WO2019128996A1 - Mesothelin-specific car-t cell expressing cd47 antibody, and use thereof - Google Patents

Abstract

Provided is a mesothelin-specific CAR-T cell expressing a CD47 antibody. The T cell: (1) comprises a coding sequence of a chimeric antigen receptor that recognizes a mesothelin antigen and a coding sequence of a CD47 antibody, and/or (2) expresses a chimeric antigen receptor recognizing the mesothelin antigen, and a CD47 antibody. While specifically targeting tumor cells highly expressing mesothelin, the T cell of the present invention can secrete a CD47 antibody, prevent immune escape of tumor cells, and recover phagocytosis of tumor cells by macrophages, thereby achieving a better anti-tumor effect.

Description

Mesothelin-specific CAR-T cells self-expressing CD47 antibody and use thereof Technical field

The present invention pertains to genetic engineering and immunology, and relates to mesothelin-specific CAR-T cells that express CD47 antibodies and uses thereof.

Background technique

Cancer has become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices of cancer, making cancer's high incidence and rejuvenation trend more and more obvious. At present, the commonly used treatments are very limited, and it is still necessary to explore a more effective treatment to improve the survival rate and quality of life of cancer patients.

Immunotherapy for malignant tumors has developed rapidly in recent years and has achieved remarkable clinical results. Since 2011, Nature and the top magazine of clinical oncology JCO have published the same article "The era of tumor immunotherapy has arrived" (Nature.2011;480(7378):480; J Clin Oncol.2011;29(36) :4828), tumor immune cell therapy ushered in a new round of research boom.

As one of the important branches of tumor immunotherapy, chimeric antigen receptor T cell therapy has achieved very good curative effect in malignant hematological tumors, and the complete remission rate of relapsed and refractory B cell leukemia is over 90%. In August 2017, the US FDA approved Novartis's tissue lecleucel chimeric antigen receptor T cell (CAR-T cell) treatment for the treatment of childhood and young adult patients with acute lymphoblastic leukemia (ALL), becoming the first approved market. CAR-T drug. Immediately in October of the same year, the US FDA announced the approval of Kite Pharma's CAR-T therapy, Yescarta, for the treatment of adult patients with certain types of large B-cell lymphoma. The approval of CAR-T drugs has taken CAR-T treatment to a new level.

A chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region. By the identification of a single-chain fragment variable (scFv) and an intracellular signal domain of an antibody-associated antigen (TAA), an immunoreceptor tyrosine-based activation Motifs, ITAM)" genetic recombination in vitro. It is then introduced into T cells by a virus or other vector system such that the genetically engineered T cells are referred to as CAR-T cells. After extensive expansion in vitro, CAR-T cells are returned to the patient and can exhibit potent anticancer effects in a non-MHC-restricted mode.

However, the efficacy of CAR-T cells in the treatment of solid tumors is still insufficient. The main reasons include: 1. The solid tumor is highly heterogeneous, lacking cell surface targets suitable for CAR-T treatment; 2. Solid tumors have a microenvironment that strongly inhibits immunity.

Mesothelin is a glycoprotein anchored to the plasma membrane by a phosphatidylinositol region (GPI), which is highly expressed in various tumor tissues and is expressed in a small amount in mesothelial cells of normal pleura, pericardium and peritoneum. The mesothelin gene encodes a 69 kDa precursor protein that is processed to form a 40 kDa membrane-bound protein and a 31 kDa detached fragment called megakaryocyte promoting factor (MPF) and released out of the cell, which we usually call Peelin refers to a fragment anchored on a membrane, which can be divided into three regions, Region I, II, and III, according to its protein structure. On the one hand, it can activate NFκB, MAPK and PI3K intracellular signaling pathways through its GPI domain to promote cell proliferation and resist apoptosis; on the other hand, interaction with its receptor CA125/MUC16 leads to abnormal cell adhesion and promotes cancer cells. Transfer. Due to the limited distribution of mesothelin in normal tissues and overexpression in a variety of malignant tumors (mesothelioma, ovarian cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, etc.), it is a potential tumor-specific therapeutic target. point.

At present, small molecule drugs and antitoxins targeting mesothelin have achieved good results, and CAR-T cell research targeting this is also in full swing, targeting mesothelin. There are 5 clinical trials for CAR-T cell therapy, ranking fourth among other targets except CD19, mainly for pancreatic cancer (NCT01897415, NCT02465983), mesothelioma (NCT01355965, NCT02414269), lung cancer and breast. Cancer (NCT02414269).

CD47 is mainly expressed on the surface of cancer cells and is generally considered to be a protective receptor for cancer cells to be protected from host immune system attack. Studies have shown that T cells and dendritic cells (DC) can exert anti-tumor effects through the CD47 blocking effect. CD47 is a type of "don't eat me" signal that inhibits macrophage function by binding to SIRP-α on the surface of macrophages. CD47 has unparalleled advantages as a target for cancer treatment: 1. It is widely expressed on the surface of various cancer cells, so it can be used to treat various types of cancer; 2. Normal cells lack the "eat me" signal, so Blocking CD47 alone does not trigger the phagocytic effect of macrophages on normal cells, so the side effects of CD47 blockers are also very small.

