Compositions and methods for treating blood cancers

Abstract

The disclosure relates to immune cells for treatment of blood cancer comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by blood cancer cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells.

Description

Attorney Ref.061250-557001WO COMPOSITIONS AND METHODS FOR TREATING BLOOD CANCERS CROSS REFERENCE TO RELATED APPLICATIONS [1] This application claims priority to U.S. provisional patent applications 63 / 592,052 filed on October 20, 2023, 63 / 550,980 filed on February 7, 2024, 63 / 683,196 filed on August 14, 2024, and 63 / 708,702 filed on October 17, 2024, the contents of each of which are incorporated herein by reference in its entireties. SEQUENCE LISTING [2] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy created on October 18, 2024, is named 061250-557001WO.xml and is 5800KB in size. BACKGROUND [3] Immune cells that are engineered with receptors to integrate signals from multiple antigens offer a route to achieve therapeutic selectivity in cancer patients. [4] In conventional adoptive cell therapies, immune cells are engineered to express specific receptors, for example chimeric antigen receptors (CARs) or T cell receptors (TCRs), which direct the activity of the immune cells to cellular targets via interaction of the receptor with a ligand expressed by the target cell. Identification of suitable target molecules remains challenging, as many targets are expressed in normal tissues which can lead to toxicity when the transplanted cells target normal tissues. [5] Selective toxicity for tumor cells is the fundamental obstacle to development of new oncology medicines. One promising strategy, called a synthetic signal-integration system, involves engineering effector cells that can integrate multiple signals derived from different profiles of antigens expressed on cancerous vs. normal cells. [6] One example of a synthetic signal-integration system involves an OR gate, by analogy to logical operations performed by machines, where either of two antigens (A-antigens) is sufficient to activate engineered effector cells. Such approaches have been designed for B cell-lineage tumors and involve two specific antigens: CD19 OR CD20 and CD19 OR CD22 (Zha et al., 2016 PMID: 27059623; Dai et al., 2020 PMID: 32245502). Another example of a synthetic signal- integration system involves AND gates in which two antigens, for example A and B, are needed to stimulate responses in engineered effector cells (Morsut et al., 2016 PMID: 26830878). A third example of a synthetic signal-integration system involves mechanisms that pair activating and inhibitory receptors which are termed NOT gates, for example, A not B. The NOT gate involves use of specific antigens to protect normal tissues via inhibitory receptors (Fedorov VD, et al., Sci Transl Med.2013; 5, 215).Attorney Ref.061250-557001WO [7] The clinical success of CAR therapy provides optimism for treating hematological malignancies. One challenge to developing CAR therapy for hematological malignancies is the lack of suitable targets. [8] There is thus a need in the art for compositions and methods useful in the treatment of blood cancers by adoptive cell therapy. SUMMARY [9] The present disclosure provides an immune cell for treatment of blood cancer, comprising an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells.

[0010] An aspect of the present disclosure provides an immune cell for treatment of blood cancer, including an inhibitor receptor including an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells.

[0011] In some embodiments, the present disclosure relates to an immune cell, wherein binding the extracellular ligand binding domain to the inhibitor antigen initiates an inhibitory signal in the immune cell which promotes sparing of the non-cancerous blood cell from killing, injuring, or distressing by the immune cell.

[0012] In some embodiments, the present disclosure relates to an immune cell, wherein the non- cancerous blood cell is a myeloid cell, a hematopoietic stem cell, a myeloid stem cell, or a myeloblast.

[0013] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor is a chimeric antigen receptor (CAR) or is a T cell receptor (TCR).

[0014] In some embodiments, the present disclosure relates to an immune cell, wherein the immune cell is a CD8+ T cell (e.g., a CD8+ CD4- T cell), a CD4+ T cell (e.g., a CD8- CD4+ T OQXX$& M SMYYM PQX`M #to$ H OQXX& MZ UZbM^UMZ` H OQXX& M ZM`a^MX WUXXQ^ #B?$ OQXX& MZ UZbM^UMZ` ZM`a^MX killer cell (iNK cell), an invariant natural killer T cell (iNKT cell), or a macrophage.

