WO2020108643A1 - Cd19-and cd70-based combined car-t immunotherapy - Google Patents
Cd19-and cd70-based combined car-t immunotherapy Download PDFInfo
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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 CD70, 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 CD70 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-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.
- CD70 is also a potential target for the treatment of malignant B-cell tumors. According to the literature, CD70 is expressed in more than 60%of non-Hodgkin's lymphomas. Moreover, a positive rate of 76%was detected when the center immunohistochemically stained 17 B-cell lymphoma patients.
- therapies that use anti-CD70 antibodies to treat B cell tumors have entered clinical trials.
- CD70 CARTs may be used in lymphoma treatment, there is no treatment using CD19 CARTs in combination with CD70 CARTs, because CD19 CARTs themselves may have significant side effects and the combined use is more uncertain.
- 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 CD70, and an application thereof.
- the present application initiates combining the two tumor targets, CD19 and CD70, possesses 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 CD70-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 CD70.
- T cells are modified with lentiviral vectors encoding antigen binding domains that bind to tumor surface antigens CD19 and CD70, thus allowing the tumor surface antigens CD19 and CD70 to specifically bind to the chimeric antigen receptors of the present application.
- CAR-T cells eliminate both tumor cells expressing CD19 and those expressing CD70, 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 CD70, respectively.
- the chimeric antigen receptor targeting CD19 may be combined with the chimeric antigen receptor targeting CD70 to express as a dual chimeric antigen receptor, i.e., the antigen binding domain binds thereof to tumor surface antigens CD19 and CD70. 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 CD70 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 CD70.
- 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 CD70 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 CD70 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 CD70.
- amino acid sequence SEQ ID No. 2 of the single-chain antibody against tumor surface antigen CD70 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 CD70 single chain antibodies.
- the amino acid mutant still functions as a CD70 single-chain antibody.
- T cells are genetically modified with the chimeric antigen receptor by lentiviral vectors.
- the CD19-and CD70-based CAR-T cells bind to tumor surface antigens CD19 and CD70, 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 has 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 CD70 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 CD70 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-CD70 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 cells.
- 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 CD70 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 mixture as 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 CD70-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 CD70, and kill tumors with a stronger effect, achieving a more significant tumor reduction effect;
- the two chimeric antigen receptors of the present application specifically recognizes tumor surface antigens CD19 and CD70 that are highly expressed in leukemia and lymphoma and have a safer and more significant effect than other chimeric antigen receptors and other tumor antigens, improving the immune effects of CAR-T cells, making CD19 escape not easy to occur, and improving the effects of treating 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 CD70 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 CD70 according to the present application;
- Figure 3 is a graph showing the flow cytometry analysis results of CD19 and CD70 of various B cell tumor cell lines
- Figure 3 (a) - Figure 3 (b) show leukemia cell line RS4-11
- Figure 3 (c) - Figure 3 (d) show lymphoma cell line OCI-Ly3
- Figure 3 (e) - Figure 3 (f) show lymphoma cell line SUD-HL8;
- Figure 4 is a graph showing the results of in vitro killing of B cell tumor cell lines by CD19-targeted and CD70-targeted chimeric antigen receptor T cells;
- Figure 5 shows the results of immunohistochemical staining of CD19 and CD70.
- the samples were tumor tissues from lymphoma patients.
- Figure 5 (a) shows the tumor tissues from a patient with central DLBCL stage IVa
- Figure 5 (b) shows the structure of tumor tissues from a patient with relapsed non-GCB DLBCL;
- Figure 6 is a statistical graph showing the expression level of antigens in tumor tissues from 17 patients with B-cell lymphoma as detected by immunohistochemical staining. The scores range from 1+ to 4+ for weak to strong expression.
