CAR-T cells targeting B7-H3 and their application in the treatment of acute myeloid leukemia

Abstract

The present invention provides CAR-T cells targeting B7-H3 and their use in the treatment of acute myeloid leukemia. Specifically, the present invention provides a CAR-T cell targeting B7-H3, which comprises a scFv targeting B7-H3, a 41BB co-stimulatory signal molecule and a CD3ζ domain. The B7-H3-CAR-T cells of the present invention have significant specific killing of B7-H3-positive AML tumor cells. Animal experimental results show that B7-H3-CAR-T cells can significantly inhibit the growth of AML tumor cells in mice, significantly prolong the survival of mice, and have significant in vivo anti-tumor effects. The B7-H3-CAR-T cells of the present invention can be used as a new treatment method for targeted treatment of AML, with great prospects for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a CAR-T cell targeting B7-H3 and an application thereof in the treatment of acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a common type of acute leukemia. It is a highly heterogeneous malignant clonal disease originating from hematopoietic tissue. Patients have a high mortality rate, a low long-term survival rate, and are prone to relapse, which seriously endangers human health and life. For decades, the treatment of AML has remained basically unchanged, with chemotherapy and hematopoietic stem cell transplantation as the main means. The classic chemotherapy regimen is still anthracyclines combined with cytarabine. Although this regimen can achieve a relatively high remission rate after treatment, patients have a high risk of drug resistance and relapse.

[0003] According to the French-American-British (FAB) classification of AML morphology, AML can be divided into eight types, M0-M7. Studies have shown that patients with M3 and M4 subtypes have better prognosis than other subtypes, and the most common (8;12) translocation in AML is associated with a good prognosis, but it is rare in the M5 subtype. At the same time, there are also reports showing that the prognosis of patients with the M5 subtype is significantly worse than that of other AML. Therefore, it is particularly urgent to develop better treatments for AML, especially for AML of the M5 subtype.

[0004] Chimeric antigen receptor T (CAR T) cell immunotherapy is to give T cells antibodies that can recognize antigens on the surface of tumor cells, thereby avoiding the restriction of MHC and inducing T cell specific activation. The modified T cells can better recognize and kill tumors than natural T cells. In recent years, CAR T therapy has made great progress in the invention of cancer treatment and is considered one of the most promising tumor treatment methods.

[0005] At present, CAR T therapy has the best efficacy and the most extensive application in the treatment of ALL. Among them, CAR targeting CD19 has achieved significant efficacy in the treatment of both adult and pediatric ALL, with a remission rate of up to 90%. CD123 and CD33 have also achieved good results in the CAR-T treatment of AML, but these two antigens are expressed in normal myeloid cells, which will inevitably cause toxic side effects to patients, and it is still a problem that cannot be ignored at present. Therefore, it is crucial to find a relatively safe and effective target, and new therapeutic targets and treatment methods need to be developed in this field. Summary of the invention

[0006] The object of the present invention is to provide a CAR-T cell targeting B7-H3 and its application in the treatment of acute myeloid leukemia.

[0007] In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the antigen binding domain (i.e., scFv) of the chimeric antigen receptor comprises a heavy chain variable region and a light chain variable region,

[0008] CDR1 shown in SEQ ID NO:6,

[0009] CDR2 shown in SEQ ID NO:7, and

[0010] CDR3 shown in SEQ ID NO:8;

[0011] And the light chain variable region includes the following complementarity determining region CDR:

[0012] CDR1' shown in SEQ ID NO:9,

[0013] CDR2' shown in SEQ ID NO:10, and

[0014] CDR3' shown in SEQ ID NO:11.

[0015] In another preferred embodiment, the antigen binding domain of the chimeric antigen receptor includes the antibody heavy chain variable region shown in SEQ ID NO:1, and the antibody light chain variable region shown in SEQ ID NO:2.

[0016] In another preferred embodiment, the antibody heavy chain variable region and the antibody light chain variable region are connected via a connecting peptide.

[0017] In another preferred embodiment, the structure of the antigen binding domain is shown in the following formula I or II:

[0018] V L -V H (I); V H -V L (II)

[0019] Among them, V H V is the variable region of the antibody heavy chain; L is the variable region of antibody light chain; “-” is a connecting peptide or peptide bond.

[0020] In another preferred embodiment, the structure of the antigen binding domain is as shown in Formula II (V H -V L ) as shown.

[0021] In another preferred embodiment, the V HThe amino acid sequence of is shown in SEQ ID NO: 1, V L The amino acid sequence is shown in SEQ ID NO: 2.

[0022] In another preferred embodiment, the connecting peptide is 1-4 consecutive sequences shown in SEQ ID NO: 4 (GGGGS), preferably 2-4, and more preferably 3.

[0023] In another preferred embodiment, the antigen binding domain binds to B7-H3, preferably human B7-H3.

[0024] In another preferred example, the heavy chain variable region and light chain variable region of the antigen binding domain are derived from humanized antibodies.

