ADGRE2 Chimeric Receptor NK Cell Composition and Method of Use

Abstract

This application provides umbilical cord blood-derived natural killer (CB-NK) cells engineered to express a chimeric receptor targeting ADGRE2. Pharmaceutical compositions, kits, and methods for treating cancer are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 380,896, filed on 25 October 2022, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, which was filed electronically in ST26(XML) format and is incorporated herein by reference in its entirety. The above ST26(XML) copy, created on 23 October 2023, is named MIL-023WO1_SL.xml and is 18,127,008 bytes in size. [Background technology]

[0003] The ADGRE2 antigen-binding domain binds to adhesion G protein-coupled receptor E2, namely the ADGRE2 antigen, also known as EMR2, CD312, VBU, or CD97. ADGRE2 is expressed on monocytes, macrophages, dendritic cells, and granulocytes. ADGRE2 is expressed on cancer cells, including acute myeloid leukemia (AML) cells. Cell-based immunotherapies (e.g., chimeric antigen receptor (CAR) T-cell therapy) have shown efficacy in treating certain cancers. However, challenges remain, including systemic cytotoxicity, target antigen-negative relapse, and logistical hurdles to produce autologous cell-based products. The need for alternative targets or targets for cell-based immunotherapies remains. [Overview of the Initiative]

[0004] The present invention provides compositions and methods for cell-based immunotherapy for individuals in need, including cell therapy in which the cells are genetically modified natural killer (NK) cells (e.g., umbilical cord blood-derived natural killer (CB-NK) cells). The modified NK cells express the ADGRE2 chimeric receptor and are particularly effective in treating autoimmune diseases and cancers such as leukemia, lymphoma, myeloma (e.g., relapsed and refractory acute myeloid leukemia).

[0005] In one embodiment, the present invention provides umbilical cord blood-derived natural killer (CB-NK) cells comprising a chimeric receptor including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to ADGRE2.

[0006] In some embodiments, the present invention provides umbilical cord blood-derived natural killer (CB-NK) cells containing a chimeric receptor, comprising an extracellular antigen-binding domain, a costimulatory domain, and a transmembrane domain that bind to ADGRE2.

[0007] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region comprising HCDR1 containing the amino acid sequence GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence ARGFTAYGMDY (SEQ ID NO: 3).

[0008] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 7.

[0009] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 9.

[0010] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 15.

[0011] In some embodiments, the extracellular antigen-binding domain includes a light chain variable region comprising LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0012] In one embodiment, the present invention provides umbilical cord blood-derived natural killer (CB-NK) cells comprising a chimeric receptor including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to ADGRE2, wherein the extracellular antigen-binding domain comprises a heavy chain variable region including HCDR1 containing the amino acid sequence GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region including LCDR1 containing the amino acid sequence SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence QQWSSNPLT (SEQ ID NO: 6).

[0013] In some embodiments, the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 14.

[0014] In some embodiments, the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 12.

[0015] In some embodiments, the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 16.

[0016] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 7, and a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 14.

[0017] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 9, and a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 12.

[0018] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 15, and a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 16.

[0019] In some embodiments, the extracellular antigen-binding domain includes a single-chain variable fragment (scFv).

[0020] In some embodiments, the extracellular antigen-binding domain includes a linker between the heavy chain variable region and the light chain variable region.

[0021] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 24.

[0022] In some embodiments, the heavy chain variable region and the light chain variable region are arranged in a VH-VL configuration from the N-terminus to the C-terminus.

[0023] In some embodiments, the extracellular antigen-binding domain includes an scFv which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 19, 20, or 68.

[0024] In some embodiments, the extracellular antigen-binding domain includes an scFv which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 19.

[0025] In some embodiments, the extracellular antigen-binding domain includes an scFv which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 20.

[0026] In some embodiments, the extracellular antigen-binding domain includes an scFv which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 68.

[0027] In some embodiments, the transmembrane domain includes a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-I polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide.

[0028] In some embodiments, the transmembrane domain contains a CD28 polypeptide. In some embodiments, the transmembrane domain contains a CD8 polypeptide.

[0029] In some embodiments, the intracellular domain further includes at least one co-stimulatory signaling region.

[0030] In some embodiments, at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a DAP-12 polypeptide, an FcεRIγ polypeptide, a CD160 polypeptide, an NTB-A polypeptide, or a combination thereof.

[0031] In some embodiments, at least one co-stimulatory signaling region comprises a CD28 polypeptide or a DAP-10 polypeptide.

[0032] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule.

[0033] In some embodiments, the chimeric receptor is a CAR.

[0034] In some embodiments, the chimeric receptor is a CAR, and the intracellular domain contains a CD3ζ polypeptide.

[0035] In some embodiments, the CD3ζ polypeptide is a modified CD3ζ polypeptide.

[0036] In some embodiments, the modified CD3ζ polypeptide comprises the native ITAM1, an ITAM2 variant containing two loss-of-function mutations, and an ITAM3 variant containing two loss-of-function mutations.

[0037] In some embodiments, natural ITAM1 comprises the amino acid sequence described in SEQ ID NO: 63.

[0038] In some embodiments, the ITAM2 variant includes the amino acid sequence described in SEQ ID NO: 65.

[0039] In some embodiments, the ITAM3 variant includes the amino acid sequence described in SEQ ID NO: 67.

[0040] In some embodiments, the modified CD3ζ polypeptide comprises the amino acid sequence described in SEQ ID NO: 63.

[0041] In some embodiments, the chimeric receptor is a CCR.

[0042] In some embodiments, CB-NK cells further include a second chimeric receptor that binds to a second target.

[0043] In some embodiments, the chimeric receptor that binds to ADGRE2 is a chimeric antigen receptor (CAR), and the second chimeric receptor is a chimeric costimulatory receptor (CCR).

[0044] In some embodiments, CB-NK cells include a chimeric antigen receptor (CAR) that binds to ADGRE2 and a second chimeric receptor that is either a chimeric antigen receptor (CAR) or a chimeric costimulatory receptor (CCR).

[0045] In some embodiments, the second chimeric receptor binds to CD123.

[0046] In one embodiment, the present invention provides a nucleic acid encoding a chimeric receptor.

[0047] In some embodiments, cells are transduced with nucleic acids encoding chimeric receptors.

[0048] In some embodiments, the chimeric receptor is constitutively expressed on the surface of the cell.

[0049] In some embodiments, the nucleic acid molecule further includes a promoter operably linked to the chimeric receptor.

[0050] In some embodiments, the promoter is either endogenous or exogenous.

[0051] In some embodiments, the exogenous promoter is selected from the group consisting of the elongation factor (EF)-1 promoter, the cytomegalovirus pre-initial promoter (CMV) promoter, the Simianvirus 40 initial promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, the metallothionein promoter, and the ubiquitin C promoter.

[0052] In some embodiments, the promoter is an induceable promoter.

[0053] In some embodiments, the inducible promoter is selected from the group consisting of the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, the IL-2 promoter, the 4-1BB promoter, the PD1 promoter, and the LAG3 promoter.

[0054] In some embodiments, the promoter is an endogenous promoter.

[0055] In some embodiments, the endogenous promoter is selected from the TCR alpha promoter, the TCR beta promoter, and the beta 2-microglobulin promoter.

[0056] In some embodiments, CB-NK cells further express exogenous IL-15.

[0057] In one embodiment, the present invention provides a composition comprising CB-NK cells expressing one or more chimeric receptors described herein.

[0058] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0059] In some embodiments, the pharmaceutical composition is approximately 20 × 10 6 ~Approx. 150×10 7 Contains 1 ADGRE2 CB-NK cell.

[0060] In some embodiments, the pharmaceutical composition is approximately 20 × 10 6 pieces, approximately 100×10 6 1, or approximately 500 x 10 6 1 piece, or approximately 150 x 10 7 Contains 1 ADGRE2 CB-NK cell.

[0061] In one embodiment, the present invention provides a method for reducing tumor volume in a subject, comprising administering ADGRE2 CB-NK cells as described herein to the subject.

[0062] In some embodiments, administration of ADGRE2 CB-NK cells by this method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

[0063] In one embodiment, the present invention provides a method for increasing or extending the survival of a subject having a tumor, comprising administering ADGRE2 CB-NK cells or a composition comprising them to the subject.

[0064] In one embodiment, the present invention provides a method for treating and / or preventing a tumor in a subject, comprising administering to the subject ADGRE2 CB-NK cells or a composition comprising them.

[0065] In some embodiments, the tumor expresses ADGRE2.

[0066] In some embodiments, the tumor is cancer.

[0067] In some embodiments, the tumor is a blood cancer.

[0068] In some embodiments, the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancies.

[0069] In some embodiments, leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotypic acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia.

[0070] In some embodiments, the leukemia is acute myeloid leukemia (AML).

[0071] In some embodiments, AML is relapsed / refractory acute myeloid leukemia (R / R AML).

[0072] In some embodiments, myeloid malignancies are selected from the group consisting of myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), myeloid / lymphoid neoplasms, myeloid / lymphoid neoplasms with eosinophilia and rearrangement of platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth factor receptor 1 (FGFR1), or PCM1-JAK2, acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasms, B lymphoblastic leukemia / lymphoma, and T lymphoblastic leukemia / lymphoma.

[0073] In some embodiments, myeloid malignancies include myelodysplastic syndrome (MDS).

[0074] In some embodiments, the subject is a human subject.

[0075] In one embodiment, the present invention provides a method for producing CB-NK cells as described herein, comprising introducing a nucleic acid molecule encoding a chimeric receptor into cells.

[0076] In one embodiment, the present invention provides umbilical cord blood-derived natural killer (CB-NK) cells comprising a chimeric receptor including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to ADGRE2, wherein the extracellular antigen-binding domain comprises a heavy chain variable region including HCDR1 containing the amino acid sequence GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region including LCDR1 containing the amino acid sequence SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence QQWSSNPLT (SEQ ID NO: 6).

[0077] In one embodiment, the present invention provides natural killer (NK) cells comprising a chimeric receptor including an extracellular antigen-binding domain that binds to ADGRE2, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain comprises a heavy chain variable region including HCDR1 containing the amino acid sequence GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region including LCDR1 containing the amino acid sequence SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence QQWSSNPLT (SEQ ID NO: 6), and the NK cells further express exogenous IL-15.

[0078] In some embodiments, the present invention relates to umbilical cord blood natural killer (CB-NK) cells comprising a chimeric receptor and a nucleic acid encoding an exogenous IL-15 polypeptide, wherein the chimeric receptor is: a) A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), An extracellular antigen-binding domain that binds to ADGRE2, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6); b) Hinge domain containing CD28 hinge polypeptide c) A transmembrane domain containing the CD28 transmembrane polypeptide; d) Co-stimulatory domains containing DAP10 polypeptide; and e) Contains an intracellular domain which is a CD3ζ polypeptide.

[0079] In some embodiments, the CD28 hinge polypeptide includes SEQ ID NO: 53.

[0080] In some embodiments, the CD28 transmembrane polypeptide includes SEQ ID NO: 54.

[0081] In some embodiments, the DAP-10 polypeptide includes SEQ ID NO: 56.

[0082] In some embodiments, the CD3ζ polypeptide includes SEQ ID NO: 55.

[0083] In some embodiments, the IL-15 polypeptide includes SEQ ID NO: 54.

[0084] In some embodiments, the present invention relates to umbilical cord blood natural killer (CB-NK) cells comprising a chimeric receptor and a nucleic acid encoding an exogenous IL-15 polypeptide, wherein the chimeric receptor is: a) A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), An extracellular antigen-binding domain that binds to ADGRE2, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6), b) Hinge domain containing CD8 hinge polypeptide, c) Transmembrane domain containing CD8 transmembrane polypeptide, d) Co-stimulatory domains containing DAP10 polypeptide, and e) Contains an intracellular domain which is a CD3ζ polypeptide.

[0085] In some embodiments, the CD8 hinge polypeptide includes SEQ ID NO: 69.

[0086] In some embodiments, the CD8 transmembrane polypeptide includes SEQ ID NO: 70.

[0087] In some embodiments, the DAP-10 polypeptide includes SEQ ID NO: 56.

[0088] In some embodiments, the CD3ζ polypeptide includes SEQ ID NO: 55.

[0089] In some embodiments, the IL-15 polypeptide includes SEQ ID NO: 54.

[0090] In some embodiments, the present invention relates to umbilical cord blood NK cells comprising a chimeric antigen receptor that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NOs: 48, 72, 75, 76, 79, or 80.

[0091] In some embodiments, the chimeric antigen receptor includes SEQ ID NO: 48.

[0092] In some embodiments, the present invention relates to umbilical cord blood natural killer (CB-NK) cells comprising a chimeric receptor and a nucleic acid encoding an exogenous IL-15 polypeptide, wherein the chimeric receptor is: (a) A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), An extracellular antigen-binding domain that binds to ADGRE2, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6); (b) Hinge domain containing CD8 polypeptide, (c) Transmembrane domains containing CD8 polypeptide; and (d) Co-stimulatory domain containing OX-40 polypeptide, Includes.

[0093] In some embodiments, the CD8 hinge polypeptide includes SEQ ID NO: 69.

[0094] In some embodiments, the CD8 transmembrane polypeptide includes SEQ ID NO: 70.

[0095] In some embodiments, the present invention relates to umbilical cord blood natural killer (CB-NK) cells comprising a chimeric receptor and a nucleic acid encoding an exogenous IL-15 polypeptide, wherein the chimeric receptor is: (a) A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), An extracellular antigen-binding domain that binds to ADGRE2, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6); (b) Hinge domain containing CD28 polypeptide, (c) Transmembrane domain containing CD28 polypeptide; and (d) Co-stimulatory domain containing OX-40 polypeptide, Includes.

[0096] In some embodiments, the CD28 hinge polypeptide includes SEQ ID NO: 53.

[0097] In some embodiments, the CD28 transmembrane polypeptide includes SEQ ID NO: 54.

[0098] In some embodiments, the OX40 polypeptide includes SEQ ID NO: 71.

[0099] In some embodiments, the IL-15 polypeptide includes SEQ ID NO: 54.