- Figure 6 (a) shows the distribution of CD19 expression
- Figure 6 (b) shows the distribution of CD70 expression;
- Figure 7 shows a flow chart of the clinical trial using CD19-targeted and CD70-targeted chimeric antigen receptor T cells
- Figure 8 shows the imaging comparison before ( Figure 8 (a) ) and after ( Figure 8 (b) ) infusion, when a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells is administered to a patient with central diffuse large B-cell lymphoma stage IV in clinical trial;
- Figures 9 is a graph showing the copy numbers of CAR genes detected in vivo in a patient with central diffuse large B-cell lymphoma stage IV after infusion of a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells in clinical trial;
- Figure 10 shows the imaging comparison before ( Figure 10 (a) ) and after ( Figure 10 (b) ) infusion, when a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells is administered to a patient with relapsed diffuse large B-cell lymphoma 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 CAR-T cells.
- the chimeric antigen receptors as used in the present application can achieve significant therapeutic effects in treating tumors, especially tumors with weak CD19 expression, and effectively prevent CD19 escape by combining CARTs expressing CD19-targeted chimeric antigen receptors and CD70-targeted chimeric antigen receptors.
- CD19 CART therapy has been developed for many years and has a good effect on the treatment of B cell acute lymphoblastic leukemia.
- 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 lymphoma 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 CD70.
- Fig. 5 (a) and Fig. 5 (b) 2 patients with diffuse large B lymphoma were taken as an example and immunohistochemically stained for tumor tissues. Compared with the control group, the tumors of these two lymphoma patients simultaneously expressed antigens CD19 and CD70. Further, statistical analysis was performed on the antigen expression levels in tumor tissues of 17 patients with B cell lymphoma as detected by immunohistochemical staining.
- Figure 6 (a) showed that 53%of patients had strong CD19 expression (3-4+) , and 47%of patients had weak or no expression of antigen CD19 (0-2+) , and Figure 6 (b) showed that 71%of patients had high expression of antigen CD70 and 29%of patients had low or no CD70 expression.
- the above data demonstrated that CD70 was indeed widely expressed in lymphoma and thus can be used in combination with CD19 CARTs.
- FIG. 3 Another patient with relapsed diffuse large B lymphoma had lesions located in the abdominal cavity before infusion.
- the infusion effect was evaluated by PET-CT after combined treatment with CD19+CD70 CART.
- the two chimeric antigen receptors of the present application specifically recognizes tumor surface antigens CD19 and CD70.
- using the combination of two types of CAR-T cells achieves better therapeutic effects and it is not easy to occur CD19 escape, allowing the disease to be easily relieved.
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Abstract
It relates 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 CD70 and an application thereof. The chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting CD70 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 CD70. Compared to the use of other single chimeric antigen receptor T cells, using the combination of CAR-T cells targeting two antigens achieves better therapeutic effects and it is not easy to occur CD19 escape, allowing 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 CD70, 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 CD70 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-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 a CD19-targeted chimeric antigen receptor 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, CD70 is also a potential target for the treatment of malignant B-cell tumors. According to the literature, CD70 is expressed in more than 60%of non-Hodgkin's lymphomas. Moreover, a positive rate of 76%was detected when the center immunohistochemically stained 17 B-cell lymphoma patients. Currently, therapies that use anti-CD70 antibodies to treat B cell tumors have entered clinical trials. All adverse reactions are in a controllable range, and a response rate of 20%was observed, indicating that CD70 can indeed be used as a target in the treatment of B-cell lymphoid tumors. Although CD70 CARTs may be used in lymphoma treatment, there is no treatment using CD19 CARTs in combination with CD70 CARTs, because CD19 CARTs themselves may have significant side effects and the combined use is more uncertain.
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 CD70, and an application thereof. The present application initiates combining the two tumor targets, CD19 and CD70, possesses 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 CD70-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 CD70.
In the present application, T cells are modified with lentiviral vectors encoding antigen binding domains that bind to tumor surface antigens CD19 and CD70, thus allowing the tumor surface antigens CD19 and CD70 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 CD70, 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 CD70, respectively. Alternatively, the chimeric antigen receptor targeting CD19 may be combined with the chimeric antigen receptor targeting CD70 to express as a dual chimeric antigen receptor, i.e., the antigen binding domain binds thereof to tumor surface antigens CD19 and CD70. 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 CD70 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 CD70, the antigen-binding domain is a single chain antibody against tumor surface antigen CD70.
