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

Abstract

The invention belongs to the field of cell biology, and particularly relates to a HER2 positive tumor targeting CAR-T containing an SHP 2N-SH 2 structural domain, a preparation method and application thereof, which are used for relieving the problem of poor curative effect of HRE2 positive expressed solid tumors in the existing research. The CAR-T extracellular domain is a nanobody targeting HER2 positive tumor, and the intracellular domain comprises an SHP2 protein domain; the SHP2 protein structural domain is an N-SH2 structural domain, and the nucleotide sequence of the SHP2 protein structural domain is shown as SEQ ID NO. 1.

Description

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

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a HER2 positive tumor targeting CAR-T containing an SHP 2N-SH 2 structural domain, a preparation method and application thereof.

Background

Human epidermal growth factor receptor 2 (Human Epidermal Growth Factor Receptor, HER 2) is a cell-derived proto-oncogene, also known as the c-erbB-2 gene, that is overexpressed and amplified in a variety of tumors. The presence of excessive HER2 protein on the surface of tumor cells promotes the division and growth of tumor cells, thereby forming HER2 positive tumors. HER2 overexpression is common in tumors such as breast cancer, gastric cancer, intestinal cancer, and the like.

CAR-T cell (CAR-T for short) therapy (Chimeric Antigen Receptor T-Cell Immunotherapy) is fully called chimeric antigen receptor T cell immunotherapy, and is a novel accurate targeted therapy for treating tumors. The research shows that the CAR-T cells can accurately, quickly, efficiently and specifically identify and kill the tumor cells, and in recent years, the CAR-T cells have good effect on assisting in the treatment of tumors through optimization and improvement, so that the CAR-T cells are a very promising research direction for novel tumor immunotherapy which is possible to cure cancers. However, CAR-T therapies are currently still controversial in the effects of different tumors, especially solid tumors, which is a challenging goal of CAR-T therapies to overcome.

Src homology 2 domain protein tyrosine phosphatase (SHP 2) is a dephosphorylase belonging to the protein tyrosine phosphatase family. The full length of SHP2 consists of two SH2 domains (N-SH 2 and C-SH 2), a conserved PTP domain and a flexible C-terminal tail. The activity of the complete SHP2 phosphatase is regulated and controlled by the change of the conformation of the complete SHP2 phosphatase, and the N-SH2 domain is combined with the PTP domain in the ground state and directly blocks the active site of the complete SHP2 phosphatase, so that the complete SHP2 phosphatase maintains a self-inhibition conformation; when the signaling protein of SHP2 is activated by the signal of tumor cells, the upstream receptor tyrosine kinase (Receptor Tyrosine Kinase, RTK) triggers pTyr (phosphorylated tyrosine) residues on PD1 to bind to both SH2 domains of SHP2, releasing the PTP domains from the self-inhibited state, transmitting the downstream signal of activated PD1, thereby inhibiting T cell activity.

In summary, the present invention will provide a novel CAR-T with improved structure that specifically targets HER2 positive expressing tumor cells to complement the shortcomings of the current studies.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide a HER2 positive tumor targeting CAR-T comprising a SHP 2N-SH 2 domain, and a preparation method and application thereof, and the specific technical scheme is as follows.

A HER2 positive tumor-targeting CAR-T, the chimeric antigen receptor portion of which comprises an extracellular domain, a transmembrane domain and an intracellular domain, the extracellular domain being a nanobody (abbreviated as HER2 nanobody) that targets a HER2 positive tumor, the intracellular domain comprising a SHP2 protein domain; the SHP2 protein structural domain is an N-SH2 structural domain, and the nucleotide sequence of the SHP2 protein structural domain is shown as SEQ ID NO. 1.

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

Further, the intracellular domain of the chimeric antigen receptor also includes a 4-1BB costimulatory molecule and a CD3 zeta truncating signal domain, wherein CD3 zeta is preferably truncated and comprises only the ITAM1 moiety.

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

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

The application of the CAR-T in preparing an anti-tumor drug, wherein the SHP 2N-SH 2 domain in the CAR-T is used for competitively combining with PD-1 to block the transmission of PD-1 and/or PD-L1 immunosuppression signals.

Further, the tumor is a solid tumor that HER2 expresses positively.