[0015] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitory antigen is any B antigen identified in Table 1.

[0016] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a binding domain as disclosed in Table 12C, Table 31, Table 39, Table 35, Table 43 and Table 51.

[0017] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1.

[0018] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising a set of complement determining regionAttorney Ref.061250-557001WO (CDR), wherein: the VH region comprises: (a) a complementarity-determining region VH-1 (CDR- H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4387- 4410 and 5876-5882; (b) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4411-4434 and 5883-5890; and (c) a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4435-4469 and 5891-5897, and the VL region comprises: (d) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4470-4495 and 5898-5902; (e) a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4496-4519 and 5905; and (f) a CDR3-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4520-4546 and 5907-5914.

[0019] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a set of CDR sequences as disclosed in a row of Table 33.

[0020] In some embodiments, the present disclosure relates to an immune cell, wherein the row of Table 33 is identified as 13P13, 3K22, 3G19, or 10H1.

[0021] In some embodiments, the present disclosure relates to an immune cell, wherein the VH region comprises the sequence of any one of SEQ ID NOs: 4547-4591 and 5915-5922, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID NOs: 4592-4636 and 5923-5930, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0022] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a VH region and a VL region comprising a set of sequences as disclosed in a row of Table 34, or sequences having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0023] In some embodiments, the present disclosure relates to an immune cell, wherein the row of Table 34 is identified as 13P13, 3K22, 3G19, or 10H1.

[0024] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 4637-4681 and 5931-5938.

[0025] In some embodiments, the present disclosure relates to an immune cell, wherein the scFv comprises a sequence as disclosed in Table 35 identified as 13P13, 3K22, 3G19, or 10H1.Attorney Ref.061250-557001WO

[0026] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 4682-4726 and 5939-5946.

[0027] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a sequence as disclosed in Table 36 identified as 13P13, 3K22, 3G19, or 10H1.

[0028] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9.

[0029] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising a set of complement determining region (CDR), wherein: the VH region comprises: (a) a complementarity-determining region VH-1 (CDR- H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4805- 4836 and 5947-5948; (b) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4837-4886 and 5949-5953; and (c) a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4887-4926 and 5955-5960, and the VL region comprises: (d) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4927-4958 and 5961-5966; (e) a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4959-4984 and 5967-5971; and (f) a CDR3-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4985-5026 and 5972-5978.

[0030] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a set of CDR sequences as disclosed in a row of Table 41.

[0031] In some embodiments, the present disclosure relates to an immune cell, wherein the row of Table 41 is identified as 14EE8 or 19M17.

[0032] In some embodiments, the present disclosure relates to an immune cell, wherein the VH region comprises the sequence of any one of SEQ ID NOs: 5027-5091 and 5979-5985, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID NOs: 5092-5156 and 5986-5992, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0033] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises a VH region and a VLAttorney Ref.061250-557001WO region comprising a set of sequences as disclosed in a row of Table 42, or sequences having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that are 100% identical thereto.

[0034] In some embodiments, the present disclosure relates to an immune cell, wherein the row of Table 42 is identified as 14EE8 or 19M17.

[0035] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the inhibitor receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 5157-5221 and 5993-5999.

[0036] In some embodiments, the present disclosure relates to an immune cell, wherein the scFv comprises a sequence as disclosed in Table 43 identified as 14EE8 or 19M17.

[0037] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 5222-5286 and 6097-6103.

[0038] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a sequence as disclosed in Table 44 identified as 14EE8 or 19M17.

[0039] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises a LILRB1 intracellular domain or a functional variant thereof.

[0040] In some embodiments, the present disclosure relates to an immune cell, wherein the LILRB1 intracellular domain comprises the sequence of SEQ ID NO: 113, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0041] In some embodiments, the present disclosure relates to an immune cell, wherein the inhibitor receptor comprises LILRB1 hinge and transmembrane domains, or functional variants thereof.