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 CD70 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 CD70 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 CD70.
The amino acid sequence (SEQ ID No. 2) of the single-chain antibody against tumor surface antigen CD70 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 CD70 single chain antibodies. The amino acid mutant still functions as a CD70 single-chain antibody.
According to the present application, T cells are genetically modified with the chimeric antigen receptor by lentiviral vectors. The CD19-and CD70-based CAR-T cells bind to tumor surface antigens CD19 and CD70, 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 has 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 CD70 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 CD70 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-CD70 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-CD70 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-CD70 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 cells.
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 CD70 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 mixture as 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 CD70; 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 CD70.
Compared with the prior art, the present application has the following beneficial effects:
(1) The CD19-and CD70-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 CD70, 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 recognizes tumor surface antigens CD19 and CD70 that are highly expressed in leukemia and lymphoma and have a safer and more significant effect than other chimeric antigen receptors and other tumor antigens, improving the immune effects of CAR-T cells, making CD19 escape not easy to occur, and improving the effects of treating 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 CD70 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 CD70 according to the present application;
Figure 3 is a graph showing the flow cytometry analysis results of CD19 and CD70 of various B cell tumor cell lines, Figure 3 (a) -Figure 3 (b) show leukemia cell line RS4-11, Figure 3 (c) -Figure 3 (d) show lymphoma cell line OCI-Ly3, and Figure 3 (e) -Figure 3 (f) show lymphoma cell line SUD-HL8;
Figure 4 is a graph showing the results of in vitro killing of B cell tumor cell lines by CD19-targeted and CD70-targeted chimeric antigen receptor T cells;
Figure 5 shows the results of immunohistochemical staining of CD19 and CD70. The samples were tumor tissues from lymphoma patients. Figure 5 (a) shows the tumor tissues from a patient with central DLBCL stage IVa, and Figure 5 (b) shows the structure of tumor tissues from a patient with relapsed non-GCB DLBCL;
Figure 6 is a statistical graph showing the expression level of antigens in tumor tissues from 17 patients with B-cell lymphoma as detected by immunohistochemical staining. The scores range from 1+ to 4+ for weak to strong expression. Figure 6 (a) shows the distribution of CD19 expression, and Figure 6 (b) shows the distribution of CD70 expression;
Figure 7 shows a flow chart of the clinical trial using CD19-targeted and CD70-targeted chimeric antigen receptor T cells;
Figure 8 shows the imaging comparison before (Figure 8 (a) ) and after (Figure 8 (b) ) infusion, when a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells is administered to a patient with central diffuse large B-cell lymphoma stage IV in clinical trial;
Figures 9 is a graph showing the copy numbers of CAR genes detected in vivo in a patient with central diffuse large B-cell lymphoma stage IV after infusion of a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells in clinical trial;
Figure 10 shows the imaging comparison before (Figure 10 (a) ) and after (Figure 10 (b) ) infusion, when a combination of CD19-targeted and CD70-targeted chimeric antigen receptor T cells is administered to a patient with relapsed diffuse large B-cell lymphoma 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 CD70 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-CD70 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-CD70 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 CAR-T cells.
Example 5 In vitro killing of malignant B cell lines with CD19 CAR-T cells combined with CD70 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, especially tumors with weak CD19 expression, and effectively prevent CD19 escape by combining CARTs expressing CD19-targeted chimeric antigen receptors and CD70-targeted chimeric antigen receptors.
(2) Cell lines from different B cell tumors, including leukemia and lymphoma, were analyzed by flow cytometry for CD19 and CD70 expression. The results were shown in Fig. 3 (a) -Fig. 3 (f) . It can be seen that CD19 and CD70 expression levels varied in different B cell tumors, suggesting that treatment with CD19 CARTs in combination with CD70 CARTs may be more effective against B cell tumors.
