments are retained. Then, contig assembly and annotation screening are performed for each cell. The selected and retained single-cell contigs are used for subsequent consensus sequence assembly in samples. For high-quality contigs in a sample, consensus sequence assembly is further performed by combining contigs from all effective cells in that sample. The results of the consensus sequence assembly and annotation are then used for subsequent BCR genotyping.

[0056] Specifically, the B cell with a complete BCR variable region sequence refers to a B cell that simultaneously possesses a complete heavy chain variable region (VH) and light chain variable region (VL) sequence, wherein the variable region sequence includes a complete complementarity determining region (CDR) sequence and a framework region (FR) sequence.

[0057] To ensure that the plasmids constructed subsequently contain the complete BCR variable region sequence, all CDR sequences of the light and heavy chains of each B cell are extracted separately and uniformly defined as "one BCR recognition sequence" for subsequent cloning frequency calculation, alignment, and screening processes. Specifically, the "one BCR recognition sequence" refers to the three CDR sequences corresponding to the light chain variable region (VL) and the heavy chain variable region (VH), namely VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3.

[0058] It should be noted here that the complete BCR variable region sequence includes the CDR sequence and the backbone region sequence. Since the CDR sequence region forms a spatial conformation complementary to the antigen epitope, the six CDRs (VH and VL) together constitute the antigen-binding site of the antibody, which determines the specificity of the antibody. The backbone region mainly plays the role of stabilizing the spatial conformation of the CDR region, and the amino acid composition and sequence do not change much. Therefore, this embodiment mainly uses the six CDR regions corresponding to one BCR for screening. However, it should be noted that the plasmid used for final construction should be the complete BCR variable region sequence including the CDRs and their corresponding backbone regions.

[0059] (3) Further analysis and screening of BCR recognition sequences obtained from peripheral blood B cells and tumor tissue infiltrating B cells from the same mouse. The specific method is as follows: based on the cloning frequency of each BCR recognition sequence, first select BCR recognition sequences that only appear in tumor tissue infiltrating B cells and are not detected in peripheral blood B cells. The number of such BCR recognition sequences is judged. When the number of BCR recognition sequences is 3 or more, the top three BCR recognition sequences are selected according to the cloning frequency from high to low. When the number of BCR recognition sequences is less than 3, supplementation is made according to the cloning frequency of the heavy chain. The sequence with the highest heavy chain frequency is selected and combined with its corresponding light chain to form a BCR recognition sequence until 3 BCR recognition sequences are supplemented.

[0060] Note: To facilitate subsequent explanations and experimental verification, this embodiment takes the selection of three clone amplification target sequences for each sample as an example. In reality, the required clone amplification sequences include, but are not limited to, three. The number of target sequences and the clone frequency threshold for sequence screening should be determined according to actual needs.

[0061] Following the above method, in this embodiment, three BCR recognition sequences were screened from each mouse, for a total of six BCR recognition sequences, which are the tumor-targeting BCR sequences. Note that during CAR plasmid construction, these six BCR sequences should be supplemented with their respective framework regions (FRs), namely VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3, VL-FR4; VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3, VH-FR4. The BCR recognition sequences screened from mouse 1 are abbreviated as M11, M12, and M13; the BCR recognition sequences screened from mouse 2 are abbreviated as M21, M22, and M23; the complete sequence information is as follows. Figure 3As shown, each complete BCR sequence contains a light chain variable region (VL) and a heavy chain variable region (VH) sequence. Each light chain variable region sequence and heavy chain variable region sequence contains 3 CDR sequences and 4 FR sequences, respectively. Specific sequences are shown in Tables 1 and 2.

[0062] Table 1. Tumor-targeting BCR sequences in tumor-bearing mice.

[0063]

[0064] Table 2. Tumor-targeting BCR sequences in tumor-bearing mice.

[0065]

[0066] Example 2

[0067] BCR-CAR-T cell preparation

[0068] 1. The six BCR sequences were synthesized into a CAR nucleic acid molecule using the whole genome: CD8a signal peptide-BCR scFv (VH-Linker-VL), CD8a hinge-Flag tag-CD8a transmembrane domain-41BB-CD3ζ. The specific amino acid sequence is shown in Table 3, and the structural diagram is shown in the figure. Figure 4 As shown in the figure. The above nucleic acid molecules were then cloned into viral vectors to obtain plasmids expressing M11-BBz-CAR, M12-BBz-CAR, M13-BBz-CAR, M21-BBz-CAR, M22-BBz-CAR, and M23-BBz-CAR, respectively.

