Patent Application
20250161448
The present application belongs to the technical field of tumor immunotherapies and, in particular, relates to a disialoganglioside 2 (GD2) chimeric antigen receptor (CAR) and use thereof.
With the development of tumor immunology theories and clinical techniques, chimeric antigen receptor T (CAR-T) cell immunotherapy has become one of the most promising tumor immunotherapies. In general, a CAR consists of a tumor-associated antigen (TAA) binding region, an extracellular hinge region, a transmembrane region and an intracellular signaling region. Generally, the CAR contains a single-chain variable fragment (scFv) region of an antibody or a domain specifically binding to a TAA, which is coupled to a cytoplasmic domain of a T-cell signaling molecule through hinge and transmembrane regions. The most common lymphocyte activating moiety includes a T-cell costimulatory domain in tandem with a moiety (for example, CD3ζ) triggering the function of a T-cell effector.
CAR-mediated adoptive immunotherapy allows CAR-modified T cells to directly recognize TAAs on target tumor cells in a non-human leukocyte antigen (HLA)-restricted manner. At present, the treatment of CAR-T achieves an exciting effect on hematological tumors, and it is proved that a second generation CD19 CAR-T has an anti-tumor efficacy on recurrent and refractory B acute lymphocytic leukemia, chronic lymphocytic leukemia and lymphoma with an overall response rate of about 50% to 90% according to different tumors.
Disialoganglioside 2 (GD2), which is widely expressed in tumors such as neuroblastoma, melanoma, glioma and sarcoma and is expressed in a low amount and limitedly in normal tissues, is an ideal tumor antigen for immunotherapies. At present, this cancer antigen is mostly used in the treatment of neuroblastoma, which is the most common extracranial solid malignant tumor in children. About 50% of the children with neuroblastoma have large-scale spread and metastasis of the tumor after the onset of the disease. Conventional surgery, chemotherapy, radiotherapy and autologous stem cell transplantation have limited therapeutic effects on this group of patients, and even after the disease is controlled to be in remission, more than 80% of the patients suffer from the recurrence of the disease and die within one year.
At present, among the immunotherapies of neuroblastoma, a relatively mature immunotherapy is antibody treatment for GD2, which achieves a preliminary clinical success. However, a published clinical research report indicates that GD2 monoclonal antibody therapy has relatively high toxicity, 52% of the patients have grades 3 to 4 toxicity reactions and some patients have neurotoxicity. In addition, the antibody is mainly present in peripheral blood after administration. It is relatively difficult for the antibody to accurately enter tumor tissues or sites where a minor tumor remains, and the antibody cannot be present in vivo for a long time. Therefore, in addition to advantages of antibody treatment, the prepared CAR GD2 CAR-T cell can accurately enter the tumor tissues and be present in vivo for a long time due to characteristics of the T cell, which can provide a more effective treatment option for children with recurrent and refractory neuroblastoma. At present, clinical reports of the treatment of neuroblastoma through GD2 CAR-T have shown a preliminary efficacy of CAR-T. However, there is still a lack of long-term observation data.
At present, an effect of CAR-T technology in treating solid tumors is not very ideal. Many scFv regions of CAR-T are derived from murine antibodies, and the murine ScFv is easily rejected by a human immune system, which causes that CAR-T cannot be present in vivo for a long time, thereby limiting a therapeutic effect. It has been reported that this is also one of the reasons of recurrence in many patients with acute lymphocytic leukemia after complete remission by using CD19 CAR-T and also causes the difficulty of retreatment.
Therefore, how to provide a humanized GD2 CAR-T with a good therapeutic effect and a long duration has become an urgent problem to be solved.
The present application provides a GD2 CAR and use thereof. CAR-T containing the GD2 CAR can remove GD2-positive solid tumors, effectively remove minor residues in bone marrow without adverse reactions and improve the safety, efficacy, memory and long-term maintenance of CAR-T in combination with an optimized T cell signaling region.
In a first aspect, the present application provides a humanized GD2 scFv. The humanized GD2 scFv has activity of binding to a GD2 antigen.
The humanized GD2 scFv has an amino acid sequence having more than 80% identity with SEQ ID NO. 1.
In the present application, the humanized GD2 scFv has been subjected to specific modification regarding humanization and structure against the antigen GD2 on the surface of tumors. The modified scFv antibody has a stronger function and better compatibility in a human body and is not easily rejected by an immune system.
In a second aspect, the present application provides a derivative antibody conjugate of the humanized GD2 scFv according to the first aspect.
In a third aspect, the present application provides a nucleic acid molecule. The nucleic acid molecule encodes the humanized GD2 scFv according to the first aspect or the derivative antibody conjugate of the humanized GD2 scFv according to the second aspect.
