CN113493526B

CN113493526B - Multiple co-stimulation signal chimeric antigen receptor and application thereof

Info

Publication number: CN113493526B
Application number: CN202110766645.6A
Authority: CN
Inventors: 金涛
Original assignee: Xinghande Biomedical Dalian Co ltd
Current assignee: Xinghande Biomedical Dalian Co ltd
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2022-10-14
Anticipated expiration: 2041-07-07
Also published as: CN113493526A

Abstract

The invention relates to the technical field of immunotherapy, in particular to a multiple co-stimulation signal chimeric antigen receptor and application thereof. A chimeric antigen receptor with multiple co-stimulating signals consisting of signal peptide and HBV _S The Ag antigen binding structure domain, the extracellular hinge region, the transmembrane region, the multiple intracellular costimulatory signal molecules and the intracellular signal transduction molecules are connected in series. When the first signal of the chimeric antigen receptor T cell is activated, the second signal CD28 multiple co-stimulatory molecules can generate strong clustering effect to kill tumor cells. Meanwhile, the multiple activation effect cannot cause strong T cell immunity and cause potential serious toxic and side effects like the activation type antibody for injecting CD 28; the chimeric antigen receptor T cell has good killing effect on HBV positive liver cancer cells and has good application prospect.

Description

Multiple co-stimulation signal chimeric antigen receptor and application thereof

Technical Field

The invention relates to the technical field of immunotherapy, in particular to a chimeric antigen receptor with multiple co-stimulation signals and application thereof.

Background

Adoptive Cell Therapy (ACT) is to transfuse processed autologous or allogeneic immune cells (mainly autologous cells) to a tumor patient to directly kill tumor cells or kill tumor cells by stimulating the immune response of the body, so as to achieve the purpose of treatment. The current adoptive cell therapy of tumors develops rapidly, and has good curative effect in clinical treatment of various malignant tumors. Tumor immune cell therapy is considered to be one of the most promising approaches to the treatment of malignant tumors.

Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC-unrestricted manner and to continuously activate expanded T cells. The treatment of tumors by chimeric antigen receptor-modified T cells is a milestone breakthrough in the field of tumor therapy in recent years. Wherein CAR-T products CTL019 (Nowa) and KTE-C19 (Kite) for treating leukemia and lymphoma are successively approved by US breakthrough therapy, and the complete remission rate of patients in clinical trials reaches 70-94%. Good therapeutic effects prompted the FDA in the united states to accelerate the drug approval process, and CD19CAR-T drugs were approved in 2017 for each of the above two companies.

Liver cancer (HCC) is one of the most common digestive system malignant tumors in China, and various treatment strategies aiming at liver cancer cannot remarkably prolong the effective life of liver cancer patients at present. In recent years, tumor immunotherapy has been rapidly developed, and cellular immunotherapy has been a breakthrough, and the development of CAR-T technology brings new eosin for the treatment of liver cancer. In the CAR-T treatment aiming at HCC, the CAR-T taking Glypican-3 (GPC 3) as a target point shows good liver cancer cell killing effect in preclinical research. However, GPC3 is highly expressed in 75% or more of HCCs, but it is not a tumor-specific antigen and is also expressed in normal tissues such as lung, kidney, female reproductive system, and the like, and a potential multi-organ off-target effect is inevitable.

Hepatitis B Virus (HBV) infection is an important factor in the induction of HCC. As a hepatotropic virus, HBV can induce the canceration of liver cells through various ways, which is also an important reason for the high incidence of HBV-related liver cancer in China. Worldwide, HBV infection-associated HCC accounts for about 50%, while nearly 90% of HCC patients in China show HBV positivity. More importantly, viral HBV surface antigen (hbsag) can be expressed on the surface of hepatocytes after infection of the host cells by HBV.

