WO2021012857A1 - Universal cell therapy product for expressed antigen recognition region, and preparation method therefor and application thereof - Google Patents

Abstract

Provided are a universal cell therapy product for an expressed antigen recognition region, and a preparation method therefor and an application thereof. According to the product, an antigen recognition region aiming at tumor cell antigens is carried on a universal cell without an HLA restriction, the antigens are degraded after treatment, and tumor cells are subjected to a series of changes until death, thereby achieving a therapeutic effect.

Description

Universal cell therapy product expressing antigen recognition region and preparation method and application thereof Technical field

The invention relates to the technical field of biomedicine, in particular to a universal cell therapy product expressing an antigen recognition region, and a preparation method and application thereof.

Background technique

Chimeric Antigen Receptor T cells (CART) are genetic modification methods that allow the single-chain variable region (scFv) of a monoclonal antibody that specifically recognizes the target antigen to be expressed on the surface of T cells. The scFv is coupled to the artificially designed T cell activation and proliferation signal threshold through the transmembrane region. Therefore, the specific recognition of the target antigen by the monoclonal antibody is combined with the function of the T cell to produce a specific killing effect, and CART can kill the target cell in a non-MHC restricted manner. B-cell non-Hodgkin's lymphoma (B-NHL) tumor cells stably express CD19 molecules on the surface, which provides a target for immunotherapy. In recent years, the application of CD19-targeted chimeric antigen receptor recombinant T cells to treat B-cell acute lymphoblastic leukemia (B-ALL) has made significant progress, and has achieved subversive therapeutic effects in the clinic. However, the preparation of CART is time-consuming and expensive. It is necessary to collect the patient's autologous T cells and expand them in vitro. However, leukemia patients generally require multiple chemotherapy. Therefore, the number of T cells is small and the quality is poor. Even some long-term chemotherapy patients cannot achieve in vitro expansion of T cells. This has set obstacles to the wide application of CART. In addition, the promotion and application of CART is also facing a huge challenge, that is, cytokine release syndrome (CRS), also known as cytokine storm. After the patient receives treatment, under the action of the costimulatory signal of the CAR molecule, T cells expand in large quantities and release a large amount of cytokines. These inflammatory mediators cause inflammatory reactions, tissue damage, and even death. Patients may even have life-threatening symptoms such as high fever, low blood pressure, tachycardia, heart failure, and liver failure. Neurotoxicity is also another major safety hazard during CART treatment. Nervous system symptoms caused by CART treatment, such as neurological disorders, expressive aphasia, muscle spasms, seizures, etc., are believed to be related to the sharp increase in cytokine levels.

The principle of CART for tumor treatment is to use chimeric antigen receptor T cells to specifically recognize tumor-associated antigen targets and transmit signals to the cells to cause T cell activation and proliferation to effectively kill tumor cells. Although T cells play a key role in CART therapy, it is also the reason that limits the wide application of CART and causes many adverse reactions.

At present, there is no general cell therapy product for the expression antigen recognition region of leukemia.

Summary of the invention

The purpose of the present invention is to address the deficiencies in the prior art and provide a universal cell therapy product for the expression antigen recognition region of leukemia.

In the first aspect, the present invention provides a cell expressing an antigen recognition region, the cell expressing the antigen recognition region is based on lymphocytes, myeloid cells, or mesenchymal stem cells, and expressed on the surface of the body cells Antibodies that can specifically recognize the surface antigens of leukemia cells and do not express co-stimulatory signals of immune cells.

As a preferred embodiment, the leukemia is selected from acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hairy cell leukemia, T cell lymphocytic leukemia, and large granular lymphocytic leukemia And adult T-cell leukemia.

As another preferred embodiment, the leukemia cell surface antigen is a CD antigen.

As another preferred embodiment, the CD antigen is selected from CD19, CD20, CD33, CD52, CD45, CD22, CD25, CD7, CD2, CD3, CD79A, CD79B, CD133 and CD13.

As another preferred embodiment, the antibody is selected from scFV, Fab, VHH and monoclonal antibodies.

As another preferred embodiment, the method for preparing the cell expressing the antigen recognition region is: constructing a recombinant expression vector for expressing the antibody, and then introducing the recombinant expression vector into the body cell.

