LILRB1-based chimeric antigen receptor

Abstract

Chimeric antigen receptors are provided that contain the hinge, transmembrane region, and / or intracellular domain of LILRB1, or functional fragments or variants thereof. Cells containing LILRB1-based receptors, as well as methods for producing and using the same, are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 946,888, filed December 11, 2019, and U.S. Provisional Patent Application No. 63 / 085,969, filed August 30, 2020, the contents of each of those applications being incorporated herein by reference in their entirety.

[0002] Inclusion by referencing the sequence list This application includes a sequence listing submitted in ASCII format via EFS-WEB, which is incorporated herein by reference in its entirety. Created on December 11, 2020, this ASCII copy is named A2BI-015-01WO_SeqList.txt and has a size of 228KB. [Background technology]

[0003] Chimeric antigen receptor (CAR) T-cell therapy and T-cell receptor (TCR) therapy have proven to be effective therapeutic approaches for a variety of diseases, particularly hematological malignancies, as well as other cancers. CAR NK cells may also have clinical applications. Conventional CARs provide stimulating signals to engineered immune cells (e.g., T cells or NK cells). In CAR-T cells, this results in cell death activity against target cells identified by the antigen-binding domain of the CAR. Inhibitory CARs (iCARs) have been developed as a means of controlling cellular activity or limiting the activity of activated CARs to specific cell types. Fedorov et al. Sci. Transl. Med. 5(215):215ra172 (2013). Inhibitory CARs generally have an intracellular domain of an inhibitory signaling molecule (such as PD-1 or CTLA-4) fused to an antigen-binding domain (e.g., single-stranded variable fragment, scFv) via a transmembrane domain and, where appropriate, a hinge domain.

[0004] Numerous alternative iCAR structures have been described in the art. However, there remains an unmet need for the identification of novel alternative inhibitory receptors and specific inhibitory receptor structures with superior performance, along with related compositions and methods of use. [Overview of the project] [Means for solving the problem]

[0005] In one embodiment, the disclosure provides a chimeric antigen receptor having the hinge, transmembrane domain, and / or intracellular domain of LILRB1, or functional fragments or variants thereof. The chimeric antigen receptor may comprise a single polypeptide or more than one polypeptide. The receptor may comprise one or more, or all of, the following: (a) the LILRB1 hinge domain or a functional fragment or variant thereof; (b) the LILRB1 transmembrane domain or a functional variant thereof; and (c) the LILRB1 intracellular domain or a functional variant thereof, e.g., the LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM) found in the polypeptide sequence of LILRB1. In some embodiments, the receptor comprises at least two ITIMs found in the polypeptide sequence of LILRB1. The ITIMs of LILRB1 are NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). The receptor may contain one, two, three, four, five, six, or more of these ITIMs in any combination that includes multiple copies of the same ITIM.

[0006] In some embodiments of the receptors of this disclosure, the intracellular domain includes both ITIMs NLYAAV (SEQ ID NO: 8) and VTYAEV (SEQ ID NO: 9). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the intracellular domain includes both ITIMs VTYAEV (SEQ ID NO: 9) and VTYAQL (SEQ ID NO: 10). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the intracellular domain includes both ITIMs VTYAQL (SEQ ID NO: 10) and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the polypeptide includes at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM including an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes the ITIMs NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), and VTYAQL (SEQ ID NO: 10). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the intracellular domain includes the ITIMs VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the intracellular domain includes the ITIMs NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 7). In some embodiments, the intracellular domain includes the sequences of SEQ ID NOs: 12-17.

[0007] In some embodiments of the receptors of this disclosure, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembrane domain comprises SEQ ID NO: 5.

[0008] In some embodiments of the receptor of this disclosure, the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof. In some embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 93. In some embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. In some embodiments, the LILRB1 hinge domain includes a sequence identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84.

[0009] In some embodiments of the receptors of this disclosure, the polypeptide comprises (a) the LILRB1 hinge domain or a functional fragment or variant thereof, and (b) the LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 20.

[0010] In some embodiments of the receptors of this disclosure, the polypeptide comprises (a) the LILRB1 transmembrane domain or a functional variant thereof, and (b) the LILRB1 intracellular domain, and / or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 21.

[0011] In some embodiments of the receptors of this disclosure, the polypeptide comprises (a) a LILRB1 hinge domain or a functional fragment or variant thereof, (b) a LILRB1 transmembrane domain or a functional variant thereof, and (c) an intracellular LILRB1 domain, and / or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0012] In some embodiments of the receptors of this disclosure, the polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the polypeptide includes a sequence that is at least 99% identical to SEQ ID NO: 20. In some embodiments, the polypeptide includes a sequence that is at least 99% identical to SEQ ID NO: 21. In some embodiments, the polypeptide includes a sequence that is at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the polypeptide includes a sequence that is identical to SEQ ID NO: 20. In some embodiments, the polypeptide includes a sequence that is identical to SEQ ID NO: 21. In some embodiments, the polypeptide includes a sequence that is identical to SEQ ID NO: 2 or SEQ ID NO: 3.

[0013] In some embodiments of the receptors of this disclosure, the polypeptide comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is an antigen-binding domain other than the LILRB1 extracellular ligand-binding protein. In some embodiments, the polypeptide comprises two or more antigen-binding domains. In some embodiments, the antigen-binding domain comprises a single-strand variable fragment (scFv). In some embodiments, the receptor comprises a second polypeptide. In some embodiments, the first polypeptide comprises the first chain of the antibody, and the second polypeptide comprises the second chain of the antibody. In some embodiments, the receptor comprises a Fab fragment of the antibody. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody, and (b) the second polypeptide comprises an antigen-binding fragment of the light chain of the antibody. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody, and (b) the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody. In some embodiments, the first polypeptide comprises the first chain of the T cell receptor (TCR), and the second polypeptide comprises the second chain of the TCR. In some embodiments, the receptor comprises an extracellular fragment of the T cell receptor (TCR). In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR, and (b) the second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, (a) the first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR, and (b) the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR. In some embodiments, the receptor comprises a single-stranded TCR. In some embodiments, the scFv comprises one of the complementarity-determining regions (CDRs) of SEQ ID NOs. 22-33. In some embodiments, the scFv comprises a sequence that is at least 95% identical to one of SEQ ID NOs. 35-46 or 125. In some embodiments, scFv includes a sequence that is at least 95% identical to one of sequence numbers 35, 39, 46, or 125. In some embodiments, scFv includes a sequence that is identical to one of sequence numbers 35-46 or 125. In some embodiments, scFv includes a sequence that is identical to one of sequence numbers 35, 39, 46, or 125.In some embodiments, the antibody heavy chain comprises one of the heavy chain CDRs of SEQ ID NOs. 25-27 or 31-33, and the antibody light chain comprises one of the light chain CDRs of SEQ ID NOs. 22-24 or 28-30. In some embodiments, the antibody heavy chain comprises a sequence that is at least 95% identical to one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the antibody light chain comprises a sequence that is at least 95% identical to one of the light chain portions of SEQ ID NOs. 35-46 or 125. In some embodiments, the antibody heavy chain comprises a sequence that is identical to one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the antibody light chain comprises a sequence that is identical to one of the light chain portions of SEQ ID NOs. 35-46 or 125. In some embodiments, the antibody heavy chain includes a sequence identical to one of the heavy chain portions of SEQ ID NOs. 35, 39, 46, or 125, and the antibody light chain includes a sequence identical to one of the light chain portions of SEQ ID NOs. 35, 39, 46, or 125.

[0014] In some embodiments of the receptors of this disclosure, the receptor comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 47-71, 77-79, 89-92, 120, or 122. In some embodiments, the receptor comprises the amino acid sequence of SEQ ID NOs: 47-71, 77-79, 89-92, 120, or 122.

[0015] In some embodiments of the receptors of this disclosure, the receptor is an inhibitory receptor.

[0016] This disclosure provides polynucleotides comprising nucleic acid sequences encoding receptors or polypeptides of the disclosure.

[0017] This disclosure provides vectors comprising the polynucleotides of this disclosure. In some embodiments, the vector further comprises a sequence encoding a promoter operably ligated to the polynucleotide.

[0018] The present disclosure provides a receptor, polynucleotide, polypeptide, or immune cell comprising the receptor of the present disclosure. In some embodiments, immune cell activation is reduced when the cell contacts an antigen or a cell expressing an antigen on its surface. In some embodiments, immune cell activation comprises the expression of a gene operably linked to an NFAT promoter. In some embodiments, the immune cell is a T cell. In some embodiments, it further comprises an activator receptor. In some embodiments, the activator receptor is a chimeric antigen receptor or a T cell receptor.

[0019] The present disclosure provides a method of producing an immune cell, comprising introducing the polynucleotide or vector of the present disclosure into the immune cell. In some embodiments, the immune cell expresses a receptor. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, immune cell activation is reduced when the cell contacts an antigen specific to a chimeric antigen receptor or a cell expressing an antigen on its surface. In some embodiments, immune cell activation comprises the expression of a gene operably linked to an NFAT promoter.

[0020] The present disclosure provides a method of treating a subject having a disease or disorder, comprising administering to the subject a plurality of immune cells of the present disclosure. In some embodiments, the disease or disorder is cancer.

[0021] The present disclosure provides a kit comprising the receptor, polypeptide, polynucleotide, vector, or immune cell of the present disclosure.

[0022] The present disclosure provides an immune cell comprising a chimeric antigen receptor comprising a polypeptide, wherein the polypeptide sequence shares at least 95% identity or at least 100% identity with SEQ ID NO: 21.

[0023] In some embodiments of the immune cells of this disclosure, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO: 3. In some embodiments, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO: 2. In some embodiments, the chimeric antigen receptor includes antigen-binding domains comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences, respectively, according to SEQ ID NOs: 22-27. In some embodiments, the chimeric antigen receptor includes antigen-binding domains comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences, respectively, according to SEQ ID NOs: 28-33. In some embodiments, the polypeptide sequence shares at least 95% or at least 100% identity with SEQ ID NO: 122. In some embodiments, the polypeptide sequence, in combination with SEQ ID NO: 2, shares at least 95% or at least 100% identity with any one of SEQ ID NOs: 35, 39, 46, or 125.

[0024] In some embodiments, the immune cells are T cells. In some embodiments, the T cells comprise chimeric antigen receptors or T cell receptors that specifically bind to a target expressed on tumor cells. In some embodiments, the T cells are etiolate receptors, ανββ integrins, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, glypican-3 (GPC3), h5T4, IL-11R, IL13R-a2, KDR, κ light chain, λ light chain, LeY, LI CAM, MAGE-A1, mesothelin, MHC-presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2 / 3, NY-ESO-1, PRAME, PSCA, PSMA, survivin, TAG-72, TEM, TERT, VEGFR2, and a chimeric antigen receptor or T cell receptor that specifically binds to a target selected from ROR1.

[0025] The present disclosure provides a method of treating and / or preventing cancer in a subject that needs it, comprising administering the immune cells of the present disclosure to the subject. In some embodiments, this method is treating and / or preventing cancer in a subject that needs it, comprising administering the immune cells of the present disclosure to the subject.

[0026] Exemplary CARs provided herein include, but are not limited to, single-chain variable fragment (scFv) CARs, Fab CARs, or other antibody-based CARs, and T cell receptor (TCR)-based CARs.

[0027] In other embodiments, the disclosure provides polynucleotides encoding such receptors, vectors for delivering such polynucleotides, and immune cells having such polynucleotides and receptors.

[0028] In a further embodiment, the disclosure provides a method for introducing a polynucleotide or vector encoding such a receptor into a cell. Advantageously, when a cell comes into contact with an antigen or a cell expressing an antigen on its surface, immune cell activation is reduced.