Preferably, the nucleic acid molecule has a nucleotide sequence having more than 80% identity with SEQ ID NO. 2.
In a fourth aspect, the present application provides a humanized GD2 CAR. The GD2 CAR includes a GD2-antigen-binding scFv domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain and an inducible suicide fusion domain.
The GD2 antigen binding scFv domain includes the humanized GD2 scFv according to the first aspect or the derivative antibody conjugate of the humanized GD2 scFv according to the second aspect.
In the present application, a CAR targeting GD2 is provided that comprises a costimulatory signaling region, a CD3ζ signaling domain and an inducible caspase 9 (iCasp9) suicide fusion domain in tandem, as well as a humanized antigen binding domain that binds to antigen GD2 on tumor surface and has been subjected to specific gene modification. Compared with other CARs, the CAR has a better binding ability to the antigen, thereby improving the effect of CAR-T.
Preferably, the transmembrane domain includes a CD28 transmembrane domain and/or a CD8α transmembrane domain. In some specific embodiments, the transmembrane domain may be selected or modified through an amino acid substitution.
According to the present application, the costimulatory signaling region is a combination of a CD28 costimulatory signaling domain and a T co-signal signaling domain. The CD28 costimulatory signaling domain and the T co-signal signaling domain may be adjusted as required by those skilled in the art, and the arrangement of the CD28 costimulatory signaling domain and the T co-signal signaling domain does not affect the CAR.
Preferably, the costimulatory signaling region includes CD28 and CD27 costimulatory signaling regions or CD28 and IL-15Ra costimulatory signaling regions.
In a specific embodiment, the CAR according to the present invention includes a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain and a CD27 costimulatory signaling domain (abbreviated herein as CD28-CD27). The CD28 costimulatory signaling domain and the CD27 costimulatory signaling domain may be linked through a linker. In a more specific embodiment, the CD28-CD27 has an amino acid sequence having more than 90% of identity with the amino acid sequence as shown in SEQ ID NO. 3. In a more specific embodiment, CD28-CD27 has an amino acid sequence as shown in SEQ ID NO. 3.
In another specific embodiment, the CAR according to the present invention includes a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain and an IL-15Ra costimulatory signaling domain (abbreviated herein as CD28-IL-15Ra). The CD28 costimulatory signaling domain and the IL-15Ra costimulatory signaling domain may be linked through a linker. In a more specific embodiment, the CD28-IL-15Ra has an amino acid sequence having more than 90% of identity with the amino acid sequence as shown in SEQ ID NO. 4. In a more specific embodiment, CD28-IL-15Ra has an amino acid sequence as shown in SEQ ID NO. 4.
Preferably, the inducible suicide fusion domain includes a caspase 9 domain fused to an FK506 binding protein (FKBP), abbreviated herein as FKBP.Casp9.
Preferably, the FKBP.Casp9 domain has an amino acid sequence having more than 90% identity with SEQ ID NO. 5.
Preferably, the GD2 CAR further includes a signal peptide and/or a 2A sequence.
According to the present application, the CAR further includes a signal peptide. The signal peptide may be a signal peptide capable of directing the transmembrane transfer of the CAR, and those skilled in the art may select a conventional signal peptide of a secretory protein gene in the art as required.
According to the present application, the inducible suicide fusion domain is in tandem with the CD3ζ signaling domain through the 2A sequence, where the 2A sequence can break a protein expressed by the inducible suicide fusion domain from a protein of the CAR, thereby causing the CAR to function; through the injection of an activator, the inducible suicide fusion domain is activated, thereby causing the CAR to be out of function.
Preferably, the signal peptide includes a Secretory signal peptide.
Preferably, the Secretory signal peptide is a signal peptide of a CD8α gene, and the Secretory signal peptide has an amino acid sequence as shown in SEQ ID NO. 6.
Preferably, the Secretory signal peptide is a signal peptide of a GM-CSFR gene, and the Secretory signal peptide has an amino acid sequence as shown in SEQ ID NO. 7.
In the present application, the CAR further includes a hinge region having an amino acid sequence of a combination of multiple GGGGS (SEQ ID NO. 10), which may be, for example, GGGGSGGGGS (SEQ ID NO. 11). The hinge region may be selected by those skilled in the art according to an actual situation, which is not particularly limited here. The presence of the hinge region does not affect the performance of the CAR of the present application.
Preferably, the GD2 CAR includes a Secretory signal peptide, a GD2 antigen binding scFv domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence and an inducible suicide fusion domain.
As a preferred technical solution, the CAR is formed by a Secretory signal peptide, a GD2 antigen binding scFv domain, a CD8α and/or CD28 transmembrane domain, CD28 and CD27 costimulatory signaling regions, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem, and the specific arrangement is as follows: Secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9.
