Application of mitochondrial transcription factor TFAM in targeting CD20 and CD19 double-target chimeric antigen receptor and CAR-T cell

Abstract

The invention discloses an application of a mitochondrial transcription factor TFAM in a CD20 and CD19 targeted double-target chimeric antigen receptor (CAR) and a CAR-T cell. Specifically, the invention provides a CD20 and CD19 double-target chimeric antigen receptor for expressing a mitochondrial transcription factor TFAM, a nucleic acid molecule of the chimeric antigen receptor, a CAR-T cell for expressing the chimeric antigen receptor and the like. The CAR-T cell disclosed by the invention can be used for remarkably improving the early depletion and functional damage of the T cell under the condition of excessive antigen stimulation, and has a remarkably improved anti-tumor effect.

Description

Application of mitochondrial transcription factor TFAM in dual-target chimeric antigen receptors targeting CD20 and CD19 and CAR-T cells Technical Field

[0001] This invention belongs to the field of immunotherapy, specifically relating to the application of mitochondrial transcription factor TFAM in dual-target chimeric antigen receptors targeting CD20 and CD19 in CAR-T cells. Background Technology

[0002] Chimeric antigen receptor (CAR) T-cell immunotherapy is an emerging cancer treatment approach in recent years. Since the first CAR-T cell therapy drug was successfully launched in the United States in 2017, CAR-T cell therapy has achieved great success in various hematologic malignancies, such as acute B-lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM). However, the performance of CAR-T cell therapy in the treatment of solid tumors is still not ideal, and it still faces many limiting factors in efficacy. For example, the high heterogeneity of antigen expression in solid tumor cells, and the significant differences in antigens between different tumor cells and even within the same tumor, make it difficult for CAR-T cells to fully recognize and attack all cancer cells, and are prone to antigen escape. In addition, the tumor microenvironment (TME) constitutes a dual physical and immune barrier: the dense extracellular matrix (ECM) hinders CAR-T cell infiltration, while immunosuppressive cells (such as Tregs and MDSCs) and cytokines (such as TGF-β and IL-10) in the TME inhibit the activity and proliferation of CAR-T cells. Existing technologies can address antigen escape by using dual-targeting binders, but there is still a lack of effective strategies to address the decline in CAR-T cell efficacy caused by the tumor microenvironment.

[0003] When T cells are exposed to the tumor microenvironment for extended periods, they gradually become exhausted, exhibiting characteristics such as decreased proliferation, reduced cytokine secretion, and weakened effector function. The mechanisms of T cell exhaustion involve multiple factors, including continuous antigen stimulation, an immunosuppressive microenvironment, and upregulation of co-inhibitory receptor expression. In CAR-T therapy, T cell exhaustion may prevent CAR-T cells from sustaining proliferation and exerting their anti-tumor effects, thereby increasing the risk of relapse.

[0004] Therefore, developing methods that can significantly slow down T cell depletion and enhance the anti-tumor effect of CAR-T cells is of great significance in this field. Summary of the Invention

[0005] This invention provides the application of mitochondrial transcription factor TFAM in dual-target chimeric antigen receptor (CAR) and CAR-T cells targeting CD20 and CD19.

[0006] The purpose of this invention is to develop novel CAR-T cells to improve premature T cell depletion and functional damage under excessive antigen stimulation, increase the proportion of CD4+ T cells, and enhance the overall efficacy of CAR-T cells.

[0007] In a first aspect of the present invention, a CD20 and CD19 dual-target chimeric antigen receptor expressing the mitochondrial transcription factor TFAM is provided, wherein the chimeric antigen receptor comprises, from the amino terminus to the carboxyl terminus, a signal peptide region, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain, and the mitochondrial transcription factor TFAM.

[0008] The signal peptide region is a CD8 leader peptide;

[0009] The antigen-binding domain includes a single-chain antibody against CD20 and a single-chain antibody against CD19.

[0010] The hinge area is the CD4 hinge area;

[0011] The transmembrane structural domain is the CD28 transmembrane region;

[0012] The co-stimulatory domain is a 4-1BB co-stimulatory signaling molecule;

[0013] The signal transduction structure domain is composed of ITM1 and CD3δ of CD3ζ connected in series, and from the N end to the C end, they are ITM1 and CD3δ of CD3ζ respectively.

[0014] In another preferred embodiment, the chimeric antigen receptor is formed by sequentially linking the following modules from the amino terminus to the carboxyl terminus: CD8 leader peptide, single-chain antibody against CD20 and single-chain antibody against CD19, CD4 hinge region, CD28 transmembrane region, 4-1BB co-stimulatory signaling molecule, ITM1 and CD3δ of CD3ζ, self-cleaving region P2A, and TFAM.

[0015] In another preferred embodiment, the ITAM1 and CD3δ of CD3ζ are composed of the ITAM1 and CD3δ of CD3ζ connected in series, and from the N end to the C end, they are in the following order: ITAM1 of CD3ζ, CD3δ.

[0016] In another preferred embodiment, the single-chain antibody (from the N-terminus to the C-terminus) has the structure shown in Formula I or Formula II:

[0017] VL-VH(I);

[0018] VH-VL(II);

[0019] In this context, the "-" sign independently represents a linking peptide or a peptide bond.

