WO2021068108A1

WO2021068108A1 - Nkg2d car-t cell, preparation therefor, and application thereof

Info

Publication number: WO2021068108A1
Application number: PCT/CN2019/109960
Authority: WO
Inventors: 曾昕; 刘江海; 李欣檑; 刘彬; 孔洋; 曾顺泽; 李玲芳
Original assignee: 成都盛世君联生物技术有限公司; 成都盛世锐科生物技术有限公司
Priority date: 2019-10-08
Filing date: 2019-10-08
Publication date: 2021-04-15

Abstract

The present invention provides a fusion protein, comprising an antigen-binding domain, a transmembrane domain, and a costimulatory signaling region. The antigen-binding domain can specifically bind to a tumor-specific antigen, that is an NKG2D ligand, and activate T cells by means of the transmembrane domain and the costimulatory signaling region. The fusion protein and an NKG2D CAR-T cell capable of expressing the fusion protein provided by the present invention can specifically kill tumor cells using the NKG2D CAR-T cell with the NKG2D ligand as a target antigen. The fusion protein and the NKG2D CAR-T cell can be used as a drug for treating tumor diseases and can be used to treat tumors in which a ligand of an NKG2D molecule is highly expressed, providing a new method for preventing or treating tumors.

Description

NKG2D CAR-T cell and its preparation and application

Technical field

The present invention relates to the field of biomedicine, in particular to NKG2D CAR-T cells and their preparation and application.

Background technique

Chimeric antigen receptor (CAR) modified immune cells use genetic engineering methods to modify immune cells to express exogenous tumor-specific genes. CAR genes mainly include extracellular recognition domains and intracellular signal transduction domains: the former is used to recognize tumor surface specific molecules and the latter is used to initiate immune cell responses after recognizing tumor surface molecules to exert cytotoxic effects.

T cells are a type of lymphocytes and play an important role in cell-mediated immunity. It differs from other lymphocytes (such as B cells and natural killer cells (NK cells)) in the presence of T cell receptors (TCR) on the cell surface. T helper cells, also known as CD4 ⁺ T or CD4 T cells, express CD4 glycoprotein on their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, such cells proliferate rapidly and secrete cytokines that can regulate immune responses. Cytotoxic T cells, also known as CD8 ⁺ T cells or CD8 T cells, express CD8 glycoprotein on the cell surface. CD8 ⁺ T cells are activated when exposed to peptide antigens presented by MHC class I molecules. Memory T cells are a subset of T cells that exist for a long time and respond to related antigens, thus providing the immune system with memories of past infections and/or tumor cells.

After genetic modification, T cells can produce chimeric antigen receptors on their surface. CAR is a protein that allows T cells to recognize specific proteins (antigens) on tumor cells. Genetically engineered CAR T cells can grow in the laboratory until their number reaches billions. The expanded CART cells can then be infused into the patient.

Natural killer (natural killer, abbreviated NK) cells are an important part of the non-specific immune system, and the key mediator cells of the innate immune system. NK cells are a kind of broad-spectrum immune cells, which have the specific function of quickly discovering and destroying abnormal cells (such as cancer or virus-infected cells), and do not need to be sensitized or HLA matching in advance to display a powerful ability to dissolve abnormal cells. active. The use of immune cells (including NK cells) to treat cancer is a new trend in recent years. This new therapy is expected to provide new hopes for curing tumors that are ineffective against traditional surgery, chemotherapy and radiotherapy.