[0025] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in the following formula III:

[0026] L-scFv-H-TM-C-CD3ζ (III)

[0027] in,

[0028] L is none or a signal peptide sequence;

[0029] scFv is a scFv targeting B7-H3;

[0030] H is the hinge region;

[0031] TM is the transmembrane domain;

[0032] C is a co-stimulatory signal molecule;

[0033] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0034] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0035] In another preferred embodiment, the L is a signal peptide derived from macrophage colony stimulating factor.

[0036] In another preferred embodiment, the H is a hinge region selected from the following histones: CD8, CD28, CD137, Fc, or a combination thereof.

[0037] In another preferred embodiment, the H is a hinge region derived from CD8.

[0038] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0039] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0040] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0041] In another preferred embodiment, C is a co-stimulatory signal molecule derived from 4-1BB.

[0042] In another preferred embodiment, the CAR (preferably the C-terminus) also includes a cell suicide element.

[0043] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleavage element.

[0044] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A.

[0045] In another preferred embodiment, the cell suicide element is selected from the following group: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0046] In another preferred embodiment, the cell suicide element is tEGFR.

[0047] In another preferred example, the amino acid sequence of tEGFR is shown in SEQ ID NO:5.

[0048] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:3.

[0049] In a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the chimeric antigen receptor (CAR) described in the first aspect of the present invention.

[0050] In another preferred embodiment, the nucleic acid molecule is isolated.

[0051] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0052] In another preferred embodiment, the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof.

[0053] In another preferred embodiment, the vector is selected from the following group: plasmid, viral vector.

[0054] In another preferred embodiment, the vector is in the form of a virus particle.

[0055] In another preferred embodiment, the vector is a lentiviral vector.

[0056] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention or a chromosome in which the exogenous nucleic acid molecule described in the second aspect of the present invention is integrated or expresses the CAR described in the first aspect of the present invention.

[0057] In another preferred embodiment, the host cells include eukaryotic cells and prokaryotic cells.

[0058] In another preferred embodiment, the host cell includes Escherichia coli.

[0059] In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell expresses the CAR described in the first aspect of the present invention.

[0060] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0061] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0062] In another preferred embodiment, the cells include T cells and NK cells.

[0063] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0064] In another preferred embodiment, CAR and cell suicide elements are co-expressed in the immune cells.

[0065] In another preferred embodiment, the engineered immune cells include T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

[0066] In the sixth aspect of the present invention, a preparation is provided, which contains the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the immune cell described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0067] In another preferred embodiment, the preparation is a liquid preparation.

[0068] In another preferred embodiment, the preparation is in the form of an injection.

[0069] In another preferred embodiment, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0070] In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably a second antibody or a chemotherapeutic agent.

[0071] In another preferred embodiment, the chemotherapeutic agent is selected from the following group: docetaxel, carboplatin, or a combination thereof.

[0072] In the seventh aspect of the present invention, there is provided a use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the immune cell described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0073] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0074] In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0075] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

[0076] In another preferred embodiment, the tumor is a B7-H3 positive tumor, preferably a tumor with high expression of B7-H3.

[0077] In another preferred embodiment, the medicine or preparation is used for preventing and / or treating acute myeloid leukemia (AML).

[0078] In the eighth aspect of the present invention, a kit for preparing the host cell described in the fourth aspect of the present invention is provided, the kit comprising a container, and the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention located in the container.

[0079] In a ninth aspect of the present invention, a method for preparing an engineered immune cell is provided, wherein the immune cell expresses the CAR according to the first aspect of the present invention, and the method comprises the following steps:

[0080] (a) providing immune cells to be modified; and

[0081] (b) transducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the immune cell, thereby obtaining the engineered immune cell.

[0082] In another preferred embodiment, the engineered immune cells are CAR-T cells or CAR-NK cells.

[0083] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.

[0084] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering an appropriate amount of the vector described in the third aspect of the present invention, the immune cell described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention to a subject in need of treatment.

[0085] In another preferred embodiment, the disease is cancer or tumor.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The expression rate of B7-H3 antigen on the surface of AML tumor cells is shown. Among them, the expression of B7-H3 on the surface of six AML tumor cells was detected by flow cytometry.

[0088] Figure 2 The structure of B7-H3-CAR and its functional validation are shown. Figure 2A shows a schematic diagram of the structure of B7-H3-CAR, which includes EF1 promoter, B7-H3 single-chain antibody, CD8 transmembrane domain, co-stimulatory domain 41-BB and intracellular signaling domain CD3ζ. Figure 2 B shows the expression of B7-H3-CAR on the surface of transfected Jurkat cells analyzed by flow cytometry. Figure 2 C shows the expression of CD25 and CD69 on the cell surface. After Jurkat and B7-H3-CAR Jurkat cells were co-cultured with HEL, AML5 and KG-1 cells at a ratio of 1:1 for 48 hours, CD25-APC and CD69-APC antibodies were used to detect the expression of CD25 and CD69 on the surface of Jurkat cells and B7-H3-CAR Jurkat cells, respectively.