[0020] In another preferred embodiment, the structure of the antigen-binding domain (or the single-chain antibody against CD20 and the single-chain antibody against CD19) is selected from the group consisting of:

[0021] (a)VH1-VL1-VH2-VL2;

[0022] (b)VH1-VL1-VL2-VH2;

[0023] (c)VL2-VH1-VL1-VH2;

[0024] (d)VH2-VH1-VL1-VL2;

[0025] (e)VL2-VL1-VH1-VH2;

[0026] (f)VH2-VL1-VH1-VL2;

[0027] (g)VL1-VH2-VL2-VH1;

[0028] (h)VH1-VH2-VL2-VL1;

[0029] (i)VL1-VL2-VH2-VH1;

[0030] (j)VH1-VL2-VH2-VL1;

[0031] Wherein, VH1 is the VH chain of the anti-CD20 single-chain antibody, and VL1 is the VL chain of the anti-CD20 single-chain antibody; VH2 is the VH chain of the anti-CD19 single-chain antibody, and VL2 is the VL chain of the anti-CD19 single-chain antibody.

[0032] In another preferred embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO:12, 16 or 18.

[0033] In another preferred embodiment, the amino acid sequence of the CD8 leader peptide is shown in SEQ ID NO:2.

[0034] In another preferred embodiment, the amino acid sequences of the VL and VH chains of the anti-CD20 single-chain antibody are shown in SEQ ID NO:4 and 6, respectively; or

[0035] The amino acid sequences of the VL and VH chains of the anti-CD20 single-chain antibody are shown in SEQ ID NO:8 and 10, respectively.

[0036] In another preferred embodiment, the amino acid sequences of the VL and VH chains of the anti-CD19 single-chain antibody are shown in SEQ ID NO:20 and 22, respectively.

[0037] In another preferred embodiment, the amino acid sequence of the linker is shown in SEQ ID NO:14.

[0038] In another preferred embodiment, the amino acid sequence of the CD4 hinge region is shown in SEQ ID NO:24.

[0039] In another preferred embodiment, the amino acid sequence of the 4-1BB co-stimulatory signaling molecule is shown in SEQ ID NO:28.

[0040] In another preferred embodiment, the amino acid sequences of ITM1 and CD3δ of CD3ζ are as shown in SEQ ID NO:30.

[0041] In another preferred embodiment, the amino acid sequence of the self-cleaving region P2A is shown in SEQ ID NO:34.

[0042] In another preferred embodiment, the amino acid sequence of the TFAM is shown in SEQ ID NO:32.

[0043] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:44 or SEQ ID NO:46.

[0044] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the chimeric antigen receptor described in the first aspect of the invention.

[0045] In a third aspect of the invention, a carrier is provided, the carrier comprising the nucleic acid molecule described in the second aspect of the invention.

[0046] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, lentiviruses, exosomes, or other vectors.

[0047] In a fourth aspect of the invention, a recombinant lentivirus is provided, which is prepared from mammalian cells transfected with the vector and helper plasmid described in the third aspect of the invention.

[0048] In a fifth aspect of the invention, a CAR-T cell is provided, the CAR-T cell expressing the dual-target chimeric antigen receptor described in the first aspect of the invention.

[0049] In a sixth aspect of the invention, a method for preparing CAR-T cells as described in the fifth aspect of the invention is provided, the method comprising the step of introducing the encoding gene of the chimeric antigen receptor as described in the first aspect of the invention or the nucleic acid molecule as described in the second aspect of the invention into T cells.

[0050] In a seventh aspect of the invention, the use of the chimeric antigen receptor described in the first aspect of the invention, the nucleic acid molecule described in the second aspect of the invention, the vector described in the third aspect of the invention, the recombinant lentivirus described in the fourth aspect of the invention, or the CAR-T cell described in the fifth aspect of the invention is provided for the preparation of a drug for treating tumors.

[0051] In another preferred embodiment, the tumor is a CD20-positive and / or CD19-positive tumor.

[0052] In another preferred embodiment, the tumor is a CD20 overexpressing and / or CD19 overexpressing tumor.

[0053] In another preferred embodiment, the tumor is selected from the group consisting of: B-cell lymphomas (such as Hodgkin lymphoma and nodular lymphoma, and non-Hodgkin lymphomas, such as diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, mantle cell lymphoma (MCL)), lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, gastric cancer, acute myeloid leukemia, or combinations thereof.

[0054] In an eighth aspect of the invention, a pharmaceutical composition is provided comprising the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, the recombinant lentivirus of the fourth aspect of the invention, or the CAR-T cell of the fifth aspect of the invention; and a pharmaceutically acceptable vector.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0056] Figure 1A shows a schematic diagram of four target CD19 / CD20 CAR domains.

[0057] Figure 1B shows four lentiviral plasmid vectors targeting the CD19 / CD20 CAR structure.

[0058] Figure 2A shows flow cytometry data of CAR molecule expression in four types of CD19 / CD20 targeted CAR-T cells.

[0059] Figure 2B shows the percentage of CD4+ / CD8+ subsets in four types of CD19 / CD20 targeted CAR-T cells.