Summary of the invention

The present invention provides a T cell: (1) a coding sequence comprising a chimeric antigen receptor recognizing a mesothelin antigen and a coding sequence of a CD47 antibody; and/or (2) expression recognition of a mesothelin antigen Chimeric antigen receptor and CD47 antibody.

In one or more embodiments, the expression cassette of the chimeric antigen receptor recognizing the mesothelin antigen and the expression cassette of the CD47 antibody are integrated into the genome of the T cell.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a signal peptide, a single-chain antibody against the mesothelioma proximal membrane end, a hinge region longer than 50 amino acid residues, and a cross Membrane region, intracellular costimulatory signal domain and intracellular signal domain.

In one or more embodiments, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably a light chain signal peptide; preferably, the light chain signal peptide The amino acid sequence is shown as amino acid residues 1 to 20 of SEQ ID NO: 1.

In one or more embodiments, the amino acid sequence of the single chain antibody is as shown in amino acid residues 21 to 270 of SEQ ID NO: 1.

In one or more embodiments, the hinge region longer than 50 amino acid residues is selected from the group consisting of a CD8 alpha hinge region, an IgD hinge region, an IgGl Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is CD8α Hinge region or IgG4Fc CH2CH3 hinge region; more preferably, the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is as shown in amino acid residues 271-498 of SEQ ID NO: 1.

In one or more embodiments, the transmembrane region is a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region. One; preferably a CD8 transmembrane region, preferably having an amino acid sequence as shown in amino acid residues 499-526 of SEQ ID NO: 1.

In one or more embodiments, the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase An intracellular domain of an inducible T cell costimulatory factor (ICOS) and a DNAX activator protein 10; preferably, the intracellular costimulatory signal domain is an intracellular domain of CD28; preferably, the amino acid sequence of the CD28 Shown as amino acid residues 527-567 of SEQ ID NO: 1.

In one or more embodiments, the intracellular signal domain is a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. ID NO: 1 is described in amino acid residues 568-679.

In one or more embodiments, the chimeric antigen receptor comprises a light chain signal peptide, a single chain antibody against mesothelin Region III, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, The intracellular domain of CD28 and the tyrosine activation motif of CD3ζ.

In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 21 to 679 of SEQ ID NO: 1, or as SEQ ID NO: 1; preferably, the inlay The coding sequence of the antigen receptor is shown as bases 61 to 2037 of SEQ ID NO: 4, or as shown in SEQ ID NO: 4.

In one or more embodiments, the amino acid sequence of the CD47 antibody is set forth in amino acid residues 21 to 367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2; preferably, the CD47 antibody The coding sequence is shown as bases 61-1101 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5.

The invention also provides a composition comprising: a vector comprising an expression cassette of a chimeric antigen receptor of the invention, the vector for integrating the expression cassette into the genome of a host cell; A vector comprising an expression cassette of a CD47 antibody for integration of the expression cassette into the genome of a host cell.

In one or more embodiments, the amino acid sequence of the CD47 antibody is set forth in amino acid residues 21 to 367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2; preferably, the CD47 antibody The coding sequence is shown as bases 61-1101 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5.

The invention also provides a kit, the kit comprising:

(1) a vector comprising an expression cassette of a chimeric antigen receptor of the present invention, which vector is used to integrate the expression cassette into the genome of a host cell;

(2) A vector comprising an expression cassette of a CD47 antibody, which vector is used to integrate the expression cassette into the genome of a host cell.

In one or more embodiments, the amino acid sequence of the CD47 antibody is set forth in amino acid residues 21 to 367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2; preferably, the CD47 antibody The coding sequence is shown as bases 61-1101 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5.

The present invention also provides a pharmaceutical composition comprising the T cell of the present invention or the T cell and the CD47 antibody expressed thereby.

The invention also provides the use of a T cell or a T cell as described herein and a CD47 antibody thereof, for the manufacture of a medicament for the treatment or prevention of a malignancy. Preferably, the cancer is a cancer in which the surface of the cancer cell abnormally expresses mesothelin, preferably 100 times or more when the expression level of mesothelin is normal on the surface of the cancer cell, and the mesothelin is uniformly distributed over the entire cell surface. Cancer; preferably, the cancer is selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer; Preferably, the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.

DRAWINGS

Figure 1: Schematic diagram of plasmid structure of Meso3CAR and CD47 antibodies.

Figure 2A: Flow detection of positive rates of αCD47-Meso3CAR T cell CAR.

Figure 2B: ELISA assay for expression of CD47 antibody to αCD47-Meso3CAR T cells.

Figure 3A: CAR-T positive rate of αCD47-Meso3 CAR T cells constructed under different ratios of Meso3CAR and CD47 antibody plasmids.

Figure 3B: Expression of CD47 antibody in αCD47-Meso3CAR T cells constructed under different ratios of Meso3CAR and CD47 antibody plasmids.

Figure 4: Flow cytometry analysis of CD47 expression of Mock, Meso3CAR and αCD47-Meso3CAR T cells.