[0089] Figure 3 B7-H3-CAR T cells showed + AML cell lines have specific killing. Figure 3 A shows the positive rate of B7-H3-CAR T cells. Among them, through flow cytometry analysis, the positive rate of B7-H3-CAR T cells is about 51.84%. Figure 3 B shows the expression of B7-H3-CAR in T cells analyzed by Western blot. Proteins were extracted from T (lane 1) and B7-H3-CAR T (lane 2) cells and separated by SDS-PAGE, and immunoblots were detected by chemiluminescence after incubation with CD3ζ chain-specific mAb and HRP-goat anti-mouse IgG. Figure 3 C shows the apoptotic ratio of target cells after 48 h of co-incubation at an effector-target ratio of 1:1. ****P<0.0001, n=3. Figure 3 D shows the secretion of cytokines (IL-2, TNF, INF-γ and granzyme B) in the supernatant after co-incubation. ****P<0.0001, ***P<0.001, **P<0.01.

[0090] Figure 4 B7-H3-CAR T cells were shown to have antitumor activity in AML-engrafted mice. Figure 4 A shows the schematic diagram of the AML transplantation model of NSG mice. Among them, 6-8 week old NSG female mice, 4 in each group, a total of three groups (PBS group, T cell group, B7-H3-CAR T cell group), were injected with 5×10 5 luciferase-GFP-HEL cells were injected into the tail vein on the fifth day. 7 An equal volume of effector cells (PBS is an equal volume of cell-free liquid). Figure 4B shows an in vivo imaging of mice, showing leukemia progression in mice. Figure 4 C shows a statistical graph of the average body weight of mice. Figure 4 D shows the mouse survival curve. Figure 4 E shows the statistical graph of mouse survival. **P<0.01, *P<0.1.

[0091] Figure 5 showed that B7-H3-CAR T cells could eliminate tumor cells in AML-transplanted mice. Figure 5 A shows the schematic diagram of the AML transplantation model of NSG mice. Among them, 6-8 week old NSG female mice, 3 mice in each group, a total of three groups (PBS group, T cell group, B7-H3-CAR T cell group), were injected with 5*10 5 HEL-luciferase-GFP cells were injected into the tail vein on the second day. 7 An equal volume of effector cells (PBS is an equal volume of cell-free liquid). Figure 5 B shows an in vivo imaging of mice, showing the progression of the disease in mice. Figure 5 C shows the proportion of tumor cells. After the red blood cells were lysed from mouse peripheral blood and bone marrow cells, they were co-incubated with CD45 and CD3 antibodies, and the proportion of tumor cells was detected by flow cytometry. ****P<0.0001, *P<0.1. Figure 5 D shows the tumor cell infiltration. The spleen and ovarian tissues of mice were taken, and B7-H3 antibody immunohistochemistry showed that at 400x and 100x fields of view, there was obvious tumor cell infiltration in the PBS group and T cell mice compared with the B7-H3-CAR T cell group. n=3.

[0092] Figure 6 B7-H3-CAR T cell safety validation was shown. Figure 6 A shows the statistical graphs of RBC, WBC, PLT and HGB after five-category detection of mouse peripheral blood. Figure 6 B shows the HE staining results of mouse tissues. The heart, liver, kidney and intestinal tissues of the mice were taken and HE stained. The mouse tissues were observed under a microscope at 100 times and 400 times magnification to see if there were any lesions. n=3.

[0093] Figure 7 AML mouse model modeling is shown. Figure 7 A shows the positive rate of luciferase-GFP-HEL cells detected by flow cytometry. Figure 7 B shows the schematic diagram of mouse AML model establishment. In which, 6-8 week old NSG female mice were injected with 5*10 5 After the cells were transfected with luciferase-GFP-HEL cells, the mice were observed. Figure 7C shows flow cytometry analysis of tumor cells in the bone marrow, peripheral blood, spleen, liver, and kidney of tumor-bearing mice.

[0094] Figure 8 The positive rate of B7-H3-CAR T cells is shown. Figure 8 A shows the positive rate of B7-H3-CAR T cells in the first mouse experiment detected by flow cytometry. Figure 8 B shows the positive rate of B7-H3-CAR T cells in the second mouse experiment detected by flow cytometry. DETAILED DESCRIPTION

[0095] After extensive and in-depth research, the inventor unexpectedly discovered a CAR-T cell targeting B7-H3 for the first time. The results of in vitro functional experiments showed that B7-H3-CAR-T cells had significant specific killing of B7-H3-positive AML tumor cells. The results of animal experiments showed that B7-H3-CAR-T cells could significantly inhibit the growth of AML tumor cells in mice, significantly prolong the survival of mice, and have significant in vivo anti-tumor effects, indicating that the B7-H3-CAR-T cells constructed by the present invention can be used as a new treatment method for targeted treatment of AML, and have great clinical application prospects. On this basis, the present invention is completed.