[0060] Figure 3A shows the results of four rounds of target cell residual detection using antigen stimulation for four types of CD19 / CD20 CAR-T cells, with effector-to-target ratios of E:T = 1:1 and E:T = 1:3.

[0061] Figure 3B shows the statistical results of CAR-T cell proliferation after four rounds of antigen stimulation using four types of CD19 / CD20 CAR-T cells at an effector-to-target ratio of E:T = 1:1.

[0062] Figure 3C shows the flow cytometry results of mitochondrial damage in four types of CAR-T cells targeted with CD19 / CD20, after four rounds of antigen stimulation at an effector-to-target ratio of E:T = 1:1.

[0063] Figure 3D shows the statistical results of mitochondrial damage in four types of CAR-T cells after four rounds of antigen stimulation using four different target CD19 / CD20 CAR-T cells at an effector-to-target ratio of E:T = 1:1.

[0064] Figure 3E shows the flow cytometry results of the surface memory phenotypes of four types of CD19 / CD20 CAR-T cells after four rounds of antigen stimulation at an effector-to-target ratio of E:T = 1:1.

[0065] Figure 3F shows the statistical results of the proportion of Tcm subtypes in CD3+, CD4+, or CD8+ cells after four rounds of antigen stimulation using four types of CD19 / CD20 CAR-T cells at an effector-to-target ratio of E:T = 1:1.

[0066] Figure 4A shows the flow cytometry results of CD4+ / CD8+ cell subsets after multiple rounds of low antigen stimulation for four types of CD19 / CD20 CAR-T cells.

[0067] Figure 4B shows the statistical results of the proportion of CD4+ cell subsets after multiple rounds of low antigen stimulation for four types of CD19 / CD20 CAR-T cells.

[0068] Figure 4C shows the 24-hour killing effect of four types of CD19 / CD20 CAR-T cells on Raji target cells after the ninth round of low antigen stimulation.

[0069] Figure 5A shows the in vivo tumor clearance imaging results of Loop CAR-T w / o TFAM in animals.

[0070] Figure 5B shows the statistical results of the Loop CAR-T w / o TFAM animal survival curves.

[0071] Figure 5C shows the results of quantitative fluorescence detection of tumor burden in animals using Loop CAR-T w / o TFAM. Detailed Implementation

[0072] Through extensive and in-depth research and screening, the inventors have, for the first time, overexpressed the mitochondrial transcription factor TFAM protein in CAR-T cells to improve premature T cell exhaustion and functional impairment under excessive antigen stimulation, increase the proportion of CD4+ T cells, and enhance the overall anti-tumor function of CAR-T cells. Experiments show that CAR-T cells overexpressing TFAM exhibit a higher proportion of CD4+ T cells in the CAR-T cell subset compared to the control group, significantly less mitochondrial damage after continuous antigen stimulation, and significantly higher CAR-T cell proliferation. Simultaneously, in vivo experiments demonstrate that overexpression of TFAM can significantly improve the therapeutic effect of CAR-T therapy. Based on these findings, this invention was completed.

[0073] the term

[0074] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0075] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101.

[0076] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0077] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0078] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0079] TFAM

[0080] Mitochondrial transcription factor A (TFAM) plays an important role in mitochondrial DNA (mtDNA) transcription, replication, assembly, and stability. TFAM is a mitochondrial transcription factor expressed by nuclear genes. The TFAM protein consists of two HMG (high mobility group) domains, HMG1 and HMG2, and a carboxyl terminus containing 25 amino acids.

[0081] Recent studies have demonstrated that TFAM also contains an LC3 interaction region (LIR) motif. HMG has a high affinity for mtDNA, enabling it to rapidly recognize and bind to upstream motifs of mitochondrial DNA promoters, promoting mitochondrial DNA transcription. TFAM can also bind tightly to mitochondrial DNA, forming a stable complex that protects mtDNA from damage and degradation. The key significance of TFAM for normal cellular physiological function lies in its role as a core regulator of mitochondrial function, directly influencing cellular survival and normal activity. For example, in cellular energy metabolism, TFAM regulates mtDNA transcription, controlling the synthesis of mitochondrial respiratory chain complexes, thereby affecting cellular energy production. TFAM also participates in the regulation of intracellular oxidative stress responses, protecting cells from oxidative damage.

[0082] Furthermore, the LC3 interaction region (LIR) motif in the TFAM protein structure interacts with the autophagy protein LC3 via the autophagosome pathway (called nucleophagy), helping to eliminate leaked mtDNA. TFAM may be a potential target for anti-inflammatory therapy.

[0083] Signal transduction domain

[0084] The chimeric antigen receptor described in this invention includes a signal transduction domain. This signal transduction domain provides a sequence for initiating intracellular signal transduction in CAR-expressing cells.

[0085] In some embodiments, the signal transduction domain includes an ITAM-containing sequence, which is or is derived from an ITAM-containing amino acid sequence of a protein having an ITAM-containing amino acid sequence. The ITAM-containing sequence includes one or more immune receptor tyrosine-based activation motifs (ITAMs). In some embodiments, the signal transduction domain includes an ITAM-containing sequence, which is or is derived from an amino acid sequence of an intracellular domain of a protein selected from the group consisting of: CD3ζ, CD3δ, CD3ε, CD3γ, CD79α, CD79β, FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIV, or DAP12. In some embodiments, the signal transduction domain includes an ITAM-containing sequence, which is or is derived from an amino acid sequence of the CD3-ζ intracellular domain.