(3) In vitro evaluation of recognition and killing effects of CAR-T cells on target cells: non-specific T cells, GD2 CAR-T cells, and CD19 CART and CD70 CART cells as prepared in the present application were co-cultured with target cells expressing CD19 and CD70 but not GD2, i.e. RS4-11 human acute lymphoblastic leukemia cell line expressing green fluorescent protein (T cell: tumor cell line = 2: 1) in a 5%CO2 incubator at 37 ℃ for 24 h;
(4) After the co-culture, cells that were apoptotic due to specific killing were stained with Annexin V and PI and analyzed by flow cytometry. After selecting target cells with green fluorescence, the percentage of cells that were negative for both PI and Annexin V, that is, the percentage of target cells that survived after 24 hours of killing was further analyzed. The lower the percentage, the better the killing effect.
The results were shown in Fig. 4. Compared with the control group T cells and GD2 CARTs, CD19 CARTs, CD70 CARTs and CD19 CARTs+CD70 CARTs of the present application had obvious killing effects on RS4-11 cells, showing that CD19 CARTs+CD70 CARTs of the present application were capable of ablating B cell tumors.
Example 6 Clinical Application of CAR-T Cells
From July 2013 to July 2016, the laboratory cooperated with 22 clinical medical centers and hospitals to treat 102 CD19-positive and chemotherapy-resistant B-ALL patients who met the enrollment criteria, including 55 children and 47 adults. Among them, 27 patients underwent allogeneic hematopoietic stem cell transplantation, and the median percentage of naive leukemia cells in the bone marrow was 14.5% (ranging from 0%to 98%) when they receiving CAR-T therapy. Among them, 69 patients’ bone marrow naive leukemia cells were less than 50%and another 33 patients’ bone marrow naive leukemia cells were more than 50%. The median time period from initial diagnosis to CAR-T cell therapy was 17 months (ranging from 2 to 164 months) .
At present, CD19 CART therapy has been developed for many years and has a good effect on the treatment of B cell acute lymphoblastic leukemia. 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 lymphoma 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 positive 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
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 CD70. As shown in Fig. 5 (a) and Fig. 5 (b) , 2 patients with diffuse large B lymphoma were taken as an example and immunohistochemically stained for tumor tissues. Compared with the control group, the tumors of these two lymphoma patients simultaneously expressed antigens CD19 and CD70. Further, statistical analysis was performed on the antigen expression levels in tumor tissues of 17 patients with B cell lymphoma as detected by immunohistochemical staining. Figure 6 (a) showed that 53%of patients had strong CD19 expression (3-4+) , and 47%of patients had weak or no expression of antigen CD19 (0-2+) , and Figure 6 (b) showed that 71%of patients had high expression of antigen CD70 and 29%of patients had low or no CD70 expression. The above data demonstrated that CD70 was indeed widely expressed in lymphoma and thus can be used in combination with CD19 CARTs.
According to the above results, clinical trials were carried out. The flow chart was shown in Fig. 7. The specific steps of the patient treatment case were as follows. The results were shown in Fig. 8 (a) -Fig. 8 (b) , Fig. 9, Fig. 10 (a) -Fig. 10 (b) .
(1) First, patients were evaluated by the hospital and laboratory, and autologous or donor white blood cells were collected if the enrollment conditions were met. Then following the standard protocol, CD3-positive T cells were screened from PBMC, activated and transduced with 4SCAR19 and 4SCAR70 (as described herein) to prepare CD19 CAR-T cells and CD70 CAR-T cells.
(2) The patients were 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 patients, the immune factor storm and CAR copy numbers were closely monitored within one month. Conclusions about treatment toxicity and final therapeutic effects were obtained by long-term clinical and laboratory evaluations. Toxicity evaluation was performed based on the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE v4.03) .
Specifically, the clinical results were as follows.
1) At present, there are two lymphoma patients in the center who were treated with CD19+CD70 CART therapy. According to the toxicity assessment of CTCAE, the two patients showed only mild fatigue and dizziness and poor appetite. Their body temperature and other blood parameters were normal. A grade 1 toxic effect was evaluated, indicating that the combination therapy with CD19+CD70 CART was highly safe.
