CN111286512A

CN111286512A - Chimeric antigen receptor targeting humanized tyrosine kinase orphan receptor 1 and uses thereof

Info

Publication number: CN111286512A
Application number: CN202010089172.6A
Authority: CN
Inventors: 王恩秀; 汪晨
Original assignee: Nanjing Aide Institute Of Immunotherapy Co ltd
Current assignee: Nanjing Aide Institute Of Immunotherapy Co ltd
Priority date: 2019-06-05
Filing date: 2020-02-12
Publication date: 2020-06-16

Abstract

The invention relates to a chimeric antigen receptor targeting humanized ROR1 and application thereof. In particular, the invention provides a nucleic acid sequence selected from the group consisting of: contains the coding sequence of human DAP12, the coding sequence of T2A, the coding sequence of humanized ROR1 single-chain antibody, and the coding sequence of human TREM1 transmembrane and intracellular region which are connected in sequence. The invention also provides a related fusion protein, a vector containing the coding sequence, and applications of the fusion protein, the coding sequence and the vector.

Description

Chimeric antigen receptor targeting humanized tyrosine kinase orphan receptor 1 and uses thereof

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a chimeric antigen receptor of a targeted tyrosine kinase orphan receptor 1 and application thereof.

Background

Tyrosine kinase-like orphan receptor 1 (ROR 1) is a transmembrane protein in the tyrosine kinase Receptor (RTK) family. Mainly exists in the process of embryonic development, and is not expressed in most normal tissues after birth, but research shows that ROR1 is overexpressed in a plurality of blood and solid malignant tumors, ROR1 is preferentially expressed in low-differentiated tumors, and ROR 1-expressing cancer cells show stronger invasive metastasis and recurrence capacity, the expression degree of the ROR1 is related to poor prognosis, and a marker gene related to epithelial-mesenchymal transition is expressed. In breast cancer and melanoma cell lines with metastasis tendency, siRNA is used for interfering ROR1 expression, or a monoclonal antibody against ROR1 is used for inhibiting migration and invasion of tumor cells, reducing expression of EMT related genes and mediating tumor cell apoptosis. Suggesting that ROR1 can be used as a potential target for treating hematological malignancy and solid tumor.

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell therapy (CAR-T) is one of the most promising tumor immunotherapy at present [ Schmitz M, et al. 10.1155/2010/956304.]. Chimeric Antigen Receptors (CARs) are a core component of CAR-T, CARs can redirect their specificity and reactivity towards selected immune cells, thus conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which makes CAR-engineered T cells capable of activating and proliferating, and thus capable of efficiently killing tumor cells, unlike normal T Cell Receptor (TCR) responses, independent of MHC restriction.

The research shows that α chain variable region of TCR is replaced by single chain variable region (scFv) of tumor specific monoclonal antibody, and the scFv is directly connected with T cell signal conducting structure domain to form Chimeric Antigen Receptor (CAR) which is expressed on the surface of T cell, tumor specific antigen can be identified by scFv, activation signal of T cell can be directly generated, T cell activation and proliferation are promoted, and tumor cell is specifically killed.

Currently, ROR1 is being used in clinical trials for breast and lung cancer, expressed on B-cell malignancies and solid tumor subpopulations, but not in healthy B-cells or normal postpartum tissues. Therefore, T cells that specifically recognize ROR1 have the potential to eliminate tumor cells and differentiate healthy tissue. To test this hypothesis, it has been reported that ROR 1-CARs specific for malignant cells were developed, and two second generation CAR molecules specifically recognized by ROR1 were constructed using the Sleeping Beauty transposon: ROR1RCD28 and ROR1RCD137, and the CAR-T can be successfully constructed, the cell can eliminate ROR1 positive tumor, and clinical tests will investigate whether the ROR1 specific CAR-T cell has specific clearance capacity for target cells while maintaining normal B cell number.