[0100] In some embodiments, the present invention relates to umbilical cord blood cells containing a chimeric antigen receptor that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NOs.

[0101] In some embodiments, the present invention provides nucleic acids encoding chimeric receptors and exogenous IL-15 polypeptides, the chimeric receptor comprising an extracellular antigen-binding domain that binds to ADGRE2, comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence GYTFTNYW (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence VYPGDGDT (SEQ ID NO: 2), and HCDR3 comprising the amino acid sequence ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region comprising LCDR1 comprising the amino acid sequence SSVSY (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence DTS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQWSSNPLT (SEQ ID NO: 6).

[0102] definition A or An: The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the articles. For example, "an element" means one or more elements.

[0103] Affinity: As used herein, the term "affinity" refers to the characteristics of the binding interaction between a binding moiety (e.g., an antigen-binding moiety (e.g., a variable domain described herein) and / or an Fc receptor-binding moiety (e.g., an FcRn-binding moiety described herein)) and a target (e.g., an antigen (e.g., ADGRE2) and / or an FcR (e.g., FcRn)), which indicates the strength of the binding interaction. In some embodiments, the measure of affinity is represented as the dissociation constant (K D ). In some embodiments, the binding moiety has a high affinity for the target (e.g., less than about 10 -7 M, less than about 10 -8 M, or less than about 10 -9 M of K D ). In some embodiments, the binding moiety has a low affinity for the target (e.g., higher than about 10 -7 M, higher than about 10 -6 M, higher than about 10 -5 M, or higher than about 10 -4 M of K D ). In some embodiments, the binding moiety has a high affinity for the target at a first pH, a low affinity for the target at a second pH, and an intermediate affinity for the target at a pH level between the first pH and the second pH.

[0104] Approximately or about: As used herein, when applied to one or more target values, the term "approximately" or "about" refers to a value similar to the referenced value. In certain embodiments, the term "approximately" or "about" refers to a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the referenced value, unless otherwise specified or apparent from the context (except when such a number exceeds 100% of the possible values).

[0105] Antibody: As used herein, the term “antibody” refers to a polypeptide comprising an amino acid sequence that provides at least one immunoglobulin variable region, e.g., an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody may comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may comprise two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments), as well as complete antibodies, e.g., intact immunoglobulins of type (and their subtypes) IgA, IgG, IgE, IgD, and IgM. The light chain of an immunoglobulin may be of kappa or lambda type.

[0106] Binding moiety: As used herein, “binding moiety” is any molecule or part of a molecule that can specifically bind to a target, for example, a target of interest (e.g., an antigen (e.g., ADGRE2) and / or an FcR (e.g., FcRn)). Examples of binding moieties include antibodies, their antigen-binding fragments, Fc regions or their Fc fragments, antibody mimetic compounds, peptides, and aptamers.

[0107] An antigen-binding fragment or antibody fragment refers to a portion of an intact antibody. An antigen-binding fragment or antibody fragment refers to a portion of an intact antibody that binds to an antigen (e.g., ADGRE2). The antigen-binding fragment may contain the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, antibody mimes, scFv, and single-chain antibodies.

[0108] Complementarity-Determining Regions (CDRs): The “CDR” of a variable domain is an amino acid residue within a variable region identified according to Kabat, Chothia, or both Kabat and Chothia's accumulations, i.e., AbM, contact and / or conformation definitions, or any method of CDR determination known in the art. The CDR of an antibody may be identified as a hypervariable region, first defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, for example, Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contact, described in MacCallum et al., J.Mol.Biol., 262:732-745, 1996. Another approach, referred to herein as the "conformation definition" of CDRs, allows for the identification of the CDR's location as a residue that contributes enthalpy to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1 156-1166, 2008. Furthermore, other CDR boundary definitions may not strictly adhere to one of the approaches described above, and may be shortened or extended in light of predictions or experimental findings that specific residues, groups of residues, or even the entire CDR do not significantly affect antigen binding, but still overlap with at least some part of Kabat's CDR.As used herein, CDR may refer to a CDR defined by any approach known in the art, including combinations of approaches. Methods used herein may utilize a CDR defined according to any of these approaches. For any given embodiment including more than one CDR, a CDR may be defined according to the definitions of Kabat, Chothia, extension, AbM, contact, and / or conformation.

[0109] Constant Region: As used herein, the term “constant region” refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. A constant region may include any or all of the following immunoglobulin domains: CH1 domain, hinge region, CH2 domain, CH3 domain (derived from IgA, IgD, IgG, IgE, or IgM), and CH4 domain (derived from IgE or IgM).

[0110] Epitope: As used herein, “epitope” is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be, for example, a polypeptide or two or more discontinuous regions of a polypeptide joined together (conformational, nonlinear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which the antibody binds may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scan assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software well-known in the art, such as Refmac and Phenix. Mutagenesis mapping studies can be achieved using any method known to those skilled in the art. For example, for a description of mutational mutation techniques, including alanine scanning mutational mutation techniques, please refer to Champe M et al, (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0111] Fc region: As used herein, the term “Fc region” refers to a dimer of two “Fc polypeptides,” each “Fc polypeptide” containing the constant region of an antibody excluding the first constant region immunoglobulin domain. In some embodiments, the “Fc region” contains two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. The “Fc polypeptide” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, as well as the last three constant region immunoglobulin domains of IgE and IgM, which may include some or all of the N-terminal flexible hinge to these domains. In the case of IgG, the “Fc polypeptide” includes the immunoglobulin domains C-gamma 2 (Cγ2) and C-gamma 3 (Cγ3), as well as the lower part of the hinge between C-gamma 1 (Cγ1) and Cγ2. While the boundaries of Fc polypeptides can vary, human IgG heavy chain Fc polypeptides are typically defined as containing residues from T223 or C226 or P230 to their carboxyl terminus, and numbering follows the EU index by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). In the case of IgA, the Fc polypeptide includes the immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower part of the hinge between C alpha 1 (Cα1) and Cα. The Fc region may be synthetic, recombinant, or generated from natural sources such as IVIG.

[0112] Genetic manipulation or genetic modification: As used herein, the terms “genetic manipulation” or “genetic modification” refer to adding additional genetic material in the form of DNA or RNA, such as DNA or RNA encoding the ADGRE2-targeted CAR polypeptide of the present invention, to the total genetic material within a cell. The terms “genetically modified cell,” “modified cell,” and “cell genetically engineered or modified to express” are used interchangeably.

[0113] Humanized antibody: As used herein, a humanized antibody is an antibody derived from a non-human species in which the protein sequence has been modified to increase their similarity to antibody variants naturally produced in humans. "Humanization" is typically applied to monoclonal antibodies developed for administration to humans (e.g., anti-ADGRE2 antibodies developed as anticancer drugs). In some embodiments, humanization is used when developing specific antibodies in a non-human immune system (e.g., antibodies in mice).

[0114] K a :When used in this specification, "K a " refers to the association rate between a specific binding site and the target for forming a binding site / target complex.

[0115] K d :When used in this specification, "K d " refers to the dissociation rate of a specific binding site / target complex.

[0116] K D :When used in this specification, "K D " refers to the dissociation constant, which is K d K a The ratio to (i.e., K d / K a It is obtained from ) and expressed as molar concentration (M). K D The value can be determined using methods well established in the art, for example, by using surface plasmon resonance or by using a biosensor system such as the Biacore® system.

[0117] Reference: The "reference" entity, system, quantity, set of conditions, etc., are compared to the test entity, system, quantity, set of conditions, etc., as described herein. For example, in some embodiments, the "reference" antibody is a control antibody that has not been manipulated as described herein.

[0118] Selective binding: As used herein, “selective binding,” “selectively binding,” “specific binding,” or “specifically binding” means, with respect to the binding portion and the target, the preferential association of the binding portion to the target and not to an entity that is not the target. Some degree of nonspecific binding may occur between the binding portion and the non-target. In some embodiments, the binding portion selectively binds to the target if the binding between the binding portion and the target is more than 2 times, more than 5 times, more than 10 times, or more than 100 times compared to the binding between the binding portion and the non-target. In some embodiments, the binding affinity is about 10 -5 Less than M, approximately 10 -6 Less than M, approximately 10 -7 Less than M, approximately 10 -8 Less than M, or about 10 -9 If the M is less than M, the binding site selectively binds to the target. In some embodiments, the molecule that specifically binds to the antigen may bind to other peptides or polypeptides with generally lower affinity, when determined by, for example, immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.

[0119] Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment" or "scFv" means V H ::VL heavy chains of immunoglobulins (e.g., mouse or human) covalently bonded to form a heterodimer (VL) H ) and light chain (V L This refers to a fusion protein of the variable region of the heavy chain (V). H ) and light chain (V L ) are directly joined or V H The N-terminus of V L At the C-terminus, or V H The C-terminus of V LThe extracellular antigen-binding domain is linked to the N-terminus by a peptide code linker (e.g., 10, 15, 20, or 25 amino acids). Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. Linkers can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917(2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety.

[0120] Subject: As used herein, the term “subject” means any subject to which diagnosis, prognosis, or treatment is desired. For example, a subject may be a mammal, such as a human or non-human primate (e.g., ape, monkey, orangutan, or chimpanzee), a dog, a cat, a guinea pig, a rabbit, a rat, a mouse, a horse, a cattle, or a dairy cow.

[0121] Target: As used herein, “target” is any molecule to which an antibody’s binding portion or its antigen-binding fragment is specifically bound. In some embodiments, the target is an antigen described herein (e.g., ADGRE2). The terms “first target” and “second target” are used herein to refer to two molecules of different molecular species, rather than two molecules of the same molecular species. For example, in some embodiments, the first target is ADGRE2 and the second target is CD123.

[0122] Therapeutic Dose: As used herein, the term “therapeutic dose” refers to the amount of a therapeutic molecule (e.g., the ADGRE2 antigen-binding domain described herein) that gives a therapeutic effect to the subject being treated in a reasonable benefit / risk ratio applicable to any medical treatment. Therapeutic effect may be objective (i.e., measurable by several tests or markers) or subjective (i.e., the subject shows signs of or feels an effect). In particular, “therapeutic dose” refers to the amount of a therapeutic molecule that is effective in treating, improving or preventing a particular disease or condition, or that is effective in demonstrating a detectable therapeutic or preventive effect, for example, by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of the symptoms of the disease. Therapeutic doses may be administered in a dosing regimen that may consist of multiple unit doses. For any particular therapeutic molecule, the therapeutic dose (and / or an appropriate unit dose within an effective dosing regimen) may vary depending, for example, the route of administration, and combinations with other pharmaceuticals. Furthermore, the specific therapeutically effective dose (and / or unit dose) for any particular subject may depend on various factors, including the disorder being treated and its severity; the activity of the specific drug used; the specific composition used; the subject's age, weight, overall health, sex, and diet; the timing of administration, route of administration, and / or rate of elimination or metabolism of the specific therapeutic molecule used; the duration of treatment; and similar factors well known in the medical field.

[0123] Treatment: As used herein, the term “treatment” (similarly “to treat” or “to treat”) refers to any administration of a therapeutic molecule (e.g., the ADGRE2 antigen-binding domain as described herein) that partially or completely reduces, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or characteristics of a particular disease, disorder, and / or condition. Such treatment may be for subjects who do not show signs of the disease, disorder, and / or condition in question, and / or for subjects who show only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who show one or more established signs of the disease, disorder, and / or condition in question.

[0124] The drawings are for illustrative purposes only and are not intended to be restrictive. [Brief explanation of the drawing]

[0125] [Figure 1A] This shows exemplary results of the in vitro cytotoxic activity of ADGRE2 CAR or CCR CB-NK against the acute myeloid leukemia cell line MOLM-13. [Figure 1B] Exemplary results of the in vitro cytotoxic activity of ADGRE2 CAR or CCR CB-NK against the acute myeloid leukemia cell line KG-1a are shown. [Figure 1C] Exemplary results of the in vitro cytotoxic activity of ADGRE2 CAR or CCR CB-NK against the acute myeloid leukemia cell line MV-4-11 are shown. [Figure 2A] This shows exemplary results of the in vivo efficacy of ADGRE2 CAR CCR CB-NK against the acute myeloid leukemia cell line MOLM-13. [Figure 2B] This shows exemplary results of the in vivo efficacy of ADGRE2 CAR or CCR CB-NK against the acute myeloid leukemia cell line KG-1a. [Figure 2C]This shows exemplary results of the in vivo efficacy of ADGRE2 CAR or CCR CB-NK against the acute myeloid leukemia cell line MV-4-11. [Figure 3A] This shows an exemplary co-stimulatory domain of a chimeric receptor examined in the MOLM-13 killing assay. [Figure 3B] This study demonstrates the ability of NK cells engineered with CD123-OX40-CCR to persistently control MOLM-13 tumor cells across numerous diverse tumor rechallenges in donor A. [Figure 3C] This study demonstrates the ability of NK cells engineered with CD123-OX40-CCR to persistently control MOLM-13 tumor cells across numerous diverse tumor rechallenges in donor B. [Modes for carrying out the invention]

[0126] This disclosure is partly based on the discovery that CB-NK cells engineered to express a chimeric receptor that binds to adhesion G protein-coupled receptor E2, ADGRE2 (e.g., human ADGRE2), exhibit target-specific cytotoxicity.

[0127] ADGRE2, also known as EMR2, CD312, VBU, or CD97, is a cell surface receptor that is a member of the adhesion G protein-coupled receptor (GPCR) family. ADGRE2 binds to the chondroitin sulfate moiety of glycosaminoglycan chains and promotes cell adhesion. ADGRE2 is expressed in monocytes, macrophages, dendritic cells, and granulocytes and plays a role in chemotaxis, cell adhesion, and degranulation. In macrophages, ADGRE2 signals via G proteins to promote the release of inflammatory cytokines, including IL-8 and TNF.

[0128] ADGRE2 is highly expressed in cancer cells, including, for example, relapsed and refractory acute myeloid leukemia. In some embodiments, ADGRE2 CB-NK cells are used to treat cancer. In some embodiments, ADGRE2 CB-NK cells are used to treat relapsed and refractory acute myeloid leukemia.