[0086] There are four molecules in the TCR complex that contain the ITAM motif: CD3γ, CD3δ, CD3ε, and CD3ζ. CD3γ, CD3δ, and CD3ε each contain one immune receptor tyrosine activation motif (ITAM), while CD3ζ contains three ITAMs. The phosphorylation pattern of the CD3ζ immune receptor tyrosine activation motif (ITAM) directly affects signal intensity. The chimeric signal transduction domain of this invention contains the phosphorylation site of the first ITAM of CD3ζ (i.e., ITAM1 of CD3ζ).

[0087] Theoretically, any signal transduction domain containing the ITAM motif in a CAR structure can activate downstream signals and perform CAR-T anti-tumor function. However, in practical applications, CAR structures constructed using CD3γ or CD3ε molecules have weak signal transduction capabilities and lack co-stimulatory integration sites, failing to meet the requirements of CAR-T for efficient activation and sustained killing.

[0088] The CD3δ molecule contains a BRS motif. In its inactive state, the BRS motif is encapsulated in a negatively charged cell membrane, which can easily affect the endocytosis of CAR molecules and cause unstable CAR expression, thereby affecting CAR-T cell function. On the other hand, phosphorylated BRS recruits the inhibitory kinase Csk through ITAM, competitively replacing Lck and inhibiting its activity, thus restoring the signal to baseline and avoiding side effects such as cytokine release syndrome (CRS).

[0089] Nucleic Acids and Vectors

[0090] This invention provides one or more nucleic acids encoding the CAR described herein. In some embodiments, the nucleic acid comprises or is composed of DNA and / or RNA.

[0091] In some embodiments, the nucleic acid is or is contained in one or more vectors. That is, the nucleotide sequence of the nucleic acid may be contained in a vector. The CAR of the present invention can be generated by transcription and translation within cells via a vector, which encodes the antigen-binding molecule, polypeptide, or CAR.

[0092] Therefore, the present invention also provides one or more vectors comprising the nucleic acids or multiple nucleic acids described in the present invention. The vectors facilitate the delivery of nucleic acids encoding the CARs described in the present invention. The vectors may be expression vectors containing elements required for expressing the CARs described in the present invention.

[0093] The nucleic acids and vectors described in this invention can be provided in purified or isolated form, i.e., separated from other nucleic acids or natural biological materials.

[0094] The nucleotide sequence may be contained in a vector (such as an expression vector). As used herein, "vector" refers to a nucleic acid molecule used to transfer exogenous nucleic acids into cells. The vector may be a vector for expressing nucleic acids in cells. The vector may include a promoter sequence operatively linked to the nucleotide sequence to be expressed. The vector may also contain a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express peptides or polypeptides in the vectors described herein.

[0095] The term "operably linked" refers to the covalent linkage of a selected nucleic acid sequence to a regulatory nucleic acid sequence (such as a promoter and / or enhancer) in such a way that the expression of the nucleic acid sequence is influenced or controlled by the regulatory sequence (thus forming an expression cassette). Therefore, if a regulatory sequence can influence the transcription of a nucleic acid sequence, then the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0096] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (such as retroviral vectors, for example, gamma-retroviral vectors (such as murine leukemia virus (MLV)-derived vectors, such as SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (such as yeast artificial chromosomes).

[0097] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements required for expressing the vector protein in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, such as a vector containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0098] Cells expressing the CAR described in this invention

[0099] In some aspects and embodiments, the present invention provides a cell comprising the CAR described herein. The CAR described herein can be used to generate cells expressing CAR, such as CAR-expressing immune cells (e.g., CAR-T cells).

[0100] It is understandable that when this article refers to a singular cell (such as "a type / described cell"), it also covers the plural / population of such cells.

[0101] Cells expressing CAR may contain or express nucleic acids encoding the CAR described in this invention. It is understood that cells expressing CAR contain the CAR they express. Similarly, it is understood that cells expressing nucleic acids encoding CAR also express and contain the CAR encoded by that nucleic acid.

[0102] Some aspects and embodiments of the present invention particularly relate to T cells comprising / expressing the CD19 and CD20 bispecific CARs described herein.

[0103] The main advantages of this invention include:

[0104] (a) CAR-T cells overexpressing the mitochondrial transcription factor TFAM of the present invention can significantly improve premature exhaustion and functional impairment of T cells under excessive antigen stimulation conditions.

