WO2020108646A1 - Cd19-and psma-based combined car-t immunotherapy - Google Patents
Cd19-and psma-based combined car-t immunotherapy Download PDFInfo
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- WO2020108646A1 WO2020108646A1 PCT/CN2019/122164 CN2019122164W WO2020108646A1 WO 2020108646 A1 WO2020108646 A1 WO 2020108646A1 CN 2019122164 W CN2019122164 W CN 2019122164W WO 2020108646 A1 WO2020108646 A1 WO 2020108646A1
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- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present application relates to the field of cellular immunotherapy for tumors, in particular to an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA, and an application thereof, and specifically to a method for constructing a chimeric antigen receptor T (CAR-T) cell technology based on tumor specific targets CD19 and PSMA and its application in anti-tumor therapy.
- CAR-T chimeric antigen receptor T
- chimeric antigen receptor T cell CAR-T
- the chimeric antigen receptor typically consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
- the CAR generally comprises a single chain fragment variable (scFv) region of an antibody or a binding domain specific for a tumor-associated antigen (TAA) , which is coupled to the cytoplasmic domain of a T cell signaling molecule via hinge and transmembrane regions.
- scFv single chain fragment variable
- TAA tumor-associated antigen
- the most common lymphocyte activation moieties include a T cell costimulatory domain in tandem with a T-cell effector function triggering (e.g. CD3 ⁇ ) moiety.
- the CAR-mediated adoptive immunotherapy allows CAR-T-transplanted T cells to directly recognize the TAAs on target tumor cells in a non-HLA-restricted manner.
- B-ALL B cell acute lymphocytic leukemia
- CLL chronic lymphocytic leukemia
- One approach to treat these patients is to genetically modify T cells to target the antigens expressed on tumor cells through the expression of CARs.
- CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen (HLA) -independent manner. Attempts in using genetically modified cells expressing CARs to treat these types of patients have achieved promising success.
- CD19 molecule is a potential target for the treatment of B lymphocyte tumors, and is also a focus in CAR research.
- the expression of CD19 is restricted to normal and malignant B cells and thus is a widely accepted CAR target for safety tests.
- T cells genetically modified with a chimeric antigen receptor targeting the CD19 molecule have achieved great success in the treatment of multiple, refractory acute B lymphocytic leukemia, they have significant poor therapeutic effects in the treatment of refractory, recurrent chronic B lymphocytic leukemia and B lymphocyte lymphoma.
- CN 104788573 A discloses a chimeric antigen receptor hCD19scFv-CD8 ⁇ -CD28-CD3 ⁇ and use thereof.
- This second-generation chimeric antigen receptor is composed of variable regions of light and heavy chains of anti-human CD19 monoclonal antibody HI19a (hCD19scFv) , a human CD8 ⁇ hinge region, human CD28 transmembrane and intracellular regions, and a human CD3 ⁇ intracellular region in tandem arrangement.
- hCD19scFv anti-human CD19 monoclonal antibody HI19a
- a human CD8 ⁇ hinge region a human CD8 ⁇ hinge region
- human CD28 transmembrane and intracellular regions and a human CD3 ⁇ intracellular region in tandem arrangement.
- the expression level of CD19 is decreased after a single infusion of CAR-T cells, causing the tumor cells to easily escape immune mechanisms.
- this second-generation CART causes a strong immune factor storm which has safety concerns.
- PSMA is also a potential target for the treatment of malignant B-cell tumors. A positive rate of 98%was detected when the center immunohistochemically stained tumors of 16 patients with B-cell lymphoma, indicating that PSMA can indeed be used as a target for the treatment of B-cell lymphoid tumors.
- the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA, and an application thereof.
- the present application initiates combining two tumor targets, CD19 and PSMA, posseses advantages including strong specificity and high targeting ability, and it can effectively improve and prolong the therapeutic effects of CARTs, shows a better therapeutic effect on surface antigens CD19 and PSMA-positive leukemia or B-cell lymphoma and can effectively avoid the off-target escape as found in single-targeted therapy.
- the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA.
- T cells are modified with lentiviral vectors encoding antigen binding domains that bind to tumor surface antigens CD19 and PSMA, thus allowing the tumor surface antigens CD19 and PSMA to specifically bind to the chimeric antigen receptors of the present application.
- CAR-T cells eliminate both tumor cells expressing CD19 and those expressing PSMA, effectively avoiding the escape of tumor cells resulting from a low antigen expression, and enhancing the long-term immune effects of CAR-T cells.
- the two chimeric antigen receptors may be a separate chimeric antigen receptor targeting CD19 and a separate chimeric antigen receptor targeting PSMA, respectively.
- the chimeric antigen receptor targeting CD19 may be combined with the chimeric antigen receptor targeting PSMA to express as a dual chimeric antigen receptor, i.e., the antigen binding domain thereof binds to tumor surface antigens CD19 and PSMA. Both cases can achieve a combination therapy of the two chimeric antigen receptors.
- the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 ⁇ signaling domain, and an inducible suicide fusion domain in tandem arrangement.
- the antigen-binding domain is a single chain antibody against tumor surface antigen CD19
- the antigen-binding domain is a single chain antibody against tumor surface antigen PSMA.
- the chimeric antigen receptor (CAR) of the genetically modified T cell and the single chain antibodies (scFv) of the antigen binding domains for CD19 and PSMA of the genetically modified T cells are exemplified below.
- the single chain antibody against tumor surface antigen CD19 has an amino acid sequence selected from any one of the group consisting of
- the amino acid sequence has the activity of a single chain antibody against tumor surface antigen CD19.
- amino acid sequence (SEQ ID No. 1) of the single-chain antibody against the tumor surface antigen CD19 is listed as follows:
- the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized CD19 single chain antibodies.
- the amino acid mutant still functions as a CD19 single-chain antibody.
- the single chain antibody against the tumor surface antigen PSMA has an amino acid sequence selected from any one of the group consisting of
- the amino acid sequence has the activity of a single chain antibody against tumor surface antigen PSMA.
- amino acid sequence SEQ ID No. 2 of the single-chain antibody against tumor surface antigen PSMA is listed as follows:
- the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized PSMA single chain antibodies.