[0042] In some embodiments, the present disclosure relates to an immune cell, wherein the LILRB1 transmembrane domain comprises the sequence of SEQ ID NO: 102, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0043] In some embodiments, the present disclosure relates to an immune cell, wherein the LILRB1 hinge domain comprises the sequence of any one of SEQ ID NOs: 103, 104, 115, 116, 117, 118, 119, 120, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.Attorney Ref.061250-557001WO

[0044] In some embodiments, the present disclosure relates to an immune cell, wherein the LILRB1 hinge, LILRB1 transmembrane, and LILRB1 intracellular domains comprise the sequence of SEQ ID NO: 108, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0045] In some embodiments, the present disclosure relates to an immune cell, wherein the immune cell further comprises an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by a blood cancer cell.

[0046] In some embodiments, the present disclosure relates to an immune cell, wherein binding the extracellular ligand binding domain to the activator antigen initiates an activating signal in the immune cell which promotes the killing, injuring, or distressing of the cancerous blood cell by the immune cell.

[0047] In some embodiments, the present disclosure relates to an immune cell, wherein the blood cancer cell is a hematopoietic cancer cell or a hematologic cancer cell.

[0048] In some embodiments, the present disclosure relates to an immune cell, wherein the blood cancer cell is a lymphoma cell, a leukemia cell (e.g., an acute myeloid leukemia (AML) cell), a myeloma cell, a Reed-Sternberg cell, a myeloproliferative neoplasm cell, or a Waldenstrom macroglobulinemia cell.

[0049] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor is a chimeric antigen receptor (CAR) or is a T cell receptor (TCR).

[0050] In some embodiments, the present disclosure relates to an immune cell, wherein the activator antigen is any A antigen identified in Table 1.

[0051] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a binding domain as disclosed in Table 4B, Table 5C, Table 6C, Table 19, Table 23, Table 27 and Table 47.

[0052] In some embodiments, the present disclosure relates to an immune cell, wherein the activator antigen expressed by the cancerous blood cell is ITGA4.

[0053] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising a set of complement determining region (CDR), wherein: the VH region comprises: (a) a complementarity-determining region VH-1 (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2704-2731; (b) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2732-2776; and (c) a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2777-2812, and the VL region comprises: (d) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2813-Attorney Ref.061250-557001WO 2846; (e) a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2847-2874; and (f) a CDR3-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2875-2909.

[0054] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 17.

[0055] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a set of CDR sequences in the row of Table 17 is identified as 16E9, 17J1, or 2F5.

[0056] In some embodiments, the present disclosure relates to an immune cell, wherein the VH region comprises the sequence of any one of SEQ ID NOs: 2910-2971, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID NOs: 2972-3033, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0057] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a VH region and a VL region comprising a set of sequences as disclosed in a row of Table 18, or sequences having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0058] In some embodiments, the present disclosure relates to an immune cell, In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a VH region and a VL region comprising a set of sequences as disclosed in a row of Table 18 identified as 16E9, 17J1, or 2F5.

[0059] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3034-3095.

[0060] In some embodiments, the present disclosure relates to an immune cell, wherein the scFv comprises a sequence as disclosed in Table 19 identified as 16E9, 17J1, or 2F5.

[0061] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 3096-3157.Attorney Ref.061250-557001WO

[0062] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 20 identified as 16E9, 17J1, or 2F5.

[0063] In some embodiments, the present disclosure relates to an immune cell, wherein the activator antigen expressed by the cancerous blood cell is CLL1.

[0064] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising a set of complement determining region (CDR), wherein: the VH region comprises: (a) a complementarity-determining region VH-1 (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5287-5326; (b) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5327-5389; and (c) a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5390-5452, and the VL region comprises: (d) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5453- 5507; (e) a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5508-5513; and (f) a CDR3-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5545-5551.

[0065] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 45.

[0066] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a set of CDR sequences as as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2 of Table 45.