Figure 5: Killing of tumor cell lines by αCD47-Meso3CAR T cells.

Figure 6: CD47 of the surface of tumor cells was blocked after co-culture of αCD47-Meso3CAR T cell supernatant with tumor cells.

Figure 7: Blocking the surface of tumor cells CD47 can enhance the phagocytosis of macrophages.

Figure 8: In vivo anti-tumor effect of αCD47-Meso3CAR T cells.

Detailed ways

Some of the terms related to the present invention are explained below.

In the present invention, the term "expression cassette" refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.

The term "coding sequence" is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "Fc", ie, fragment crystallizable (Fc) of an antibody, refers to a peptide located at the end of the handle of the "Y" structure of the antibody molecule, comprising the CH2 and CH3 domains of the heavy chain constant region of the antibody, and is an antibody and effect. The site of molecular or cellular interactions.

The term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules. The costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell. B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.

The term "linker" or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.

The term "specifically binds" refers to the reaction between an antibody or antigen-binding fragment and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) means that the antibody is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, Affinity (KD) of 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or less binds to the antigen. "Specific recognition" has a similar meaning.

The term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The term "effective amount" refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.

The term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition described herein.

The term "subject" or "patient" may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.

The term "chimeric antigen receptor" (CAR) is an artificially engineered receptor that anchors specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), allowing immune cells to recognize tumor antigens. Or viral antigens and cells that kill tumor cells or virus infection. CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region. Generally, a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity. The polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.

The term "single-chain antibody" (scFv) refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability. In certain embodiments, the single chain antibody of interest (scFv) is from an antibody of interest. Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies. The antibody can be secreted or membrane anchored.

By constructing a Meso3CAR T cell which expresses CD47 antibody, the tumor cell which specifically targets mesothelin is simultaneously secreted, can secrete CD47 antibody, eliminate immune escape of tumor cells, and restore macrophage to tumor cells. Phagocytosis for better anti-tumor effects. In addition, the Fc fragment of the CD47 antibody designed by the present invention is a mutant IgG4Fc, which avoids binding to the γ-2 receptor on the surface of dendritic cells and is recognized and phagocytized by macrophages, so that the self-expressing CD47 antibody CAR-T The cells function without causing an AICD response.

Accordingly, the present invention provides a CD47 antibody comprising a CD47 ligand and an IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 139-367 of SEQ ID NO: 2; preferably, the coding sequence thereof is SEQ ID NO: 5 base sequence of 415-1101 Shown.

In certain embodiments, the amino acid sequence of the CD47 ligand is represented by amino acid residues 21 to 138 of SEQ ID NO: 2; preferably, the coding sequence thereof is at base 61-414 of SEQ ID NO: The base sequence is shown.

In certain embodiments, the CD47 antibody further comprises a light chain signal peptide. In certain embodiments, the CD47 antibody, from the N-terminus to the C-terminus, in turn comprises a light chain signal peptide, a CD47 ligand, and an IgG4 Fc. In certain embodiments, the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 2; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 5 base sequence of 1-60 is shown.

In certain embodiments, the amino acid sequence of the CD47 antibody is set forth in amino acid sequence 21-367 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.

The invention also encompasses a coding sequence for the CD47 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof. In certain embodiments, the coding sequence of the CD47 antibody comprises the sequence set forth in bases 61-1101 of SEQ ID NO: 5, preferably the sequence set forth in SEQ ID NO: 5.

The present invention also encompasses a nucleic acid construct comprising the coding sequence of a CD47 antibody of the present invention or a complement thereof. Preferably, the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement into a host cell.

The invention also provides a host cell comprising a nucleic acid construct as described herein.

The invention also provides the use of the CD47 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignant tumor, particularly a tumor associated with CD47, including but not limited to Various malignant tumors.

The present invention also provides a T cell which is modified by a mesothelin CAR gene and can express a CD47 antibody, and the T cell can stably express a mesothelin CAR gene and a CD47 antibody at a high level, and the exogenously expressed mesothelin CAR gene can be Accurate targeting of mesothelin antigen, enhance the proliferation of T cells and secretion of cytokines, enhance the killing of tumor cells by CAR-T cells, and exert anti-tumor effects by enhancing immune response. At the same time, the exogenously expressed CD47 antibody can eliminate the immune escape of tumor cells, restore the phagocytosis of macrophages to tumor cells, promote the apoptosis of tumor cells, and exert an anti-tumor immune response. In addition, the exogenous mesothelin CAR gene and the CD47 antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stably and continuously expressing in T cells. The T cells of the present invention which stably express the mesothelin CAR gene and the CD47 antibody gene at a high level can be used for the treatment of various mesothelin-expressing malignant tumors.

The CAR of the invention typically contains an optional signal peptide sequence, an scFv that recognizes a mesothelin antigen, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

A signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures. The signal peptide can be a secreted signal peptide or a membrane-bound signal peptide. In certain embodiments of the invention, the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide or a light chain signal peptide; more preferably a light chain signal peptide. The amino acid sequence of the light chain signal peptide can be as shown in amino acid residues 1-20 of SEQ ID NO: 1; in certain embodiments, the coding sequence is as shown in bases 1 to 60 of SEQ ID NO: 4. .