The invention is directed to novel CARs constructed to target ROR1, the invention uses humanized ROR1(huUC961) single chain antibodies, huUC961 has high affinity. huUC961 is a humanized form of a monoclonal antibody targeting ROR1, and has therapeutic and diagnostic effects on tumors. The invention modifies CAR-T cells and plays a CAR-T cell anti-tumor role aiming at ROR1 targets. The invention lays a good foundation for clinical experiments and clinical treatment.

With the accumulation and continued sophistication of the experience of CAR-T cell therapy, there is increasing interest in its use in malignancies. Under the environment, the pace is to be accelerated, and the CAR-T cell therapy is promoted to rapidly advance on the way of treating malignant tumors by applying and developing clinical tests by utilizing the existing work foundation, research and development teams and medical teams.

Disclosure of Invention

In a first aspect, the present invention provides a nucleic acid sequence selected from the group consisting of:

(1) the polypeptide contains a coding sequence of a human natural killer activated receptor related protein (DAP12), a coding sequence of T2A, a coding sequence of an anti-tyrosine kinase orphan receptor 1 single-chain antibody and a coding sequence of transmembrane and intracellular regions of a human myeloid triggering receptor (TREM1) which are connected in sequence.

In one or more embodiments, the coding sequence for the signal peptide preceding the coding sequence for the anti-ROR 1 single chain antibody is as shown in nucleotide sequence 412-474 of SEQ ID NO: 1. In one or more embodiments, the coding sequence of the anti-ROR 1 single-chain antibody is shown in the nucleotide sequence at position 481-1194 of SEQ ID NO. 1. In one or more embodiments, the nucleic acid sequence of human DAP12 is set forth in nucleic acids SEQ ID NO. 1, positions 1-339. In one or more embodiments, the nucleic acid sequence of T2A is as shown in SEQ ID NO 1 at position 355-405. In one or more embodiments, the nucleic acid sequence of the transmembrane and intracellular region of human TREM1 is as set forth in SEQ ID NO 1, nucleic acids 1231-1368. In a second aspect, the invention provides a fusion protein selected from the group consisting of:

(2) the fusion protein comprises a fusion protein of human DAP12, T2A, anti-tyrosine kinase orphan receptor 1 single-chain antibody, human TREM1 transmembrane and intracellular regions which are connected in sequence, and a fusion protein which is formed by substituting, deleting or adding one or more amino acids in a limited amino acid sequence and retains the activity of activated T cells;

preferably, the anti-ROR 1 monoclonal antibody huUC 961.

In one or more embodiments, the nucleic acid sequence further comprises a coding sequence for a signal peptide prior to the coding sequence for the anti-ROR 1 single chain antibody. In one or more embodiments, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:2 amino acids 138-158. In one or more embodiments, the amino acid sequence of the anti-ROR 1 single chain antibody is shown as amino acids 161-398 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of human DAP12 is set forth in SEQ ID NO. 2 amino acids 1-113. In one or more embodiments, the amino acid sequence of T2A is depicted as amino acids 119-135 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the transmembrane and intracellular region of human TREM1 is depicted as amino acids 411-455 of SEQ ID NO 2.

In a third aspect, the invention provides a nucleic acid vector comprising a nucleic acid sequence as described herein.

In one or more embodiments, the nucleic acid construct is a lentiviral vector comprising a nucleic acid sequence as described herein, and optionally a selectable marker.

In a fourth aspect, the invention provides a lentivirus comprising a nucleic acid vector as described herein, preferably comprising said lentivirus vector.

In a fifth aspect, the invention provides a genetically modified T cell comprising a nucleic acid sequence as described herein, or comprising a nucleic acid vector as described herein, or infected with a lentivirus as described herein, or stably expressing a fusion protein as described herein.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified T cell as described herein.

In a seventh aspect, the invention provides the use of a nucleic acid sequence, fusion protein, nucleic acid vector or lentivirus as described herein in the preparation of an activated T cell.