[0096] the term

[0097] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0098] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0099] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0100] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0101] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0102] B7-H3

[0103] B7-H3, also known as CD276, was discovered in 2001 and is mostly expressed on the cell surface, such as activated dendritic cells, monocytes, T cells, B cells and NK cells. Studies have shown that B7-H3 can stimulate the proliferation and killing of T cells, and may selectively stimulate the signal receptors of T cells. At the same time, B7-H3 is also a tumor-associated antigen that plays an important role in tumor progression and metastasis. The survival rate of patients with negative B7-H3 protein expression is higher than that of patients with positive B7-H3 protein expression. Many studies have shown that B7-H3 is abnormally expressed in human malignant tumors, including melanoma, leukemia, breast cancer, prostate, etc. Studies have found that the vast majority of cancer patients have abnormally high expression of B7-H3 in tumor tissues of 60%-93%, while B7-H3 expression in normal healthy tissues is very limited. B7-H3 is one of the most strongly expressed B7 family molecules in AML, with the highest expression in M3 and M5 subtypes.

[0104] Literature reports show that B7-H3 is not expressed in immune cells. In addition, most of the literature related to B7-H3 proves that B7-H3 is related to tumor progression and metastasis. More importantly, B7-H3 has been found to be highly expressed in tumor-related stromal cells, fibroblasts, and tumor angiogenesis. Therefore, targeted B7-H3 therapy is expected to break the inhibition of the tumor microenvironment on drugs and is a new target that has received increasing attention in recent years. Currently, several research groups have constructed

[0105] B7-H3-CAR T cells have shown encouraging tumor killing activity in preclinical studies of various solid tumors such as pancreatic cancer, childhood neuroblastoma, and osteosarcoma. The present invention constructs a B7-H3-CAR-T cell with 4-1BB as the costimulatory domain and a safety switch, a truncated EGFR molecule (EGFRt), which exhibits good cytotoxicity in in vitro and mouse model experiments against AML tumor cell lines.

[0106] Chimeric Antigen Receptor

[0107] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0108] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0109] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting B7-H3. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0110] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv specifically recognizes B7-H3.

[0111] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0112] The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0113] Carrier

[0114] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0115] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0116] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0117] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0118] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0119] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0120] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0121] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0122] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0123] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0124] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0125] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0126] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0127] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0128] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0129] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0130] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0131] Therapeutic applications of CART

[0132] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker B7-H3, synergistically activate T cells, and induce a T cell immune response, thereby significantly improving its efficiency in killing tumor cells.

[0133] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0134] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0135] In one embodiment, the CAR-T cells of the present invention can undergo a robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-B7-H3 CAR-T cells cause a specific immune response against cells expressing B7-H3.

[0136] Although the data disclosed herein specifically discloses lentiviral vectors comprising anti-B7-H3 scFv, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

[0137] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0138] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0139] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

[0140] In a preferred embodiment, the CAR-T cells of the present invention can be used to treat acute myeloid leukemia.

[0141] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0142] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0143] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0144] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0145] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0146] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0147] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0148] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0149] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0150] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0151] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0152] The main advantages of the present invention include:

[0153] (a) The B7-H3-CAR-T cells constructed in the present invention have significant in vitro anti-tumor effects on AML cell lines.

[0154] (b) The B7-H3-CAR-T cells constructed in the present invention have significant in vivo anti-tumor effects on AML cell line HEL tumor-bearing mice.

[0155] (c) The results of mouse experiments showed that the B7-H3-CAR-T cells constructed by the present invention had good safety.

[0156] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are usually 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0157] General Materials and Methods:

[0158] 1. Cells

[0159] KG-1, MOLM-16, and Jurkat cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and HEL, THP-1, HL-60, and AML5 cell lines were kindly provided by the laboratory of Professor Yun Zhao, Tang Zhongying Hematology Research Center, Soochow University.

[0160] The culture medium used for HEL cells, Jurkat cells and AML5 cells is RP-MI1640 supplemented with 10% FBS. The culture medium used for KG-1 cells is IMDM supplemented with 10% FBS. Peripheral blood mononuclear cells (PBMC) were extracted from normal subjects using the Ficoll method, T cells were activated with Transact, and cultured with TexMACSTMGMP culture medium supplemented with IL-7 and IL-15. All cells were cultured in a cell culture incubator containing 5% CO2 and 37°C. The extracted T cells were transfected after 48 hours of in vitro activation, and toxicity experiments were performed when the number of cell proliferation was sufficient.

[0161] 2. Construction of CAR

[0162] The present application constructs a second-generation CAR, in which the B7-H3-scFv sequence is constructed onto a lentiviral vector, which includes B7-H3-scFv, CD8 hinge region and transmembrane region, 41BB intracellular region sequence and CD3ζ sequence, and human EGFR sequence.

