CN116355859B - HER2 positive tumor targeting CAR-T containing SHP 2N-SH 2 domain, preparation method and application - Google Patents

Abstract

The present invention belongs to the field of cell biology, and specifically relates to a CAR-T targeting HER2-positive tumors containing SHP2 N-SH2 domain and its preparation method and application, which are used to alleviate the ineffectiveness of solid tumors with HRE2-positive expression in existing studies. good question. The CAR-T extracellular domain of the present invention is a nanobody targeting HER2-positive tumors, and the intracellular domain includes a SHP2 protein domain; the SHP2 protein domain is an N-SH2 domain, and its nucleotide sequence is as shown in SEQ ID NO: 1.

Description

CAR-T targeting HER2-positive tumors comprising SHP2 N-SH2 domain and its preparation Methods and Applications

technical field

The invention belongs to the field of cell biology, and in particular relates to a CAR-T targeting HER2-positive tumors comprising a SHP2 N-SH2 domain, a preparation method and an application thereof.

Background technique

Human Epidermal Growth Factor Receptor 2 (HER2) is a cell-derived proto-oncogene, also known as c-erbB-2 gene, which is overexpressed and amplified in a variety of tumors. Excessive HER2 protein on the surface of tumor cells will promote the division and growth of tumor cells, and then form HER2-positive tumors. HER2 overexpression is common in breast cancer, gastric cancer, intestinal cancer and other tumors.

CAR-T cell (CAR-T for short) therapy (Chimeric Antigen Receptor T-Cell Immunotherapy) is called chimeric antigen receptor T cell immunotherapy, which is a new type of precise targeted therapy for tumor treatment. Studies have found that CAR-T cells can accurately, quickly, efficiently and specifically recognize and kill tumor cells. In recent years, through optimization and improvement, they have achieved good results in assisting the treatment of tumors. It is a very promising and potentially curable New tumor immunotherapy research direction for cancer. However, there are still controversies about the effect of CAR-T therapy on different tumors, especially in solid tumors. Solid tumors are a challenging target for CAR-T therapy.

Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2) is a dephosphorylase that belongs to the protein tyrosine phosphatase family. The full length of SHP2 consists of two SH2 domains (N-SH2 and C-SH2), a conserved PTP domain and a flexible C-terminal tail. The complete SHP2 phosphatase activity is regulated by its own conformational changes. In the ground state, the N-SH2 domain binds to the PTP domain and directly blocks its active site, maintaining a self-inhibitory conformation; when the signal protein of SHP2 is activated by the signal of tumor cells After activation, the upstream receptor tyrosine kinase (Receptor Tyrosine Kinase, RTK) will trigger the pTyr (phosphorylated tyrosine) residue on PD1 to bind to the two SH2 domains of SHP2, and the PTP domain will be released from the self-inhibited state Released to transmit and activate PD1 downstream signals, thereby inhibiting T cell activity.

In summary, the present invention will provide a structurally improved novel CAR-T that specifically targets HER2-positive tumor cells to supplement the deficiencies of existing research.

Contents of the invention

In view of this, the main purpose of the present invention is to provide a CAR-T targeting HER2-positive tumors comprising the SHP2 N-SH2 domain and its preparation method and application. The specific technical scheme is as follows.

A CAR-T targeting HER2-positive tumors, the chimeric antigen receptor part includes an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain is a nanobody targeting HER2-positive tumors (referred to as HER2 nanobody for short), the intracellular domain includes a SHP2 protein domain; the SHP2 protein domain is an N-SH2 domain, and its nucleotide sequence is shown in SEQ ID NO:1.

Further, the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO:2.

Further, the intracellular domain of the chimeric antigen receptor also includes 4-1BB co-stimulatory molecule and CD3ζ truncated signal domain, wherein CD3ζ is preferably truncated and only contains ITAM1 part.

Further, the chimeric antigen receptor also includes a CD8 hinge region.

Further, the CAR-T expresses any one of the chimeric antigen receptors described above.

The application of the above-mentioned CAR-T in the preparation of anti-tumor drugs, the SHP2 N-SH2 domain in the CAR-T is used to competitively bind to PD-1 and hinder the transmission of PD-1 and/or PD-L1 immunosuppressive signals .

Further, the tumor is a solid tumor with positive expression of HER2.

Further, the solid tumor includes ovarian cancer, breast cancer, gastric cancer or intestinal cancer.

The preparation method of the above-mentioned CAR-T comprises the following steps:

1) Synthesize the CAR structural sequence including nanobody targeting HER2-positive tumors, CD8 hinge region, transmembrane region, 4-1BB co-stimulatory molecule, CD3ζ truncated signal domain and SHP2 N-SH2 domain;

2) Double-digest the plasmid vector and the synthesized CAR structure with restriction endonuclease, connect with T4 ligase, perform transformation and screening culture, and sequence and identify the obtained positive clones;

3) Combine the synthetic CAR with T cells.