In an eighth aspect, the invention provides the use of a nucleic acid sequence, fusion protein, nucleic acid vector, lentivirus, or genetically modified T cell, or a pharmaceutical composition thereof, as described herein, in the preparation of a medicament for the treatment of a ROR 1-mediated disease.

In one or more embodiments, the ROR 1-mediated disease is brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-hodgkin's lymphoma, uterine cancer. In one or more embodiments, the ROR 1-mediated disease is osteosarcoma, breast cancer, leukemia, lung cancer, ovarian cancer.

Drawings

FIG. 1: schematic representation of pELNS-ROR 1CAR lentiviral expression vector.

FIG. 2: ROR1-CAR structural schematic diagram

FIG. 3: ROR1 CAR-T and NTD proliferation status

FIG. 4: ROR1 CAR-T Co-culture cytokine secretion with target cells

FIG. 5: ROR1 CAR-T killing effect

Detailed Description

The present invention provides a Chimeric Antigen Receptor (CAR) targeting humanized ROR 1. The CAR contains sequentially linked human DAP12, T2A, anti-ROR 1 single chain antibody, human TREM1 transmembrane and intracellular domains.

anti-ROR 1 single chain antibodies suitable for use in the present invention may be derived from a variety of anti-ROR 1 monoclonal antibodies known in the art.

Thus, in certain embodiments, anti-ROR 1 chain antibodies suitable for use in the invention contain a heavy chain that specifically recognizes human ROR 1. Optionally, the light chain variable region and the heavy chain variable region may be linked together by a linker sequence. Such single chain antibodies that may be exemplified include, but are not limited to, D10, 2a2, 6D4, 4a5, UC 961. In certain embodiments, the monoclonal antibody is huUC 961.

The fusion proteins of the present invention, such as human DAP12, T2A, anti-ROR 1 single chain antibodies, human TREM1 transmembrane and intracellular domains, etc., are formed and may be directly linked to each other or may be linked through a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, such as 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like.

It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce 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, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins.

The invention also includes a CAR represented by the amino acid sequence at positions 1-455 or a mutant of the CAR represented by SEQ ID NO. 2. These mutants include: an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity (e.g., activating T cells) of the CAR. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.

Mutants also include amino acid sequences having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence shown in positions 1-455 of SEQ ID No. 2 or in the amino acid sequence shown in SEQ ID No. 2, which usually refer to within 1-10, such as 1-8, 1-5 or 1-3, substitutions are preferably conservative substitutions, e.g., conservative substitutions with amino acids that are similar or analogous in performance do not normally alter the function of the protein or polypeptide.

The invention includes nucleic acid sequences encoding the fusion proteins of the invention. The nucleic acid sequences of the invention may be in the form of DNA or in the form of RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of the nucleic acid sequences encoding the fusion proteins, i.e., nucleotide sequences which encode the same amino acid sequence but differ in nucleotide sequence.

The nucleic acid sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleic acid sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the nucleic acid sequence encoding the fusion proteins described herein is as set forth in nucleic acids 1-1368 of SEQ ID NO: 1.

The invention also relates to nucleic acid vectors comprising the nucleic acid sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The nucleic acid sequences of the invention can be manipulated in a variety of ways to ensure expression of the fusion protein (CAR). The nucleic acid vector may be manipulated prior to its insertion into the vector depending on the identity or requirements of the expression vector. Techniques for altering nucleic acid sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate 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 which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The nucleic acid sequences of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning: Alabortory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and 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 (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

For example, in certain embodiments, the invention employs a lentiviral vector comprising a nucleic acid sequence as described herein, and optionally a selectable marker.

An example of a suitable promoter is the elongation growth factor-1 α (EF-1 α) promoter sequence Another example of a suitable promoter is immediate early Cytomegalovirus (CMV). however, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the EB virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.

Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, for example, mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing nucleic acids into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a nucleic acid of interest into a host cell include the use of DNA and RNA vectors. Chemical means of introducing nucleic acids into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing nucleic acids into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from retroviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art.