T cell activation requires stimulation by two signals, two T cell activation-related signals. Wherein, the T cell surface TCR-CD3 complex is combined with antigen peptide-MHC molecule to provide a first signal for activating T cells and determine the killing specificity of the T cells; the co-stimulatory molecule (e.g., CD 28) on the surface of the T cell binds to the corresponding ligand (e.g., B7) and provides a second signal for T cell activation, promoting T cell activation, proliferation and survival. However, the lack or reduced expression of the first signal stimulator (e.g., MHC molecules) and the second signal ligand (e.g., B7) in tumor cells may not effectively provide signals related to T cell activation, and thus may not activate T cell immune responses.

The chimeric antigen receptor CAR activates ITAM (immunoreceptor type-based activation motion) signaling of intracellular signals CD3 ζ or Fc ε RI γ through Single chain antibody fragments (scFv) that specifically recognize tumor antigens extracellularly. However, the first generation CAR receptors lack the costimulatory signal of T cells, resulting in only transient effects of T cells, short residence time in the body and low cytokine secretion. Second and third generation CARs combine two signals required for T cell activation, linking the second signal, CD28 or/and the 4-1BB intracellular signaling region directly to the CD3 zeta molecule, thereby circumventing the barrier to tumor cells that normally lack the second signal, e.g., B7, etc., to cause T cell inactivation. After the first signal and the second signal are combined, the activation, proliferation and killing capacity of the T cells are greatly improved, the curative effect of the T cells is greatly improved, and based on the understanding of the current activation mechanism of the T cells, CD28, 4-1BB molecules can provide the second activation signal and further strengthen TCR/CD3 signals.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide a multiple co-stimulatory signal chimeric antigen receptor and application thereof, and aims to solve the problem that potential multi-organ off-target effect exists in the traditional chimeric antigen receptor T cell when liver cancer is treated.

In order to realize the purpose, the invention provides the following technical scheme:

a chimeric antigen receptor with multiple co-stimulatory signals is composed of signal peptide and HBV _S The Ag antigen binding domain, the extracellular hinge region, the transmembrane region, the multiple intracellular costimulatory signal molecules and the intracellular signal transduction molecules are connected in series.

Preferably, the signal peptide is a membrane protein signal peptide;

the HBV _S The Ag antigen binding domain is HBV _S Based on Ag antibody, constructing heavy chain variable region and light chain variable region, and connecting them by short peptide;

the extracellular hinge region is selected from one or more of an IgG1FC CH2CH3 hinge region, an IgG2FC CH2CH3 hinge region, an IgG4FC CH2CH3 hinge region, a CD28 hinge region and a CD8 hinge region;

the transmembrane region is selected from one or more of a CD28 transmembrane region, a CD8 transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region and a DAP10 transmembrane region;

the intracellular costimulatory signal molecule is selected from one or more of the group consisting of a CD28 intracellular domain, a CD134/OX40 intracellular domain, a CD137/4-1BB intracellular domain, a LCK intracellular domain, an ICOS intracellular domain, and a DAP10 intracellular domain;

the intracellular signaling molecule is a CD3 ζ intracellular domain.

More preferably, said HBV _S The nucleic acid sequence of the Ag antibody is shown in SEQ ID NO. 1;

the extracellular hinge region is an IgG FC hinge region, and the nucleic acid sequence of the extracellular hinge region is shown in SEQ ID NO. 2;

the transmembrane region is a CD28 transmembrane region, and the nucleic acid sequence of the transmembrane region is shown as SEQ ID NO. 3;

the intracellular co-stimulatory signaling molecule is a 1XCD28 intracellular domain, a 2XCD28 intracellular domain, and/or a 3XCD28 intracellular domain; 1XCD28 intracellular domain nucleic acid sequence is shown as SEQ ID NO. 4; the 2XCD28 intracellular domain nucleic acid sequence is shown as SEQ ID NO. 5; the 2XCD28 intracellular domain nucleic acid sequence is shown as SEQ ID NO. 6;

the nucleic acid sequence of the intracellular domain of CD3 zeta is shown in SEQ ID NO. 7.