[0069] Table 3 shows the amino acid sequences of CAR nucleic acid molecules synthesized from six BCR sequences.

[0070]

[0071] 2. After constructing and extracting the CAR plasmid, 293T17 cells were transduced using calcium phosphate transfection reagent to produce the virus. The viral supernatant was collected after 48 hours. T cells were obtained by grinding and sorting the spleens of healthy mice. Then, the T cells were activated using anti-CD3 / CD28 activation magnetic beads. After 50 hours of activation, the viral supernatant was added to the T cells for centrifugation infection. The infection MOI value was 10-30. After the infection was completed, the medium was replaced with fresh 10% RIPM1640 medium and placed in an incubator for normal cell culture and expansion.

[0072] The six types of CAR-T cells prepared in this embodiment are M11-BBz-CAR-T, M12-BBz-CAR-T, M13-BBz-CAR-T, M21-BBz-CAR-T, M22-BBz-CAR-T, and M23-BBz-CAR-T (abbreviated as M11, M12, M13, M21, M22, and M23). The results are as follows... Figure 5 As shown.

[0073] Example 3

[0074] Effects of BCR-CAR-T cells on B16 melanoma cells

[0075] B16-WT cells were selected as the target cells, and untransduced primary T cells were used as a negative control. Six types of BCR-BBz-CAR-T cells were seeded in 96-well U-shaped plates at different effector-to-target ratios (1, 3, 9), with three replicates per group. Specifically, 1E+5 tumor cells were added to each well, and the corresponding number of untransduced T cells or CAR-T cells were added according to the different effector-to-target ratios. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in an incubator (37℃, 5% CO2) for 40 hours. Cell apoptosis was detected by flow cytometry. Results are as follows: Figure 6 As shown, after 40 hours of co-culture, all six types of BCR-CAR-T cells were able to kill B16 tumor cells to varying degrees.

[0076] Simultaneously, the supernatant from the co-incubation group with an effector-target ratio of 1 was collected for ELISA to detect the secretion of effector factors IFNγ and IL-2. The results are as follows: Figure 7 As shown, the BCR-CAR-T cells constructed using the method of this invention can all specifically secrete high levels of effector factors on B16 tumor cells.

[0077] The above results indicate that the BCR-CAR-T cells constructed using the method of this invention have the ability to specifically kill B16 melanoma tumor cells and secrete high levels of effector factors IFNγ and IL-2 to combat B16 melanoma cells.

[0078] Example 4

[0079] Effects of BCR-CAR-T cells on a B16-WT tumor mouse model

[0080] Melanoma tumor cells suspended in PBS were injected subcutaneously into the groin area of ​​the dorsal thigh of female C57BL / 6J mice. After confirming subcutaneous tumor formation on day 6, 35 tumor-forming female C57BL / 6J mice were randomly divided into 7 groups of 5 mice each: a control group (untransduced T cells group, UTD, intravenous infusion of untransduced T cells in saline) and a BCR-CAR-T group (6 groups in total). Each group received intravenous infusion of 6 different CAR-T cells prepared in Example 2 at a dose of 1×10⁻⁶. 7 Cells / animal, each group was infused with cells once, and the day of cell infusion was recorded as day 0 of the experiment. Tumor volume was measured every three days thereafter, and tumor growth was monitored until the end of the experiment.

[0081] The results are as follows Figure 8 As shown, compared with the control group (UTD), the tumor volume of mice in all six BCR-CAR-T cell groups (referred to as M11, M12, M13, M21, M22, and M23) was reduced to varying degrees. During the experiment, all mice in the control group died on day 15, while mice in the six BCR-CAR-T cell groups remained stable at the end of the experiment. These results indicate that the BCR-CAR-T cells constructed using the method of this invention can effectively inhibit tumor growth in tumor-bearing mice and significantly prolong their survival.

[0082] To further confirm their tumor-inhibiting effects, a second, repeated animal experiment was conducted using M12-BBz-CAR-T and M21-BBz-CAR-T cells, which showed significant tumor-inhibiting effects in the first animal experiment. Healthy 6-week-old C57BL / 6J mice were treated with tumor-bearing cells (using the same method as above), and then the two types of CAR-T cells (at the same dosage) were reinfused into the mice. Tumor volume in the tumor-bearing mice was observed and measured. The results of the repeated experiment still showed that both CAR-T cells had significant tumor-inhibiting effects (e.g., ...). Figure 9 (As shown).

