WO2019020088A1 - Chimeric antigen receptor-modified t cell targeting mesothelin and use thereof - Google Patents

Abstract

The invention provides chimeric antigen receptors that target mesothelin and uses thereof. The chimeric antigen receptor provided by the invention comprises a membrane protein signal peptide, an anti-mesothelin proximal membrane single-chain antibody, a hinge region of 50 amino acid residues or more, a transmembrane region, and a costimulatory stimulation from the N-terminus to the C-terminus. Signaling molecule intracellular domain and immunoreceptor tyrosine activation motif. The present invention also provides a T cell expressing the chimeric antigen receptor, which can specifically kill a tumor cell strain with high expression of mesothelin, and has little or no killing effect on a tumor cell line which is not expressed, and has a very high Good specificity.

Description

Chimeric antigen receptor-modified T cell targeting mesothelin and use thereof Technical field

The present invention belongs to genetic engineering and immunology, and relates to a chimeric antigen receptor-modified T cell targeting mesothelin and use 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%. 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 and lacks cell surface targets suitable for CAR-T treatment. 2. Solid tumors have a microenvironment that strongly suppresses 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).

CAR-T cells targeting mesothelin have shown a certain effect in various cancers, but they are not effective for ovarian cancer with high expression of mesothelin. The reason may be that the current CAR-T cells targeting mesothelin are mostly the mesothelin N-terminal I region (Region I, 296-390, distal membrane end) as the recognition site, but at the same time ovarian cancer. The interaction of CA125/MUC16 protein with mesothelin in the serum of patients also passes through this site, and they compete with CAR-T cells for the binding of mesothelin, Region I, which affects the efficacy.

Summary of the invention

Accordingly, provided herein is an IgG4 Fc CH2CH3 hinge region, the amino acid sequence of which is set forth in SEQ ID NO: 6.

Also provided herein are coding sequences for the IgG4 Fc CH2CH3 hinge region, including the complements thereof.

In one or more embodiments, the coding sequence for the IgG4 Fc CH2CH3 hinge region is set forth in bases 817-1500 of SEQ ID NO: 14.

Also provided herein is a chimeric antigen receptor (CAR), which comprises a signal peptide, an anti-mesothelin single-chain antibody, and a hinge region of 50 amino acid residues or more in length from the N-terminus to the C-terminus. The transmembrane region, the intracellular domain of the costimulatory signaling molecule, and the immunoreceptor tyrosine activation motif.

In one or more embodiments, the signal peptide is a membrane protein signal peptide.

In one or more embodiments, the signal peptide is selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide.

In one or more embodiments, the signal peptide is a CD8 signal peptide having the amino acid sequence set forth in SEQ ID NO: 1.

In one or more embodiments, the anti-mesothelin single chain antibody is a specific antibody to the mesothelin III region.

In one or more embodiments, the amino acid sequence of the mesothelin III region is set forth in SEQ ID NO:12.

In one or more embodiments, the amino acid sequence of the anti-mesothelin III region single chain antibody is set forth in SEQ ID NO:3.

In one or more embodiments, the anti-mesothelin single chain antibody is a specific antibody to the mesothelin I region, and the chimeric antigen receptor comprises an IgG4 Fc CH2CH3 hinge region.

In one or more embodiments, the amino acid sequence of the mesothelin I region is set forth in SEQ ID NO:11.

In one or more embodiments, the amino acid sequence of the single chain antibody to the anti-mesothelin I region is set forth in SEQ ID NO:4.

In one or more embodiments, the hinge region is a CD8 hinge region, the amino acid sequence of which is set forth in SEQ ID NO: 5.

In one or more embodiments, the hinge region is an IgG4 Fc CH2CH3 hinge region, the amino acid sequence of which is set forth in SEQ ID NO: 6.

In one or more embodiments, the transmembrane region is selected from the group consisting 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. One or more of them.