2) One patient with central diffuse large B lymphoma achieved tumor remission one month after infusion. The MRI image of the brain tumor before infusion was shown in Figure 8 (a) . The MRI report one month after CD19+CD70 CART infusion, as shown in Figure 8 (b) , showed that the mass (the lymphoma lesion) in the temporoparietal lobe at the edge of the postoperative cavity in the right temporal lobe disappeared (as indicated by the arrow) , and the patient achieved remission. Figure 9 showed the detected copy numbers of CD19 CARTs and CD70 CARTs in peripheral blood after the patient received infusion. It has been one year since the patient received infusion and the tumor has not relapsed.
3) Another patient with relapsed diffuse large B lymphoma had lesions located in the abdominal cavity before infusion. The infusion effect was evaluated by PET-CT after combined treatment with CD19+CD70 CART. The abdominal lymphoma lesion before infusion had a tumor size of 1.6×1.1 cm and a SUV max of 6.9, as shown in Figure 10 (a) . Two months after CART infusion, the abdominal mass disappeared and no high-metabolic lesions were observed, as shown in Figure (b) , and a complete remission was assessed by the physician.
In summary, the two chimeric antigen receptors of the present application specifically recognizes tumor surface antigens CD19 and CD70. Compared to the use of other single chimeric antigen receptor T cells, using the combination of two types of CAR-T cells achieves better therapeutic effects and it is not easy to occur CD19 escape, allowing 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 CD70.
- The immune cell mixture according to claim 1, wherein the chimeric antigen receptor targeting CD19 and the chimeric antigen receptor targeting CD70 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 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 CD70, the antigen-binding domain is a single chain antibody against tumor surface antigen CD70.
- 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 CD70 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 CD70.
- 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 CD3ζ signaling 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 CD70 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 CD70 is obtained by connecting a Secretory signal peptide, a CD70 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 CD70 is Secretory-CD70 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 CD70 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 CD70 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 CD70; orb) a mixture of immune cells expressing a chimeric antigen receptor targeting CD19 and an immune cell expressing a chimeric antigen receptor targeting CD70.
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| CN110592023B (en) * | 2019-09-11 | 2020-09-04 | 浙江蓝盾药业有限公司 | Anti CD70CAR-T cell and preparation method and application thereof |
| WO2021055437A1 (en) * | 2019-09-16 | 2021-03-25 | The General Hospital Corporation | Cd70 targeted chimeric antigen receptor (car) t cells and uses thereof |
| CN110804099B (en) * | 2019-10-31 | 2020-06-16 | 浙江蓝盾药业有限公司 | Human CD70 antigen binding polypeptides |
| WO2022116952A1 (en) * | 2020-12-01 | 2022-06-09 | 苏州克睿基因生物科技有限公司 | Antigen-binding protein targeting cd70 and use thereof |
| CN113292652A (en) * | 2021-06-17 | 2021-08-24 | 南京蓝盾生物科技有限公司 | anti-CD 70 antibodies with enhanced ADCC effect and uses thereof |
| CN116120451A (en) * | 2021-06-17 | 2023-05-16 | 南京蓝盾生物科技有限公司 | anti-CD 70 internalizing antibodies, antibody conjugates and uses thereof |
| CN113292653B (en) * | 2021-06-17 | 2022-11-11 | 南京蓝盾生物科技有限公司 | anti-CD 70 antibodies with enhanced ADCP effect and uses thereof |
| CN114369622A (en) * | 2021-12-31 | 2022-04-19 | 西安桑尼赛尔生物医药有限公司 | Bispecific universal CAR-T cells simultaneously targeting CD7 and CD19 and methods of making the same |
| CN116903757A (en) * | 2023-07-21 | 2023-10-20 | 杭州荣谷生物科技有限公司 | CD70 nanobody and double-targeting chimeric antigen receptor |
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