The scFv recognizing the mesothelin antigen described herein may be a single chain antibody directed against mesothelin antigen as is well known in the art. Preferably, the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody directed against the mesothelin near membrane end amino acid sequence. Preferably, the anti-mesothelin single chain antibody described herein is a single chain antibody directed against Region I or III of mesothelin. Preferably, the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody against the amino acid sequence of mesothelin, Region I or III. In certain embodiments, the amino acid sequence of mesothelin Region I is set forth in SEQ ID NO: 6; the amino acid sequence of mesothelin Region III is set forth in SEQ ID NO: 7. An exemplary amino acid sequence of a single chain antibody against mesothelin, Region I, is set forth in SEQ ID NO: 8. The amino acid sequence of an exemplary single-chain antibody against mesothelin-Region III is shown in amino acid residues 21 to 270 of SEQ ID NO: 1, and an exemplary coding sequence thereof is SEQ ID NO: 4, item 61- The nucleotide sequence of 810 is shown. Herein, mesothelin refers to a mesothelin fragment anchored on a membrane unless otherwise specified.

Herein, the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes. The hinge region suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4. . The hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer. In certain embodiments, a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein. The amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is shown in amino acid residues 271-498 of SEQ ID NO: 1, and the coding sequence of the exemplary IgG4 FcCH2CH3 hinge region is SEQ ID NO: 4 at positions 811-1494. Show.

The transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region Preferably, the amino acid sequence thereof is set forth in 499-526 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1495-1578 of SEQ ID NO: 4.

The intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably the amino acid sequence thereof is set forth in amino acid residues 527-567 of SEQ ID NO: 1, exemplary The coding sequence is shown as bases 1579-1701 of SEQ ID NO:4.

The intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain As described in SEQ ID NO: 1 at amino acid residues 568-679; in certain embodiments, the coding sequence is set forth in SEQ ID NO: 4, 1702-2037.

In certain embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, an optional light chain signal peptide, an anti-mesothelin Region III scFv, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, The intracellular domain of CD28 and the CD3 intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 21 to 679 of SEQ ID NO: 1. In certain embodiments, the chimeric antigen receptor further comprises a light chain signal peptide, preferably the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 1-20 of SEQ ID NO: 1.

It will be understood that the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.

Forming the above-described various portions of the chimeric antigen receptor herein, such as a signal peptide, a light chain variable region and a heavy chain variable region of an anti-mesothelin single-chain antibody, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and The intracellular signal domains and the like may be directly connected to each other or may be connected by a linker sequence. The linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.

It will be appreciated that in gene cloning procedures, it is often desirable to design a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, promote expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein. Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino or carboxy terminus of a CAR herein may also contain one or more polypeptide fragments as a protein tag. Any suitable label can be used in this article. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used to purify proteins.

Also included herein are polynucleotide sequences encoding the chimeric antigen receptor. The polynucleotide sequence herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.

The polynucleotide sequences described herein can generally be obtained by PCR amplification. In particular, primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO:4.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the CD47 antibody, and one or more operably linked to the sequences Regulatory sequence. In certain embodiments, the nucleic acid construct is an expression cassette.

The control sequence can be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used herein.

In certain embodiments, the nucleic acid construct is a vector. In particular, the coding sequences for CARs herein or the coding sequences for CD47 antibodies can be cloned into a wide variety of vectors, such as, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector can be an expression vector. The expression vector can be provided to the cells in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism. For example, in certain embodiments, the invention employs a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein or a coding sequence for a CD47 antibody And optional optional markers.

Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered. The use of an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

In certain embodiments, various promoter sequences disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1α promoter shown in SEQ ID NO: 5 of the application. TCEF promoter containing CD3e enhancer, mCMV enhancer, hCMV enhancer and EF1α promoter as shown in SEQ ID NO: 7; mCMV-containing enhancer, hCMV enhancer and inclusion as shown in SEQ ID NO: a CCEFI promoter of the EF1α promoter containing a subunit; a TEFI promoter comprising a CD3e enhancer and an intron-containing EF1α promoter represented by SEQ ID NO: 3; and a CD3e enhancer represented by SEQ ID NO: , the mCMV enhancer, the hCMV enhancer, and the TCEFI promoter of the intron-containing EF1α promoter. The entire contents of this application are herein incorporated by reference.

The selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by the viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes such as neo and the like. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.

In certain embodiments, the coding sequence of a chimeric antigen receptor described herein and the coding sequence of a CD47 antibody are separately cloned into a vector (also referred to as an integration vector) for integration of a nucleic acid sequence of interest into the genome of a host cell. Medium, especially transposon vectors. In certain embodiments, the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty. Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon. The transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When a transposase from a different transposition system is used, the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art. . Between the 5' LTR and the 3' LTR is the expression cassette for the CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for the CAR or antibody, and the polyA tailing signal sequence.