Further, the sequences of primers for amplifying the SHP2 N-SH2 domain are shown in SEQ ID NO:3 and SEQ ID NO:4.

Beneficial technical effect

The present invention provides a novel CAR-T targeting HRE2-positive tumor cells with improved structure. The SHP2 N-SH2 domain in the CAR-T structure of the present invention can be used to competitively bind PD-1, hindering PD-1 and / or the transmission of PD-L1 immunosuppressive signals, thereby maintaining the immune activity state of T lymphocytes, and maintaining the killing effect on HER2-positive tumor cells.

Secondly, although current studies have shown that the pTyr residue on PD1 can bind to the two SH2 domains of SHP2. However, the experiment of the present invention proves that a single SHP2 N-SH2 fragment can not exert ideal anti-tumor efficacy, which is characterized in that when the effect-to-target ratio E:T=1:1 (E represents Effector cell, i.e. effector cell; T represents Target cell , that is, tumor cells), after 24 hours of continuous killing, the CAR-T cells prepared by the present invention can already see the obvious effect of killing/killing tumors, while the single SHP2 N-SH2 fragment has almost no anti-tumor effect Effect. It can be seen that the CAR-T cells prepared by the present invention have great potential in the field of immunotherapy.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

Figure 1 is a schematic diagram of the CAR structure (Her2 VH-BBZ _(short) -P2A-SHP2 N-SH2) synthesized by the present invention;

Fig. 2 is a result graph of the positive expression rate of CAR synthesized by the present invention (APC is a fluorescent dye, and H represents height);

Figure 3 is the effect diagram of CAR-T cell killing (LDH) in vitro;

Figure 4 is an effect diagram of the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:1;

Figure 5 is a graph showing the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:1;

Figure 6 is an effect diagram of the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:2.5;

Figure 7 is a graph showing the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:2.5;

Figure 8 is an effect diagram of the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:5;

Figure 9 is a graph showing the continuous killing of tumor cells and CAR-T cells at an effect-to-target ratio of 1:5.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of the range 1 to 6 should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc. , and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the scope.

Example 1

This embodiment provides a method for preparing CAR-T cells.

1. Construction of CAR expression vector:

1) The CAR structure is composed of extracellular HER2 nanobody, CD8 hinge region, transmembrane region, intracellular 4-1BB co-stimulatory molecule, CD3ζ truncated signal domain and SHP2 N-SH2 domain, and the structural fragments are prepared by gene synthesis .

2) Double-digest the Pwpxld vector and the CAR structural fragment with restriction endonucleases EcoR I and BamH I respectively, connect with T4 ligase, transform and screen for culture, sequence and identify the positive clones obtained, and finally obtain HER2 CAR-Pwpxld expression vector and SHP2 N-SH2-Pwpxld control vector (the control vector only contains SHP2 N-SH2 fragment). The full name of the CAR synthesized in the present invention is Her2 VH-BBZ _(short) -P2A-SHP2 N-SH2, and its structure is shown in Figure 1.

The sequences of the fragments synthesized/amplified in this example are shown in Table 1.

Table 1

2. Lentiviral packaging:

1) Resuscitate and culture 293T cells, and carry out virus packaging when the confluence reaches about 80%.

2) Add chloroquine solution according to the amount of medium (DMEM+10%FBS), so that the final concentration is 100 μmol/L.

3) Virus packaging system (10cm dish): 50μl CaCl ₂ , 20μg constructed core plasmid, 10μg Pspax2, 5μg PMD2.0G, 500μl Nuclease-free water (nuclease-free water), 500μl 2X BBS.

4) Replace with fresh pre-warmed medium (DMEM+10%FBS) in about 10 hours.

5) Collect and culture the virus fluid for 48h and 72h, and filter with a 0.22μm filter to remove impurities.

6) Ultracentrifuge, resuspend the concentrated virus with 1/400 DPBS of the virus stock solution, aliquot according to the subsequent dosage, and freeze at -80°C for later use.

3. Peripheral blood cell separation:

1) Incubate the T cell culture square flask (T25) one day in advance and incubate overnight at 4°C. T cell medium containing anti-human CD3 and CD8D monoclonal antibodies: 3ml DPBS, 4μg/mL CD3, 4μg/mL CD28.

2) Take appropriate amount of fresh blood from healthy volunteers.

3) Take a 15mL BD tube, add 5mL Ficoll, and slowly add 5mL fresh blood.

4) Centrifuge at room temperature, increase by 1 and decrease by 1, 1000g, 30min.

5) After centrifugation, carefully aspirate the peripheral blood lymphocytes in the middle layer (white).

6) Add three times the volume of pre-warmed DPBS to wash, 300g, 10min.

7) Optionally add 5mL red blood cell lysate, lyse at room temperature for 5min, and add an equal volume of pre-warmed DPBS.