[0163] 3. Virus Packaging and T / Jurkat Cell Transfection

[0164] Lentivirus was prepared according to conventional methods in the prior art. After 48 h of T cell activation, Jurkat cells were placed in a 48-well plate (5 × 10 5 50 μl of virus was added to each well (cells / well), the final system was 100 μl, and 1000 ul of culture medium was added for culture 15 hours after transfection.

[0165] 4. Flow analysis

[0166] Six AML cell lines were collected, namely AML5, KG-1, HEL, HL-60, THP-1 and MOLM-16. The cells were centrifuged and the supernatant was discarded. The cells were washed once with PBS at pH 7.4, incubated with B7-H3 antibody with FITC fluorescence (1:1000) for 30 minutes, washed twice with PBS, and the expression of B7-H3 on the cell surface was detected by flow cytometry.

[0167] Approximately 2×10 B7-H3-CAR Jurkat cells, Jurkat cells, B7-H3-CAR T cells, and T cells were taken out of the cultured 6 The cells were co-incubated with 100ul of EGFR antibody diluted 1:1000 (final antibody concentration: 1ug / ml) at 37°C for 20min, washed twice with PBS, and Jurkat cells or T cells were used as control cells. The B7-H3-CAR positivity rate of B7-H3-CAR Jurkat and B7-H3-CAR T cells was analyzed by flow cytometry.

[0168] Jurkat cells and B7-H3-CAR Jurkat cells co-incubated with KG-1, HEL and AML5 cells were incubated with CD25-APC and CD69-APC antibodies for 20 minutes, respectively, and the expression of CD25 and CD69 was detected by flow cytometry.

[0169] 5.WESTERN test

[0170] Take about 1×10 6 T cells and B7-H3-CAR T cells were lysed and separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore). After blocking with 5% skim milk for 1 hour, it was incubated in mouse anti-human CD3ζ antibody at 4°C overnight. Subsequently, after incubation with HRP-goat anti-mouse IgG at room temperature for 2 hours, the developing solution was added to the membrane and exposed in a dark room.

[0171] 6. Detection of the toxic effect of B7-H3 CAR T cells on tumor cells

[0172] Take 1×10 5After centrifugation, KG-1, HEL and AML5 tumor cells were incubated with 100 μl CFSE (1:1000) at 37°C for 15 minutes, washed twice with PBS, and added to a 24-well plate with effector cells at a ratio of 1:1, and the solution was replenished to 2 ml. After 48 hours, the cells were centrifuged and the supernatant was taken for cytokine detection. The precipitate was washed once with PBS and the proportion of apoptotic tumor cells was detected using the Annexin V-APC / 7-AAD apoptosis detection kit (LK-AP105-100; Lianke Bio, Hangzhou, China, http: / / www.liankebio.com).

[0173] 7. Cytokine Detection

[0174] The amount of cytokines produced by T cells and B7-H3-CAR T cells in the supernatant after killing was detected using a flow cytometry bead array (CBA). The human granzyme B CBA Flex Set D7 kit (catalog number #560304), human TNF Flex Set D9 kit (catalog number #558273), human IL-2Flex Set A4 kit (catalog number #558270) and human IFN-γ CBA Flex Set E7 kit (catalog number #558269) purchased from BD were used for detection.

[0175] 8. AML Mouse Model

[0176] The model was established using 6-8 week old NOD-Prkdcscidll2rgtm1 / Bcgen (B-NSG) female mice purchased from Biocytogen. 5×10 5 The tumor burden was detected by IVIS-spectrum after 2 or 5 days. The bioluminescence of mice was recorded 10 min after intraperitoneal injection of 150 mg / kg D-luciferin substrate. 1×10 7 100 μl of mouse peripheral blood, femoral bone marrow, ground organs, lysed red blood cells, incubated cells with anti-human CD45 antibody for flow cytometry analysis to detect tumor cells.

[0177] 9. Immunohistochemical Analysis

[0178] After the mice were killed, the spleen and ovarian tissues were taken, embedded in paraffin, and sliced ​​(3 μm thick) for immunohistochemical analysis. After the paraffin sections were dewaxed and rehydrated, they were immersed in 0.01M citrate buffer, microwaved for 5 minutes, and then blocked with a methanol solution containing 0.3% hydrogen peroxide. Staining was performed with B7-H3 antibody (1:300 dilution) and biotin-labeled secondary antibody, and finally a fresh DAB colorimetric solution was added. The infiltration of tumor cells in the tissue was observed. After the paraffin sections were dewaxed and rehydrated, they were stained with hematoxylin-and-eosin to observe the pathological changes of the tumor tissue.

[0179] 10. Statistical analysis

[0180] GraphPad Prism5 software was used, and t-tests were used to compare the two groups. ****P<0.0001, ***P<0.001, **P<0.01 and *P<0.05 indicated statistically significant differences.

