CN113321743A

CN113321743A - Chimeric antigen receptor of targeting lysyl oxidase 1 and application thereof

Info

Publication number: CN113321743A
Application number: CN202110752178.1A
Authority: CN
Inventors: 代小勇; 苏明
Original assignee: Hainan Precision Medical Technology Co ltd
Current assignee: Hainan Precision Medical Technology Co ltd
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2021-08-31
Anticipated expiration: 2041-07-02
Also published as: CN113321743B

Abstract

The invention relates to a lysyl oxidase 1-targeted chimeric antigen receptor and application thereof, and particularly provides an isolated chimeric antigen receptor which comprises a binding domain, a transmembrane domain and an intracellular signal transduction domain of anti-lysyl oxidase 1, wherein the binding domain of the anti-lysyl oxidase 1 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity determining regions 1-3, and the light chain variable region comprises light chain complementarity determining regions 1-3. The invention also provides an immune effector cell comprising the chimeric antigen receptor of the invention. The immune effector cells of the invention have significant tumor-inhibiting effects.

Description

Chimeric antigen receptor of targeting lysyl oxidase 1 and application thereof

Technical Field

The invention relates to the field of biotechnology and biomedicine, in particular to a chimeric antigen receptor of targeting lysyl oxidase 1 and application thereof.

Background

1.1 Breast cancer

Breast cancer is the most common malignant tumor in women, the incidence rate of the breast cancer is the first malignant tumor in the world, and the breast cancer is also one of the malignant tumors with the highest mortality rate. According to the american cancer society, over 138 ten thousand women worldwide suffer from breast cancer, accounting for 23% of new cases of cancer in women, over 45 ten thousand people die of breast cancer, accounting for 14% of the total number of cancer deaths, and the incidence of breast cancer is higher in developed countries such as western europe and north america than in less developed countries in 2008. In 2016, the number of new cancer cases in the United states is estimated to be about 168.5 ten thousand, and the number of cancer deaths is 59.5 ten thousand, wherein breast cancer is the most common malignant tumor in women, and the number of new cancer cases is about 24.6 ten thousand, which accounts for 29 percent of the total malignant tumor cases in women; about 4.05 million people die from the disease, accounting for 14% of cancer deaths in women. In China, the incidence rate of breast cancer is on the rise obviously in recent years. Between 1998 and 2007, the annual growth in the incidence of breast cancer in urban and rural women was 3.9% and 6.3%, respectively. According to the statistics published by the Chinese anticancer society, about 21 ten thousand new breast cancer cases are published in China, the incidence rate is increased by 3 percent per year, and the breast cancer cases are the top of the major cities of Beijing, Shanghai, Tianjin, Guangzhou and the like. From 1991 to 2000, the mortality rate of breast cancer patients in China has increased by 38.9%, and the breast cancer patients become malignant tumors with the fastest increase of the mortality rate and become the biggest threat to the health of women.

Breast cancer has long been recognized as a systemic disease, and the treatment mainly comprises systemic multidisciplinary comprehensive treatment, including surgical treatment, auxiliary radiotherapy and chemotherapy, biological targeted treatment and endocrine treatment. With the popularization of mammary gland molybdenum target X-ray radiography examination, the development of new surgical operation technology, the update of chemoradiotherapy technology, the popularization of biological targeted therapy and the like, the treatment of the breast cancer has made great progress, and the cure rate is obviously improved. However, since breast cancer is a highly heterogeneous malignant tumor, it still causes great treatment difficulty, and even in early-stage breast cancer patients, about 30% of patients fail to treat and have metastasis. Metastasis is the leading cause of death in breast cancer patients, and once recurrent metastasis occurs, the cure rate is lower than 5%, and the prognosis is poor. Therefore, how to carry out effective targeted therapy aiming at the recurrence and the metastasis of the breast cancer has important practical significance for improving the survival rate and the life quality of breast cancer patients and improving the prognosis.

1.2 Lysyl Oxidase (LOX)

LOX is a copper (Cu) dependent amino oxidase whose gene is located in human autosome 5q23.1 and consists of seven exons and six introns. LOX is a matrix remodeling enzyme that plays an important role in the formation and repair of extracellular matrix, and is a key enzyme in the extracellular matrix that stabilizes the extracellular matrix during the initial phase of collagen and elastin polymerization. In Cu²⁺With the assistance of LOX, it oxidizes specific amino acid residues on collagen and elastin in the extracellular matrix and covalently cross-links them, modulating the tensile strength of the tissue, resisting hydrolysis by collagenase. LOX, when bound to a ligand, exerts biological effects in a variety of target cells, including regulation of cell signaling and gene transcription, effects on cell motility migration and cell adhesion, and the like.