[0029] Further aspects and embodiments of the present invention are provided in the following detailed description. This disclosure provides, for example, the following: [Section 1] A chimeric antigen receptor comprising a polypeptide, wherein the polypeptide is a) LILRB1 hinge domain or its functional fragment or variant, b) LILRB1 transmembrane domain or a functional variant thereof, c) An intracellular domain comprising the LILRB1 intracellular domain or at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). A chimeric antigen receptor containing one or more of the following. [Section 2] The receptor according to claim 1, wherein the polypeptide comprises a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). [Section 3] The receptor according to item 2, wherein the intracellular domain comprises both ITIMs of NLYAAV (SEQ ID NO: 8) and VTYAEV (SEQ ID NO: 9). [Section 4] The receptor according to claim 3, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 12. [Section 5] The receptor according to item 2, wherein the intracellular domain comprises both ITIMs of VTYAEV (SEQ ID NO: 9) and VTYAQL (SEQ ID NO: 10). [Section 6] The receptor according to claim 5, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 13. [Section 7] The receptor according to item 2, wherein the intracellular domain comprises both ITIMs of VTYAQL (SEQ ID NO: 10) and SIYATL (SEQ ID NO: 11). [Section 8] The receptor according to item 5, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 14. [Section 9] The receptor according to claim 1, wherein the polypeptide comprises an intracellular domain containing at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). [Section 10] The receptor according to claim 2, wherein the intracellular domain comprises the ITIM of NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), and VTYAQL (SEQ ID NO: 10). [Section 11] The receptor according to item 10, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 15. [Section 12] The receptor according to claim 2, wherein the intracellular domain comprises the ITIM of VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). [Section 13] The receptor according to item 12, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 16. [Section 14] The receptor according to claim 2, wherein the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). [Section 15] The receptor according to item 14, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence number 17. [Section 16] The receptor according to claim 15, wherein the intracellular domain comprises a sequence that is at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 7). [Section 17] The receptor according to item 1, wherein the intracellular domain comprises the sequences of sequence numbers 12-17. [Section 18] The receptor according to any one of claims 1 to 17, wherein the polypeptide comprises the LILRB1 transmembrane domain or a functional variant thereof. [Section 19] The receptor according to item 18, wherein the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 5. [Section 20] The receptor according to item 19, wherein the LILRB1 transmembrane domain comprises SEQ ID NO: 5. [Section 21] The receptor according to any one of claims 1 to 20, wherein the polypeptide comprises the LILRB1 hinge domain or a functional fragment or variant thereof. [Section 22] The receptor according to claim 21, wherein the LILRB1 hinge domain or its functional fragment or variant comprises a sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 93. [Section 23] The receptor according to claim 21, wherein the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 93. [Section 24] The receptor according to claim 21, wherein the LILRB1 hinge domain or its functional fragment or variant comprises a sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. [Section 25] The receptor according to claim 21, wherein the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. [Section 26] The polypeptide described above, a) LILRB1 hinge domain or its functional fragment or variant, and b) The LILRB1 transmembrane domain or its functional variant A receptor, including any one of items 1 to 25. [Section 27] The receptor according to claim 26, wherein the polypeptide comprises a sequence that is at least 95% identical to sequence number 20. [Section 28] The polypeptide described above, a) The LILRB1 transmembrane domain or its functional variant, and b) The LILRB1 intracellular domain and / or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM being independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11), comprising an intracellular domain A receptor, including any one of items 1 to 27. [Section 29] The receptor according to claim 28, wherein the polypeptide comprises a sequence that is at least 95% identical to sequence number 21. [Section 30] The receptor according to item 28, wherein the polypeptide comprises the sequence of SEQ ID NO: 21. [Section 31] The polypeptide described above, a) LILRB1 hinge domain or its functional fragment or variant, b) LILRB1 transmembrane domain or its functional variants, and c) The LILRB1 intracellular domain and / or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM being independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11), comprising an intracellular domain The receptors described in item 1, including the receptors described in item 1. [Section 32] The receptor according to claim 1, wherein the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 3. [Section 33] The receptor according to claim 1, wherein the polypeptide comprises a sequence that is at least 99% identical to sequence number 20. [Section 34] The receptor according to claim 1, wherein the polypeptide comprises a sequence that is at least 99% identical to sequence number 21. [Section 35] The receptor according to claim 1, wherein the polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 3. [Section 36] The receptor according to claim 1, wherein the polypeptide comprises a sequence identical to sequence number 20. [Section 37] The receptor according to claim 1, wherein the polypeptide comprises a sequence identical to sequence number 21. [Section 38] The receptor according to claim 1, wherein the polypeptide comprises a sequence identical to SEQ ID NO: 2 or SEQ ID NO: 3. [Section 39] The receptor according to item 1, wherein the polypeptide comprises an antigen-binding domain. [Section 40] The receptor according to item 39, wherein the antigen-binding domain is an antigen-binding domain other than the LILRB1 extracellular ligand-binding protein. [Section 41] The receptor according to item 39, wherein the polypeptide comprises two or more antigen-binding domains. [Section 42] An inhibitory receptor, as described in any one of items 1 to 41. [Section 43] The receptor according to item 39, wherein the antigen-binding domain comprises a single-stranded variable fragment (scFv). [Section 44] The receptor according to item 1, comprising a second polypeptide. [Section 45] The receptor according to claim 44, wherein the first polypeptide comprises a first chain of an antibody, and the second polypeptide comprises a second chain of the antibody. [Section 46] The receptor described in item 44, comprising an antibody Fab fragment. [Section 47] a) The first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody, b) The second polypeptide comprises an antigen-binding fragment of the light chain of the antibody, The receptor described in item 44. [Section 48] a) The first polypeptide comprises an antigen-binding fragment of the light chain of the antibody, b) The second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody, The receptor described in item 44. [Section 49] The receptor according to claim 44, wherein the first polypeptide comprises the first chain of a T cell receptor (TCR), and the second polypeptide comprises the second chain of the TCR. [Section 50] The receptor described in item 44, comprising an extracellular fragment of a T cell receptor (TCR). [Section 51] a) The first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR, b) The second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR, The receptor described in item 50. [Section 52] a) The first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR, b) The second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR, The receptor described in item 50. [Section 53] The receptor described in item 1, including a single-stranded TCR. [Section 54] The receptor according to item 43, wherein the scFv includes one of the complementarity-determining regions (CDRs) of sequence numbers 22 to 33. [Section 55] The receptor according to claim 43, wherein the scFv comprises a sequence that is at least 95% identical to one of sequence numbers 35-46 or 125. [Section 56] The receptor according to item 43, wherein the scFv comprises a sequence identical to any one of sequence numbers 35-46 or 125. [Section 57] The receptor according to claim 47 or 48, wherein the heavy chain of the antibody comprises one of the heavy chain CDRs of SEQ ID NOs. 25-27 or 31-33, and the light chain of the antibody comprises one of the light chain CDRs of SEQ ID NOs. 22-24 or 28-30. [Section 58] The receptor according to claim 47 or 48, wherein the heavy chain of the antibody comprises a sequence that is at least 95% identical to any one heavy chain portion of SEQ ID NOs. 35-46 or 125, and the light chain of the antibody comprises a sequence that is at least 95% identical to any one light chain portion of SEQ ID NOs. 35-46 or 125. [Section 59] The receptor according to claim 47 or 48, wherein the heavy chain of the antibody comprises a sequence identical to any one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the light chain of the antibody comprises a sequence identical to any one of the light chain portions of SEQ ID NOs. 35-46 or 125. [Section 60] The receptor described in item 1, comprising an amino acid sequence that is at least 95% identical to one of sequence numbers 47-71, 77-79, 89-92, 120, or 122. [Section 61] The receptor described in item 1, comprising the amino acid sequence of sequence numbers 47-71, 77-79, 89-92, 120, or 122. [Section 62] A polynucleotide comprising a nucleic acid sequence encoding a receptor as described in any one of items 1 to 61. [Section 63] A vector containing the polynucleotides described in item 62. [Section 64] The vector according to claim 63, further comprising a sequence encoding a promoter operably linked to the polynucleotide. [Section 65] Immune cells comprising a receptor as described in any one of items 1 to 61, a polynucleotide as described in item 62, or a vector as described in item 63 or 64. [Section 66] The immune cells according to claim 65, wherein when the cells come into contact with the antigen or cells expressing the antigen on their surface, immune cell activation is reduced. [Section 67] Immune cells according to claim 65 or 66, wherein immune cell activation includes the expression of a gene operably linked to the NFAT promoter. [Section 68] A T cell, which is an immune cell as described in any one of items 65 to 67. [Section 69] An immune cell according to any one of items 65-68, further comprising an activator receptor. [Section 70] The immune cell according to item 69, wherein the activator receptor is a chimeric antigen receptor or a T cell receptor. [Section 71] A method for producing immune cells, comprising introducing a polynucleotide as described in item 62, or a vector as described in item 63 or 64, into the immune cells. [Section 72] The method according to item 69, wherein the immune cells express the receptor. [Section 73] The method according to claim 69 or 70, wherein the cells are immune cells. [Section 74] The method according to item 71, wherein the immune cell is a T cell. [Section 75] The method according to any one of claims 69 to 72, wherein when the cells come into contact with an antigen specific to the chimeric antigen receptor, or with cells expressing the antigen on their surface, immune cell activation is reduced. [Section 76] The method according to item 73, wherein immune cell activation includes the expression of a gene operably linked to an NFAT promoter. [Section 77] A method for treating a subject having a disease or disorder, comprising administering to the subject a plurality of immune cells as described in any one of paragraphs 65 to 70. [Section 78] The method according to paragraph 77, wherein the disease or disorder is cancer. [Section 79] A kit comprising a receptor as described in any one of items 1 to 61, a polynucleotide as described in item 62, a vector as described in item 63 or 64, or an immune cell as described in any one of items 65 to 68. [Section 80] Immune cells comprising a polypeptide-containing chimeric antigen receptor, wherein the polypeptide sequence shares at least 95% or 100% identity with SEQ ID NO: 21. [Section 81] The immune cell according to claim 80, wherein the polypeptide sequence shares at least 95% or 100% identity with SEQ ID NO: 3. [Section 82] The immune cell according to claim 80, wherein the polypeptide sequence shares at least 95% or 100% identity with SEQ ID NO: 2. [Section 83] The immune cell according to item 81, wherein the chimeric antigen receptor comprises an antigen-binding domain containing the sequences CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, respectively, according to SEQ ID NOs. 22-27. [Section 84] The immune cell according to item 81, wherein the chimeric antigen receptor comprises an antigen-binding domain containing the sequences CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, respectively, according to SEQ ID NOs. 28-33. [Section 85] The immune cell according to claim 80, wherein the polypeptide sequence shares at least 95% or 100% identity with SEQ ID NO: 122. [Section 86] A T cell, which is an immune cell as described in any one of sections 80-83. [Section 87] The immune cells according to claim 84, wherein the T cells include a chimeric antigen receptor or a T cell receptor that specifically binds to a target expressed on tumor cells. [Section 88] The aforementioned T cells include ethiolate receptor, ανββ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, glypican-3 (GPC3), h5T4, IL-11R, IL13R-a2, KDR, κ light chain, λ light chain, LeY, L1 Immune cells as described in item 87, comprising a chimeric antigen receptor or T cell receptor that specifically binds to a target selected from CAM, MAGE-A1, mesothelin, MHC-presenting peptide, MUC1, MUC16, NCAM, NKG2D ligand, Notchl, Notch2 / 3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEM, TERT, VEGFR2, and ROR1. [Section 89] A method for treating and / or preventing cancer in a subject requiring such treatment, comprising administering the immune cells described in item 87 to the subject. [Section 90] A method for treating and / or preventing cancer in a subject requiring such treatment, comprising administering the immune cells described in paragraph 88 to the subject. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows an explanatory diagram of the domain arrangement in one embodiment, which includes a ligand-binding domain (LBD), a hinge, a transmembrane domain (TM), and an intracellular signaling domain (ICD). [Figure 2-1] Figures 2A and 2B illustrate the domain arrangement in embodiments having a ligand-binding domain (LBD), hinge, transmembrane domain (TM), and intracellular signaling domain (ICD). If the ligand-binding domain contains two peptides, for example, a T cell receptor-derived heterodimer LDB, each peptide may be fused with the hinge, TM, and intracellular domain (Figure 2A). Alternatively, only one peptide of the ligand-binding domain may be fused with the hinge, TM, and intracellular domain (Figure 2B). [Figure 2-2] Same as above. [Figure 3] Figure 3 shows four exemplary embodiments of immune cells having activator scFv-based chimeric antigen receptors (CARs) [101 and 102] or activator T cell receptors [103 and 104] and inhibitory scFv-based CARs [101 and 103] or inhibitory TCR-based CARs [102 and 104]. [Figure 4] Figure 4 shows the luminescence (relative luminescence intensity, RLU) in an NFAT-based reporter assay for the shown construct at various concentrations (MP1, microliters, μM) of MAGE-A3 activating peptide 1 in the presence of 50 μM NY-ESO-1 peptide. [Figure 5] Figure 5 shows the luminescence figures (RLU) in an NFAT-based reporter assay at various concentrations of MAGE-A3 peptide 1 (MP1, nM) for the given construct in the presence of various concentrations (μM) of NY-ESO-1 peptide. [Figure 6] Figure 6 shows the luminescence figures (RLU) in an NFAT-based reporter assay at various concentrations of MAGE-A3 peptide 1 (MP1, nM) for the given construct in the presence of various concentrations (μM) of NY-ESO-1 peptide. [Figure 7] Figure 7 shows the luminescence figures (RLU) in an NFAT-based reporter assay at various concentrations of MAGE-A3 peptide 1 (MP1, nM) for the given construct in the presence of 50 μM NY-ESO-1 peptide. [Figure 8] Figure 8 shows the RLU (Ritual Lumen Activation) in an NFAT-based reporter assay at various concentrations of MAGE-A3 peptide 1 (MP1, nM) for the given construct in the presence of 50 μM NY-ESO-1 peptide. [Figure 9] Figure 9 shows the luminescence figures (RLU) in an NFAT-based reporter assay for the shown construct at various concentrations of MAGE-A3 peptide 1 (MP1, nM) in the presence of 5 μM NY-ESO-1 peptide. [Figure 10] Figure 10 shows the luminescence figures (RLU) in an NFAT-based reporter assay for the shown construct at various concentrations of MAGE-A3 peptide 1 (MP1, nM) in the presence of 50 μM NY-ESO-1 peptide. [Figure 11] Figure 11 shows the luminescence figures (RLU) in an NFAT-based reporter assay at various concentrations of MAGE-A3 peptide 1 (MP1, nM) for the given construct in the presence of 50 μM NY-ESO-1 peptide. [Figure 12-1]Figure 12A is a graph showing the effect of the LIR-1 hinge on the ability of the HLA-A*02 scFv inhibitory receptor to block KRAS TCR-mediated Jurkat cell activation. H: hinge, T: transmembrane domain, ICD: intracellular domain, s: short. The LIR-1 construct is described in more detail in Figure 12B. Humanized PA2.1 and humanized BB7.2, which have a shorter LIR-1 hinge, block as well as the original, longer hinge. [Figure 12-2] Figure 12B is a graph and table showing the EC50 shift (+ / -HLA-A*02 target cells) of Jurkat cells expressing the KRAS TCR activator and the HLA-A*02 scFv LIR-1 inhibitory receptor, as shown in the table below. [Figure 13-1] Figure 13A is a graph showing the effect of the LIR-1 hinge on the ability of the HLA-A*02 inhibitory receptor to block KRAS TCR-mediated Jurkat cell activation. H: hinge, TM: transmembrane domain, ICD: intracellular domain, s: short, tr: truncated. The LIR-1 construct is described in more detail in Figure 13B. Mouse PA2.1, which has a slightly longer hinge, functions similarly to the original LIR-1 hinge in the T2-Jurkat assay. [Figure 13-2] Figure 13B is a graph and a pair of tables showing the EC50 shift (+ / -HLA-A*02 target cells) of Jurkat cells expressing the KRAS TCR activator and the HLA-A*02 scFv LIR-1 inhibitory receptor, with the hinge length indicated on the left in the table below. [Figure 14-1] Figure 14A shows a schematic diagram of the T2-Jurkat experiment used to evaluate the blocker construct. [Figure 14-2]Figure 14B shows graphs, tables, and figures illustrating the effects of various NY-ESO-1 scFv LBD blocker modules (PD-1, CTLA-4, LIR-1) on the EC50 of the MAGE-A3 CAR activator (MP1-LBD 1-CAR), as measured by MAGE peptide titration of cells loaded with a fixed concentration (50 μM) of NY-ESO-1 blocker peptide. In each of Figures 14B–14F, the NFAT-luciferase signal of Jurkat cells transfected with either the activator CAR alone or in combination with each blocker receptor was assayed after 6 hours of co-culture with T2 cells loaded with the activator and blocker peptide. Baseline (Jurkat only) varied depending on the various activator-only constructs and could be particularly high in the presence of the CAR; in most cases, expression of the blocker receptor without its ligand suppressed baseline. The activator peptide concentration varied from 0 to 10⁻⁶ and up to 10² μM, and the luminescence measurements ranged from 0 to 80,000 RLU. [Figure 14-3] Figure 14C shows graphs, tables, and figures illustrating the effect of various scFv LBDs (ESO, MP1 LBD 1, MP1 LBD 2, HPV E6 LBD 1, HPV E6 LBD 2, HPV E7) on the EC50 of the MAGE-A3 CAR activator (MP1-CAR) when the corresponding blocker peptide is loaded at a fixed (50 μM) peptide concentration, as in Figure 14B. RLU = relative luminescence; error bars indicate ±SD (n=2). Activator peptide concentrations varied from 0 to 10⁻³ and 10² μM, and luminescence measurements ranged from 0 to 100,000 RLU. [Figure 14-4]Figure 14D shows graphs, tables, and figures illustrating the effect of LIR-1 blocker receptors with NY-ESO-1 scFv LBD on the EC50 of various MAGE-A3 CAR activators (MP1-LBD 1-CAR or MP2-CAR) when loaded with 50 μM NY-ESO-1 blocker peptide. Activator peptide concentrations varied from 0 to 10⁻⁴ and 10² μM, and luminescence measurements ranged from 0 to 80,000 RLU. [Figure 14-5] Figure 14E shows graphs, tables, and figures illustrating the effect of LIR-1 blocker receptors with NY-ESO-1 scFv LBD on the EC50 of various TCR activators (MP1-TCR, MP2-TCR, HPV E6-TCR) when loaded with 50 μM NY-ESO-1 blocker peptide. Activator peptide concentrations varied from 0 to 10⁻⁴ and 10² μM, and normalized luminescence measurements ranged from 0 to 150 RLU. [Figure 14-6] Figure 14F shows graphs, tables, and figures illustrating the effect of LIR-1 blocker receptors with NY-ESO-1 TCR LBD on the EC50 of MAGE-A3 CAR and TCR activators (MP1-LBD 1-CAR, MP1-TCR) when loaded with 50 μM NY-ESO-1 blocker peptide. RLU = relative luminescence; error bars indicate ±SD (n=2). Activator peptide concentrations varied from 0 to 10⁻⁷ and 10¹ μM, and normalized luminescence measurements ranged from 0 to 150 RLU. [Figure 15] Figure 15 is a graph showing the effects of blocker peptide loading (50 μM each of NY-ESO-1, MAGE-A3, HPV E6, and HPV E7) on activated MAGE-A3 CAR. MP2-CAR[0 μM], EC50=44 nM; MP2-CAR[50 μM HPVp2], EC50=495 nM. RLU = Relative Luminescence; Error bars indicate ±SD (n=2). [Figure 16-1]Figure 16A is a series of graphs showing the NFAT-luciferase signaling of Jurkat cells transfected with either the activator MAGE-A3 CAR alone or in combination with various amounts of the NY-ESO-1 scFv LBD blocker (the DNA ratio of the activator and blocker receptor components is shown on the left as A:B, i.e., activator receptor:blocker receptor after 6 hours of co-culture with T2 cells loaded with the activator and blocker peptides. The T2 cells were loaded with titrations of the activator MAGE-A3 peptide and a fixed amount of the blocker NY-ESO-1 peptide. The activator peptide and / or blocker peptide concentrations ranged from 0 to 10⁻⁶ and up to 10² μM, and the luminescence measurements ranged from 0 to 200,000 RLU. Figure 16B shows the signaling of Jurkat cells transfected with either the activator MAGE-A3 CAR alone or in combination with various amounts of the NY-ESO-1 scFv This is a series of graphs showing the NFAT-luciferase signaling of Jurkat cells transfected with one of the following combinations of LBD blockers. T2 cells were loaded with titrated amounts of the blocker NY-ESO-1 peptide and fixed amounts of the activator MAGE-A3 peptide exceeding the Emax concentration (approximately 0.1 mM). Activator peptide and / or blocker peptide concentrations ranged from 10⁻⁵ to 10² μM, and luminescence measurements ranged from 0 to 200,000 RLU. [Figure 16-2]Figure 16C is a series of graphs and two tables showing the NFAT-luciferase signals of Jurkat cells transfected with either the activator MAGE-A3 CAR alone or in combination with various amounts of the NY-ESO-1 scFv LBD blocker. The x-value blocker NY-ESO-1 peptide concentration from Figure 16B was normalized to a constant activator MAGE peptide concentration used for each curve and plotted on the x-axis. The ratio of the blocker peptide to the activator peptide required for 50% blocking (IC50) is shown for each curve. For all DNA ratios, the required B:A peptide ratio was less than 1, indicating that for this pair of activator CAR and blocker, a similar (or less) amount of blocker pMHC antigen is required on the target cell to block the activator pMHC antigen. Activator peptide and / or blocker peptide concentrations ranged from 10⁻¹⁰ to 10⁴ μM, and luminescence measurements ranged from 0 to 200,000 RLU. [Figure 16-3] Figure 16D is a table and graph showing that blocking CD19CAR activators are possible using pMHC blockers with a blocker pMHC antigen density similar to that required to activate pMHC CARs. NFAT-luciferase signaling in Jurkat cells transfected with either activator CD19CAR alone or in combination with various amounts of NY-ESO-1 blockers (shown DNA ratios) after 6 hours of co-culture with T2 cells loaded with the blocker peptide expressing endogenous levels of CD19 antigen. IC50 is estimated from inhibition curves ranging from 0.1 to 1.0 mM, corresponding to approximately 1,500–3,500 pMHC / cell. RLU = relative luminescence; error bars indicate ±SD (n=2). [Figure 17-1]Figure 17A is a graph showing the effect of the NY-ESO-1-LIR-1 blocker on the EC50 of activated MAGE-A3 CAR (MP1-CAR) when loaded with various concentrations of the NY-ESO-1 blocker peptide. The EC50 shift increases as the concentration of the blocker peptide (NY-ESO-1) increases. The shift in the presence of a negative control HPV peptide (which binds to HLA-A*02 but not to the NY-ESO-1 blocker scFv) is routinely observed and is thought to be caused by competition for the control peptide for the binding site on the T2 HLA-A*02 molecule, which reduces the number of activator targets. Activator concentrations ranged from 0 to 10⁻⁴ and up to 10² μM, and luminescence measurements ranged from 0 to 140,000 RLU. [Figure 17-2] Figure 17B is a graph showing the effect of modified LIR-1 blocker receptors containing either NY-ESO-1 scFv LBD and no ICD, or containing a mutated ICD, on the EC50 of MAGE-A3 CAR activator (MP2-CAR) loaded with 10 μM NY-ESO-1 blocker peptide. Activator concentrations ranged from 0 to 10⁻⁴ and up to 10² μM, and luminescence measurements ranged from 0 to 140,000 RLU. [Figure 17-3]Figures 17C–17E are a series of graphs showing the effects of various NY-ESO-1 scFv LBD blocker receptors (CTLA-4 (Figure 17C), PD-1 (Figure 17D), and LIR-1 (Figure 17E)) on the EC50 of the MAGE-A3 CAR activator (MP1-LBD 1-CAR) with or without blocker stimulation. NFAT-luciferase signaling in Jurkat cells transfected with either the activator CAR alone or in combination with each blocker after 6 hours of co-culture with T2 cells loaded with the peptide. T2 cells were loaded with a titration of the activator MAGE peptide and tested with or without an additional fixed amount (50 μM) of the NY-ESO-1 blocker peptide. RLU = relative luminescence; error bars indicate ±SD (n=2). Activator concentrations ranged from 0 to 10⁻⁶ and up to 10² μM, and luminescence measurements ranged from 0 to 100,000 RLU. [Figure 17-4] Same as above. [Figure 17-5] Same as above. [Figure 18-1] Figure 18A is a figure and a pair of graphs showing that Jurkat cells transfected with HPV E7-CAR or HPV E7-CAR&A2-LIR-1, co-cultured with beads displaying various ratios of activator (HPV E7) and blocker (NY-ESO-1) antigens, exhibit blocking in the cis state but not in the trans state. [Figure 18-2] Figure 18B is a graph showing how the HLA-A*O2-LIR-1 blocker receptor blocks the CD19-CAR activator at various activator-to-blocker ratios. The ratios ranged from 0 to 10, and the luminescence (RLU) ranged from 0 to 70,000. [Figure 18-3] Figure 18C is a graph showing the surface expression of the titrated HLA-A*02(A2)LIR-1 blocker receptor. [Figure 18-4]Figure 18D is a graph showing that scFv for HLA-A*02 can also function as an activator when fused with an activator CAR. T2 cells expressing endogenous HLA-A*02 act as targets. RLU = relative luminescence; error bars indicate ±SD (n=2). [Figure 19-1] Figure 19A is a graph showing the effect of LIR-1 blocker receptors with NY-ESO-1 scFv LBD on the EC50 of various TCR activators (MP1-TCR, MP2-TCR, HPV E6-TCR) when loaded with NY-ESO-1 blocker peptide. For Figure 14E, each set was normalized to the Emax of the curve showing the response of the activator alone. Activator concentrations ranged from 0 to 10⁻⁴ and up to 10² μM, and luminescence measurements ranged from 0 to 140,000 RLU. [Figure 19-2] Figure 19B is a graph showing the effect of LIR-1 blocker receptors with NY-ESO-1 TCR LBD on the EC50 of MAGE-A3 CAR and TCR activators (MP1-LBD 1-CAR, MP1-TCR). Each set is normalized to the Emax of the curve showing the response of the activator only. RLU = relative luminescence; error bars indicate ±SD (n=2). Activator concentrations ranged from 0 to 10⁻⁷ and up to 10¹ μM, and luminescence measurements ranged from 0 to 140,000 RLU. [Figure 20-1] Figure 20A is a graph showing that primary T cells (donor 1) transduced with HPV E7-TCR activator and ESO-LIR-1 blocker shift the EC50 by approximately 25 × in a primary T cell killing assay (HPV E7 TCR EC50 = 0.044 nM; HPV E7 TCR + ESO-LIR-1, EC50 = 1.1 nM). The assay was performed using MCF7 target cells loaded with peptides in a 3:1 E:T ratio. Luciferase measurement refers to living target cells at 48 hours. [Figure 20-2]Figure 20B is a graph showing that HLA-A*02-LIR-1 blocks the NY-ESO-1 CAR activator in Jurkat cells at various activator:blocker DNA ratios, using T2 target cells loaded with the NY-ESO-1 peptide. RLU = relative luminescence; error bars indicate ±SD (n=2). [Figure 21-1] Figure 21A is a series of images and graphs showing that primary T cells (Donor 1) transduced with CD19 CAR activator and HLA-A2 blocker distinguish "tumor" cells from "normal" cells in an in vitro cytotoxicity assay and exhibit selective killing of "tumor" cells in a 3:1 E:T mixed target cell assay. The images shown were captured at 72 hours. Untransduced, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1 cells are shown. Measurements were taken between 0 and 150 hours, with normalized fluorescent protein intensity (GFP or RFP) ranging from 0 to 10. [Figure 21-2] Figure 21B is a series of graphs showing that primary T cells (Donor 1) selectively kill tumor cells in the Incucyte imaging assay, similarly at various tumor-to-"normal" cell ratios, with an E:T ratio of 3:1. RLU values ​​were normalized for a target cell mixture grown in the absence of primary T cells. Untransduced, CD19CAR T cells, and CD19-CAR T+A2-LIR-1 are shown. Measurements were taken between 0:00 and 150:00, and normalized fluorescent protein intensities (GFP or RFP) ranged from top row, left to right: 0–3×10⁷, 0–2×10⁷, 0–1.6×10⁷, 0–7×10⁶, 0–2×10⁶; bottom row, left to right: 0–7×10⁵, 0–2×10⁵, 0–4×10⁵, 0–6×10⁵, 0–7×10⁵. [Figure 21-3]Figure 21C is a series of graphs showing that primary T cells (donor 1) selectively kill tumor cells in quantitative targeted cell lysis and IFNγ secretion, similarly at various tumor-to-"normal" cell ratios, with an E:T ratio of 3:1. Untransduced, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1 cells are shown. [Figure 22-1] Figure 22A is a pair of graphs showing that Jurkat cells transfected with MSLN LBD1-CAR or MSLN LBD1-CAR&A2-LIR-1, co-cultured with K562 cells expressing MSLN or MSLN&HLA-A*02, exhibit blockade of activation by high-density antigens with A2-LIR-1 blockers only in the presence of HLA-A*02. [Figure 22-2] Figure 22B is a pair of graphs showing that the killing of endogenous MSLN+HeLa cells by MLSN LBD1-CAR T cells is blocked in the presence of HLA-A*02, which has the A2-LIR-1 blocker. [Figure 22-3] Figure 22C shows a pair of graphs illustrating the killing of endogenous MSLN+HeLa cells by MLSN LBD2-CAR T cells. The effect of A2-LIR-1 blockers on T cell killing is partially regulated by the activator LBD. [Figure 23] Figure 23 is a graph showing that in the absence of the blocker antigen, the LIR-1 blocker receptor has little effect on the activator's killing efficacy. In the absence of the NY-ESO-1 blocker antigen, primary T cells (donor 1) transduced with HPV E7-TCR activator and ESO-LIR-1 blocker exhibit similar killing efficacy to primary T cells transduced with HPV E7-TCR activator alone. Luciferase measurement refers to living target cells. RLU = relative luminescence; error bars indicate ±SD (n=2). [Figure 24-1]Figure 24A is a pair of graphs showing that A2-LIR-1 blocks Jurkat activation in A2+ Raji cells but not in WT Raji cells. The histogram shows that HLA-A*02 is expressed only in Raji A2 "normal" cells, while Raji WT "tumor" cells and Raji A2+ "normal" cells have identical CD19 surface expression. [Figure 24-2] Figure 24B is a graph showing that A2-LIR-1 blocks Jurkat activation in A2+Raji cells but not in WT Raji cells. Jurkat cells transfected with either CD19 or CD19+A2-LIR-1 were co-cultured with either WT(A2-)Raji cells or A2+Raji cells in various cell ratios. RLU = relative luminescence; error bars indicate ±SD (n=2). [Figure 25-1] Figures 25A and 25B are a series of images demonstrating the reversibility of blockade by the LIR-1 inhibitory receptor, respectively. Primary T cells (donor 2) transduced with CD19 CAR activator and HLA-A*02 blocker showed reversible blockade (Figure 25A) and activation (Figure 25B) after three rounds of antigen exposure (AB-A-AB and A-AB-A) in a 3:1 E:T in vitro cytotoxicity assay. The primary T cell cytotoxicity assay was replicated with three HLA-A*02-negative donors. The images shown were captured at 72 hours. [Figure 25-2] Same as above. [Figure 25-3]Figures 25C and 25D are a pair of graphs showing the quantification of target cell lysis (Figure 25C) and IFNγ (Figure 25D) in response to repeated exposure to multiple rounds of normal and target cells (T cells from donor 2) with an E:T ratio of 3:1, respectively. The conditions shown are untransduced, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1. Error bars indicate ±SEM (n=2). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; these were determined using two-way ANOVA followed by Tukey's multiple comparison test. In this experiment, cytotoxicity remained robust, but the IFNγ response decreased over time. [Figure 25-4] Same as above. [Figure 26-1] Figures 26A and 26B are a pair of graphs, respectively, showing cytotoxic T cell killing and IFNγ secretion in co-culture with another donor (Donor 3). Transduced T cells with the cytotoxic CD19 CAR activator and HLA-A2 blocker showed reversible blockade in cytotoxic assays and IFNγ after multiple rounds of antigen exposure, in a 9:1 E:T ratio. We observed that T cell viability and activity from this donor decreased over time during culture. The conditions shown are untransduced, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1. The results for cytotoxicity (Figure 26A) and IFNγ (Figure 26B) correspond to Figures 25C and 25D. Error bars indicate ±SEM (n=2). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; these were determined using two-way ANOVA followed by Tukey's multiple comparison test. [Figure 26-2] Same as above. [Figure 27-1] Figure 27A is a graph showing that primary T cells transduced with CD19 CAR activator and HLA-A*02 blocker proliferate approximately 20-fold after 10 days of CD3 / 28 stimulation. [Figure 27-2]Figure 27B shows an experiment demonstrating that CAR-T cells expressing the LIR-1 blocker receptor selectively kill tumors in a xenograft model. HLA-A*02 NSG mice were subcutaneously administered either "tumor cells" (A2-negative Raji cells) or "normal cells" (A2-positive Raji cells). When the Raji xenograft reached an average size of approximately 70 mm3, primary T cells (human, HLA-A*02-negative donor 4) were injected into the tail vein. [Figure 27-3] Figures 27C and 27E are pairs of graphs showing readings from caliper measurement (Figure 27C), human T cell count in peripheral blood by flow cytometry (Figure 27D), and survival (Figure 27E), respectively. Error bars in C and D indicate ±SEM (n=7). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; these were determined using two-way ANOVA followed by Tukey's multiple comparison test. [Figure 27-4] Same as above. [Figure 27-5] Same as above. [Figure 28-1] Figure 28A shows a series of graphs illustrating flow cytometry analysis of primary T cells after enrichment and proliferation, prepared for mouse tail vein injection. Tumor volume was measured at specific days after T cell injection, starting 10 days before injection (-10) and continuing until 40 days after T cell injection (40). Tumor volume ranged from 0 to 2500 mm³. [Figure 28-2] Figure 28B is a series of graphs showing tumor measurements taken with calipers, plotted for individual mice within each group. [Figure 28-3] Same as above. [Figure 28-4] Figure 28C shows a series of graphs illustrating the correlation between huCD3+ T cells in mouse blood and tumor growth. The graphs compare huCD3+ T cells to tumor volume 10 and 17 days after T cell injection. [Figure 28-5]Figure 28D is a pair of graphs showing the number of huCD4+ and huCD8+ T cells in peripheral blood, as determined by flow cytometry. Out-of-focus circles represent mice transplanted with "normal" cells; filled circles represent mice transplanted with tumor cells. Samples with fewer than 100 cells were excluded from the analysis. Error bars indicate ±SEM (n=6 in the CD19+ / A2-Raji group treated with CD19-CAR+A2-LIR-1 T cells, and n=7 in all other groups). [Figure 29-1] Figure 29A shows a series of images illustrating the histological analysis of T cell infiltration within the tumor. Representative images of tumor specimens collected, sectioned, and stained for huCD3 at the end of the study are shown. [Figure 29-2] Figure 29B is a graph showing the quantification of T cell infiltration using ImageJ. T cell infiltration was significantly higher in T cells with CD19-CAR or CD19-CAR+A2-LIR-1 in CD19+ / A2- tumors compared to untransduced cells. However, within CD19+ / A2+ tumors, CD19-CAR+A2-LIR-1 T cells did not show a significant difference compared to untransduced cells. There was also a significant decrease in CD19-CAR+A2-LIR-1 T cell infiltration between CD19+ / A2- tumors and CD19+ / A2+ tumors. Qualitatively, CD19-CAR+A2-LIR-1 T cells were not dominant in CD19+ / A2+ tumors compared to CD19-CAR-only T cells; however, this difference was not statistically significant. Saline samples were similarly quantified to show background staining levels. The data sets were analyzed using a standard one-way ANOVA, while individual pairs between "tumor" and "normal" were analyzed using independent t-tests. ns = not significant, *p < 0.05, **p < 0.01. [Modes for carrying out the invention]