In one or more embodiments, the transmembrane region is a CD8 transmembrane region, the amino acid sequence of which is preferably set forth in SEQ ID NO: 7.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule is selected from one or more of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule is the CD28 intracellular domain, the amino acid sequence of which is set forth in SEQ ID NO: 8.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule is the CD137 intracellular domain, the amino acid sequence of which is set forth in SEQ ID NO:9.

In one or more embodiments, the immunoreceptor tyrosine activation motif is a tyrosine activation motif of CD3ζ and/or FcεRIγ.

In one or more embodiments, the immunoreceptor tyrosine activating motif is a CD3 ζ tyrosine activation motif, the amino acid sequence of which is set forth in SEQ ID NO: 10.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-mesothelin III region single chain antibody, a CD8 hinge region or an IgG4 Fc CH2CH3 hinge region, and a CD8 span. Membrane region, CD28 intracellular domain and tyrosine activation motif of CD3ζ.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-mesothelin III domain single-chain antibody, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, and a CD28 Intracellular domain and tyrosine activation motif of CD3ζ.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-mesothelin I region single chain antibody, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, The intracellular domain of CD137 and the tyrosine activation motif of CD3ζ.

In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 13.

Also provided herein are coding sequences for chimeric antigen receptors herein, including complements thereof.

In one or more embodiments, the nucleotide sequence of the coding sequence is set forth in SEQ ID NO: 14.

Also provided herein is a nucleic acid construct comprising a coding sequence for a chimeric antigen receptor described herein.

In one or more embodiments, the nucleic acid construct is an expression vector for expression of a chimeric antigen receptor as described herein.

In one or more embodiments, the expression vector is a transposon vector.

In one or more embodiments, the transposon vector contains a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5, and Ty.

Also provided herein is a recombinant host cell comprising a coding sequence for a chimeric antigen receptor described herein or a nucleic acid construct described herein.

In one or more embodiments, the host cell is a mammalian cell.

In one or more embodiments, the host cell is a T cell.

In one or more embodiments, the host cell is a primary cultured T cell.

In one or more embodiments, the cell expresses a chimeric antibody receptor as described herein.

Also provided herein is the use of: (1) the use of the IgG4 Fc CH2CH3 hinge region or its coding sequence for the preparation of a chimeric antigen receptor or a coding sequence thereof described herein; (2) a chimeric antigen receptor as described herein. Use of the coding sequence for the preparation of a recombinant expression vector; (3) use of the nucleic acid construct described herein in the preparation of a recombinant host cell; and (4) preparation of a recombinant host cell described herein for the treatment or prevention of cancer Use in medicine.

In one or more embodiments, the cancer is selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer, or prostate cancer.

Also provided herein is a pharmaceutical composition comprising a recombinant host cell as described herein.

DRAWINGS

Figure 1A: Schematic diagram of various chimeric antigen receptor gene structures for mesothelin.

Figure 1B: Vector plasmid map (pNB328-meso3CAR).

Figure 2: Western blot analysis of CAR gene expression in meso3CAR-T cells with two different signal peptides.

Figure 3: Comparison of killing of meso3CAR-T containing CD8 hinge region and mutant IgG4 Fc hinge region to cervical cancer cell line Hela, ovarian cancer cell line SK-OV-3 and gastric cancer cell HGC-27.

Figure 4: IL-2, IL-4, IL-6, IL-10, TNF- and IFN- cells stimulated by mesothelin antigen for meso1CAR-T cells and meso3CAR-3-T cells in different regions of mesothelin Changes in factor secretion.

Figure 5: CA125/MUC16 positive cell line (OVCAR-3) and negative cell line (PANC-1) with high expression of mesothelin for meso1CAR-T cells and meso3CAR-3-T cells in different regions of mesothelin Killing results.

Figure 6: Therapeutic effect of meso1CAR-T cells and meso3CAR-3-T cells on a transplanted tumor model of CA125/MUC16-positive OVCAR-3 ovarian cancer mice.