[0083] Example 5

[0084] Effects of BCR-CAR-T cells on YUMM1.7 tumor cells

[0085] To investigate the universality of BCR-based CAR-T cells in killing tumor cells, another type of melanoma cell, YUMM1.7, was selected as the target cell. (YUMM1.7 is a cell line containing several of the most common key mutations in human melanoma (BRAF)). <v600e>The BCR-CAR-T cells (such as PTEN deletion) were precisely introduced into mouse genes to create an artificially induced melanoma model that highly mimics the molecular characteristics of human BRAF-mutant melanoma. Untransduced primary T cells were used as a negative control. The six BCR-CAR-T cells constructed in Example 2 were seeded into 96-well U-shaped plates at different effector-to-target ratios (1, 3), with three replicates per group. Specifically, 1 × 10⁻⁶ tumor cells were placed in each well. 5 Cells were added in appropriate numbers of untransduced T cells or CAR-T cells according to different effector-to-target ratios. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in an incubator (37℃, 5% CO2) for 40 hours. Cells were then collected, and apoptosis of tumor cells was measured by flow cytometry. Results are as follows: Figure 10 As shown.

[0086] After 40 hours of co-culture, all six different BCR-CAR-T cell lines were able to kill YUMM1.7 tumor cells to varying degrees. Simultaneously, the supernatant from the co-incubation group with an effector-to-target ratio of 1 was collected for ELISA to detect the secretion of effector factors IFN-γ and IL-2. The results are as follows: Figure 11 As shown, the BCR-CAR-T cells provided by this invention specifically secrete high levels of effector factors in YUMM1.7 tumor cells.

[0087] The above results indicate that the BCR-CAR-T cells provided by this invention have the ability to specifically kill YUMM1.7 tumor cells and secrete high levels of effector factors IFNγ and IL-2 to combat YUMM1.7 cells.

[0088] Example 6

[0089] Effects of BCR-CAR-T cells on YUMM1.7 tumor model mice

[0090] YUMM1.7 tumor cells suspended in PBS were injected subcutaneously into the groin area of ​​the dorsal thigh of female C57BL / 6J mice. After confirming subcutaneous tumor formation on day 6, 35 tumor-forming female C57BL / 6J mice were randomly divided into 7 groups of 5 mice each: a control group (untransduced T cells, UTD, intravenous infusion of untransduced T cells with saline) and a BCR-CAR-T group (6 groups in total). The respective groups received intravenous infusion of corresponding CAR-T cells at a dose of 1×10⁻⁶. 7 Cells / animal, each group was infused with cells once, and the day of cell infusion was recorded as day 0 of the experiment. Tumor volume was measured every three days thereafter, and tumor growth was monitored until the end of the experiment.

[0091] The results are as follows Figure 12 As shown, compared with the control group (UTD), except for M23, the other five BCR-CAR-T cell groups (referred to as M11, M12, M13, M21, and M22) all showed varying degrees of inhibitory effects on mouse tumor growth. During the experiment, all mice in the control group died on day 14, while mice in the six BCR-CAR-T groups remained stable at the end of the experiment. These results indicate that the BCR-CAR-T cells constructed using the method of this invention can effectively inhibit tumor growth in melanoma mice and significantly prolong the survival of tumor-bearing mice.