- the amino acid mutant still functions as a PSMA single-chain antibody.
- T cells are genetically modified with the chimeric antigen receptor by lentiviral vectors.
- the CD19-and PSMA-based CAR-T cells bind to tumor surface antigens CD19 and PSMA, exhibiting a stronger tumor-killing effect.
- the transmembrane domain is a CD28 transmembrane domain and/or a CD8 ⁇ transmembrane domain.
- the transmembrane domain can be selected or modified by amino acid substitution.
- the costimulatory signaling region is any one selected from the group consisting of a CD28 signaling domain, a CD27 signaling domain or a CD137 signaling domain, or a combination of at least two thereof.
- a person skilled in the art can adjust the arrangement of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain according to requirements. Different arrangements of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain will not affect the chimeric antigen receptor.
- the present application employs the order of CD28-CD27.
- the fourth-generation CAR comprises an inducible suicide fusion domain which contains a Caspase 9 domain having the amino acid sequence as shown in SEQ ID NO. 3, which is as follows:
- the inducible suicide fusion domain is connected in tandem with the CD3 ⁇ signaling domain via a 2A sequence.
- the 2A sequence will cause the protein expressed by the inducible suicide fusion domain to cleave off from the chimeric antigen receptor protein, thereby allowing the chimeric antigen receptor to exert its function.
- the suicide fusion domain can be activated by injecting an activator, thereby causing the T cells expressing the chimeric antigen receptor to die to lose their functions.
- the chimeric antigen receptor further comprises a signal peptide which is capable of directing transmembrane transfer of the chimeric antigen receptor.
- a signal peptide which is capable of directing transmembrane transfer of the chimeric antigen receptor.
- the signal peptide is a Secretory signal peptide, which is the signal peptide for gene GMCSFR and may have the amino acid sequence as shown in SEQ ID NO. 8, which is as follows: MLLLVTSLLLCELPHPAFLLIP.
- the Secretory signal peptide is a signal peptide for CD8a gene, and the Secretory signal peptide has the amino acid sequence as shown in SEQ ID NO. 9, which is as follows: MALPVTALLLPLALLLHAARP.
- the chimeric antigen receptor of the present application may further comprise a hinge region.
- the hinge region may be selected by those skilled in the art according to actual situation, and is not particularly limited herein. The presence of a hinge region will not affect the performance of the chimeric antigen receptor of the present application.
- the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises a signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 ⁇ signaling domain, a 2A sequence and an inducible suicide fusion domain in tandem arrangement.
- the chimeric antigen receptor is obtained by connecting a Secretory signal peptide, a CD19 antigen-binding domain and/or a PSMA antigen-binding domain, CD8 ⁇ and/or CD28 transmembrane domain (s) , a CD28 extracellular signaling domain, a CD28 intracellular signaling domain, a CD27 intracellular signaling domain, a CD3 ⁇ intracellular signaling domain, a 2A sequence and a FBKP.
- Casp9 domain in tandem. Specifically, the arrangement is as follows:
- Casp9 has the amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence that shares more than 90%homology therewith.
- the amino acid sequence as shown in SEQ ID NO. 4 is as follows:
- Casp9 has the nucleotide sequence as shown in SEQ ID NO. 5 or an nucleotide sequence that shares more than 95%homology therewith.
- the nucleic acid sequence as shown in SEQ ID NO. 5 is as follows:
- the chimeric antigen receptor Secretory-PSMA scFv-CD28-CD27-CD3 ⁇ -2A-FBKP has the amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence that shares more than 90%homology therewith.
- the amino acid sequence as shown in SEQ ID NO. 6 is as follows:
- Casp9 has the nucleotide sequence as shown in SEQ ID NO. 7 or an nucleotide sequence that shares more than 95%homology therewith.
- the nucleic acid sequence as shown in SEQ ID NO. 7 is as follows:
- the chimeric antigen receptor further comprises a promoter, which is any one of the group consisting of EF1a, CMV-TAR and CMV, or a combination of at least two thereof.
- the chimeric antigen receptor is expressed by transducing the nucleic acid encoding the same into T cell.
- the transduction is performed by transduction into T cells via any one of the group consisting of a viral vector, an eukaryotic expression plasmid and an mRNA sequence, or a combination of at least two thereof, preferably by transduction into T cells via a viral vector.
- the viral vector is any one of the group consisting of a lentiviral vector and a retroviral vector, or a combination of at least two thereof, preferably a lentiviral vector.
- the present application provides a recombinant lentivirus mixture comprising a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting CD19 and packaging helper plasmids pNHP and pHEF-VSVG and a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting PSMA and packaging helper plasmids pNHP and pHEF-VSVG.
- the recombinant lentivirus can efficiently immunize cells including T cells to prepare targeting T cells.
- the mammalian cell is any one of the group consisting of a 293 cell, a 293T cell and a TE671 cell, or a combination of at least two thereof.
- the present application provides a pharmaceutical composition comprising the immune cell mixture as described in the first aspect and/or the recombinant lentivirus mixtureas described in the second aspect.
- the present application provides use of the immune cell mixture as described in the first aspect, the recombinant lentivirus mixture as described in the second aspect or the pharmaceutical composition as described in the third aspect for the preparation of chimeric antigen receptor T cells, immune competent cells or tumor therapeutics.
- the antigen receptor T cells have a good targeting effect and are capable of releasing low dose of immune factors, having a property of low toxic reaction.
- the tumor is a blood-associated neoplastic disease and/or a solid tumor.
- the neoplastic disease is selected from, but not limited to, leukemia.
- the present application provides a method for treating a tumor comprising administrating to a subject in need thereof a therapeutically effective amount of
- the CD19-and PSMA-based CAR-T cells obtained by genetically modifying T cells with the chimeric antigen receptors of the present application bind to tumor surface antigens CD19 and PSMA, and kill tumors with a stronger effect, achieving a more significant tumor reduction effect;
- the two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and PSMA that are highly expressed in leukemia and lymphoma and has a safer and more significant effect than other chimeric antigen receptors and other tumor antigens, thereby improving the immune effects of CAR-T cells, making CD19 escape not easy to occur, being easy to reach the lesion site, and improving the therapeutic effects for diseases.