Thus, in certain embodiments, the invention also provides a lentivirus for activating T cells, the virus comprising a lentiviral vector as described herein and a corresponding packaging gene, such as gag, pol, and vsvg.

Thus, in certain embodiments, the invention provides a genetically modified T cell comprising a nucleic acid sequence as described herein, or comprising a lentiviral vector as described herein, or infected with a lentivirus as described herein, or prepared by a method as described herein, or stably expressing a fusion protein as described herein.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and sustained at high levels in the blood and bone marrow for extended amounts of time, and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate into a central memory-like state in vivo upon encountering and subsequently depleting target cells expressing a surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific for the antigen-binding portion in the CAR.

Thus, the diseases that can be treated with the CARs, their coding sequences, nucleic acid vectors, expression vectors, viruses, and CAR-T cells of the invention are preferably ROR 1-mediated diseases.

In particular, herein, "ROR 1-mediated diseases" include various types of brain, bladder, breast, cervical, colorectal, liver, kidney, lymphoma, leukemia, lung, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian, prostate, pancreatic, renal, skin, thymoma, sarcoma, non-hodgkin's lymphoma, uterine cancer, among others. In certain embodiments, the ROR 1-mediated disease is osteosarcoma, breast cancer, leukemia, lung cancer, ovarian cancer.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as relevant cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise CAR-T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Dosage of individual cells/kg body weight. The T cell composition may also be administered multiple times at these doses. Cells can be purified by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New eng.j. of med.319:1676,1988) and (4) application. Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

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

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment may be in conjunction with radiation or chemotherapeutic agents known in the art for the treatment of ROR1 mediated diseases.

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1 chimeric antigen receptor preparation

The invention provides a chimeric antigen receptor targeting humanized ROR 1. The design sequentially comprises DAP12, T2A, humanized ROR1 single-chain variable region and TREM1, and the structure of the design is shown in figure 1.

The plasmid pELNS-DAP12-T2A was stored by Nanjing Katsui medicine science and technology Co., Ltd, or was constructed according to the method disclosed in the literature (EnxiuWang et al. Generation of patent T-cell Immunology for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreppers. 2015, Cancer Immunology Research,3(7):815), ROR1 scFv-TREM1 gene synthesis was synthesized and provided by Biotech Co., Ltd, DAP was produced by DAP Bio-Tech Co., Shanghai, plasmid LNpES-12-T2A and the synthesized gene fragment were separately digested by double digestion with SalI (from Takara), the digestion was performed as described, and the digestion was followed by ligation to obtain a lentiviral vector expressing a Chimeric antigen receptor:

5 mu.L of lentiviral vector was transformed into E.coli TOP10 competent cells (purchased from Nanjing Ande high Biotech Co., Ltd.), and after culturing at 37 ℃ for 16h, monoclonals were picked up, and after culturing at 37 ℃ for 12h, plasmids were extracted with a plasmid extraction kit (purchased from Takara Co., Ltd.), and the specific method is described in the specification.

Example 2 Lentiviral packaging

The slow virus is packaged by a calcium phosphate method, and the method comprises the following specific steps:

293T cells were passaged every other day, 5X 10 cells were seeded per T150 cell flask⁶And (4) cells. After 48 hours, the cell number should reach 20-25 million/vial. In the case of 1T 150 cell culture flask, the cells were gently washed twice with about 15ml of 1 XPBS, 3ml of 0.25% pancreatin-2.21 mM EDTA was added until the cells were exfoliated, 12ml of 10% (wt) FBS (from Gibico) DMEM medium (from corning) was added to the exfoliated cells, the cells were collected and transferred to a sterile centrifuge tube, 1000rpm was applied, the cell was centrifuged for 10 minutes, the supernatant was aspirated, and the pellet was resuspended in 10ml of 10% (wt) FBS DMEM medium. Cell count, 12X 10 from cell concentration⁶The required volume of each cell, cells and 25ml of 10% (wt) FBS DMEM medium were combined, placed in a T150 cell flask, and shaken gently to distribute the cells evenly to the bottom of the cell flask at 37 deg.C and cultured overnight in a 5% CO2 incubator.