In certain embodiments, the transposase is a transposase from a piggybac transposition system. Thus, in these embodiments, the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively. In certain embodiments, the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference. In certain embodiments, the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7 SEQ ID NO: 4. In certain embodiments, the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.

The promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence. In certain embodiments, the expression of the transposase coding sequence is controlled using a CMV promoter. The sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.

In certain embodiments, the vector of the present invention comprising a coding sequence for a chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7. The coding sequence of the chimeric antigen receptor of the present invention can be prepared by a method conventional in the art and cloned into a suitable vector.

In certain embodiments, the vector for integrating a gene of interest into the genome of a host cell does not contain a transposase coding sequence. For example, such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector. Typically, such vectors are used to integrate the coding sequence for the CD47 antibody and the signal peptide coding sequence (e.g., the coding sequence for the light chain signal peptide) into the genome of the host cell. The amino acid sequence of an exemplary light chain signal peptide is shown in amino acid residues 1-20 of SEQ ID NO: 1, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 2 bases 1-60. Shown.

In certain embodiments, a T cell modified with a mesothelin CAR gene and capable of expressing a CD47 antibody described herein can be transduced into a transposon for integration into a chimeric antigen receptor coding sequence in the T cell genome. A vector encoding the sequence of the enzyme, and a vector for the integration of the coding sequence of the CD47 antibody described herein into the T cell genome without the transposase coding sequence.

Preferably, the T cell is transformed into a vector containing a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a backbone vector and constructed using a pS328 vector (with no transposase coding sequence compared to pNB328) as a backbone vector. A vector containing a CD47 antibody coding sequence. In certain embodiments, the coding sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 4; the coding sequence of the CD47 antibody is as set at bases 61-1488 of SEQ ID NO: 5. In certain embodiments, in the vector comprising the coding sequence for a CD47 antibody, the signal peptide of the CD47 antibody is a light chain signal peptide. The amino acid sequence of an exemplary light chain signal peptide can be shown as amino acid residues 1-20 of SEQ ID NO: 1; the coding sequence of an exemplary light chain signal peptide is nucleus 1 to 60 of SEQ ID NO: 5. The nucleotide sequence is shown. More specifically, in certain embodiments, the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome in turn contains a 5' LTR, a promoter, a light chain signal peptide a coding sequence, a coding sequence for a scFv recognizing a mesothelin antigen (preferably a coding sequence for the scFv recognizing mesothelin Region III), a coding sequence for the IgG4 Fc CH2CH3 hinge region, a coding sequence for the CD8 transmembrane region, and a CD28 intracellular domain Coding sequence, coding sequence for the CD3 intracellular signal domain, polyA tailing signal sequence, coding sequence for 3'LTR and transposase, and promoter thereof; said integration of the CD47 antibody described herein in the T cell genome The vector containing the transposase coding sequence of the coding sequence contains, in sequence between the 5'LTR and the 3' LTR, a promoter, a coding sequence for the light chain signal peptide, a coding sequence for the CD47 antibody, and a polyA tailing signal sequence.

Preferably, at the time of transfection, the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD47 antibody coding sequence is from 1 to 7:1 to 7, preferably from 1 to 3:1 to 3, preferably 1:1. ～3, more preferably 1:1 to 2, still more preferably 1:1.

Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In certain embodiments, the vector is transfected into a cell of interest using electroporation.

The cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokines. T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL). In certain embodiments, the T cell can be derived from a PBMC of a B cell malignancy patient. In certain embodiments, the T cell is a primary cultured T cell.

The invention also provides a composition comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a CD47 antibody described herein. Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.

The invention also provides a kit comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a CD47 antibody described herein, or a composition described herein. A reagent or instrument for transferring the vector into a cell can also be provided in the kit.

As described herein, the expression cassette contains at least a suitable promoter and a PolyA tailing signal sequence in addition to the coding sequence comprising a chimeric antigen receptor or a CD47 activating antibody.

The invention also provides a pharmaceutical composition comprising a T cell or a T cell as described herein and a CD47 antibody expressed thereby. Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions. The pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of the T cell can be determined according to factors such as the condition of the patient.

The invention also provides the use of a T cell or a pharmaceutical composition thereof described herein or the T cell and the CD47 antibody expressed thereby for the preparation of a medicament for the treatment or prevention of a malignancy. The invention also provides a method of treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the invention. The cancer suitable for the treatment or prevention of the T cells described herein is preferably a mesothelin-positive cancer, specifically a cancer having abnormal expression of mesothelin on the surface of the cancer cell, such as 100 when the expression level of mesothelin on the surface of the cancer cell is normal. A cancer that is more than twice as large and mesothelin is evenly distributed throughout the cell surface. Specifically, such cancer may be selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer; more preferably The cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed, such as ovarian cancer.

Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased through the market.