- Figure 1 is a diagram showing the synthetic gene sequence map of the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA according to the present application;
- Figure 2 is a diagram showing the mechanism for using chimeric antigen receptors targeting CD19 in combination with chimeric antigen receptors targeting PSMA according to the present application;
- Figure 3 shows the results of immunohistochemical staining of CD19 and PSMA.
- the samples were tumor tissues from lymphoma patients.
- Figure 3 (a) shows the tumor tissues of a patient with follicular lymphoma
- Figure 3 (b) shows the tumor tissues of a patient with diffuse large B lymphoma;
- Figure 4 is a statistical graph showing the expression levels of antigens in tumor tissues from 16 patients with B-cell lymphoma as detected by immunohistochemical staining. The scores range from 1+ to 4+ for weak to strong expression.
- Figure 4 (a) shows the distribution of CD19 expression
- Figure 4 (b) shows the distribution of PSMA expression;
- Figure 5 is a flow chart showing the clinical trial using CD19-targeted chimeric antigen receptor T cells combined with PSMA-targeted chimeric antigen receptor T cells to treat B cell leukemia/lymphoma;
- Figures 6 is a graph showing the copy numbers of CAR genes detected in vivo in a patient with gastric mucosa-associated lymphoma after infusion of a combination of CD19-targeted and PSMA-targeted chimeric antigen receptor T cells in clinical trial.
- reagents or instruments used herein which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.
- Casp9 had the nucleotide sequence as shown in SEQ ID NO. 5, and
- Casp9 had the nucleotide sequence as shown in SEQ ID NO. 7.
- virus supernatant was filtered with a 0.45 ⁇ m low protein-binding filter, and the virus was divided into small portions and stored at -80 °C;
- lentiviral vectors at a titer of 10 6 to 10 7 transducing units can be produced by transduced cells per ml media.
- the virus supernatant was added to the Centricon filter tube or the like, then centrifuged at 2500g for 30 minutes;
- the activated T cells were seeded into a culture dish, and concentrated lentiviruses containing target genes were added, centrifuged at a centrifugal force of 100 g for 100 minutes, then cultured at 37 °C for 24 hours, and AIM-V media containing cell culture factors were added, after 2-3 days of culture, the cells were harvested and counted to produce available CAR-T cells.
- the chimeric antigen receptors as used in the present application can achieve significant therapeutic effects in treating tumors and effectively prevent CD19 escape by combining CAR-T cells expressing CD19-targeted chimeric antigen receptors and PSMA-targeted chimeric antigen receptors.
- Non-specific T cells or CAR-T cells with different signaling domains including GD2 CARTs, CD19 CARTs, PSMA CARTs and CD19 CARTs + PSMA CARTs of the present application were co-cultured with RS4-11 in an incubator at 37 degrees, 5%CO 2 for 24 hours. The proportion of viable RS4-11 cells was recorded 24 hours after the culture.
- CD19 CARTs, PSMA CARTs and CD19 CARTs + PSMA CARTs of the present application had an obvious killing effect on RS4-11 cells. After 24 hours, although the killing effect of CD19 CARTs + PSMA CARTs was slightly weaker than that of CD19 CARTs, but it was stronger than that of PSMA CARTs. It was shown that the CD19 CARTs + PSMA CARTs of the present application was capable of ablating leukemia cells.
- CD19 CART therapy has been developed for many years and has a good effect on the treatment of B cell acute lymphoblastic lymphocyte tumors.
- lymphoma we found that it failed to achieve more than 85%complete remission like leukemia. Only partial remission or stable tumor were observed in many patients. So statistical analysis was performed on patients who had been treated with single-targeted CD19 CART therapy in the center and subjected to CD19 immunohistochemical staining for their tumors by the center rather than a third party institution. The results were shown in Table 1 below.
- Tumor tissues obtained from surgeries were fixed, sealed with wax, and the section was placed on a slide, and stained for tumor antigens with antibodies specific for CD19 and PSMA. Before the infusion, the research center immunohistochemically stained the tumor sections. The results were shown in Figure 3 (a) - Figure 3 (b) , showing strong positive for targets CD19 and PSMA. Compared to the control group, these two patients with tumors simultaneously expressed antigens CD19 and PSMA. Further, statistical analysis was performed on the antigen expression levels in tumor tissues of 10 patients with B cell lymphoma as detected by immunohistochemical staining.
- Figure 4 (a) showed that 44%of patients had strong CD19 expression (3-4+) , and 56%of patient had weak or no expression of antigen CD19 (0-2+) ;
- Figure 4 (b) showed that 38%of patients had high expression of antigen PSMA and 62%of patients had low or no PSMA expression.
- PSMA was indeed widely expressed in lymphoma and thus can be used in combination with CD19 CARTs.
- CART cells were prepared according to the method in Example 4, and infused into the patient in two batches. CD19 CART cells were infused at a total dose of 1 ⁇ 10 6 CART cells per kilogram of body weight, and PSMA CART cells were infused at a total dose of 1 ⁇ 10 6 CART cells per kilogram of body weight.
- the two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and PSMA.
- using the combination of two types of CAR-T cells achieves better therapeutic effects, makes CD19 escape not easy to occur, and allows the disease to be easily relieved.
Abstract
Provided is an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA and an application thereof. The chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, and an inducible suicide fusion domain in tandem arrangement. The chimeric antigen receptors specifically recognize tumor surface antigens CD19 and PSMA. Compared to using other single chimeric antigen receptor T cells, using the combination of CAR-T cells targeting two antigens achieves better therapeutic effects, makes CD19 escape not easy to occur, and allows the disease to be easily relieved.
Description
The present application relates to the field of cellular immunotherapy for tumors, in particular to an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA, and an application thereof, and specifically to a method for constructing a chimeric antigen receptor T (CAR-T) cell technology based on tumor specific targets CD19 and PSMA and its application in anti-tumor therapy.
