CHIMERIC ANTIGEN RECEPTOR COMPRISING CO-STIMULATORY RECEPTOR AND APPLICATION THEREOF

Abstract

Provided by the present invention is a chimeric antigen receptor comprising a co-stimulatory receptor, the chimeric antigen receptor having a structure of scFv(X)-(Y)CD3zeta-2A-(Z); X comprises a tumor targeting antibody or a ligand or receptor capable of specifically binding to a tumor; Y is an intracellular region of the co-stimulatory receptor, and Z is a co-stimulatory receptor that is selected from among ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIMI, SLAM, CD2, CD226. Further provided by the present invention are CAR-T cells that are constructed by means of a recombinant expression vector of the described chimeric antigen receptor, a preparation method therefor and an application thereof. The CAR-T cells described in the present invention significantly improve the tumor-killing abilities and amplification abilities thereof.

Claims

1. A chimeric antigen receptor comprising a co-stimulatory receptor, wherein said chimeric antigen receptor has a structure of scFv(X)-(Y)CD3zeta-2A-(Z); wherein X comprises a tumor-targeting antibody or a ligand or receptor capable of specifically binding to a tumor; Y is an intracellular domain of a co-stimulatory receptor, and said co-stimulatory receptor is selected from a group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226; and Z is a co-stimulating receptor, and said co-stimulatory receptor is selected from a group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226.

2. The chimeric antigen receptor comprising a co-stimulatory receptor according to claim 1, wherein said X is selected from a group consisting of anti-CD19 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-EGFRVIII antibody, anti-PSMA antibody, anti-BCMA antibody, anti-CD22 antibody, and anti-CD30 antibody.

3. The chimeric antigen receptor comprising a co-stimulatory receptor according to claim 1, wherein said X is anti-CD20 antibody, anti-CD19 antibody or EGFR antibody, said Y is 4-1BB, said Z is one selected from a group consisting of OX40, HVEM, ICOS, CD27, and 4-1BB.

4. The chimeric antigen receptor comprising a co-stimulatory receptor according to claim 3, wherein said scFv(X)-(Y)CD3zeta is anti-CD20 scFv with a sequence of SEQ ID No.1; anti-CD19 scFv with a sequence of SEQ ID No.11, and anti-EGFR scFv with a sequence of SEQ ID No.12.

5. The chimeric antigen receptor comprising a co-stimulatory receptor according to claim 3, wherein said OX40 has a sequence of SEQ ID No.2; said HVEM has a sequence of SEQ ID No.3; said ICOS has a sequence of SEQ ID No.4; said CD27 has a sequence of SEQ ID No.5; and/or, said 4-1BB has a sequence of SEQ ID No.6.

6. The chimeric antigen receptor comprising a co-stimulatory receptor according to claim 3, wherein said 2A has a sequence of SEQ ID No.7; SEQ ID No.8; SEQ ID No.9 or SEQ ID No.10.

7. A CAR-T cell constructed by a recombinant expression vector of said chimeric antigen receptor according to any one of claims 1-4.

8. A method of preparing said CAR-T cell according to claim 5, comprising the following steps: step 1, construction of lentiviral vector and production of virus; incorporating 2A between scFv(X)-(Y)CD3zeta and Z to form a fusion protein, adding a lentiviral vector to both ends of the fusion protein, and co-transfecting with a lentiviral packaging plasmid to obtain an scFv(X)-(Y)CD3zeta-2A-(Z) virus.

9. The method of preparing said CAR-T cell according to claim 8, wherein the method comprises the following steps: step 2, preparation of scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cell; culturing purified human PBMC, and infecting the T cell isolated from said PBMC with the scFv(X)-(Y)CD3zeta-2A-(Z) virus obtained in Step 1, subjecting them to cell expansion under suitable conditions to prepare the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cell.

10. The method of preparing said CAR-T cell according to claim 8, wherein said construction of lentiviral vector and production of virus comprises: incorporating 2A between scFv(X)-(Y)CD3zeta and Z by overlap PCR to form a fusion protein, and adding restriction sites to both ends of the fusion protein to clone a lentiviral vector; subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with a lentiviral packaging plasmid; after a predetermined time period, collecting a supernatant, filtering, centrifuging to concentrate the virus to obtain an scFv(X)-(Y)CD3zeta-2A-(Z) virus.