NKG2D is an activated receptor of NK cells, which can recognize MHC-I molecules and plays an important role in natural immunity. NKG2D is involved in the recognition of virus-infected cells and the killing of tumor cells by NK. NKG2D belongs to the C-type lectin-like receptor family, because the receptor encoded by this gene exists in the NKG2 (natural killer group 2) complex. The NKG2 gene complex is located on human chromosome 12. NKG2D is a type II transmembrane protein. NKG2D needs to bind some adaptor proteins to complete signal transduction through charged residues in the transmembrane region. Human NKG2D can bind 10kDa DNAX activating protein (DAP10). DAP10 contains YXXM motif (Tyr-X-X-Meth) in the cytoplasm which can recruit phosphatidylinositol trihydroxy kinase (PI3K) and growth factor receptor binding protein-2. All NK cells, most NKT cells, and macrophages express NKG2D. In addition, NKG2D also exists on the surface of CD8+ T cells. Under normal conditions, human and mouse CD4+ T cells do not express NKG2D. However, in patients, the proportion of CD4+ T cells expressing NKG2D has increased significantly. NKG2D can bind to many different ligands, which belong to major histocompatibility complex class I (MHC-I) related proteins. Another family of human NKG2D ligands are MIC-A and MIC-B. Both MIC-A and MIC-B are polymorphic. Currently, MIC-A has 61 alleles and MIC-B has 30 alleles. The ligands of NKG2D molecules are not expressed or expressed very little in normal cells, but when the cells are infected or become cancerous, the expression of these ligands will increase significantly.

Celyad used NKG2D's recognition of MICA and MICB to fuse NKG2D receptor with CD3ζ to construct NKG2D CAR (NKR-2). When NKR-2 transfected NK cells or CD8+T cells contact the NKG2D ligand on the tumor cells, they will be activated and kill the tumor cells. In October 2017, Celyad announced that the first CR (complete remission) occurred in patients with relapsed and refractory AML who were treated with NKR-2. The technical advantages of NKR-2CART include: 1) NKG2D is derived from the human body, and the NKG2D CAR-T formed is very low immunogenic, which prolongs the efficacy cycle of NKR-2; 2) NKG2D receptors can be recognized in most hematomas and Eight different ligands (MICA, MICB and ULBP1-6) expressed on the surface of solid tumor cells give NKR-2 a broad spectrum of tumor treatment; 3) Celyad's Phase I clinical trial results show that NKR-2 is in AML The safety and tolerability of the treatment of patients with MM and MM are good, indicating that although NKG2D has a wide variety of ligands, there is no obvious off-target effect.

Although NKR-2 has a good effect, it also has some shortcomings: 1) NKG2D is mainly expressed on NK cells, but the number of NK cells in the blood of tumor patients is small, and expansion is difficult, and it is difficult to produce NKG2D CAR-NK; 2 ) The expression level of NKG2D on CD8+T is low. Viral transfection can promote its expression on CD8+T. However, whether it is the expression of NKG2D or the activation of T cells after binding with ligand, it depends on intracellular DAP10. The concentration of NKR-2CAR-T will affect the efficacy of NKR-2CAR-T to a certain extent; 3) The number of CD4+T cells accounts for about 50% of the total number of T cells, but since NKG2D and DAP10 are not expressed on CD4+T, NKR- 2Not applicable to CD4+ T cells.

Summary of the invention

In view of these problems, the present invention optimizes and improves NKR-2. The improved NKG2D CAR-T (NKG92) only retains the extracellular 92-216 region (ligand binding region) of the NKG2D receptor, and is connected to the transmembrane domain and costimulatory signal transduction region through its C216 end to form a fusion protein. Exemplarily, the present invention forms a fusion protein by connecting the hinge-transmembrane (TM) region of CD8a, the costimulatory region of 4-1BB, and the signal region of CD3zeta. The data shows that NKG92 transfection can significantly increase the expression of NKG2D 92-216 on the cell membrane surface in both CD4+ T cells (NKG2D negative) and CD4+ T cells (NKG2D low expression); NKG92 transfected T cells can significantly kill A variety of tumor cells died, including hematoma and solid tumors. In view of this, the present invention provides a fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein, and preparation and application thereof. The cells can specifically recognize and kill tumors and have more efficient tumor killing activity.

One aspect of the present invention provides a fusion protein comprising NKG2D (92-216) or a homologous sequence thereof. Preferably, the fusion protein is a chimeric antigen receptor (CAR) and comprises an antigen binding domain , Transmembrane domain and intracellular signal transduction domain (preferably costimulatory signal transduction area), the antigen binding domain can specifically bind to tumor-specific antigen NKG2D ligand, and pass the transmembrane domain and intracellular signal The conduction domain (preferably a costimulatory signal conduction area) activates T cells, and the antigen-binding domain therein comprises or is NKG2D (92-216) or its homologous sequence (preferably with at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity and has binding NKG2D ligand Active homologous sequence).