With the development of immunology theory and clinical technology for tumors, chimeric antigen receptor T cell (CAR-T) immunotherapy has become one of the most promising immunotherapies for cancer treatment. The chimeric antigen receptor (CAR) typically consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The CAR generally comprises a single chain fragment variable (scFv) region of an antibody or a binding domain specific for a tumor-associated antigen (TAA) , which is coupled to the cytoplasmic domain of a T cell signaling molecule via hinge and transmembrane regions. The most common lymphocyte activation moieties include a T cell costimulatory domain in tandem with a T-cell effector function triggering (e.g. CD3ζ) moiety. The CAR-mediated adoptive immunotherapy allows CAR-T-transplanted T cells to directly recognize the TAAs on target tumor cells in a non-HLA-restricted manner.
Most patients with B-cell malignancies including B cell acute lymphocytic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) will die from their disease. One approach to treat these patients is to genetically modify T cells to target the antigens expressed on tumor cells through the expression of CARs. CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen (HLA) -independent manner. Attempts in using genetically modified cells expressing CARs to treat these types of patients have achieved promising success.
CD19 molecule is a potential target for the treatment of B lymphocyte tumors, and is also a focus in CAR research. The expression of CD19 is restricted to normal and malignant B cells and thus is a widely accepted CAR target for safety tests. Although T cells genetically modified with a chimeric antigen receptor targeting the CD19 molecule (CD19 CAR-T) have achieved great success in the treatment of multiple, refractory acute B lymphocytic leukemia, they have significant poor therapeutic effects in the treatment of refractory, recurrent chronic B lymphocytic leukemia and B lymphocyte lymphoma.
CN 104788573 A discloses a chimeric antigen receptor hCD19scFv-CD8α-CD28-CD3ζ and use thereof. This second-generation chimeric antigen receptor is composed of variable regions of light and heavy chains of anti-human CD19 monoclonal antibody HI19a (hCD19scFv) , a human CD8α hinge region, human CD28 transmembrane and intracellular regions, and a human CD3ζintracellular region in tandem arrangement. In this patent, the expression level of CD19 is decreased after a single infusion of CAR-T cells, causing the tumor cells to easily escape immune mechanisms. In addition, this second-generation CART causes a strong immune factor storm which has safety concerns.
In addition, according to the actual statistics on the treatment of lymphoma with a fourth generation CD19-targeted chimeric antigen receptor accomplished by our center, among 9 patients, 4 patients showed CD19 strong positive in tumor tissue immunohistochemical staining and achieved complete remission after receiving CD19-targeted chimeric antigen receptor therapy, while among another 5 patients that showed weak CD19 expression, only 1 achieved remission after the therapy, 2 only achieved partial remission, 1 maintained stable disease and 1 had progressive disease. This result indicates that the treatment with only CD19-targeted chimeric antigen receptors is difficult.
Therefore, for CD19-negative relapse, as well as CD19 low-expression B-cell tumors, it is particularly important to combine another potential chimeric antigen receptor to address the problem of easy mutation and low expression of CD19. In addition to CD19, PSMA is also a potential target for the treatment of malignant B-cell tumors. A positive rate of 98%was detected when the center immunohistochemically stained tumors of 16 patients with B-cell lymphoma, indicating that PSMA can indeed be used as a target for the treatment of B-cell lymphoid tumors.
Therefore, it is particularly important to find a chimeric antigen receptor that is highly specific and highly targeted and can effectively improve the therapeutic effects of CARTs.
SUMMARY
In view of the fact that the current single-targeted CAR-T therapies for treating tumors don’t have a desired long-term effect, and of the influence of tumor microenvironment on the therapeutic effects of the CAR-T technique, the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA, and an application thereof. The present application initiates combining two tumor targets, CD19 and PSMA, posseses advantages including strong specificity and high targeting ability, and it can effectively improve and prolong the therapeutic effects of CARTs, shows a better therapeutic effect on surface antigens CD19 and PSMA-positive leukemia or B-cell lymphoma and can effectively avoid the off-target escape as found in single-targeted therapy.
To achieve this purpose, the present application uses the following technical solutions:
In one aspect, the present application provides an immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA.
In the present application, T cells are modified with lentiviral vectors encoding antigen binding domains that bind to tumor surface antigens CD19 and PSMA, thus allowing the tumor surface antigens CD19 and PSMA to specifically bind to the chimeric antigen receptors of the present application. Thereby, CAR-T cells eliminate both tumor cells expressing CD19 and those expressing PSMA, effectively avoiding the escape of tumor cells resulting from a low antigen expression, and enhancing the long-term immune effects of CAR-T cells.
In the present application, the two chimeric antigen receptors may be a separate chimeric antigen receptor targeting CD19 and a separate chimeric antigen receptor targeting PSMA, respectively. Alternatively, the chimeric antigen receptor targeting CD19 may be combined with the chimeric antigen receptor targeting PSMA to express as a dual chimeric antigen receptor, i.e., the antigen binding domain thereof binds to tumor surface antigens CD19 and PSMA. Both cases can achieve a combination therapy of the two chimeric antigen receptors.
According to the present application, the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, and an inducible suicide fusion domain in tandem arrangement.
Preferably, in the case of a chimeric antigen receptor targeting CD19, the antigen-binding domain is a single chain antibody against tumor surface antigen CD19, and in the case of a chimeric antigen receptor targeting PSMA, the antigen-binding domain is a single chain antibody against tumor surface antigen PSMA.
According to the present application, the chimeric antigen receptor (CAR) of the genetically modified T cell and the single chain antibodies (scFv) of the antigen binding domains for CD19 and PSMA of the genetically modified T cells are exemplified below.
In a specific embodiment, the single chain antibody against tumor surface antigen CD19 has an amino acid sequence selected from any one of the group consisting of
(I) the amino acid sequence as shown in SEQ ID NO. 1;
(II) an amino acid sequence that shares ≥90%, preferably ≥95%, more preferably ≥98%, most preferably ≥99%homology with the amino acid sequence as shown in SEQ ID NO. 1;
(III) an amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO. 1; and
the amino acid sequence has the activity of a single chain antibody against tumor surface antigen CD19.