Detailed ways

Therefore, we designed a novel CAR-T cell targeting mesothelin, with the Region III (near membrane end) of the mesenchyme as the recognition site. Since this epitope is present at the proximal membrane end, we designed two CAR structures with different hinge lengths, one is the transmembrane hinge region formed by the combination of the CD8 hinge region and the CD8 transmembrane region, and one is composed of IgG4 Fc CH2CH3. A transmembrane hinge region formed by a combination of a hinge region and a CD8 transmembrane region. In addition, since the IgG4 Fc fragment present on CAR is easily recognized by monocytes/macrophages to cause T cell AICD reaction, we modified the IgG4 Fc CH2CH3 hinge region on CAR to satisfy our CAR fragment and recognize antigen well. It does not cause an AICD reaction, thereby achieving good recognition of antigen and cell killing.

The chimeric antigen receptor (CAR) herein contains a signal peptide, an anti-mesoside single-chain antibody, a hinge region of 50 amino acids or more in length, a transmembrane region, and a total of N-terminal to C-terminal. Stimulation of the intracellular domain of the signaling molecule and the immunoreceptor tyrosine activation motif.

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 herein is a membrane protein signal peptide, which may be selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide. In a preferred embodiment, the invention employs a CD8 signal peptide. The amino acid sequence of an exemplary CD8 signal peptide can be as set forth in SEQ ID NO: 1.

Single-chain antibody (scFv) means an antibody connected by a light chain variable region (V _L region) and the amino acid sequence of a heavy chain variable region (V _H region) from the amino acid sequence via a hinge, an antibody fragment having antigen binding ability. The anti-meso single chain antibody described herein may be a single chain antibody to 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: 11; the amino acid sequence of mesothelin Region III is set forth in SEQ ID NO: 12. An exemplary amino acid sequence of a single-chain antibody against mesothelin, Region I, is set forth in SEQ ID NO:4. An exemplary amino acid sequence of a single-chain antibody against mesothelin-Region III is set forth in SEQ ID NO: 3. Herein, mesothelin refers to a mesothelin fragment anchored on a membrane unless otherwise specified.

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 and antigenic epitope of the antibody. Complementary complementarity. A hinge region suitable for use herein may be selected from any 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 or A plurality of hinge regions preferably having a length of 50 amino acid residues or more, more preferably 80 amino acid residues or more. In certain embodiments, a CD8 alpha hinge region and an IgG4 Fc CH2CH3 hinge region are used herein. The amino acid sequence of an exemplary CD8 alpha hinge region is set forth in SEQ ID NO: 5. The amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is set forth in SEQ ID NO: 6.

The transmembrane region can be selected from one or more of the CD28 transmembrane region, the CD8 transmembrane region, the CD3ζ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region, and the DAP10 transmembrane region. Preferably, the transmembrane region for the chimeric antigen receptor herein is a CD8 transmembrane region. The amino acid sequence of an exemplary CD8 transmembrane region can be as set forth in SEQ ID NO: 7.

The intracellular domain of the costimulatory signaling molecule can be selected from one or more of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10. In certain embodiments, the intracellular domain of CD28 or the intracellular domain of CD137 is used. The amino acid sequence of an exemplary CD28 intracellular domain can be set forth in SEQ ID NO: 8; the amino acid sequence of an exemplary CD137 intracellular domain can be set forth in SEQ ID NO: 9.

The immunoreceptor tyrosine activation motif can be a tyrosine activation motif of CD3ζ and/or FcεRIγ. An amino acid sequence of an exemplary CD3 ζ tyrosine activating motif can be set forth in SEQ ID NO: 10.

Forming the above-described various portions of the chimeric antigen receptor herein, such as the CD8 signal peptide, the light chain variable region and the heavy chain variable region of the anti-meso single chain antibody, the hinge region, the transmembrane region, and the intracellular domain of the costimulatory signaling molecule And the immunoreceptor tyrosine activation motif and the like may be directly linked to each other or may be linked 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.