[0031] This disclosure describes receptors having one or more domains from leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, sometimes referred to as LIR1 or LIR-1). Numerous receptors, engineered cells, and their uses are contemplated herein. The inventors have found that chimeric receptors comprising one or more LILRB1 domains, including an antigen-binding domain and an intracellular LILRB1 domain, can inhibit immune cell signaling even in the presence of activated chimeric antigen receptors (CARs) or T cell receptors (TCRs).

[0032] As used herein, the terms “chimeric antigen receptor” or “CAR” may refer, for example, to an artificial T cell receptor, chimeric T cell receptor, or chimeric immune receptor, and encompass engineered receptors that conjugate artificial specificity to specific immune effector cells, such as helper T cells (CD4+), cytotoxic T cells (CD8+), or NK cells. CARs may be used to confer the specificity of monoclonal antibodies to T cells, thereby generating a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, CARs induce cell specificity to tumor-associated antigens. In some embodiments, CARs include an intracellular signaling domain, a transmembrane domain, and an extracellular domain including an antigen-binding region. In some embodiments, CARs include a fusion of a single-strand variable fragment (scFv) or scFab derived from a monoclonal antibody, fused to the transmembrane domain and the intracellular signaling domain. The fusion may also include a hinge. Either heavy-light (HL) and light-heavy (LH) scFv may be used. The specificity of the CAR design may derive from the receptor ligand (e.g., a peptide). Depending on the type of intracellular domain, the CAR may be an activating receptor or an inhibitory receptor. In some embodiments, for example, if the CAR is an activating receptor, the CAR includes domains for further co-stimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10, and / or 0X40. In some embodiments, the molecule may include a co-stimulatory molecule, a reporter gene for imaging (e.g., for positron emission tomography), a gene product for conditionally removing T cells upon addition of a prodrug, a homing receptor, a cytokine, and a cytokine receptor, which may be co-expressed with the CAR. As used herein, the characteristic attributed to a chimeric antigen receptor may be understood to refer to the receptor itself or a host cell containing the receptor.

[0033] As used herein, “TCR,” sometimes also referred to as the “TCR complex” or “TCR / CD3 complex,” refers to a protein complex comprising one or more of the TCR alpha chain, TCR beta chain, and, sometimes referred to as subunits, immutable CD3 chains (zeta, gamma, delta, and epsilon). The TCR alpha and beta chains can disulfide-bond to each other and function as heterodimers to bind to peptide-MHC complexes. When the TCR alpha / beta heterodimer engages with peptide-MHC, it induces a structural change in the TCR complex at the associated immutable CD3 subunits, leading to their phosphorylation and association with downstream proteins, thereby transmitting the primary stimulus signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma form a heterodimer, and the two CD3 zeta form a homodimer.

[0034] The term "stimulus" refers to a primary response that is induced, but not limited to, by the binding of a stimulating domain or stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, and mediates a signaling event, such as signaling mediated by the TCR / CD3 complex. Stimuli can mediate changes in the expression of certain molecules and / or rearrangements of the cytoskeleton, among other things.

[0035] The terms “stimulating molecule” or “stimulating domain” refer to a molecule or portion thereof that, when naturally expressed by T cells, provides a primary cytoplasmic signaling sequence that modulates the activation of the TCR complex in a stimulating manner for at least some aspects of the T cell signaling pathway. The TCR alpha and / or TCR beta chains of the wild-type TCR complex do not contain stimulating domains and require association with a CD3 subunit, such as CD3 zeta, to initiate signaling. In one embodiment, the primary stimulating signal is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-bound major histocompatibility complex (MHC), thereby mediating a T cell response, including, but not limited to, proliferation, activation, and differentiation. One or more stimulating domains described herein may be fused to the intracellular portion of any one or more subunits of the TCR complex, including TCR alpha, TCR beta, CD3 delta, CD3 gamma, and CD3 epsilon.

[0036] As used herein, “stimulatory signal-providing domain” means any domain that can directly or indirectly provide a stimulatory signal to enhance or increase the effectiveness of signaling mediated by the TCR complex in order to enhance at least some aspects of T cell signaling. A stimulatory signal-providing domain can provide this signal directly; for example, a stimulatory signal-providing domain is a primary stimulatory domain or a co-stimulatory domain. Alternatively, or in addition, a stimulatory signal-providing domain can act indirectly. For example, a domain can be a scaffold for recruiting a stimulatory protein to the TCR, or it can provide enzymatic activity, such as kinase activity, that acts via a downstream target to provide a stimulatory signal.

[0037] As used herein, “inhibitory signal-providing domain” means any domain that can directly or indirectly provide an inhibitory signal that inhibits or reduces the effectiveness of signaling mediated by the TCR complex. An inhibitory signal-providing domain can reduce or block, whole or partially, at least some aspects of T cell signaling or function. An inhibitory signal-providing domain can provide this signal directly; for example, an inhibitory signal-providing domain provides a primary inhibitory signal. Alternatively, or in addition, a stimulating signal-providing domain can act indirectly. For example, a domain can recruit further inhibitory proteins to the TCR or provide enzymatic activity that acts via downstream targets to provide an inhibitory signal.

[0038] Scope: Throughout this disclosure, various aspects of the invention may be presented in the form of scopes. It should be understood that descriptions in the form of scopes are for convenience and brevity only and should not be interpreted as inflexible limitations on the scope of the invention. Accordingly, scope descriptions should be considered to have all possible sub-scopes specifically disclosed and the individual numbers within those scopes. For example, a scope description such as 1-6 should be considered to have the specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95-99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the range width.

[0039] Generally, “sequence identity” or “sequence homology” refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their “percent identity.” Percent identity of two sequences, whether nucleic acids or amino acid sequences, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Percent identity can also be determined by comparing sequence information using the Advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health, for example. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and is discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. This program can be used to determine the percentage identity of proteins over their entire length. Default parameters are provided, for example, to optimize searches on short query sequences in the blastp program. The desired range of sequence identity is approximately 80% to 100% and integer values ​​in between.Typically, the percentage identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0040] As used herein, “subsequence” means a length of consecutive amino acids or nucleotides that form part of a sequence described herein. When aligned with a full-length sequence, a subsequence may be identical to a portion of the full-length sequence, or may be less than 100% identical to the portion of the full-length sequence to which it is aligned (e.g., 50% and 90% identical to the whole sequence).

[0041] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecule compounds, etc., that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from within the organism (i.e., naturally produced by the organism).

[0042] When a polynucleotide is placed in a functional relationship with another polynucleotide, it is "operably ligated" to that polynucleotide. For example, a promoter or enhancer is operably ligated to a coding sequence when it affects the transcription of the sequence. When a coding polynucleotide is operably ligated, the peptide is "operably ligated" to another peptide, and preferably they are within the same open reading frame.

[0043] A "promoter" is a sequence of DNA required to turn a gene on or off. Promoters are located immediately upstream of and / or overlapping with the transcription start site and are typically between 100 and several hundred base pairs in length.

[0044] All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of any conflict, including any definitions herein, this application shall prevail. However, any reference to any reference, article, publication, patent, patent gazette, and patent application cited herein shall not and should not be construed as any form of acknowledgment or suggestion that they constitute valid prior art or form part of the common general knowledge in any country of the world.

[0045] In this explanation, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated. The term "approximately" when it immediately precedes a number or value means that the number or value is within a range of plus or minus 10%.

[0046] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0047] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) This disclosure describes receptors having one or more domains from leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, or LIR1). Numerous receptors, engineered cells, and their uses are contemplated herein.

[0048] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as leukocyte immunoglobulin-like receptor B1, along with ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2, LIR-1, MIR-7, and PIR-B, are members of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to subfamily B class of LIR receptors. These receptors contain 2-4 extracellular immunoglobulin domains, a transmembrane domain, and 2-4 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells, transmitting a negative signal that inhibits the stimulation of the immune response. LILRB1 is thought to play a role in regulating inflammatory responses and cytotoxicity, and limiting autoreactivity. Multiple transcript variants encoding different isoforms of LILRB1 exist, all of which are intended to be within the scope of this disclosure.

[0049] In some embodiments of receptors having one or more domains of LILRB1, one or more domains of LILRB1 include an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the sequence or subsequence of SEQ ID NO: 1. In some embodiments, one or more domains of LILRB1 include an amino acid sequence that is identical to the sequence or subsequence of SEQ ID NO: 1. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the sequence or subsequence of SEQ ID NO: 1. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence that is identical to the sequence or subsequence of SEQ ID NO: 1.

[0050] In some embodiments of receptors having one or more domains of LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the sequence or partial sequence of SEQ ID NO: 34.

[0051] In some embodiments of receptors having one or more domains of LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or partial sequence of SEQ ID NO: 34.