The second purpose of the invention is to provide a coding gene which codes targeted HBV _S A chimeric antigen binds to the receptor, and its nucleic acid sequence is shown in SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

The third purpose of the invention is to provide a vector, wherein the vector comprises the coding gene.

A fourth object of the present invention is to provide a cell comprising the chimeric antigen receptor described above, or comprising the vector described above; the cells are T cells or other immune cells.

The fifth purpose of the invention is to provide the application of the cell modified by the chimeric antigen receptor in preparing the drugs for treating HBV-induced diseases, wherein the diseases are hepatitis B, liver cirrhosis or liver cancer.

The seventh purpose of the invention is to provide a medicine, which comprises the chimeric antigen receptor modified cell and nucleotide analogs or interferon, wherein the nucleotide analogs comprise lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoproxil and cladribine.

The eighth purpose of the invention is to provide a preparation method of the chimeric antigen receptor modified cell, which comprises the following steps:

(1) Synthesizing a DNA fragment for coding the chimeric antigen receptor of any one of claims 1 to 3 by gene, adding double enzyme cutting sites at the upstream and the downstream, and inserting the synthesized gene into a pCDH vector to form a plasmid after double enzyme cutting;

(2) Will be resistant to HBV _S Transfecting expression plasmids of the Ag chimeric antigen receptor to 293T cells, packaging to obtain virus particles, and performing centrifugal concentration to obtain a lentivirus concentrated solution;

(3) Infecting cells with lentivirus concentrate to obtain targeted HBV _S Ag chimeric antigen receptor modified cells.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention uses HBV _S Ag is used as a target to design a chimeric antigen receptor T cell, and cancer cells are killed by virtue of the recognition of the Ag on HBV, so that the chimeric antigen receptor T cell can be prevented from off-target and damaging any organ except the liver to the maximum extent, and a new exploration direction is provided for treating HCC by the chimeric antigen T cell; and the constructed target HBV of the invention _S The Ag chimeric antigen receptor T cell is proved to have good killing effect on HBV positive liver cancer cells through phenotype analysis and in vitro killing experiments, and has good application prospect.

When the first signal of the chimeric antigen receptor T cell is activated, the second signal CD28 multiple co-stimulatory molecules can generate strong clustering effect to kill tumor cells. Meanwhile, the multiple activation effect does not cause strong T cell immunity and cause potential serious toxic and side effects like the activation type antibody of CD 28.

Drawings

FIG. 1 is a schematic diagram of a 1XCD28/2XCD28/3X28 HBV-targeted CAR vector;

FIG. 2 is a flow chart of detecting the expression of CAR targeting HBV by using anti-human IgG antibody or anti-human Fab antibody respectively in the presence of 1XCD28/2XCD28/3X 28;

FIG. 3 is a CAR-T streaming expression statistic plot for 1XCD28/2XCD28/3X28 targeted HBV;

FIG. 4 is 1XCD28/2XCD28/3X28 HBV-targeted CAR-T depleted phenotype expression;

FIG. 5 is 1XCD28/2XCD28/3X28 HBV-targeted CAR-T memory cell phenotype expression;

FIG. 6 is a 1XCD28/2XCD28/3X28 HBV-targeted CAR-T versus target cell killing scenario;

FIG. 7 shows the 1XCD28/2XCD28/3X28 HBV-targeted secretion of CAR-T versus target cell cytokines.

Detailed Description

In the following, the technical solutions of the present invention are described clearly and completely, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: construction of vectors expressing CAR lentiviruses

HBV CAR-1XCD28 DNA fragment, HBV CAR-2XCD28 DNA fragment and HBV CAR-3XCD28 DNA fragment are synthesized by whole gene, the synthesized gene is inserted into pCDH vector after double enzyme digestion to construct plasmid, and the constructed vector is shown in figure 1;

in this example, the nucleic acid writes for HBV CAR-1XCD28, HBV CAR-2XCD28 and HBV CAR-3XCD28 are shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10, respectively.