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

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

1) Synthesizing a CAR structural sequence comprising a nanobody targeting a HER2 positive tumor, a CD8 hinge region, a transmembrane region, a 4-1BB costimulatory molecule, a CD3 zeta truncated signal domain, and a SHP 2N-SH 2 domain;

2) Respectively carrying out double enzyme digestion on the plasmid vector and the synthesized CAR structure 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) The synthesized CAR was bound to T cells.

Further, the primer sequences for amplifying the SHP 2N-SH 2 domain are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

Beneficial technical effects

The invention provides a novel structure-improved CAR-T targeting HRE2 positive tumor cells, and the SHP 2N-SH 2 structural domain in the CAR-T structure can be used for competitively combining with PD-1 to block the transmission of PD-1 and/or PD-L1 immunosuppression signals, so that the immunocompetence state of T lymphocytes is maintained, and the tumor cells positively expressed by HER2 are continuously kept to have a killing effect.

Second, although studies have now shown that the pTyr residues on PD1 can bind to both SH2 domains of SHP 2. However, the experiment proves that the single SHP 2N-SH 2 fragment cannot exert ideal anti-tumor efficacy, and is characterized in that when the effective Target ratio E is T=1:1 (E represents Effector cell, namely Effector cell; T represents Target cell, namely tumor cell), after 24 hours of continuous killing, the CAR-T cell prepared by the invention can show obvious effect of killing/killing tumor, and the single SHP 2N-SH 2 fragment hardly exerts anti-tumor effect. It can be seen that the CAR-T cells prepared by the method have great potential in the field of immunotherapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 shows the synthesized CAR structure of the present invention (Her 2 VH-BBZ _(short) -P2A-SHP 2N-SH 2) schematic;

FIG. 2 is a graph showing the results of the positive expression rate of the synthesized CAR according to the present invention (APC is a fluorescent dye, H represents height);

FIG. 3 is a graph of the in vitro killing (LDH) effect of CAR-T cells;

FIG. 4 is a graph showing the effect of continuous killing of tumor cells with CAR-T cells at an effective target ratio of 1:1;

FIG. 5 is a graph of statistical tumor cell to CAR-T cell continuous killing at an effective target ratio of 1:1;

fig. 6 shows that tumor cells and CAR-T cells were at 1:2.5, an effect graph of continuous killing condition under the effective target ratio;

fig. 7 is a graph of statistical tumor cells versus CAR-T cells at 1:2.5, a plot of continuous killing at an effective target ratio;

fig. 8 shows that tumor cells and CAR-T cells were at 1:5, an effect graph of continuous killing condition under the effective target ratio;

fig. 9 is a graph of statistical tumor cells versus CAR-T cells at 1: graph of continuous killing at effective target ratio of 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

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

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered as having specifically disclosed sub-ranges 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., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Example 1

The present example provides a method of preparing a CAR-T cell.

Construction of car expression vector:

1) The CAR structure consists of extracellular HER2 nanobody, CD8 hinge region, transmembrane region, intracellular 4-1BB costimulatory molecule, CD3 zeta truncated signal domain and SHP 2N-SH 2 structural domain, and the structural fragment is prepared by adopting a gene synthesis method.

2) The restriction endonucleases EcoR I and BamH I are used for respectively carrying out double digestion on the Pwpxld vector and the CAR structural fragment, T4 ligase is used for connection, transformation and screening culture are carried out, and the obtained positive clone is subjected to sequencing identification, so that the HER2 CAR-Pwpxld expression vector and the SHP 2N-SH 2-Pwpxld control vector (the control vector only contains the SHP 2N-SH 2 fragment) are finally obtained. The CAR synthesized by the invention is totally called Her2 VH-BBZ _(short) P2A-SHP 2N-SH 2, the structure of which is shown in FIG. 1.

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

TABLE 1

2. Lentivirus packaging:

1) And recovering and culturing 293T cells, and packaging the viruses when the fusion degree reaches about 80%.

2) Chloroquine solution was added in the amount of medium (DMEM+10% FBS) to give a final concentration of 100. Mu. Mol/L.

3) The virus packaging system (10 cm dish) is: 50 μl CaCl ₂ 20. Mu.g of the constructed core plasmid, 10. Mu.g of Pspax2, 5. Mu.g of PMD2.0G, 500. Mu.l of nucleic-free wafer (Nuclease free water), 500. Mu.l of 2 XBBS.