Cells were observed to reach a cell density of approximately 80% -90% at which time transfection was initiated by gently aspirating the medium 30-60 minutes prior to transfection. Plasmid DNA and calcium chloride solution were mixed, and one T150 vial was used as an example, 28ug pRSV.rev (available from Invitrogen), 28ug pGAG-Pol (available from Invitrogen), 11ug pVSVG (available from Invitrogen), and 23ug recombinant lentivirus expression plasmid ROR1CAR were added to 1.5ml of calcium chloride solution and mixed well. Adding 1.5ml of BBS solution into a 15ml sterile centrifuge tube, uniformly mixing the DNA-calcium chloride solution with a 1ml gun head, dropwise adding the mixture into the BBS solution, rapidly mixing the mixture uniformly for 15-20 ℃, and incubating the mixture for 25-30 minutes at room temperature. The DNA-calcium chloride-BBS mixture (available from Shanghai Bintian Biotechnology Co., Ltd.) was added dropwise to the T150 bottle uniformly using a 5ml pipette. Culturing in a cell culture box containing 5% carbon dioxide at 37 deg.C for 6 hr. And changing the liquid after 6 h. The plate was gently shaken several times to suspend some calcium phosphate precipitate sufficiently, the culture solution containing calcium phosphate precipitate was aspirated, 20ml of fresh 5% (wt) FBS DMEM culture solution was added, and the culture was continued.

The 293T cell culture supernatant transfected the previous day was collected into a centrifuge tube, centrifuged at 1000rpm for 5 minutes, labeled, and stored in a 4 ℃ freezer. 20ml of 5% (wt) FBS DMEM medium previously preheated was added to the cell flask and the cell culture was continued overnight in the cell incubator at 37 ℃. The viral supernatant was collected a second time (48 h/day). The supernatants from both collections were pooled together and filtered through a 0.45 μm filter to remove cellular debris. Centrifuging at 12000-

Example 3 viral infection of T cells

Collecting peripheral blood by using a blood collection tube containing an anticoagulant, naturally settling for about 30min at room temperature (18-25 ℃), collecting upper plasma, centrifuging the collected upper plasma for 10min at 5000r/min, adding the collected upper plasma to a lymphocyte separation solution (purchased from Tianjin Shangjing Biotechnology Limited liability company) according to a volume ratio of 1:1, carrying out gradient centrifugation for 3000r/min, centrifuging for 30min, and after centrifugation, layering a centrifugal tube from top to bottom: the first layer is a plasma layer; the second layer is a lymphocyte leucocyte layer; the third layer is a transparent separation liquid layer; the fourth layer of red blood cells. Sucking lymphocyte leucocyte membrane layer, washing with PBS for 2 times, centrifuging twice at 1500r/min for 10min, resuspending cells with PBS, adding 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581 complete culture medium to culture human peripheral blood mononuclear cells. Freshly prepared mononuclear cell PBMC were cultured in complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581, IL-2 from R & D Systems, KBM581 from Corning, CD3/CD28 Dynabeads immunomagnetic beads (from invitrogen) added on day 0 to activate T cells, lentiviral infection was performed the first 3 days, 0.25MOI corresponding lentiviral vector was added, uninfected T lymphocytes were used as a blank, after 48h the medium was changed to complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM581, and culture was continued for 7-9 days.

Example 4 Effect of viral infection of CAR-T cells on cell proliferation

After lentivirus infection of T cells, the T cells were counted every 1-2 days in complete medium containing a volume fraction of 5% autologous plasma +300IU/ml recombinant human IL-2+ KBM 581. T lymphocyte growth was then observed, and the results are shown in FIG. 3. The results show that the cells can still form typical proliferation clone masses after being infected by the CAR-expressing virus, and the proliferation curves of the cells are drawn by counting the cells, so that the proliferation capacity of the ROR1 CAR-T cells is slightly weaker than that of T cells (NTD in figure 3) which are not infected by the virus.