Example 2: CAR-expressing lentiviral packaging

D10 cell complete culture preparation: DMEM, 10 FBS, 1%.

Day 0:293T cells are less than 20 generations and do not overgrow; at 2X 10 ⁷ Spreading 150mm dish, 20mL D10 culture medium, mixing cells uniformly, and culturing overnight at 37 ℃;

day 1: when the fusion degree of the 293T cells reaches 60-80%, performing transfection, wherein the time from plate laying to transfection is not more than 24h;

lentivirus plasmid complexes were prepared as in Table 1 below

TABLE 1 materials required for plasmid complexes

Container with a lid

Area of

Number of cells

Volume of

Master plasmid

RRE

REV

VSVG

PEI

OptiMEM

15cm ³

151cm ²

2×10 ⁷

20mL

18ug

10ug

7ug

70uL

1mL+1mL

While gently swirling the plasmid, adding PEIpro dropwise, mixing well and standing at room temperature for 15min to form a compound; slowly adding the plasmid-PEI complex into 150mm 293T cells, fully and uniformly mixing, and culturing for 6h in an incubator;

day 1: the 293T cells transfected for 6h are gently changed into 20mL of fresh D10 culture medium;

day 3: collecting the virus supernatant after 48h of transfection, temporarily storing in a refrigerator at 4 ℃, and continuously adding 20mL of D10 culture medium;

day 4: collecting virus supernatant after transfection for 72h, and mixing with the virus supernatant after collection and transfection for 48 h; centrifuging at 4 deg.C and 3000g for 10min, or filtering with 0.45um filter to remove debris, retaining supernatant, concentrating virus, or concentrating with 100K ultrafilter cup, and adding filtered supernatant into the filter cup;

centrifuging at 4 ℃ and 3000g to the volume of the virus to be concentrated, taking out the centrifugal device after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, and inversely buckling the filter cup on the sample collecting cup;

centrifuging at 4 deg.C and 3000g for 2min to obtain virus concentrated solution in the sample collection cup, collecting concentrated solution, and packaging at-70 deg.C.

Example 3: preparation of 1X28/2X28/3X28 HBV CAR-T

Separating to obtain relatively pure CD3+ T cells, and adjusting cell concentration to 1 × 10 with T cell-containing culture medium ⁶ Per mL; inoculating 1 mL/hole of cells to anti-human 50ng/mL CD3 antibody and 50ng/mL CD28 antibody, adding 400IU/mL interleukin 2, and stimulating and culturing for 24h to infect viruses;

after cell infectionObserving cell concentration every day, and timely supplementing T cell culture solution containing IL-2400IU/mL to maintain the density of T cells at 5 × 10 ⁵ mL, expanding cells;

after 5d of cell infection, 1X28/2X28/3X28 targeted HBV CAR expression is detected by anti-human IgG antibody or anti-human Fab antibody in a flow mode, and the results are shown in FIG. 2 and FIG. 3;

as can be seen from FIGS. 2 and 3, the CAR positive rate was above 70%, indicating that 1X28/2X28/3X28 HBV CAR-T can be successfully and stably expressed.

Example 4: flow cytometry detection of CAR-T depletion phenotype expression

After 10 days of preparation of 1X28/2X28/3X28 HBV CAR-T, CD4, CD8, PD-1, LAG-3, TIM-3 antibodies were stained simultaneously, and CAR-T cell depletion phenotype was detected in each group by flow assay, with the results shown in FIG. 4;

as can be seen from FIG. 4, the depletion phenotype of 2X28 HBV CAR-T is expressed the highest, both LAG-3 and PD-1 are more positive than those of other groups, and the depletion indexes of other groups are all below 20%, indicating that 2 CD28 costimulatory signals can exhaust T cells earlier.