In certain embodiments, the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-meso scFv, a CD8 hinge region, a CD8 transmembrane region, a CD28 intracellular domain, and a CD3 ζ tyrosine. Activation motif.

In other embodiments, the chimeric antigen receptors herein comprise a CD8 signal peptide, an anti-meso scFv, an IgG4 FcCH2CH3 hinge region, a CD8 transmembrane region, a CD28 intracellular domain, and a CD3 tyrosine tyrosine activation from N-terminus to C-terminus. Motif.

Preferably, the anti-meso scFv is a single chain antibody directed against the mesothelin III region. Preferably, when the mesothelin single chain antibody is a single chain antibody directed against the mesothelin I region, the chimeric antigen receptor comprises an IgG4 FcCH2CH3 hinge region.

The amino acid sequence of an exemplary chimeric antigen receptor can be as set forth in SEQ ID NO: 13.

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: 14.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding a chimeric antigen receptor as described herein, and one or more regulatory sequences operably linked to the sequences.

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 of the CARs herein can be cloned into a variety of types 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, and optionally a Selected tag.

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: 1 of the application. The TEF promoter containing the CD3e enhancer and the EF1α promoter shown in SEQ ID NO: 2; the TCEF promoter containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer and the EF1α promoter shown in SEQ ID NO: a CCEFI promoter comprising the mCMV enhancer, the hCMV enhancer and the intron-containing EF1α promoter represented by SEQ ID NO: 4; the CD3e enhancer and the intron-containing SEQ ID NO: 5 The TEFI promoter of the EF1α promoter; and the TCEFI promoter containing the CD3e enhancer, the mCMV enhancer, the hCMV enhancer, and the intron-containing EF1α promoter shown in SEQ ID NO: 5. 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, an expression vector eukaryotic expression vector, particularly a transposon vector, is used herein. 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' ITR) of the corresponding transposon and the 3' inverted terminal repeat (3' ITR) of the corresponding transposon. For example, in certain embodiments, a nucleic acid construct or expression vector herein comprises, in sequence from 5' to 3', a transposon 5' inverted terminal repeat (5' ITR), a CD8 signal peptide coding sequence, an anti-meso scFv Coding sequence, CD8 hinge region coding sequence or IgG4 Fc CH2CH3 hinge region coding sequence, CD8 transmembrane region coding sequence, CD28 intracellular domain coding sequence, CD3 ζ tyrosine activation motif coding sequence and transposon 3' inverted end Repeat sequence (3'ITR). The transposon vector may also contain a transposase coding sequence and a promoter that controls expression of the transposase coding sequence. In certain embodiments, the eukaryotic expression vector is a pNB328 vector.

The vectors herein can be introduced into host cells by conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like. In certain embodiments, the nucleic acid constructs herein are introduced into a host cell using electroporation. Specifically, the recombinant plasmid is transferred to the host cell of interest by electrokinetic electrokinetics.

Host cells suitable for use herein can be mammalian cells well known in the art, preferably T cells, including various types of T cells from various sources. For example, T cells can be derived from PBMC in patients with B cell malignancies. In certain embodiments, the T cell is a primary cultured T cell.

Accordingly, also included herein is a recombinant host cell comprising a coding sequence for a chimeric antigen receptor described herein or a nucleic acid construct described herein; and/or the recombinant host cell expresses a Chimeric antigen receptor. The recombinant host cell can be a host cell as described above that has been introduced into a vector described herein.

The IgG4 Fc CH2CH3 hinge region and its coding sequences for use in the chimeric antigen receptors herein are also included within the scope of the disclosure. More specifically, the IgG4 Fc CH2CH3 hinge region and its coding sequence (including complementary sequences) having an amino acid sequence such as SEQ ID NO: 6 are included herein.