As another preferred embodiment, the nucleotide sequence for expressing the antibody is shown in SEQ ID NO: 3.

In the second aspect, the present invention provides the application of the cells expressing the antigen recognition region in the preparation of drugs for treating leukemia.

In the third aspect, the present invention provides a preparation containing the cells expressing the antigen recognition region, and a pharmaceutically acceptable carrier, diluent or excipient.

In a fourth aspect, the present invention provides a method for preparing the cell expressing the antigen recognition region, the preparation method is: constructing a recombinant expression vector expressing the antibody, and then introducing the recombinant expression vector into the body cell.

The advantages of the present invention are:

The present invention provides a universal cell product and a new strategy for using the universal cell product to treat leukemia, that is, a universal cell that does not have HLA restriction carries an antigen recognition region for tumor cell antigens, and the antigen is degraded after treatment, The tumor cells undergo a series of changes until they die, which can then play a role in the treatment of leukemia.

The principle of CART treatment is to recognize tumor cells and activate effector T cells to kill tumors.

Mesenchymal stem cells (MSCs) are a type of adult pluripotent stem cells with multiple differentiation potentials that are derived from the mesoderm and neuroectoderm and do not express hematopoietic-related markers. They are used in lung and respiratory diseases, cardiovascular diseases, and Diseases, nervous system diseases, autoimmune diseases, regenerative medicine and tissue engineering have been widely studied and applied. There are also many studies on the relationship between MSCs and tumors. Current studies have shown that MSCs may inhibit or promote tumor growth. The promotion or inhibition of MSCs on tumor biological behavior may be the result of a combination of multiple factors. However, the current research on the mechanism of MSCs acting on tumors is not sufficient, with greater complexity and uncertainty.

Compared with CART treatment or MSCs treatment, the treatment method of the present invention is essentially different.

Experiments have confirmed that after the universal cells expressing the antigen recognition region of the present invention are in contact with tumor cells in vivo, the target antigen of tumor cells completely disappears, which further leads to spontaneous apoptosis of tumor cells, and significantly inhibits tumor progression in leukemia model mice. The therapeutic effect is very effective. Significantly.

Compared with the CART treatment strategy, the present invention can not only reduce the burden of critically ill patients, shorten the cell preparation cycle, reduce cell preparation and transportation costs, and benefit more leukemia patients, but there is no costimulatory signal in the antigen recognition area of the present invention, and the effector cells are not A large number of cytokines are produced, so there will be no cytokine storm and no neurotoxicity, so it is safer and more reliable in future clinical use.

This program can be aimed at different tumor antigen targets, so it can be widely used in the treatment of different tumors. The present invention can use a wide variety of universal cell types, including lymphocytes, myeloid cells and mesenchymal stem cells, and other universal cell types can also be used. Therefore, the present invention lays a foundation for follow-up research, brings hope for a wide range of clinical use, and has dual guiding significance for scientific research and clinical practice.

Description of the drawings

Figure 1 is a schematic diagram of CD19CAR-Jurkat, CD133CAR-Jurkat, scFv-Jurkat, scFv-K562, and scFv-MSC cells.

Figure 2 shows the CD19 expression of target cells SEM, REH and effector cells CD19CAR-Jurkat, CD133CAR-Jurkat, scFv-Jurkat, scFv-K562, and scFv-MSC for different periods of time.

Figure 3 shows the changes in the CD19-PI3K-AKT-cMyc signaling pathway after the target cells REH, SEM and effector cells scFv-K562 were co-cultured for 24 hours.

Figure 4 is a schematic diagram of the CD19-PI3K-AKT-cMyc signaling pathway.

Figure 5 shows the apoptosis of target cells after SEM, REH and effector cells scFv-Jurkat, scFv-K562, and scFv-MSC are co-cultured for different times.

Figure 6 shows the in vivo therapeutic effect of effector cell scFv-MSC.

Figure 7 is a map of pCDH-MCS-T2A-copGFP-MSCV virus vector.