In recent years, more and more researchers found that lysyl oxidase 1(LOX-1) is closely related to invasion and metastasis of malignant tumors through research. The over-expression of LOX-1 in cancer cells can promote the invasiveness of the cells, resist anoikis of the cancer cells and increase the motor capacity and the invasiveness of the cells, and the LOX-1 antisense oligonucleotide can inhibit the invasive metastatic capacity of the cancer cells. Research shows that the expression of LOX-1 protein in breast cancer tissues is obviously higher than that of normal breast tissues beside cancer and benign lesion tissues of breast, and the breast cancer with axillary lymph node metastasis is higher than that without axillary lymph node metastasis. Weiwei and the like inoculate MDA-MB-231 cells transfected with LOX-RNAi-LV into a nude mouse by establishing a nude mouse breast cancer orthotopic transplantation tumor model, measuring the volume and the mass of the transplantation tumor after tumor formation and comparing the volume and the mass, and finds that the interference of the expression of LOX-1 in the MDA-MB-231 breast cancer cells can inhibit the growth and the invasion of the nude mouse breast cancer transplantation tumor.

1.3 chimeric antigen receptor T cell immunotherapy techniques

Chimeric antigen receptor T cell immunotherapy technology is also known as CAR-T cell therapy. CAR-T is known as chemomeric antigen T-cell Immunotherapy, and is expressed on T cells of patients after antibodies aiming at antigens of specific tumors are combined with CD3 zeta and Fc epsilon RI gamma in the T cells, amplified in vitro, screened and transfused into the patients in large quantity. The basic principle is to use the patient's own immune cells to eliminate cancer cells, which is a cell therapy. CARs are composed of an intracellular signaling region (e.g., CD3 ζ and CD28), a transmembrane region, and an extracellular antigen binding region of a T Cell Receptor (TCR). Recombinant CARs rely on antigen, but not on MHC presentation, effectively avoiding the immune escape mechanism of MHC downregulation. Based on the differences in intracellular signaling domain structure, CARs can be divided into four generations, the first generation of CAR structure has only one intracellular signaling component (TCR/CD3 zeta chain and fcsri gamma chain), is able to recognize target antigen and activate T cells, has no co-stimulatory molecules, is unable to transduce proliferation signal and induce cytokine production, and T cells are unable to proliferate, thus having little tumor killing effect. Second generation CARs have added to them a costimulatory molecule, such as CD28, CD27, 41BB (CD137), OX40(CD134) or inducible costimulatory molecule (ICOS), that continues to proliferate and release cytokines even in the absence of exogenous costimulatory molecules. While the second generation CARs greatly improved T cell antitumor efficacy, the killing ability of T cells was not fully turned on, and third generation CARs comprising two costimulatory molecules (CD28, 4-1BB, and CD3 ζ) were born. Mouse experiments show that the third-generation CAR can enhance secretion of cytokines and inhibit tumor growth.

The CAR-T still has partial obstacles in the treatment practice of the solid tumor due to the influence of various factors such as complex microenvironment, various immune escape mechanisms, complicated tumor antigen targets, large volume of the solid tumor, difficult homing of CAR-T cells to tumor tissues and the like. Ovarian cancer is the worst-prognosis gynecological malignancy, and the clinical diagnosis and treatment of ovarian cancer has not progressed in a breakthrough manner for nearly half a century, and is one of the solid tumors for which CAR-T treatment was first attempted. Second and third generations of CAR-T are now in clinical development. CAR-T cell multicenter clinical studies are currently underway worldwide against different solid tumor antigenic targets, representative antigenic targets also include: mesothelin for the treatment of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer; CEA is used for treating lung cancer, colon cancer, gastric cancer, breast cancer and pancreatic cancer; MUC-1 can be used for treating hepatocarcinoma, lung cancer, pancreatic cancer, colon cancer, gastric cancer, and breast cancer; GPC3 for treating liver cancer; EGFR-vIl for glioma, head and neck tumors; PSMA is used for prostate cancer and the like. At present, with the successful application of CAR-T technology in blood tumor, CAR-T cell technology enters a new era and brings new hope for tumor patients. However, despite the good therapeutic effect of CAR-T cells in hematological tumors, difficulties are faced in the application of solid tumors, such as lack of specific targets, inability of CAR-T cells to effectively infiltrate into tumor tissues, and inhibitory effect of the immunosuppressive microenvironment on CAR-T cells. The final aim of CAR-T cell therapy is to cure tumors, so that novel high-specificity tumor surface antigen targets are searched or a plurality of targets are selected for therapy, migration and infiltration of CAR-T cells to tumor tissues can be promoted, the immunosuppressive property of a tumor microenvironment is relieved, and the anti-tumor effect of the CAR-T cells in solid tumors is improved.