Also included herein are the uses of various amino acid sequences, nucleic acid sequences, recombinant host cells, and the like as mentioned hereinbefore. In particular, the use herein includes the IgG4 Fc CH2CH3 hinge region and/or its coding sequence for the preparation of a chimeric antigen receptor and/or a coding sequence thereof; the coding sequence of the chimeric antigen receptor is Use of a recombinant expression vector; use of the nucleic acid construct for the preparation of a recombinant host cell; and use of the recombinant host cell for the preparation of a medicament for the treatment or prevention of cancer. In certain embodiments, the IgG4 Fc CH2CH3 hinge region and/or coding sequence thereof, the chimeric antigen receptor and/or coding sequence thereof, and the nucleic acid construct are prepared for use in the treatment or prevention of cancer The use of recombinant host cells.

A cancer suitable for treatment or prevention of a CAR or a cell expressing the same as described herein is preferably a mesothelin-positive cancer, and specifically includes a cancer having abnormal expression of mesothelin on the surface of a cancer cell. Specifically, such cancers may be selected from the group consisting of mesothelioma, 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. In certain embodiments, the single-chain antibody of the present invention has an excellent killing effect against a CAR of the mesothelin III region or a cell expressing the same, and a cancer which simultaneously expresses CA125/MUC16, such as ovarian cancer, therefore, Such CARs or their expression cells are particularly useful for the treatment or prevention of cancers that simultaneously express mesothelin and CA125/MUC16.

Also provided herein is a kit comprising the recombinant expression vector described herein. The kit may also contain reagents suitable for the transfer of the recombinant expression vector into a cell, and optionally instructions for those skilled in the art to transfer the recombinant expression vector into a cell.

Also provided herein is a pharmaceutical composition comprising a recombinant host cell described herein and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a carrier well known in the art for cell administration, including but not limited to the pNB328 vector.

Methods of treating or preventing cancer are also included herein, the method comprising the step of administering a recombinant host cell or pharmaceutical composition described herein to a subject in need thereof. The method of administration can be a method commonly used in cell therapy. The dose administered can be considered based on factors such as the sex, age, disease, and physical condition of the patient.

The mesoCAR-T cells provided herein can specifically kill tumor cell lines with high expression of mesothelin, and have little or no killing effect on tumor cell lines which are not expressed, indicating that they have good specificity. In addition, meso3CAR-T cells targeting the mesothelin III region provide mesothelin III region, which is not affected by CA125/MUC16, and therefore has a special tumor/cancer that simultaneously expresses mesothelin and CA125/MUC16. Good killing effect. In the OVCAR-3 ovarian cancer mouse xenograft model, the tumor suppressive ability of meso3CAR-T cells was significantly stronger than that of meso1CAR-T cells. Therefore, the meso3CAR-T provided herein is particularly useful in the adoptive immunocyte treatment of CA125/MUC16 and mesothelin-expressing 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-mesoCAR

Synthetic coding contains a signal peptide (CD8 signal peptide or light chain signal peptide), an antigen recognition single chain antibody (anti-meso3 scFv or anti-meso1 scFv), a hinge region (CD8 hinge region or IgG4 hinge region), CD8 transmembrane region , a co-stimulation of the intracellular domain of the signaling molecule and the exogenous gene of the mesoCAR of the immunoreceptor tyrosine activation motif (named meso3CAR-1, meso3CAR-2, meso3CAR-3, and meso1CAR, respectively, as shown in Figure 1A), A polyclonal cleavage site (BglII-XbaI-EcoRI-BamHI) was introduced upstream of it, and a restriction enzyme site (SalI-NheI-HindIII-SpeI) was inserted downstream of it, and it was commissioned by Shanghai Jierui Biotech Co., Ltd. to assemble it. The recombinant plasmid pNB328-mesoCAR was constructed by using the pNB328-EF1α vector digested with EcoR1+SalI (pNB328 containing EF1α promoter, and CN 201510812654.9 for pNB328 vector), and named as pNB328-meso3CAR-1 and pNB328-meso3CAR-2, respectively. , pNB328-meso3CAR-3 and pNB328-meso1CAR, the schematic structure of which is shown in Figure 1B. In Fig. 1B, "EF1 promoter" is the EF1 promoter; "meso3CAR" represents any of the above three antigen-recognizing single-chain antibodies as any anti-meso3 scFv mesoCAR, and replaced with meso1CAR, the plasmid is pNB328-meso1CAR.

The amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 1; the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO: 2; the amino acid sequence of anti-meso3 scFv is shown in SEQ ID NO: 3; anti-meso1 The amino acid sequence of the scFv is shown in SEQ ID NO: 4; the amino acid sequence of the hinge region of CD8 is shown in SEQ ID NO: 5; the amino acid sequence of the hinge region of IgG4 is shown in SEQ ID NO: 6; SEQ ID NO: 7; the amino acid sequence of the intracellular domain of the costimulatory signal molecule is set forth in SEQ ID NO: 8; the amino acid sequence of the immunoreceptor tyrosine activating motif is set forth in SEQ ID NO: 10.

Example 2: MesoCAR-T cell construction targeting mesothelin

Peripheral blood mononuclear cells (PBMCs) were isolated from the Shanghai Cell Therapy Production Center. 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 rpm for 3 min, the supernatant was discarded, physiological saline was added, 1200 rpm, and centrifuged for 3 min. Discard the physiological saline and repeat this step; take two 1.5ml centrifuge tubes, add 5×106 cells per tube, number a, b, 1200rmp, centrifuge for 3min, discard the supernatant, and take the electrotransfer kit (from Lonza). a, b tube proportionally added electroporation reagent a total of 100ul, a tube was added 6ug of the constructed recombinant plasmid pNB328-mesoCAR, resuspended and mixed cells, b tube was added 6ug control plasmid (ie pNB328 empty plasmid); the mixture was transferred to electrical rotation In the cup, put the electro-rotation instrument, select the required procedure, and perform electric shock; use the micro-pipette in the kit to transfer the electrically-transferred cell suspension to the six-well plate with the culture solution (AIM-V with 2% FBS) The culture solution was mixed, placed in a 37 ° C, 5% CO ₂ incubator, and after six hours, the stimulating factors IL-2 and anti-CD3/anti-CD28 were added, and cultured at 37 ° C, 5% CO _{2 for} 3 to 4 days. Observe the growth of T cells and obtain T cells expressing the mesoCAR gene.

Example 3: Comparison of expression levels of different signal peptide meso3CAR-T

The two meso3 CAR-T cells (meso3CAR-1 and meso3CAR-2) prepared in Example 2 were collected and washed twice with physiological saline. 160 μl of cell lysate was added and placed on ice for 10 min. After the cells were fully lysed, 12000 rmp, 4 Centrifuge for 10 min at ° C and collect the supernatant. Add 40ul 5×loading Buffer, cook at 100°C for 10min, and place on ice for 5min. The expression of the foreign gene was detected by western blot using a mouse anti-human CD3 antibody (purchased from abcam), a GAPDH antibody (purchased from Beyotime), and a HRP goat anti-mouse secondary antibody (purchased from Jackson).

As a result, as shown in Fig. 2, meso3CAR-1 using a light chain antibody signal peptide was not expressed normally.

Example 4: Comparison of killing function of meso3CAR-T in different extracellular hinge regions

The effector cells and target cells matched by MHC class I were selected, and the two meso3 CAR-T cells (meso3CAR-2 and meso3CAR-3) obtained in Example 2 were detected by Essen's real-time label-free cell function analyzer (RTCA). The in vitro killing activity, 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: cervical cancer cell Hela, ovarian cancer cell SK-OV-3, gastric cancer HGC-27 (purchased from the American Type Culture Collection ATCC) were plated at 10 ⁴ cells/50 μl per well in the 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 3. The meso3CAR-3 cells using the mutant IgG4 Fc hinge region were significantly more potent against tumor cells than meso3 CAR-2 cells using the CD8 hinge region as well as control T cells.