Example 5 detection of cytokine secretion by Virus-infected CAR-T cells

(1) Cytokine detection was performed by the method of Elisa using a kit of R & D.

(2) And (3) diluting the standard: preparing 7 centrifuge tubes of 1ml, numbering in sequence, firstly adding 500 mu L of standard substance diluent into each centrifuge tube, then adding 500 mu L of original concentration standard substance into 1 numbered centrifuge tube, fully and uniformly mixing, then adding 500 mu L of standard substance into a second centrifuge tube in the centrifuge tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a third centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a fourth centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a fifth centrifugal tube, and fully and uniformly mixing; then taking 500 mu L of the centrifugal tube, adding the centrifugal tube into a sixth centrifugal tube, and fully and uniformly mixing; then, 500. mu.L of the suspension was added to a seventh centrifuge tube and mixed well.

(3) And standard substance holes are formed in the enzyme-labeled coating plate, and 100 mu L of standard substances with different concentrations are sequentially added, wherein each concentration is 2-3 parallel holes.

(4) Sample adding: respectively arranging blank holes (the blank control hole is replaced by water, the enzyme labeling reagent and the biotin-labeled antibody are operated as before) and sample holes to be detected, adding 100 mu L of sample in the sample holes to be detected on the enzyme labeling coated plate, adding the sample to the bottom of the plate hole of the enzyme labeling plate, keeping the hole wall untouched as far as possible, gently shaking and uniformly mixing

(5) And (3) incubation: standing at room temperature for incubation for 2h

(6) Washing: discarding liquid, spin-drying, adding 200 μ L of washing solution into each well, standing for 30s, discarding, repeating for 3 times, and patting to dry

(7) Adding an antibody: adding 100 mu L of detection antibody on the enzyme-labeled coated plate

(8) And (3) incubation: same operation (5)

(9) Washing: same operation (6)

(10) Marking: 100 μ L of horseradish peroxidase-labeled streptavidin was added to each well

(11) And (3) incubation: incubating at room temperature in dark for 20min

(12) Washing: same operation (6)

(13) Color development: adding 100 μ L of color development liquid into each well, shaking gently, mixing, incubating at room temperature in dark for 20min

(14) And (4) terminating: stop solution (50. mu.L) was added to each well to stop the reaction

(15) And (3) determination: the blank value is used for zero calibration, the absorbance (OD value) of each hole is measured in sequence at the wavelength of 450nm, and the measurement is carried out within 15min after the stop solution is added.

The target cells with different antigen expression levels are selected to be co-cultured with ROR1 CAR-T, the IFN-gamma level secreted by ROR1 CAR-T in response to antigen stimulation is detected, the MG63(ROR1 is highly expressed) and 293T (ROR1 negative) are selected as target cells, so that the ROR1 CAR-T specifically secretes the IFN-gamma when being stimulated by ROR1 antigen, and the result reflects that ROR1CAR produces different responses to the target cells with different antigen expression levels. ROR1 CAR-T secreted significantly IFN- γ when co-cultured with ROR1 high expressing target cell MG63 (fig. 4), indicating that ROR1 CAR-T had a response effect on antigen positive tumor cells.

Example 6 evaluation of the killing Effect of virally infected CAR-T cells in vitro

(1) MG63 cells (ROR1 high expression cell line), 293T (ROR1 negative cell line) and ROR1 CAR-T cells as target cells are respectively cultured.