[0129] cell In one embodiment, the present invention provides umbilical cord blood natural killer (CB-NK) cells genetically engineered to express an ADGRE2-targeted chimeric receptor (e.g., a CAR or CCR polypeptide as described herein). CB-NK cells are immune effector cells. "Immune effector cell" means any cell of the immune system having one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC). As described herein, CB-NK cells are transformed with a polynucleotide encoding a chimeric receptor (e.g., a CAR or CCR construct), resulting in the expression of the chimeric receptor on the cell surface. The CB-NK cells described herein exhibit antitumor properties.

[0130] A method for producing CB-NK cells expressing the chimeric receptors envisioned herein is provided. In one embodiment, the method comprises transfecting or transfecting CB-NK cells isolated from an organism so that the CB-NK cells express one or more chimeric receptors envisioned herein. In certain embodiments, CB-NK cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to an organism. In further embodiments, CB-NK cells are first activated and stimulated to proliferate in vitro before being genetically modified to express the chimeric receptors. In this regard, CB-NK cells can be cultured before and / or after genetic modification (i.e., transfecting or transfecting to express the ADGRE2-targeted chimeric receptor envisioned herein).

[0131] For example, in one embodiment, CB-NK cells are transduced with a viral vector encoding the chimeric receptor polypeptide of the present invention. In one embodiment, the viral vector is a viral vector such as a lentiviral vector or an AAV vector. In another example, CB-NK cells are transfected with a nucleic acid molecule encoding the chimeric receptor polypeptide of the present invention, such as mRNA, cDNA, or DNA.

[0132] Cell supply source According to the present invention, in some embodiments, immune effector cells are obtained from a subject to be expanded and genetically modified to express the chimeric receptor polypeptide of the present invention.

[0133] Immune effector cells may be autologous ("self") or non-autologous ("non-self," e.g., allogeneic, syngeneic, or heterogeneous). As used herein, "autologous" means cells derived from the same subject. As used herein, "allogeneic" means cells of the same species but genetically different from the cells being compared. As used herein, "synogeneic" means cells of a different subject that are genetically identical to the cells being compared. As used herein, "heterogeneous" means cells of a different species than the cells being compared. In some embodiments, the cells are autologous.

[0134] In some embodiments, the immune effector cells are NK cells. NK cells may originate from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, or bone marrow. NK cells may originate from umbilical cord blood mononuclear cells. NK cells may be CD56+ NK cells.

[0135] In some embodiments, any immunoeffector cell line available in the art may be used. For example, NK cells may be derived from the NK-92 cell line.

[0136] In some embodiments, cells transduced with polynucleotides encoding the chimeric receptor constructs described herein are expanded. In some embodiments, the cells are expanded by culturing for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days).

[0137] In some embodiments, cells are expanded in a suitable medium containing one or more interleukins that result in a cell increase of at least 100-fold, at least 150-fold, and at least 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) over a 14-day expansion period, as measured by methods such as flow cytometry.

[0138] In some embodiments, the population of modified immune effector cells for cancer treatment includes the ADGRE2-targeted chimeric receptor as intended herein. For example, the population of modified immune effector cells is a population of NK cells.

[0139] Chimeric receptor In one embodiment, the present invention relates to cells expressing an ADGRE2-targeted chimeric receptor polypeptide and a polynucleotide encoding it. The ADGRE2-targeted chimeric receptor comprises an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, and a costimulatory domain that specifically bind to ADGRE2. In some embodiments, the chimeric receptor further comprises at least one intracellular activation / signaling domain. The ADGRE2-targeted chimeric receptor may further comprise one or more additional polypeptides, such as a cytokine (e.g., IL-15). Each component within the chimeric receptor may be linked by one or more linker sequences.

[0140] In some embodiments, the ADGRE2-targeted chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the ADGRE2-targeted chimeric receptor is a chimeric costimulatory receptor (CCR). In some embodiments, the chimeric receptor is a TCR-like fusion molecule.

[0141] Chimeric antigen receptor (CAR) In certain embodiments, the chimeric receptor is a CAR. A CAR is an engineered receptor that implants or confers desired specificity to immune effector cells (e.g., CB-NK cells). In some embodiments, the CAR consists of an extracellular antigen-binding domain (e.g., scFv) which is fused to a transmembrane domain, which is then fused to a cytoplasmic / intracellular signaling domain. In some embodiments, the CAR further includes an intracellular signaling domain from a co-stimulatory molecule (e.g., CD28, DAP10, 4-IBB, ICOS, OX40). In some embodiments, the CAR includes a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is CD28 or DAP10. In some embodiments, the co-stimulatory molecule is CD28. In some embodiments, the co-stimulatory molecule is DAP10. If the CAR includes a co-stimulatory domain, additional signaling is added to the effector cells. In some embodiments, the CAR includes both co-stimulation (e.g., CD28 or 4-IBB) and activation (CD3ζ). In some embodiments, the CAR includes multiple co-stimulatory (e.g., CD28 and 4-IBB) domains and an activation (CD3ζ) signal.

[0142] Chimeric costimulatory receptor (CCR) In certain embodiments, the chimeric receptor is a CCR. The CCRs of this disclosure bind to an antigen (e.g., ADGRE2) and provide a co-stimulatory signal, but do not provide an activation signal on their own. In certain embodiments, the CCR does not contain a CD3ζ polypeptide. The CCR provides a co-stimulatory signal, e.g., a CD28-like signal, in the absence of a native co-stimulatory ligand on the antigen-presenting cell. Combined antigen recognition, i.e., using a CCR in combination with a CAR, can enhance the responsiveness of T cells to dual-antigen-expressing T cells, thereby improving selective tumor targeting. Kloss et al. describe a strategy that integrates combined antigen recognition, split signaling, and critically balanced T cell activation and co-stimulatory intensity to generate T cells that eliminate target cells expressing the combined antigens while preserving cells expressing each antigen individually (the entire content is incorporated by reference, Kloss et al., Nature Biotechnology (2013);31(1):71-75). In this approach, T cell activation requires CAR-mediated recognition of one antigen, while co-stimulation is independently mediated by a CCR specific to a second antigen. To achieve tumor selectivity, the combined antigen recognition approach reduces the efficiency of T cell activation to a level where it becomes ineffective without rescue by simultaneous CCR recognition of the second antigen.

[0143] In certain embodiments, the CCR includes a co-stimulatory signaling region comprising an extracellular antigen-binding domain that binds to an antigen (e.g., ADGRE2), a transmembrane domain, and an intracellular domain or fragment thereof of at least one co-stimulatory molecule. In certain embodiments, the CCR alone does not deliver an activation signal to immune-responsive cells. Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, and NKGD2. In certain embodiments, the co-stimulatory signaling region of the CCR includes an intracellular domain or fragment thereof of a co-stimulatory signaling molecule. In certain embodiments, one co-stimulatory signaling molecule is CD28. In certain embodiments, one co-stimulatory signaling molecule is 4-1BB. In certain embodiments, one co-stimulatory signaling molecule is OX40. In certain embodiments, the co-stimulatory signaling region of the CCR includes an intracellular domain or fragment thereof of a first co-stimulatory signaling molecule and an intracellular domain or fragment thereof of a second co-stimulatory signaling molecule. In certain embodiments, the first and second co-stimulus signaling molecules are CD28 and 4-1BB.

[0144] Similar to CARs, the extracellular antigen-binding domain of CCRs can be a fusion protein with scFv, Fab, F(ab)2, or a heterologous sequence that forms the extracellular antigen-binding domain of the CCR.

[0145] TCR-like fusion molecule In certain embodiments, the chimeric receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HIT-CARs," for example, disclosed in International Patent Application PCT / US19 / 017525, which is incorporated herein by reference in whole), T cell receptor fusion structures (TRuCs) (for example, disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response,” Nature Communications volume 10, Article number: 2087 (2019), which is incorporated herein by reference in whole), synthetic T cell receptors and antigen receptors (STARs) (for example, disclosed in Liu et al. Science Translational Medicine (2021);13(586):eabb5191, which is incorporated herein by reference in whole), and antibody-T cell receptors (AbTCRs) (for example, disclosed in Xu et al. Cell Discovery, which is incorporated herein by reference in whole). Examples include (disclosed in 2018)4:62), and T cell antigen couplers (TACs) (for example, the entirety of which is incorporated by reference, as disclosed in Helsen et al. Nature Communications (2018);9:3049).

[0146] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain including an extracellular antigen-binding domain and a constant domain, and the TCR-like fusion molecule binds to the antigen in an HLA-independent manner. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of native or modified TRAC polypeptide, native or modified TRBC polypeptide, native or modified TRDC polypeptide, native or modified TRGC polypeptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a native or modified TRBC polypeptide. In certain embodiments, the constant domain can form homodimers or heterodimers with another constant domain. In certain embodiments, the antigen-binding chain can bind to a CD3ζ polypeptide. In certain embodiments, when the antigen-binding chain binds to an antigen (e.g., ADGRE2), it can activate the CD3ζ polypeptide bound to the antigen-binding chain. In certain embodiments, activation of the CD3ζ polypeptide can activate immune-responsive cells. In certain embodiments, the TCR-like fusion molecule can integrate with the CD3 complex to provide HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule can be dimerized with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule includes a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding moiety or fragment thereof of an antibody, or an antigen-binding moiety of a TCR.

[0147] In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or more immunoglobulin variable regions. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the heavy chain variable region (VH) of the antibody. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region (VL) of the antibody. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule can dimerize with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the VH of the antibody, and the VH can dimerize with another extracellular antigen-binding domain comprising the VL of the antibody to form a variable fragment (Fv). In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the VL of the antibody, and the VL can dimerize with another extracellular antigen-binding domain comprising the VH of the antibody to form a variable fragment (Fv).

[0148] ADGRE2 antigen-binding domain The antigen-binding domain of the chimeric receptor described herein specifically binds to the ADGRE2 antigen. In some embodiments, the anti-ADGRE2 chimeric receptor binds to human ADGRE2. ADGRE2 has been described in the art, for example, in Uniprot sequence reference Q9UHX3-1. In some embodiments, human ADGRE2 includes an EGF-like 1 domain, an EGF-like 2 domain, an EGF-like 3 domain, an EGF-like 4 domain, an EGF-like 5 domain, and a GPS domain.

[0149] The ADGRE2-binding domain may be any protein (e.g., an ADGRE2 binder) that binds to ADGRE2 or a portion of ADGRE2. The ADGRE2-binding domain may be an antibody that specifically binds to human ADGRE2, or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments that specifically bind to ADGRE2 may be monoclonal antibodies, single-specific antibodies, humanized antibodies, human antibodies, single-chain antibodies, domain-specific antibodies, single-domain antibodies, domain deletion antibodies, scFc fusion proteins, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, CDR-transplanted antibodies, antibody fragments such as Fab, Ftab^ fragments, Fab' fragments, F(ab)2 fragments, Fv fragments, single-chain Fv(scFv) fragments, Fd fragments, dAb fragments, diabodies, nanobodies, bivalent nanobodies, shark variable IgNAR domains, VHH antibodies, camelid antibodies, and minibodies.

[0150] In some embodiments, the appropriate heavy chain constant region is derived from IgG1, IgG2, or IgG4. The light chain may include a light chain variable domain and a light chain constant domain. The light chain constant domain may include either a kappa light chain or a lambda light chain. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain may be further divided into variability regions called complementarity-determining regions (CDRs), which are typically interspersed with more conserved regions called framework regions (FRs). Such heavy chain and light chain variable domains may each include three CDRs and four framework regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. One or more of these may be manipulated as described herein. The amino acid assignments to each domain follow the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989). As used herein, CDR refers to the heavy chain (HCDR1, HCDR2, HCDR3) and the light chain (LCDR1, LCDR2, LCDR3), respectively.

[0151] In some embodiments, the ADGRE2 binder includes three heavy chain complementarity determination regions (HCDRs) namely HCDR1, HCDR2, and HCDR3 in the heavy chain variable region (VH), and / or three light chain complementarity determination regions (LCDRs) namely LCDR1, LCDR2, and LCDR3 in the light chain variable region (VL). In some embodiments, the ADGRE2 binder includes VH and / or VL.

[0152] In some embodiments, anti-ADGRE2 scFv comprises the amino acid sequence described in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 68. In some embodiments, anti-ADGRE2 scFv comprises SEQ ID NO: 19.

[0153] In some embodiments, the anti-ADGRE2 scFv includes VH and VL disclosed in WO2017 / 087800, which are incorporated by reference in whole.

[0154] Embodiments of the present invention include antibodies and antigen-binding fragments comprising CDRs found in the VH and VL domains described herein, which are identified using conventional numbering systems such as IMGT, Kabat, and Chothia numbering systems. Such numbering systems are well known in the art. In certain embodiments, the CDRs are identified or numbered according to the IMGT numbering system.

[0155] An example of an ADGRE2 antigen-binding domain In some embodiments, the ADGRE2 antigen-binding domain or fragment thereof described herein includes the heavy chain variable region (VH) complementarity-determining region (CDR) sequences VH CDR1:GYTFTNYW (SEQ ID NO: 1), VH CDR2:VYPGDGDT (SEQ ID NO: 2), and VH CDR3:ARGFTAYGMDY (SEQ ID NO: 3).

[0156] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7).

[0157] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9).

[0158] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11).

[0159] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13).

[0160] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSAST (SEQ ID NO: 15).

[0161] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17).

[0162] In some embodiments, the heavy chain variable region includes the amino acid sequence QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30).

[0163] In some embodiments, the ADGRE2 antigen-binding domain comprises a variable heavy chain amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NOs: 7, 9, 11, 13, 15, 17, or 30.

[0164] In some embodiments, the ADGRE2 antigen-binding domain includes a heavy chain variable region amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NOs. 7, 9, 11, 13, 15, 17, or 30, and also includes one or more of the VH CDR1, vHCDR2, and / or vHCDR3 sequences described herein.