[0067] In some embodiments, the present disclosure relates to an immune cell, wherein the VH region comprises the sequence of any one of SEQ ID NOs: 5587-5658, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID NOs: 5659-5730, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0068] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a VH region and a VL region comprising a set of sequences as disclosed in a row of Table 46, or sequences having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto. In some embodiments, wherein the row of Table 46 is identified as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2.Attorney Ref.061250-557001WO

[0069] In some embodiments, the present disclosure relates to an immune cell, herein the extracellular ligand binding domain of the activator receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 5731-5802.

[0070] In some embodiments, the present disclosure relates to an immune cell, wherein the scFv comprises a sequence as disclosed in Table 47 identified as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2.

[0071] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 5803-5874.

[0072] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 48 identified as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2.

[0073] In some embodiments, the present disclosure relates to an immune cell, wherein the activator antigen expressed by the cancerous blood cell is CD33.

[0074] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising a set of complement determining region (CDR), wherein: the VH region comprises: (a) a complementarity-determining region VH-1 (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1512-1618; (b) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1619-1725; and (c) a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1726-1832, and the VL region comprises: (d) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1833- 1947; (e) a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1948-2062; and (f) a CDR3-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2063-2177.

[0075] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 6B. In some embodiments, the row of Table 6B is identified as Activator 6, Activator 37, or Activator 100.

[0076] In some embodiments, the present disclosure relates to an immune cell, wherein the VH region comprises the sequence of any one of SEQ ID NOs: 1290-1396 and 2442-2449, or aAttorney Ref.061250-557001WO sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID NOs: 1397-1511 and 2450-2457, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

[0077] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises a VH region and a VL region comprising a set of sequences as disclosed in a row of Table 6A, or sequences having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto. In some embodiments,

[0078] In some embodiments, the present disclosure relates to an immune cell, the row of Table 6A is identified as Activator 6, Activator 37, or Activator 100.

[0079] In some embodiments, the present disclosure relates to an immune cell, wherein the extracellular ligand binding domain of the activator receptor comprises an antibody fragment or a single chain Fv antibody fragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2458-2580.

[0080] In some embodiments, the present disclosure relates to an immune cell, wherein the scFv comprises a sequence as disclosed in Table 6C identified as Activator 6, Activator 37, or Activator 100.

[0081] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 2581-2703.

[0082] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 6D identified as Activator 6, Activator 37, or Activator 100.

[0083] In some embodiments, the present disclosure relates to an immune cell, wherein the immune cell is modified to reduce or eliminate expression of the B2M gene product.

[0084] In some embodiments, the present disclosure relates to an immune cell, wherein expression of the B2M gene product is reduced or eliminated using a short hairpin RNA (shRNA).

[0085] In some embodiments, the present disclosure relates to an immune cell, wherein the shRNA O[Y\^U_Q_ M RU^_` _Q]aQZOQ& TMbUZS R^[Y / q `[ -q QZP M _Q]aQZOQ O[Y\XQYQZ`M^e `[ `TQ 7,A YFB65 MZP M _QO[ZP _Q]aQZOQ& TMbUZS R^[Y / q `[ -q QZP M _Q]aQZOQ O[Y\XQYQZ`M^e `[ `TQ RU^_` _Q]aQZOQ& wherein the first sequence and second sequence form the shRNA.Attorney Ref.061250-557001WO

[0086] The present disclosure provides an immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is ITGA4 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3034- 3095 or comprising the CDR sequences as disclosed in a row of Table 17; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDR sequences as disclosed in a row of Table 33.

[0087] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 17 identified as 16E9, 17J1, or 2F5 and wherein the inhibitor receptor comprises a sequence as disclosed in Table 33 identified as 13P13, 3K22, 3G19, or 10H1.

[0088] The present disclosure provides an immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is ITGA4 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3034- 3095 or comprising the CDR sequences as disclosed in a row of Table 17; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 5157-5221 and 5993-5999 or comprising the CDR sequences as disclosed in a row of Table 41.

[0089] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 17 identified as 16E9, 17J1, or 2F5Attorney Ref.061250-557001WO and wherein the inhibitor receptor comprises a sequence as disclosed in Table 41 identified as 14EE8 or 19M17.