Disclosure of Invention

The invention provides a chimeric antigen receptor, which comprises an amino acid sequence shown in SEQ ID.1, a nucleic acid for encoding the chimeric antigen receptor, a vector comprising the nucleic acid, an immune effector cell comprising the chimeric antigen receptor, the nucleic acid molecule and/or the vector, a method for preparing the immune effector cell, a composition comprising the immune effector cell and application of the chimeric antigen receptor.

The chimeric antigen receptor of the invention at least comprises the following beneficial effects: (1) stably express on the surface of immune cells (such as T cells) with high expression level; (2) has stronger ability of killing LOX-1 positive target cells; (3) promoting secretion of cytokines by immune cells, e.g., enhancing the ability of T cells to secrete cytokines (INF-gamma or IL-6) by at least 1-fold, 2-fold or more than 3-fold; (4) has no hemolysis, does not cause hemolysis or coagulation of red blood cells; (5) no blood vessel irritation, no local or systemic abnormality after administration; (6) no tumorigenicity, no tumorigenicity in vivo or in vitro; (7) prolonging the survival time of tumor patients; (8) effectively relieving the disease condition of tumors (such as breast cancer); and, (9) high safety with low risk of causing side effects (e.g., cytokine release syndrome or CAR-T cell-related encephalopathy syndrome).

In one aspect, the present invention provides an isolated chimeric antigen receptor comprising a binding domain of anti-lysyl oxidase 1, a transmembrane domain, and an intracellular signaling domain, the binding domain of anti-lysyl oxidase 1 comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1(CDR-H1), heavy chain complementarity determining region 2(CDR-H2), and heavy chain complementarity determining region 3(CDR-H3), and a light chain variable region (VL) comprising light chain complementarity determining region 1(CDR-L1), light chain complementarity determining region 2(CDR-L2), and light chain complementarity determining region 3(CDR-L3),

the heavy chain complementarity determining region 1 has a sequence shown in SEQ ID No.11

SerSerAsnTrpTrpSer SEQ ID No.11；

The heavy chain complementarity determining region 2 has a sequence shown in SEQ ID No.13

GluIleTyrHisSerGlySerThrAsnTyrAsnProSerLeuLysSer SEQ ID No.13；

The heavy chain complementarity determining region 3 has a sequence shown in SEQ ID No.15

LeuLeuGlyGlyIleAlaGlyArgTrpPheAspPro SEQ ID No.15；

The light chain complementarity determining region 1 has a sequence shown in SEQ ID No.4

ArgAlaSerGlnSerValSerSerHisLeuAla SEQ ID No.4；

The light chain complementarity determining region 2 has a sequence shown in SEQ ID No.6

AspThrSerThrArgAlaThr SEQ ID No.6；

The light chain complementarity determining region 3 has a sequence shown in SEQ ID No.8

GlnGlnTyrGlySerSerProGlyTyrThr SEQ ID No.8。

Further, the binding domain of the anti-lysyl oxidase 1 comprises a single-chain variable fragment (scFv) of the anti-lysyl oxidase 1, and the series of the single-chain variable fragments of the anti-lysyl oxidase 1 is shown as SEQ ID NO. 2.

Further, a basal signaling domain selected from at least one of CD3 ζ, TCR ζ, FcRI γ, FcRI β, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, CD278(ICOS), Fc ∈ RI, DAP10, DAP12, or CD66d is included in the intracellular signaling domain.

Further, the intracellular signaling domain further comprises at least one co-stimulatory molecule domain, the co-stimulatory molecule is selected from the group consisting of 4-1BB, CD, OX, CD, CDS, ICAM-1, B-H, ICOS, GITR, BAFFR, LIGHT, HVEM, KIRDS, SLAMF, NKp, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, GAITX, CD11, ITGB, CD, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM, SLAMF, CD, TAITCEACAM, TAM, CD160, PAG, SLGL, SLAG, SLMG-6, SLAMF, SLAMD, NKG2, TNFR, TRANCE, TAM, CD-1, CD-SLAMBB, SLAMBR, SLAMF, SLAMBR, SLAMD, CD-1, and SLAMBR. Preferably, two co-stimulatory molecule domains are included.

Further, the transmembrane domain is selected from the group consisting of a transmembrane domain of CD8, CD3 epsilon, the alpha, beta or zeta chain of a T cell receptor, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD 154.