Exemplarily, the NKG2D ligand is selected from one or more of major histocompatibility complex class I (MHC-I) molecules, MIC-A and MIC-B.

Optionally, the extracellular 92-216 region of the NKG2D receptor of the present invention is SEQ ID NO: 3 (preferably, its coding sequence is SEQ ID NO: 4), and optionally the extracellular 92-216 region The 216 region was replaced with the homology sequence of the ligand binding region.

Exemplarily, the transmembrane domain is selected from the group consisting of α, β or ζ chains of T cell receptors, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45 One or more transmembrane domains in the group consisting of CD80, CD86, CD134, CD137, CD152, CD154 and ICOS; preferably, the transmembrane domain is a CD8 transmembrane domain; and/or ,

The intracellular signal conduction domain (preferably a costimulatory signal conduction area) is selected from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, One or a combination of two or more of CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C, and B7-H3; preferably, the total The stimulus signal transduction region contains 4-1BB and CD3ζ intracellular domains.

Exemplarily, the structure of the fusion protein is NKG2D(92-216)-CD8αhinge-CD8TM-4-1BB-CD3ζ, and the fusion protein can specifically recognize NKG2D ligand.

Optionally, between the extracellular antigen binding domain and the transmembrane domain is a hinge domain, and preferably the hinge domain is derived from CD8α.

In some embodiments of the present invention, residue 216 of NKG2D (92-216) in the fusion protein (preferably CAR) of the present invention is connected as the C-terminus to the hinge domain or the N-terminus of the transmembrane domain. The homologous sequence of NKG2D (92-216) or its uncovered sequence is similar to the N-terminal connection of the hinge domain or transmembrane domain.

In some specific embodiments of the present invention, the ^{amino acid sequence of the NKG2D(92-216)-CD8αhinge-CD8 TM} -4-1BB-CD3ζ fusion protein is shown in SEQ ID NO:1 or its homologous sequence.

Exemplarily, the homology between the homologous sequence and the original sequence is about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% Or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% Or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% Or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above , 99.7% or more, 99.8% or more, or 99.9% or more.

Another aspect of the present invention provides a nucleotide sequence expressing the above-mentioned fusion protein.

Exemplarily, the nucleotide sequence is shown in SEQ ID NO: 2 or a degenerate sequence thereof.

Exemplarily, the homology between the degenerate sequence and the original sequence is about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% Or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% Or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% Or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above , 99.7% or more, 99.8% or more, or 99.9% or more.

Another aspect of the present invention provides a NKG2D CAR-T cell, which can express the above-mentioned fusion protein.

In a specific embodiment of the present invention, the fusion protein and/or the NKG2D CAR-T cell can effectively kill and/or kill lung cancer cells, breast cancer cells, colon cancer cells, breast cancer cells, and lung cancer cells Wait.

Preferably, the CART cells of the present invention are CD4+ T cells or a cell mixture containing CD4+ T cells and CD8+ T cells.

Another aspect of the present invention also provides a method for preparing the above-mentioned NKG2D CAR-T cell, which includes the following steps:

(1) Synthesize and amplify the NKG2D-CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion protein gene, and ^{clone the NKG2D-CD8αhinge-CD8 TM} -4-1BB-CD3ζ fusion protein gene onto a lentiviral expression vector;

(2) Use the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect 293T cells, package and prepare the lentivirus; and

(3) Use the lentivirus obtained in step (2) to infect NK-92 cells to obtain CAR-T cells.

The present invention also provides the application of the above-mentioned fusion protein and/or NKG2D CAR-T cells in the preparation of drugs for the treatment and/or prevention of cancer.

Exemplarily, the application of the fusion protein and/or NKG2D CAR-T cells in the preparation of drugs for the treatment of tumors and related diseases that highly express NKG2D ligands.