[0181] Example 1 Expression of B7-H3 in AML cell lines

[0182] Six AML cell lines were selected and their B7-H3 expression was detected by flow cytometry.

[0183] The streaming results are as follows Figure 1 As shown, the B7-H3 expression rate of HEL cells is 96.4%, the B7-H3 expression rate of AML5 cells is 52.69%, the B7-H3 expression rate of THP-1 cells is 40.67%, the B7-H3 expression rate of MOLM-16 cells is 25.35%, the B7-H3 expression rate of HL-60 cells is 16.43%, and the B7-H3 expression rate of KG-1 cells is 11.74%. Finally, HEL, AML5, and KG-1 cells (high, medium, and low expression of B7-H3) were selected for later experiments.

[0184] Example 2 Construction and specificity verification of B7-H3-CAR

[0185] First, a CAR vector targeting B7-H3 was constructed. It is a second-generation CAR containing a B7-H3 single-chain antibody and a 41BB co-stimulatory domain, and co-expresses EGFR as a detection tag for CAR. The specific structure is as follows Figure 2 A, and the amino acids are shown in SEQ ID NO: 3. After lentiviral packaging, Jurkat cells were transfected to construct B7-H3-CAR-Jurkat cells that can stably and highly express B7-H3-CAR. The CAR positive rate of B7-H3-CAR-Jurkat cells detected by flow cytometry was as high as 97.19% ( Figure 2 B).

[0186] The pre-transfected Jurkat cells and the transfected B7-H3-CAR Jurkat cells were co-cultured with HEL, AML5 and KG-1 cells at a 1:1 effector-target ratio for 48 hours. The results showed that after co-culture with tumor cells that highly expressed B7-H3 antigen, the expression of CD25 and CD69 on the surface of B7-H3-CAR Jurkat cells was higher than that of Jurkat cells that were not transfected with CAR. However, after co-culture with tumor cells that lowly expressed B7-H3, there was no significant difference in the expression of CD25 and CD69 on the surface of B7-H3-CAR Jurkat cells and Jurkat cells ( Figure 2 C).

[0187] The above results indicate that the B7-H3-CAR-Jurkat cells constructed in the present invention can be specifically activated by B7-H3 positive tumor cells.

[0188] Example 3 In vitro killing of B7-H3-positive tumor cells by B7-H3-CAR T cells

[0189] In order to further demonstrate the in vitro biological function of the B7-H3-CAR constructed in Example 2, B7-H3-CAR-T cells were prepared, and the positive rate of flow cytometry was approximately 51.84% ( Figure 3 A). Western blotting showed that B7-H3-CAR fusion protein was expressed in B7-H3-CAR T cells ( Figure 3 B), which includes two endogenous CD3ζ of 16kD and 28kD expressed by all T cells (lane 1, lane 2), and one exogenous CD3ζ expressed by CAR-T cells (56kD in lane 2).

[0190] After T cells and prepared B7-H3-CAR T cells were co-incubated with HEL, AML5 and KG-1 cells at a 1:1 effector-target ratio for 48 hours, apoptosis of tumor cells was detected. The results showed that compared with T cells, B7-H3-CAR T cells had a good toxic effect on tumor cells with high and moderate expression of B7-H3, while there was no significant difference for tumor cells with low expression of B7-H3. This shows that B7-H3-CAR T cells have a strong toxic effect on AML cells expressing B7-H3 ( Figure 3 C).

[0191] Subsequently, the secretion of cytokines in the supernatant was detected. Consistent with the killing results, the cytokine secretion of B7-H3-CAR T cells co-incubated with tumor cells with high and moderate expression of B7-H3 was significantly higher than that of other groups ( Figure 3D). This further illustrates that the B7-H3-CAR T cells constructed by the present invention can specifically kill AML tumor cells expressing B7-H3.

[0192] Example 4 B7-H3-CAR T cells can prolong the survival of tumor-bearing mice

[0193] To further evaluate the anti-tumor activity of B7-H3-CAR T cells in vivo, luciferase-GFP-HEL cells were successfully constructed ( Figure 7 A).

[0194] First, establish an AML mouse tumor model. Take 6-8 week old NSG female mice and inject 5×10 5 luciferase-GFP-HEL cells were used to observe the mice. After about 20 days, they developed depression, hind limb paralysis, emaciation, and weight loss. Figure 7 B). After natural death, the bone marrow, peripheral blood, spleen, liver, and kidney of the mice were collected for flow cytometry analysis of the proportion of tumor cells ( Figure 7 C) Experiments have shown that mice injected with 5×10 5 AML model mice can be successfully established with only one tumor cell.