In another aspect, the present invention also provides an isolated nucleic acid molecule encoding a chimeric antigen receptor of the present invention, which encodes the chimeric antigen receptor described above.

In another aspect, the invention also provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid sequence encoding the anti-lysyl oxidase 1 binding domain is as set forth in SEQ ID No. 2.

In another aspect, the invention also provides a vector comprising a nucleic acid molecule according to the invention.

Further, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

In another aspect, the present invention also provides an immune effector cell comprising the chimeric antigen receptor, the nucleic acid molecule and/or the vector of the present invention.

In certain embodiments, the immune effector cell is selected from the group consisting of: t lymphocytes and natural killer cells.

In certain embodiments, the surface of the immune effector cell expresses the chimeric antigen receptor of the invention.

In another aspect, the present invention also provides a method of preparing an immune effector cell, comprising the steps of: transducing the vector of the invention into an immune effector cell.

In another aspect, the present invention also provides an anti-tumor pharmaceutical composition comprising the immune effector cell of the present invention.

In another aspect, the invention also provides the use of said chimeric antigen receptor, said nucleic acid molecule, said vector and/or said immune effector cell for the manufacture of a medicament for the treatment of a disease or disorder associated with the expression of lysyl oxidase 1, or for increasing cytokine secretion.

In certain embodiments, the disease or disorder associated with expression of lysyl oxidase 1 comprises hematological and solid tumors.

In certain embodiments, the solid tumor comprises breast cancer, liver cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, brain glioma.

In certain embodiments, the cytokine is selected from at least one of IFN- γ, IL-6, and granzyme B.

In yet another aspect, the invention provides a method of treating a tumor, said method comprising the step of administering said chimeric antigen receptor, said nucleic acid molecule, said vector and/or said immune effector cell to a subject.

Further, the immune effector cell is derived from a subject, the vector of the invention is transduced into the immune effector cell in vitro, and the obtained immune effector cell is administered to the subject.

Other aspects and advantages of the present invention will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present invention have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present invention enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Advantageous effects

The invention obtains an antigen binding fragment of the target LOX-1 by screening, and applies the antigen binding fragment to a chimeric antigen receptor, wherein the antigen binding receptor is expressed on the surface of an immune cell. The immune effector cells have a significant tumor-inhibiting effect.

Drawings

FIG. 1 Structure diagram of a chimeric antigen receptor targeted to LOX-1;

FIG. 2 is a graph of the effect of example 3 targeting LOX-1 chimeric antigen receptor T cells on the killing effect of tumor cells;

FIG. 3 is a graph of the IL-6 secretion levels of the chimeric antigen receptor T cells targeting LOX-1 of example 4;

FIG. 4 is a graph of the level of IFN- γ secretion from chimeric antigen receptor T cells targeting LOX-1 of example 4;

FIG. 5 is a graph of the level of granzyme B secretion from the chimeric antigen receptor T cells targeting LOX-1 of example 4;

FIG. 6 is a graph of the effect of chimeric antigen receptor T cells targeting LOX-1 on mouse tumor volume in example 5;

note:^#is Non-T group compared with PBS control group;^*is the CAR-LOX-1T group compared to the PBS control group.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are consecutively linked by a short flexible polypeptide linker and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, a scFv can have VL and VH variable regions, e.g., in any order relative to the N-terminus and C-terminus of a polypeptide, can comprise a VL-linker-VH or can comprise a VH-linker-VL.

In some embodiments of the invention, the intracellular signaling region comprises a primary signaling domain, for example, the primary signaling domain of CD 3-zeta. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and/or CD 28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain) | transmembrane domain and an intracellular signaling domain (which comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule). In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

Example 1: LOX-1 humanized antibody scFV screen

1.1 establishment of a permanent high LOX-1 expressing 293T cell line: 293T-LOX-1+/+:

(1) vigorous growing human 293T cells were selected at 5X 10 days before transfection⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

(2) the transfection was performed the next day by diluting 3. mu.g of plasmid with 200. mu.L of opti-MEM medium and 6. mu.L of Lipofectamine2000 with 200. mu.L of opti-MEM medium in one well of a 6-well plate, gently mixing them, and then leaving them at room temperature for 5 minutes;

(3) gently mixing the two tube dilutions, standing at room temperature for 20 minutes, and gently adding 600. mu.L of opti-MEM medium to the mixed dilution;

(4) gently rinsing the cells to be transfected once with PBS, then gently adding the mixed diluent into the culture hole, and placing the culture hole in a carbon dioxide incubator for culture;

(5) after culturing for 4-6 h, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

(6) after 48 hours, a culture medium containing 1 mu g/mL puromycin (puromycin) is selected for screening; obtaining the 293T cell line which stably expresses LOX-1 after the cell is not dead any more.