Exemplarily, the cancer is blood cancer, lymphoma, breast cancer, cervical cancer, colon cancer, or lung cancer.

The present invention also provides a pharmaceutical composition, which comprises the above-mentioned NKG2D CAR-T cell, and optionally, pharmaceutically acceptable excipients.

The invention provides a fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein. The fusion protein can specifically bind to the tumor-specific antigen NKG2D ligand, and activate the T cell through the transmembrane domain and the costimulatory signal transduction region. The NKG2D CAR-T cells are constructed by using NKG2D receptors for CAR-T cells. The fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein use NKG2D ligand as the target antigen, which can specifically kill tumor cells. It can be used as a therapeutic drug for tumor diseases and is used for tumors with high expression of NKG2D ligand. Treatment provides a new method for the prevention and treatment of tumors.

Description of the drawings

Figure 1 shows a schematic diagram of the CAR in the NKG92-CART structure provided by an embodiment of the present invention, where A is a structure containing a CD8a hinge-transmembrane (TM) domain and a 4-1BB co-stimulatory domain fused with the CD3ζ signaling region Schematic diagram of the structure of the NKG92-CAR construct of NKG2D 92-216; B is the structure of the NKG2D 92-216 homodimer; C is formed on the surface of transduced T cells through the conserved cysteine in the hinge domain of CD8a NKG92 homodimer.

Fig. 2 (Fig. 2A and Fig. 2B) shows the results of NKG2D CAR-NK flow cytometric detection of the positive rate of CAR cells provided by an embodiment of the present invention, wherein Fig. 2A is the control group; Fig. 2B is the experimental group.

Figure 3 (Figure 3A and Figure 3B) is a FACS analysis of NKG2D expression on the cell surface 5 days after the Lentiviral vector encoded by NKG2D-CAR was used to transduce T cells, where A is a graph and B is a histogram.

Figure 4 is a diagram of the experimental results of NKG2D CAR-T cells killing different tumor cells provided by the embodiments of the present invention, in which (from left to right) are respectively directed against myeloid leukemia cells K562, lymphoma raji, and breast cancer cells MDA-MB- 231. Experimental results of lung cancer cells A549, breast cancer BT474 cells and cervical cancer Hela.

Detailed ways

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those of ordinary skill in the technical field described in the present invention.

Specifically, the nucleotide sequence encoding the NKG2D-CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion protein of the present invention is any DNA sequence capable of encoding the fusion protein, preferably, the sequence is SEQ ID NO: 2 or its complementary sequence. On the other hand, the nucleotide sequence encoding the NKG2D-CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion protein of the present invention can be hybridized with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions. And the polynucleotide or its complementary sequence encoding the fusion protein;

The "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions, and high stringency conditions, and preferably high stringency conditions. Exemplarily, "low stringency conditions" may be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide; “medium stringency conditions” may be 40°C, 5×SSC, 5× The conditions of Denhardt solution, 0.5% SDS, 52% formamide; "high stringency conditions" can be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide. Those skilled in the art should understand that the higher the temperature, the more highly homologous polynucleotides can be obtained. In addition, those skilled in the art can select a comprehensive result formed by multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration that affect the stringency of hybridization to achieve the corresponding stringency.

In addition, the hybridizable polynucleotide can also be calculated by FASTA, BLAST and other homology search software with default parameters set by the system, which has about 60% or more of the polynucleotide encoding sequence number 6 , About 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or Above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or Above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or Polynucleosides of above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above identity acid.

For the identity of the nucleotide sequence, the algorithm rule BLAST of Karlin and Altschul can be used (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873, 1993) to make sure. Programs BLASTN and BLASTX based on BLAST algorithm rules have been developed (Altschul SF, et al: J Mol Biol 215:403, 1990). When using BLASTN to analyze the base sequence, if the parameters are score=100, wordlength=12; in addition, when using BLASTX to analyze the amino acid sequence, if the parameters are score=50, wordlength=3; when using the BLAST and Gapped BLAST programs, use each The system of the program can set default parameter values.

Unless otherwise specified, "encoding nucleotides" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The nucleotide sequence encoding the protein may include introns.