[0090] The present disclosure provides for an immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is CLL1 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 5731- 5802 or comprising the CDR sequences as disclosed in a row of Table 45; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDR sequences as disclosed in a row of Table 33.

[0091] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 45 identified as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2 and wherein the inhibitor receptor comprises a sequence as disclosed in Table 33 identified as 13P13, 3K22, 3G19, or 10H1.

[0092] The present disclosure provides for immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is CLL1 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 5731- 5802 or comprising the CDR sequences as disclosed in a row of Table 45; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos: 5157-5221 and 5993-5999 or comprising the CDR sequences as disclosed in a row of Table 41.Attorney Ref.061250-557001WO

[0093] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 45 identified as 2N24, 14L11, 7F2, 7G3, 9O11, 20I21, 3F10, or 7F2 and wherein the inhibitor receptor comprises a sequence as disclosed in Table 41 identified as 14EE8 or 19M17.

[0094] The present disclosure provides for immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is CD33 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2458- 2580 or comprising the CDR sequences as disclosed in a row of Table 6B; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDR sequences as disclosed in a row of Table 33.

[0095] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 6B identified as 1A4, 2H20, or 1C20 and wherein the inhibitor receptor comprises a sequence as disclosed in Table 33 identified as 13P13, 3K22, 3G19, or 10H1.

[0096] The present disclosure provides for immune cell for treating a blood cancer, the immune cell comprising an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen expressed by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand binding domain specific to an inhibitor antigen expressed by non-cancerous blood cells, wherein the activator antigen expressed by the cancerous blood cell is CD33 and wherein the activator receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2458- 2580 or comprising the CDR sequences as disclosed in a row of Table 6B; and wherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 and wherein the inhibitor receptor comprises an extracellular ligand binding domain comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to, or that is 100% identicalAttorney Ref.061250-557001WO to a sequence selected from the group consisting of SEQ ID NOs: 5157-5221 and 5993-5999 or comprising the CDR sequences as disclosed in a row of Table 41.

[0097] In some embodiments, the present disclosure relates to an immune cell, wherein the activator receptor comprises a sequence as disclosed in Table 6B identified as 1A4, 2H20, or 1C20 and wherein the inhibitor receptor comprises a sequence as disclosed in Table 41 identified as 14EE8 or 19M17.

[0098] In some embodiments, the present disclosure relates to an immune cell, wherein the cancer is leukemia, lymphoma, myeloma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), myeloid leukemia, Non-Hodgkin lymphoma, Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma, myeloproliferative neoplasms (MPNs), juvenile myelomonocytic leukaemia (JMML), Juvenile Chronic Myeloid Leukaemia (JCML), or Chronic Myelomonocytic Leukaemia of Infancy.

[0099] The present disclosure provides for an activator receptor expressed by an immune cell.

[0100] In some embodiments, the present disclosure relates to an immune cell, expresses an activator receptor but not an inhibitor receptor.

[0101] In some embodiments, the present disclosure relates to a polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising one or more polynucleotide sequences encoding the activator receptor. In some embodiments, the present disclosure relates to a method of making an immune cell therapy, comprising one or more polynucleotides comprising one or more polynucleotide sequences encoding the inhibitor receptor.

[0102] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of the immune cells of the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

[0103] In some embodiments, the present disclosure relates to a method for treating a blood cancer in a subject, the method comprising administering to the subject a therapeutically amount of the pharmaceutical composition.

[0104] In some embodiments, the present disclosure relates to a method for treating blood cancer in a subject, wherein the immune cell is autologous to the subject or is allogeneic to the subject.

[0105] In some embodiments, the present disclosure relates to a method for treating a blood cancer in a subject, the method comprising administering to the subject a therapeutically amount of an immune cell disclosed herein.