(2) Collecting target cells and effector cells, centrifuging at 1500rpm/min for 5min, discarding supernatant

(3) Resuspension of target and effector cells in 10% FBS +1640 complete Medium

(4) Using a real-time cell analysis System (RTCA), 50. mu.L 1640 medium was added to the E-Plate16 in air

(5) Determining normal contact of selected holes using RTCA baseline detection

(6) Setting the effective target ratio to be 0:1, 1:1, 5:1, 10:1

(7) E-Plate16 was removed and 100. mu.L of the well-mixed target cell suspension was added to each well in an effective target ratio so that the number of each cell was 10⁴cells/100μL。

(8) E-Plate16 was placed in an incubator at 37 ℃ under 5% CO2 overnight

(9) The next day, E-Plate16 was removed, 50. mu.L of the corresponding effector cells were added, and the killing rate 8h after the addition of the effector cells was calculated.

(10)

The results of the detection are shown in FIG. 5. ROR1 CAR-T showed significant killing effect on ROR1 antigen positive tumor cells. The results of fig. 4 and fig. 5 show that the results of in vitro killing experiments show that the CAR-T cells targeting the humanized tyrosine kinase orphan receptor 1 have significant anti-tumor activity, and the CAR design is beneficial to clinical application.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing Aide immunotherapy research institute Co., Ltd

<120> chimeric antigen receptor targeting humanized tyrosine kinase orphan receptor 1 and use thereof

<130>2019

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>1368

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>1

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360

agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatggcctta 420

ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag gccgggatcc 480

caggtgcagc tgcaggagtc cggccctgga ctggtgaagc catcccagac cctgtctctg 540

acctgcacag tgtctggcta cgccttcaca gcctataaca tccactgggt gaggcaggca 600

ccaggacagg gactggagtg gatgggcagc ttcgaccctt acgatggcgg cagctcctat 660

aatcagaagt ttaaggaccg cctgaccatc agcaaggata catccaagaa ccaggtggtg 720

ctgaccatga caaatatgga cccagtggat accgccacat actattgcgc ccggggctgg 780

tactattttg actactgggg ccacggcacc ctggtgacag tgtctagcgg cggaggaggc 840

tctggcggag gaggcagcgg cggaggaggc tccgacatcg tgatgaccca gacaccactg 900

agcctgccag tgaccccagg agagcctgcc tccatctctt gtagggccag caagtccatc 960

tctaagtacc tggcctggta tcagcagaag ccaggacagg caccaaggct gctgatctac 1020

agcggctcca ccctgcagtc cggcatcccc cctagattct ctggcagcgg ctatggcaca 1080

gactttaccc tgacaatcaa caatatcgag tctgaggacg ccgcctacta tttctgtcag 1140

cagcacgatg agagccctta cacctttggc gagggcacaa aggtggagat caaggctagc 1200

ggtggcggag gttctggagg tgggggttcc atcaacctta caaatgtgac agatatcatc 1260

agggttccgg tgttcaacat tgtcattctc ctggctggtg gattcctgag taagagcctg 1320

gtcttctctg tcctgtttgc tgtcacgctg aggtcatttg taccctag 1368

<210>2

<211>455

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>2

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys Val Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

165 170 175

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ala Phe Thr Ala Tyr

180 185 190

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

195 200 205

Gly Ser Phe Asp Pro Tyr Asp Gly Gly Ser Ser Tyr Asn Gln Lys Phe

210 215 220

Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val

225 230 235 240

Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys

245 250 255

Ala Arg Gly Trp Tyr Tyr Phe Asp Tyr Trp Gly His Gly Thr Leu Val

260 265 270

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

290 295 300

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Ile

305 310 315 320

Ser Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

325 330 335

Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Pro Arg

340 345 350

Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn

355 360 365

Ile Glu Ser Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Asp Glu

370 375 380

Ser Pro Tyr Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys Ala Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asn Leu Thr Asn Val

405 410 415

Thr Asp Ile Ile Arg Val Pro Val Phe Asn Ile Val Ile Leu Leu Ala

420 425 430

Gly Gly Phe Leu Ser Lys Ser Leu Val Phe Ser Val Leu Phe Ala Val

435 440 445

Thr Leu Arg Ser Phe Val Pro

450 455

Claims

1. A nucleic acid sequence selected from the group consisting of: contains the coding sequence of human DAP12, the coding sequence of T2A, the coding sequence of anti-tyrosine kinase orphan receptor 1 single-chain antibody, and the coding sequence of human TREM1 transmembrane and intracellular region which are connected in sequence.