The term "lentivirus" refers to the genus of the Retroviridae family, which can effectively infect acyclic and post-mitotic cells; they can transmit a significant amount of genetic information into the host cell's DNA, so that they are the best gene delivery vector. One of the effective methods.

The term "promoter" is defined as a DNA sequence that is required to initiate specific transcription of a polynucleotide sequence and is recognized or guided by the synthetic machinery of the cell.

The term "specifically binds" refers to recognizing a specific antigen but not substantially recognizing or binding other molecules in the sample.

The term "carrier" is a composition of matter, which includes an isolated nucleic acid, and which can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

The term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, colorectal cancer, liver cancer, lung cancer, and so on.

As used herein, "comprising" is synonymous with "including", "containing" or "characterized by", and is inclusive or open-ended, and does not exclude additional unstated elements or method steps. Any expression of the term "comprising" herein, especially when describing the method, use or product of the present invention, should be understood to include essentially consisting of the components or elements or steps and consisting of the components or elements or The products, methods, and uses of the steps. The present invention exemplarily described herein can suitably be practiced in the absence of any one or more elements, one or more limitations that are not specifically disclosed herein.

The terms and expressions used herein are used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or parts thereof, but various modifications should be recognized It is possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can adopt modifications and variations of the concepts disclosed herein, and such modifications and variations are deemed to be as defined by the appended claims. Defined within the scope of the invention.

Although the present invention uses NKG2D (92-216) or its homologous sequence as the antigen binding domain in the examples of CAR, those skilled in the art will understand that NKG2D (92-216) or its homologous sequence can be further truncated Source sequence to prepare CAR-T cells that can kill tumors more efficiently. For example, 1-20 amino acid residues can be deleted in the amino acid sequence of NKG2D (92-216), or some amino acid residues can be replaced and modified.

The English names appearing in this article are not case-sensitive; NKG2D CAR-T and nkg2D-car T have the same meaning, which means CAR-T cells expressing NKG2D molecules; CD8 ^TM means transmembrane domain.

In order to illustrate the present invention more clearly, a detailed description will now be given in conjunction with the following embodiments, but these embodiments are merely exemplary descriptions of the present invention and cannot be construed as limiting the present application.

Example 1 Preparation of lentiviral vector

Gene synthesis NKG2D-CD8 ^TM -4-1BB-CD3ζ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 1, the gene sequence is shown in SEQ ID NO: 2, and its structure diagram is shown in Figure 1a, Figure 1b and Figures 1c) The NKG2D-CD8 ^TM -4-1BB-CD3ζ fusion gene sequence was transformed and connected to the PWPXLD-kana vector by restriction digestion, and the upstream of the gene was the EF-1α promoter. The vector is transformed into E. coli strain Stbl3, kanamycin is screened, a positive clone is obtained, the plasmid is extracted, and the clone is identified by restriction enzyme digestion to obtain the target gene transfection vector, that is, the PWPXLD plasmid vector containing the CAR gene fragment. The lentiviral packaging helper plasmids psPax2 and PMD2.0G were transformed into DH5α, ampicillin screened, and plasmids were extracted.

Example 2 Preparation of Lentivirus

Inoculate ^{10ml of 3x10 5} cells/ml 293T cells in a new 10cm petri dish, and prepare the following solution after 24h:

CaCl ₂ : 2.5M;

2×BBS: 50mM BES, 280mM NaCl, 1.5mM Na ₂ HPO4;

Plasmid system: PWPXLD plasmid vector containing CAR gene fragment: 9μg; psPax2: 9μg; and PMD2.0G: 4.5μg.

Add the plasmid in the plasmid system to 0.45 mL sterile ddH ₂ O, add 50 μL CaCl ₂ solution; add 500 μL 2×BBS dropwise and keep the solution vortexed; leave it at room temperature for 15-30 minutes.