Alternatively, the CAR is formed by a Secretory signal peptide, a GD2 antigen binding scFv domain, a CD8α and/or CD28 transmembrane domain, a CD28 and IL-15Ra costimulatory signaling regions, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem, and the specific arrangement is as follows: Secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9.
In the present application, the CAR further includes a promoter. The promoter is EF1a or any highly expressed promoter.
In a fifth aspect, the present application provides a nucleic acid molecule. The nucleic acid molecule encodes the GD2 CAR according to the fourth aspect.
In a sixth aspect, the present application provides a viral vector. The viral vector includes at least one copy of the nucleic acid molecule according to the fifth aspect.
In the present application, the viral vector can effectively modify immune cells to prepare targeting cells.
Preferably, the viral vector includes a lentiviral vector or a retroviral vector, preferably the lentiviral vector.
In a seventh aspect, the present application provides a recombinant virus, which is obtained by co-transferring the viral vector according to the sixth aspect and a packaging helper plasmid into a mammalian cell.
Preferably, the packaging helper plasmid includes pNHP and pHEF-VSVG.
Preferably, the mammalian cell includes any one of a 293 cell, a 293T cell or a TE671 cell.
In an eighth aspect, the present application provides a CAR-T cell. The CAR-T cell expresses the GD2 CAR according to the fourth aspect.
In the present application, the CAR-T cell has a good targeting and killing effect while releasing a low level of immune factor, showing response properties with low toxicity and high immune killing. The schematic mechanism diagram of the CAR-T cell is shown in
Preferably, the CAR-T cell is prepared through transferring the nucleic acid molecule according to the fifth aspect into an immune cell.
Preferably, the transferring is performed via any one of a viral vector, an eukaryotic expression plasmid or mRNA, preferably a viral vector.
Preferably, the CAR-T cell is prepared through transferring the nucleic acid molecule according to the fifth aspect into a T cell via a viral vector.
Preferably, the CAR-T cell is a T cell modified by the viral vector according to the sixth aspect.
In a ninth aspect, the present application provides a composition. The composition includes any one of a combination of at least two of the humanized GD2 scFv according to the first aspect, the derivative antibody conjugate of the humanized GD2 scFv according to the second aspect, the GD2 CAR according to the fourth aspect, the recombinant virus according to the seventh aspect or the CAR-T cell according to the eighth aspect.
In a tenth aspect, the present application provides use of any one of a combination of at least two of the humanized GD2 scFv according to the first aspect, the derivative antibody conjugate of the humanized GD2 scFv according to the second aspect, the GD2 CAR according to the fourth aspect, the recombinant virus according to the seventh aspect, the CAR-T cell according to the eighth aspect or the composition according to the ninth aspect in the preparation of a medicine for treating a tumor.
Preferably, the tumor includes a tumor expressing a GD2-specific antigen.
Preferably, the tumor includes a nervous system tumor expressing the GD2-specific antigen.
Preferably, the tumor includes neuroblastoma.
Compared with the existing art, the present application has the beneficial effects below.
(1) The CAR of the present application has been subjected to specific gene modification on the costimulatory signaling region of the humanized CAR that targets the antigen GD2 on the tumor surface. The modified CAR specifically binds to GD2 and has a better response effect so that the CAR-T cell has a stronger immune response to the tumor, and compared with other GD2 CARs, the CAR has a better long-term effect.
(2) The CAR-T cell of the present application has higher safety and persistence than other GD2 CAR-T cells. Even in a case of a cytokine release syndrome (CRS) caused by an overly strong immune response in a patient, the CAR-T cell can be removed by a drug that induces CAR-T cell apoptosis due to the presence of apoptosis-inducing mechanism. After the CAR-T cell of the present application is infused, a long-term presence of CAR-T can be detected in vivo, which proves that the CAR-T cell has a long-term effect and can achieve an effect of long-term remission of the patient.
(3) A preparation related to the humanized antibody of the present application can play a role in all GD2-positive diseases and has actually been applied to a patient with stage IV neuroblastoma expressing a tumor-specific target GD2. For a patient with minor residues in bone marrow, the preparation has reduced clinical side effects and improved safety and can effectively remove minor residues that are not sensitive to chemotherapy. In addition, GD2 CAR-T is also applied to a patient with glioma in combination with other target CAR-T, and a long-term presence of GD2 CAR-T is detected in the patient, which is conducive to maintaining long-term remission.
To further elaborate on the technical means adopted and effects achieved in the present application, the present application is further described below in conjunction with examples and drawings. It is to be understood that the specific examples set forth below are intended to explain the present application and not to limit the present application.
Experiments without specific techniques or conditions specified in the examples are conducted according to techniques or conditions described in the literature in the art or a product specification. The reagents or instruments used herein without manufacturers specified are conventional products commercially available from proper channels.