The amino acid sequence (SEQ ID No. 1) of the single-chain antibody against the tumor surface antigen CD19 is listed as follows:
According to the present application, the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized CD19 single chain antibodies. The amino acid mutant still functions as a CD19 single-chain antibody.
In a specific embodiment, the single chain antibody against the tumor surface antigen PSMA has an amino acid sequence selected from any one of the group consisting of
(I) the amino acid sequence as shown in SEQ ID NO. 2;
(II) an amino acid sequence that shares ≥90%, preferably ≥95%, more preferably ≥98%, most preferably ≥99%homology with the amino acid sequence as shown in SEQ ID NO. 2;
(III) an amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO. 2; and
the amino acid sequence has the activity of a single chain antibody against tumor surface antigen PSMA.
The amino acid sequence (SEQ ID No. 2) of the single-chain antibody against tumor surface antigen PSMA is listed as follows:
According to the present application, the amino acid sequence that shares more than 90%homology or the amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids can be replaced by other single chain antibodies or humanized PSMA single chain antibodies. The amino acid mutant still functions as a PSMA single-chain antibody.
According to the present application, T cells are genetically modified with the chimeric antigen receptor by lentiviral vectors. The CD19-and PSMA-based CAR-T cells bind to tumor surface antigens CD19 and PSMA, exhibiting a stronger tumor-killing effect.
According to the present application, the transmembrane domain is a CD28 transmembrane domain and/or a CD8α transmembrane domain. In some particular embodiments, the transmembrane domain can be selected or modified by amino acid substitution.
According to the present application, the costimulatory signaling region is any one selected from the group consisting of a CD28 signaling domain, a CD27 signaling domain or a CD137 signaling domain, or a combination of at least two thereof. A person skilled in the art can adjust the arrangement of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain according to requirements. Different arrangements of the CD28 signaling domain, CD27 signaling domain and CD137 signaling domain will not affect the chimeric antigen receptor. The present application employs the order of CD28-CD27.
According to the present application, the fourth-generation CAR comprises an inducible suicide fusion domain which contains a Caspase 9 domain having the amino acid sequence as shown in SEQ ID NO. 3, which is as follows:
According to the present application, the inducible suicide fusion domain is connected in tandem with the CD3ζ signaling domain via a 2A sequence. The 2A sequence will cause the protein expressed by the inducible suicide fusion domain to cleave off from the chimeric antigen receptor protein, thereby allowing the chimeric antigen receptor to exert its function. While the suicide fusion domain can be activated by injecting an activator, thereby causing the T cells expressing the chimeric antigen receptor to die to lose their functions.
According to the present application, the chimeric antigen receptor further comprises a signal peptide which is capable of directing transmembrane transfer of the chimeric antigen receptor. A person skilled in the art can select a signal peptide conventional in the art according to requirements. The signal peptide is a Secretory signal peptide, which is the signal peptide for gene GMCSFR and may have the amino acid sequence as shown in SEQ ID NO. 8, which is as follows: MLLLVTSLLLCELPHPAFLLIP.
Preferably, the Secretory signal peptide is a signal peptide for CD8a gene, and the Secretory signal peptide has the amino acid sequence as shown in SEQ ID NO. 9, which is as follows: MALPVTALLLPLALLLHAARP.
The chimeric antigen receptor of the present application may further comprise a hinge region. The hinge region may be selected by those skilled in the art according to actual situation, and is not particularly limited herein. The presence of a hinge region will not affect the performance of the chimeric antigen receptor of the present application.
According to the present application, the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises a signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence and an inducible suicide fusion domain in tandem arrangement.
As a preferable technical solution, the chimeric antigen receptor is obtained by connecting a Secretory signal peptide, a CD19 antigen-binding domain and/or a PSMA antigen-binding domain, CD8α and/or CD28 transmembrane domain (s) , a CD28 extracellular signaling domain, a CD28 intracellular signaling domain, a CD27 intracellular signaling domain, a CD3ζ intracellular signaling domain, a 2A sequence and a FBKP. Casp9 domain in tandem. Specifically, the arrangement is as follows:
Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9;
Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9.
In a specific embodiment, the chimeric antigen receptor Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 has the amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence that shares more than 90%homology therewith. The amino acid sequence as shown in SEQ ID NO. 4 is as follows:
In a specific embodiment, the chimeric antigen receptor Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 has the nucleotide sequence as shown in SEQ ID NO. 5 or an nucleotide sequence that shares more than 95%homology therewith. The nucleic acid sequence as shown in SEQ ID NO. 5 is as follows:
In a specific embodiment, the chimeric antigen receptor Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 has the amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence that shares more than 90%homology therewith. The amino acid sequence as shown in SEQ ID NO. 6 is as follows:
In a specific embodiment, the chimeric antigen receptor Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 has the nucleotide sequence as shown in SEQ ID NO. 7 or an nucleotide sequence that shares more than 95%homology therewith. The nucleic acid sequence as shown in SEQ ID NO. 7 is as follows:
In the present application, the chimeric antigen receptor further comprises a promoter, which is any one of the group consisting of EF1a, CMV-TAR and CMV, or a combination of at least two thereof.
According to the present application, the chimeric antigen receptor is expressed by transducing the nucleic acid encoding the same into T cell.
According to the present application, the transduction is performed by transduction into T cells via any one of the group consisting of a viral vector, an eukaryotic expression plasmid and an mRNA sequence, or a combination of at least two thereof, preferably by transduction into T cells via a viral vector.
Preferably, the viral vector is any one of the group consisting of a lentiviral vector and a retroviral vector, or a combination of at least two thereof, preferably a lentiviral vector.
In a second aspect, the present application provides a recombinant lentivirus mixture comprising a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting CD19 and packaging helper plasmids pNHP and pHEF-VSVG and a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting PSMA and packaging helper plasmids pNHP and pHEF-VSVG.
In the present application, the recombinant lentivirus can efficiently immunize cells including T cells to prepare targeting T cells.
According to the present application, the mammalian cell is any one of the group consisting of a 293 cell, a 293T cell and a TE671 cell, or a combination of at least two thereof.
In a third aspect, the present application provides a pharmaceutical composition comprising the immune cell mixture as described in the first aspect and/or the recombinant lentivirus mixtureas described in the second aspect.
