Search

KR-20260066103-A - Chimeric Antigen Receptor Construct and Uses

KR20260066103AKR 20260066103 AKR20260066103 AKR 20260066103AKR-20260066103-A

Abstract

The present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof, comprising i) an extracellular domain comprising an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) an extracellular domain comprising an antigen-binding region that binds to CD19, wherein the nucleotide sequence encoding the CAR is operably linked to a promoter, and the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP). The present invention also relates to a chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule and a method for treating cancer using the polynucleotide molecule and/or the chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule.

Inventors

  • 후터, 그레고어
  • 드 아키누 두스 산투스 마르팅스, 토마스

Assignees

  • 유니버시타트 바셀

Dates

Publication Date
20260512
Application Date
20240829
Priority Date
20230831

Claims (15)

  1. As a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof, i) comprising an extracellular domain comprising an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) comprising an extracellular domain comprising an antigen-binding region that binds to CD19, and The nucleotide sequence encoding the above CAR is operably linked to a promoter, and The polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP).
  2. In paragraph 1, The above polynucleotide molecule is a polynucleotide molecule further comprising a nucleotide sequence encoding a self-cleaving peptide.
  3. In paragraph 2, The above polynucleotide molecules are arranged in the following order from the 5' end to the 3' end: a) Promoter; b) i) an extracellular domain comprising an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof, comprising an extracellular domain comprising an antigen-binding region that binds to CD19, wherein the nucleotide sequence encoding the chimeric antigen receptor (CAR) or a fragment thereof is a nucleotide sequence operably linked to the promoter of a); c) a nucleotide sequence encoding a self-cleaving peptide; and d) Nucleotide sequence encoding a heterologous signal peptide fused to the signal regulatory protein gamma (SIRPγ)-associated protein (SGRP). A polynucleotide molecule containing
  4. In any one of paragraphs 1 through 3, A heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP) is a signal peptide selected from the group consisting of interleukin 2 (IL-2) signal peptide, interleukin 4 (IL-4) signal peptide, interleukin 9 (IL-9) signal peptide, and interferon gamma (IFNγ) signal peptide, and preferably a polynucleotide molecule selected from the group consisting of human IL-2 signal peptide, human IL-4 signal peptide, human IL-9 signal peptide, and human IFNγ signal peptide.
  5. In any one of paragraphs 1 through 3, The heterologous signaling peptide fused to the signal regulatory protein gamma (SIRPγ)-associated protein (SGRP) is a polynucleotide molecule that is a human interleukin 2 (IL-2) signaling peptide.
  6. In any one of paragraphs 1 through 5, A heterologous signal peptide is a polynucleotide molecule fused to the N-terminal region of a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP).
  7. In any one of paragraphs 1 through 6, A polynucleotide molecule in which a promoter operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof is an elongation factor 1 alpha (EF1A) promoter or an elongation factor 1 alpha shortening (EFS) promoter, preferably an EF1A promoter.
  8. In any one of paragraphs 1 through 7, A chimeric antigen receptor (CAR) or a fragment thereof comprises i) an extracellular domain comprising an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) an extracellular domain comprising an antigen-binding region that binds to CD19, wherein the CAR or the fragment thereof further comprises a CD8α leader, a CD8α hinge and transmembrane domain, a TNF receptor superfamily member 9 (4-1BB) co-stimulatory domain and a CD3ζ signaling domain, a polynucleotide molecule.
  9. In paragraph 8, i) an extracellular domain containing an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII) is a single-chain variable fragment (scFv); or ii) an extracellular domain containing an antigen-binding region that binds to CD19 is a single-chain variable fragment (scFv), a polynucleotide molecule.
  10. In any one of paragraphs 1 through 9, A polynucleotide molecule comprising the sequence described in SEQ ID NO. 1 or the sequence described in SEQ ID NO. 2.
  11. An amino acid sequence comprising a chimeric antigen receptor (CAR) or a fragment thereof, comprising an extracellular domain containing an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII), The nucleotide sequence encoding the above chimeric antigen receptor (CAR) is operably linked to a promoter, and The above-mentioned polynucleotide molecule is an amino acid sequence further comprising a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP) comprising the sequence described in SEQ ID NO. 3.
  12. An amino acid sequence comprising a chimeric antigen receptor (CAR) or a fragment thereof, comprising an extracellular domain including an antigen-binding region that binds to CD19, The nucleotide sequence encoding the above CAR is operably linked to a promoter, and The above-mentioned polynucleotide molecule is an amino acid sequence further comprising a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP) comprising the sequence described in SEQ ID NO. 4.
  13. A construct comprising a polynucleotide molecule of any one of claims 1 to 10.
  14. A chimeric antigen receptor (CAR)-T cell comprising a T cell expressing a polynucleotide molecule of any one of claims 1 to 10 and/or a construct of claim 13.
  15. In Paragraph 14, The chimeric antigen receptor (CAR)-T cells are intended for use in a method for treating cancer in a subject who has epidermal growth factor receptor (EGFR)-related cancer or CD19-related cancer, the method comprising administering a therapeutically effective amount of the chimeric antigen receptor (CAR)-T cells of claim 14 to the subject.