A humanized GD2 scFv was provided. The humanized GD2 scFv had activity of binding to a GD2 antigen.
The humanized GD2 scFv had an amino acid sequence shown in SEQ ID NO. 1.
Two types of CARs were provided. The structure diagram of the two types of CARs is shown in
One CAR was formed by a Secretory signal peptide, a GD2 antigen binding scFv domain, a CD28 transmembrane domain, CD28 and CD27 costimulatory signaling regions, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem, and the specific arrangement is as follows: Secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9.
The other CAR was formed by a Secretory signal peptide, a GD2 antigen binding scFv domain, a CD28 transmembrane domain, a CD28 and IL-15Ra costimulatory signaling regions, a CD3ζ signaling domain, a 2A sequence and a caspase 9 domain in tandem, and a specific arrangement is as follows: Secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9.
The Secretory signal peptide had an amino acid sequence shown in SEQ ID NO. 6.
The CD28 transmembrane domain had a sequence shown in SEQ ID NO. 7.
In the construct shown in Secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9, “CD28-CD27” included a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain and a CD27 costimulatory signaling domain.CD28-CD27 had an amino acid sequence shown in SEQ ID NO. 3.
In the construct shown in Secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9, “CD28-IL-15Ra” included a CD28 hinge region, a CD28 transmembrane domain, a CD28 signaling domain and an IL-15Ra signaling domain.CD28-IL-15Ra had an amino acid sequence shown in SEQ ID NO. 4.
The CD3ζ signaling domain had a sequence shown in SEQ ID NO. 8.
The 2A sequence had a sequence shown in SEQ ID NO. 9.
The caspase 9 domain had an amino acid sequence shown in SEQ ID NO. 5.
Two types of lentiviral vectors encoding the two types of CARs in Example 2 were prepared.
The backbone vector of the lentiviral vector was pTYF. For details, see publications such as Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd Ed., pp 303-318, Ed. Jeffrey Morgan, Humana Press, Inc.; Cui, Y. and Chang, L.-J. (2003) Detection and selection of lentiviral vector transduced cells. “Methods in Molecular Biology Vol. 229: Lentivirus Gene Engineering Protocols” pp 69-85, Ed. Maurizio Federico, Humana Press, Inc; Oka, M. Chang, L.-J., Costantini, F., and Terada, N. (2005) Lentivirus mediated gene transfer in embryonic stem cells. Series: “Methods in Molecular Biology” Embryonic Stem Cells 2.
A plasmid map is shown in
Two types of recombinant lentiviruses were prepared. The two types of recombinant lentiviruses were obtained by co-transferring the lentiviral vector in Example 3 and packaging helper plasmids into a mammalian cell. Steps are described below.
Cell debris was removed through centrifugation (1000 g, 5 min) to obtain the virus supernatant, the virus supernatant was filtered by a 0.45 μm low protein binding filter, and the viruses were divided and stored at −80° C.
Generally, the transduced cells may produce lentiviral vectors having a titer of 106 to 107 transduction units per milliliter of the culture medium.
2. Concentrating Lentiviruses with Centrifugal Filter
Two types of CAR-T cells expressing the CARs in Example 2 were prepared. The preparation method is described below.
The activated T cells were suspended in a culture solution, and 10 μg/mL of polybrene (Sigma) was added to the culture solution. The culture solution was AIM-V containing cell culture factors IL-2, IL-7 and IL-15 (all purchased from PeproTech), and the concentrated lentiviruses in Example 5 were separately added to the culture solution. After centrifugation at 100 g for 100 min at room temperature, the cells were cultured for 24 h at 37° C., and a culture solution was added. After four days of culture, the cells were harvested and counted. After two days of culture, the cells were infused into a patient.
Moreover, the green fluorescence of remaining target cells was quantified by flow cytometry and statistically analyzed. The results are shown in
The above experiment was repeated more than three times.
In conjunction with the results of
In conclusion, the GD2 CAR described in the present application has a better response effect and a better long-term effect. The GD2 CAR is applied to the patient with stage IV neuroblastoma expressing the tumor-specific target GD2. For the patient with minor residues in bone marrow, the GD2 CAR has a smaller clinical side effect and higher safety and can effectively remove minor residues that are not sensitive to chemotherapy. In addition, GD2 CAR-T can also be applied to the treatment for the patient with glioma in combination with other target CAR-T, and the presence of GD2 CAR-T can be successfully monitored in the patient for a long time, which is conducive to maintaining long-term remission.
The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that the implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients, selections of specific manners, etc., all fall within the protection scope and the disclosure scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210197703.2 | Mar 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/079018 | 3/1/2023 | WO |