1.2 panning, amplification, purification, sequencing and identification of the LOX-1 humanized antibody scFV

First screening: laying on 6-well plate 1X 10⁷293T-LOX-1^+/+A cell; dissolving BSA with PBST to a concentration of 3%, 300. mu.L/well, blocking at 37 ℃ for 2h, and washing the plate for 3 times; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 3 times; adding glycine buffer solution, shaking gently for 10min at room temperature, sucking out the eluent, and adding Tris-HCl buffer solution for neutralization reaction. mu.L of the eluate was added to 5mL of TG1 strain in the logarithmic growth phase, infected at 37 ℃ for 30min, and then added to a medium containing antibiotics, and cultured overnight at 37 ℃. Taking the culture solution at 4 ℃, 4000rpm, centrifuging for 15min, collecting supernatant, adding 5mL of PEG/NaCl, carrying out ice bath for 30min, carrying out ice bath at 4 ℃, 8000rpm, centrifuging for 15min, discarding supernatant, carrying out heavy suspension by using 1mL of PBS, carrying out centrifugation at 4 ℃, 12000rpm for 10min, and obtaining precipitate, namely the phage antibody particles.

And (3) second screening: laying on 6-well plate 1X 10⁷293T-LOX-1^+/+A cell. Washing the plate 3 times with 3% skimmed milk powder; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 5-8 times; the rest of the operations are the same as the first round of screening.

And (3) third screening: laying on 6-well plate 1X 10⁷293T-LOX-1^+/+A cell. Blocking with 3% BSA, and washing the plate 3 times; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 5-8 times; the rest of the operations are the same as the first round of screening.

After screening, TG1 bacteria were infected with phage, plated on petri dishes, 50 single clones were randomly picked from the plates after cultivation, positive clones were screened by ELISA using soluble human LOX-1 as antigen, and sequencing was performed. According to the sequencing results, 10 sequences were selected for subsequent experiments.

1.3 expression and analysis of target antibodies

Extracting positive clone plasmid and transforming to colibacillus competent cell, inducing antibody protein expression with 100mM IPTG, and purifying target antibody protein by adopting a mode of mutually matching membrane separation and resin separation. And (3) detecting the affinity by using a Fortebio biomolecule interaction platform, and selecting the antibody with the highest affinity for constructing a chimeric antigen receptor.

1.4 antibody sequence analysis

The amino acid sequence structure of the scFv antibody is determined, and the amino acid sequence is placed in a structure database to perform a search for a homologous structure and an alignment analysis of the antibody sequence structure, thereby determining the complementarity determining region (CDR region) thereof. The sequence is as follows, as shown below,

TABLE 1 LOX-1 humanized scFV DNA sequences

TABLE 2 LOX-1 humanized scFV protein sequences

TABLE 3 LOX-1 humanized CDRs protein sequences

Example 2: preparation of CAR-T cells

2.1 preparation of vectors containing chimeric antigen vectors

The CAR LOX-1 nucleotide sequence is obtained through amplification by a PCR method, enzyme cutting sites are introduced at two ends of a target sequence through primer design, double enzyme cutting reaction is carried out on a target gene segment and a lentiviral vector plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro, and incubation is carried out for 60min at 37 ℃. After the completion of the digestion reaction, the nucleic acid fragment was purified by agarose gel (using a gel purification kit of Taraka), and the specific procedures were performed according to the kit instructions. Incubating the target nucleic acid molecule after gel purification for 10-12h at 16 ℃ under the action of T4 ligase, transferring the obtained ligation product into DH5 alpha competent cells by electric shock, transferring the obtained competent cells into a liquid LB culture medium without antibiotic, culturing for 2h at 37 ℃ by a shaking table at 200rpm, then centrifuging for 2min at 4000rpm, then centrifugally suspending the cells in a fresh LB culture medium, uniformly coating the bacterial liquid in a solid LB culture medium containing the antibiotic, culturing overnight in a 37 ℃ culture box, picking and identifying positive clones, and storing the positive clone strains in a refrigerator at-70 ℃.