[0052] receptor In various embodiments, a chimeric antigen receptor is provided, comprising a polypeptide, wherein the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof, a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain, or at least one or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising one or more intracellular domains independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0053] intracellular domain This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises an intracellular domain. In some embodiments, the intracellular domain is the LILRB1 intracellular domain or a functional variant thereof.

[0054] As used herein, “intracellular domain” refers to the cytoplasmic or intracellular domain of a protein, such as a receptor, that interacts with the inside of a cell and performs cytoplasmic functions. As used herein, “cytoplasmic function” refers to the function of a protein or protein complex that is performed in the cytosol of a cell. For example, intracellular signaling cascades are cytoplasmic functions.

[0055] As used herein, “immune receptor tyrosine-based inhibitory motif” or “ITIM” refers to a conserved amino acid sequence having a consensus sequence such as S / I / V / LxYxxI / V / L (SEQ ID NO: 124) found in the cytoplasmic tails of many inhibitory receptors of the immune system. After inhibitory receptors possessing an ITIM interact with their ligands, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol phosphatase called SHIP.

[0056] In some embodiments, the polypeptide includes an intracellular domain comprising at least one immune receptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least three ITIMs, at least four ITIMs, at least five ITIMs, or at least six ITIMs. In some embodiments, the intracellular domain has one, two, three, four, five, or six ITIMs.

[0057] In some embodiments, the polypeptide includes an intracellular domain comprising at least one ITIM selected from the group of ITIMs consisting of NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0058] In further specific embodiments, the polypeptide comprises at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0059] In some embodiments, the intracellular domain includes both ITIM sequences NLYAAV (SEQ ID NO: 8) and VTYAEV (SEQ ID NO: 9). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the intracellular domain includes or is essentially identical to a sequence that is identical to SEQ ID NO: 12.

[0060] In some embodiments, the intracellular domain includes both ITIM sequences VTYAEV (SEQ ID NO: 9) and VTYAQL (SEQ ID NO: 10). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the intracellular domain includes or is essentially identical to a sequence that is identical to SEQ ID NO: 13.

[0061] In some embodiments, the intracellular domain includes both ITIM sequences VTYAQL (SEQ ID NO: 10) and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the intracellular domain includes or is essentially identical to a sequence that is SEQ ID NO: 14.

[0062] In some embodiments, the intracellular domain includes ITIMs of NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), and VTYAQL (SEQ ID NO: 10). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the intracellular domain includes a sequence that is identical to or essentially identical to SEQ ID NO: 15.

[0063] In some embodiments, the intracellular domain includes ITIMs of VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the intracellular domain includes a sequence that is identical to or essentially the same as SEQ ID NO: 16.

[0064] In some embodiments, the intracellular domain includes ITIM sequences NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the intracellular domain includes a sequence that is identical to or essentially identical to SEQ ID NO: 17.

[0065] In some embodiments, the intracellular domain includes a sequence that is at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 7). In some embodiments, the intracellular domain includes or is essentially identical to the LILRB1 intracellular domain (SEQ ID NO: 7).

[0066] The LILRB1 intracellular domain or its functional variants may have at least one, at least two, at least four, at least four, at least five, at least six, at least seven, or at least eight ITIMs. In some embodiments, the LILRB1 intracellular domain or its functional variants may have two, three, four, five, or six ITIMs.

[0067] In certain embodiments, the polypeptide comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0068] In certain embodiments, the polypeptide comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0069] In certain embodiments, the polypeptide comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0070] In certain embodiments, the polypeptide comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0071] In certain embodiments, the polypeptide comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0072] In certain embodiments, the polypeptide comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0073] In certain embodiments, the polypeptide comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0074] In some embodiments, the intracellular domain includes a TCR alpha intracellular domain. In some embodiments, the intracellular domain includes a TCR alpha intracellular domain and a LILRB1 intracellular domain, as described herein. In some embodiments, the TCR alpha intracellular domain includes Ser-Ser. In some embodiments, the TCR alpha intracellular domain is encoded by the TCCAGC sequence.

[0075] In some embodiments, the intracellular domain includes a TCR beta intracellular domain. In some embodiments, the intracellular domain includes a TCR beta intracellular domain and a LILRB1 intracellular domain, as described herein. In some embodiments, the TCR beta intracellular domain includes an amino acid sequence that has at least 80% identity, at least 90% identity, or is identical to the sequence of MAMVKRKDSR (SEQ ID NO: 94). In some embodiments, the TCR beta intracellular domain includes or is essentially derived from MAMVKRKDSR (SEQ ID NO: 94). In some embodiments, the TCR beta intracellular domain is encoded by the sequence of ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 95).

[0076] transmembrane domain This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a transmembrane domain. In some embodiments, the transmembrane domain is the LILRB1 transmembrane domain or a functional variant thereof.

[0077] As used herein, “transmembrane domain” refers to a protein domain that extends across the cell membrane. Transmembrane domains are typically composed primarily of nonpolar amino acids and can traverse the lipid bilayer once or multiple times. Transmembrane domains usually contain an alpha-helix, a configuration that maximizes internal hydrogen bonding.

[0078] Transmembrane domains isolated from or derived from any source are assumed to be within the scope of the fusion proteins of this disclosure. In certain embodiments, the polypeptide comprises the LILRB1 transmembrane domain or a functional variant thereof.

[0079] In some embodiments, the LILRB1 transmembrane domain or its functional variant contains a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembrane domain or its functional variant contains a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembrane domain contains a sequence that is identical to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembrane domain is essentially derived from a sequence that is identical to SEQ ID NO: 5.

[0080] In some embodiments of the chimeric antigen receptors of this disclosure, the transmembrane domain is not the LILRB1 transmembrane domain. In some embodiments, the transmembrane domain is associated with one of the other domains of the fusion protein, or is isolated from or derived from the same protein as one of the other domains of the fusion protein.

[0081] Transmembrane domains may originate from either natural or recombinant sources. If the source is natural, the domains may originate from any membrane-binding or transmembrane protein. Exemplary transmembrane domains may include, for example, the alpha, beta, or zeta chains of the TCR, the transmembrane regions of CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0082] In some embodiments, the transmembrane domain includes a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain includes an amino acid sequence that has or is identical to the sequence VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96) with at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and at least 99% identity. In some embodiments, the TCR alpha transmembrane domain includes or is essentially derived from VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96). In some embodiments, the TCR alpha transmembrane domain is encoded by the sequence GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 97).

[0083] In some embodiments, the transmembrane domain includes a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain includes an amino acid sequence that has or is identical to the sequence TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 98) with at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and at least 99% identity. In some embodiments, the TCR beta transmembrane domain includes or is essentially derived from TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 98). In some embodiments, the TCR beta transmembrane domain is encoded by the sequence ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 99).

[0084] In some embodiments, the TCR alpha and / or TCR beta transmembrane domains include one or more mutations that attenuate or disable the interaction of the TCR with the TCR CD3 subunit. In some embodiments, the TCR alpha transmembrane domain includes an R253L mutation. In some embodiments, the TCR beta transmembrane domain includes a K288L mutation.

[0085] In some embodiments, the transmembrane domain includes the CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain includes an amino acid sequence that has or is identical to the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 100) with at least 80% identity, at least 90% identity, at least 95% identity, and at least 99% identity. In some embodiments, the CD28 transmembrane domain includes or is essentially derived from FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 100). In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence that has or is identical to the sequence TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 101) with at least 80% identity, at least 90% identity, at least 95% identity, and at least 99% identity.

[0086] In some embodiments, the transmembrane domain may attach to an extracellular chimeric antigen receptor, such as an antigen-binding domain or a ligand-binding domain, via a hinge, such as a hinge derived from a human protein. For example, in some embodiments, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0087] Hinged Domain This disclosure provides a chimeric antigen receptor comprising a polypeptide sequence. In some embodiments, the polypeptide comprises a hinge domain. In some embodiments, the hinge domain is the LILRB1 hinge domain or a functional variant thereof.

[0088] The LILRB1 protein has four immunoglobulin (Ig)-like domains, called D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain includes the LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain includes a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18. In some embodiments, the LILRB1 D3D4 domain includes or is essentially derived from SEQ ID NO: 18.

[0089] In some embodiments, the polypeptide comprises the LILRB1 hinge domain or a functional fragment or variant thereof. In embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19. In embodiments, the LILRB1 hinge domain or a functional fragment or variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19.

[0090] In some embodiments, the LILRB1 hinge domain includes a sequence identical to sequence number 4, sequence number 18, or sequence number 19.

[0091] In some embodiments, the LILRB1 hinge domain essentially consists of a sequence identical to sequence number 4, sequence number 18, or sequence number 19.

[0092] In some embodiments, the chimeric antigen receptors, polypeptides of this disclosure include hinges that are not isolated from or derived from LILRB1.

[0093] In some embodiments, the hinge is isolated from or derived from CD8α or CD28. In some embodiments, the CD8α hinge includes an amino acid sequence that has or is identical to the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102) with at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity. In some embodiments, the CD8α hinge includes TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102). In some embodiments, the CD8α hinge is essentially derived from TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102). In some embodiments, the CD8α hinge is encoded by a nucleotide sequence that has or is identical to the sequence accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 103) and has at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity.

[0094] In some embodiments, the CD28 hinge includes an amino acid sequence that has or is identical to the sequence CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 104) with at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity. In some embodiments, the CD28 hinge includes or is essentially derived from CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 104). In some embodiments, the CD28 hinge is encoded by a nucleotide sequence that has or is identical to the sequence tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 105) and has at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity.

[0095] LILRB1 domain combinations In some embodiments, the chimeric antigen receptor of the present disclosure comprises a polypeptide containing one or more LILRB1 domains or their functional equivalents. For example, in some embodiments, the polypeptide comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain and an intracellular domain.

[0096] In certain embodiments, the polypeptide comprises the LILRB1 hinge domain or a functional fragment or variant thereof, and the LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 20. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO: 20.

[0097] In further embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, as well as a LILRB1 intracellular domain and / or at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM comprises an intracellular domain selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, as well as a LILRB1 intracellular domain and / or at least two ITIMs, wherein each ITIM comprises an intracellular domain independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0098] In some embodiments, the polypeptide includes a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide includes a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 21. In some embodiments, the polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the polypeptide includes a sequence that is identical to SEQ ID NO: 21.

[0099] In preferred embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional fragment or variant thereof, a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain, and / or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM comprising an intracellular domain independently selected from LYAAV (SEQ ID NO: 8), VTYAE (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

[0100] In some embodiments, the polypeptide includes a sequence that is at least 95% identical to sequence number 2 or sequence number 3, or a sequence that is at least 99% identical to sequence number 2 or sequence number 3, or a sequence that is identical to sequence number 2 or sequence number 3.

[0101] In some embodiments, the polypeptide includes a sequence that is at least 99% identical to sequence number 20, or a sequence that is at least 99% identical to sequence number 20, or a sequence that is identical to sequence number 20.

[0102] In some embodiments, the polypeptide includes a sequence that is at least 99% identical to sequence number 21, or a sequence that is at least 99% identical to sequence number 21, or a sequence that is identical to sequence number 21.

[0103] Extracellular domain This disclosure provides a chimeric antigen receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a ligand-binding domain, such as an antigen-binding domain. Suitable antigen-binding domains include, but are not limited to, antibody-derived antigen-binding domains, antibody fragments, scFvs, and T-cell receptor-derived antigen-binding domains. All forms of antigen-binding domains known in the art are assumed to be within the scope of this disclosure.

[0104] As used herein, “extracellular domain” refers to the extracellular portion of a protein. For example, each of the TCR alpha and beta chains contains an extracellular domain that includes constant and variable regions involved in peptide-MHC recognition. The “extracellular domain” may also include, for example, a fusion domain of a fusion between an additional domain capable of binding to and targeting a specific antigen and the endogenous extracellular domain of the TCR subunit.

[0105] As used herein, the term “antibody” refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins or fragments thereof of polyclonal or monoclonal origin and may be derived from natural or recombinant sources.

[0106] The terms “antibody fragment” or “antibody-binding domain” refer to an antibody, or at least a portion of a recombinant variant thereof, that contains an antigen-binding domain, i.e., an antigen-determining variable region of an intact antibody sufficient to give the antibody fragment recognition and specific binding to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-stranded (sc)Fv ("scFv") antibody fragments, linear antibodies, single-domain antibodies (abbreviated as "sdAb") (either VL or VH), camel VHH domains, and multispecific antibodies formed from antibody fragments.

[0107] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked via a short, flexible polypeptide linker, allowing it to be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it originates.

[0108] In the context of antibodies, a "heavy chain variable region" or "VH" (or, in the case of a single-domain antibody, e.g., a nanobody, "VHH") refers to a heavy chain fragment containing three CDRs interposed between adjacent stretches, known as framework regions. These framework regions are generally more conserved than the CDRs and form a scaffold to support them.

[0109] Unless otherwise specified, as used herein, scFv may have VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and scFv may contain a VL-linker-VH or a VH-linker-VL.

[0110] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa ("Κ") and lambda ("λ") light chains refer to the two main antibody light chain isotypes.

[0111] The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. The term should also be interpreted to mean an antibody or an amino acid sequence that identifies an antibody, which is produced by the synthesis of a DNA molecule encoding the antibody, and which expresses an antibody protein, and which is obtained using recombinant DNA or amino acid sequence technologies that are available and well known in the art.

[0112] In some embodiments, for example, in these embodiments in which the receptor comprises first and second polypeptides, the antigen-binding domain is isolated from or derived from the extracellular domain of the T cell receptor (TCR) or an antibody.

[0113] In preferred embodiments, the polypeptide includes antigen-binding domains, e.g., antigen-binding domains other than the LILRB1 antigen-binding protein. An exemplary embodiment of a receptor having a single antigen-binding domain is shown in Figure 1. The chimeric antigen receptors intended by this disclosure have two, three, four, or more antigen-binding domains. The antigen-binding domains may be provided on the same or different chains of the chimeric antigen receptor. In embodiments, the chimeric antigen receptor is a DARIC, for example, as described in Leung et al. JCI Insight. 2019 Jun 6; 4(11):e124430, WO2015017214A1; and WO2017156484A1.

[0114] In some embodiments, the receptor is an inhibitory chimeric antigen receptor (iCAR). Various methods and compositions suitable for use as disclosed herein include those provided in US2018 / 0044399A1;WO2018148454A1; and WO2017087723A1, each of which is incorporated herein for all purposes.

[0115] In some embodiments, the antigen-binding domain includes a single-stranded variable fragment (scFv).

[0116] In some embodiments, the receptor comprises a second polypeptide. The disclosure provides a receptor having two polypeptides, each having a portion of a ligand-binding domain (e.g., a heterodimeric LDB such as TCRα / β- or Fab-based LBD) and each having an intracellular domain, as shown in Figure 2A. The disclosure further provides a receptor having two polypeptides, each having a portion of a ligand-binding domain (e.g., a heterodimeric LDB such as TCRα / β- or Fab-based LBD), where one portion of the ligand-binding domain is fused to a hinge or transmembrane domain, while the other portion of the ligand-binding domain does not have an intracellular domain, as shown in Figure 2B. Further variations include receptors in which each polypeptide has a hinge domain and each polypeptide has both a hinge and a transmembrane domain. In some embodiments, the hinge domain is absent. In other embodiments, the hinge domain is a membrane-proximal extracellular region (MPER), such as the LILRB1 D3D4 domain. In any of the embodiments disclosed herein, domains may be fused adjacent to one another by linkers between them.

[0117] In some embodiments, the first polypeptide comprises the first chain of the antibody, and the second polypeptide comprises the second chain of the antibody.

[0118] In some embodiments, the receptor comprises a Fab fragment of the antibody. In embodiments, the antigen-binding fragment of the antibody's heavy chain and the second polypeptide comprise an antigen-binding fragment of the antibody's light chain. In embodiments, the first polypeptide comprises an antigen-binding fragment of the antibody's light chain and the second polypeptide comprises an antigen-binding fragment of the antibody's heavy chain.

[0119] In some embodiments, the first polypeptide comprises the first chain of the T cell receptor (TCR), and the second polypeptide comprises the second chain of the TCR. In embodiments, the receptor comprises an extracellular fragment of the T cell receptor (TCR). In embodiments, the first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR, and the second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR, and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

[0120] In some embodiments, the receptor includes a single-stranded TCR, such as, but not limited to, those disclosed in WO2017091905A1.

[0121] Exemplary antigen-binding domain Various single variable domains known in the art or disclosed herein are suitable for use in the embodiments. Such scFvs include, for example, HLA-A by peptide-independent means, but are not limited to * The following mouse and humanized scFv antibodies bind to O2 (complementarity-determining regions are underlined): [ka] [ka] [Table 1]

[0122] In some embodiments, the scFv includes one of the complementarity-determining regions (CDRs) of SEQ ID NOs. 22-33. In some embodiments, the scFv includes a sequence that is at least 95% identical to one of SEQ ID NOs. 22-33. In some embodiments, the scFv includes a sequence that is identical to one of SEQ ID NOs. 22-33. In some embodiments, the antibody heavy chain includes one of the heavy chain CDRs of SEQ ID NOs. 25-27 or 31-33, and the antibody light chain includes one of the light chain CDRs of SEQ ID NOs. 22-24 or 28-30. In some embodiments, the antibody heavy chain includes a sequence that is at least 95% identical to one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the antibody light chain includes a sequence that is at least 95% identical to one of the light chain portions of SEQ ID NOs. 35-46 or 125. In some embodiments, the heavy chain includes all of SEQ ID NOs. 25-27, and the light chain includes all of SEQ ID NOs. 22-24. In some embodiments, the heavy chain includes all of sequence numbers 31-33, and the light chain includes all of sequence numbers 28-30.

[0123] In some embodiments, the antibody heavy chain includes a sequence identical to one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the antibody light chain includes a sequence identical to one of the light chain portions of SEQ ID NOs. 35-46 or 125.

[0124] In some embodiments, the ScFv includes a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to one of sequence numbers 35-46 or 125.

[0125] B lymphocyte and T lymphocyte attenuator (BTLA) domains In some embodiments, the polypeptide comprises B lymphocyte and T lymphocyte attenuator (BTLA) hinge domains, transmembrane domains, intracellular domains, or functional variants, derivatives, or combinations thereof.

[0126] In some embodiments, the polypeptide includes a BTLA intracellular domain. In some embodiments, the BTLA intracellular domain includes the sequence RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 87). In some embodiments, the BTLA intracellular domain includes SEQ ID NO: 87, or a sequence having at least 95% identity thereto. In some embodiments, the BTLA intracellular domain is essentially derived from SEQ ID NO: 87.

[0127] In some embodiments, the BTLA transmembrane domain and intracellular domain include a sequence that is at least 95% identical to the sequence of Sequence ID No. 88. In some embodiments, the BTLA transmembrane domain and intracellular domain include or are essentially derived from the sequence of Sequence ID No. 88.