[0165] In some embodiments, the ADGRE2 antigen-binding domain contains the same heavy chain variable region amino acid sequence as SEQ ID NOs. 7, 9, 11, 13, 15, 17, or 30. In certain embodiments, V H It contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence described in SEQ ID NOs. 7, 9, 11, 13, 15, 17, or 30. For example, V HIt contains amino acid sequences that are approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or homologous to the amino acid sequences described in SEQ ID NOs. In some embodiments, the anti-ADGRE2 antigen binding domain includes 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions for SEQ ID NOs.

[0166] As will be understood by those skilled in the art, any such heavy chain constant domain sequence can be readily combined by molecular biological techniques with any framework region, CDR, or constant domain, or a portion thereof, which may be present in any form of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art, or which may be provided herein or otherwise known in the art, including a constant domain.

[0167] The present invention further provides an ADGRE2 antibody or fragment thereof comprising one or more light chain variable regions containing various designated sequences, including light chain complementarity determining regions LCDR1-3. In various embodiments, a molecule having the designated light chain variable regions is provided together with the heavy chain sequence as discussed above. In certain embodiments, the CDRs are identified according to the IMGH numbering system.

[0168] Accordingly, in one embodiment, the present invention provides an ADGRE2 antigen-binding domain or fragment thereof, comprising a light chain variable region having complementarity-determining region (CDR) sequences of LCDR2 containing the amino acid sequences of SSVSY (SEQ ID NO: 4) and DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0169] In some embodiments, the ADGRE2 antigen-binding domain or a fragment thereof includes an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NOs: 8, 10, 12, 14, 16, 18, or 31, and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NOs: 7, 9, 11, 13, 15, 17, or 30.

[0170] In some embodiments, the VL region includes an amino acid sequence that is at least 95% identical to sequence numbers 8, 10, 12, 14, 16, 18, or 31.

[0171] In some embodiments, the ADGRE2 antigen-binding domain or a fragment thereof It contains a light chain variable region (VL) with the amino acid sequence EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8).

[0172] In some embodiments, the light chain variable includes the amino acid sequence EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10).

[0173] In some embodiments, the light chain variable includes the amino acid sequence EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12).

[0174] In some embodiments, the light chain variable includes the amino acid sequence EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14).

[0175] In some embodiments, the light chain variable includes the amino acid sequence QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16).

[0176] In some embodiments, the light chain variable includes the amino acid sequence EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18).

[0177] In some embodiments, the light chain variable includes the amino acid sequence EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31).

[0178] In some embodiments, the ADGRE2 antigen-binding domain comprises a light chain amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NOs: 8, 10, 12, 14, 16, 18, or 31.

[0179] In some embodiments, the ADGRE2 antigen-binding domain includes a light chain amino acid sequence having at least about 85%, about 90%, about 95%, about 98%, or about 99% sequence identity to SEQ ID NOs: 8, 10, 12, 14, 16, 18, or 31, and also includes one or more of the VL CDR1, vLCDR2, and / or vLCDR3 sequences described herein.

[0180] In some embodiments, the anti-ADGRE2 antibody or a fragment thereof contains the same light chain amino acid sequence as SEQ ID NOs: 8, 10, 12, 14, 16, 18, or 31. In some embodiments, the ADGRE2 antigen binding domain contains 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or two or fewer amino acid substitutions compared to SEQ ID NOs: 8, 10, 12, 14, 16, 18, or 31.

[0181] As will be understood by those skilled in the art, any such light chain CDR sequence can be readily combined by molecular biological techniques with any framework region, CDR, or constant domain, or a portion thereof, that may be present in any form of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art, including any other antibody sequence or domain provided herein or otherwise known in the art, including any constant domain.

[0182] In some embodiments, the ADGRE2 antigen-binding domain described herein comprises a light chain containing any light chain constant domain sequence, for example, a light chain constant sequence known to those skilled in the art. As those skilled in the art will recognize, the light chain constant domain may be a kappa light chain constant domain or a lambda light chain constant domain. In certain embodiments, the light chain constant domain disclosed herein is a kappa light chain constant domain. In various embodiments, the ADGRE2 antigen-binding domain described herein comprises a light chain constant domain.

[0183] Accordingly, in one embodiment, the present invention provides an ADGRE2 antigen-binding domain or fragment thereof, comprising a heavy chain variable region having complementarity-determining region (CDR) sequences of VH CDR1:GYTFTNYW (SEQ ID NO: 1), VH CDR2:VYPGDGDT (SEQ ID NO: 2), and VH CDR3:ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region having complementarity-determining region (CDR) sequences of LCDR2 containing the amino acid sequences of SSVSY (SEQ ID NO: 4) and DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0184] The table below shows exemplary heavy chain variable region (VH) and light chain variable region (VL) sequences of the humanized ADGRE2 antibodies disclosed herein.

[0185] [Table 1-1]

[0186] [Table 1-2]

[0187] In various embodiments, the ADGRE2 antigen-binding domain disclosed herein is a homodimeric monoclonal antibody. In various embodiments, the ADGRE2 antigen-binding domain disclosed herein is a heterodimeric antibody. In various embodiments, the ADGRE2 antigen-binding domain is, for example, a typical antibody, or a diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, scFv, TandAb, scFv, Fab, Fab2, Fab3, F(ab')2, or any combination thereof.

[0188] In some embodiments, the present disclosure provides a fusion protein comprising one or more variable domains or manipulated antibodies, or a portion thereof, as described herein, and one or more additional polypeptides.

[0189] Exemplary single-chain variable fragment In some embodiments, the ADGRE2 binder according to the invention may be an scFv that specifically binds to ADGRE2. A "single-chain Fv" or "scFv" consists of a VH domain and a VL domain of an antibody, where these domains are present in a single polypeptide chain in either direction (e.g., VL-VH or VH-VL). Typically, an scFv may take the form of VH-linker-VL or VL-linker-VH.

[0190] The linker connecting the VH chain and the VL chain may consist of an amino acid sequence of (GGGGS)n (where n is an integer of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the linker contains an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one of sequence numbers 24-27.

[0191] In some embodiments, anti-ADGRE2 scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0192] In some embodiments, the linker includes GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 25).

[0193] In some embodiments, the linker includes GGGGSGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 26).

[0194] In some embodiments, the linker includes GGGGSGGGGSGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 27).

[0195] In some embodiments, the present disclosure provides a single-chain variable fragment. In some embodiments, the scFv is a human scFv. The terms “single-chain variable fragment” or “scFv” are used to refer to V H ::VL heavy chains of immunoglobulins (e.g., mouse or human) covalently bonded to form a heterodimer (VL) H ) and light chain (V L This refers to a fusion protein of the variable region of the heavy chain (V). H ) and light chain (V L ) are directly joined or V H The N-terminus of V L At the C-terminus, or V H The C-terminus of V L The N-terminus of the extracellular antigen-binding domain is linked by a peptide code linker (e.g., 10, 15, 20, or 25 amino acids). Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. Linkers can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917(2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the linker is a G4S linker (GGGGS-SEQ ID NO: 45).

[0196] Alternatively, scFv may be derived from Fab (for example, not from an antibody obtained from a Fab library). In certain embodiments, the ADGRE2 antigen-binding domain or a fragment thereof is Fab. In certain embodiments, the Fab is crosslinked. In certain embodiments, the ADGRE2 antigen-binding domain or a fragment thereof is F(ab)2. Any of the aforementioned molecules can be included in a fusion protein with a heterologous sequence to form an anti-ADGRE2 antigen antibody or its antigen-binding fragment.

[0197] In certain embodiments, the ADGRE2 antigen-binding domain or a fragment thereof is at least about 1 × 10⁻¹⁶ -6 M, at least about 1 × 10 -7M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 The dissociation constant (K) of M D ) binds to ADGRE2 (e.g., human ADGRE2). In certain embodiments, the ADGRE2 binding domain or a fragment thereof is at least about 2 × 10⁻¹⁶ -8 Dissociation constant of M (KK) D ) binds to ADGRE2 (e.g., human ADGRE2). In certain embodiments, the ADGRE2 binding domain or a fragment thereof is approximately 2 × 10 -8 M ~ approx. 8×10 -9 The dissociation constant (K) of M D It binds to ADGRE2 (for example, human ADGRE2).

[0198] In some embodiments, the ADGRE2 antigen-binding domain or a fragment thereof has a dissociation constant (K) of approximately 1 nM to 50 nM, approximately 5 nM to 30 nM, approximately 5 nM to 25 nM, or approximately 8 nM to 20 nM. D ) binds to ADGRE2 (e.g., human ADGRE2). In some embodiments, the ADGRE2 antigen-binding domain or fragment thereof has a dissociation constant (K) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, and at least about 5 nM. D It binds to ADGRE2 (for example, human ADGRE2).

[0199] In some embodiments, anti-ADGRE2 scFv includes a heavy chain variable region and / or a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 68.

[0200] In some embodiments, the ADGRE2 antigen-binding domain or fragment thereof includes conservative sequence modifications (e.g., the ADGRE2 antigen-binding domain or fragment thereof described herein). In some embodiments, conservative sequence modifications are amino acid modifications that do not significantly affect or alter the binding properties of the ADGRE2 antigen-binding domain or fragment thereof of this disclosure (e.g., an antibody or fragment thereof), which include an amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into an anti-ADGRE2 antibody or fragment thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as their charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues within the CDR region can be substituted with other amino acid residues from the same group, and the modified antibody can be tested for retained function. In certain embodiments, one or fewer, two or fewer, three or fewer, four or five or fewer residues within a specific sequence or CDR region are modified.

[0201] In some embodiments, the light and / or heavy chains of anti-ADGRE2 scFv include a signal peptide or signal sequence. The terms signal peptide and signal sequence are used interchangeably herein. In some embodiments, the signal peptide is the amino acid sequence MDWTWRILFLVAAATGAHS (SEQ ID NO: 50), MALPVTALLLPLALLLHA (SEQ ID NO: 51), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 33), MYRMQLLSCIALSLALVTNS (SEQ ID NO: 34), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 35), MALPVTALLLPLALLLHAARP (SEQ ID NO: 36), MKWVTFISLLFSSAYS (SEQ ID NO: 37), MDSKGSSQKGSRLLLLLVVSNLL The amino acid sequence contains at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity with LCQGVVS (SEQ ID NO: 38), MDMRVPAQLLGLLLLWLPDTRC (SEQ ID NO: 28), or MEFGLSWVFLVALLRGVQC (SEQ ID NO: 29).

[0202] In some embodiments, the signal peptide includes MDWTWRILFLVAAATGAHS (SEQ ID NO: 50). In some embodiments, the signal peptide includes MALPVTALLLPLALLLHA (SEQ ID NO: 51). In some embodiments, the signal peptide includes METDTLLLWVLLLWVPGSTG (SEQ ID NO: 33). In some embodiments, the signal peptide includes MYRMQLLSCIALSLALVTNS (SEQ ID NO: 34). In some embodiments, the signal peptide includes METPAQLLFLLLLWLPDTTG (SEQ ID NO: 35). In some embodiments, the signal peptide includes MALPVTALLLPLALLLHAARP (SEQ ID NO: 36). In some embodiments, the signal peptide includes MKWVTFISLLFSSAYS (SEQ ID NO: 37). In some embodiments, the signal peptide includes MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 38). In some embodiments, the signal peptide includes MDMRVPAQLLGLLLLWLPDTRC (SEQ ID NO: 28). In some embodiments, the signal peptide comprises MEFGLSWVFLVALLRGVQC (SEQ ID NO: 29).

[0203] In some embodiments, anti-ADGRE2 scFv is QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK(Sequence ID 19)(scFv "K"); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK(Sequence ID 20)(scFv「B」); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGS GGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21) (scFv “N”); QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK(Sequence ID 22)(scFv"A"); or Contains the amino acid sequence QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSQIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (Sequence ID 68).

[0204] In some embodiments, anti-ADGRE2 scFv comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity with SEQ ID NOs.

[0205] In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 19, 20, 21, 22, or 68. In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 19. In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 20. In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 21. In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 22. In some embodiments, anti-ADGRE2 scFv includes the amino acid sequence described in SEQ ID NO: 68.

[0206] Measurement of the interaction between the binding site and the target The binding properties of the antibodies or fragments thereof described herein (e.g., the ADGRE2 antigen-binding domain described herein) to a target (e.g., ADGRE2) can be measured by methods known in the art, such as: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis, and scanning mutagenesis. The binding interaction between the antibody and ADGRE2 and / or FcRn can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling either of the interacting substances. A change in mass at the binding surface of the BIA chip (indicating a binding event) results in a change in the refractive index of light near the surface. The change in refractive index generates a detectable signal and is measured as an indicator of real-time reactions between biomolecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705; and online materials provided by BIAcore International AB (Uppsala, Sweden). In addition, the KinExA® (bound equilibrium exclusion method) assay, available from Sapidyne Instruments (Boise, Id.), can also be used.

[0207] Information from SPR indicates the binding of the binding portion to the target (e.g., the ADGRE2 antigen-binding domain to ADGRE2) and the equilibrium dissociation constant (K). D ), and K on and K offIt can be used to provide accurate and quantitative measurements of kinetic parameters, including K. Such data can be used to compare different molecules. Information from SPR can also be used in the development of structure-activity relationships (SARs). For example, the kinetic and equilibrium binding parameters of a specific binding site to a target can be evaluated at various pH levels. At a specific pH level, specific binding parameters, such as high affinity, low affinity, and slow K, can be evaluated. off It is possible to identify variant amino acids at a given position that correlate with [the given expression].

[0208] In some embodiments, the Specified provides chimeric receptors that bind to human ADGRE2 with higher affinity than ADGRE2 of another species. In certain embodiments, antibodies or fragments thereof that bind to human ADGRE2 with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more higher affinity than ADGRE2 of another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion, are provided herein. In certain embodiments, a chimeric antigen receptor comprising an ADGRE2 binder that binds to human ADGRE2 binds to an ADGRE2 protein of another species with less than 10%, 15%, or 20% of the binding of the antibody or fragment to the human ADGRE2 protein, as measured, for example, by radioimmunoassay, surface plasmon resonance, or binding equilibrium exclusion.