11. The method of preparing said CAR-T cell according to claim 8, wherein said preparation of said scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cell comprises: isolating T cells from human PBMC for purification, inoculating into a culture plate under suitable stimulation conditions, culturing them for a predetermined period of time, infecting said T cells with the scFv(X)-(Y)CD3zeta-2A-(Z) virus produced in Step 1, and subjecting them to cell expansion under suitable stimulation conditions, after 2 rounds of expansion under stimulation, the obtained cells are the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

12. A formulation, comprising said CAR-T cell according to claim 7.

13. A method of treating or preventing tumors, comprising administrating said chimeric antigen receptor according to any one of claims 1-6 or said CAR-T cell according to claim 7 to the subject in need of.

14. The method of treating or preventing tumors according to claim 13, wherein said tumor is selected from the group consisting of a hematological tumor, a solid tumor, and a combination thereof.

15. The method of treating or preventing tumors according to claim 13, wherein said tumor comprises Burkitt lymphoma (BL), acute lymphoblastic leukemia (ALL), and/or lung cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 is an illustrative schematic view showing the molecular structure of chimeric antigen receptor (CAR) including the third signal receptor in embodiments of the present invention;

[0082] FIG. 2 is a schematic view showing the virus titer measured after 293 cells were infected with BBZ-2A-OX40 virus in an embodiment of the present invention;

[0083] FIG. 3 is a schematic view showing the virus titer measured after 293 cells were infected with BBZ-2A-HVEM virus in an embodiment of the present invention;

[0084] FIG. 4 is a schematic view showing the virus titer measured after 293 cells were infected with BBZ-2A-ICOS virus in an embodiment of the present invention;

[0085] FIG. 5 is a schematic view showing the virus titer measured after 293 cells were infected with BBZ-2A-CD27 virus in an embodiment of the present invention;

[0086] FIG. 6 is a schematic view showing the virus titer measured after 293 cells were infected with BBZ-2A-4-1BB virus in an embodiment of the present invention;

[0087] FIG. 7 is a schematic view showing the results of phenotypic analysis of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention;

[0088] FIG. 8 is a schematic view showing the results of phenotypic analysis of BBZ CAR-T cell and BBZ-2A-HVEM CAR-T cell in an embodiment of the present invention;

[0089] FIG. 9 is a schematic view showing the results of phenotypic analysis of BBZ CAR-T cell and BBZ-2A-ICOS CAR-T cell in an embodiment of the present invention;

[0090] FIG. 10 is a schematic view showing the results of phenotypic analysis of BBZ CAR-T cell and BBZ-2A-CD27 CAR-T cell in an embodiment of the present invention;

[0091] FIG. 11 is a schematic view showing the results of phenotypic analysis of BBZ CAR-T cell and BBZ-2A-4-1BB CAR-T cell in an embodiment of the present invention;

[0092] FIG. 12 is a schematic view showing the expansion ability of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention;

[0093] FIG. 13 is a schematic view showing the tumor killing ability of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell, BBZ-2A-CD27 CAR-T cell and BBZ-2A-ICOS CAR-T cell in an embodiment of the present invention;

[0094] FIG. 14 is a schematic view showing the number of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in the bone marrow in an embodiment of the present invention;

[0095] FIG. 15 is a schematic view showing the survival ability of mouse model administrated BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention;

[0096] FIG. 16 is a schematic view showing the amplification ability of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention;

[0097] FIG. 17 is a schematic view showing the tumor killing ability of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention;

[0098] FIG. 18 is a schematic view showing the in vivo anti-tumor ability of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Terms

[0099] To make the disclosure easier to understand, some terms are firstly defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meanings given below. Other definitions are set forth throughout the application.

[0100] As used herein, the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition as determined by those skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0101] As used herein, the term “administering” refers to the physical introduction of a product of the invention into a subject using any one of various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as by injection or infusion.

[0102] As used herein, the term “antibody” (Ab) may comprise, but is not limited to, an immunoglobulin that specifically binds an antigen and contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen binding parts thereof. Each H chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1 CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed within more conservative regions called framework regions (FR). Each VH and VL contains three CDRs and four FRs, which are arranged from amino terminal to carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0103] As used herein, the term “Single-chain Fv” also abbreviated as “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, please refer to Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0104] As used herein, the term “chimeric antigen receptor (CAR)” generally refers to an antigen receptor fused by fusing an antigen binding region of an antibody which recognizes a tumor associated antigen (TAA) or a binding fragment of other target molecules with an “immune receptor tyrosine-based activation motifs (ITAM, typically CD3ζ or FccRIγ) of an intracellular signal domain. For example, the basic structure of CAR can include an antigen binding domain of a tumor-associated antigen (TAA) or other target molecules (typically, an scFv originated from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an immunoreceptor tyrosine-based activation motif (ITAM) of an intracellular immune receptor.