Example 1: Construction of recombinant plasmid pNB328-Meso3CAR, PS328-αCD47

Entrusted commercial company to synthesize Meso3CAR (containing CD8 signal peptide, anti-mesothelin single-chain antibody, IgG4CH2CH3 hinge region, CD8 transmembrane region, CD28 intracellular domain and CD3ζ tyrosine activation motif, its nucleotide sequence is SEQ. ID NO: 4, the encoded amino acid sequence is as shown in SEQ ID NO: 1) and the mutant αCD47 gene (the nucleotide sequence thereof is shown as SEQ ID NO: 5; the encoded amino acid sequence is SEQ ID NO: 2 </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The recombinant plasmids constructed between the EcoRI and SalI cleavage sites of the pNB328 vector were designated as pNB328-Meso3CAR and pS328-αCD47, respectively. The structural pattern of the constructed recombinant plasmid is shown in Figure 1.

A recombinant plasmid of the wild type αCD47 gene (nucleotide sequence shown in SEQ ID NO: 9; encoded amino acid sequence shown in SEQ ID NO: 3) was constructed in the same manner using the pS328 vector, and designated as pS328-αCD47-wt. The vector structure is similar to pS328-αCD47, except that the antibody coding sequence therein.

Example 2: Construction of αCD47-Meso3CAR T cells

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation. The PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells. The suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rmp for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rmp for 3 min to abandon the physiology. Saline, and repeat this step; take 1.5ml centrifuge tube, add 5 × 10 ⁶ cells per tube, centrifuge at 1200rmp for 3min, discard the supernatant, take the electro-transfer kit (from Lonza company), add 100ul of electro-rotation reagent proportionally, add pNB328 -Meso3CAR, pS328-αCD47 plasmid each 4ug, transfer the mixture to the electric rotor, put into the electro-rotation instrument, select U-014 program, electric shock; use the micro-pipette in the kit to transfer the cell suspension to the electric In a six-well plate of good culture solution (AIM-V medium containing 2% FBS), mix, place at 37 ° C, incubate in a 5% CO ₂ incubator, and add stimulating factors IL-2 and anti-CD3 six hours later. /anti-CD28, 37 ° C, continue to culture for 4 to 5 days, observe the growth of T cells, and obtain αCD47-Meso3CAR T cells.

Using the same method, wt-αCD47-Meso3 CAR T cells were constructed using 4 ug each of pNB328-Meso3CAR and pS328-αCD47-wt plasmid.

Using the same method, Meso3CAR T cells were constructed using pNB328-Meso3CAR alone (6 ug). Using the same method, Mock T cells were constructed using the pNB328 vector (6 ug).

Example 3: Detection of expression of CAR and CD47 antibodies in αCD47-Meso3CAR T cells

1. Flow detection of CAR T cell positive rate

The αCD47-Meso3CAR T cells prepared in Example 2 were collected and divided into two portions, each of which was 1×10 ⁶ cells, washed twice with physiological saline, resuspended in 100 ul of physiological saline, and one ug of mesothelin antigen was added in one portion. - Biotin, the other is not added, incubate at 4 ° C for 30 minutes. The saline was washed twice, and the cells were resuspended again with 100 ul of physiological saline, and 1 ul of streptomycin-PE antibody was added thereto, and incubated at 4 ° C for 30 minutes. The saline was washed twice and detected by the machine, and the T cells of the empty plasmid (pNB328) were electroporated as a control. The result is shown in Figure 2A.

2. ELISA was used to detect the expression level of αCD47-Meso3 CAR T cells prepared in Example 2.

1 Dilute the CD47 antigen to 0.5 ug/ml (5 ul + 1 ml coating solution) with a coating solution, and coat 100 ul/well of the enzyme-labeled reaction plate at 4 ° C overnight.

2 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

3 100 μl of blocking solution was added to each well and incubated at 37 ° C for 1 hour.

4 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

5 Add samples and standards, 100 ul / well, set up duplicate wells and control wells, incubate for 1 hour at 37 °C.

6 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

7 Blocking solution IgG Fc-HRP was diluted 1:30000, 100 ul/well, and incubated at 37 ° C for 45 minutes.

8 Wash 5 times with PBST for 3 minutes each time, pat dry with absorbent paper, 200 ul / well.

9 Add coloring solution TMB, 100 ul / well, and develop light at 37 ° C for 10-15 min.

10 The stop solution was added to stop the reaction, 50 ul / well.

酶标仪上450nm处测OD值，绘制标准曲线，计算CD47抗体浓度。

The OD value was measured at 450 nm on a microplate reader, a standard curve was drawn, and the CD47 antibody concentration was calculated.

The result is shown in Figure 2B.

Example 4: Comparison of the positive rate and antibody secretion of αCD47-Meso3CAR T cells under different ratios of pNB328-Meso3CAR and pS328-αCD47 plasmids

The amount of pNBS328-Meso3CAR and pS328-αCD47 plasmids were set to 1 ug+7 ug, 2 ug+6 ug, 3 ug+5 ug, 4 ug+4 ug, 5 ug+3 ug, 6 ug+2 ug, 7 ug +1 ug, respectively, for CAR T The cell was constructed and constructed in the same manner as in Example 2.

The positive rate of CAR-T cells and the amount of antibody secreted by the seven ratios were detected (the detection method was the same as in Example 3).