2.2 preparation of Lentiviral vectors

Recovering the correctly sequenced and stored bacterial liquid, inoculating the recovered bacterial liquid to 20mL of liquid LB culture medium containing antibiotics at 37 ℃ and 200rpm of a shaking table, culturing overnight, centrifugally collecting thalli at 4000rpm, extracting plasmids by adopting a plasmid extraction kit of Takara company, and carrying out the specific operation steps according to the kit operation instructions. And (5) enzyme digestion identification, and whether the target sequence is correct or not. Simultaneously, 293T cells were cultured at 37 ℃ with 5% CO₂After stable cultivation for 2-3 generations under the condition, 1 × 10⁶The 293T cells were inoculated into six-well plates at 37 ℃ with 5% CO₂Culturing under the condition to logarithmic growth phase, and replacing fresh culture medium. Mixing the target nucleic acid plasmid and the VSVG envelope protein plasmid according to the ratio of 3:1, adding an Opti-MEM culture medium to make up the volume of 5mL, dropwise adding the mixed solution of the plasmids and the transfection reagent into 293T cell culture solution, and dropwise adding 5% CO at 37 DEG C₂Culturing under the condition for 48 h. After completion of the culture, the supernatant was collected by centrifugation at 2500rpm for 20min, and cell debris and other impurities were removed by filtration using a 0.45 μm filter and a 0.22 μm filter in this order to obtain a lentivirus carrying the CAR LOX-1 gene.

2.3 preparation of CAR-T cells

Healthy volunteers were recruited, 100mL of peripheral blood was collected, separated by flow cytometry and CD4 in the peripheral blood was obtained⁺、CD8⁺T cell, regulated CD4⁺：CD8⁺The ratio is 1:1 for subsequent use. The obtained T cells were inoculated in RPMI1640 medium containing 10% FBS, and subcultured for 3 to 5 passages in preparation for reaching a stable state. T cells were cultured using 6-well plates, seeded 5X 10 cells per well⁶Cell line, 5% CO at 37 ℃₂Culturing in logarithmic growth phase under the condition, adding slow virus carrying CAR LOX-1 gene, incubating at 37 deg.C for 3 hr, discarding supernatant, washing with fresh culture medium for 3-5 times, and washing with 5% CO at 37 deg.C₂Culturing under the condition, continuing culturing for 5-7 days, and collecting CAR-LOX-1T cells for subsequent experiments.

Example 3 CAR-T cells in vitro anti-tumor Effect

To verify the antitumor effect of the CAR-T cells of interest, they were first verified in vitro cell experiments for preliminary screening. In the cytological experiment, the breast cancer cell MDA-MB-231 cell is selected as an experimental object. Collecting MDA-MB-231 cell strain (stored in laboratory) stored in liquid nitrogen, rapidly recovering in warm water, adding DMEM culture medium, mixing, centrifuging at 2000rpm for 5min to collect cells, washing with fresh culture medium for 3 times, adding DMEM containing 10% FBS, culturing at 37 deg.C and 5% CO₂Culturing under the condition for 3-5 generations. CAR-LOX-1T cells obtained after transfection of lentivirus were mixed with MDA-MB-231 cells at 1:1, 2:1, 4:1, 8:1, 16:1 ratios, while PBS and untransfected T cells (Non-T) were used as controls at the same ratio. Inoculating the above mixed cells into 6-well culture plate with 5% CO₂And culturing in an incubator at 37 ℃ for 24 hours, and detecting the killing effect of the tumor cells. The specific detection mode is as follows: after the mixed culture is completed, 10 mul CCK-8 is added into each well, after incubation for 2h at 37 ℃, the wavelength of 450nm is detected by a microplate reader, and the OD value is measured, wherein the killing rate is [1- (experiment group OD value-effector cell control group OD value)/target cell control group OD value ═]X 100%. As shown in FIG. 2, the CAR-LOX-1T cells constructed in the present invention can effectively exert tumor cell inhibitory activityWith, CAR-LOX-1T treatment group had significant differences compared to PBS and untransfected groups, and as the mixing ratio increased, the anti-tumor effect gradually increased, with "dose-dependence". This demonstrates that the targeted LOX-1 antibody screened and obtained in the present invention can effectively bind to the target antigen, mediating the exertion of anti-tumor effects.

Example 4 detection of secreted cytokines after Co-culture of CAR LOX-1T cells with target cells

Taking out the mixed culture supernatant sample of each hole in the embodiment 3 from a refrigerator at the temperature of-20 ℃, and melting at room temperature; ELISA kits (manufactured by Nanjing) are used, samples are processed according to the instruction, and the secretion levels of IFN-gamma, IL-6 and granzyme B in each group are detected. As shown in FIGS. 3, 4, and 5, the CAR-LOX-1T treated group was significantly increased in the secretion of IFN-. gamma., IL-6, and granzyme B after coculture with MDA-MB-231 cells, compared to PBS and untransfected groups, and the secretion was gradually increased with increasing mixing ratio, showing "dose dependence".