[0105] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0106] Example 1: Preparation of CD20-CD19-4-1BB-TFAM CAR-T cells

[0107] (1) Construction of lentiviral overexpression vector: The sequence contains antibody sequences targeting CD19 and CD20 and the fusion gene of the mitochondrial transcription factor TFAM. The connection sequence of the chimeric antigen receptor structure is shown in Figure 1A. Furthermore, the main structures in the chimeric antigen receptor are as follows:

[0108] (a) CD8 precursor peptide, whose nucleotide and amino acid sequences are shown in SEQ ID NO:1 and 2, respectively;

[0109] (b) Anti-CD19 SCFV, the amino acid sequences of its VL and VH are shown in SEQ ID NO:20 and 22, respectively;

[0110] (c) Anti-CD20 SCFV, the amino acid sequences of its VL and VH are shown in SEQ ID NO:4 and 6, respectively, or in SEQ ID NO:8 and 10, respectively;

[0111] (d) CD4 hinge region, whose nucleotide and amino acid sequences are shown in SEQ ID NO:23 and 24, respectively;

[0112] (e) CD28 transmembrane region, whose nucleotide and amino acid sequences are shown in SEQ ID NO:25 and 26, respectively;

[0113] (f)4-1BB, whose nucleotide and amino acid sequences are shown in SEQ ID NO:27 and 28, respectively;

[0114] (g) The amino acid sequence of the intracellular signal transduction region, CD3ΔZD (i.e., the tandem CD3ζ of ITM1 and CD3δ), is shown in SEQ ID NO:30;

[0115] (h)P2A, whose nucleotide and amino acid sequences are shown in SEQ ID NO:33 and 34, respectively;

[0116] (i) The TFAM fusion gene, whose nucleotide and amino acid sequences are shown in SEQ ID NO:31 and 32, respectively;

[0117] The fusion gene was artificially synthesized by Shanghai Qingke Biotechnology Co., Ltd. using conventional genetic engineering methods and cloned into the pLenti-EF1a overexpression vector. Plasmid extraction was also performed by Shanghai Qingke Biotechnology Co., Ltd., and the plasmid was stored at -80℃ for later use. The plasmid map is shown in Figure 1B.

[0118] The constructed lentiviral expression vectors were named as follows:

[0119] Tan-CD20-CD19-CART (or Tan CAR-T);

[0120] Tan-CD20-CD19-CART-TFAM (or Tan CAR-T TFAM);

[0121] Loop-CD20-CD19-CART (or Loop CAR-T);

[0122] Loop-CD20-CD19-CART-TFAM (or Loop CAR-T TFAM).

[0123] (2) Preparation, packaging and concentration of lentiviruses

[0124] 293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Once the cells reached the logarithmic growth phase, they were digested with trypsin, centrifuged, counted, and resuspended. 20 million total cells were then seeded into a new 15 cm dish, mixed, and transferred to a 37°C, 5% CO2 incubator for overnight culture until the virus was packaged.

[0125] Three plasmids, pMD2.G, pRSV-Rev, and pMDLg pRRE, along with the lentiviral overexpression vector, were mixed thoroughly in 1.5 ml of serum-free and antibiotic-free Opti-MEM medium at a specific ratio. 128 μL of transfection reagent PEI was added to 1372 μL of serum-free and antibiotic-free Opti-MEM medium and mixed thoroughly. The 1.5 ml mixtures of the above reagents were then thoroughly combined and incubated at room temperature for 12 minutes. The lentiviral packaging system is shown in Table 1 below.

[0126] Table 1

[0127] pMD2.G 4.16 μg, pRSV-Rev 10.4 μg, pMDLg, pRRE 3.38 μg, target plasmid 16.6 μg, total 34.54 μg surface

[0128] Take 12 ml of serum-free and antibiotic-free Opti-MEM culture medium and add it to the 3 ml mixture described above. Gently pipette and mix thoroughly. Add the final mixture to a 15 cm dish of 293T cells, mix well, and incubate at 37°C with 5% CO2 for 5.5 hours. Discard the supernatant, add 30 ml of high-glucose DMEM medium containing 2% fetal bovine serum, and incubate at 37°C with 5% CO2. Collect the supernatant of 293T cells after 48 hours.

[0129] Approximately 30 ml of 293T cell supernatant was collected in a 50 ml centrifuge tube. After centrifugation at 3000 rpm for 10 minutes, the supernatant was collected and filtered through a 0.25 μm filter flask. The filtrate was transferred to a 35 ml ultracentrifuge tube. This tube was then transferred to a high-speed metal centrifuge tube, vacuum-sealed, and centrifuged at 25000 rpm for 2 hours. The vacuum was removed, the ultracentrifuge tube was removed with forceps, the supernatant was aspirated, and 160 μL of serum-free, antibiotic-free, and cytokine-free Lonza medium was added. The tube was then incubated overnight at 4°C to thaw the virus particles. The next day, the virus suspension was gently pipetted, aliquoted, and the concentrated virus solution was transferred to a -80°C freezer for cryopreservation.

[0130] (3) Sorting, activation and infection of human peripheral blood pan-T cells

[0131] PBMC cell isolation (provided by Shanghai Saili Biotechnology Co., Ltd.)

[0132] Leukopark disinfects the outer surface of the incision and transfers it to a 50ml centrifuge tube, with each tube containing no more than 20ml. Take out 20ul of cell suspension, dilute it 50 times, and count the cells. Take a new 50ml centrifuge tube, add 20ml of lymphocyte separation medium to the lower layer, and add 15-17ml of diluted apheresis blood to the upper layer to form density stratification.