[0105] As used herein, the term “binding domain” generally refers to a domain that (specifically) binds to a given target epitope or a given target site of a target molecule (e.g., an antigen), interacts with the given target epitope or the given target site, or recognizes the given target epitope or the given target site.

[0106] As used herein, the term “specific binding” generally refers to a measurable and reproducible interaction, such as, the binding between a target and an antibody, which can determine the presence of a target in the presence of heterogeneous populations of molecules (including biomolecules). For example, antibodies that specifically bind to targets (which can be epitopes) are antibodies that bind the target(s) with greater compatibility, affinity, easiness, and/or duration than other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved in proteins of different species. In another embodiment, the specific binding includes but is not limited to exclusive binding.

[0107] As used herein, the term “transmembrane domain” generally refers to a polypeptide or protein which is encoded at a DNA level by an exon including at least an extracellular region, a transmembrane region, and an intracellular region. The transmembrane domain generally includes three different structural regions: N-terminal extracellular region, middle conserved transmembrane extension region, and C-terminal cytoplasmic region. The transmembrane domain may further include an intracellular region or a cytoplasmic region.

[0108] As used herein, the term “hinge region” generally refers to a region located between the binding domain and the transmembrane domain in the CAR structure. The hinge region usually comes from IgG family, such as IgG1 and IgG4, and some from IgD and CD8. Generally, the hinge region has a certain degree of flexibility, which affects the spatial constraints between the CAR molecule and its specific target, thereby affecting the contact between CAR T cells and tumor cells.

[0109] As used herein, the term “costimulatory” generally refers to a source of the second signal of lymphocyte activation, which is usually generated by an interaction of costimulatory molecules on the surface of immune cells (between T cells/B cells or between antigen presenting cells/T cells) involved in adaptive immunity with their receptors. For example, the complete activation of T cells depends on dual signaling and the action of cytokine. The first signal of T cell activation is derived from the specific binding of its receptors with the antigens, that is, the recognition of T cells to the antigens; and the second signal of T cell activation is derived from the costimulatory molecule, that is, the interaction of the costimulatory molecules of the antigen presenting cells with the corresponding receptors on the surfaces of T cells.

[0110] As used herein, the term “costimulatory domain” generally refers to an intracellular portion of the corresponding receptor of the costimulatory molecule, which can transduce a costimulatory signal (also known as the second signal). For example, in CAR-T cells, the costimulatory domain derived from CD137 (or receptors of other costimulatory molecules) can be activated after the binding of the extracellular binding domain in the CAR structure with the corresponding antigen, thereby transducing a costimulatory signal.

[0111] As used herein, the term “primary signal transduction domain” generally refers to an amino acid sequence within a cell that can generate signals which promote the immune effector function of CAR-containing cells such as CAR-T cells. Examples of the immune effector functions in, e.g., CAR-T cells can include cell lysis activity and auxiliary activity, including cytokine secretion. In some embodiments, the primary signal transduction domain transduces the effector functional signals and directs the cells to perform the specialization function. Although the primary signal transduction domain can be used in its entirety, it is not necessary to use the entire chain in many cases. As for the use of a truncated portion of the primary signal transduction domain, such truncated portion can be used to replace the intact chain, as long as it can transduce the effector functional signals. The term “primary signal transduction domain” is thus intended to encompass any truncated portion of an intracellular signal transduction domain that is sufficient to transduce the effector functional signals. For example, in CAR-T cells, the primary signal transduction domain derived from CD3zeta.

[0112] As used herein, the term “tumor” generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor can be a solid tumor or a non-solid tumor. In some embodiments, a visible lump that can be detected by clinical examinations such as, X-ray radiography, CT scanning, B-ultrasound or palpation can be called solid tumor, while a tumor that cannot be seen or touched by X-ray, CT scanning, B-ultrasound and palpation, such as leukemia, can be called non-solid tumor.