[0128] Signal peptide In some embodiments, the polypeptide includes a signal peptide. For example, the polypeptide includes the VK1 signal peptide. In some embodiments, the signal peptide is an N-terminal signal peptide. In some embodiments, the signal peptide includes a sequence that is at least 95% identical to the sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide includes the sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide is encoded by a sequence that is at least 95% identical to the sequence of ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQ ID NO: 129), or a sequence that is identical thereto.

[0129] antigen Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, can act as an antigen to the LILRB1-based receptor described herein. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded solely by a full-length nucleotide sequence of a gene. Furthermore, those skilled in the art will understand that an antigen does not have to be encoded by a "gene" at all. It is readily apparent that antigens may be generated or synthesized, or drawn from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or liquids containing other biological components.

[0130] In some embodiments, the antigen-binding domain is etiolate receptor, ανββ integrin, TNF receptor superfamily member 17 (BCMA), CD276 molecule (B7-H3), natural killer cell cytotoxic receptor 3 ligand 1 (B7-H6), carbonic anhydrase 9 (CAIX), CD19 molecule (CD19), transmembrane 4-domain A1 (CD20), CD22 molecule (CD22), TNF receptor superfamily member 8 (CD30), CD33 molecule (CD33), CD37 molecule (CD37), CD44 molecule (CD44), CD44v6, CD44v7 / 8, CD70 molecule (CD70), interleukin 3 receptor subunit alpha (CD123), syndecane 1 (CD138), L1 cell adhesion molecule (CD171), CEA cell adhesion molecule (CEA), delta-like canonical Notch ligand 4 (DLL4), epithelial cell adhesion molecule (E GP-2), epithelial cell adhesion molecule (EGP-40), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor receptor (EGFR), EGFR family including ErbB2 (HER2), EGFRvIII, epithelial cell adhesion molecule (EPCAM), EPH receptor A2 (EphA2), EpCAM, fibroblast activating protein alpha (FAP), folate receptor alpha (FBP), fetal acetylcholine receptor, frizzled class receptor 7 (Fzd7), diganglioside GD2 (GD2), ganglioside GD3 (GD3), glypican-3 (GPC3), trophoblast glycoprotein (h5T4), interleukin-11 receptor subunit alpha (IL-11R), interleukin-13 receptor subunit alpha 2 (IL13R-a2), kinase insertion domain receptor (KDR), κ light chain, λ light chain, LeY, L1 cell adhesion molecule (L1It specifically binds to targets selected from CAM, MAGE-A1, mesothelin, MHC-presenting peptides, cell surface-bound mucin 1 (MUC1), cell surface-bound mucin 16 (MUC16), neuronal cell adhesion molecule 1 (NCAM), killer cell lectin-like receptor K1 (NKG2D) ligand, Notch1, Notch2 / 3, NY-ESO-1, PRAME nuclear receptor transcription regulator (PRAME), prostate stem cell antigen (PSCA), folate hydrolase 1 (PSMA), Survivin, TAG-72, TEM, telomerase reverse transcriptase (TERT), kinase insertion domain receptor (VEGFR2), and receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0131] In some embodiments, the antigen-binding domain specifically binds to targets selected from CD33, CD38, human leukocyte antigens (HLA), organ-specific antigens, blood-brain barrier-specific antigens, epithelial-mesenchymal transition (EMT) antigens, E-cadherins, cytokeratins, opioid-binding proteins / cell adhesion molecules (OPCMLs), HYLA2, colorectal cancer deletion (DCC), scaffold / matrix adhesion region-binding protein 1 (SMAR1), cell surface glycans, and mucin-type O-glycans.

[0132] In some embodiments, the extracellular domain of the LILRB1-based receptor described herein includes an antigen-binding domain specific to the antigen lost through loss of heterozygosity in the target cell.

[0133] As used herein, “Loss of Heterozygosity (LOH)” refers to a genetic change that occurs frequently in cancer, resulting in the deletion of one of two alleles, leaving a single allele (hemizygous) locus.

[0134] In some embodiments, LILRB1-based receptors include an antigen-binding domain specific to minor histocompatibility antigens (MiHAs). MiHAs are peptides derived from proteins containing non-synonymous differences between alleles, presented by a common HLA allele. Non-synonymous differences may arise from SNPs, deletions, frameshift mutations, or insertions in the coding sequence of the gene encoding the MiHA. Exemplary MiHAs may be about 9–12 amino acids long and can bind to MHC class I and MHC class II proteins. Binding of a TCR to an MHC complex presenting a MiHA can activate T cells. The genetic and immunological properties of MiHAs are known to those skilled in the art, and specific MiHAs are described in PCT / US2020 / 045228 (its contents are incorporated by reference).

[0135] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to the antigen lost in target cancer cells through Y chromosome loss.

[0136] In some embodiments, the LILRB1-based receptor contains an antigen-binding domain specific to the HLA class I allele. Major histocompatibility complex (MHC) class I is a protein complex that presents antigens to cells of the immune system, triggering an immune response. The human leukocyte antigens (HLAs) corresponding to MHC class I are HLA-A, HLA-B, and HLA-C. HLA-E is known in the art as a non-classical MHC class I molecule. In some embodiments, the antigen for the LILRB1-based receptor contains the HLA class I allele. In some embodiments, the HLA class I allele is lost in target cells, such as cancer cells, through loss of heterozygosity (LOH).

[0137] HLA-A is a group of human leukocyte antigens (HLAs) of the major histocompatibility complex (MHC) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. Its receptor is a heterodimer containing a heavy α chain and a smaller β chain. The α chain is encoded by HLA-A variants, while the β chain (β2-microglobulin) remains unchanged. There are thousands of HLA-A variants, all of which are within the scope of this disclosure.

[0138] In some embodiments, LILRB1-based receptors contain an antigen-binding domain specific to the HLA-B allele. The HLA-B gene has many possible variations (alleles). Hundreds of versions (alleles) of the HLA-B gene are known, each of which is assigned a specific number (e.g., HLA-B27).

[0139] In some embodiments, LILRB1-based receptors include an antigen-binding domain specific to the HLA-C allele. HLA-C belongs to the HLA class I heavy chain paralog. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin).

[0140] In some embodiments, the HLA class I allele results in widespread or ubiquitous RNA expression.

[0141] In some embodiments, the HLA class I allele has known or generally high minor allele frequencies.

[0142] In some embodiments, the HLA class I allele does not require a peptide-MHC antigen when it is recognized, for example, by the pan-HLA ligand-binding domain.

[0143] In some embodiments, the LILRB1-based receptor includes an antigen-binding domain specific to the HLA-A allele. In some embodiments, the HLA-A allele is HLA-A* Includes 02. HLA-A * Various single variable domains known in the Art or disclosed herein that combine with and recognize 02 are suitable for use in embodiments and are described herein.

[0144] In some embodiments, the antigen-binding domain is HLA-A * 02 Specifically binds to the antigen. In some embodiments, the antigen-binding domain is HLA-A * 02 Specifically binds to the antigen in a peptide-independent manner.

[0145] Polynucleotides and vectors In other embodiments, the Disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the receptor of the Disclosure. In some embodiments, the polynucleotide encodes one or more of the LILRB1 hinge domain, the LILRB1 transmembrane domain, and the LILRB1 intracellular domain, or functional derivatives or fragments thereof.

[0146] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to one of SEQ ID NOs: 1-7 or 12-21. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to one of SEQ ID NOs: 47-71, 77-79, 89-92, 120, or 122. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to the heavy chain or light chain portion of one of SEQ ID NOs: 35-46 or 125. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is at least 95% identical to the heavy chain or light chain portion of one of SEQ ID NOs: 35, 39, 46, or 125. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide that is identical to the heavy chain or light chain portion of one of SEQ ID NOs: 35, 39, 46, or 125. In another embodiment, the Disclosure provides a vector comprising a polynucleotide encoding the receptor of the Disclosure.

[0147] In some embodiments, the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 121 or 123. In some embodiments, the polynucleotide comprises SEQ ID NO: 121 or 123.

[0148] In some embodiments, the polynucleotide includes sequences of the LILRB1 hinge, transmembrane, and intracellular domain. In some embodiments, the polynucleotide includes a sequence that is at least 95% identical to sequence number 126. In some embodiments, the polynucleotide includes the sequence of sequence number 126.

[0149] Retrovirus vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term, stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have an additional advantage over onchoretrovirus vectors, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0150] The expression of native or synthetic nucleic acids encoding receptors is typically achieved by operably ligating the nucleic acid encoding the receptor or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation in eukaryotes. A typical cloning vector contains transcription and translation terminators, start sequences, and promoters useful for controlling the expression of a desired nucleic acid sequence.

[0151] Polynucleotides encoding receptors can be cloned into several types of vectors. For example, polynucleotides can be cloned into vectors that include plasmids, phagemids, phage derivatives, animal viruses, and cosmids, without limitation. Particularly targeted vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0152] Furthermore, expression vectors can be supplied to cells, such as immune cells, in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains an origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers that can function in at least one organism (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0153] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Several adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0154] In some embodiments, the vector includes a promoter. The vector may also include additional regulatory elements. These additional regulatory elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located 30–110 base pairs (bp) upstream of the start site, although some promoters have recently been shown to also include functional elements downstream of the start site. The spacing between promoter elements is flexible so that promoter function is preserved when elements are reversed or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decrease. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0155] One example of a suitable promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but are not limited to, the monkey virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, and, in addition, human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of inductive promoters provides a molecular switch that has the ability to turn on the expression of the polynucleotide sequence it operably ligates to when such expression is desired, or turn off when such expression is not desired. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0156] To evaluate receptor expression, the expression vector introduced into cells may also contain either or both a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells intended to be transfected or infected via a viral vector. In other embodiments, the selection marker may be vested on a separate DNA fragment and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0157] Methods for introducing genes into cells and expressing them are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0158] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Methods for creating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0159] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0160] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersions such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery medium is liposomes (e.g., artificial membrane vesicles).

[0161] Various assays can be performed to confirm the presence of recombinant DNA sequences in host cells, whether by methods used to introduce exogenous nucleic acids into host cells or by other methods used to expose cells to the inhibitors of the present invention. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and “biochemical” assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting) or by assays described herein for identifying active ingredients within the scope of the present invention.

[0162] Manipulated cells In another embodiment, the Disclosure provides a nucleic acid sequence or vector encoding the receptor of the Disclosure and / or an immune cell expressing the receptor of the Disclosure.

[0163] In embodiments, immune cell activation is reduced when the cell comes into contact with an antigen of the LILRB1-based receptor of this disclosure, or with a cell expressing that antigen on its surface. In embodiments, immune cell activation includes the expression of a gene operably linked to the NFAT promoter. Immune cell activation and / or inhibition of activation can be measured by various other methods known in the art. In some embodiments, the immune cell includes additional exogenous receptors, such as activator receptors, including chimeric antigen receptors (CARs) or TCRs.

[0164] In this embodiment, the immune cells are T cells.

[0165] As used herein, the term “immune cells” refers to cells involved in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytes such as neutrophils, monocytes, and macrophages; polymophonuclear leukocytes such as natural killer cells, neutrophils, eosinophils, and basophils; and mononuclear cells such as monocytes, macrophages, and mast cells. Immune cells that play a role in acquired immunity include lymphocytes such as T cells and B cells.

[0166] As used herein, “T cell” refers to a type of lymphocyte that originates from bone marrow progenitor cells that develop in the thymus. There are several distinct types of T cells that develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, and stem memory T cells. Different types of T cells can be distinguished by those skilled in the art based on the expression of their markers. Methods for distinguishing between T cell types will be readily apparent to those skilled in the art.

[0167] Method for creating manipulated cells In another embodiment, the Disclosure provides a method comprising introducing the polynucleotides of the Disclosure into cells, optionally using the vectors of the Disclosure. In an embodiment, the resulting cells express a LILRB1-based receptor encoded by the polynucleotide. In an embodiment, the cells are immune cells. In an embodiment, the immune cells are T cells.

[0168] A method for transforming a population of immune cells, such as T cells, with the vectors of this disclosure will be readily apparent to those skilled in the art. For example, CD3+ T cells can be isolated from PBMCs using the CD3+ T cell negative isolation kit (Miltenyi) according to the manufacturer's instructions for use. The T cells are isolated at a rate of 1 × 10⁶ in X-Vivo 15 medium supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1:1 cell-to-bead ratio) and 300 units / mL of IL-2 (Miltenyi). 6 Cells can be cultured at a density of 100 cells / mL. After 2 days, T cells can be transduced with a viral vector, such as a lentiviral vector, using methods known in the art. In some embodiments, the viral vector is transduced at an infection multiplicity (MOI) of 5. The cells can then be cultured for an additional 5 days before enrichment with IL-2 or other cytokines, e.g., a combination of IL-7 / 15 / 21. Methods for isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells will be readily apparent to those skilled in the art. Although this method depicts a promising approach, it should be noted that these methodologies are rapidly evolving. For example, excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth, producing a highly transduced cell population with >99% CD3+.

[0169] Methods for activating and culturing populations of T cells comprising the receptors, polynucleotides, or vectors of this disclosure will be readily apparent to those skilled in the art.

[0170] Before or after genetic modification, T cells can generally be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Patent Application Publication No. 2006 / 0121005.

[0171] In some embodiments, the T cells of the disclosure are expanded and activated in vitro. Generally, the T cells of the disclosure are expanded in vitro by contact with a surface to which an agent that stimulates CD3 / TCR complex - related signals and a ligand that stimulates costimulatory molecules on the surface of the T cell are attached. In particular, a T cell population can be stimulated as described herein, such as by contact with an anti - CD3 antibody. For costimulation of accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, a population of T cells can be contacted with an anti - CD3 antibody and an anti - CD28 antibody under conditions appropriate to stimulate the proliferation of T cells. Anti - CD3 and anti - CD28 antibodies can be used to stimulate the proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti - CD28 antibodies include 9.3, B - T3, XR - CD28 (Diaclone, Besancon, France), and they can be used such that other methods generally known in the art can also be used [Berg et al., Transplant Proc. 30(8):3975 - 3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319 - 1328, 1999; Garland et al., J. Immunol Meth. 227(1 - 2):53 - 63, 1999].

[0172] In some embodiments, the primary stimulation signal and the co-stimulation signal for T cells can be provided by different protocols. For example, the agents that provide each signal can be in solution or can be linked to a surface. When linked to a surface, the agent can be linked to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent can be linked to a surface and the other agent can be in solution. In some embodiments, the agent that provides the co-stimulation signal is bound to the cell surface, and the agent that provides the primary activation signal is in solution or linked to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agent can be in the form of a solution and then cross-linked to a surface such as a cell that expresses an Fc receptor, or an antibody, or another binding substance that binds to the agent. In this context, for example, reference is made to US Patent Application Publications Nos. 20040101519 and 20060034810 regarding artificial antigen-presenting cells (aAPCs) contemplated for use in activating and proliferating T cells in the present invention.

[0173] In some embodiments, the two agents are immobilized on beads, either on the same bead (i.e., “cis”) or on separate beads (i.e., “trans”). As an example, the agent that provides the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent that provides the co-stimulation signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead at an equal molecular quantity. In one embodiment, a 1:1 ratio of each antibody bound to beads for CD4+ T cell proliferation and T cell growth is used. In some embodiments, the ratio of CD3:CD28 antibodies bound to beads is in the range from 100:1 to 1:100, including all integer values therebetween. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1.

[0174] Particle-to-cell ratios ranging from 1:500 to 500:1, including any integer value in between, can be used to stimulate T cells or other target cells. As will be readily apparent to those skilled in the art, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, smaller beads may bind fewer cells, while larger beads may bind more cells. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1, including any integer value in between, and in further embodiments, the ratio ranges from 1:9 to 9:1, including any integer value in between, which can also be used to stimulate T cells. In some embodiments, a cell-to-bead ratio of 1:1 is used. Those skilled in the art will understand that various other ratios may be suitable for use in the present invention. In particular, the ratio varies depending on the particle size as well as the cell size and type.

[0175] In a further embodiment of the present invention, cells such as T cells are combined with active substance-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, the active substance-coated beads and cells are not separated before culture, but are cultured together. In a further embodiment, the beads and cells are first enriched by the application of a force such as magnetism, resulting in an increase in the ligation of cell surface markers, thereby inducing cell stimulation.

[0176] For example, cell surface proteins can be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 proteins are attached to come into contact with T cells. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28T paramagnetic beads in a 1:1 ratio) are combined in a buffer. Again, it will be readily apparent to those skilled in the art that any cell concentration can be used. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, a concentration higher than 100 million cells / ml is used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, cell concentrations ranging from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In yet another embodiment, concentrations of 125 million or 150 million cells / ml may be used. In one embodiment, 1 × 10 6 Cells cultured at a density of 1 cell / mL are used.

[0177] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any integer value in between. In other embodiments, the beads and T cells are cultured together for 2 to 3 days. Suitable conditions for T cell culture include a suitable medium (e.g., basal medium or RPMI medium 1640, or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Examples of culture media include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or some cytokines sufficient for T cell growth and proliferation. In some embodiments, the medium comprises X-VIVO-15 medium supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml of IL-2 (Miltenyi).

[0178] T cells are maintained under conditions necessary to support their growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2 added).

[0179] In some embodiments, the T cells containing the receptors of this disclosure are autologous. Prior to proliferation and genetic modification, the source of the T cells is obtained from a subject. Immune cells such as T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments of the present invention, any of the T cell lines available in the art may be used. In certain embodiments of the present invention, T cells can be obtained from a single unit of blood collected from a subject using any of the techniques known to those skilled in the art, such as Ficoll® isolation.

[0180] In some embodiments, cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granule cells, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or deficient in many but not all divalent cations. As will be readily apparent to those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter CytoMate, or Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions for use. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components may be removed from the apheresis sample, and the cells may be resuspended directly in the culture medium.

[0181] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, by centrifugation through a PERCOLL® gradient or by countercurrent centrifugation elution. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 conjugated beads for a sufficient time to positively select the desired T cells.

[0182] The enrichment of immune cell populations, such as T cell populations, by negative selection can be achieved using a combination of antibodies directed to surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0183] For the isolation of a desired population of immune cells by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) can vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) in order to ensure maximum contact between cells and beads.

[0184] In some embodiments, cells may be incubated on a rotor at either 2–10°C or room temperature, at various rates, and for various lengths of time.

[0185] PBMCs from which T cells for stimulation, or immune cells such as T cells, are isolated, can also be frozen after the washing step. While not bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granule cells, and to some extent monocytes, from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Many cryopreservation solutions and parameters are known in the art and useful in this regard, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or a medium containing 31.25% Plasmalyte-A, 31.25% 5% dextrose, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell cryopreservation medium containing, for example, Hespan and PlasmaLyte A, after which the cells are frozen to -80°C at a rate of 1° per minute and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing, in addition to immediate uncontrolled freezing at -20°C or in liquid nitrogen, may also be used.