[0195] Luciferase-GFP-HEL cells were injected into the tail vein of 12 B-NSG mice (divided into 3 groups, 4 mice in each group) Figure 4 A), each mouse was injected with 5×10 5 On the fifth day, the proportion of tumor cells in the mice was detected by live imaging, which was equivalent to a mid-to-late stage AML case model. Then, the same amount of PBS / T / B7-H3-CAR T was injected into the tail vein (the positive rate was about 58.19%). Figure 8 The survival of the mice was observed, and the tumor load in the mice was detected every few days using in vivo imaging. The in vivo imaging showed that the tumor progression in the B7-H3-CAR T group was significantly weaker than that in the other two groups ( Figure 4 B). The average body weight of mice in the PBS and T groups dropped sharply around 30 days, and the mice died one after another, while the body weight of mice in the B7-H3 group remained nearly stable ( Figure 4 C). After all the mice died, their survival time was counted. The results showed that the survival time of mice in the B7-H3-CAR T group was significantly longer than that in the PBS group and the T cell group, and there was a statistical difference ( Figure 4 D, Figure 4 E). This indicates that B7-H3-CAR T cells can effectively inhibit the growth of tumor cells in tumor-bearing mice, and B7-H3-CAR T cells have a significant in vivo anti-tumor effect on B7-H3-positive AML cells.

[0196] Example 5 B7-H3-CAR T cells can reduce tumor burden in peripheral blood and bone marrow of tumor-bearing mice

[0197] In order to further demonstrate the in vivo anti-tumor effect of B7-H3-CAR T cells, a group of mouse models were established ( Figure 5 A). On the second day after tumor cell injection, in vivo imaging showed that tumor cells infiltrated the bone marrow, indicating that the modeling was successful. 7 T cells and B7-H3-CAR T cells (CAR positive rate was 52.82%, Figure 8 B) were injected into mice, and then live imaging was performed every 4-5 days. The results showed that compared with the PBS group and T group mice, the tumor growth of the B7-H3-CAR T group mice was significantly inhibited ( Figure 5 B).

[0198] On day 20, the mice in the PBS group showed symptoms such as weight loss, mental depression, and quadriplegia, so all mice were killed, and peripheral blood and bone marrow cells were collected to detect the proportion of tumor cells using flow cytometry ( Figure 5 C) The results showed that the tumor burden of mice in the B7-H3-CAR T group was significantly lower than that of the other two groups of mice.

[0199] Immunohistochemical analysis of the spleen and ovarian tissues of mice showed that there was obvious tumor cell infiltration in the tissues of mice in the PBS group and T cell group, while there was almost no or only a few tumor cell infiltration in the tissues of mice in the B7-H3 group. Therefore, it was proved that B7-H3-CAR T cells can effectively reduce the development of tumor cells in mice.

[0200] Example 6 B7-H3-CAR T does not produce toxicity to normal cells

[0201] The scFv sequence used in the present invention has high affinity, a faster dissociation constant, higher killing activity and safety. The sequence can simultaneously recognize mouse B7-H3 molecules, so the safety of the sequence can be demonstrated by detecting the activity of normal cells in mice.

[0202] Peripheral blood routine tests were performed on the blank group, PBS group, T cell group, and B7-H3-CAR T cell group of mice (mouse model of Example 5). The results showed that there was no significant difference between the B7-H3-CAR T group and the blank group in terms of white blood cells, red blood cells, platelets, and hemoglobin ( Figure 5D). Moreover, the HE staining results also showed that compared with healthy mice, the heart, liver, kidney and intestine of the B7-H3-CAR T cell group mice did not have lesions. Therefore, the above experiments prove that the B7-H3-CAR T developed by the present invention is a CAR-T product with high safety.

[0203] discuss

[0204] AML is an aggressive hematopoietic stem cell malignancy that is very difficult to treat. In the past decade or so, with the emergence of some new treatments, the life expectancy of AML patients has improved, but due to drug resistance and refractoryness, AML is still one of the most deadly and difficult to cure malignant cancers. In addition, AML patients have very limited treatment options and a poor prognosis. They cannot be cured after chemotherapy and are prone to relapse, so new treatments are urgently needed. CAR T therapy has made great progress in both hematological tumors and solid tumors, and is considered one of the most promising tumor treatments.

[0205] At present, most CAR T treatments for AML require bone marrow ablation before treatment because most targets are also highly expressed in normal myeloid cells, which greatly complicates the treatment method and increases many unnecessary risks. Therefore, it is important to find an antigen that is selectively expressed only in malignant cells and lacks in normal cells. An article has statistically shown that about 40% of AML patients will express B7-H3, and studies have also shown that B7-H3 expression was detected in >50% of cells in 31 pancreatic tumor specimens, while B7-H3 expression was not detected in normal pancreatic tissue specimens. The expression of B7-H3 is regulated by RNA transcription. It is a surface immunoregulatory glycoprotein that can inhibit natural killer cells and T cells. Although B7-H3 transcription products are widely expressed in human solid tumors and normal tissues, B7-H3 protein is preferentially expressed only in tumor tissues. Some scholars have also pointed out that B7-H3 protein is only expressed in a few tissues and cells, including activated lymphocytes and tumor cells. Therefore, although B7-H3 is only expressed in some AML, it is an excellent therapeutic target for AML because it does not cause toxicity to the hematopoietic system.