[0113] As used herein, the term “pharmaceutically acceptable diluent” or “pharmaceutically acceptable excipient” generally refers to a pharmaceutically acceptable substance, composition, or vehicle involved in carrying, storing, transferring, or administering a cell preparation, e.g., liquids, semi-solid or solid fillers, diluents, osmotic agents, solvent, or encapsulating substances. The pharmaceutically acceptable diluent or excipient can include a pharmaceutically acceptable salt, wherein the term “pharmaceutically acceptable salt” includes salts of active compounds prepared by using a relatively nontoxic acid or base, e.g., sodium chloride, depending on the cell nature of the present application. The pharmaceutically acceptable carrier can further include organic acids (e.g., lactic acid), bioactive substances (e.g., polypeptides, antibodies, and the like) and antibiotics (e.g., penicillin, streptomycin), etc. The pharmaceutically acceptable carrier can further include a hydrogel, such as, a hydrogel containing polyacrylamide. The pharmaceutically acceptable diluent or excipient can include storage solution, cryopreservation solution, injection, etc., which can be used for cells. In general, the pharmaceutically acceptable diluent or excipient can maintain the activity of the cells carried by the carrier without hindering its therapeutic efficacy. The pharmaceutically acceptable diluent or excipient can also contribute to the storage, transportation, proliferation and migration of cells, and is suitable for clinical application.

[0114] As used herein, the term “subject” generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey. In some embodiments, said subject is a human.

[0115] As used herein, the term “include/including” or “comprise/comprising” generally refers to encompassing clearly specified features, but does not exclude other elements.

[0116] The present invention provides a chimeric antigen receptor including a co-stimulatory receptor having a structure of scFv(X)-(Y)CD3zeta-2A-(Z); wherein X is a tumor-targeting antibody or other protein; Y is an intracellular domain of a co-stimulatory receptor, and said co-stimulatory receptor is selected from a group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226; Z is a co-stimulatory receptor, and said co-stimulatory receptor is a group consisting of selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226. The present invention also relates to a CAR-T cell constructed by a recombinant expression vector of any one of the aforesaid chimeric antigen receptor and a preparation method therefor, a formulation including the CAR-T cell, and a use of the CAR-T cell.

[0117] Hereinafter the embodiments of the present invention are further described with reference to the accompanying drawings and examples. The following examples are only for more clearly illustrating the technical solutions of the present invention, but not for limiting the protective scope of the present invention.

[0118] The chimeric antigen receptor (CAR) molecules including a co-stimulatory receptor used in the following examples of the present invention are BBZ-2A-OX40, BBZ-2A-HVEM, BBZ-2A-ICOS, BBZ-2A-CD27, and BBZ-2A-4-1BB, respectively, and their structures are shown in FIG. 1.

Example 1—Preparation of 20BBZ-2A-OX40 CAR-T Cell

[0119] The preparation of the 20BBZ-2A-OX40 CAR-T cell in this example includes the following steps:

[0120] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-OX40 and production of virus

[0121] incorporating 2A (SEQ ID No. 7) sequence between scFv-antihCD20-20BBZ (SEQ ID No. 1) and OX40 (SEQ ID No.2) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone the pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-OX40 virus (briefly, 20BBZ-2A-OX40 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 2.

[0122] 2. Preparation of 20BBZ-2A-OX40 CAR-T cell and 20BBZ CAR-T cell

[0123] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-OX40 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZ-2A-OX40 CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 7. It can be seen that the obtained cells are CAR-POSITIVE.

Example 2—Preparation of 20BBZ-2A-HVEM CAR-T Cell

[0124] The preparation of the 20BBZ-2A-HVEM CAR-T cell in in this example includes the following steps:

[0125] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-HVEM and production of virus

[0126] incorporating 2A (SEQ ID No. 8) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and HVEM (SEQ ID No.3) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-HVEM virus (briefly, 20BBZ-2A-HVEM virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus). Infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 3.

[0127] 2. Preparation of 20BBZ-2A-HVEM CAR-T cell and 20BBZ CAR-T cell

[0128] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells were infected with 20BBZ virus and 20BBZ-2A-HVEM virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZ-2A-HVEM CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 8. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 3—Preparation of 20BBZ-2A-ICOS CAR-T Cell

[0129] The preparation of the 20BBZ-2A-ICOS CAR-T cell in this example includes the following steps:

[0130] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-ICOS and production of virus

[0131] incorporating 2A (SEQ ID No. 9) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and ICOS (SEQ ID No.4) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-ICOS virus (briefly, 20BBZ-2A-ICOS virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 4.

[0132] 2. Preparation of 20BBZ-2A-ICOS CAR-T cell and 20BBZ CAR-T cell

[0133] purifying human PBMCs by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-ICOS virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZ-2A-ICOS CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 9. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 4—Preparation of 20BBZ-2A-CD27 CAR-T Cell

[0134] The preparation of 20BBZ-2A-CD27 CAR-T cell in this example includes the following steps:

[0135] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-CD27 and production of virus

[0136] incorporating 2A (SEQ ID No. 10) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and CD27 (SEQ ID No.5) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-CD27 virus (briefly, 20BBZ-2A-CD27 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 5.