[0186] Signal transduction assays In some embodiments, immune cell activation is reduced when the cell comes into contact with an antigen corresponding to the LILRB1-based receptor of this disclosure or with a cell expressing that antigen on its surface. In some embodiments, immune cell activation includes the expression of a gene operably linked to an NFAT promoter. Nuclear factors of activated T cells (NFATs) are a family of transcription factors that have been shown to be important in the immune response. The NFAT transcription factor family consists of five members: NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATs play a role in regulating inflammation.

[0187] As used herein, an NFAT promoter is a promoter that controls (i.e., activates or suppresses) NFAT when it is expressed in a cell. NFAT-targeted promoters are described in Badran, BM et al. (2002) J. Biological Chemistry Vol. 277: 47136-47148 and contain NFAT consensus sequences such as GGAAA.

[0188] Methods for evaluating the effect of receptor activation on gene expression are known in the art and include the use of reporter genes, the expression of which can be quantified. Reporter genes are used to identify potentially transfected or transduced cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a polypeptide-encoding gene that is not present in or expressed by the recipient organism and whose expression is manifested by several readily detectable properties, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, a construct having the smallest 5' facile region that exhibits the highest level of expression of the reporter gene is identified as a promoter. Such a promoter region can be ligated to a reporter gene and used to evaluate the action of an agent for its ability to regulate promoter-driven transcription. In an exemplary embodiment, an NFAT promoter operably ligated to a reporter gene is used to evaluate the expression of the receptor of this disclosure during NFAT signaling.

[0189] Pharmaceutical composition The present disclosure provides immune cells comprising a receptor based on LILRB1 of the present disclosure, and a pharmaceutical composition comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0190] Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0191] Method of treating a disease Provided herein is a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of immune cells comprising a receptor based on LILRB1 described herein. In some embodiments, the immune cells further comprise an activator receptor such as an activator CAR or TCR.

[0192] Additional methods of treating a subject, and combinations of activator receptors combined with inhibitory receptors, are described in PCT / US202, the contents of which are incorporated herein by reference in their entirety.

[0193] In some embodiments, the subject requiring it has cancer. In some embodiments, a method of treating the subject involves administering a plurality of immune cells comprising the LILRB1 receptor of the Disclosure to the subject. In some embodiments, the plurality of immune cells further comprises an activator receptor such as CAR or TCR. In some embodiments, the CAR or TCR comprises an antigen-binding domain specific to the cancer antigen. The cancer antigen-specific activator receptor may comprise an antigen-binding domain isolated or drawn from any antibody or an antigen-binding domain known in the Art, the antigen-binding domain of which includes urelumab, utomirumab, olecumab, naptumomab, ascrinbakumab, tacutuzumab, nesbakumab, vanucizumab, belimumab, tavarmab, tibrizumab, verantamab, igovomab, olegobomab, sophi Tuzumab, mogamulizumab, tarakotsuzumab, tavolimab, bonrelorizumab, ipilimumab, duvortuxizumab, blinatumomab, coltuximab, denintuzumab, inebilizumab, ronkastuximab, taplitumomab, ibritumomab, obinutuzumab, okalatuzumab, ocrelizumab, ofatumumab, rituximab, tositumomab, bertuzumab, samarizumab, vectumomab, epratuzumab, inotuzumab, moxetumomab, pinatuzumab, gomiliximab, lumiliximab, camidanrumab, basiliximab, inolimomab, daclizumab, varlilumab, enobrituzumab, ombrutamab, brentuximab, iratsutumumab, gemtuzumab, lintuzumab, vadasutuximab, rirotomab, otlertuzumab tetulomab, daratumumab, isatuximab, bivatuzumab, abituzumab, intetumumab, rorbotuzumab, itolizumab, cusatuzumab, borsetuzumab, milatuzumab, polatuzumab, iladatuzumab, galixima, artumomab, arcitumomab, rabetsuzumab, sibisatamab,Zolbetuximab, lacunotuzumab, cabiralizumab, emuctuzumab, dimcirumab, rezirumab, ochirimab, mabrilimumab, tremelimumab, ulocuplumab, tepoditamab, lovalpituzumab, demcizumab, dorodizumab, parsatuzumab, cetuximab, depatuxizumab, futuximab, imugatuzumab, laprituximab , matsuzumab, necitumumab, nimotuzumab, panitumumab, zaltumumab, modotuximab, amibantamab, tomzotuximab, losatuxizumab, adecatumumab, citatuzumab, edrecolomab, oportuzumab, solitomab, tucotsuzumab, katsumakisomab, ifabotuzumab, darigotuzumab, elgemtumab, rumretuzumab, patrizumab, cerivantuzumab Xenoctuzumab, Apurutuzumab, Bemarituzumab, Bantikutuzumab, Dinutuximab, Eclomeximab, Mitumomab, Codrituzumab, Glenbatumumab, Zatuximab, Ertumaxomab, Margetuximab, Timigutuzumab, Gancotamab, Pertuzumab, Trastuzumab, Ficlatuzumab, Rilotumumab, Terisotuzumab, Emibetuzumab mab), ixtumumab, darotuzumab, figtumumab, ganitumumab, lobatumumab, teprotumumab, flotetuzumab, bermekimab, sergutuzumab, boroximab, etalacizumab, relatrimab, carlumab, amatuximab, cribatuzumab, gatipotuzumab, pemtumomab, cantuzumab, pankomab, racotumomab, bronchicutuzumab, tarextumab, besenkumab, camrelizumab, cetrerimab, nivolumab,Examples include, but are not limited to, pembrolizumab, pidilizumab, semiprimab, spartalizumab, atezolizumab, abelumab, durvalumab, cirmtuzumab, tenatumomab, fresolimumab, brodalumab, bevacizumab, ranibizumab, varisacumab, faricimab, ikrucumab, alacizumab, and ramucirumab.

[0194] In some embodiments, the LILRB1-based receptor of the present disclosure includes an antigen-binding domain specific for an antigen that is lost in cancer cells through loss of heterozygosity. In some embodiments, the antigen is a minor histocompatibility antigen (MiHA). In some embodiments, the antigen is an HLA class I allele. In some embodiments, the HLA class I allele includes HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA class I allele includes HLA-E. In some embodiments, the HLA class I allele is the HLA-A * 02 allele. In some embodiments, the antigen is not expressed in target cells due to loss of the Y chromosome. In some embodiments, the antigen specific for the LILRB1-based receptor is the HLA-A * 02 antigen.

[0195] In some embodiments, the subject requiring it has cancer. Cancer is a disease in which abnormal cells divide uncontrollably and spread to nearby tissues. In some embodiments, cancer includes humoral tumors or solid tumors. Exemplary humoral tumors include leukemia and lymphoma. Further cancers that are humoral tumors may occur in the blood, bone marrow, and lymph nodes, and these may include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin lymphoma, melanoma, and multiple myeloma. Examples of leukemia include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia. Exemplary solid tumors include sarcomas and carcinomas. Cancer may occur substantially in organs in the body, such as the blood, bone marrow, lungs, breasts, colon, bones, central nervous system, pancreas, prostate, and ovaries. Further cancers that are solid tumors include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell carcinoma, kidney cancer, head and neck cancer, pharyngeal cancer, squamous cell carcinoma that forms in the moist mucosal lining of the nose, mouth, and pharynx, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the conditions treated by the methods described herein are metastases of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, gastric cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, kidney cancer cells, head or neck cancer cells, pharyngeal cancer cells, squamous cell carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

[0196] Any cancer in which multiple cancer cells express a first activator ligand and do not express a second inhibitor ligand is conceived within the scope of this disclosure. For example, CEA-positive cancers that can be treated using the methods described herein include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia.

[0197] Treating cancer can result in a reduction in tumor size. This reduction in tumor size may also be called "tumor regression." Preferably, after treatment, the tumor size is reduced by 5% or more compared to its size before treatment; more preferably by 10% or more; more preferably by 20% or more; more preferably by 30% or more; more preferably by 40% or more; even more preferably by 50% or more; and most preferably by more than 75%. Tumor size can be measured by any reproducible measuring means. Tumor size can be measured as the diameter of the tumor.

[0198] Treating cancer can result in a reduction in tumor volume. Preferably, after treatment, the tumor volume is reduced by 5% or more compared to its size before treatment; more preferably, by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably by more than 75%. Tumor volume can be measured by any reproducible measuring means.

[0199] Treating cancer results in a reduction in the number of tumors. Preferably, after treatment, the number of tumors decreases by 5% or more compared to the number before treatment; more preferably, the number of tumors decreases by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably by more than 75%. The number of tumors can be measured by any reproducible measuring means. The number of tumors can be measured by counting tumors visible to the naked eye or at a specific magnification. Preferably, the specific magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

[0200] Treating cancer can result in a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions decreases by 5% or more compared to the number before treatment; more preferably, the number of metastatic lesions decreases by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably by more than 75%. The number of metastatic lesions can be measured by any reproducible measuring means. The number of metastatic lesions can be measured by counting metastatic lesions visible to the naked eye or at a specific magnification. Preferably, the specific magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

[0201] Treating cancer can result in an increase in the mean survival time of the treated population compared to the population that received the carrier alone. Preferably, the mean survival time increases by more than 30 days; more preferably, more than 60 days; more preferably, more than 90 days; and most preferably, more than 120 days. The increase in the mean survival time of the population can be measured by any reproducible means. For example, the increase in the mean survival time of the population can be measured by calculating the mean length of survival for the population after the start of treatment with the active compound. The increase in the mean survival time of the population can also be measured, for example, by calculating the mean length of survival for the population after the completion of the first round of treatment with the active compound.

[0202] Treating cancer can result in an increase in the mean survival time of the treated population compared to the untreated population. Preferably, the mean survival time increases by more than 30 days; more preferably, more than 60 days; more preferably, more than 90 days; and most preferably, more than 120 days. The increase in mean survival time of the population can be measured by any reproducible means. The increase in mean survival time of the population can be measured, for example, by calculating the mean length of survival for the population after the start of treatment with the active compound. The increase in mean survival time of the population can also be measured, for example, by calculating the mean length of survival for the population after the completion of the first round of treatment with the active compound.

[0203] Treating cancer may result in an increase in the mean survival time of the treated population compared to a population treated with monotherapy with a drug other than the compound of the present invention, or with a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof. Preferably, the mean survival time increases by more than 30 days; more preferably, more than 60 days; more preferably, more than 90 days; and most preferably, more than 120 days. The increase in mean survival time of the population can be measured by any reproducible means. The increase in mean survival time of the population can be measured, for example, by calculating the mean length of survival for the population after the start of treatment with the active compound. The increase in mean survival time of the population can also be measured, for example, by calculating the mean length of survival for the population after the completion of the first round of treatment with the active compound.

[0204] Treating cancer may result in a reduction in mortality in the treated population compared to a population that received the carrier alone. Treating cancer may result in a reduction in mortality in the treated population compared to a population that did not receive treatment. Treating cancer may result in a reduction in mortality in the treated population compared to a population that received monotherapy with a drug other than the compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof. Preferably, the mortality reduction is greater than 2%; more preferably greater than 5%; more preferably greater than 10%; most preferably greater than 25%. The reduction in mortality in the treated population can be measured by any reproducible means. The reduction in population mortality can be measured, for example, by calculating the mean disease-related deaths per unit time after the start of treatment with the active compound for the population. The reduction in population mortality can also be measured, for example, by calculating the mean disease-related deaths per unit time after the completion of the first round of treatment with the active compound for the population.

[0205] Treating cancer can result in a reduction in tumor growth rate. Preferably, after treatment, the tumor growth rate is reduced by at least 5% compared to the pre-treatment number; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. Tumor growth rate can be measured by any reproducible measuring means. Tumor growth rate can be measured by the change in tumor diameter per unit time.

[0206] Treating cancer can result in a reduction in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably less than 10%; more preferably less than 20%; more preferably less than 30%; more preferably less than 40%; more preferably less than 50%; even more preferably less than 50%; most preferably less than 75%. Tumor regrowth can be measured by any reproducible means. Tumor regrowth is measured, for example, by measuring the increase in tumor diameter after the previous tumor reduction that occurred after treatment. A reduction in tumor regrowth is indicated by the inability of the tumor to recur after treatment is discontinued.

[0207] Treating or preventing cell proliferation disorders may result in a decrease in the rate of cell proliferation. Preferably, after treatment, the rate of cell proliferation decreases by at least 5%; more preferably at least 10%; more preferably at least 20%; more preferably at least 30%; more preferably at least 40%; more preferably at least 50%; even more preferably at least 50%; most preferably at least 75%. The rate of cell proliferation can be measured by any reproducible measuring means. The rate of cell proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0208] Treating or preventing cell proliferation disorders may result in a decrease in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells decreases by at least 5%; more preferably at least 10%; more preferably at least 20%; more preferably at least 30%; more preferably at least 40%; more preferably at least 50%; even more preferably at least 50%; most preferably at least 75%. The proportion of proliferating cells can be measured by any reproducible measurement means. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of mitotic cells relative to the number of non-dividing cells in a tissue sample. The proportion of proliferating cells may be equivalent to the mitotic index.

[0209] Treating or preventing cell proliferation disorders may result in a reduction in the size of the cell proliferation area or zone. Preferably, after treatment, the size of the cell proliferation area or zone is reduced by at least 5% compared to its size before treatment; more preferably by at least 10%; more preferably by at least 20%; more preferably by at least 30%; more preferably by at least 40%; more preferably by at least 50%; even more preferably by at least 50%; most preferably by at least 75%. The size of the cell proliferation area or zone can be measured by any reproducible measuring means. The size of the cell proliferation area or zone can be measured as the diameter or width of the cell proliferation area or zone.

[0210] Treating or preventing cell proliferation disorders may result in a reduction in the number or percentage of cells with abnormal appearance or morphology. Preferably, after treatment, the number of cells with abnormal morphology is reduced by at least 5% compared to their size before treatment; more preferably by at least 10%; more preferably by at least 20%; more preferably by at least 30%; more preferably by at least 40%; more preferably by at least 50%; even more preferably by at least 50%; most preferably by at least 75%. Abnormal cell appearance or morphology can be measured by any reproducible measuring means. Abnormal cell morphology can be measured by microscopic observation, for example, using a tissue culture inverted microscope. Abnormal cell morphology can take the form of nuclear pleiomorphism.

[0211] Kits and manufactured products This disclosure provides kits and products comprising polynucleotides and vectors encoding receptors described herein. In some embodiments, the kit includes items such as vials, syringes, and instructions for use.

[0212] In some embodiments, the kit includes a polynucleotide or vector comprising a sequence encoding one or more chimeric antigen receptors of the Disclosure. For example, the polynucleotide or vector comprises a sequence of one or more LILRB1 domains described herein.

[0213] In some embodiments, the kit includes a plurality of immune cells containing the chimeric antigen receptor described herein. In some embodiments, the plurality of immune cells include a plurality of T cells.

[0214] [Table 2-1] [Table 2-2] [Table 2-3]

[0215] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8]

[0216] This description includes numerous exemplary configurations, methods, parameters, etc. However, it should be recognized that such descriptions are provided as exemplary embodiments, rather than as limitations to the scope of this disclosure. [Examples]

[0217] [Example 1] LILRB1-based inhibitory scFv-CAR compared to PD-1, KIR3DL2, and KIR3DL3 NY-ESO-1 responsive inhibitory constructs were created by fusing the NY-ESO-1 ligand-binding scFv domain (C-266) to the domains of receptors including the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1, LILRB1 (LILRB1); killer cell immunoglobulin-like receptor 3DL2, KIR3DL2; killer cell immunoglobulin-like receptor 3DL3, KIR3DL3; and / or B lymphocyte and T lymphocyte attenuators (BTLAs). The gene segments were joined using Golden Gate cloning and inserted downstream of the eF1α promoter contained in the lentiviral expression plasmid (pLenti1).

[0218] As reporter cells, Jurkat cells encoding the NFAT luciferase reporter were maintained in RPMI medium supplemented with 10% FBS, 1% Pen / Strep, and 0.4 mg / ml G418 / Genetesin. T2 cells (ATCC CLR-1992) were maintained in IMDM medium + 20% FBS and 1% Pen / Strep. To evaluate each construct, Jurkat cells were transfected using a 100 μL format Neon electroporation system (Thermo Fisher) with the following settings, according to the manufacturer's protocol: 3 pulses, 1500 V, 10 msec.

[0219] Cotransfection was performed using either 3 μg of activated CAR construct (C-563) or TCR construct (CT-139) and 3 μg of inactivated CAR construct or empty vector (pLenti0) per 1 million cells, and the cells were recovered in RPMI medium supplemented with 20% thermally inactivated FBS and 0.1% Pen / Strep.

[0220] The peptides MAGE-A3(FLWGPRALV) (SEQ ID NO: 106) and modified NY-ESO-1(SLLMWITQV) (SEQ ID NO: 107) were synthesized using Genscript. The activated peptide, MAGE-A3, was sequentially diluted 5-fold, starting at 50 μM. The inactivated peptide, NY-ESO-1, was diluted to 50 μM, 5 μM, 0.5 μM, or 0.05 μM. A fixed amount of each was added to the serial dilutions of MAGE-A3, and subsequently loaded onto 10,000 T2 cells in 15 μL of RPMI supplemented with 1% BSA and 0.1% Pen / Strep. The cells were incubated in Corning® 384-well low-flange white flat-bottom polystyrene TC-treated microplates. The following day, 10,000 Jurkat cells were resuspended in 15 μL of RPMI supplemented with 10% thermally inactivated FBS and 0.1% Pen / Strep, added to T2 cells loaded with peptides, and co-cultured for 6 hours. Jurkat luminescence was evaluated using the ONE-Step Luciferase Assay System (BPS Bioscience). The assay was performed using technical duplicates.

[0221] LILRB1 compared to PD-1, KIR3DL2, and KIR3DL3 Figure 4 shows the testing of the constructs shown in Table 1. The data show that scFv-LILRB1 inhibits CAR activation in trans. Since constructs with the LILRB1 domain inhibit signal transduction at the higher concentration of the MAGE-A3 activator peptide, the data demonstrate that CARs with the LILRB1-derived hinge, transmembrane domain, and intracellular domain are superior to CARs generated with the same domains derived from PD-1, KIR3DL2, or KIR3DL3.

[0222] [Table 4]

[0223] Inhibition by LILRB1-based CARs requires antigen recognition via their ligand-binding domain. Figure 5 shows the testing of constructs selected from Table 1. The data indicate that LILRB1 inhibits signaling in a dose-dependent manner in trans. Increasing the concentration of the inhibitory peptide NY-ESO-1 shifts the response to the activating peptide MAGE-A3 downward. This suggests that the inhibitory effect of LILRB1-based CARs depends on the engagement of the antigen to which the ligand-binding domain exhibits specificity.