4) Fresh pre-warmed medium (dmem+10% fbs) was changed for about 10 h.

5) The virus liquid cultured for 48 hours and 72 hours is collected, and the impurities are removed by filtration through a 0.22 mu m filter membrane.

6) Ultracentrifugation, re-suspending concentrated virus with DPBS 1/400 of virus stock, and sub-packaging at-80deg.C for use.

3. Peripheral blood cell separation:

1) T cell culture flasks (T25) were incubated one day in advance and overnight at 4 ℃. T cell culture medium contains anti-human CD3 and CD8D monoclonal antibodies: 3mL DPBS, 4. Mu.g/mL CD3, 4. Mu.g/mL CD28.

2) An appropriate amount of fresh blood was drawn from healthy volunteers.

3) A15 mL BD tube was taken, 5mL Ficoll was added, and 5mL fresh blood was gently added.

4) Centrifuging at normal temperature, and lifting by 1 to 1000g for 30min.

5) After centrifugation, the intermediate (white) peripheral blood lymphocytes are carefully aspirated.

6) Three volumes of pre-warmed DPBS were added for washing, 300g,10min.

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

8) Cell count, centrifuge at normal temperature, 100g,10min.

9) Appropriate amount of T cell culture medium (X-vivo, 5% AB serum, 400IU IL2) was added at 2X 10-6/mL to resuspend the cells.

10 The antibody incubation liquid in the T25 square bottle is sucked, the T cells resuspended by the T cell culture liquid are added, and the normal culture is carried out for about 48 hours at 37 ℃.

car-T cell preparation:

1) T cell culture plates were incubated one day in advance and overnight at 4 ℃. A24-well plate was used, and 0.5mL of DPBS and 10. Mu.l of Retronectin (1 mg/mL) were added to each well.

2) The RetroNectin solution was blotted off.

3) 1mL of 2% BSA solution was added to each well and the wells were blocked at 37℃for 30min.

4) The blocking solution was aspirated and washed once with DPBS.

5) Mu.l 400 Xvirus, 300. Mu. l X-vivo complete medium, 32℃and 1000g up and down 2 were added to each well (non-adherent well plate) and centrifuged for 2h.

6) 2-2.5X 10X 6T cells activated for 48h are added into each hole, the X-vivo complete culture medium is supplemented to 1 mL/hole, the temperature is 32 ℃, and the centrifugation is carried out for 15min by 300g 2 g.

7) The CAR positive expression rate is detected through flow after normal culture for about 72 hours at 37 ℃, and is shown in figure 2. The results showed that the positive expression rate of the CAR constructed according to the present invention was 75.7%.

Example 2

This example provides an in vitro kill test.

1) Killing of 96-well plates: tumor cells 1 x 10≡4/well, CAR-T cells constructed with the invention and control T cells containing SHP 2N-SH 2 alone were plated at 1:1, 2.5:1, 5:1 effective target ratio to tumor cells, 3 duplicate wells per effective target ratio, and blank T cell (Mock) controls were made.

This example uses the Skov3-mCherry cell line, a cell model with a broad representation.

2) And continuously detecting the in vitro killing condition of the CAR-T cells by using a living cell dynamic detector.

3) Culture supernatants were taken around 24h to detect LDH to calculate tumor lysis.

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

Figure 3 shows that tumor lysis gradually increased with increasing effective target ratio, demonstrating that CAR-T cells exert greater killing efficacy with increasing effective target ratio.

Figures 4-9 show the continuous killing effect at different effective target ratios. It can be seen that even at an effective Target ratio (also called E/T ratio), E: t=1:1 (E stands for Effector cell, i.e. Effector cell; T stands for Target cell, i.e. tumor cell), the number of tumor cells in the field of view after 24 hours of continuous killing was significantly smaller than that of the control group. The CAR-T cell constructed by the invention has obvious killing effect on tumor cells positively expressed by HER 2; whereas control SHP 2N-SH 2T cells alone showed little anti-tumor effect. In addition, the CAR-T can maintain the killing efficacy for 48 hours continuously, which shows that the efficacy has better persistence.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by 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.