Example 5 CAR-T cells anti-tumor Effect in vivo

NSG female mice of 6-8 weeks old are selected and bred in a clean laboratory environment. The MDA-MB-231 cells were revived and cultured. Each mouse was injected subcutaneously with 1X 10⁶Each MDA-MB-231 cell was injected with 100. mu.L of cell suspension. Then the growth state of the mouse and the development condition of tumor tissues are observed every day, and obvious tumor masses appear after about 3 to 5 days, which indicates that the tumor mouse model is successfully established. The tumor-bearing mice were randomly divided into 5 groups, namely, normal saline group and T cell control group Non-T (Non-transfected T cells were injected, 2X 10)⁷Cell/cell), CAR-LOX-1T low dose group (5 × 10)⁶Cell/cell), CAR-LOX-1T medium dose group (1 × 10)⁷Cell/cell), CAR-LOX-1T high dose group (2 × 10)⁷Cell/cell) was treated and tumor volume was measured at 2 and 4 weeks after treatment, respectively. As shown in FIG. 6, all of the CAR-LOX-1T-treated groups inhibited tumor growth, and at the fourth week, the CAR-LOX-1T-treated group showed significant difference in tumor volume from the control group, with the inhibition effect being most significant in the high dose group. Illustrating the CAR-obtained by the preparation of the present inventionLOX-1T cells have tumor-inhibiting effects and are long lasting.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

SEQUENCE LISTING

<110> Hainan precision medical science and technology Limited

<120> chimeric antigen receptor targeting lysyl oxidase 1 and use thereof

<130> CP121010601C

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 753

<212> DNA

<213> Artificial sequence

<400> 1

gaaattgtgt tgacgcagtt tccaggcacc ctgtcttttg tctccagggg aaagagccac 60

cctctcctgc agggccagtc agagtgttag tagccactta gcctggtacc agcagaggcc 120

tggccagcct cccaggctcc tcatctatga tacatccacc agggccactg gtatccccgc 180

caggttcagt ggcagtgggt ctgggacaga gttcactctc accatcagca gcctggagtc 240

tgaagatttt gcagtgtatt actgtcagca gtatggtagc tcaccggggt acacttttgg 300

ccaggggacc aagctggaga tcaaacgttc cggaggtcga ccataacttc gtataatgta 360

tactatacga agttatcctc gagcggtacc caggtgcagc tgcagcagtc gggcccagga 420

ctggtgaagc cttcggggac cctgtccctc acctgcgctg tctctggtgg ctccatcagc 480

agtagtaact ggtggagttg ggtccgccag cccccaggga aggggctgga gtggattggg 540

gaaatctatc atagtgggag caccaactac aacccgtccc tcaagagtcg agtcaccata 600

tcagtagaca agtccaagaa ccagttctcc ctgaagctga gctctgtgac cgccgcggac 660

acggccgtgt attactgtgc gagactcctg ggggggatag cggggaggtg gttcgacccc 720

tggggccagg gaaccctggt cactgtctcc tca 753

<210> 2

<211> 249

<212> PRT

<213> Artificial sequence

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Glu Ile Val Leu Thr Gln Phe Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser His

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Thr Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Gly

85 90 95

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

100 105 110

Arg Pro Leu Arg Ile Met Tyr Thr Ile Arg Ser Tyr Pro Arg Ala Val

115 120 125

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

130 135 140

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

145 150 155 160

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

165 170 175

Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

180 185 190

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

195 200 205

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Leu Leu Gly Gly Ile Ala Gly Arg Trp Phe Asp Pro Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser

245

<210> 3

<211> 23

<212> PRT

<213> Artificial sequence

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Glu Ile Val Leu Thr Gln Phe Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<400> 4

Arg Ala Ser Gln Ser Val Ser Ser His Leu Ala

1 5 10

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence

<400> 5

Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 6

<211> 7

<212> PRT

<213> Artificial sequence

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Asp Thr Ser Thr Arg Ala Thr

1 5

<210> 7

<211> 32

<212> PRT

<213> Artificial sequence

<400> 7

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence

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Gln Gln Tyr Gly Ser Ser Pro Gly Tyr Thr