[0224] LILRB1 CAR inhibits signal transduction via the T cell receptor (TCR). Figure 6 shows the constructs selected from Table 1, specifically the TCR testing specific to the activating peptide MAGE-A3, rather than the CARs used in previous experiments. LILRB1-based inhibitory CARs dose-dependently inhibit TCR-mediated signaling.

[0225] LIRLB1 CAR inhibition is conserved in the presence of two of the four native ITIMs. Figure 7 shows the analysis of the LILRB1 intracellular domain with an inactivating mutation in the ITIM motif of LILRB1. A tyrosine-to-phenylalanine (Y→F) mutation was used to inactivate the ITIM, indicated by the number of asterisks in Table 2.

[0226] [Table 5]

[0227] Inactivation of all four ITIMs resulted in a non-functional inhibitory CAR (C2182). Inactivation of only two of the four ITIMs preserved the inhibitory function of the CAR at the tested concentrations (C1760 and C1762). Inactivation of all four ITIMs (C1759) demonstrates that the ITIMs are necessary for the inhibitory function. If all four ITIMs are mutated, the molecule loses its inhibitory function. If only two of the four ITIMs are mutated, the inhibitory activity is retained.

[0228] Figure 8 shows the testing of LILRB1 ITIMs in combinations different from those of the natural LILRB1 intracellular domain. As shown in Table 3, CARs with a total of 4 or 6 copies of the third and fourth ITIMs of LILRB1 achieve inhibitory activity comparable to that of the natural LILRB1 intracellular domain. Inhibitory activity is also observed when only one copy of the third and fourth ITIMs is used (C2179), or when two copies of each ITIM are used (C2180), or when multiple copies are used (C2302 or C2180).

[0229] [Table 6] [Example 2]

[0230] The LILRB1 hinge and transmembrane domain enhance the inhibitory activity of BTLA-based inhibitory CARs. BTLA-based CARs and LILRB1 / BTLA-based CARs inhibit NFAT-mediated signaling. The B lymphocyte and T lymphocyte attenuator (BTLA), also known as CD272 (surface antigen classification 272), interacts with B7 homolog B7H4. Unlike CTLA-4 and PD-1, it is also a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14). Inhibitory signaling via BTLA occurs in response to the binding of B7H4 or TNFRSF14.

[0231] The full-length BTLA protein was cloned into a construct containing an extracellular scFv domain (C2220). Constructs in which the extracellular domain of BTLA was replaced with that of LILRB1 (C2219), and constructs in which the extracellular (extracell) domain and transmembrane domain of BTLA were replaced with those of LILRB1 (C2218), were generated and tested, as shown in Table 4 and Figure 9. The LILRB1-BTLA fusion exhibits inhibitory signaling comparable to that of LILRB1-based CARs.

[0232] [Table 7] [Example 3]

[0233] LILRB1 hinge and membrane penetration compared to CD8 or CD28 LILRB1-based CARs with LILRB1 hinges and transmembrane regions are superior to those with CD8 hinges and CD28 transmembrane regions. As shown in Table 5 and Figure 10, LILRB1-based CARs were compared with CARs that possess a LILRB1 intracellular domain but have a CD8 hinge and a CD28 transmembrane domain.

[0234] [Table 8]

[0235] The LILRB1 hinge and transmembrane region yield remarkably better results than constructs with the CD8 hinge and CD28 transmembrane region. Emin is reduced. The overall dynamic range is increased. Inhibitory power is increased.

[0236] [Table 9] [Example 4]

[0237] TCR-based inhibitory chimeric antigen receptors Construct design and cloning High-affinity anti-HLA-A*02:01 / NY-ESO-1 1G4α95:LY T cell receptor (TCR) mutants were used to create NY-ESO-1 responsive inhibitory constructs. Charged residues in the TM of TCRα (R253 and K258) and TCRβ (K288) were mutated to leucine. Then, LILRB1 ITIM (residues 484-650) was added to the mutant TCRα or TCRβ. Anti-HLA-A*02:01 / MAGE-A3 single-stranded variable fragments (scFv) were generated in-house. The anti-HLA-A*02:01 / MAGE-A3 chimeric antigen receptor (CAR) used in this study contains anti-HLA-A*02:01 / MAGE-A3 scFv, CD8 hinge, CD28 TM, and intracellular domains (ICD) of CD28, 41BB, and CD3ζ. All fragments, including those containing the 5' and 3' BsmBI sites, were amplified using Q5 polymerase (New England Biolabs) and digested with DpnI (Thermo Scientific) at 37°C for 60 minutes. The resulting PCR fragments were purified using Nucleospin gel and a PCR purification kit (Macherey-Nagel). Plasmids were assembled using Golden Gate in a reaction mixture containing BsmBI (Thermo Scientific), T4 DNA ligase (Thermo Scientific), 10 mM ATP, and 1× FastDigest buffer (Thermo Scientific).

[0238] Jurkat NFAT Activation Assay Jurkat T lymphocytes containing the firefly luciferase gene under the control of the nuclear factor of activator T cells (NFAT) transcription factor (BPS Bioscience) were co-transfected with plasmids encoding the TCR and / or scFv fusion construct using the Neon transfection system (Thermo Fisher). Electropermeable cells were incubated in RPMI medium supplemented with 20% fetal bovine serum (FBS) (HIA-FBS) and 0.1% penicillin-streptomycin (P / S) (Gibco) after thermal inactivation at 56°C for 60 minutes. TAP-deficient T2 lymphoblasts (ATCC RL-1992) were loaded with RPMI supplemented with 1% BSA and 0.1% P / S (Gibco) containing 50 μM NY-ESO-1 peptide in addition to various amounts of modified NY-ESO-1 peptide (SLLMWITQV) (SEQ ID NO: 107), MAGE-A3 peptide (FLWGPRALV) (SEQ ID NO: 106), or 50 μM NY-ESO-1 peptide in addition to various amounts of MAGE-A3 peptide. The peptides used in the assay were synthesized to a purity of >95% as assessed by mass spectrometry (Genscript). 18 hours after transfection, 0.8 × 10⁶ peptides per ml were added to RPMI supplemented with 10% HIA-FBS and 1% P / S. 6 Jurkat was resuspended in cells. In a 384-well plate, 12,000 Jurkat cells per well were co-cultured with 12,000 T2 cells loaded with peptides at 37°C and 5% CO2 for 6 hours. NFAT-mediated luciferase production was measured by adding 15 μL of ONE-Step luciferase assay reagent (BPS Bioscience). After 20 minutes, luminescence was detected using a plate reader (Tecan).

[0239] TCR-based inhibitory CARs using the LILRB1 intracellular domain As shown in Table 7 and Figure 11, co-expression of TCR-based CARs that either possess only the extracellular domain of the TCR or also possess the transmembrane domain of the TCR with mutations to polar residues results in functionally inhibitory CARs.

[0240] [Table 10]

[0241] TCR-based CARs trans-inhibit the anti-HLA-A*02:01 MAGE-A3 CAR. Jurkat-NFAT luciferase reporter cells were either (1) transfected with anti-HLA-A*02:01 / MAGE-A3 CAR (C563) alone, (2) co-transfected with MAGE-A3 CAR (C563) and TCRα(R253L / K258L)-LILRB1 fusion and TCRβ(K288L)-LILRB1 ICD fusion TCR inhibitory fusion constructs (C2156+C2157), or (3) co-transfected with MAGE-A3 CAR and TCRαECD / TCRβECD-LIRT1(TM+ICD) fusion (C2057+C2058) TCR inhibitory fusion constructs. The effects of two inhibitory mutants on NFAT activation were measured by co-culturing transfected Jurkat cells with T2 cells loaded with 50 μM NY-ESO-1 peptide combined with various amounts of MAGE-A3 peptide. The data are summarized in Table 8.

[0242] [Table 11]

[0243] The truncated TCR alpha or TCR beta extracellular domains (ECDs) inhibit CAR activation in conjunction with the LILRB1™-ICD fusion. When TCR transmembrane mutations cause loss of TCR-CD3 subunit interaction, the TCR alpha or TCR beta extracellular domains (ECDs), along with the TCR™-LILRB1 ICD fusion, also acted as inhibitory CARs. Experiments demonstrate that TCR α and β chains can be used to generate inhibitory chimeric antigen receptors by interfering with the recruitment of stimulants by the CD3 subunit. [Example 5]

[0244] Methods for Examples 6-14 cell culture Jurkat cells encoding the NFAT-luciferase reporter were obtained from BPS Bioscience. All other cell lines used in this study were obtained from ATCC. During culture, Jurkat cells were maintained in RPMI medium supplemented with 10% FBS, 1% Pen / Strep, and 0.4 mg / ml G418 / Genetesin. T2, MCF7, Raji, K562, and HeLa cells were maintained as suggested by ATCC. "Normal" Raji cells were generated by transduction of Raji cells with HLA-A*02 lentivirus (custom-made lentivirus, Alstem) at MOI5. HLA-A*02-positive Raji cells were sorted using a FACSMelody cell sorter (BD).

[0245] Plasmid construction NY-ESO-1-responsive inhibitory constructs were created by fusing NY-ESO-1 scFv LBDs to the domains of receptors including the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1, LILRB1 (LIR-1), programmed cell death protein 1, PDCD1 (PD-1), or cytotoxic T lymphocyte protein 4, CTLA4 (CTLA-4). All activated CAR constructs contained scFvs fused to the CD8α hinge, CD28™, and ICDs of CD28, 4-1BB, and CD3 zeta. CD19-activated CAR scFvs were obtained from FMC63 mouse hybridomas. MSLN-activated CAR scFvs were obtained from the described human M5 (LBD1) and humanized SS1 (LBD2). The gene segments were joined using Golden Gate cloning and inserted downstream of the human EF1α promoter contained in the lentiviral expression plasmid.

[0246] Jurkat cell transfection Jurkat cells were transiently transfected using a 100 μL format Neon electroporation system (Thermo Fisher Scientific) with the following settings: 3 pulses, 1500 V, 10 msec. Co-transfection was performed with 1–3 μg of activator CAR or TCR construct and 1–3 μg of scFv or TCR alpha / TCR beta LIR-1 blocker construct or empty vector per 1e6 cells, and the cells were recovered in RPMI medium supplemented with 20% thermally inactivated FBS and 0.1% Pen / Strep. To confirm blocker surface expression, Jurkat cells were stained with 10 μg / mL streptavidin-PE-HLA-A*O2-pMHC tetramer in PBS containing 1% BSA for 60 minutes at 4°C 18–24 hours after transfection and characterized by flow cytometry (BD FACSCanto II).

[0247] Jurkat-NFAT-luciferase activation research MAGE-A3(MP1;FLWGPRALV;SEQ ID NO: 106), MAGE-A3(MP2;MPKVAELVHFL;SEQ ID NO: 108), HPV E6(TIHDIILECV;SEQ ID NO: 109), HPV E7(YMLDLQPET;SEQ ID NO: 110) peptides and modified NY-ESO-1 ESO(ESO;SLLMWITQV;SEQ ID NO: 107) were synthesized by Genscript. Activating peptides were serially diluted, starting at 50 μM. The blocker peptide NY-ESO-1 was diluted to 50 μM (unless otherwise specified), added to the serially diluted activating peptides, and subsequently loaded onto 1e4 T2 cells in 15 μL of RPMI supplemented with 1% BSA and 0.1% Pen / Strep, and incubated in Corning® 384-well low-flange white flat-bottom polystyrene TC-treated microplates. The following day, 1e4 Jurkat cells were resuspended in 15 μL of RPMI supplemented with 10% thermally inactivated FBS and 0.1% Pen / Strep, added to T2 cells loaded with peptides, and co-cultured for 6 hours. Jurkat luminescence was evaluated using the ONE-Step Luciferase Assay System (BPS Bioscience). For assays involving high-density targets, Jurkat cells were similarly transfected and co-cultured with tumor cells expressing the target antigen at various Jurkat:tumor cell ratios. Assays were performed using technical duplicates.

[0248] Transduction, proliferation, and enrichment of primary T cells Leukopak was purchased from AllCells®. The collection protocol and donor informed consent were approved by the Institutional Relations Board (IRB) under strict supervision. HIPAA compliance and approved protocols were also followed. Frozen PBMCs were thawed in a 37°C water bath and cultured at 1e6 cells / mL in LymphoONE (Takara) containing 1% human serum. They were activated using 1:100 T cells TransAct (Miltenyi) supplemented with IL-15 (10 ng / ml) and IL-21 (10 ng / ml). After 24 hours, lentiviruses were added to the PBMCs at an MOI of 5. Activator and blocker receptors were co-introduced simultaneously for each lentivirus at an MOI of 5. The PBMCs were cultured for a further 2-3 days to allow cells to proliferate under TransAct stimulation. After proliferation, blocker-positive T cells were enriched from transduced activator and blocker primary T cells by positive selection using anti-PE microbeads (Miltenyi) according to the manufacturer's instructions. Briefly, primary T cells were incubated with 10 μg / mL streptavidin-PE-HLA-A*O2-pMHC tetramer in MACS buffer (PBS solution of 0.5% BSA + 2 mM EDTA) at 4°C for 60 minutes. Cells were washed three times with MACS buffer and passed through an LS column (Miltenyi) to separate blocker-positive cells (a mixture of blocker-only cells and activator + blocker cells) from non-transduced activator-only cells.

[0249] In vitro cytotoxicity studies of primary T cells For cytotoxic studies using pMHC targets, enriched primary T cells were incubated for 48 hours with 2e3 MCF7 cells expressing Biosettia luciferase, loaded with the target peptide titration in a 3:1 effector:target ratio as described above. The number of living MCF7 cells expressing luciferase was quantified using the Biosettia luciferase reporter assay system (Promega). For cytotoxic studies using non-pMHC targets, enriched primary T cells were incubated for up to 6 days with 2e3 WT Raji cells ("tumor" cells) or HLA-A*02 transduced Raji cells ("normal" cells) in a 3:1 effector:target ratio. WT "tumor" Raji cells stably expressing GFP and sea urchin luciferase (Biosettia) or HLA-A*02 transduced "normal" Raji cells stably expressing RFP and firefly luciferase (Biosettia) were imaged together with unlabeled primary T cells using the IncuCyte live cell imager. The fluorescence intensity of living Raji cells over time was quantified using IncuCyte imaging software. For reversibility studies, enriched primary T cells were co-cultured with "normal" or "tumor" Raji cells for 3 days and imaged. After 3 days, T cells were isolated from the remaining Raji cells using CD19-negative selection and re-seeded with new "normal" or "tumor" Raji cells as described. In separate wells, at 72 hours, living luciferase-expressing Raji cells were quantified using a dual luciferase reporter assay system (Promega). In studies where IFNγ secretion was assessed, the supernatant collected after 48 hours of co-culture was tested for IFNγ using the BD Human IFNγ Flex Kit, following the manufacturer's strict instructions.

[0250] Mouse xenotransplantation research Frozen PBMCs were thawed in a 37°C water bath and rested overnight in serum-free TexMACS medium (Miltenyi) before activation. PBMCs were activated at 1.5e6 cells / mL using TexMACS medium supplemented with T Cell TransAct (Miltenyi) and IL-15 (20 ng / ml) and IL-21 (20 ng / ml). After 24 hours, lentivirus was added to the PBMCs at MOI5. PBMCs were cultured for a further 8-9 days to allow cell proliferation under TransAct stimulation. Post-proliferation, T cells were enriched for A2-LIR-1 for a further 2-5 days using anti-PE microbeads (Miltenyi) against streptavidin-PE-HLA-A*O2-pMHC before in vivo injection. The enriched T cells were also examined for the expression of CD19 scFv activator and HLA-A*02 LIR-1 blocker by sequential staining with CD19-Fc (1:100; R&D Systems) and goat anti-human IgGFITC (1:200; Invitrogen) for the activators, and 10 μg / mL streptavidin-APC-HLA-A*02-pMHC for the blockers using flow cytometry (BD FACSCanto II).

[0251] In vivo experiments were conducted by Explora BioLabs using a protocol approved by the Institutional Animal Care Committee (IACUC). Female NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg.(HLA-A / H2-D / B2M)1Dvs / SzJ(NSG-HLA-A2 / HHD) mice, 5-6 weeks old, were purchased from The Jackson Labs. The animals were acclimatized to the housing environment for at least 3 days prior to the start of the study. 2e6 WT Raji cells or HLA-A*02 transdextrinsed Raji cells were subcutaneously injected into the right flank of the animals in a volume of 100 μL. Tumors averaged 70 mm. 3When (V=L×W×W / 2) was reached, animals were randomized into 5 groups (n=7) and administered either 2e6 or 1e7 T cells via tail vein. Tumor measurements were performed three times per week after T cell injection, and blood was collected for flow analysis on days 10 and 17. One animal in the WT Raji group that received 1e7 CD19-CAR+A2-LIR-1 T cells was excluded from the study for tail vein injection failure and subsequent flow cytometry confirmation of the absence of human T cells in the blood. At each time point, after RBC lysis, human T cells in the blood were quantified by flow cytometry (BD FACSCanto II). Cells were stained with anti-mouse CD45-FITC (clone 30-F11), anti-human CD3-PE (clone SK7), anti-human CD4-APC (clone OKT4), and anti-human CD8-PerCP-Cy5.5 (clone RPA-T8). All antibodies were obtained from Biolegend and used at a 1:100 dilution. Dead cells were excluded from the analysis using DAPI (Invitrogen). For histopathological analysis, tumor samples were fixed, sectioned, and stained for huCD3 (clone EP449E). Image quantification was performed using ImageJ software.

[0252] statistical analysis Statistical analysis was performed using GraphPad Prism software. Unless otherwise specified, all peptide and cell titration studies are presented as mean ± standard deviation (SD), while in vitro and in vivo studies using primary T cells are presented as mean ± standard error of the mean (SEM). Peptide and cell titration curves were fitted using a four-parameter nonlinear regression analysis. EC50 values ​​were calculated directly from the curves. All other groups of data were analyzed using standard two-way ANOVA followed by Tukey's multiple comparison test, unless otherwise specified. [Example 6]

[0253] Assay the effect of the LIR-1 hinge on activity blockade. The effect of various LIR-1 hinges on the ability of the HLA-A*02 scFv LIR-1 inhibitory receptor to block killing by Jurkat cells expressing the KRAS TCR activator was assayed using the Jurkat NFat luciferase assay described above. Humanized PA2.1 scFv LIR-1 receptors and humanized BB7.2 scFv LIR-1, which have shorter LIR-1 hinges, were assayed in Jurkat cells as described above. The results are shown in Figures 12A-12B. Jurkat cells were transfected with the KRAS TCR activator receptor and / or HLA-A*02 scFv LIR-1 inhibitory receptors (humanized PA2.1 or humanized BB7.2) with various LIR-1-derived hinges and co-cultured with T2 target cells that were HLA:A11-positive or HA:A11 and HLA:A02-positive. Inhibitory receptors with shorter and longer hinges behaved similarly (Figures 12A–12B). Mouse PA2.1 scFv and inhibitory receptors with slightly longer hinges were also assayed and functioned similarly to the shorter LIR-1 hinge in the T2-Jurkat assay (Figures 13A–13B). The hinge sequence is shown in black in Figures 12B and 13B, where gray SS in the sequence in Figure 13B represents the linker between the antigen-binding domain and the hinge, and gray VIGIL represents the initiation of the LIR-1 transmembrane domain. The hinge, transmembrane domain, and intracellular domain of the inhibitory receptor were all derived from LIR-1. Figures 12A–12B and 13A–13B demonstrate that the LIR-1 hinge length can be altered without negatively affecting the LIR-1 inhibitory receptor. Shorter hinges can offer advantages when packaging nucleic acid sequences encoding LIR-1 inhibitory receptors into lentiviral vectors for delivery.