In a fourth aspect, the present application provides use of the immune cell mixture as described in the first aspect, the recombinant lentivirus mixture as described in the second aspect or the pharmaceutical composition as described in the third aspect for the preparation of chimeric antigen receptor T cells, immune competent cells or tumor therapeutics.
In the present application, the antigen receptor T cells have a good targeting effect and are capable of releasing low dose of immune factors, having a property of low toxic reaction.
Preferably, the tumor is a blood-associated neoplastic disease and/or a solid tumor. The neoplastic disease is selected from, but not limited to, leukemia.
In another aspect, the present application provides a method for treating a tumor comprising administrating to a subject in need thereof a therapeutically effective amount of
a) an immune cell expressing both a chimeric antigen receptor targeting CD19 and a chimeric antigen receptor targeting PSMA; or
b) a mixture of an immune cell expressing a chimeric antigen receptor targeting CD19 and an immune cell expressing a chimeric antigen receptor targeting PSMA.
Compared with the prior art, the present application has the following beneficial effects:
(1) The CD19-and PSMA-based CAR-T cells obtained by genetically modifying T cells with the chimeric antigen receptors of the present application bind to tumor surface antigens CD19 and PSMA, and kill tumors with a stronger effect, achieving a more significant tumor reduction effect;
(2) The two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and PSMA that are highly expressed in leukemia and lymphoma and has a safer and more significant effect than other chimeric antigen receptors and other tumor antigens, thereby improving the immune effects of CAR-T cells, making CD19 escape not easy to occur, being easy to reach the lesion site, and improving the therapeutic effects for diseases.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram showing the synthetic gene sequence map of the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA according to the present application;
Figure 2 is a diagram showing the mechanism for using chimeric antigen receptors targeting CD19 in combination with chimeric antigen receptors targeting PSMA according to the present application;
Figure 3 shows the results of immunohistochemical staining of CD19 and PSMA. The samples were tumor tissues from lymphoma patients. Figure 3 (a) shows the tumor tissues of a patient with follicular lymphoma, and Figure 3 (b) shows the tumor tissues of a patient with diffuse large B lymphoma;
Figure 4 is a statistical graph showing the expression levels of antigens in tumor tissues from 16 patients with B-cell lymphoma as detected by immunohistochemical staining. The scores range from 1+ to 4+ for weak to strong expression. Figure 4 (a) shows the distribution of CD19 expression, and Figure 4 (b) shows the distribution of PSMA expression;
Figure 5 is a flow chart showing the clinical trial using CD19-targeted chimeric antigen receptor T cells combined with PSMA-targeted chimeric antigen receptor T cells to treat B cell leukemia/lymphoma;
Figures 6 is a graph showing the copy numbers of CAR genes detected in vivo in a patient with gastric mucosa-associated lymphoma after infusion of a combination of CD19-targeted and PSMA-targeted chimeric antigen receptor T cells in clinical trial.
In order to further illustrate the technical measures adopted by the present application and the effects thereof, the technical solutions of the present application are further described below with reference to the accompanying drawings and specific embodiments, and however, the present application is not limited to the scope of the embodiments. In the examples, techniques or conditions, which are not specifically indicated, are performed according to techniques or conditions described in the literature of the art, or according to product instructions.
The reagents or instruments used herein, which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.
Example 1 Construction of chimeric antigen receptors
(1) The Secretory signal peptide, CD19 or PSMA antigen-binding domain, CD8α and/or CD28 transmembrane domain, CD28 signaling domain, CD27 signaling domain, CD3ζ signaling domain, 2A sequence and Caspase 9 domain, as shown in Figure 1, i.e. Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 and Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 were synthesized by whole gene synthesis. Specifically,
Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 had the nucleotide sequence as shown in SEQ ID NO. 5, and
Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9 had the nucleotide sequence as shown in SEQ ID NO. 7.
Example 2 Lentiviral packaging
(1) 293T cells were used and cultured for 17-18 hours;
(2) Fresh DMEM containing 10%FBS was added;
(3) The following reagents were added to a sterile centrifuge tube: the DMEM taken for each well and helper DNA mix (pNHP, pHEF-VSV-G) and pTYF DNA vector, vortexed and shaken;
(4) Superfect or any transgenic material was added to the centrifuge tube, left for 7-10 minutes at room temperature;
(5) To each culture cells the DNA-Superfect mixture in the centrifuge tube was added, vortexed and mixed;
(6) Incubated in a 37 ℃, 3%CO
2 incubator for 4-5 hours ;
(7) The supernatant was removed from the culture medium, the culture was rinsed with 293 cell media, and media was added for further culture;
(8) The plate was placed back into the incubator with 3%CO
2 for overnight incubation. The next morning, transduction efficiency was observed with a fluorescence microscope.
Example 3 Purification and Concentration of Lentivirus
1) Virus purification
Cell debris were removed by centrifuging at 1000 g for 5 minutes to obtain virus supernatant. The virus supernatant was filtered with a 0.45 μm low protein-binding filter, and the virus was divided into small portions and stored at -80 ℃;
Typically, lentiviral vectors at a titer of 10
6 to 10
7 transducing units can be produced by transduced cells per ml media.
2) Concentration of lentivirus with a Centricon filter or the like
(1) The virus supernatant was added to the Centricon filter tube or the like, then centrifuged at 2500g for 30 minutes;
(2) The filter tube was shaken, then centrifuged at 400 g for 2 minutes, and the concentrated virus was collected to a collection cup. Finally, the virus was collected from all tubes into a single centrifuge tube.
Example 4 Transduction of CAR-T cells
The activated T cells were seeded into a culture dish, and concentrated lentiviruses containing target genes were added, centrifuged at a centrifugal force of 100 g for 100 minutes, then cultured at 37 ℃ for 24 hours, and AIM-V media containing cell culture factors were added, after 2-3 days of culture, the cells were harvested and counted to produce available CAR-T cells.
