KR-20260064752-A - Chimeric ILT receptor composition and method

Abstract

A chimeric ILT receptor (CIR) is provided, comprising a targeting region from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD). The ICD comprises a signaling region (e.g., CD3 zeta (CD3ζ), DAP10, DAP12) and optionally a co-stimulation region (e.g., MyD88, TIR domain, etc.). Additionally, a nucleic acid encoding the target CIR (e.g., an expression vector) and genetically modified cells expressing the target CIR (e.g., immune cells such as NK cells, NK-T cells, T cells, iNKT cells, macrophages, etc.) are provided.

Inventors

• 베일 조셉 헨리
• 드엉 미린 티
• 박 지현
• 오그나르 라파엘
• 웨인-홉슨 사이먼

Assignees

• 엔킬트 테라퓨틱스 인코포레이티드

Dates

Publication Date

20260507

Application Date

20240725

Priority Date

20230728

Claims (20)

1. As a chimeric receptor protein, (a) a targeting region targeting HLA-G, comprising the D1-D2 extracellular domains of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) As an intracellular domain (ICD), it includes a signaling region capable of transmitting a signal into an immune effector cell immediately after binding to HLA-G of the targeting region to derive effector cell function, and A chimeric receptor protein comprising a domain in which the signaling region comprises a co-stimulation region comprising a MyD88 polypeptide.
2. A chimeric receptor protein according to claim 1, wherein the MyD88 polypeptide comprises an amino acid sequence having 85% or more sequence identity with the sequence of SEQ ID NO. 27.
3. A chimeric receptor protein according to claim 1 or 2, wherein the MyD88 polypeptide is fused to a CD40, 4-1BB, or HVEM co-stimulation domain.
4. In paragraph 3, the chimeric receptor protein in which the MyD88 polypeptide is fused to a 4-1BB co-stimulation domain.
5. A chimeric receptor protein according to any one of claims 1 to 4, wherein the signaling domain comprises a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof.
6. In paragraph 4, the chimeric receptor protein, wherein the signaling region contains a CD3ζ signaling domain.
7. A chimeric receptor protein according to claim 1 or 2, wherein the signaling region comprises a DAP12 signaling domain.
8. In claim 7, a chimeric receptor protein in which the signaling region does not include a CD3ζ signaling domain.
9. A chimeric receptor protein according to claim 1 or 2, wherein the signaling domain comprises a DAP12 signaling domain and a CD3ζ signaling domain.
10. A polypeptide as an intracellular domain (ICD) polypeptide comprising a signaling region capable of transmitting a signal to an immune effector cell to derive effector cell function, wherein the signaling region comprises (i) a CD3ζ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a co-stimulation region comprising a Toll/Interleukin-1 Receptor/Resistance Protein (TIR) domain.
11. In claim 10, an ICD polypeptide in which the TIR domain is a TLR2 TIR domain, a TLR3 TIR domain, or an IL18R1 TIR domain.
12. In claim 10, an ICD polypeptide comprising an amino acid sequence in which the TIR domain has at least 85% sequence identity with the TLR2 TIR domain of SEQ ID NO. 111.
13. An ICD polypeptide according to claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR3 TIR domain of SEQ ID NO. 113.
14. In claim 10, an ICD polypeptide comprising an amino acid sequence in which the TIR domain has at least 85% sequence identity with the IL18R1 TIR domain of SEQ ID NO. 109.
15. An ICD polypeptide according to any one of claims 10 to 15, wherein the signal transduction region comprises the CD3ζ signal transduction domain.
16. In any one of claims 10 to 15, the co-stimulating polypeptide, (a) a targeting region targeting HLA-G, comprising the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) The above ICD polypeptide ICD polypeptide composed of a chimeric receptor protein including
17. As a chimeric receptor protein, (a) a targeting region targeting HLA-G, comprising the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) As an intracellular domain (ICD), it includes a signaling region capable of transmitting a signal into the immune effector cell immediately after binding to HLA-G of the targeting region to derive effector cell function, and A chimeric receptor protein comprising a signaling domain that includes a DAP10 signaling domain or a DAP12 signaling domain.
18. A chimeric receptor according to claim 17, wherein the DAP10 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO. 4.
19. A chimeric receptor according to claim 17, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO. 4.
20. A chimeric receptor according to any one of claims 17 to 19, wherein the signaling region further comprises a CD3ζ signaling domain.