[0254] [Table 12-1] [Table 12-2] [Table 12-3] [Example 7]

[0255] Comparison of LIR-1, CTLA-4, and PD-1 inhibitory receptors NY-ESO-1-responsive inhibitory constructs were created by fusing NY-ESO-1 scFv LBDs to the domains of receptors including the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1, LILRB1 (LIR-1), programmed cell death protein 1, PDCD1 (PD-1), or cytotoxic T lymphocyte protein 4, CTLA4 (CTLA-4). The MAGE-A3 activated CAR construct contained scFv fused to the CD8α hinge, CD28™, and the intracellular domains (ICDs) of CD28, 4-1BB, and CD3 zeta. The gene segments were joined using Golden Gate cloning and inserted downstream of the human EF1α promoter contained in a lentiviral expression plasmid.

[0256] Since peptide-MHC (pMHC) targets for both activator and blocker receptors allow for convenient quantification of the system's pharmacology, we initially used pMHC targets (Figure 14A). Specifically, we used a single-stranded fragment variable (scFv) bound to HLA-A*02-NY-ESO-1 (SLLMWITQC / V) as the inhibitor receptor ligand-binding domain (LBD), and a second scFv for HLA-A*02-MAGE-A3 (FLWGPRALV) pMHC (Gallo, unpublished) as part of the third-generation activator receptor CAR. Jurkat effector cells expressing luciferase upon NFAT activation were used to read activator sensitivity, and EC50 values ​​reported the maximum half-volume activator peptide concentration required for the response. Both the intracellular domains (ICDs) of PD-1 and CTLA-4 mediated a shift in activation EC50 in approximately 10 × fewer Jurkat cells, as measured by titration of peptides loaded onto T2 cells as a stimulus (Figure 14B).

[0257] We screened various potential inhibitor (blocker) receptor constructs and discovered that the LIR-1 blocker construct exhibited stronger blocking properties than PD-1 and CTLA-4. This blocker receptor contains the intracellular domain, transmembrane (TM) domain, and hinge domain of the LIR-1 (LILRB1) receptor, one of several LIR-family molecules encoded by the human genome. The LIR-1 blocker (hereinafter referred to as LIR-1) fused to NY-ESO-1 LBD mediated an EC50 shift of >5,000× (Figure 14B, Figures 17A-17D). Titration of unrelated control HLA-A*O2 binding peptides provided an estimate of the shift caused by competition for loaded peptides on T2 cells for available HLA molecules, with the contribution to the full shift estimated to be typically less than approximately 10× (Figure 15). For the EC50 shift values ​​reported here, the comparison was typically with the EC50 of the activator-only construct. Furthermore, for a given pair of activator / blocker receptors, the midpoint of the inhibition titration was nearly constant, dependent on the ratio of the activating peptide to the blocking peptide, and likely directly correlated with the target-antigen ratio (Figures 16A–16D). The target concentrations explored in most of these experiments are estimated to be in the range of approximately 1,000–10,000 copies / cell. [Example 8]

[0258] LIR-1 inhibitory receptor having multiple scFv ligand-binding domains The activity of LIR-1 inhibitory receptors was tested with various antigen-binding domains specific to other pMHC targets. For four different pMHC targets, a total of six different scFvs implanted on LIR-1 mediated dramatic shifts in EC50 ranging from 10 to 1,000 × (Figure 14C). Regarding its interaction with activator receptors, the LIR-1 receptor was also robust, and its blocking behavior was applicable to multiple targets and scFvs (Figure 14D). Blockade was ligand-dependent (Figure 17A), but many LIR-1 constructs produced lower basal / tonic signaling when paired with specific activator receptors. The EC50 shift depended on the presence of a fused ICD, as LIR-1 constructs that completely lost the ICD or contained mutations in key elements of the ICD were ineffective (Figure 17B). However, ligand-independent blocker activity had little effect on ligand-absent activated EC50 (Figures 16A-16D). LIR-1 inhibitory receptors are modular and adaptive ligand-dependent systems that function across multiple target and antigen-binding domains. [Example 9]

[0259] LIR-1 inhibitory domains fused to TCR alpha and TCR beta LIR-1 inhibitory receptors were tested when fused to TCR alpha and TCR beta subunits, or in combination with TCR activator receptors. The TCRs were directed to three different pMHC targets: two derived from MAGE-A3 and one from HPV (see methods above). In all cases, LIR-1 significantly shifted the activated EC50, estimated to be in the >1,000× range (Figure 14E; Figure 19A). Furthermore, the NY-ESO-1 TCR LBD also produced a substantial EC50 shift when fused with LIR-1 (Figure 14F; Figure 19B). Indeed, all combinations of NY-ESO-1(SLLMWITQV)scFv or TCR with MAGE-A3(FLWGPRALV)CAR or TCR showed a significant shift. Therefore, LIR-1 inhibitory receptors exhibited modularity encompassing both CARs and TCRs. [Example 10]

[0260] LIR-1 inhibitory receptors respond to target antigens that are present in cis position to the activator receptor target antigen. The ability of LIR-1 blocker receptors to inhibit activation by activator receptors when activator and inhibitor targets are presented in cis orientation was assayed. In the first assay, a simplified stimulus consisting of beads approximately the size of the cell (approximately 2.8 μm in diameter) on which the targets were loaded was used. Jurkat cells expressing activator and blocker receptors were activated only by beads containing A (activator) targets and not by beads with dual A / B (activator / blocker) targets (Figure 18A). Interestingly, effector cells were activated by a mixture of A+ and B+ beads, even when A+ beads accounted for only 20% of the total. This demonstrates that cells expressing activator and blocker receptors are (i) blocked from activation by blocker receptors when the targets are present in cis orientation on the same surface, and (ii) activated by individual A+ beads in an excess of B+ beads. [Example 11]

[0261] LIR-1 inhibitory receptors and cell surface antigens We assayed the ability of the LIR-1 inhibitor receptor to block activation in response to non-pMHC targets, representing surface antigens that can extend to a region of 100,000 epitopes / cell. We tested scFv, which peptide-dependently binds to either the B cell marker CD19, the solid tumor antigen mesothelin (MSLN), or HLA-A*02. In these cases, the target antigen concentration was not controlled, as in the case of pMHC or exogenous peptides. Instead, we varied the ratio of activator to blocker expression using various DNA concentrations in transient transfection assays. While assay sensitivity prevented exploration of the full range of EC50 shifts, shifts of Emax greater than 10 × were observed. These experiments demonstrated that the properties of the LIR-1 receptor in the dual receptor system were generally the same for high-density targets (Figure 18B; Figures 16-16D, Figure 21A), and that the blocker receptor blocked A receptor activation to the extent reflected by its relative surface level on effector cells (Figure 18C). The LIR-1 receptor also exhibited modular behavior in this high-antigen-density setting. When scFv for HLA-A*02 fused to the activator or blocker receptor, it acted as either an activator (Figure 18D) or a blocker, respectively. The LIR-1 receptor is sufficiently flexible to accommodate both low and high target densities, thereby allowing for optimization for pMHC targets and non-pMHC surface antigens in principle. [Example 12]

[0262] LIR-1 inhibitory receptors in primary T cells The ability of the LIR-1 receptor to block primary T cell activation was assayed. pMHC targets were used first. After enriching transduced T cells via physical selection, engineered T cells expressing activator and blocker receptors were assayed using target cells engineered to express luciferase as live cell readout information. When HPV TCR was used as the activator, NY-ESO-1 scFv fused to LIR-1 shifted the cell number vs. peptide concentration curve by approximately 25 × in MCF7 tumor cells loaded with the peptide (Figure 20A; Figure 23). Thus, the behavior of the LIR-1 receptor demonstrated in the Jurkat cell activation assay was extended to primary T cell function, including cytotoxicity.

[0263] To establish proof of concept, the HLA-A*02 LIR-1 construct was shown to function as a blocker in Jurkat cells, in the presence of a pMHC-dependent activator, along with T2 target cells (Figure 20B). CD19 scFv was used as the CAR activator. This activator / blocker pair was shown to function as robustly together as other pairs previously tested in Jurkat cells (Figure 18B). CD19-positive HLA-A*02-negative Raji cells were used to model tumor cells differentially expressing LIR-1 receptor ligands. The same cell line stably expressing the HLA-A*02 gene was used to model corresponding normal cells (Figure 24A). When target cells expressed only CD19, the cell line activated Jurkat cells, but not when they expressed both CD19 and HLA-A*02; that is, the HLA-A*02 blocker receptor blocked CD19-CAR activation in a ligand-dependent manner (Figure 24B).

[0264] The CD19 / HLA-A*02 receptor pair also acted on primary T cells (Figure 21). Manipulated T cells killed CD19-expressing Raji cells in the absence of HLA-A*02 expression. Raji cells expressing both CD19 and HLA-A*02 were blocked from cytotoxicity. Importantly, primary T cells harboring this receptor pair distinguished CD19+ "tumor" cells in mixed cultures from CD19+ / HLA-A*02+ "normal" cells. These results mirrored those from the bead experiment described above, but in a more complex cellular setup where the cytotoxicity of effector cells was concentrated on "tumor" targets, even when surrounded by "normal" cells. The degree of selectivity was impressive, considering that neither receptor had undergone planned optimization for maximum selectivity. This behavior was confirmed with a second antigen, MSLN (Figure 22B). [Example 13]

[0265] Reversibility of inhibition by LIR-1 inhibitory receptors The LIR-1 inhibitory receptor was tested for its reversible function, i.e., its ability to cycle from blockade to activation and then blockade. Effector T cells expressing the LIR-1 receptor and activator receptor were tested to see if they could function reversibly and repeatedly. Effector cells were co-cultured with Raji cells to mimic either tumor (CD19+) cell encounters or normal (CD19+ / HLA-A*O2+) cell encounters. After each round of exposure to target cells, the Raji cells were removed from the culture and a new population of target cells was introduced. Cytotoxicity and gamma interferon (IFNγ) were measured at the end of each round. T cells functioned as required by this type of cell therapy in both blockade-kill-blockade and kill-blockade-kill permutations (Figures 25A-25D). Their cells cycled reversibly from blockade to cytotoxic and back again, depending on the target cells to which they were exposed. These results demonstrate that T cells expressing activator receptors and LIR-1 inhibitory receptors can incorporate signals from normal and tumor cells, thus not becoming immobile in a single state (blocked or activated) but rather switching between them. In addition, these experiments were replicated with primary T cells from multiple donors, despite their complexity, heterogeneity, and diversity among donors (Figures 21A-D, 25A-25D; 26A-26B). This demonstrates the robust function of the LIR-1 inhibitory receptor. [Example 14]

[0266] Human T cells expressing activator receptors and LIR-1 blocker receptors that selectively target cancer cells in a mouse model. Using standard CD3 / CD28 stimulation, we assayed the ability of primary T cells to manipulate CD19 / HLA-A*02 activator / blocker pairs, enabling T cells to proliferate to large numbers in vitro (Figure 27A; Figure 28A). Thus, T cells expressing the activator and LIR-1 receptor can be proliferated to sufficient numbers for animal experiments and ultimately for patient use.

[0267] To demonstrate the selective killing of CD19+ tumor cells, the CD19 / HLA-A*02 activator / blocker combination was tested in vitro while preserving CD19+ / HLA-A*02+ cells in a mouse xenograft cancer model (Figure 27B). The same two Raji cell lines described above, one CD19+ and the other CD19+ / HLA-A*02+, were injected into the flanks of immunodeficient (NGS-HLA-A2.1) mice. Engineered T cells were injected at two doses, 2e6 T cells (not shown) or 1e7 T cells. Tumor growth and persistence of the transplanted T cells were analyzed over time. Tracking by the number of transplanted T cells, only CD19+ tumor cells were killed in the mouse and tumor controls, promoting host mouse survival (Figures 27C - 27E; Figures 28B - 28C). CD19+ / HLA-A*02+ cells designed to model normal cells were unaffected. The human CD4+ / CD8+ cell ratio in the blood of mice retaining engineered cells tracked with control counterparts, i.e., in "tumor" transplanted mice, was CD4+ cells > CD8+ cells as in CD19 CAR positive control mice, and in "normal" transplanted mice, CD4+ cells < CD8+ cells as in mice retaining non-transduced T cells (Figure 28D). Also, huCD3+ cells within the tumor (Figures 29A - 29B) were inversely correlated with tumor volume, and engineered T cells expressing the two receptors behaved like non-transduced T cells in "normal" grafts (low infiltrates) and like CD19 CAR in "tumor" grafts (high infiltrates). Finally, the mice appeared normal with respect to typical clinical observations (Table 10). Together, these results demonstrated that the LIR-1 inhibitory receptor can function in vivo to inhibit the activator receptor in a simplified setting designed to mimic the various normal and tumor cell types that would be encountered in a patient.

[0268]

Table 13-1

Table 13-2

[0269] Tumor cells and "normal" cells were injected on day 0 of the study, and treatment began on day 10 of the study. Clinical observation (CO) was performed 3 times / week, focusing on poor health, stress, and pain. Following IACUC guidelines, tumors >2000 mm 3 If the condition reached a certain point, the mouse was euthanized. Codes: N=normal; 19A=abnormal tumor [N=necrotic, O=open], EUT=euthanized. Severity codes: 0=absent, 1=moderate, 2=severe.

Claims

1. A chimeric antigen receptor comprising a polypeptide, wherein the polypeptide is a) LILRB1 hinged domain b) LILRB1 transmembrane domain c) A chimeric antigen receptor comprising a LILRB1 intracellular domain containing at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), each ITIM being selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11), wherein the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 2 or 3, and the binding of the antigen-binding domain inhibits nuclear factor (NFAT)-mediated activation of activated T cells.

2. The receptor according to claim 1, wherein the LILRB1 intracellular domain comprises at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM being selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

3. The LILRB1 intracellular domain, i.) Includes both NLYAAV (SEQ ID NO: 8) and VTYAEV (SEQ ID NO: 9) ITIM; ii.) Includes both ITIMs VTYAEV (sequence number 9) and VTYAQL (sequence number 10); iii.) Includes both ITIMs VTYAQL (sequence number 10) and SIYATL (sequence number 11); iv.) Includes ITIMs for NLYAAV (sequence number 8), VTYAEV (sequence number 9), and VTYAQL (sequence number 10); v.) Including ITIMs VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11); or vi.) The receptor according to claim 2, comprising ITIMs of NLYYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

4. The receptor according to claim 1, wherein the intracellular domain comprises a sequence that is at least 95% identical to sequence numbers 7 or 12-17.

5. The receptor according to claim 1, wherein the polypeptide comprises an intracellular domain comprising at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM being independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

6. The receptor according to claim 1, wherein the intracellular domain comprises the sequences of SEQ ID NOs: 12 to 17.

7. The receptor according to any one of claims 1 to 6, wherein the LILRB1 transmembrane domain comprises a sequence that is at least 95% identical to SEQ ID NO:

5.

8. The LILRB1 hinge domain contains a sequence that is at least 95% identical to sequence number 4, sequence number 18, sequence number 19, sequence number 80, sequence number 81, or sequence number 93, or The receptor according to any one of claims 1 to 7, wherein the LILRB1 hinge domain includes a sequence identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO:

93.

9. The receptor according to any one of claims 1 to 8, wherein the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 20, or a sequence that is at least 95% identical to SEQ ID NO:

21.

10. The polypeptide contains an sequence that is at least 95% identical to sequence number 2 or sequence number 3, or The polypeptide contains a sequence that is at least 99% identical to sequence number 20, or The polypeptide contains a sequence that is at least 99% identical to sequence number 21, or The polypeptide contains an sequence that is at least 99% identical to sequence number 2 or sequence number 3, or The polypeptide contains an sequence identical to sequence number 20, or The polypeptide contains an sequence identical to sequence number 21, or The polypeptide contains an sequence identical to sequence number 2 or sequence number 3, or The receptor according to claim 1, wherein the polypeptide comprises an antigen-binding domain.

11. The receptor according to claim 1, wherein the polypeptide comprises one or more antigen-binding domains.

12. The receptor according to claim 11, wherein one or more antigen-binding domains include a single-stranded variable fragment (scFv).

13. The receptor according to claim 12, wherein the scFv includes one complementarity-determining region (CDR) from any one of SEQ ID NOs. 22 to 33, and the scFv includes a sequence that is at least 95% identical to any one of SEQ ID NOs. 35 to 46 or 125.

14. The receptor according to claim 12 or 13, wherein the scFv comprises a heavy chain having a sequence that is at least 95% identical to any one of the heavy chain portions of SEQ ID NOs. 35-46 or 125, and the scFv comprises a light chain having a sequence that is at least 95% identical to any one of the light chain portions of SEQ ID NOs. 35-46 or 125.

15. The receptor according to any one of claims 1 to 14, which is an inhibitory receptor.

16. The receptor according to claim 15, wherein the inhibitory receptor comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 47-71, 77-79, 89-92, 120, or 122, or comprises the amino acid sequence of SEQ ID NOs: 47-71, 77-79, 89-92, 120, or 122.

17. A polynucleotide comprising a nucleic acid sequence encoding the receptor according to any one of claims 1 to 16.

18. A vector comprising the polynucleotide described in claim 17.

19. An immune cell comprising a receptor according to any one of claims 1 to 16, a polynucleotide according to claim 17, or a vector according to claim 18.

20. The immune cell according to claim 19, which is a T cell.

21. The immune cell according to claim 19 or 20, further comprising an activator receptor.

22. The immune cell according to claim 21, wherein the activator receptor is a chimeric antigen receptor or a T cell receptor.

23. A method for producing manipulated immune cells, comprising introducing the polynucleotide described in claim 17 or the vector described in claim 18 into immune cells.

24. A pharmaceutical composition comprising immune cells according to any one of claims 19 to 22 for use in the treatment of a disease or disorder, wherein the treatment comprises administering a plurality of the immune cells to a target.

25. The use of immune cells according to any one of claims 19 to 22 in the manufacture of a pharmaceutical product for treating a patient suffering from cancer.

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