[0133] Carefully transfer to a centrifuge, centrifuge at 600g at room temperature for 25 minutes, increment 4 and decrement 5, transfer the white membrane layer cells (~10ml) to a new 50ml centrifuge tube, add 40ml washing buffer, invert 5 times to mix the cells, centrifuge at 600g at room temperature for 10 minutes, increment 9 and decrement 9, and collect the cells;

[0134] Discard the supernatant suspension, resuspend the precipitate in 10 ml washing buffer, and transfer it to a new 50 ml centrifuge tube.

[0135] Combine 4 tubes into 1 tube;

[0136] Invert the tube 5 times to mix the cells, take 20 μL, dilute it 50 times, count the cells, and calculate the PBMC cell isolation yield.

[0137] (4) Pan-T cell sorting

[0138] Collect cells by centrifuging at 400g at room temperature for 10 min, resuspend the cells with washing buffer, and adjust the cell density to 50 million / ml. Add pan-T isolation antibody cocktail at a ratio of 50ul / ml, mix the cells, and incubate at room temperature for 5 min.

[0139] Add Rapidsphere at a ratio of 40 μL / mL, mix the cells, and incubate at room temperature for 3 min. Insert a magnet, let stand for 10 min, and aspirate the supernatant into a new 50 mL centrifuge tube. Take a sample, dilute and count the cells, and calculate the Pan-T separation yield.

[0140] Cells were collected by centrifugation at 400g at room temperature for 10 min. The cells were resuspended in cryopreservation buffer (90% fetal bovine serum and 10% DMSO) and aliquoted into cryovials at a density of 50 million / vial and 20 million / vial. The cells were then transferred to a cryopreservation box and stored at -80°C overnight, and then transferred to a liquid nitrogen tank for long-term storage.

[0141] (5) Pan-T activation and infection

[0142] Pan-T cells were taken from liquid nitrogen, thawed in a 37°C water bath, and transferred to a 15ml centrifuge tube. 9ml of preheated Lonza medium at 37°C was added, and the mixture was mixed well. 20ul of AOPI reagent was mixed with the cells and counted. The mixture was centrifuged at 300g for 6 minutes.

[0143] Resuspend the cells in Lonza medium containing 2% human serum albumin and 300 IU / ml IL2, and adjust the cell concentration to 2 million / ml.

[0144] Wash the anti-CD3 / CD28 magnetic beads once with Lonza medium containing 2% human serum albumin and 300 IU / ml IL2, and centrifuge at 2000 rpm for 5 minutes.

[0145] Following a T-cell:magnetic bead ratio of 1:1, the washed anti-CD3 / CD28 magnetic beads were thoroughly mixed with T cells and transferred to 6-well plates. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0146] (6) Lentiviral infection

[0147] Specifically, the following steps are included:

[0148] Take the activated T cells, gently disperse them, transfer them to a 15ml centrifuge tube, and place them on a magnetic rack for 3 minutes. Use a pipette to collect the supernatant and transfer it to a new 15ml centrifuge tube. Count the cells, adjust the cell density to 1 million / ml, and seed 1ml / well in a 12-well plate.

[0149] Calculate the required viral load based on an MOI of 3. The formula is as follows: Required viral load = (MOI × number of cells) / viral titer;

[0150] Remove the virus at -80℃, thaw it rapidly in a 37℃ incubator, add the required amount of virus according to the calculation, mix well, and incubate for 24 hours in a 37℃, 5% carbon dioxide incubator.

[0151] After 24 hours, the cells were counted, and the cell density was adjusted to 0.5 million / ml with fresh Lonza medium containing 300 IU / ml IL2. The cells were then cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0152] 24 hours later, the cells were counted, the cell density was adjusted to 0.5 million / ml with fresh Lonza medium containing 300 IU / ml IL2, and the cells were transferred to T75 culture flasks and cultured for 48 hours.

[0153] Cells were counted, and a portion of the cells were used to detect the expression of CAR molecules on the cell surface using flow cytometry. The cell density was then adjusted to 1 million / ml with fresh Lonza medium containing 300 IU / ml IL2 and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0154] Cells were counted, and a portion of the cells were used to detect the expression of CAR molecules on the cell surface using flow cytometry. After centrifugation at 300g for 6 minutes, the supernatant was removed, and cryopreservation solution was prepared with 90% fetal bovine serum and 10% DMSO. 50 million cells were cryopreserved in each tube, placed in a cryopreservation box, and stored overnight at -80°C. The next day, the cells were transferred to liquid nitrogen for cryopreservation until use.

[0155] (6) Flow cytometry was used to detect the proportion of CAR-T cells and the percentages of CD4 and CD8 cells.

[0156] Specifically, the following steps are included:

[0157] Preparation of cell suspension: Collect expanded cultured cells, count them, take 0.1 million cells and add them to a U-shaped 96-well plate, centrifuge at 300g for 6 minutes;

[0158] Prepare antibody mixture: 0.2ul / test Percp Anti-CD3, 0.25ul / test APC Anti-FMC63, 0.2ul / test PE / CY7 Anti-CD4, 0.2ul / test APC / CY7 Anti-CD8, gently mix with a 1ml pipette, add 50ul to the centrifuged cells (after removing the supernatant), and incubate at room temperature in the dark for 1 hour.