Example 5 In vitro killing of malignant B cell lines with CD19 CAR-T cells combined with PSMA CAR-T cells
(1) As can be seen from Figure 2, the chimeric antigen receptors as used in the present application can achieve significant therapeutic effects in treating tumors and effectively prevent CD19 escape by combining CAR-T cells expressing CD19-targeted chimeric antigen receptors and PSMA-targeted chimeric antigen receptors.
(2) In vitro evaluation of recognition and killing effects of dual CAR-T cells on target cells: non-specific T cells, GD2 CAR-T cells, and CD19 CART and PSMA CART cells as prepared in the present application were co-cultured with target cells expressing CD19 and PSMA but no GD2, i.e. RS4-11 human acute lymphoblastic leukemia cell line, lymphoma cell line OCI-Ly3 and lymphoma cell line SUD-HL8 expressing GFP (T cell: tumor cell line = 3: 1) in a 5%CO
2 incubator at 37 ℃for 24 h;
(3) Non-specific T cells or CAR-T cells with different signaling domains, including GD2 CARTs, CD19 CARTs, PSMA CARTs and CD19 CARTs + PSMA CARTs of the present application were co-cultured with RS4-11 in an incubator at 37 degrees, 5%CO
2 for 24 hours. The proportion of viable RS4-11 cells was recorded 24 hours after the culture.
Compared with the control group T cells and GD2 CARTs, CD19 CARTs, PSMA CARTs and CD19 CARTs + PSMA CARTs of the present application had an obvious killing effect on RS4-11 cells. After 24 hours, although the killing effect of CD19 CARTs + PSMA CARTs was slightly weaker than that of CD19 CARTs, but it was stronger than that of PSMA CARTs. It was shown that the CD19 CARTs + PSMA CARTs of the present application was capable of ablating leukemia cells.
Example 6 Clinical Application of CD19 and PSMA CAR-T Cells
Sample: a 75-year-old female, diagnosed with gastric mucosa-associated lymphoma for two and a half years. Lung metastases were developed after several cycles of chemotherapy. Rituximab was used and lung tumors still progressed with left pleural effusion. Subsequent intrathoracic injection of rituximab and bevacizumab showed no significant decrease in tumor size and no significant change in lung lesions, and a small amount of pleural effusion remained.
At present, CD19 CART therapy has been developed for many years and has a good effect on the treatment of B cell acute lymphoblastic lymphocyte tumors. However, in the treatment of lymphoma, we found that it failed to achieve more than 85%complete remission like leukemia. Only partial remission or stable tumor were observed in many patients. So statistical analysis was performed on patients who had been treated with single-targeted CD19 CART therapy in the center and subjected to CD19 immunohistochemical staining for their tumors by the center rather than a third party institution. The results were shown in Table 1 below. Among the 9 patients, 4 patients showed CD19 strong expression and achieved complete remission, while among another 5 patients who showed weak CD19 expression, only 1 achieved complete remission, and another 4 only achieved partial remission, stable disease or progressive disease, suggesting that the expression level of CD19 affected the therapeutic effects of CD19 CART. For this limitation, we envisaged to combine CD19 CART with other targets for treatment.
Table 1
Note: n=9 is the number of patients that has been stained for CD19 and received only single-targeted CD19 therapy, based on the number of people in the immunohistochemical table.
Example 7 Identification of antigenic CART targets by staining the antigens of tumor cells
Tumor tissues obtained from surgeries were fixed, sealed with wax, and the section was placed on a slide, and stained for tumor antigens with antibodies specific for CD19 and PSMA. Before the infusion, the research center immunohistochemically stained the tumor sections. The results were shown in Figure 3 (a) -Figure 3 (b) , showing strong positive for targets CD19 and PSMA. Compared to the control group, these two patients with tumors simultaneously expressed antigens CD19 and PSMA. Further, statistical analysis was performed on the antigen expression levels in tumor tissues of 10 patients with B cell lymphoma as detected by immunohistochemical staining. Figure 4 (a) showed that 44%of patients had strong CD19 expression (3-4+) , and 56%of patient had weak or no expression of antigen CD19 (0-2+) ; Figure 4 (b) showed that 38%of patients had high expression of antigen PSMA and 62%of patients had low or no PSMA expression. The above data demonstrated that PSMA was indeed widely expressed in lymphoma and thus can be used in combination with CD19 CARTs.
Clinical trials were carried out according to the above results. The flow chart is shown in Figure 5. The specific steps of a treatment case in a patient are as follows. The results are shown in Figure 6.
(1) First, white blood cells concentrate was collected from the patient, and CD3-positive T cells were isolated therefrom. CART cells were prepared according to the method in Example 4, and infused into the patient in two batches. CD19 CART cells were infused at a total dose of 1×10
6 CART cells per kilogram of body weight, and PSMA CART cells were infused at a total dose of 1×10
6 CART cells per kilogram of body weight.
(2) The patient was pretreated with cyclophosphamide and fludarabine prior to infusion. On average, 1×10
6 cells per kilogram of body weight were infused. The quality of white blood cells and CAR-T cells, the gene transduction rate, the expansion of CAR-T cells and the number of effectively infused CAR-T cells were evaluated and recorded.
(3) After the infusion of CAR-T cells into the patient, the immune factor storm (cytokine release syndrome, CRS) and CAR copy numbers were closely monitored within one month. The specific copy number results were shown in Figure 6.
Specifically, the clinical results were as follows.
1) Symptoms like weakness and poor appetite were observed within three weeks after the infusion, while no other discomfort was observed. On the 83rd day after the infusion, tumors were reduced by half as detected by CT, without pleural effusion. Life was normal, and the CA125 was normal.
2) The condition was still normal and stable 7 months after the infusion. As can be seen from Figure 6, on 21st day after the infusion, 1.58%CD19 CART cells and 0.25%PSMA CART cells were detected as peak values in the peripheral blood, respectively; and 0.15%CD19 CART cells and 0.02%PSMA CART cells were detected in the peripheral blood, respectively even on the 42nd day after the infusion, indicating that both CART cells were well expanded in vivo.
3) A return visit was made three months after the infusion. The patient's physical condition was stable and her physiological condition was good. Currently, it is not necessary to relay on other drugs for maintenance.