[0137] 2. Preparation of 20BBZ-2A-CD27 CAR-T cell and 20BBZ CAR-T cell

[0138] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-CD27 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZ-2A-CD27 CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 10. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 5—Preparation of 20BBZ-2A-4-1BB CAR-T Cell

[0139] The preparation of the 20BBZ-2A-4-1BB CAR-T cell in this example includes the following steps:

[0140] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-4-1BB and production of virus

[0141] incorporating 2A (SEQ ID No. 7) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and 4-1BB (SEQ ID No.6) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-4-1BB virus (briefly, 20BBZ-2A-4-1BB virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 6.

[0142] 2. Preparation of 20BBZ-2A-4-1BB CAR-T cell and 20BBZ CAR-T cell

[0143] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-4-1BB virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZ-2A-4-1BB CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 11. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 6—Comparison of Expansion Abilities of 20BBZ CAR-T Cell and 20BBZ-2A-OX40 CAR-T Cell

[0144] 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell prepared in Step 2 of Example 1 were continuously cultured for 14 days (as the 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T were obtained on day 2 in FIG. 12), and stimulated with artificial antigen presenting cell once every 6 days. The cells were counted, and the results are shown in FIG. 12. It can be seen from the figure that 20BBZ-2A-OX40 CAR-T cell has enhanced proliferation ability as compared with 20BBZCAR-T cell.

Example 7—Comparison of Tumor-Killing Abilities of 20BBZ CAR-T Cell and 20BBZ-2A-OX40 CAR-T Cell

[0145] 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell obtained in Step 2 of Example 1, 2OBBZ-2A-ICOS CAR-T cell obtained in Step 2 of Example 3, and 20BBZ-2A-CD27 CAR-T cell obtained in Step 2 of Example 4 were inoculated into a 96-well plate. For 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell, Raji tumor cells were added at a CAR-T: tumor cell ratio of 1:0.5, 1:1, 1:2, 1:4; For 20BBZ CAR-T cell and 20BBZ-2A-CD27 CAR-T cell, Raji tumor cells were added at a CAR-T:tumor cell ratio of 1:0.5, 1:1, 1:1.5, and 1:2; For 20BBZ CAR-T cell and 20BBZ-2A-ICOS CAR-T cell, Raji tumor cells were added at a CAR-T:tumor cell ratio of 1:0.5, 1:1, 1:1.5, and 1:2. After 24 hours, the survival rates of tumor cells were compared, and the results are shown in FIG. 13. It can be seen from the figure that 20BBZ-2A-OX40/ICOS/CD27 CAR-T cell has similar tumor killing ability as compared with 20BBZ CAR-T cell, and some CAR-T including the co-stimulatory receptor has a stronger tumor killing ability.

Example 8—Comparison of Anti-Tumor Ability and In Vivo Survival Ability of 20BBZ CAR-T Cell and 20BBZ-2A-OX40 CAR-T Cell

[0146] 10.sup.6 Nalm-6 tumor cells were intravenously inoculated into B-NDG mice, which were treated with 10.sup.7 2 OBBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells after 6 days. The mice were observed for their survival rates, and some mice were detected for the level of tumor cells and CAR-T cells in their marrow on Day 7. The results are shown in FIG. 14 and FIG. 15, respectively. It can be seen from the figure that 20BBZ-2A-OX40 CAR-T cell, as compared with 20BBZ CAR-T cell, significantly prolongs the survival of mice, and expanded more in vivo.

[0147] It can be seen from the aforesaid examples that the present invention constructs a novel CAR-T cell including a co-stimulatory receptor, which significantly increases the activation ability, survival ability, expansion ability of the CAR-T cells in tumors, as compared with the current CAR-T technology in clinic use, and has a more superior anti-tumor therapeutic effect.

Example 9 Preparation of 19BBZ-2A-OX40 CAR-T Cell

[0148] The preparation of the 19BBZ-2A-OX40 CAR-T cell in this example includes the following steps:

[0149] 1. Construction of lentiviral vector pCDH-MSCVEF-19BBZ-2A-OX40 and production of virus

[0150] Incorporating 2A (SEQ ID No. 7 of US2021/0169932A1) sequence between scFv-anti-hCD19-BBZ (SEQ ID No.11) and OX40 (SEQ ID No.2) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone the pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large-scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-19BBZ-2A-OX40 virus (briefly, 19BBZ-2A-OX40 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-19BBZ virus (briefly, 19BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer.