[0209] Hinge area In certain embodiments, the chimeric receptor (e.g., CAR or CCR) polypeptide of the present invention may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. The chimeric receptor polypeptide may include a hinge domain to ensure an appropriate distance between the antigen-binding domain and the cell surface, or to mitigate steric hindrance that may adversely affect the antigen-binding or effector function of chimeric receptor gene-modified CB-NK cells. For example, the hinge domain can position the antigen-binding domain away from the effector cell surface, enabling appropriate intercellular contact, antigen binding, and activation.

[0210] In some embodiments, the hinge domain is of a specific length, such as 10-50, 10-40, 10-30, 10-20, 10-15, 20-50, 20-40, 20-30, 15-50, 15-45, 15-30, 15-20, 12-20, 12-15, or 15-20 amino acid lengths.

[0211] In some embodiments, the hinge domain is derived from a human CD28 hinge domain. As a non-limiting example, the hinge domain of the chimeric receptor polypeptide of the present invention comprises a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 53: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In certain specific embodiments, the hinge domain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 53.

[0212] In some embodiments, the hinge domain is derived from a human CD8 hinge domain. As a non-limiting example, the hinge domain of the chimeric receptor polypeptide of the present invention comprises a CD8 hinge domain having the amino acid sequence SEQ ID NO: 69:TTTPAPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD. In some embodiments, the hinge domain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 69.

[0213] Alternatively, and by choice, one or more hinge domains or spacer domains derived from other proteins may be used. As used herein, the term “spacer domain” refers to a region that moves the antigen-binding domain away from the effector cell surface, enabling proper intercellular contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6: 412-419). Spacer domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources. For example, hinge domains and spacer domains may be derived from human IgG hinge domains, CD8a hinge domains, or Fc domains of human immunoglobulins that bind to Fc receptors (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE).

[0214] In some embodiments, the ADGRE2-targeted chimeric receptors envisioned herein may include a modified hinge domain and / or spacer domain. The modified hinge domain and / or spacer domain may include a portion of a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions) in length of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) and up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or a portion of a naturally occurring hinge region containing a core hinge region (length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids).

[0215] transmembrane domain In various embodiments, the extracellular antigen-binding domain and the intracellular signaling domain of the ADGRE2-targeted chimeric receptor (CAR or CCR) of the present invention may be fused by a transmembrane domain. The transmembrane domain may also immobilize the chimeric receptor on the cell membrane of CB-NK cells. The transmembrane domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane domain may be derived from a native, synthetic, semi-synthetic, or recombinant source. In another embodiment, for example, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell.

[0216] In some embodiments, the transmembrane domain of the ADGRE2-targeted CAR or CCR of the present invention is derived from the transmembrane domain of human CD28. For example, the CD28 transmembrane domain of the ADGRE2-targeted CAR polypeptide may contain the amino acid sequence of SEQ ID NO: 54:FWVLVVVGGVLACYSLLVTVAFIIFWV. In some embodiments, the transmembrane domain contains a sequence that is at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 54.

[0217] In some embodiments, the transmembrane domain of the ADGRE2-targeted CAR or CCR of the present invention is derived from the transmembrane domain of human CD8. For example, the CD8 transmembrane domain of the ADGRE2-targeted CAR polypeptide may contain the amino acid sequence of SEQ ID NO: 70:IYIWAPLAGTCGVLLLSLVITLYCNL. In some embodiments, the transmembrane domain contains a sequence that is at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 70.

[0218] Alternatively, examples of transmembrane domains of CARs include the alpha, beta, or zeta chains of T cell receptors, or the transmembrane regions (multiple) of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0219] Cytoplasmic domain The cytoplasmic region of the CAR polypeptide contains an intracellular activation signaling domain. The intracellular signaling domain of the chimeric antigen receptor is responsible for activating at least one of the normal effector functions of immune cells engineered to express the chimeric antigen receptor. The term "effector function" generally refers to the specialized functions of differentiated cells. The effector functions of immune cells (e.g., CB-NK cells) can include cytolytic activity, cytotoxic activity, or helper activity including cytokine secretion for killing tumor cells. In this context, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. In some embodiments, the intracellular signaling domain is derived from the intracellular signaling domain of a natural activation protein. Examples of such natural activation proteins include the zeta chain of the T cell receptor or its homologs (e.g., eta, delta, gamma, epsilon), the MB1 chain, B29, Fc RIII, Fc RI, CD3ζ, CD28, CD27, 4-1BB, DAP10, OX40 and other signaling molecules, and natural receptors such as other similar molecules. Usually, the entire intracellular signaling domain is used, but often it is not necessary to use the entire intracellular polypeptide. To the extent that truncated portions of the intracellular signaling domain are used, such truncated portions can be used in place of the intact chain as long as they transmit the effector function signal. Thus, the term "intracellular signaling domain" is meant to include any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal upon CAR binding to the target.

[0220] In a preferred embodiment, the intracellular signaling domain of the ADGRE2-targeted CAR polypeptide of the present invention is the human CD3ζ intracellular signaling domain. An example of the CD3ζ intracellular signaling domain is the amino acid sequence of SEQ ID NO: 55:

[0221] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 55). In certain specific embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 55.

[0222] In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutant ITAM domain whose activity is altered (e.g., increased or decreased) compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, e.g., a primary intracellular signaling domain comprising an optimized and / or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, or four or more ITAM motifs. In some embodiments, the human CD3ζ intracellular signaling domain comprises ITAM1, ITAM2, and ITAM3 motifs. In some embodiments, the modified CD3ζ polypeptide comprises a native ITAM1 motif, a variant ITAM2 motif comprising two loss-of-function mutations, and a variant ITAM3 motif comprising two loss-of-function mutations.

[0223] In some embodiments, the ITAM1 motif comprises SEQ ID NO: 62: QNQLYNELNLGRREEYDVLDKR. In some embodiments, the variant ITAM1 motif comprises SEQ ID NO: 63: QNQLFNELNLGRREEFDVLDKR.

[0224] In some embodiments, the ITAM2 motif includes sequence number 64:QEGLYNELQKDKMAEAYSEIGMK. In some embodiments, the variant ITAM2 motif includes sequence number 65:QEGLFNELQKDKMAEAFSEIGMK.

[0225] In some embodiments, the ITAM3 motif includes SEQ ID NO: 66:HDGLYQGLSTATKDTYDALHMQ. In some embodiments, the variant ITAM3 motif includes SEQ ID NO: 67:HDGLFQGLSTATKDTFDALHMQ.

[0226] In some embodiments, a CAR polypeptide may comprise one or more costimulatory signaling domains. A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a congenital binding partner on an immune cell that mediates a costimulatory response (such as proliferation, but not limited to these) by an immune cell by specifically binding to a costimulatory ligand.

[0227] In some embodiments, the ADGRE2-targeted CAR or CCR of the present invention includes a DAP10-derived co-stimulatory domain. An example of a DAP10-derived co-stimulatory domain is the amino acid sequence of SEQ ID NO: 56: LCARPRRSPAQEDGKVYINMPGRG. In certain embodiments, the DAP10 co-stimulatory domain includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 56.

[0228] In some embodiments, the ADGRE2-targeting CAR or CCR of the present invention includes a co-stimulatory domain derived from OX40. An example of an OX40-derived co-stimulatory domain is the amino acid sequence of SEQ ID NO: 71: ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI. In certain embodiments, the OX40 co-stimulatory domain includes an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 71.

[0229] CD28-derived co-stimulatory domains can be used in the ADGRE2-targeted CAR polypeptide of the present invention. An example of a CD28 co-stimulatory domain is the amino acid sequence of SEQ ID NO: 57: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 57).

[0230] In addition to the DAP10-derived costimulatory domain and the CD3ζ intracellular signaling domain, the ADGRE2-targeted CAR or CCR polypeptides described herein may comprise one or more costimulatory domains and / or one or more intracellular activation signaling domains.The co-stimulatory domain and intracellular activation signaling domain include, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD28, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), CDS-gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (member of the tumor necrosis factor superfamily; TNFSF1,4), NKG2C, 2B4, Igα (CD79a), Fcγ receptor, MHC class I molecules, TNF receptor proteins, and immunoglobulin-like proteins. Proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CDSα, CDSβ, 11.2β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE It may be derived from ligands that specifically bind to RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0231] Other components In some embodiments, one or more other polypeptides and / or proteins can be incorporated into the ADGRE2-targeted CAR construct as described herein. The additional proteins and polypeptides can be utilized for any function, such as the activation of any cells expressing the CAR polypeptide and / or CAR.

[0232] In some embodiments, the ADGRE2-targeted CAR polypeptide described herein may further comprise one or more cytokines. The CAR and other proteins may be separated, for example, by a cleavable 2A sequence.

[0233] In some embodiments, the cytokine IL-15 is incorporated into the ADGRE2-targeted CAR construct. IL-15 is a pro-inflammatory cytokine important for the differentiation and proliferation of T cells and NK / T cells, and for the development of dendritic cells. An example of IL-15 is the amino acid sequence of SEQ ID NO: 58: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID 58).

[0234] In some embodiments, the IL15 protein incorporated into the ADGRE2-targeted CAR construct contains an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99% identical to SEQ ID NO: 58.

[0235] The IL-15 peptide is encoded by the nucleic acid sequence of SEQ ID NO: 59:

[0236] atgcgcattagcaagccccacctgcggagcatcagcatccagtgctacctgtgcctgctgctgaacagccacttcctgaccgaggccggcatccacgtgttcatcctgggctgcttcagcgcc ggactgcccaagaccgaggccaactgggtgaacgtgatcagcgacctgaagaagatcgaggacctgatccagagcatgcacatcgacgccaccctgtacaccgagagcgacgtgcaccccagct gcaaggtgaccgccatgaagtgctttctgctggaactgcaggtgatcagcctggaaagcggcgacgccagcatccacgacaccgtggagaacctgatcatcctggccaacaacagcctgagcag caacggcaacgtgaccgagagcggctgcaaagagtgcgaggaactggaagagaagaacatcaaagagtttctgcagagcttcgtgcacatcgtgcagatgttcatcaacaccagc (SEQ ID NO: 59).

[0237] In some embodiments, a CAR polypeptide and another protein (e.g., IL15) within the same construct are intended to be generated into two distinct polypeptides, and cleavable 2A sequences (e.g., T2A, F2A, and E2A) may be utilized. 2A self-cleaving peptides (i.e., 2A peptides) are a class of peptides 18–22AA long that can induce ribosome skipping during protein translation in cells. These peptides share the core sequence motif of DxExNPGP and are found in a wide range of viral families. They assist in the generation of polyproteins by preventing ribosomes from forming peptide bonds. Members of the 2A peptides are named after the viruses in which they were first described. For example, F2A, the first 2A peptide to be described, originates from the foot-and-mouth disease virus. The name "2A" itself derives from the gene numbering system of this virus.

[0238] In some embodiments, the non-cleavable peptide is E2A. As a non-limiting example, E2A includes the sequence GPQCTNYALLKLAGDVESNPGP (SEQ ID NO: 60). In some embodiments, the cleavable peptide is positioned between the CAR polypeptide and IL-15.

[0239] The ADGRE2-targeted CARs described herein may further include a signal peptide. The signal peptide may contain 3-30, 3-20, 3-15, 5-30, 5-20, 5-15, 10-30, 10-20, or 10-15 amino acid residues. An example of a signal peptide is the sequence of SEQ ID NO: 61:MEFGLSWLFLVAILKGVQC.

[0240] In some embodiments, the ADGRE2-targeted CAR comprises a signal peptide, an ADGRE2-binding domain, a hinge and transmembrane domain, a DAP10-costimulatory domain, and at least one intracellular signaling domain.

[0241] In some embodiments, the ADGRE2-targeted CCR comprises a signal peptide, an ADGRE2-binding domain, a hinge and a transmembrane domain, and an OX40-costimulatory domain.

[0242] Linker The ADGRE2-targeted CAR or CCR contemplated herein may include linker residues between various domains. In some embodiments, the ADGRE2-targeted CAR contemplated herein may include one, two, three, four, or five, or more linkers. In certain embodiments, the length of the linker is from about 1 to about 30 amino acids, from about 1 to about 25 amino acids, from about 5 to about 30 amino acids, from about 5 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any intervening amino acid length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids in length. One of ordinary skill in the art will recognize that in the design of the CAR in certain embodiments, all or in part may include a flexible linker, and as a result, the linker can include not only a flexible linker but also one or more moieties that provide a less flexible structure to provide the desired CAR structure.

[0243] Exemplary ADGRE2-targeted chimeric receptor constructs The exemplary ADGRE2-targeted chimeric receptors disclosed herein are expressed as polypeptide constructs comprising a chimeric receptor and one or more other components. In some embodiments, the invention provides a nucleic acid molecule encoding the following from the N-terminus to the C-terminus: Signal sequence (SS)-ADGRE2 binder-linker-CD28 (hinge)-linker-CD28 (TM)-DAP10 (costimulatory)-CD3z-E2A-sIL15.

[0244] In some embodiments, the nucleic acid molecule encodes the following from the N-terminus to the C-terminus: SS-ADGRE2 binder-linker-CD8 (hinge)-linker-CD8 (TM)-DAP10 (costimulatory)-CD3ζ-E2A-sIL15.

[0245] In some embodiments, the nucleic acid molecule encodes the following from the N-terminus to the C-terminus: ADGRE2 Binder-Linker-CD8 (Hinge)-CD8(TM)-OX40 CCR-T2A-sIL15.

[0246] In some embodiments, the nucleic acid molecule encodes the following from the N-terminus to the C-terminus: ADGRE2 Binder-Linker-CD28(Hinge)-CD28(TM)-OX40 CCR-T2A-sIL15.

[0247] In some embodiments, the nucleic acid molecule encodes an ADGRE2-targeted CAR or CCR polypeptide construct comprising an amino acid sequence selected from SEQ ID NOs. 48, 72-82.

[0248] [Table 2-1]

[0249] [Table 2-2]

[0250] [Table 2-3]

[0251] [Table 2-4]

[0252] [Table 2-5]

[0253] [Table 2-6]

[0254] [Table 2-7]

[0255] [Table 2-8]

[0256] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 48 or 72–82.

[0257] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 48.

[0258] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 72.

[0259] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 73.

[0260] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 74.