Detailed ways

The specific implementation manners provided by the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

1 Cell preparation

The present invention designs CD19CAR and CD19 antigen recognition regions based on the CTL019 sequence, and obtains the heavy chain (VH) and light chain (VH) and light chain (VH) encoding the variable region of the antibody from the hybridoma cell line HB-12346 that secretes the IgG1 monoclonal antibody AC133 that specifically recognizes CD133. VL) cDNA sequence, spliced synthesis can bind to CD133 antigen recognition region, introduce membrane positioning signal (CD8 leader), hinge region (CD8 hinge), transmembrane region (CD8 transmenber), costimulatory signal (CD28, 4-1BB, CD3) -zata) (see Table 1 for the sequence). Entrusted Shenggong Bioengineering (Shanghai) Co., Ltd. to synthesize the entire expression cassette, amplified by PCR, introduced restriction sites EcoRI and BamHI, and inserted pCDH-MCS-T2A-copGFP-MSCV virus vector after restriction (Figure 7, purchased from System Biosciences, the article number is CD523A-1) EcoRI-BamHI sites, after the correct sequencing, use the promega company plasmid extraction kit to extract the plasmid. Lentivirus packaging: use pSPAX2, pMD2G virus packaging system, Lenti-X cells, transfect with CaCl ₂ transfection reagent, after harvesting the virus, purify it. Genetic modification of cell lines: The obtained virus infects Jurkat E6.1, K562 and primary mesenchymal stem cell MSCs, and stable transgenic cell lines are obtained: CD19CAR-Jurkat, CD133CAR-Jurkat, scFv-Jurkat, scFv-K562, scFv- MSC. The schematic diagram is shown in Figure 1.

Table 1

2 Western Blot detection of CD19 and CD133 antigen expression after co-culture of effector cells and target cells

Effector cells: CD19CAR-Jurkat, CD133CAR-Jurkat, scFv-Jurkat, scFv-K562, scFv-MSC, 1×10 ^{6 each} , co-cultured with target cells: REH or SEM (ALL cell line) at 12 In the well plate, a control group, namely Jurkat, K562, and MSC were co-cultured with target cells at a ratio of 1:1. After each effector cell and target cell were incubated at 37 degrees Celsius for 0h, 2h, 4h, 6h, and 8h, the cells were collected in a 1.5ml EP tube to prepare a protein lysate. The scFv-MSC and target cells were co-cultured for 0h, 12h, 24h, 36h, 48h, and the cells were collected in a 1.5ml EP tube to prepare protein lysate. Western Blot was used to detect CD19, CD133 expression and internal reference GAPDH expression (CD19 antibody was purchased from ABclonal, item number: A2577; CD133 antibody was purchased from SantaCruz company, item number: 30220; GAPDH antibody was purchased from Sigma company, item number: G8795). The results are shown in Figure 2. After effector cells and target cells are co-cultured, the CD19 on the surface of the target cells gradually decreases until it disappears completely, indicating that ALL cells will escape the CD19 antigen under the action of CD19-scFv, and the CD19 antigen will be blocked. Complete degradation, this result will inevitably lead to molecular changes in target cells; in addition, CD133CAR-Jurkat cells lead to the disappearance of CD133 antigen on the surface of SEM cells, indicating that under the action of the antigen recognition area, the degradation of the antigen after escape occurs is a widespread phenomenon. We can develop new treatments for such antigens and strive to break through the limitations of CART use.

3 Western Blot detects the changes in the downstream signaling pathway of CD19 after co-cultivation of scFv-K562 and REH and SEM

Effector cells scFv-K562 and target cells REH, SEM, 1×10 ^{6 each} , co-cultured in a 12-well plate at 1:1, and a control group was set up, that is, K562 and target cells were co-cultured at a ratio of 1:1, 24h Then collect the cells in a 1.5ml EP tube to prepare a protein lysis solution. Detect the protein expression of CD19-PI3K-AKT-cMyc signaling pathway (PI3K antibody was purchased from CST company, item number: 4249; AKT and p-AKT antibody were purchased from CST company, item number: 4685 and 4060; c-Myc antibody was purchased from SantaCruz Company, item number: sc-40). The results are shown in Figure 3. When CD19 is degraded, the downstream PI3K, p-Akt, and c-Myc expressions are all down-regulated. Previously, c-Myc was reported to be closely related to the proliferation and survival of ALL leukemia cells. After c-Myc was knocked down, the survival time of ALL tumor-bearing mice was significantly improved. Therefore, after the co-culture of effector cells and target cells, CD19-degraded ALL occurred. Cells are very likely to undergo spontaneous apoptosis.