The results are shown in Figures 3A and 3B. Under the ratio of 4ug + 4ug, the positive rate of CAR-T and the amount of antibody secreted can be at a good level.

Example 5: Detection of CD47 expression in Mock, Meso3CAR and αCD47-Meso3CAR T cells

The Mock T cells, Meso3CAR T cells and αCD47-Meso3CAR T cells obtained in Example 2 were collected, and the expression of PD1 was detected using the flow antibody FITC-mouse anti-human CD47 of BD, and the flow method was the same as in Example 3.

As a result, as shown in Fig. 4, the CD47 antibody secreted by αCD47-Meso3CAR T can block the expression of CD47 on the cell surface.

Example 6: Killing effect of Mock, Meso3CAR and αCD47-Meso3CAR T cells on tumor cells

Three EGFR-positive cells of gastric cancer cell line Hgc27, ovarian cancer cell line SKOV3 and pancreatic cancer cell line ASPC-1 were selected as target cells, and the real-time unlabeled cell function analyzer (RTCA) of Essen was used to detect the obtained in Example 2. The in vitro killing activity of Mock, Meso3CAR and αCD47-Meso3CAR T cells, the specific steps are as follows:

(1) Zero adjustment: add 50μl DMEM or 1640 culture solution to each well, put it into the instrument, select step 1, and adjust to zero;

(2) Target cell plating: gastric cancer cell line Hgc27, ovarian cancer cell line SKOV3, and pancreatic cancer cell line ASPC-1 (purchased from American Type Culture Collection ATCC) were plated at 10 ⁴ cells/50 μl per well in a test electrode. Place in the plate for a few minutes, wait for the cells to stabilize, then put them into the instrument, start step 2, and culture the cells;

(3) Adding effector cells: After 24 hours of target cell culture, pause step 2, add effector cells, 50 μl per well, set the target ratio to 4:1, and transfer the Mock T cells into pNB328 empty vector as control. 3. After continuing to co-culture for 24 hours, observe the cell proliferation curve.

The result is shown in Figure 5. The in vitro killing activity of Meso3CAR and αCD47-Meso3CAR T cells was significantly higher than that of Mock T cells, and the expression of CD47 antibody did not affect the killing function of CAR-T cells.

Example 7: αCD47-Meso3CAR T cell culture supernatant can block tumor cell surface CD47

The culture supernatant of αCD47-Meso3CAR T cells obtained in Example 2 was co-cultured with gastric cancer cell line Hgc27, ovarian cancer cell line SKOV3, and pancreatic cancer cell line ASPC-1, respectively. After 24 hours, tumor cells were collected and CD47 expression was detected. The control was not co-cultured with the αCD47-Meso3CAR T cell supernatant. The flow detection method is the same as above.

As a result, as shown in Fig. 6, the CD47 antibody in the supernatant of αCD47-Meso3CAR T cells blocked tumor cells from expressing CD47.

Example 8: Blocking the surface of tumor cells CD47 can enhance the phagocytosis of macrophages

1. Isolation and culture of macrophages: Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation, placed in a 5% CO ₂ incubator at 37 ° C for 4 h, washed with pre-warmed medium. Unattached cells were added to AIM-V medium and rhGM-CSF (final concentration 1000 U/ml). The medium was changed by half a day and cultured for 7 days to obtain adherent cells and macrophages.

2. Phagocytosis of tumor cells by macrophages: tumor cells were stained blue with Hoechst dye, and macrophages were stained red with CM-Dil. For specific staining methods, see the dye instructions. The stained two cells were mixed and divided into two, one portion was added to the Meso3CAR T cell culture supernatant obtained in Example 2 as a control, and one portion was added to the αCD47-Meso3 CAR T cell culture supernatant obtained in Example 2, confocal. The phagocytosis was observed under a microscope, and the phagocytic efficiency was counted by the number of cells.

The results showed that the phagocytosis of macrophages in the culture supernatant of αCD47-Meso3CAR T cells was significantly higher than that in the control group. The statistical results are shown in Figure 7.

Example 9: In vivo anti-tumor effect of αCD47-Meso3CAR T cells.

Twenty NSC mice of 4-6 weeks old were purchased and divided into 5 groups, 4 in each group, inoculated with lung cancer cell line H23, 1×10 ⁷ each. After 10 days of tumor formation, PBS (100 ul) was injected into the tail vein respectively. Mock, Meso3CAR, wt-αCD47-Meso3CAR, αCD47-Meso3CAR T cells (1×10 ⁷ cells/cell) obtained in Example 2 were observed, and tumor volume was observed and recorded.

The results showed that PBS, Mock, wt-αCD47-Meso3CAR T cells had no therapeutic effect on the tumor model, while αCD47-Meso3CAR T cells had a good anti-tumor effect, as shown in Fig. 8.