[0261] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 75.

[0262] In some embodiments, an ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 76.

[0263] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 77.

[0264] In some embodiments, an ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 78.

[0265] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 79.

[0266] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 80.

[0267] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 81.

[0268] In some embodiments, the ADGRE2-targeted CAR or CCR polypeptide construct comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100%, identical to any one of the amino acid sequences of SEQ ID NO: 82.

[0269] Polynucleotide encoding the ADGRE2-targeted chimeric receptor In another embodiment, the present invention provides nucleic acid molecules encoding one or more chimeric receptor polypeptides as described herein. As used herein, the terms “nucleic acid molecule” and “polynucleotide” are interchangeable. In some embodiments, the nucleic acid molecule is provided as a messenger RNA (mRNA) molecule. In other embodiments, the nucleic acid molecule is provided as a DNA construct. In some embodiments, the DNA construct is a non-viral vector such as a plasmid, cosmid, or artificial chromosome. In other embodiments, the DNA construct is a virus-based vector such as AAV, lentivirus, and retrovirus.

[0270] Accordingly, the present invention provides polynucleotides encoding ADGRE2 chimeric receptor polypeptides (e.g., CARs or CCRs). In some embodiments, the chimeric receptor is a CAR comprising an ADGRE2 binding domain (e.g., a human anti-ADGRE2 binding domain), a hinge, a transmembrane domain, a DAP10 costimulatory domain, and an intracellular signaling domain including a CD3ζ primary signaling domain. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding one or more polypeptides incorporated into the CAR construct. For example, the polynucleotide further comprises a nucleic acid sequence encoding a cytokine such as IL-15. In some embodiments, the polynucleotide further comprises a nucleic acid sequence of a single peptide and / or linker sequence (e.g., E2A).

[0271] In some embodiments, the chimeric receptor is a CCR comprising an ADGRE2-binding domain (e.g., a human anti-ADGRE2-binding domain), a hinge, a transmembrane domain, and an intracellular domain.

[0272] In some embodiments, the ADGRE2 binding domain is an anti-ADGRE2 binding domain as described herein. In some embodiments, the anti-ADGRE2 binding domain is encoded by any one of the nucleic acid sequences of SEQ ID NOs. 30–35. In some embodiments, the nucleic acid sequence encoding the anti-ADGRE2 binding domain of the chimeric receptor comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of the SEQ ID NOs. 30–35 sequences. Exemplary nucleic acid sequences encoding the ADGRE2 binding domain include sequences of sequence number 35, or sequences that are approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of sequence number 35.

[0273] The polynucleotide encoding the CAR may be codon-optimized. In some embodiments, the polynucleotide includes at least one modified nucleotide. In other embodiments, the polynucleotide includes only unmodified nucleotides.

[0274] The polynucleotides encoding the CAR polypeptide of the present invention can be obtained using recombinant methods known in the art, for example, by screening libraries obtained from cells expressing the CAR construct, by extracting the CAR construct from vectors known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the polynucleotides can also be produced synthetically in vitro.

[0275] In alternative embodiments, polynucleotide sequences encoding various components of the ADGRE2-targeted CAR may be located on different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vectors, e.g., lentiviral vectors. For example, (i) sequences encoding antigen-binding members may be located on a first nucleic acid, e.g., a first vector, and (ii) sequences encoding intracellular signaling members may be located on a second nucleic acid, e.g., a second vector.

[0276] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to sequence numbers 49 and 83-86.

[0277] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 49.

[0278] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 83.

[0279] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 84.

[0280] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 85.

[0281] In some embodiments, the polynucleotide encoding the ADGRE2 chimeric receptor includes a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 86.

[0282] [Table 3-1]

[0283] [Table 3-2]

[0284] [Table 3-3]

[0285] [Table 3-4]

[0286] [Table 3-5]

[0287] [Table 3-6]

[0288] [Table 3-7]

[0289] In some embodiments, the polynucleotide encoding the CAR or CCR of the present invention is an mRNA molecule. The mRNA may further include poly(A) sequences, for example, sequences containing 50-5000, 100-5000, 50-2000, 100-2000, 50-1000, or 100-1000 adenines.

[0290] Pharmaceutical compositions and preparations The present invention provides a composition comprising an ADGRE2-targeted chimeric receptor and CB-NK cells expressing a nucleic acid molecule encoding it. The composition contemplated herein may comprise one or more ADGRE2-targeted chimeric receptor polypeptides, polynucleotides, vectors comprising them, or genetically modified CB-NK cells. The composition is a pharmaceutical composition, but is not limited thereto.

[0291] In some embodiments, the composition comprises one or more cells modified to express one or more ADGRE2-targeted chimeric receptor polypeptides as described herein. In some embodiments, a pharmaceutical composition comprising an effective amount of cells, a composition comprising immune effector cells (e.g., NK cells) as described herein, is provided. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and cells expressing the ADGRE2-targeted CAR polypeptide intended herein.

[0292] "Pharmacologically acceptable carriers, diluents, or excipients" include, without limitation, any adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that are approved by the U.S. Food and Drug Administration for use in humans or farmed animals. Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth, malt, gelatin, talc, cocoa butter, waxes, animal and vegetable fats; paraffin, silicone, bentonite, silicic acid, zinc oxide, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as agar, magnesium hydroxide, and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, and any other suitable substances used in pharmaceutical formulations.

[0293] In some embodiments, the pharmaceutical composition comprises an effective amount of CAR-expressing immune effector cells (e.g., CB-NK cells) as intended herein. As used herein, the term “effective amount” refers to an amount of genetically modified therapeutic cells (e.g., NK cells) that is effective in achieving a beneficial or desirable preventive or therapeutic outcome, including a clinical outcome (e.g., anti-cancer).

[0294] A pharmaceutical composition comprising a population of CB-NK cells modified to express the ADGRE2-targeted chimeric receptor of the present invention may include a buffer, e.g., neutral buffered saline, phosphate-buffered saline; a carbohydrate, e.g., glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid, e.g., glycine; an antioxidant; a chelating agent, e.g., EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The composition is preferably formulated for parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration.

[0295] The composition may be a liquid composition. If the liquid pharmaceutical composition is a solution, suspension, or other similar form, it may contain one or more of the following components: sterile diluents, e.g., water for injection, saline (preferably saline), Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic mono or diglycerides that can be used as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol, or other solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates or phosphates; and tonicity modifiers, e.g., sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

[0296] In some embodiments, the CB-NK cells discussed herein and the compositions intended herein are formulated in the form of pharmaceutically acceptable cell culture media. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture media is a serum-free medium. Serum-free media offer several advantages over serum-containing media, including a simpler and more clearly defined composition, lower levels of contaminants, elimination of potentially infectious substances, and lower cost. In various embodiments, serum-free media are free of animal-derived components and can optionally be protein-free. Optionally, the medium may contain recombinant proteins that are biopharmaceutically acceptable. A “animal-derived component-free” medium refers to a medium in which the components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-derived component-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a “protein-free” medium is defined as substantially protein-free.

[0297] In other embodiments, the compositions comprising immunoeffector cells described herein are formulated in the form of a solution comprising a cryopreservation medium. For example, a cryopreservation medium comprising a cryopreserving agent may be used to maintain high cell viability after thawing.

[0298] In some embodiments, the composition comprises an effective amount of immunoeffector cells modified to express the ADGRE2-targeted chimeric receptor polypeptide described herein, either alone or in combination with one or more therapeutic agents. Thus, the CB-NK cell composition expressing the chimeric receptor can be administered alone or in combination with other known cancer treatments such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, and photodynamic therapy.

[0299] Package and kit Any of the compositions described herein may be included in a package and / or kit for clinical use. In non-limiting examples, cells, reagents for producing cells, vectors, and reagents for producing vectors and / or components thereof may be included in a kit. In certain embodiments, NK cells may be included in a kit, and they may or may not express an ADGRE2 chimeric receptor containing (a) the CD28 hinge, optionally a cytokine (e.g., IL-15), or optionally a suicide gene. Such a kit may or may not have one or more reagents for manipulating the cells. Such reagents may include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. One or more nucleotides encoding chimeric receptors, suicide gene products, and / or cytokines may be included in a kit. Proteins such as cytokines or antibodies (including monoclonal antibodies) may be included in a kit. Nucleotides encoding components of the manipulated chimeric receptor may be included in a kit containing reagents for producing it.

[0300] Methods of use and treatment In some embodiments, CB-NK cells containing the ADGRE2 chimeric receptor described herein (e.g., a chimeric receptor containing the ADGRE2 antigen-binding domain described herein) are used in methods for treating one or more ADGRE2-related conditions. In some embodiments, CB-NK cells containing the ADGRE2 chimeric receptor are used as pharmaceuticals. ADGRE2-related conditions may, non-limitingly, include conditions caused by ADGRE2 expression, conditions involved in symptoms attributable to ADGRE2 expression as a whole or in part, or conditions known to occur in association with ADGRE2 expression.

[0301] In some embodiments, the present invention provides a method for treating cancer, comprising administering CB-NK cells containing the ADGRE2 chimeric receptor. Cancer is a broad group of various diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and proliferation can lead to the formation of malignant tumors, which can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. In some embodiments, “cancer” or “cancer tissue” includes solid tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, cancer is acute myeloid leukemia.

[0302] In some embodiments, lymphoma includes acute lymphoblastic leukemia (ALL), AIDS-associated lymphoma, ALK-positive large B-cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), classical Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, intravascular large B-cell lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, lymphomatoid granulomatosis, and lymphomatous granulomatosis. The group consists of pa-plasmacytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), nodal marginal zone B-cell lymphoma (NMZL), nodular lymphocytosis-dominant Hodgkin lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, splenic marginal zone lymphoma (SMZL), and Waldenström macroglobulinemia. In some embodiments, the lymphoma is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-Hodgkin lymphoma. In some embodiments, the lymphoma is non-Hodgkin lymphoma. In some embodiments, the cancer is relapsed and refractory acute myeloid leukemia.

[0303] In certain embodiments, the tumor is cancer. In certain embodiments, the tumor is a hematological malignancy. In certain embodiments, the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancies. Non-limiting examples of hematological malignancies include multiple myeloma, leukemia, and lymphoma. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotypic acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. Lymphoma may be Hodgkin lymphoma or non-Hodgkin lymphoma. Non-limiting examples of myeloid malignancies include myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), myeloid / lymphoid neoplasms (e.g., myeloid / lymphoid neoplasms with eosinophilia and platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth receptor 1 (FGFR1), or myeloid / lymphoid neoplasms with PCM1-JAK2), acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasms, B lymphoblastic leukemia / lymphoma, and T lymphoblastic leukemia / lymphoma. In certain embodiments, myeloid malignancies include myelodysplastic syndromes.

[0304] In certain embodiments, the tumor is a B-cell malignancy. Non-limiting examples of B-cell malignancies include B-cell lymphoma (BCL), B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter transformation, and CNS lymphoma. Examples of B-cell lymphomas include B-cell non-Hodgkin lymphoma (NHL) and B-cell Hodgkin lymphoma.

[0305] In various embodiments, administration of CB-NK cells containing the ADGRE2 chimeric receptor described herein results in a reduction in the prevalence, frequency, level, and / or amount of one or more symptoms or biomarkers of the ADGRE2-related conditions described herein or known in the art, for example, a reduction of at least about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, 95%, about 99%, or about 100% of one or more symptoms or biomarkers compared to previous measurements or reference values ​​in the subject.

[0306] In some embodiments, administration of CB-NK cells containing the ADGRE2 chimeric receptor to cancer patients results in a significant reduction or improvement of one or more cancer symptoms or biomarkers compared to reference chimeric receptor-expressing cells, such as cells expressing an ADGRE2 chimeric receptor that cross-competes with ADGRE2 binding, under equivalent conditions.

[0307] In some embodiments, CB-NK cells containing the ADGRE2 chimeric receptor (e.g., including the ADGRE2 antigen-binding domain described herein) can be physically introduced into a target using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, for example, by injection or infusion. As used herein, the term “parenteral administration” means a method of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered by parenteral routes, including topical, epidermal, or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Furthermore, administration may be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0308] In another aspect of the present invention, methods for targeting ADGRE2, and for using cells and compositions containing it, are provided, among other things.

[0309] The ADGRE2-targeted chimeric receptor and genetically modified CB-NK cells expressing the ADGRE2-targeted chimeric receptor described herein provide an improved adoptive immunotherapy method for use in the prevention, treatment, and improvement of ADGRE2-associated conditions. The genetically modified immune effector cells expressing the ADGRE2-targeted chimeric receptor described herein provide an improved immunotherapy method for use in increasing the cytotoxicity of target cancer cells or reducing the number of target cancer cells.

[0310] In some embodiments, a type of cell therapy is provided in which NK cells are genetically modified to express the chimeric receptor polypeptide of the present invention that targets ADGRE2-expressing cancer cells, and these NK cells are injected into a recipient in need. The injected cells can kill disease-causing cells in the recipient, such as tumor cells. These NK cells expressing the ADGRE2-targeted chimeric receptor can undergo robust in vivo cell expansion and can persist for extended periods.

[0311] In some embodiments, methods are provided for treating cancers such as ADGRE2-associated cancers in subjects requiring treatment, the methods comprising administering a therapeutically effective amount of a composition, such as those described herein, to the subject requiring treatment. The therapeutically effective amount of a composition containing genetically modified therapeutic cells may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of stem cells and progenitor cells to induce a desired response in the individual. The therapeutically effective amount is also the amount in which the therapeutically beneficial effect outweighs the toxic or adverse effects of the virus or transduced therapeutic cells.