Description

Chimeric Antigen Receptor Construct and Uses The present invention relates to a polynucleotide molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a fragment thereof, comprising i) an extracellular domain comprising an antigen-binding region that binds to epidermal growth factor receptor variant III (EGFRvIII); or ii) an extracellular domain comprising an antigen-binding region that binds to CD19, wherein the nucleotide sequence encoding the CAR is operably linked to a promoter, and the polynucleotide molecule further comprises a nucleotide sequence encoding a heterologous signal peptide fused to a signal regulatory protein gamma (SIRPγ)-associated protein (SGRP). The present invention also relates to a chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule and a method for treating cancer using the polynucleotide molecule and/or the chimeric antigen receptor (CAR)-T cell expressing the polynucleotide molecule. Glioblastoma (GBM) is an aggressive, malignant primary brain tumor resistant to current standard of care (SOC). Complete surgical resection is often impossible due to the difficult-to-access localization and invasive nature of GBM. Furthermore, chemotherapy and radiation therapy regimens are not curative and invariably lead to recurrent disease. With a survival time of approximately 15 months for GBM patients, this highlights the need for a significant breakthrough in effective medical treatment 1-3 . While chimeric antigen receptor (CAR) T cell-based immunotherapy has demonstrated remarkable results in the clinical treatment of hematological cancers, developing effective CAR T cell therapies for solid tumors remains a challenging task 4,5 . A critical limitation of CAR T cells is the lack of known tumor-specific surface antigens and their heterogeneous expression profiles within the GBM 6. One of the most well-studied target antigens in the GBM is EGFRvIII, a tumor-specific form of EGFR mutation expressed in approximately 40% of GBM cases 7,8 . Although EGFRvIII mutations are expressed only in tumor cells, they occur in conjunction with EGFR amplification during clonal evolutionary events in GBM development, resulting in the formation of EGFRvIII mosaic tumors 8,9 . A clinical trial of anti-EGFRvIII CAR T cells for relapsed GBM (NCT01454596) demonstrated safety and transient efficacy, but failed to elicit a long-term therapeutic response due to adaptive resistance and antigen evasion 10 . Conversely, targeting more uniformly expressed GBM-related antigens is controversial due to the potential risk of toxicity caused by on-target/off-tumor cross-reaction 11,12 . Immuno-checkpoint inhibitors have recently shown promising responses in solid tumors 13. However, the highly immunosuppressive tumor microenvironment (iTME) of the GBM severely limits the efficacy of immune checkpoint blockades (ICBs) 14. Therefore, to effectively target these tumors with immunotherapeutic approaches, it is important to understand the complex context-dependent interactions between the GBM and the surrounding iTME 15 . The most important and numerous immune cells residing in the GBM are pro-inflammatory and proliferative brain-resident microglia, peripheral monocyte-derived macrophages, polymorphonuclear myeloid-derived suppressor cells, and Tregs 16,17 . Glioma-associated macrophages and microglia (GAMs), which are the major immune cell populations within the GBM-iTME, contribute substantially to GBM progression 11. Microglia are the brain's specialized phagocytic cells. They play a crucial role in the brain's innate immune surveillance and exert a powerful influence on outcomes and responses to pathological conditions through the secretion of cytokines, chemokines, and growth factors 18,19 . The phagocytic activity of both microglia and macrophages is regulated particularly through the CD47-SIRPα phagocytic axis, in which SIRPα expressed on the surfaces of microglia and macrophages interacts with the widely expressed CD47 transmembrane protein to inhibit phagocytosis 20,21 . Therefore, CD47 is an innate immune checkpoint utilized by tumor cells as a 'don't eat me' signal, which leads to immune evasion by tumor cells through reduced recognition by phagocytes 11,22 . It has been demonstrated that CD47 blockade induces a potent in vivo anti-tumor response by restoring GAM phagocytosis in GBM-transplanted mice 23-25 . However, clinical studies of systemic monotherapy using CD47 blockade have only recently begun evaluating efficacy against solid tumors and are showing promising clinical activity 26,27 . Data published from these clinical trials suggest overall safety and significant activity, but at the same time, low intratumoral bioavailability and treatment-related toxicity are also reported 28 . Therefore, current treatment options for GBM are far from satisfactory, and there is still a high medical need to provide effective treatments for patients. The present invent