Claims (10)

A T cell, wherein: (1) a coding sequence comprising a chimeric antigen receptor that recognizes a mesothelin antigen and a coding sequence of a CD47 antibody; and/or (2) expression of a chimera that recognizes a mesothelin antigen Antigen receptor and CD47 antibody; Preferably, the expression cassette of the chimeric antigen receptor recognizing the mesothelin antigen and the expression cassette of the CD47 antibody are integrated into the genome of the T cell. The T cell according to claim 1, wherein the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, an optional signal peptide, a single-chain antibody against the mesothelial proximal membrane end, and a length of 50 A hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain above the amino acid residue. The T cell of claim 2, wherein the chimeric antigen receptor has one or more of the following characteristics: The signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably a light chain signal peptide; preferably, the amino acid sequence of the light chain signal peptide is SEQ ID NO: 1 Shown at amino acid residues 1-20; The amino acid sequence of the single-chain antibody is shown as amino acid residues 21 to 270 of SEQ ID NO: 1; The hinge region longer than 50 amino acid residues is selected from the group consisting of a CD8α hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is a CD8α hinge region or an IgG4 Fc CH2CH3 hinge More preferably, the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is as shown in amino acid residues 271-498 of SEQ ID NO:1; The transmembrane region is one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region a region, preferably having an amino acid sequence as shown in amino acid residues 499-526 of SEQ ID NO: 1; The intracellular costimulatory signal domain includes the intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, and inducible T cell costimulatory factor ( ICOS) and the intracellular domain of DNAX activator protein 10; preferably, said intracellular costimulatory signal domain is the intracellular domain of CD28; preferably, the amino acid sequence of said CD28 is SEQ ID NO: 1 527- 567 amino acid residues are shown; and The intracellular signal domain is a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ ID NO: 1 at positions 568-679 Said by amino acid residues. The T cell according to claim 2 or 3, wherein the chimeric antigen receptor has one or more of the following characteristics: The coding sequence of the signal peptide is shown in bases 1-60 of SEQ ID NO: 4; The coding sequence of the single-chain antibody is shown in nucleotide sequence 61-810 of SEQ ID NO: 4; The coding sequence of the hinge region is represented by the nucleotide sequence of positions 811-1494 of SEQ ID NO: 4; The coding sequence of the transmembrane region is represented by bases 1495-1578 of SEQ ID NO: 4; The coding sequence of the intracellular costimulatory signal domain is represented by bases 1579-1701 of SEQ ID NO: 4; The coding sequence for the intracellular signal domain is set forth in SEQ ID NO: 4, 1702-2037. The T cell according to claim 1 or 2, wherein the chimeric antigen receptor comprises a light chain signal peptide, a single-chain antibody against mesothelin Region III, and IgG4 Fc CH2CH3 from N-terminal to C-terminal. Hinge region, CD8 transmembrane region, CD28 intracellular domain and tyrosine activation motif of CD3ζ; Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 21 to 679 of SEQ ID NO: 1; preferably, the coding sequence of the chimeric antigen receptor is SEQ ID NO: 4 The bases are shown at positions 61-2037. The T cell according to any one of claims 1 to 5, wherein the CD47 antibody comprises a CD47 ligand and an IgG4 Fc sequence; wherein the amino acid sequence of the IgG4Fc is SEQ ID NO: 2, pp. 139-367 The amino acid residue is shown; Preferably, the amino acid sequence of the CD47 ligand is as shown in amino acid residues 21 to 138 of SEQ ID NO: 2; Preferably, the antibody further comprises a signal peptide sequence, preferably a light chain signal peptide, more preferably, the amino acid sequence of the light chain signal peptide is as shown in amino acid residues 1-20 of SEQ ID NO: 2; Preferably, the amino acid sequence of the CD47 antibody is shown in amino acid sequence 21-367 of SEQ ID NO: 2, or as shown in SEQ ID NO: 2; Preferably, the coding sequence of the CD47 antibody is shown as bases 61-1101 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5. A composition comprising: (1) A vector comprising the expression cassette of the chimeric antigen receptor as defined in any one of claims 2 to 5, which is for integrating the expression cassette into the genome of the host cell; (2) A vector comprising the expression cassette of the CD47 antibody as defined in claim 6, which is for integrating the expression cassette into the genome of a host cell. A kit comprising: (1) A vector comprising the expression cassette of the chimeric antigen receptor as defined in any one of claims 2 to 5, which is for integrating the expression cassette into the genome of the host cell; (2) A vector comprising the expression cassette of the CD47 antibody as defined in claim 6, which is for integrating the expression cassette into the genome of a host cell. A pharmaceutical composition comprising the T cell of any one of claims 1 to 6 or a CD47 antibody comprising the T cell and the expression thereof. The use of the T cell or the T cell of any one of claims 1 to 6 and the CD47 antibody expressed thereby for the preparation of a medicament for treating or preventing malignancy; preferably, the cancer is abnormal for a cancer cell surface thereof The cancer expressing mesothelin is preferably a cancer which is more than 100 times the expression level of mesothelin on the surface of cancer cells is normal, and mesothelin is uniformly distributed throughout the cell surface; preferably, the cancer is selected from the group consisting of: Adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer; more preferably, the cancer is mesothelin and CA125 /MUC16 is a cancer that is highly expressed at the same time.

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