[0312] According to the present invention, the pharmaceutical composition containing CB-NK cells described herein is 10 per kg of body weight 4 ~10 10 Individual cells, preferably 10 per kg of body weight 5 ~10 6 It can be generally stated that the composition can be administered in doses of 10 cells (including all integer values ​​within their range). The number of cells depends on the final intended use of the composition and also on the type of cells contained therein. For the uses provided herein, the cells are generally in volumes of 1 liter or less, 950 mL or less, 900 mL or less, 850 mL or less, 800 mL or less, 750 mL or less, 700 mL or less, 650 mL or less, 600 mL or less, 500 mL or less, and even 250 mL or 100 mL or less. Therefore, the desired cell density is typically 10 6 Cells / ml exceeding 10 7 Cells / ml exceeding 10 8cells / ml, generally 10 9 or more. Clinically appropriate numbers of immune cells can be distributed over multiple infusions that cumulatively result in 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 or more cells. In some embodiments, particularly where all of the infused cells are redirected to a specific target antigen, lower numbers of cells in the range of 10 6 / kilogram (10 6 to 10 11 cells per patient) can be administered. The compositions can be administered multiple times at dosages within these ranges.

[0313] In some embodiments, immune effector cells such as NK cells expressing the ADGRE2-targeted chimeric receptor of the invention are administered to a subject in need thereof at a dose of at least 0.1×10 5 cells, at least 0.5×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 0.5×10 7 cells, at least 1×10 7 cells, at least 0.5×10 8 cells, at least 1×10 8 cells, at least 0.5×10 9 cells, at least 1×10 9 cells, at least 2×10 9 cells, at least 3×10 9 cells, at least 4×10 9 cells, at least 5×10 9 cells, or at least 1×10 10 cells.

[0314] As a non-limiting example, NK cells engineered to express the ADGRE2-targeted CAR of the present invention are approximately 1 × 10⁶ 6 ~2×10 7 pieces, or approximately 1 x 10 6 ~Approx. 1.5×10 7 pieces, or approximately 1 x 10 6 ~1 × 10 7 pieces, or approximately 5 x 10 6 ~2×10 7 pieces, or approximately 5 x 10 6 ~1.5×10 7 pieces, or approximately 5 x 10 6 ~1 × 10 7 It is administered in doses of individual cells.

[0315] In some embodiments, the amount of CB-NK cells expressing the ADGRE2-targeted chimeric receptor (e.g., CAR or CCR) of the present invention is administered to a subject at a dose of at least 0.1 × 10⁶ per kg of body weight. 4 Each cell, at least 0.5 × 10⁶ per kg of body weight 4 Each cell, at least 1 x 10⁶ per kg of body weight 4 Each cell, at least 5 x 10⁶ per kg of body weight 4 Each cell, at least 1 x 10⁶ per kg of body weight 5 Each cell, at least 0.5 × 10⁶ per kg of body weight 6 Each cell, at least 1 x 10⁶ per kg of body weight 6 Each cell, at least 0.5 × 10⁶ per kg of body weight 7 Each cell, at least 1 x 10⁶ per kg of body weight 7 Each cell, at least 0.5 × 10⁶ per kg of body weight 8 Each cell, at least 1 x 10⁶ per kg of body weight 8 Each cell, at least 2 × 10⁶ per kg of body weight 8 Each cell, at least 3 x 10⁶ per kg of body weight 8 Each cell, at least 4 x 10⁶ per kg of body weight 8 Each cell, at least 5 x 10⁶ per kg of body weight 8 Each cell, at least 1 x 10⁶ per kg of body weight 9 per cell, or at least per kg of body weight10 These are individual cells. In a particular embodiment, there are approximately 1 × 10⁶ cells per kg of body weight. 6 NK cells per kg of body weight: approximately 1 x 10⁶ 8 Approximately 2 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.9 x 10⁶ 8 Approximately 3 x 10¹⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.8 x 10⁶ 8 Approximately 4 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.7 × 10⁶ 8 Approximately 5 x 10⁶ NK cells per kg of body weight 6 NK cells per kg of body weight: approximately 0.6 × 10⁶ 8 NK cells, or approximately 5 x 10⁶ cells per kg of body weight. 6 Each NK cell is approximately 0.5 × 10⁻⁶ 8 Individual NK cells are administered to the target.

[0316] In some embodiments, the amount of CB-NK cells expressing ADGRE2-targeted chimeric receptors (e.g., CAR or CCR) administered to the target is approximately 20 × 10⁻¹⁴. 6 ~Approx. 150×10 7 It is an individual cell.

[0317] In some embodiments, the amount of CB-NK cells expressing ADGRE2-targeted chimeric receptors (e.g., CAR or CCR) administered to the target is approximately 20 × 10⁻¹⁴. 6 pieces, approximately 100×10 6 1, or approximately 500 x 10 6 1 piece, or approximately 150 x 10 7 It is an individual cell.

[0318] It is recognized that multiple administrations of the compositions intended herein may be necessary to obtain the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, over periods of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or over the lifetime of the subject requiring treatment.

[0319] The compositions described herein may be administered by any convenient method, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. In preferred embodiments, the compositions are administered parenterally. As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, injections and infusions of intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intratracheal, subcutaneous, subepidermal, intraarticular, subarachnoid, intraspinal, and intrasternal methods. In one embodiment, the compositions intended herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0320] Combination therapy ADGRE2-targeted chimeric receptors and immunoeffector cells expressing them can be used in combination with other known drugs and therapies. ADGRE2-targeted chimeric receptor therapy and at least one additional therapeutic agent can be administered simultaneously, in the same or different compositions, or sequentially. In the case of sequential administration, the chimeric receptor-expressing cells described herein may be administered first, followed by the additional agent, or the order of administration may be reversed. Administration may occur at intervals ranging from simultaneous to several minutes, several days, several weeks, or several months.

[0321] In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., drugs intended to reduce the occurrence and / or severity of side effects of the treatment, such as anti-nausea agents).

[0322] In some embodiments, additional therapies may be other specific anti-cancer therapies such as radiotherapy, surgery (e.g., mammary gland tumor removal and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic viruses, or combinations thereof.

[0323] A wide variety of chemotherapeutic agents can be used in combination with the compositions of the present invention. "Chemotherapeutic agent" is used to refer to compounds or compositions administered in the treatment of cancer.

[0324] Additional immunotherapies may be used in combination with or in conjunction with the compositions and methods described herein. Examples of immunotherapeutic agents include antibodies, antibody-drug conjugates, cancer vaccines, immune effector cells, and immune checkpoint inhibitors.

[0325] The compositions and methods of the present invention may be used in combination with surgery. Approximately 60% of individuals with cancer undergo several types of surgery, including preventive, diagnostic, or staging, curative, and palliative surgeries. Post- and / or pre-operatively, patients who require it may be treated with immune effector cells.

[0326] In some embodiments, other agents may be used in combination with specific embodiments of the present invention to improve the therapeutic effect of the treatment. Exemplary therapeutic agents that can be combined with the compositions of the present invention include small molecule enzyme inhibitors, antitransfer agents, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other activators and adjuvants.

[0327] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. Furthermore, substances, methods, and examples are illustrative and not intended to be limiting. Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and substances may be used in the practice or testing of the present invention, but appropriate methods and substances are described herein. [Examples]

[0328] The following examples illustrate some preferred ways of creating and carrying out the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0329] Example 1. ADGRE2 CAR or CCR CB-NK cells exhibit in vitro cytotoxic activity. Umbilical cord blood (CB) units for the study were obtained from the MD Anderson Cancer Center Cord Blood Bank. CB mononuclear cells were isolated from frozen CB units by Ficoll density gradient centrifugation. CB NK cells were then isolated using the EasySep Human NK Isolation Kit (Stem Cell Technologies catalog no. 17955). The isolated CB-NK cells were activated on day 0 by co-culture with irradiated uAPC cells and grown in NK medium [StemSpan® SFEM II medium (STEMCELL Technologies catalog no. 09655) + 2 mM L-glutamine (1% GIBCO L-glutamine 200 mM REF 25030-081) + 10% GIBCO thermally inactivated fetal bovine serum Certified One Shot® Performance Plus (REF A38400-02)] containing 200 IU / mL recombinant IL-2 (Miltenyi 130-097-743). During expansion, cells were supplied with fresh IL-2 every two days. On day 6, cells were transduced by spinoculation with RD114 virus. On day 8, cells were stimulated with a second round of uAPC addition and supplied with IL-2 every two days until day 15, when they were ready for in vivo or in vitro testing.

[0330] Acute myeloid leukemia (AML) cell lines MOLM-13 and KG-1a were transduced with viruses carrying only the firefly luciferase gene under a constitutive promoter and a selectable puromycin resistance gene. MV-4-11 cells were transduced with viruses carrying only the firefly luciferase gene and GFP gene under a constitutive promoter and a selectable puromycin resistance gene. The resulting cells were initially cultured in the presence of puromycin to select a population highly enriched with luciferase. Luciferase-expressing MOLM-13, KG-1a, and MV-4-11 cells were typically cultured in the following media:

[0331] MOLM-13: GIBCO RPMI 1640 (REF 11875-093) + 20% GIBCO heat-inactivated fetal bovine serum Certified One Shot™ Performance Plus (REF A38400-02)

[0332] KG-1a: GIBCO IMDM (REF 12440-053) + 10% GIBCO heat-inactivated fetal bovine serum Certified One Shot™ Performance Plus (REF A38400-02)

[0333] MV-4-11: GIBCO IMDM (REF 12440-053) + 10% GIBCO heat-inactivated fetal bovine serum Certified One Shot™ Performance Plus (REF A38400-02)

[0334] 2-20 × 10 per 1 mL 6 Cells growing in the logarithmic growth phase at a typical density of viable cells were collected by centrifugation at 350 × g for 4 minutes. The supernatant was aspirated and removed, and the cell pellet was resuspended in 10-20 mL of IL-2-free NK cell medium with 2 mM L-glutamine (1% GIBCO L-glutamine 200 mM REF 25030-081) and 10% GIBCO thermally inactivated fetal bovine serum Certified One Shot® Performance Plus (REF A38400-02) added to STEMCELL Technologies StemSpan® SFEM II medium (catalog no. 09655). AML cell density and viability were counted using a NucleoCounter NC-200® cell counter with a Via1-Casette®. The AML cells were then diluted with additional NK cell medium, and the viable cell density was 333.3 × 10⁶ per mL. 3The cells were separated into individual cells. The AML cells were dispensed into Corning white 384-well flat-bottom poly-D-lysine treated plates (#354661) using a MultiDrop® Combi reagent dispenser with a standard dispensing cassette, achieving a seeding density of 10,000 live cells per well. The resulting plates were incubated in a water jack incubator at 37°C in a 5% CO2 atmosphere for 1–2 hours.

[0335] Count transduced or untransduced umbilical cord blood NK effector cells, dilute them in NK cell medium (without IL-2), and determine the viable cell density: 3 × 10⁶ cells per mL. 6 The concentration was adjusted to cells / mL. Effector cells were pipetteed into a 384-well V-bottom polypropylene plate (Greiner, catalog no. 781280) and titrated in 1:3 serial dilutions with NK medium (without IL-2) in four stops. The serially diluted effector cells were transferred to plates containing the required AML cell lines, and the effector-to-target ratios (E:T) were set to 3:1, 1:1, 0.3:1, and 0.1:1, respectively, resulting in final effector cell counts of 30,000, 10,000, 3,333, and 1,111 cells, respectively. The resulting co-culture plates were incubated at 37°C and 5% CO2 for 20-24 hours. After incubation, Promega ONE-Glo® Luciferase Assay System reagent (Promega#E6120) was added to each plate via MultiDrop® Combi.

[0336] Next, the luminescence signal from each well is determined using a BMG LABTECH PHERAstar FSX plate reader equipped with the LUM Plus optical module. The signal from each well is normalized against the mean signal of a control consisting only of AML cell lines. This normalized signal is then inverted to obtain a specific killing percentage determined by the decrease in AML luminescence.

[0337] Figures 1A–1C show exemplary results of the in vitro cytotoxic activity of ADGRE2 CAR or CCR CB-NK against acute myeloid leukemia cell lines MOLM-13 (Figure 1A), KG-1a (Figure 1B), and MV-4-11 (Figure 1C).

[0338] Example 2. ADGRE2 CAR CB-NK cells demonstrate in vivo efficacy. This example demonstrates the efficacy of ADGRE2 CAR-expressing CB-NK cells against acute myeloid leukemia cells in vivo.

[0339] NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) were purchased from The Jackson Laboratory. Female mice aged 10–12 weeks were used in this study. All animals received 1.5 Gy of whole-body X-ray irradiation using Precision X-Ray SmART+ (North Branford, CT, USA), and 24 hours later, each animal received 0.5 × 10⁶ doses. 6 MOLM-13-RFluc, 5 x 10 6 10 MV4-11-eGFP / luc, and 5 × 10 6Individual KG-1a-luc cells (in 200 μl PBS) were injected intravenously (iv). Tumors were allowed to grow for 2 days (MOLM-13-RFluc), 3 days (MV4-11-eGFP / luc), and 6 days (KG-1a-luc). Mice were randomly assigned to groups (n=4 mice / group) using individual whole-body bioluminescence imaging (BLI) signals and BW data acquired and reported by Studylog (Studylog Systems [Pacifica, CA, USA]). On days 3, 4, and 7 after tumor transplantation (MOLM-13-RFluc, MV4-11-eGFP / luc, and KG-1a-luc, respectively), CAR-expressing NK (i.e., CAR CB-NK) cells and non-transduced NK (UTD-NK) cells were injected intravenously into the tail vein of mice at appropriate concentrations in 200 μl PBS / animal. To observe tumor volume and the efficacy of the test substance, mice were imaged twice weekly (MOLM-13-RFluc model) and once weekly (MV4-11-eGFP / luc and KG-1a-luc models), and body weight (BW) was measured twice weekly. On the day of imaging, mice were intraperitoneally (IP) injected with 200 μL of RediJect D-luciferin (150 mg / kg BW) (Perkin Elmer, MA, USA), and IVIS® SpectrumCT (Perkin Elmer, MA) was performed in the abdominal position. The total flux (photons / second) for the entire mouse was quantified using Living Image software (PerkinElmer, USA). Data were analyzed using Graph-Pad Prism 8 and expressed as mean ± SEM (n=4).

[0340] Figures 2A-2C show exemplary results of the in vivo efficacy of ADGRE2 CAR CCR CB-NK against acute myeloid leukemia cell lines MOLM-13 (Figure 2A), KG-1a (Figure 2B), and MV-4-11 (Figure 2C).