1 5 10

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence

<400> 9

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

1 5 10

<210> 10

<211> 30

<212> PRT

<213> Artificial sequence

<400> 10

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

1 5 10 15

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

20 25 30

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<400> 11

Ser Ser Asn Trp Trp Ser

1 5

<210> 12

<211> 14

<212> PRT

<213> Artificial sequence

<400> 12

Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10

<210> 13

<211> 16

<212> PRT

<213> Artificial sequence

<400> 13

Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 14

<211> 32

<212> PRT

<213> Artificial sequence

<400> 14

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

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence

<400> 15

Leu Leu Gly Gly Ile Ala Gly Arg Trp Phe Asp Pro

1 5 10

<210> 16

<211> 11

<212> PRT

<213> Artificial sequence

<400> 16

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

Claims

1. An isolated chimeric antigen receptor comprising a binding domain of anti-lysyl oxidase 1, a transmembrane domain, and an intracellular signaling domain, wherein the binding domain of anti-lysyl oxidase 1 comprises a heavy chain variable region comprising heavy chain complementarity determining region 1, heavy chain complementarity determining region 2, and heavy chain complementarity determining region 3, and a light chain variable region comprising light chain complementarity determining region 1, light chain complementarity determining region 2, and light chain complementarity determining region 3, wherein,

the light chain complementarity determining region 1 has a sequence shown in SEQ ID No. 4;

the light chain complementarity determining region 2 has a sequence shown in SEQ ID No. 6;

the light chain complementarity determining region 3 has a sequence shown in SEQ ID No. 8;

the heavy chain complementarity determining region 1 has a sequence shown in SEQ ID No. 11;

the heavy chain complementarity determining region 2 has a sequence shown in SEQ ID No. 13;

the sequence of the heavy chain complementarity determining region 3 is shown as SEQ ID No. 15;

preferably, the binding domain of anti-lysyl oxidase 1 comprises a single-chain variable fragment of anti-lysyl oxidase 1;

more preferably, the series of the single-chain variable fragments of the anti-lysyl oxidase 1 is shown as SEQ ID NO. 2.

2. The isolated chimeric antigen receptor according to claim 1, wherein a basal signaling domain selected from at least one of CD3 ζ, TCR ζ, FcRI γ, FcRI β, CD3 γ, CD3 δ, CD3 e, CD5, CD22, CD79a, CD79b, CD278(ICOS), fceri, DAP10, DAP12, or CD66d is comprised in the intracellular signaling domain;

preferably, the intracellular signaling domain further comprises at least one co-stimulatory molecule domain, the co-stimulatory molecule is selected from the group consisting of 4-1BB, CD, OX, CD, CDS, ICAM-1, B-H, ICOS, GITR, BAFFR, LIGHT, HVEM, KIRDS, SLAMF, NKp, CD alpha, CD beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, GAITX, CD11, ITGB, CD, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM, SLAMF, CD, TAITACAM, TAM, CD160, PAG, SLGL, SLAG-2, NKG2, TNFR, TRANCE/RANKL, SLAMF, CD, SLAMBR, CD-1, CD-19, SLAMBR, SLAMF, SLAMBR, and SLAMBR; more preferably, two co-stimulatory molecule domains are included.

3. The isolated chimeric antigen receptor according to claim 1, wherein the transmembrane domain is selected from the group consisting of the transmembrane domains of CD8, CD3 epsilon, the alpha, beta or zeta chain of the T cell receptor, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD 154.

4. An isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein said nucleic acid molecule encodes the chimeric antigen receptor of any one of claims 1-3;

preferably, the nucleic acid sequence encoding the anti-lysyl oxidase 1 binding domain is as shown in SEQ ID No. 2.

5. A vector comprising the nucleic acid molecule of claim 4;

preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

6. An immune effector cell, wherein the immune effector cell comprises at least one of the chimeric antigen receptor of any one of claims 1-3, the nucleic acid molecule of claim 4, and the vector of claim 5;

preferably, the immune effector cell is selected from at least one of a T lymphocyte and a natural killer cell;

preferably, the immune effector cell surface expresses the chimeric antigen receptor of claim 1.

7. A method of producing an immune effector cell, comprising the steps of: transferring the vector of claim 5 into an immune effector cell;

preferably, the immune effector cell is selected from at least one of a T lymphocyte and a natural killer cell.

8. An anti-tumor pharmaceutical composition comprising the immune effector cell of claim 6.

9. Use of the chimeric antigen receptor of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5 and/or the immune effector cell of claim 6 for the preparation of a medicament for the treatment of a disease or disorder associated with the expression of lysyl oxidase 1, or for increasing cytokine secretion;

preferably, the disease or disorder associated with expression of lysyl oxidase 1 includes hematological and solid tumors; more preferably, the solid tumor comprises breast cancer, liver cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, brain glioma;

preferably, the cytokine is selected from at least one of IFN-gamma, IL-6 and granzyme B.

10. A method of treating a tumor, comprising the step of administering to a subject the chimeric antigen receptor of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5, and/or the immune effector cell of claim 6;

preferably, the immune effector cell is derived from a subject, and the vector of the present invention is transduced into the immune effector cell in vitro, and the obtained immune effector cell is administered to the subject.