[0106] In some embodiments, the present disclosure relates to a method for treating a blood cancer in a subject, wherein the immune cell is autologous to the subject or is allogeneic to the subject.Attorney Ref.061250-557001WO

[0107] In some embodiments, the present disclosure relates to use of the immune cells disclosed herein in a method of manufacturing a medicament for treating blood cancer.

[0108] In some embodiments, the present disclosure relates to a kit comprising the immune cell of the present disclosure or a pharmaceutical composition comprising the immune cells of this disclosure and a pharmaceutically acceptable carrier, diluent or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0110] FIG.1A is a diagram depicting engineering a cell using an mRNA lipid nanoparticle (LNP).

[0111] FIG.1B is a graph showing the expression of CD33 in non-cancerous “normal” HSCs, AML stage cells, and non-cancerous “normal” monocytes.

[0112] FIGs. 2A and 2B are illustrative designs of cellular logic systems to integrate multiple- antigen inputs.

[0113] FIG.2A shows venn diagrams depicting classes of multi-antigen receptor systems that have been constructed in immune effector cells to integrate inputs (logic gates). Teal represents the antigen combination on target cells that activates killing by the engineered immune cells.

[0114] FIG. 2B depicts an illustrative NOT-gate engineered receptor system. Cells, receptors and antigens involved in tumor-selective killing. The engineered receptor NOT-gate system incorporates an inhibitory receptor (blocker) which inhibits T cell activation when engaged with the blocker antigen present on normal cells but not when the blocker antigen is absent on cancer cells. Engineered Receptor System = immune cells expressing both activator and inhibitor receptors.

[0115] FIG. 3 shows a schematic depicting an illustrative model for how an engineered receptor system functions as a proximity detector to integrate antigen inputs locally in real time. Cells expressing an engineered receptor system are able to spare normal cells based on detection of activator / blocker antigens on the same vs. different surfaces. Only when activator and blocker antigens are in close proximity (i.e. on the same surface) will the CAR be blocked. Therefore, even cells expressing an engineered receptor system interacting with many normal cells through blocker binding will not be prevented from killing a cancer cell.

[0116] FIG.4 is a diagram depicting properties of cells expressing an engineered receptor system of therapeutic relevance. Cells expressing an engineered receptor system can selectively kill tumor cells while in a sea of normal tissue but also spare nearby normal cells while surrounded by tumor; reversibility: Cells expressing an engineered receptor system can kill tumor cells after encountering normal cells and also spare normal cells after encountering and killing tumor cells (and back-and- forth).Attorney Ref.061250-557001WO

[0117] FIG. 5 is a schematic of an illustrative algorithm to identify A and B antigens for activator and inhibitor receptors for blood cancers.

[0118] FIG.6 is a graph depicting A antigen SPN expression (left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0119] FIG. 7 is a graph depicting A antigen CD45 expression (left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0120] FIG.8 is a graph depicting A antigen LFA-1 expression (left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0121] FIG. 9 is a graph depicting A antigen SPN expression (top left panel) and A antigen CD33 expression (bottom left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0122] FIG.10 is a graph depicting A antigen SPN expression (top left panel) and A antigen CD45 expression (bottom left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0123] FIG.11 is a graph depicting A antigen SPN expression (top left panel) and A antigen FLT3 expression (bottom left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0124] FIG.12 is a graph depicting A antigen CD33 expression (top left panel) and A antigen FLT3 expression (bottom left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is aAttorney Ref.061250-557001WO graph depicting B antigen (PECAM-1) expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0125] FIG. 13 shows an anti-CD19 CAR, anti-CD20 CAR, or tandem anti-CD19 / anti-CD20 activator CAR functioning as an OR gate in combination with an HLA-A*02 inhibitor receptor module. The plot depicts HLA*A*02 blocker IC50 measurements when paired with anti-CD19, anti- CD20, or both anti-CD19 and anti-CD20 tandem activators in Jurkat cells, with titration of HLA- A*02 mRNA in ng, showing HLA-A*02-dose-dependent blocking.