In summary, the two chimeric antigen receptors of the present application specifically recognize tumor surface antigens CD19 and PSMA. Compared to using other single-targeted chimeric antigen receptor T cells, using the combination of two types of CAR-T cells achieves better therapeutic effects, makes CD19 escape not easy to occur, and allows the disease to be easily relieved.
The applicant states that detailed methods of the present application are demonstrated in the present application through the above embodiments, however, the present application is not limited to the above detailed methods, and does not mean that the present application must rely on the above detailed methods to implement. It should be apparent to those skilled in the art that, for any improvement of the present application, the equivalent replacement of the raw materials of the present application, the addition of auxiliary components, and the selection of specific modes, etc., will all fall within the protection scope and the disclosure scope of the present application.
Claims (15)
- An immune cell mixture comprising an immune cell genetically modified with a chimeric antigen receptor targeting CD19 and an immune cell genetically modified with a chimeric antigen receptor targeting PSMA.
- The immune cell mixture according to claim 1, wherein the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3ζsignaling domain, and an inducible suicide fusion domain in tandem arrangement.
- The immune cell mixture according to claim 2, wherein in the case of a chimeric antigen receptor targeting CD19, the antigen-binding domain is a single chain antibody against tumor surface antigen CD19; and in the case of a chimeric antigen receptor targeting PSMA, the antigen-binding domain is a single chain antibody against tumor surface antigen PSMA.
- The immune cell mixture according to claim 3, wherein the single chain antibody against tumor surface antigen CD19 has an amino acid sequence selected from any one of the group consisting of(I) the amino acid sequence as shown in SEQ ID NO. 1;(II) an amino acid sequence that shares ≥90%, preferably ≥95%, more preferably ≥98%, most preferably ≥99%homology with the amino acid sequence as shown in SEQ ID NO. 1;(III) an amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO. 1; andthe amino acid sequence has the activity of a single chain antibody against the tumor surface antigen CD19.
- The immune cell mixture according to claim 3, wherein the single chain antibody against tumor surface antigen PSMA has an amino acid sequence selected from any one of the group consisting of(I) the amino acid sequence as shown in SEQ ID NO. 2;(II) an amino acid sequence that shares ≥90%, preferably ≥95%, more preferably ≥98%, most preferably ≥99%homology with the amino acid sequence as shown in SEQ ID NO. 2;(III) an amino acid sequence that is obtained by modifying, substituting, deleting or adding one or several amino acids to the amino acid sequence as shown in SEQ ID NO. 2; andthe amino acid sequence has the activity of a single chain antibody against the tumor surface antigen PSMA.
- The immune cell mixture according to any one of claims 2-5, wherein the transmembrane domain is a CD28 transmembrane domain and/or a CD8α transmembrane domain;preferably, the costimulatory signaling region is any one selected from the group consisting of a CD28 signaling domain, a CD27 signaling domain or a CD137 signaling domain, or a combination of at least two thereof;preferably, the inducible suicide fusion domain comprises a caspase 9 domain;preferably, the caspase 9 domain has the amino acid sequence as shown in SEQ ID NO. 3;preferably, the inducible suicide fusion domain is connected in tandem with the CD3ζsignaling domain via a 2A sequence.
- The immune cell mixture according to any one of claims 1-6, wherein the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting PSMA each comprises a signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence and an inducible suicide fusion domain in tandem arrangement;preferably, the chimeric antigen receptor targeting CD19 is obtained by connecting a Secretory signal peptide, a CD19 antigen-binding domain, CD8α and/or CD28 transmembrane domain (s) , a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem; and the chimeric antigen receptor targeting PSMA is obtained by connecting a Secretory signal peptide, a PSMA antigen-binding domain, CD8α and/or CD28 transmembrane domain (s) , a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem;preferably, the chimeric antigen receptor targeting CD19 is Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9; and the chimeric antigen receptor targeting PSMA is Secretory-PSMA scFv-CD28-CD27-CD3ζ-2A-FBKP. Casp9;preferably, the chimeric antigen receptor targeting CD19 has the amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence that shares more than 90%homology therewith;preferably, the chimeric antigen receptor targeting PSMA has the amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence that shares more than 90%homology therewith.
- The immune cell mixture according to any one of claims 1-7, wherein the chimeric antigen receptor is transduced into T cells by nucleic acid sequence encoding the same for expression;preferably, the transduction is performed by transduction into T cells via any one of the group consisting of a viral vector, an eukaryotic expression plasmid and an mRNA sequence, or a combination of at least two thereof, preferably by transduction into T cells via a viral vector;preferably, the viral vector is any one of the group consisting of a lentiviral vector and a retroviral vector, or a combination of at least two thereof, preferably a lentiviral vector.
- A recombinant lentivirus mixture, comprising a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting CD19 and packaging helper plasmids pNHP and pHEF-VSVG, and a recombinant lentivirus which is obtained by transducing mammalian cells with a viral vector comprising a nucleotide sequence encoding a chimeric antigen receptor targeting PSMA and packaging helper plasmids pNHP and pHEF-VSVG.
- The recombinant lentivirus mixture according to claim 9, wherein the mammalian cell is any one of the group consisting of a 293 cell, a 293T cell and a TE671 cell, or a combination of at least two thereof.
- A pharmaceutical composition comprising the immune cell mixture according to any one of claims 1-8 and/or the recombinant lentivirus mixture according to claim 9 or 10.
- Use of the immune cell mixture according to any one of claims 1-8, the recombinant lentivirus mixture according to claim 9 or 10 or the pharmaceutical composition according to claim 11 for the preparation of chimeric antigen receptor T cells, immune competent cells or tumor therapeutics.
- The use according to claim 12, wherein the tumor is a blood-associated neoplastic disease.
- The use according to claim 13, wherein the blood-associated neoplastic disease is leukemia or lymphoma.
- A method for treating a tumor comprising administrating to a subject in need thereof a therapeutically effective amount ofa) an immune cell expressing both a chimeric antigen receptor targeting CD19 and a chimeric antigen receptor targeting PSMA; orb) a mixture of an immune cell expressing a chimeric antigen receptor targeting CD19 and an immune cell expressing a chimeric antigen receptor targeting PSMA.
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