4 The Annexin-V labeling method detects the apoptosis-inducing effect of scFv-Jurkat, scFv-K562 and scFv-MSC cells on REH and SEM cells

The effector cells scFv-Jurkat, scFv-K562, and scFv-MSC were labeled with celltrace yellow (ThermoFisher, C34567), and the target cells REH and SEM were labeled with CFSE (Sigma, 21888), and co-cultured in a 24-well plate at 1:1. in. At 24h, 48h, 72h and 96h, some cells were taken, 50μl Annexin-V (BD, 550475) system was used to label apoptotic cells, and 200μl Bounding Burrer was added 15 minutes later, and tested on the machine. The results are shown in Figure 5. As the number of days increases, the target cells REH and SEM gradually begin to apoptotic, indicating that the CD19 signaling pathway is inhibited, leading to the disappearance of downstream c-Myc, because c-Myc is closely related to the survival and proliferation of ALL leukemia cells. Therefore, ALL leukemia cells spontaneously go to apoptosis.

5 In vivo experiments

The NOD/SCID transplanted tumor mice with 1.5×10 ⁶ tumor-forming SEM-GL cells, as a research model of ALL leukemia, were treated by tail vein injection on the third day after tumor cell inoculation, and control group 1 was treated with PBS. The control group 2 was treated with 1.5×10 ⁶ MSC cells, and the experimental group was treated with 1.5×10 ⁶ scFv-MSC cells. The treatment was performed twice a week for a total of 6 treatments. On the 10th, 15th, 20th, and 27th days, live imaging was performed to monitor the distribution and progress of tumor cells. The results are shown in Figure 6, the tumor progression of the scFv-MSC cell treatment group was significantly slower than that of the control group, and the progression of the MSC cell treatment group was no different from that of the control group, indicating that scFv-MSC can have a significant therapeutic effect in vivo.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be considered This is the protection scope of the present invention.

Claims (10)

A cell expressing an antigen recognition region is characterized in that the cells expressing the antigen recognition region are lymphocytes, myeloid cells or mesenchymal stem cells as the body cells, and the expression on the surface of the body cells can specifically recognize Antibodies to the surface antigens of leukemia cells, and do not express the costimulatory signals of immune cells. The cell expressing the antigen recognition region of claim 1, wherein the leukemia is selected from acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hairy cell leukemia, T cell Lymphocytic leukemia, large granular lymphocytic leukemia and adult T-cell leukemia. The cell expressing the antigen recognition region of claim 1, wherein the surface antigen of the leukemia cell is a CD antigen. The cell expressing an antigen recognition region according to claim 3, wherein the CD antigen is selected from the group consisting of CD19, CD20, CD33, CD52, CD45, CD22, CD25, CD7, CD2, CD3, CD79A, CD79B, CD133 and CD13. The cell expressing the antigen recognition region of claim 1, wherein the antibody is selected from the group consisting of scFV, Fab, VHH and monoclonal antibodies. The cell expressing the antigen recognition region according to claim 1, wherein the preparation method of the cell expressing the antigen recognition region is: constructing a recombinant expression vector expressing the antibody, and then introducing the recombinant expression vector into the The body cell. The cell expressing the antigen recognition region according to claim 5, wherein the nucleotide sequence for expressing the antibody is shown in SEQ ID NO: 3. The use of the cell expressing the antigen recognition region of any one of claims 1-7 in the preparation of a medicine for treating leukemia. A preparation, characterized in that the preparation contains the cell expressing the antigen recognition region according to any one of claims 1-7, and a pharmaceutically acceptable carrier, diluent or excipient. The method for preparing a cell expressing an antigen recognition region according to any one of claims 1-7, wherein the preparation method is: constructing a recombinant expression vector expressing the antibody, and then introducing the recombinant expression vector into the The body cell.

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