[0341] Example 3. OX40 Co-stimulatory Domain CCR In this example, the effects of exemplary co-stimulatory domains within a CCR construct (a chimeric receptor lacking an intracellular CD3z domain) were investigated using an in vitro repeated antigen stimulation assay.

[0342] Umbilical cord blood (CB) units for the study were obtained from the MD Anderson Cancer Center Cord Blood Bank. CB mononuclear cells were isolated from frozen CB units by Ficoll density gradient centrifugation. Subsequently, CB NK cells were isolated using the EasySep Human NK Isolation Kit (Stem Cell Technologies catalog no. 17955). The isolated CB-NK cells were activated on day 0 by co-culture with irradiated uAPC cells and grown in NK medium [StemSpan® SFEM II medium (STEMCELL Technologies catalog no. 09655) + 2 mM L-glutamine (1% GIBCO L-glutamine 200 mM REF 25030-081) + 10% GIBCO thermally inactivated fetal bovine serum Certified One Shot® Performance Plus (REF A38400-02)] containing 200 IU / mL recombinant IL-2 (Miltenyi 130-097-743). During expansion, cells were supplied with fresh IL-2 every two days. On day 6, cells were transduced by spinoculation with RD114 virus. CD123 tool binder 26292 was used for each design. Cells were stimulated with a second round of uAPC addition on day 8 and supplied with IL-2 every two days until use in in vivo or in vitro testing on day 15.

[0343] The acute myeloid leukemia (AML) cell line MOLM-13 was transduced with a virus carrying only the firefly luciferase gene under a constitutive promoter and a selectable puromycin resistance gene. The resulting cells were initially cultured in the presence of puromycin to select a population highly enriched with luciferase. Luciferase-expressing MOLM-13 cells were typically cultured in the following media:

[0344] MOLM-13: GIBCO RPMI 1640 (REF 11875-093) + 20% GIBCO heat-inactivated fetal bovine serum Certified One Shot™ Performance Plus (REF A38400-02)

[0345] MOLM-13 cells, growing in the logarithmic growth phase at a typical density of 2–20 × 10⁶ viable cells per mL, were collected by centrifugation at 350 × g for 4 minutes. The supernatant was aspirated and the cell pellet was resuspended in 10–20 mL of IL-2-free NK cell medium. AML cell density and viability were counted using a NucleoCounter NC-200™ cell counter with a Via1-Casette™. Subsequently, the AML cells were resuspended in NK cell medium containing 2x recombinant IL-2 (200 IU / mL) (Miltenyi 130-097-743) and recombinant IL-15 (2 ng / mL) (Peprotech 200-15-10UG) to a viable cell density of 4 × 10⁵ cells / mL. 100 μL of AML cells (40,000 cells total per well) were dispensed into Corning white 96-well flat-bottom non-cell culture sterile plates using a MultiDrop® Combi reagent dispenser and a standard dispensing cassette. The resulting plates were incubated in a water jack incubator at 37°C in a 5% CO2 atmosphere for 2–3 hours.

[0346] Transduced and untransduced CB-NK effector cells were counted and diluted in NK cell medium (without IL-2 / 15) to a viable cell density of 4 × 10⁵ cells per mL. 100 μL of effector cells (40,000 cells total per well) were pipetteed into a 96-well plate containing AML to achieve an E:T ratio of 1:1. The co-culture plates were placed in a humidified incubator at 37°C with 5% CO₂.

[0347] On days 2, 5, 7, 9, 12, and 14 after the start of co-culture, remove the effector-target co-culture plates from the incubator and gently shake them using a MultiDrop Combi. Mix each well using a Dynamic Devices Lynx liquid handler equipped with a 96SV head and 200uL Filtered Tip Racks (one rack per co-culture plate) to completely resuspend the target and effector cells. Next, transfer 20uL of cell suspension samples from each plate to a 384-well V-bottom polypropylene plate (Greiner) and perform flow cytometry analysis. Add FACS staining mixes (1:75 BioLegend anti-nectin-2-PE (final ratio 1:125, clone TX31) and 1:75 BioLegend anti-CD56-BV711 (final ratio 1:125, clone HCD56)) to each well. Incubate the staining mixes at 4°C for 30 minutes. After 30 minutes, the cells are washed with FACS buffer. After washing, Citox blue staining mixture (1:1000, Invitrogen) is added to each well, and the plate is read using Novoocyte Advanteon. After sampling for FACS analysis, the remaining NK cells are centrifuged and the supernatant is removed. 40,000 fresh AML cells are added to NK medium containing 100 IU / mL IL-2 and 1 ng / mL IL-15 for re-challenge. The plate is then returned to 37°C, 5% CO2 incubation until the next time point.

[0348] For flow cytometry analysis, cells were corrected for anti-CD56-BV711 and anti-Nectin-2-PE using UltraComp Plus eBeads (Invitrogen), and cells were used with Sytox Blue. Each sample was gated to exclude cell debris using SSC-H vs FSC-H, then gated to exclude cell clumps and double cells using FSC-A vs FSC-H. Live cells were then gated using FSC-H vs Sytox Blue-H. In wells containing MOLM-13 target cells, live cells were gated using anti-Nectin-2-PE-H vs FSC-H, with large cells and Nectin-2+ cells gated as targets and small cells and Nectin-2- cells gated as effectors. The number of live target and live effector cells at each time point was quantified to monitor the ability to continue controlling the tumor over time in each subsequent challenge.

[0349] Figure 3A shows the co-stimulatory molecules screened with the chimeric receptor construct (construct without intracellular CD3z). Figures 3B and 3C illustrate the ability of OX40-CCR-engineered NK cells to persistently control MOLM-13 tumor cells across various tumor rechallenges. OX40-co-stimulated CCRs were demonstrated to potently control MOLM-13 tumor cells.

[0350] Other Embodiments While many embodiments of the present invention are described herein, this disclosure and examples may be modified to provide other methods and compositions of the present invention. It will be understood that the scope of the present invention should be defined by the appended claims, in addition to the specific embodiments presented as examples. All references cited herein are incorporated by reference in their entirety.

Claims

1. Umbilical cord blood-derived natural killer (CB-NK) cells comprising a chimeric receptor including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to ADGRE2, wherein the extracellular antigen-binding domain is A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), A light chain variable region comprising LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6), The aforementioned CB-NK cells.

2. CB-NK cells according to claim 1, wherein the extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 7, 9, or 15.

3. CB-NK cells according to claim 1, wherein the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 14, 12, or 16.

4. The extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in Sequence ID No.

7. The CB-NK cell according to claim 1, wherein the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO:

14.

5. The extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in Sequence ID No.

9. The CB-NK cell according to claim 1, wherein the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO:

12.

6. The extracellular antigen-binding domain includes a heavy chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in Sequence ID No.

15. The CB-NK cell according to claim 1, wherein the extracellular antigen-binding domain includes a light chain variable region which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO:

16.

7. The CB-NK cell according to claim 1, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv).

8. The CB-NK cell according to any one of claims 1 to 7, wherein the extracellular antigen-binding domain includes a linker between the heavy chain variable region and the light chain variable region.

9. The CB-NK cell according to claim 8, wherein the linker contains the amino acid sequence of SEQ ID NO:

24.

10. The CB-NK cell according to claim 8 or 9, wherein the heavy chain variable region and the light chain variable region are arranged in a VH-VL configuration from the N-terminus to the C-terminus.

11. CB-NK cells according to any one of claims 1 to 10, comprising scFv whose extracellular antigen-binding domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NOs: 19, 20, and 68.

12. CB-NK cells according to any one of the prior claims, wherein the transmembrane domain comprises CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-I polypeptide, LAG-3 polypeptide, 2B4 polypeptide, or BTLA polypeptide.

13. The CB-NK cell according to claim 12, wherein the transmembrane domain contains a CD28 polypeptide.

14. The CB-NK cell according to claim 12, wherein the transmembrane domain contains a CD8 polypeptide.

15. The CB-NK cell according to any one of the prior claims, wherein the intracellular domain further comprises at least one costimulatory signaling region.

16. The CB-NK cell according to any one of the prior claims, wherein the chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule.

17. The CB-NK cell according to claim 16, wherein the chimeric receptor is CAR.

18. The CB-NK cell according to claim 17, wherein the intracellular domain contains a CD3ζ polypeptide.

19. The CB-NK cell according to claim 18, wherein the CD3ζ polypeptide is a modified CD3ζ polypeptide.

20. The CB-NK cell according to claim 19, wherein the modified CD3ζ polypeptide comprises native ITAM1, an ITAM2 variant containing two loss-of-function mutations, and an ITAM3 variant containing two loss-of-function mutations.

21. The CB-NK cell according to claim 20, wherein the natural ITAM1 comprises the amino acid sequence described in Sequence ID No.

63.

22. The CB-NK cell according to claim 20 or 21, wherein the ITAM2 variant comprises the amino acid sequence described in Sequence ID No.

65.

23. The CB-NK cell according to any one of claims 20 to 22, wherein the ITAM3 variant comprises the amino acid sequence described in SEQ ID NO:

67.

24. The CB-NK cell according to claim 23, wherein the modified CD3ζ polypeptide comprises the amino acid sequence described in SEQ ID NO:

55.

25. The CB-NK cell according to claim 16, wherein the chimeric receptor is CCR.

26. The CB-NK cell according to any one of the prior claims, further comprising a second chimeric receptor that binds to a second target.

27. The CB-NK cell according to claim 26, wherein the chimeric receptor that binds to ADGRE2 is a chimeric antigen receptor (CAR), and the second chimeric receptor is a chimeric costimulatory receptor (CCR).

28. CB-NK cells according to any one of the prior claims, comprising the nucleic acid encoding the chimeric receptor.

29. The CB-NK cell according to any one of claims 1 to 28, wherein the cell is transduced with the nucleic acid encoding the chimeric receptor.

30. The CB-NK cell according to any one of claims 1 to 29, wherein the chimeric receptor is constitutively expressed on the surface of the cell.

31. The CB-NK cell according to any one of claims 28 to 30, further comprising a promoter operably linked to the chimeric receptor, wherein the nucleic acid is further amplified.

32. The CB-NK cell according to claim 31, wherein the promoter is endogenous or exogenous.

33. The CB-NK cell according to claim 32, wherein the exogenous promoter is selected from the group consisting of the elongation factor (EF)-1 promoter, the cytomegalovirus pre-initial promoter (CMV) promoter, the Simianvirus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, the metallothionein promoter, and the ubiquitin C promoter.

34. The CB-NK cell according to claim 31, wherein the promoter is an induceable promoter.

35. The CB-NK cell according to claim 34, wherein the inducible promoter is selected from the group consisting of the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, the IL-2 promoter, the 4-1BB promoter, the PD1 promoter, and the LAG3 promoter.

36. The CB-NK cell according to claim 31, wherein the promoter is an endogenous promoter.

37. The CB-NK cell according to claim 31, wherein the endogenous promoter is selected from the TCR alpha promoter, the TCR beta promoter, and the beta 2-microglobulin promoter.

38. The CB-NK cells according to any one of the prior claims, wherein the CB-NK cells further express exogenous IL-15.

39. A composition comprising CB-NK cells according to any one of claims 1 to 38.

40. The composition according to claim 39, which further comprises a pharmaceutically acceptable carrier.

41. Approximately 20×10 ６ ~Approx. 150×10 ７ The composition according to claim 39 or 40, comprising a single cell.

42. Approximately 20×10 ６ pieces, approximately 100 x 10 ６ pieces, approximately 500 x 10 ６ 1 piece, or approximately 150 x 10 ７ A composition according to any one of claims 39 to 41, comprising a single cell.

43. A method for reducing tumor volume in a subject, comprising administering to the subject CB-NK cells according to any one of claims 1 to 38, or a composition according to any one of claims 39 to 42.

44. The method according to claim 43, wherein the method reduces the number of tumor cells in the target, reduces the size of the tumor, and / or eradicates the tumor.

45. A method for increasing or extending the survival period of a subject having a tumor, comprising administering to the subject CB-NK cells according to any one of claims 1 to 38, or a composition according to any one of claims 39 to 42.

46. A method for treating and / or preventing a tumor in a subject, comprising administering to the subject CB-NK cells according to any one of claims 1 to 38, or a composition according to any one of claims 39 to 42.

47. The method according to any one of claims 43 to 46, wherein the tumor expresses ADGRE2.

48. The method according to any one of claims 43 to 47, wherein the tumor is cancerous.

49. The method according to any one of claims 43 to 47, wherein the tumor is a hematological carcinoma.

50. The method according to any one of claims 43 to 47, wherein the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancy.

51. The method according to claim 50, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia.

52. The method according to claim 51, wherein the leukemia is acute myeloid leukemia (AML).

53. The method according to claim 52, wherein the AML is relapsed / refractory acute myeloid leukemia (R / R AML).

54. The method according to claim 50, wherein the myeloid malignancy is selected from the group consisting of myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), myeloid / lymphoid neoplasm, eosinophilia and myeloid / lymphoid neoplasm with rearrangement of platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth factor receptor 1 (FGFR1), or PCM1-JAK2, acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, B lymphoblastic leukemia / lymphoma, and T lymphoblastic leukemia / lymphoma.

55. The method according to claim 50, wherein the myeloid malignant tumor includes myelodysplastic syndrome (MDS).

56. The method according to any one of claims 43 to 55, wherein the subject is a human subject.

57. A method for producing CB-NK according to any one of claims 1 to 38, comprising introducing a nucleic acid molecule encoding the chimeric receptor into the cell.

58. Umbilical cord blood natural killer (CB-NK) cells containing a chimeric receptor and a nucleic acid encoding an exogenous IL-15 polypeptide, wherein the chimeric receptor is CB-NK cells comprising an extracellular antigen-binding domain that binds to ADGRE2, comprising a heavy chain variable region including HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

59. A nucleic acid encoding a chimeric receptor and an exogenous IL-15 polypeptide, wherein the chimeric receptor is A heavy chain variable region comprising HCDR1 containing the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3), The nucleic acid comprises an extracellular antigen-binding domain that binds to ADGRE2, comprising a light chain variable region including LCDR1 containing the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 containing the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 containing the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

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