[0092] In summary, this invention is the first to propose a method for constructing BCR-CAR-T cells using natural tumor-specific BCR sequences as the antigen recognition region of CAR-T cells. BCR-CAR-T cells obtained using this method exhibit excellent anti-tumor effects against different tumor types, both in vivo and in vitro. They can effectively inhibit tumor growth, secrete high levels of effector factors IFNγ and IL-2, thereby activating immune cells and enhancing anti-tumor capabilities.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening tumor-targeting BCRs, characterized in that, The method is performed according to the following steps: (1) Extract B cells from in vitro samples from individuals, analyze them using high-throughput single-cell sequencing and BCR immune repertoire information, and screen out B cells with complete BCR variable region sequences; Specifically, the assembly and consensus sequence assembly and annotation methods for the immune repertoire are as follows: When performing data analysis using SeekOne® Tools software, clean reads are first compared with known V(D)J fragments. Paired reads that align to at least 15 bp of the fragments are retained. Then, contig assembly and annotation screening are performed for each cell. The selected and retained single-cell contigs are used for subsequent consensus sequence assembly in samples. For high-quality contigs in a sample, consensus sequence assembly is further performed by combining contigs from all effective cells in that sample. The results of the consensus sequence assembly and annotation are then used for subsequent BCR genotyping. Specifically, the B cell with a complete BCR sequence refers to a B cell that simultaneously possesses complete heavy chain variable region (VH) and light chain variable region (VL) sequences, wherein the variable region sequence includes a complete complementarity determining region (CDR) sequence and a framework region (FR) sequence. (2) Further analysis and screening of BCR recognition sequences obtained from peripheral blood-derived B cells and tumor tissue-infiltrating B cells were performed. The specific method was as follows: All CDR sequences from the light chain variable region (VL) and heavy chain variable region (VH) of each B cell were extracted individually and uniformly defined as "one BCR recognition sequence" for subsequent cloning frequency calculation, comparison, and screening. Based on the cloning frequency of each BCR recognition sequence, BCR recognition sequences that appear only in tumor tissue infiltrating B cells but are not detected in peripheral blood B cells were first selected. The BCR recognition sequences that meet the above requirements were used as the first screening results, with n sequences as the first screening results. When n < 3, the sequences were then sorted according to the cloning frequency of the heavy chain, and the top m sequences by heavy chain frequency were selected and combined with their corresponding light chains to form BCR recognition sequences. Where n + m = 3, n = 0, 1, 2, 3, and m = 0, 1, 2, 3. Specifically, the BCR identification sequence includes three CDR sequences corresponding to the light chain variable region (VL) and the heavy chain variable region (VH), namely VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3. (3) The three BCR recognition sequences obtained according to the method in step (2) are supplemented with their corresponding skeleton region sequences to form the tumor-targeting BCR sequences.

2. A CAR-T cell based on tumor-targeting BCR, characterized in that, The CAR-T cells are obtained by modifying T cells using genetic engineering techniques, with the tumor-targeting BCR sequence derived from a single individual serving as the antigen recognition domain of the chimeric antigen receptor (CAR).

3. The CAR-T cell based on tumor-targeting BCR as described in claim 2, characterized in that, The tumor-targeting BCR sequence is obtained by screening according to the method described in claim 1.

4. The method for constructing CAR-T cells based on tumor-targeting BCR as described in claim 1, characterized in that, The method includes the following steps: I. Tumor-targeting BCR sequences obtained by screening according to the method described in claim 1 II. Construction of chimeric antigen receptor CAR The CAR nucleic acid molecule is synthesized from the whole genome. The CAR contains, from the N-terminus to the C-terminus, an antigen recognition domain targeting tumor antigens, a transmembrane domain, a co-stimulatory domain, and an activation domain. III. Construction of CAR-T cells based on tumor-targeting BCR The synthesized CAR was cloned into a viral vector to obtain a plasmid expressing a CAR targeting the tumor antigen. After extracting the CAR plasmid, 293T17 cells were transduced using calcium phosphate transfection reagent to produce the virus. The viral supernatant was collected after 48 hours. T cells were isolated and then activated using anti-CD3 / CD28 activation magnetic beads. After 50 hours of activation, the viral supernatant was added to the T cells for centrifugation infection with an infection MOI of 10-30. After the infection was completed, the medium was replaced with fresh 10% RIPM1640 medium and placed in an incubator for normal cell culture and expansion, thus obtaining CAR-T cells based on tumor-targeting BCR.

5. The method for constructing tumor-targeting BCR-based CAR-T cells as described in claim 4, characterized in that, The antigen recognition domain targeting tumor antigens in step (ii) is constructed from the three BCR recognition sequences obtained in step (i).

6. The method for constructing tumor-targeting BCR-based CAR-T cells as described in claim 4, characterized in that, The transmembrane domain in step (ii) can be derived from a natural polypeptide or it can be artificially designed; the artificially designed transmembrane domain is a polypeptide that mainly includes hydrophobic residues such as leucine and valine; preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthesized transmembrane domain; alternatively, a short oligopeptide linker or polypeptide linker, such as a linker with a length of 2 to 10 amino acids, can be provided between the transmembrane domain and the intracellular domain; in one embodiment, a linker sequence having a glycine-serine continuous sequence can be used, optionally (G4S)3 or (G4S)4.

7. The method for constructing tumor-targeting BCR-based CAR-T cells as described in claim 4, characterized in that, The co-stimulatory domains in step (ii) consist of one or more of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40, and DAP10.

8. The method for constructing CAR-T cells based on tumor-targeting BCR as described in claim 4, characterized in that, The activation domains in step (ii) include one or more of CD3ζ, CD3γ, CD3δε, or CD3ε.

9. The use of the CAR-T cells constructed based on tumor-targeting BCR as described in claim 2 in the preparation of immunotherapy drugs.

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