Description

Chimeric ILT receptor composition and method Cross-reference The present application claims priority to U.S. Provisional Application No. 63/516,288 filed July 28, 2023, and U.S. Provisional Application No. 63/607,881 filed December 8, 2023, the full text of which is incorporated herein by reference. Inclusion by reference to a sequence list provided as an XML file A sequence list is provided herein as "NKLT-002WO_SEQ_LIST.xml", a sequence list XML with a size of 141,995 bytes, created on July 19, 2024. The full contents of the sequence list XML are incorporated herein by reference. I. Introduction Immunotherapy is useful for specifically targeting diseased cells. Such treatments may have potential curative effects for both malignant and non-malignant pathologies. For example, donor lymphocyte infusion, allogeneic T cells, and allogeneic Natural Killer (NK) cells can be used to control the outgrowth of leukemia. Additionally, genetic modifications can direct the specificity of immune cells, including T cells, Natural Killer (NK) cells, γ/d T cells, iNK-T cells, and macrophages, toward specific target cell populations for therapeutic purposes. For example, Chimeric Antigen Receptor (CAR-) T cells can be used to redirect T cell specificity toward tumor-associated cell surface molecules independent of the presentation of peptide antigens to the T cell receptor (TCR). CAR proteins can also be designed to be expressed in other immune cells, including NK cells, γ/d T cells, iNK-T cells, and macrophages. In these cases, CAR-mediated recognition of target proteins concentrates the cytotoxic potential of these cells, which typically use so-called innate receptors to recognize foreign substances or diseased tissues. Numerous preclinical and clinical studies have demonstrated the utility of CAR technology expressed in T cells, NK cells, iNK-T cells, and macrophages, resulting in the market approval of six therapies for B-cell-derived tumors. Modifications of CAR technology that alter the binding mechanism for target recognition can improve the affinity and specificity of engineered cells toward target proteins. For example, the antibody:antigen binding mechanism present in CAR proteins can be replaced with a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein, which is a modified or natural protein for expression in immune cells to recognize multiple ligands (MICA, MIC-B, ULBP 1 to 5) for NKG-2D that can be expressed in diseased tissues in various combinations. Further examples described in this patent are the expression of chimeric ILT receptors or CIRs engineered to target HLA-G expressed in tumors. CIR proteins are engineered to have a binding moiety derived from ILT2 or ILT4, which are major receptors for immunosuppressive HLA-G. Binding of the natural receptor domain (ILT2 or ILT4) to HLA-G improves targeting of naturally expressed HLA-G as seven protein isoforms derived from alternative mRNA splice products. Antibody/scFv CAR approaches for HLA targeting may be limited by the loss of epitopes in one or more HLA-G isoforms. T cells are the most commonly used cell vehicles in targeted anticancer CAR therapy. Due to the graft-versus-host disease (GvHD) risk associated with allogeneic T cell products, these cells are generally used as autologous or patient-derived products. Cell types lacking TCR expression can mitigate the risk of autologous GvHD, reduce production costs, and be delivered to patients more quickly. Increasingly, anticancer cell products are being developed from alternative cell types, including NK cells, iNK-T cells, and macrophages. NK cell and iNK-T CAR-based products aim for target-specific cell activation by frequently incorporating mechanisms that promote IL-15 signaling, often utilizing CD28.CD3ζ or 4-1BB.CD3ζ signaling strategies. While these constructs promote target-specific NK cell and iNK-T cell activation, CAR mechanisms are designed to mimic T cell activation by APCs, particularly dendritic cells, whereas NK cells and iNK-T cells are activated by mechanisms distinct from T cells. This raised the possibility that NK cell-based therapies using chimeric proteins may not achieve optimal performance with existing CAR-T cell construct designs. In particular, it is necessary to improve NK cell activation to promote the sustainability of NK cell products, facilitate the eradication of the patient's heavy tumor burden, and maintain surveillance for tumor evasion and recurrence. II. Overview Modifications of CAR technology that alter the binding mechanism for target recognition can improve the affinity and specificity of engineered cells toward target proteins. For example, the antibody:antigen binding mechanism present in CAR proteins can be replaced with a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein, which is a modified or natural protein for expression in immune cells to recognize multiple ligands (MICA, MIC-B, ULBP 1