[0159] Washing: Add 200 μL of PBS buffer to each well, centrifuge at 300g for 6 minutes, wash twice, resuspend cells in 120 μL of PBS buffer, and analyze.

[0160] Data were analyzed using FlowJo V-10.10 software, and the results are expressed as the positive rate of APC Anti-FMC63 (Figure 2A) and the proportions of CD4 and CD8 (Figure 2B).

[0161] The results are shown in Figures 2A and 2B, respectively. The results showed that in tandem CAR-T, the proportion of CAR+ cells in CAR-T cells without TFAM was 76.6%, while the proportion of CAR+ cells in the TFAM overexpression group was 64.9%; in loop CAR-T, the proportion of CAR+ cells in CAR-T cells without TFAM was 68.0%, while the proportion of CAR+ cells in the TFAM overexpression group was 57.8% (Figure 2A). This indicates that TFAM overexpression does not affect the expression level of CAR molecules in CAR-T cells.

[0162] Furthermore, in tandem CAR-T, the proportion of CD4+ cells in CAR-T cells without TFAM was 56.0%, while in the TFAM-overexpressing group it was 53.2%; in loop CAR-T, the proportion of CD4+ cells in CAR-T cells without TFAM was 56.5%, while in the TFAM-overexpressing group it was 53.5% (Figure 2B). This demonstrates that TFAM overexpression does not significantly affect the proportion of CD4+ cells in CAR-T cells.

[0163] Example 2: Detection of the in vitro antitumor effect of CD20-CD19-CAR-T cells

[0164] CD20-CD19-CAR-T cell continuous killing experiment against Raji-Luciferase cells

[0165] (1) Detection of antitumor killing effect by high antigen stimulation

[0166] The Tan-CD20-CD19-CAR-T cells, Tan-CD20-CD19-CART-TFAM cells, Loop-CD20-CD19-CART cells, and... prepared in Example 1 were used...

[0167] Loop-CD20-CD19-CART-TFAM and untreated (non-treated, NT) control cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and transferred to 9 ml of preheated Lonza medium containing 300 IU / ml IL-2 and 2% human serum albumin. Cells were counted, centrifuged at 300g for 6 minutes, and the cell density was adjusted to 4 million / ml. The cells were then transferred to T25 culture flasks and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0168] Then, cell counting was performed, and corresponding CAR-T cells and 0.2 million Raji-Luciferase cells were co-cultured in 24-well plates at different ratios (E:T = 3:1, E:T = 1:1). After 72 hours, the killing effect was detected by flow cytometry (BV421 Anti-CD3, PE Anti-CD22 0.2ul / test 50ul).

[0169] If no CD22-positive cells are detected, it means that the target cells have been completely cleared. The cell count is restarted, and the cells are re-plated according to the above-mentioned effective target ratio for the second round of killing. This process is repeated until the CAR-T cell viability drops below 50%, the target cells cannot be cleared, and the CAR-T cells stop proliferating.

[0170] The fourth round of killing assays was performed on target cells (Figure 3A), followed by continuous stimulation of CAR-T cell proliferation (Figure 3B), CD4 / CD8 ratio, mitochondrial damage (Figures 3C and 3D), and CAR-T cell phenotype (Figure 3E), which were then statistically analyzed. Mitochondrial assay: MitoTracker Deep Red FM (1:10000) and MitoTracker Green FM (1:5000) were diluted in PBS buffer. 100 μL of each solution was added to the cell pellet after washing twice with 0.1 million μL of PBS buffer. The mixture was gently pipetted with a 1 mL pipette and incubated at room temperature in the dark for 30 minutes. After washing twice with PBS buffer, the cells were resuspended in 120 μL of PBS and analyzed.

[0171] The results are shown in Figures 3A-3F. Figure 3A shows that overexpression of TFAM enhances the killing ability of CAR-T cells against target cells; Figure 3B shows that under high antigen stimulation, overexpression of TFAM significantly upregulates the proliferation ability of CAR-T cells; Figures 3C and 3D show that overexpression of TFAM significantly reduces mitochondrial damage in CAR-T cells; Figures 3E and 3F show that under high antigen stimulation, the proportion of Tcm in CAR-T cells overexpressing TFAM is significantly higher than that in the control group, indicating that overexpression of TFAM can improve the persistence of CAR-T cells in vitro.

[0172] (2) Low antigen stimulation antitumor killing detection

[0173] The Tan-CD20-CD19-CAR-T cells, Tan-CD20-CD19-CART-TFAM cells, Loop-CD20-CD19-CART cells, and... prepared in Example 1 were used...

[0174] Loop-CD20-CD19-CART-TFAM and NT cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and transferred to 9 ml of preheated Lonza medium containing 300 IU / ml IL-2 and 2% human serum albumin. Cells were counted, centrifuged at 300g for 6 minutes, and the cell density was adjusted to 4 million / ml. The cells were then transferred to T25 culture flasks and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0175] Then, cell counting was performed, and CAR-T cells and 2 million Raji-Luciferase cells were taken separately according to...