2. The nucleic acid sequence as claimed in claim 1, wherein the nucleic acid sequence further comprises a nucleic acid sequence of a signal peptide before the coding sequence of the anti-tyrosine kinase orphan receptor 1 single-chain antibody, preferably, the nucleic acid sequence of the signal peptide is shown as the 412-474 th nucleic acid of SEQ ID NO. 1; and/or the nucleic acid sequence of the anti-tyrosine kinase orphan receptor 1 antibody is shown as the polynucleic acid at 481-1194 of SEQ ID NO. 1; and/or the nucleic acid sequence of the human DAP12 is shown as the 1 st-339 st polynucleic acid of SEQ ID NO 1; and/or the nucleic acid sequence of the T2A is shown as the 355-405 th nucleic acid of SEQ ID NO. 1; and/or the nucleic acid sequence of the transmembrane and intracellular region of said human TREM1 is as shown in SEQ ID NO:1, position 1231-1368.

3. A fusion protein selected from the group consisting of: a fusion protein containing human DAP12, T2A, anti-tyrosine kinase orphan receptor 1 single-chain antibody, human TREM1 transmembrane and intracellular regions which are connected in sequence; and a derivative fusion protein in which one or more amino acids are substituted, deleted or added in the defined amino acid sequence and which retains the activity of activated T cells; preferably, the anti-humanized tyrosine kinase orphan receptor 1 monoclonal antibody is huUC 961.

4. The fusion protein of claim 3, wherein the fusion protein has one or more of the following characteristics: the fusion protein also contains a signal peptide at the N end of the anti-tyrosine kinase orphan receptor 1 antibody, preferably, the amino acid sequence of the signal peptide is shown as the amino acid at the 138 th and 158 th positions of SEQ ID NO. 2; the amino acid sequence of the anti-tyrosine kinase orphan receptor 1 single-chain antibody is shown as the amino acid 161-398 of SEQ ID NO. 2; the amino acid sequence of the human DAP12 is shown as amino acids 1-113 of SEQ ID NO. 2; the amino acid sequence of the T2A is shown as the 119 th and 135 th amino acids of SEQ ID NO 2; the amino acid sequence of the transmembrane and intracellular region of the human TREM1 is shown as amino acids 411-455 of SEQ ID NO: 2.

5. A nucleic acid vector comprising the nucleic acid sequence of any one of claims 1-2; preferably, the nucleic acid vector is a lentiviral vector comprising the nucleic acid sequence of any one of claims 1-2.

6. A lentivirus comprising the nucleic acid vector of claim 5, preferably said lentivirus vector.

7. A genetically modified T-cell or a pharmaceutical composition comprising a genetically modified T-cell, wherein the cell comprises the nucleic acid sequence of any one of claims 1-2, or comprises the nucleic acid vector of claim 5, or is infected with the lentivirus of claim 6, or stably expresses the fusion protein of claim 4.

8. Use of the nucleic acid sequence of any one of claims 1-2, the fusion protein of any one of claims 3-4, the nucleic acid vector of claim 5, or the lentivirus of claim 7 for the preparation of activated T cells.

9. Use of the nucleic acid sequence of any one of claims 1-2, the fusion protein of any one of claims 3-4, the nucleic acid vector of claim 5, the lentivirus of claim 6, or the genetically modified T cell of claim 7, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a tyrosine kinase orphan receptor 1-mediated disease; preferably, the tyrosine kinase orphan receptor 1 mediated disease is brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-hodgkin lymphoma, uterine cancer, preferably osteosarcoma, breast cancer, leukemia, lung cancer, ovarian cancer.