[0151] 2. Preparation of 19BBZ-2A-OX40 CAR-T cell and 19BBZ CAR-T cell

[0152] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with 19BBZ virus and 19BBZ-2A-OX40 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are 19BBZCAR-T cell and 19BBZ-2A-OX40 CAR-T cell for subsequent experiments and phenotypic analysis.

Example 10 Preparation of EGFRBBZ-2A-OX40 CAR-T Cell

[0153] The preparation of the EGFRBBZ-2A-OX40 CAR-T cell in this example includes the following steps:

[0154] 1. Construction of lentiviral vector pCDH-MSCVEF-EGFRBBZ-2A-OX40 and production of virus

[0155] incorporating 2A (SEQ ID No. 7 of US2021/0169932A1) sequence between scFv-anti-hEGFR-BBZ (SEQ ID No.12) and OX40 (SEQ ID No.2 of US2021/0169932A1) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone the pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293X. After 48 and 72 hours, collecting the supernatant, filtering it by a 0.45 μm filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-EGFRBBZ-2A-OX40 virus (briefly, EGFRBBZ-2A-OX40 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-EGFRBBZ virus (briefly, EGFRBBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer.

[0156] 2. Preparation of EGFRBBZ-2A-OX40 CAR-T cell and EGFRBBZ CAR-T cell

[0157] purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28 antibody. After 2 days, infecting the cells with EGFRBBZ virus and EGFRBBZ-2A-OX40 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them by artificial antigen presenting cell or anti-hCD3/28 antibody every 6 days. After 2 rounds of stimulation, the obtained cells are EGFRBBZCAR-T cell and EGFRBBZ-2A-OX40 CAR-T cell for subsequent experiments and phenotypic analysis.

Example 11 Comparison of Expansion Abilities of 19BBZ CAR-T Cell and 19BBZ-2A-OX40 CAR-T Cell

[0158] 19BBZ CAR-T cell and 19BBZ-2A-OX40 CAR-T cell prepared in Step 2 of Example 2 were continuously cultured for 18 days (as the 19BBZ CAR-T cell and 19BBZ-2A-OX40 CAR-T cell were obtained on day 2 in FIG. 12), and stimulated with artificial antigen presenting cell once every 6 days. The cells were counted, and the results are shown in FIG. 16. It can be seen from the figure that 19BBZ-2A-OX40 CAR-T cell has enhanced proliferation ability as compared with 19BBZCAR-T cell.

Example 12 Comparison of Tumor-Killing Abilities of 19BBZ CAR-T Cell and 19BBZ-2A-OX40 CAR-T Cell

[0159] 19BBZ CAR-T cell and 19BBZ-2A-OX40 CAR-T cell obtained in Step 2 of Example 2 were inoculated into a 96-well plate, and Raji tumor cells were added at a CAR-T:tumor cell ratio of 1:1, and 0.5:1. After 24 hours, the survival rates of tumor cells were compared, and the results are shown in FIG. 17. It can be seen from the figure that 19BBZ-2A-OX40 has stronger tumor killing ability as compared with 19BBZ CAR-T cell.

Example 13 Comparison of Anti-Tumor Ability In Vivo of EGFRBBZ CAR-T Cell and EGFRBBZ-2A-OX40 CAR-T Cell

[0160] 10.sup.6 A549 tumor cells were subcutaneously inoculated into on the right flank of B-NDG mice, which were treated with 10.sup.7 EGFRBBZ CAR-T cells and EGFRBBZ-2A-OX40 CAR-T cells after 6 days. Tumor volumes were measured twice a week along three orthogonal axes (length, width, and height) and tumor volumes calculated using the equation (length×width×height)/2. The results are shown in FIG. 18. It can be seen from the figure that EGFRBBZ-2A-OX40 CAR-T cell, as compared with EGFRBBZ CAR-T cell, significantly reduced the tumor volumes in mice, indicating enhanced antitumor activity in vivo.

[0161] Hereinbefore the specific embodiments of the present invention are described in details. However, they are only used as examples, and the present invention is not limited to the specific embodiments as described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are encompassed in the scope of the present invention. Therefore, all the equal transformations and modifications without departing from the spirit and scope of the present invention should be covered in the scope of the present invention.