Example 5: Comparison of cytokine release by meso1CAR and meso3CAR under specific stimulation of mesothelin antigen

The 96-well plate was coated with 2 ug/ml of mesothelin antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 × 10 ⁵ of the meso1CAR, meso3 CAR-3 cells prepared in Example 2 and the control Mock T cells were added. (Transferred into pNB328 empty vector), the cell supernatant was collected after 24 hours of culture. With ^{BD TM CBA Human Th1 / Th2 Cytokine} Kit II detected by the three T-cell secretion of cytokines following stimulation mesothelin antigen, the following steps:

(1) Mixed human IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ capture magnetic beads, vortex and mix to capture magnetic beads, add 50 ul of mixed magnetic capture after each tube Bead

(2) Add 50 ul of Th1/Th2 cytokine standard (double dilution 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml) , 20 pg/ml, 0 pg/ml) and 50 ul of the sample to be tested (diluted 2 times by dilution).

(3) adding 50 ul of human Th1/Th2-II-PE detection antibody per tube;

(4) Incubate for 3 hours at room temperature in the dark;

(5) Add 1 ml of washing buffer to each tube, centrifuge at 200 for 5 min, and discard the supernatant;

(6) Resuspend the cells by adding 300 ul of washing buffer per tube, transfer to a flow tube, and measure the fluorescence value by flow cytometry;

As shown in Fig. 4, the secretion of various cytokines in meso1CAR-T and meso3CAR-3-T cells was significantly higher than that in Mock T cells, and meso3CAR-3 T cells were higher than meso1 CAR-T cells.

Example 6: Comparison of the activity of meso1CAR and meso3CAR killing CA125 positive or negative cells

The effector cells and target cells matched by MHC class I were selected, and the in vitro killing activity of the two mesoCAR-T cells was detected by Essen's real-time label-free cell function analyzer (RTCA). 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: ovarian cancer cell OVCAR-3 with high expression of CA125 and pancreatic cancer cell PANC-1 ( purchased from American Type Culture Collection ATCC) with no expression of CA125 were selected and plated at 10 ⁴ cells/50 μl per well. Place the plate containing the detection electrode, leave it for a few minutes, wait for the cell to stabilize, then put it into the instrument, start step 2, and culture the cells;

(3) Adding effector cells: After the target cells were cultured for 24 hours, the step 2 was suspended, and the effector cells were added, 50 μl per well, and the target ratio was set to 4:1, respectively, and the Mock T cells of the empty plasmid were used as a control, and step 3 was started. After continuing to co-culture for 24 hours, observe the cell proliferation curve;

The results are shown in Figure 5. For pancreatic cancer cell PANC-1 that does not express CA125, both mesoCAR-T cells have a certain killing effect, meso3CAR-3-T cells are slightly stronger than meso1CAR-T cells; for high expression CA125 The ovarian cancer cells OVCAR-3, meso1CAR-T cells have almost no killing effect, while meso3CAR-3-T cells still have good killing.

Example 7: Comparison of the activity of meso1CAR and meso3CAR in killing CA125-positive OVCAR-3 ovarian cancer xenografts

Fifteen NGF completely immunodeficient mice of 1:4 to 6 weeks old, with an average weight of 22-27 g, were provided by Beijing Weitongda Biotechnology Co., Ltd., and were raised in SPF animal laboratory.

2: Human ovarian cancer cell line OVCAR-3-luc was cultured in vitro, adherent growth cells in logarithmic growth phase, 0.25% trypsin digestion, centrifugation, collecting cells, resuspending in PBS solution, centrifugation at 1000 rpm for 2 minutes at room temperature, discarding supernatant After resuspending in PBS, the cells were collected by centrifugation, and the cell suspension concentration was adjusted to 5 × 10 ⁷ /ml.