Add the mixed liquid to the medium, gently mix the medium to make the transfection system evenly distributed, and replace the 2% FBS DMEM medium after 18 hours. After 48 hours, the culture supernatant was collected, and the supernatant was centrifuged at 1000 xg for 10 min to remove cell debris, and filtered with a 0.45 μm filter. The filtered virus solution was added to one-third volume of the virus solution TAKR lentivirus concentration reagent concentrator, inverted and mixed, and then allowed to stand overnight at 4°C. After overnight, the virus solution was centrifuged at 4°C at 1500g 45min, the supernatant was discarded, resuspended in PBS and aliquoted, and stored at -80°C.

Example 3 Preparation of NKG2D CAR-T cells

1. Prepare Ficoll-PaquePlus with the same volume as the peripheral blood in the centrifuge tube and let it stand at room temperature;

2.【Day 0】Peripheral blood collection;

3. Immediately add the collected peripheral blood to the centrifuge tube along the tube wall. Centrifuge at 840g for 20 minutes at 20°C; set the lifting speed to the slowest one;

4. Place the plasma components in a new sterile test tube and inactivate at 56°C for 30 minutes;

5. Wash PBMC twice with normal saline, centrifuge at 500g for 7 minutes at 20°C;

6. Centrifuge inactivated plasma, 4000rpm, 20℃, 6min, transfer, 4℃ storage;

7. Resuspend PBMC in 4ml of normal saline and count, centrifuge at 400g for 7 minutes at 4℃;

8. Configure sorting buffer: 40mM EDTA (1ml), PBS (38ml), 20% BSA (1ml); pre-cool at 4℃;

9. Discard the supernatant, 40μL/10 ⁷ cells plus buffer, 10μL/10 ⁷ cells plus Antibody Cocktail, mix well at 4℃, and incubate for 5min;

10. 30μL/10 ⁷ cells plus buffer and 20μL/10 ⁷ cells plus Microbead Cocktail, mix well and incubate at 4℃ for 10min;

11. T cell culture medium preparation: culture medium (MACS); cytokine (IL-2 (200U/ml)); plasma (5% inactivated);

12. Put the LS column on the magnetic separator and add 3mL buffer;

13. Pass PBMC through LS column, wash with 3mL buffer, and collect unbound remaining liquid;

14. 300g, 7min, centrifuge;

15. Discard the supernatant, resuspend the medium, count, and calculate the recovery rate;

16. Add 1×10 ⁶ pcs/ml to the 12-well plate;

17. Add T cell stimulating factor (dynabeads magnetic beads with buffer removed), cell: magnetic beads = 1:2;

18. Cultivate at 37°C and 5% CO ₂ ; test for bacterial culture of blood samples and cell culture fluid;

19. Pre-lay a 12-well plate and add fibronectin solution diluted with saline to a final concentration of 5μg/cm ² at 4°C overnight;

20.【Day 1】Lentivirus infects T lymphocytes;

21. Preparation of blocking solution: 20% BSA + normal saline = 2% BSA;

22. Discard the fibronectin solution in the well plate, and seal with blocking solution for 30 minutes;

23. Wash the board with physiological saline once;

24. Preparation of virus liquid: 750μl/4.5cm ² , plating;

25. 37℃, let stand for 4-6 hours;

26. Take T cells, ^{add 1x10 6} /mL to the well plate; culture at 37°C, 5% CO _2;

27. [Day 3] Record the state of T cells, adjust the T cell concentration to 5×10 ⁵ /ml, and replace all with fresh medium;

28. [Day 5] Record the T cell status, adjust the T cell concentration to 5×10 ⁵ /ml, and use FACS analysis to detect the CAR expression of CAR-T cells (see Figure 3A and Figure 3B);

29. LDH in vitro cytotoxicity test paving;

30. Cell culture fluid bacteria, fungi, endotoxin, mycoplasma detection experiment;

31. [Day 6] Record the state of T cells and adjust the concentration of T cells to 5×10 ⁵ /ml;

32. LDH in vitro cytotoxicity test results detection and data analysis;

33. [Day 8] Record the state of T cells, adjust the T cell concentration to 5×10 ⁵ /ml, use FACS analysis to detect the number of CD4+T and CD8+T cells on the surface of CAR-T cells, see Figure 2, where Figure 2A And 2B are the number of CD4+T and CD8+T cells before the infection (day 0) and after the infection (day 8), respectively.