[0176] In a 1:1 E:T ratio, Raji-Luciferase cells were co-cultured with the cells. For the first five rounds, 2 million Raji-Luciferase cells were added every 48 hours, along with an equal volume of fresh 1640 medium containing 10% fetal bovine serum. From the sixth round onwards, half of the cell suspension was discarded for each stimulation, and the same volume of fresh 1640 medium containing 10% fetal bovine serum was added until the CAR-T cell expansion rate was less than 1. Flow cytometry was used to analyze the CD4 / CD8 ratio of CAR-T cells in each round (Figures 4A and 4B). The ninth round of CAR-T cells was then used to culture Raji-Luciferase cells in different ratios (E:T = 4:1, E:T = 2:1, E:T = 1:1, E:T = 1:2) in 96-well plates. After 24 hours, the luciferase reporter gene (promega) was detected. The luciferase reporter gene detection system uses tumor cell lysis rate to represent the killing activity of the corresponding CAR-T cells (as shown in Figure 4C).

[0177] The results are shown in Figures 4A-4C. Figures 4A and 4B show that under continuous low antigen stimulation, the proportion of CD4 in TFAM-overexpressing CAR-T cells was higher than that in the control group, indicating that TFAM can maintain the excessive depletion of CD4 cell subsets; Figure 4C shows that TFAM overexpression can enhance CAR-T killing ability.

[0178] Example 3: Detection of the in vivo antitumor effect of CD20-CD19-TFAM-CAR-T cells

[0179] A hematologic malignancy orthotopic tumor model was established by inoculating Raji-Luc cells (human Burkitt's lymphoma cells, provided by Shanghai Lidi Biotechnology Co., Ltd.) into the tail vein of NCG mice. The pharmacodynamic effects of CAR-T cell therapy on this tumor model were then evaluated. Female NCG mice, 6-8 weeks old and weighing approximately 18-22g, were purchased from Jiangsu Jicui Pharmaceutical Technology Co., Ltd.

[0180] Cells in the logarithmic growth phase were resuspended in PBS, counted, and inoculated at a rate of 0.5 million cells / mouse, with a cell volume of 0.2 mL / mouse, via tail vein inoculation. Three days after inoculation, mice were selected for pharmacodynamic grouping based on total fluorescence flux, randomly divided into three groups of three mice each, with the grouping day designated as day 0. During the period, mouse weight was measured twice weekly, and mice were imaged every 7 days using a small animal in vivo imaging system. Tumor burden differences among the three groups were compared, mouse mortality times were recorded, and survival curves were plotted.

[0181] The results are shown in Figures 5A-5C. The results indicate that overexpression of TFAM can enhance the in vivo antitumor effect of CAR-T cells.

[0182] The sequence information of this invention is shown in Table A.

[0183] Table A

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A chimeric antigen receptor expressing the mitochondrial transcription factor TFAM, targeting CD20 and CD19, characterized in that, The chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide region, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain, and the mitochondrial transcription factor TFAM; the signal peptide region is the CD8 leader peptide; the antigen-binding domain includes single-chain antibodies against CD20 and CD19; the hinge region is the CD4 hinge region; the transmembrane domain is the CD28 transmembrane region; the co-stimulatory domain is a 4-1BB co-stimulatory signaling molecule; and the signal transduction domain is composed of CD3ζ ITAM1 and CD3δ tandemly, and from the N-terminus to the C-terminus, they are sequentially: CD3ζ ITAM1, CD3δ.

2. The chimeric antigen receptor as described in claim 1, characterized in that, The single-chain antibody has the structure shown in Formula I or Formula II from the N-terminus to the C-terminus: VL-VH(I); VH-VL(II); wherein the "-" is independently a linking peptide or peptide bond.

3. The chimeric antigen receptor as described in claim 1, characterized in that, The structure of the antigen-binding domain is selected from the group consisting of: (a) VH1-VL1-VH2-VL2; (b) VH1-VL1-VL2-VH2; (c) VL2-VH1-VL1-VH2; (d) VH2-VH1-VL1-VL2; (e) VL2-VL1-VH1-VH2; (f) VH2-VL1-VH1-VL2; (g) VL1-VH2-VL2- VH1; (h)VH1-VH2-VL2-VL1; (i)VL1-VL2-VH2-VH1; (j)VH1-VL2-VH2-VL1; wherein, VH1 is the VH chain of an anti-CD20 single-chain antibody, VL1 is the VL chain of an anti-CD20 single-chain antibody; VH2 is the VH chain of an anti-CD19 single-chain antibody, and VL2 is the VL chain of an anti-CD19 single-chain antibody.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor as described in claim 1.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4.

6. A recombinant lentivirus, characterized in that, The recombinant lentivirus was prepared from mammalian cells transfected with the vector and helper plasmid as described in claim 5.

7. A CAR-T cell, characterized in that, The CAR-T cells express the dual-target chimeric antigen receptor as described in claim 1.

8. A method for preparing CAR-T cells according to claim 7, characterized in that, The method includes the step of introducing the encoding gene of the chimeric antigen receptor of claim 1 or the nucleic acid molecule of claim 4 into T cells.

9. The use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the recombinant lentivirus of claim 6, or the CAR-T cell of claim 7, characterized in that, Used to prepare drugs for treating tumors.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the recombinant lentivirus of claim 6, or the CAR-T cell of claim 7; and a pharmaceutically acceptable vector.

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