[0126] FIG.14 is a plot showing HLA-A*02 inhibitor receptor module IC50 when paired with anti- CD19, anti-CD20, or both anti-CD19 and anti-CD20 activator tandem CAR, as measured by titration of HLA-A*02 mRNA in ng. K562 (CD19+) target cells, Raji (CD20+ CD19 Knock Out [KO]) target cells, and Raji (CD20+ CD19+) target cells were transfected with titrated amounts of HLA-A*02 mRNA.

[0127] FIG. 15 is a chart depicting illustrative expression results obtained through the analysis of normal cell, tumor cell, and tumor cell line RNA-Seq datasets to generate B antigen candidates.

[0128] FIG. 16 shows a schematic depicting an illustrative single mRNA encoding a cleavable fusion protein comprising an inhibitor (blocker) receptor operably linked to an activator receptor. Illustrative examples of hinge (H), transmembrane (TM), and intracellular domain components of the activator and blocker receptors are shown.

[0129] FIG. 17A is a flow cytometry graph depicting an activator CAR and inhibitor receptor expression on engineered T cells. Three days after fresh PBMCs were activated with TransAct™, B2M was knocked-out via CRISPR / CAS9 gene editing. After 7 days of recovery, unedited cells were depleted using anti-activator antigen antibody, W6 / 32. mRNA encoding empty vector, activator (C2172), or a bicistronic vector encoding the activator and inhibitor (blocker) (C6177) was transfected 2 days after enrichment and used in functional assays.

[0130] FIG. 17B U_ M RX[c Oe`[YQ`^e S^M\T PQ\UO`UZS D^[`QUZ'@ MZP <[M` n'Y[a_Q >S< Qd\^Q__U[Z on engineered T cells. Three days after fresh PBMCs were activated with TransAct™, B2M was knocked-out via CRISPR / CAS9 gene editing. After 7 days of recovery, unedited cells were depleted using anti-activator antigen antibody, W6 / 32. mRNA encoding empty vector, activator (C2172), or a bicistronic vector encoding the activator and inhibitor (blocker) (C6177) was transfected 9 days after enrichment and used in functional assays.

[0131] FIG. 18 is a graph depicting IFNgamma secretion by primary T cells after co-culture with HeLa target cells. After mRNA transfection encoding the activator (C2172) and blocker (C6177), T cells were co-cultured with HeLa target cells transfected with increasing amounts of mRNA encoding blocker receptor mRNA. T cells co-cultured with HeLa targets that received no blocker receptor mRNA (closed squares, 6177 no) produced a consistent amount of IFNgamma whereas T cells co-Attorney Ref.061250-557001WO cultured with blocker receptor mRNA (open squares, 6177 A2) were inhibited in IFNgamma production. The half maximal inhibitory concentration (IC50) is provided in the table along with the interpolated number of surface blocker receptor molecules from QiFi Kit analysis of HeLa target cells...

[0132] FIG. 19 is a panel of graphs showing the ratio of SPN / MME mRNA expression in non- cancerous “normal” HSCs, AML cells, non-cancerous “normal” monocytes, neutrophils, B cells, and T cells (left panel) and the ratio of SPN / MME protein expression in AML cells and non-cancerous “normal” HSCs (right panel).

[0133] FIG.20 is a panel of graphs showing SPN and MME mRNA expression level in transcripts per million (TPM) in AML cell lines, AML bulk tumor cells, non-cancerous “normal” HSCs, non- cancerous “normal” monocytes, neutrophils, B cells, and T cells (left panel) and SPN and MME protein expression level in label-free quantification (LFQ) units in AML cells and non-cancerous “normal” HSCs (right panel). “LOD” denotes the estimated limit of detection.

[0134] FIG. 21 is a graph depicting A antigen SPN expression (left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen MME expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0135] FIG. 22 is a graph depicting A antigen SPN expression (left panel) measured in transcripts per million (TPM) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a graph depicting B antigen FCGR3A expression measured in TPM AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu).

[0136] FIG. 23 shows a schematic depicting an illustrative engineered receptor system NOT-gate. The engineered receptor NOT-gate system incorporates an inhibitory receptor (blocker) which inhibits T cell activation when engaged with a HLA class I “B” antigen present on normal HSC cells. The B antigen matches the haplotype of transplanted stem cells.