3: OVCAR-3-luc cells were inoculated subcutaneously into the right flank of the mouse, 0.1 ml/mouse. After 10 days of inoculation, tumor size was observed by a live imager, and NSG immunodeficient mice were randomly divided into 3 groups. The route of administration is direct intratumoral injection.

4: The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 10 days.

The results are shown in Fig. 6. Meso3CAR-3-T cells have a good therapeutic effect in a mouse model of ovarian cancer mice with high expression of CA125.

Although specific embodiments of the invention have been described in detail. Those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

A chimeric antigen receptor characterized in that, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a membrane protein signal peptide, a single-chain antibody against the mesothelial proximal membrane end, and a length of 50 amino acid residues or more. The hinge region, the transmembrane region, the intracellular domain of the costimulatory signaling molecule, and the immunoreceptor tyrosine activation motif. The chimeric antigen receptor according to claim 1, wherein the anti-mesothelin single-chain antibody is a specific antibody of the mesothelin III region; Preferably, the amino acid sequence of the mesothelin III region is as shown in SEQ ID NO: 12; More preferably, the amino acid sequence of the single-chain antibody against the mesothelin III region is set forth in SEQ ID NO: 3. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric antigen receptor has one or more of the following characteristics: The signal peptide is selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide; preferably, the signal peptide is a CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO: 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 CD8 hinge region is set forth in SEQ ID NO: 5; the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is set forth in SEQ ID NO: 6; The transmembrane region is selected from one or more 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; The transmembrane region is a CD8 transmembrane region, the amino acid sequence of which is set forth in SEQ ID NO: 7. The intracellular domain of the costimulatory signaling molecule is selected from one or more of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10; preferably, the costimulatory signaling molecule The intracellular domain is the CD28 intracellular domain, the amino acid sequence thereof is shown in SEQ ID NO: 8, or the intracellular domain of the costimulatory signaling molecule is the CD137 intracellular domain, and the amino acid sequence thereof is SEQ ID NO: 9. As shown; and The immunoreceptor tyrosine activation motif is a tyrosine activation motif of CD3ζ and/or FcεRIγ; preferably, the immunoreceptor tyrosine activation motif is a CD3ζ tyrosine activation motif, an amino acid thereof The sequence is shown in SEQ ID NO: 10. A chimeric antigen receptor according to any one of claims 1 to 3, wherein The chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-mesothelin III region single-chain antibody, a CD8 hinge region or an IgG4 FcCH2CH3 hinge region, a CD8 transmembrane region, a CD28 intracellular domain, and a CD3ζ Tyrosine activation motif; or Preferably, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 13. A polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence encoding the chimeric antigen receptor of any one of claims 1 to 4; (2) (1) a complementary sequence of the polynucleotide sequence; Preferably, the polynucleotide sequence is selected from the polynucleotide sequence set forth in SEQ ID NO: 14 or its complement. A nucleic acid construct comprising the polynucleotide sequence of claim 5; Preferably, the nucleic acid construct is an expression vector; More preferably, the expression vector is a eukaryotic expression vector, preferably containing a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 and Ty. A recombinant host cell comprising the nucleic acid construct of claim 6, or the chimeric antigen receptor of any one of claims 1-4; Preferably, the host cell is a mammalian cell; more preferably, the host cell is a T cell; more preferably, the host cell is a primary cultured T cell. Use of the chimeric antigen receptor of claims 1-4 and/or its coding sequence and/or the nucleic acid construct of claim 6 for the preparation of a recombinant host cell for the treatment or prevention of cancer; and claims Use of the recombinant host cell of claim 7 for the preparation of a medicament for the treatment or prevention of cancer. The use according to claim 8, wherein the cancer is a cancer whose surface of the cancer cell abnormally expresses mesothelin; 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 a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed. A pharmaceutical composition comprising the recombinant host cell of claim 7 and a pharmaceutically acceptable carrier.

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