Example 5: flow cytometry detection of CAR-T memory cell phenotypic expression

After 10 days of 1X28/2X28/3X28 HBV CAR-T preparation, CD4, CD8, CD45RA and CD62L antibodies were stained simultaneously, and the phenotype of CAR-T memory cells in each group was detected by flow assay, the results are shown in FIG. 5;

as can be seen from FIG. 5, the 2X28 HBV CAR-T has the lowest memory cell phenotype, the CD62L expression of CAR-T of 1 CD28 or 3 CD28 co-stimulatory domains is around 80%, and the memory stem cell proportion (CD 45RA and CD62L double positive) is above 60%, while the CAR-T memory phenotype of 2 CD28 co-stimulatory domains is generally above 10% lower, indicating that 2 CD28 co-stimulatory signals can reduce the T cell memory phenotype.

Example 6: killing of target cells after coculture of CAR-T cells with target cells

Preparation of M10 cell complete medium: MEM, 10% FBS, 1% sodium longitudinally, 1% hepes, 1% NEAA, prepared and placed in a 4 ℃ refrigerator for use;

preparation of a culture plate: taking a 96-hole adherent culture plate with a transparent bottom white edge, and using a Collagen1: coating 30min with 30 diluent at 37 ℃, washing for 2 times by PBS for later use;

preparing target cells: digesting target cells, centrifuging for 5min at 500g, removing supernatant, suspending cells with M10 medium, sampling, counting, and diluting the cell suspension to 10 ⁵ /mL；

Target cell plating: according to 10 ⁴ Inoculating 96-well plates in each well and 100uL in each well, uniformly mixing, and putting into a carbon dioxide incubator for culture;

co-culture killing: taking differentiated 96-well plate to culture target cells (the target cells are according to the proportion of 5 multiplied by 10) ⁴ Calculated per well), supernatant is removed, 50uL of M10 culture medium is added into the well, the amount of CAR-T cells is calculated according to the target ratio of killing effect, the required amount of CAR + cells are taken, and the CAR-T cells are diluted to the proper concentration to ensure that 100uL of CAR-T cells are added into the well, wherein the required description is as follows: for example, the effective target ratio is 3:1,CAR-T cells diluted to 1.5X 10 ⁴ /mL；

Placing into a carbon dioxide incubator at 37 ℃, co-culturing for 2d at 5 percent of CO2, adding a luciferase assay System at 50 uL/hole, fully shaking, and detecting by using an enzyme-linked immunosorbent assay (ELISA) instrument, wherein the detection result is shown in figure 6;

the counting method comprises the following steps: killing efficiency = (1-average of Target cells/average of individual cells) × 100%;

as can be seen from FIG. 6, both 1X28 and 2X28 HBV CAR-T can kill target cells efficiently with a positive correlation of killing efficiency to target-effective ratio, but 3X28 HBV CAR-T has little killing, suggesting that 3 CD28 co-stimulatory domains may over-activate T cells disabling cells.

Example 7: cytokine secretion by CAR-T cells after coculture with target cells

Opening the lyophilized standard (C), adding 2.0ml of Assay Diluent (G), and equilibrating at room temperature for at least 15min;

taking 10 EP tubes marked as S1-S10, and diluting the standard substance;

calculating the total amount of the microsphere mixed solution and the volumes of 6 detection microspheres (A1-A6) according to the total amount of the sample;

sequentially taking the capture microspheres A1-A6, fully shaking, uniformly mixing into an EP tube, and marking for later use after uniform mixing;

sampling the sample tube and the standard tube, and adding 20 mul/tube detection microsphere mixed solution into all detection tubes;

taking a fresh sample or re-dissolving the sample at-80 ℃ and storing the sample to room temperature, and centrifuging for 10min at 400 g;

taking 50 mu l of sample and standard substance to corresponding EP tubes;