Example 4 Evaluation of the killing effect of NKG2D CAR-T cells on tumors in vitro

LDH test method

The "medium" components mentioned below are all MACS+200U/mL cytokine interleukin-2, LDH=lactate dehydrogenase, and the special reaction reagents are all from the CytoTox96 kit produced by Promega.

1: Take 4 mL of target cells and CAR T cells that have grown to Day 5 and add them to a 5 mL flow tube respectively, and centrifuge at 300 g, 3 min, and 20°C;

2: Discard the supernatant, resuspend the cells in 4mL medium, centrifuge 300g, 3min, 20℃; repeat once

3: Discard the supernatant, resuspend the cells in 4 mL of medium, count them, adjust the target cell concentration to 2×10 ⁵ /mL with the medium, and adjust the CAR T cells to 4×10 ⁵ /mL;

4: Set 200 microliters of medium as the blank group, 100 microliters of CAR T cells + 100 microliters of medium for spontaneous group A, take 100 microliters of target cells + 100 microliters of medium for spontaneous group B, and 100 microliters of CAR T cells + 100 microliters of target cells are the experimental group; another 200 microliters of medium is set as the volume correction group, and 100 microliters of target cells + 100 microliters of medium is the maximum release group; each group has 3 parallel data points; set separately Incubate in a 96-well U-shaped plate at 37°C and 5% CO ₂ for 24 hours;

5: Detection of maximum release: add 20 microliters of LDH maximum release reagent to each well in the maximum release group and volume correction group, respectively, at 37°C, 5% CO ₂ , and incubate for 45 min;

6: Preparation of LDH detection reagents: take LDH buffer, fully dissolve at 37°C, add 12ml to the LDH detection reagent bottle, and mix well in the dark;

7: Take out the 96-well U-shaped plate, mix the wells, and centrifuge at 500g for 3min;

8: Take an ELISA test 96-well plate, and add 50 μl/well of LDH test reagent in the dark;

9: Take 50 microliters/well from the centrifuged 96-well U-shaped plate and add it to the corresponding ELISA test 96-well plate; (do not suck the cells) (protect from light);

10: Put the 96-well ELISA test plate into the microplate reader, shake and mix for 30 minutes;

11: Add 50 μl/well of LDH reaction termination reagent to the 96-well ELISA test plate in the dark;

12: Put the 96-well ELISA test plate into the microplate reader, shake for 10s and then measure the wavelength at 492nm, exclude 620nm above, and record the reading;

13: Calculate the kill rate: kill rate = (experimental group-spontaneous group A-spontaneous group B + blank group) / (maximum release value-volume correction value-spontaneous group B + blank group)

14: Graphical analysis, as shown in Figure 4.

It can be clearly seen from Figure 4 that the CART of the present invention can have a significant curative effect or inhibitory effect on myeloid leukemia, breast cancer, lung cancer, breast cancer and cervical cancer.

The foregoing descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. within.

Claims

The fusion protein is characterized in that it comprises NKG2D (92-216) or its homologous sequence. Preferably, the fusion protein is a chimeric antigen receptor (CAR) and comprises an antigen binding domain, a transmembrane domain and Intracellular signaling domain (preferably a costimulatory signaling domain), the antigen binding domain can specifically bind to tumor-specific antigen NKG2D ligand, and through the transmembrane domain and intracellular signaling domain (preferably co-stimulatory) Stimulating signal transduction region) activates T cells, and wherein the antigen binding domain comprises or is NKG2D (92-216) or its homologous sequence (preferably with at least 80%, at least 81%, at least 82%, at least 83%). %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical and having homologous sequences that bind NKG2D ligand activity) .
The fusion protein of claim 1, wherein the NKG2D ligand is selected from the group consisting of major histocompatibility complex class I (MHC-I) molecules, MIC-A and MIC-B.
The fusion protein of claim 1 or 2, wherein the transmembrane domain is selected from the group consisting of α, β or ζ chains of T cell receptors, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16 One or more transmembrane domains of CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS; preferably, the transmembrane domain Is the CD8 transmembrane domain; and/or,