[0206] Studies have shown that relying solely on T cells is not enough to fight cunning tumor cells, but the B7-H3-CAR T cells of the present invention can effectively and specifically kill AML cells expressing B7-H3 ( Figure 3 C). B7-H3-CAR T cells also have a significant tumor-clearing effect in mice. In vivo imaging showed that the number of tumor cells in the B7-H3-CAR T group mice was significantly lower than that in the PBS group and the T cell group ( Figure 4B, 5B). Moreover, even for AML, a disease with particularly rapid progression, B7-H3-CAR T cells can significantly prolong the survival of mice ( Figure 4 D, 4E), reducing the proportion of tumor cells in peripheral blood, bone marrow and tissues of mice ( Figure 5 C, 5D). Therefore, the B7-H3-CAR T of the present invention can be used to treat AML patients with refractory relapse and expression of B7-H3 antigen.

[0207] In addition, the safety of B7-H3-CAR T was verified by testing whether it would produce off-target toxicity to normal cells in mice. Peripheral blood routine tests showed that B7-H3-CAR T did not produce toxic effects on normal cells in mice ( Figure 6 A). Moreover, HE staining results also showed that B7-H3-CAR T cells did not damage important organs of mice ( Figure 6 B). Therefore, the B7-H3-CAR T of the present invention is a safe CAR-T product.

[0208] In summary, the present invention not only proves that B7-H3-CAR T can effectively and specifically eliminate AML tumor cells expressing B7-H3 antigen in vitro and in vivo, but also proves that it has good safety, laying the foundation for the clinical use of B7-H3-CAR T cells to treat AML. Compared with the currently most commonly used CD33-CAR T and CD123-CAR T cells, which require patients to undergo myeloablation before use, the use of B7-H3-CAR T not only has the potential to prolong the survival of patients with relapsed and refractory AML, but also simplifies the actual use of CAR T therapy in clinical practice and reduces patient risks.

[0209] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0210] The sequences involved in the sequence table of this application are as follows:

[0211] Sequence Listing <110> Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd. <120> CAR-T cells targeting B7-H3 and their application in the treatment of acute myeloid leukemia <130> P2020-0883 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <400> 2 Gln Ile Val Leu Thr Gln Ser Pro Ala Asn Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Asn 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 492 <212> PRT <213> Artificial Sequence <400> 3 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Thr Tyr Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys 50 55 60 Arg Leu Glu Trp Val Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr 65 70 75 80 Ala Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg 85 90 95 Asn Ile Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala 100 105 110 Met Tyr Tyr Cys Val Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Asn Met Ser Ala Ser Leu 165 170 175 Gly Glu Arg Val Thr Met Thr Cys Ala Ala Ser Ser Ser Val Ser Ser 180 185 190 Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu 195 200 205 Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe 210 215 220 Asn Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met 225 230 235 240 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser 245 250 255 Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Gly Gly Gly Gly Ser 1 5 <210> 5 <211> 335 <212> PRT <213> Artificial Sequence <400> 5 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <400> 6 Gly Phe Thr Phe Ser Thr Tyr 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <400> 7 Leu Ser Gly Gly Thr 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <400> 8 Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <400> 9 Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <400> 10 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <400> 11 Gln Tyr His Arg Ser Pro Trp Thr 1 5

Claims

1. Use of an active ingredient for preparing a medicament for treating acute myeloid leukemia, wherein the active ingredient is selected from the group consisting of: (i) a T cell expressing a chimeric antigen receptor (CAR) that binds to B7-H3, wherein the antigen binding domain of the chimeric antigen receptor comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region includes the following complementarity determining regions CDR: CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8; And the light chain variable region includes the following complementarity determining region CDR: CDR1' shown in SEQ ID NO:9, CDR2' shown in SEQ ID NO:10, and CDR3' shown in SEQ ID NO:11; (ii) a preparation comprising the T cells and a pharmaceutically acceptable carrier.

2. The use according to claim 1, It is characterized in that The antigen binding domain includes the antibody heavy chain variable region shown in SEQ ID NO: 1, and the antibody light chain variable region shown in SEQ ID NO:

2.

3. The use according to claim 1, It is characterized in that The structure of the chimeric antigen receptor is shown in Formula III below: L-scFv-H-TM-C-CD3ζ (III) in, L is none or a signal peptide sequence; scFv is a scFv targeting B7-H3; H is the hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

4. The use according to claim 1, It is characterized in that The CAR also includes a cell suicide element.

5. The use according to claim 4, It is characterized in that The cell suicide element is tEGFR.

6. The use according to claim 1, It is characterized in that The amino acid sequence of the CAR is shown in SEQ ID NO:3.

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