adding 20 mul/tube of Human Th1/Th2-II PE Detection Reagent (B) into all Detection tubes, and incubating for 180min at room temperature in a dark place;

all samples were taken out, 1ml WashBuffer (F) was added, 400g was centrifuged for 10min;

discarding the supernatant, adding 200 μ l of Wash Buffer to resuspend the sample, and detecting on a computer, wherein the detection result is shown in FIG. 7;

as can be seen from FIG. 7, both 1X28 and 2X28 HBV CAR-T can secrete large amounts of IFNg and TNF while they can kill target cells effectively, but 3X28 HBV CAR-T secretes less, indicating that 3 CD28 co-stimulatory domains can over-activate T cells to disable cells and reduce the ability to secrete cytokines.

From the above, when the first signal of the chimeric antigen receptor T cell of the invention is activated, the second signal CD28 multiple co-stimulatory molecule can generate strong clustering effect to kill tumor cells.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A chimeric antigen receptor with multiple co-stimulatory signals, which is characterized by comprising a signal peptide,HBV _S The Ag antigen binding domain, the extracellular hinge region, the transmembrane region, the multiple intracellular costimulatory signal molecules and the intracellular signal transduction molecules are connected in series to form the structure; wherein the coding targets HBV _S The nucleic acid sequence of the coding gene of the A chimeric antigen binding receptor is shown in SEQ ID NO. 9.

2. The chimeric antigen receptor according to claim 1, wherein the signal peptide is a membrane protein signal peptide;

the extracellular hinge region is selected from one or more of an IgG1FCCH2CH3 hinge region, an IgG2FCCH2CH3 hinge region, an IgG4FCCH2CH3 hinge region, a CD28 hinge region, and a CD8 hinge region;

the intracellular costimulatory signal molecule is selected from one or more of the group consisting of the CD28 intracellular domain, the CD134/OX40 intracellular domain, the CD137/4-1BB intracellular domain, the LCK intracellular domain, the ICOS intracellular domain, and the DAP10 intracellular domain;

the intracellular signaling molecule is a CD3 ζ intracellular domain.

3. The chimeric antigen receptor according to claim 2, wherein the HBV is _S The nucleic acid sequence of the Ag single-chain antibody is shown in SEQ ID NO. 1;

the extracellular hinge region is an IgG1FC hinge region, and the nucleic acid sequence of the extracellular hinge region is shown as SEQ ID No. 2;

the transmembrane region is a CD28 transmembrane region, and the nucleic acid sequence of the transmembrane region is shown in SEQ ID NO. 3;

the CD3 zeta intracellular domain nucleic acid sequence is shown as SEQ ID NO. 7.

4. A vector comprising the encoding gene of claim 1.

5. A cell comprising the chimeric antigen receptor of any one of claims 1-3, or comprising the vector of claim 4.

6. The cell of claim 5 which is a T cell or other immune cell.

7. Use of a cell modified by a chimeric antigen receptor according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of a disease caused by HBV, wherein the disease is hepatitis B, cirrhosis or liver cancer.

8. A medicament comprising the chimeric antigen receptor-modified cell of any one of claims 1 to 3 and a nucleotide analog or interferon.

9. A method for preparing a chimeric antigen receptor modified cell is characterized by comprising the following steps:

(1) Synthesizing a DNA fragment encoding the chimeric antigen receptor of any one of claims 1 to 3 by gene, adding double enzyme cutting sites at the upstream and downstream, and inserting the synthesized gene into a pCDH vector to form a plasmid after double enzyme cutting;

(2) Will resist HBV _S Transfecting expression plasmids of the Ag chimeric antigen receptor to 293T cells, packaging to obtain virus particles, and performing centrifugal concentration to obtain a lentivirus concentrated solution;

(3) The slow virus concentrated solution infects cells to obtain targeted HBV _S Ag chimeric antigen receptor modified cells.