The intracellular signal conduction domain (preferably a costimulatory signal conduction area) is selected from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, One or a combination of two or more of CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3; preferably, the total The stimulating signal transduction region contains 4-1BB and CD3ζ intracellular domains; and/or,

The amino acid sequence of NKG2D (92-216) includes or is the sequence shown in SEQ ID NO: 3 or its homologous sequence; and/or

Between the extracellular antigen binding domain and the transmembrane domain is a hinge domain, and preferably the hinge domain is derived from CD8α.
The fusion protein according to claim 3, characterized in that its structure is NKG2D-CD8αhinge-CD8 TM -4-1BB-CD3ζ, wherein the NKG2D can specifically bind to tumor-specific NKG2D ligands, preferably, The NKG2D ligand is one or more of major histocompatibility complex class I (MHC-I) molecules, MIC-A and MIC-B.
The fusion protein according to claim 4, wherein the amino acid sequence of the NKG2D-CD8 TM α hinge-CD8-4-1BB-CD3ζ fusion protein comprises or is the sequence shown in SEQ ID NO:1 or its homologous sequence ( It is preferably at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% , At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or Homologous sequences with at least 99.9% identity and CAR activity).
A nucleotide sequence encoding the fusion protein of any one of claims 1-5.
The nucleotide sequence of claim 6, wherein the nucleotide sequence is shown in SEQ ID NO: 2 or its degenerate sequence.
The isolated NKG2D CAR-T cell is characterized in that the NKG2D CAR-T cell can express NKG2D (92-216) or its homologous sequence (preferably, the NKG2D The amino acid sequence of (92-216) is shown in SEQ ID NO: 3; more preferably, its coding sequence includes or consists of the sequence shown in SEQ ID NO: 4), or expresses any one of claims 1-5 The fusion protein, or express the fusion protein encoded by the nucleotide sequence of claim 6 or 7, preferably, the NKG2D CAR-T cells are CD4+ T cells or contain CD4+ T cells and CD8 A cell mixture of +T cells, wherein the ratio of CD4+T cells to CD8+T cells in the cell mixture is 1:2-2:1, preferably 1:1.5-1.5:1, more preferably 1:1.
The fusion protein of any one of claims 1 to 5, and/or the fusion protein encoded by the nucleotide sequence of claim 6 or 7, and/or the NKG2D CAR-T cell of claim 8, It can effectively kill and/or kill lung cancer cells, breast cancer cells, colon cancer cells, breast cancer cells, cervical cancer cells, lymphoma cancer cells and/or lung cancer cells.
The method for preparing NKG2D CAR-T cells according to claim 8, which comprises the following steps:

(1) Synthesize and amplify the nucleotide encoding the NKG2D-CD8αhinge-CD8 TM -4-1BB-CD3ζ fusion protein of any one of claims 4 or 5 or 7, and clone the nucleotide into Lentiviral expression vector;

(2) Use the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect cells, package and prepare the lentivirus; and

(3) Infect T-92 cells with the lentivirus obtained in step (2) to obtain CAR-T cells.
A pharmaceutical composition comprising the fusion protein according to any one of claims 1-5, and/or the fusion protein encoded by the nucleotide sequence of claims 6-7, and/or the right The NKG2D CAR-T cell according to claim 8, and/or the NKG2D CAR-T cell prepared according to claim 10.
The pharmaceutical composition of claim 11, further comprising pharmaceutically acceptable excipients.
The fusion protein of any one of claims 1-5, and/or the fusion protein encoded by the nucleotide sequence of claims 6-7, and/or the NKG2D CAR-T of claim 8 Cells, and/or use of the NKG2D CAR-T cells prepared in claim 10 in the preparation of drugs for the treatment and/or prevention of cancer; preferably, the cancers are tumors and related diseases that highly express NKG2D ligands.
The use according to claim 13, wherein the cancer is lung cancer, breast cancer, blood cancer, lymphoma, cervical cancer, or colon cancer.