8) Cell counting, centrifugation at room temperature, 100g, 10min.

9) Add an appropriate amount of T cell culture medium (X-vivo, 5% AB serum, 400IU IL2) at 2*10^6/mL to resuspend the cells.

10) Aspirate the antibody incubation solution in the T25 square bottle, add T cells resuspended in T cell culture medium, and culture at 37°C for about 48 hours.

4. Preparation of CAR-T cells:

1) Incubate the T cell culture plate one day in advance and incubate overnight at 4°C. Using a 24-well plate, add 0.5ml DPBS and 10μl RetroNectin (1mg/mL) to each well.

2) Aspirate the RetroNectin solution.

3) Add 1 mL of 2% BSA solution to each well, and block at 37°C for 30 minutes.

4) Aspirate the blocking solution and wash once with DPBS.

5) Add 200 μl 400X virus and 300 μl X-vivo complete medium to each well (non-adhesive well plate), centrifuge at 1000 g for 2 hours at 32°C.

6) Add 2-2.5*10^6 T cells activated for about 48 hours to each well, make up to 1 mL/well with complete X-vivo medium, and centrifuge at 32°C for 15 minutes at 300 g.

7) After normal culture at 37°C for about 72 hours, the positive expression rate of CAR was detected by flow cytometry, as shown in Figure 2. The results showed that the positive expression rate of the CAR constructed in the present invention was 75.7%.

Example 2

This example provides an in vitro killing assay.

1) 96-well plate killing: tumor cells 1*10^4/well, using the CAR-T cells constructed by the present invention and control T cells containing only SHP2 N-SH2, respectively according to the effect-to-target ratio of tumor cells 1:1 , 2.5:1, and 5:1 were plated, and each effect-target ratio was replicated in 3 wells, and blank T cells (Mock) were used as controls.

This example uses the Skov3-mCherry cell line, which is a widely representative cell model.

2) Continuously detect the killing status of CAR-T cells in vitro using a living cell dynamic detector.

3) Take the culture supernatant at about 24 hours to detect LDH to calculate the tumor lysis rate.

The experimental results are shown in Figure 3-Figure 9.

Figure 3 shows that as the effector-target ratio increases, the tumor lysis rate gradually increases, indicating that CAR-T cells exert a greater killing effect as the effector-target ratio increases.

Figure 4-Figure 9 shows the serial killing effect under different effect-to-target ratios. It can be seen from this that even in the effect-to-target ratio (also known as the E/T ratio), when E:T=1:1 (E stands for Effector cell, that is, effector cell; T stands for Target cell, that is, tumor cell), in After 24 hours of continuous killing, the number of tumor cells in the field of view was also significantly less than that of the control group. It shows that the CAR-T cells constructed in the present invention have obvious killing effect on HER2-positive tumor cells; while the control alone SHP2 N-SH2 T cells have almost no anti-tumor effect. In addition, the CAR-T of the present invention can maintain the killing effect for 48 hours continuously, indicating that its effect has better persistence.

Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.

Claims (4)

1. A CAR-T targeting a HER2 positive expressing solid tumor, the chimeric antigen receptor portion of which comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain is a nanobody targeting a HER2 positive expressing solid tumor, and the intracellular domain comprises a synthetic SHP2 protein domain; the synthesized SHP2 protein structural domain is an N-SH2 structural domain, and the nucleotide sequence of the synthetic SHP2 protein structural domain is shown as SEQ ID NO. 1; the intracellular domain of the chimeric antigen receptor further comprises a 4-1BB costimulatory molecule and a CD3ζ truncated signaling domain; the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 2.

2. Use of the CAR-T of claim 1 for the preparation of an anti-solid tumor drug, wherein the SHP 2N-SH 2 domain in the CAR-T is used to competitively bind to PD-1, blocking the transmission of PD-1 and/or PD-L1 immunosuppressive signals.

3. The use of claim 2, wherein the solid tumor comprises ovarian, breast, gastric or intestinal cancer.

4. The method of preparing CAR-T according to claim 1, comprising the steps of:

1) Synthesizing a CAR structural sequence comprising a nanobody targeting a HER2 positive expressing solid tumor, a CD8 hinge region, a transmembrane region, a 4-1BB co-stimulatory molecule, a CD3 ζ truncated signal domain, and a SHP 2N-SH 2 domain, wherein the primer sequence for amplifying the SHP 2N-SH 2 domain shown in SEQ ID NO:1 is as set forth in SEQ ID NO:3 and SEQ ID NO:4 is shown in the figure;

2) Respectively carrying out double enzyme digestion on a plasmid vector and a synthesized CAR structure sequence by using restriction enzymes, carrying out transformation and screening culture after being connected by using T4 ligase, and carrying out sequencing identification on the obtained positive clone;

3) Expressing the CAR structural sequence synthesized in step 2) with T cells.