[0137] FIG. 24 is a graph depicting expression measured in transcripts per million (TPM) of two different A antigens (left top and bottom panels) in AML subtype cells, CD3+ cells, CD19+ cells, HSCs, monocytes (Mono), and neutrophils (Neu). The right panel is a schematic depicting an illustrative cell expressing an engineered receptor system in contact with a cell comprising an antigen combination on target cells that inhibits killing by the engineered immune cells.

[0138] FIG. 25 shows a schematic depicting an illustrative cell expressing an engineered receptor system in contact with a tumor cell comprising an antigen combination that activates killing by the engineered immune cells (left panel). The schematic also depicts an illustrative cell expressing anAttorney Ref.061250-557001WO engineered receptor system in contact with a transplanted Hemopoietic Stem Cell (HSC) comprising an antigen combination that inhibits killing by the engineered immune cells (right panel).

[0139] FIG.26 is a panel of flow cytometry plots showing the analysis of CD33 expression in K562 cells in which CD33 was knocked out and then a titration of CD33 mRNA was transfected. CD33 antibody clone WM53 used for staining. CD33 molecule count (molecules / cell) was interpolated using the QIFIKIT™ for quantitative analysis from Agilent.

[0140] FIG. 27 is a panel of flow cytometry plots showing anti-CD33 CAR expression in Jurkat reporter cells. Anti-CD33 CAR expression was detected via staining with recombinant CD33 protein that was pre-labeled with Phycoerythrin (PE).

[0141] FIG. 28A is a graph showing CD33 responsive CAR expressing Jurkat reporter cells co- culture with K562 target cells that were transfected with a titration of CD33 mRNA. CD33 molecules / cell was interpolated using the QIFIKIT™ for quantitative analysis from Agilent (right panel).

[0142] FIG. 28B is a chart showing surface CD33 molecule levels in molecules / cell analyzed in blood cancer cell lines.

[0143] FIG.29 is a graph showing Jurkat cells transfected with an anti-CD33 CAR plasmid and an anti-CD16 Blocker 1 plasmid and co-cultured with K562 cells that overexpressed CD33 and were transfected with a titration of CD16B mRNA.

[0144] FIG. 30A is a graph showing CD16B mRNA transfected into either wild type K562 cells (“Low Endogenous CD33”), K562 cells transfected with 200 ng CD33 mRNA (“Medium CD33”), or K562 cells transfected with 500 ng CD33 mRNA (“High CD33”).

[0145] FIG. 30B is a panel of graphs showing the titration of CD16B mRNA at different levels of CD33 expression.

[0146] FIG. 30C is a graph showing Jurkat cells transfected with an anti-CD33 CAR “CAR 1” plasmid and an anti-CD16 Blocker 1 plasmid and co-cultured with K562 cells that overexpressed CD33 and were transfected with a titration of CD16B mRNA.

[0147] FIG. 30D is a graph showing Jurkat cells transfected with an anti-CD33 CAR “CAR 2” plasmid and an anti-CD16 Blocker 1 plasmid and co-cultured with K562 cells that overexpressed CD33 and were transfected with a titration of CD16B mRNA.

[0148] FIG.31A is a diagram of an anti-CD33 activator CAR and anti-CD16 Blocker 1 experiment in primary T cells.

[0149] FIG.31B is a chart showing QIFIKIT™ from Agilent analysis to estimate number of CD33 and CD16B molecules on the surface of the K562 cell line. K562 cells were engineered to model “normal” tissue by overexpressing CD33 and CD16B and K562 cells that only overexpressed CD33 were engineered to model “tumor” tissue.Attorney Ref.061250-557001WO

[0150] FIG. 31C is a graph showing Jurkat cells transfected with anti-CD33 CAR plasmid and an anti-CD16 Blocker 1 plasmid and co-cultured with engineered K562 target cells with a titration of Effector:Target (E:T) ratios. Jurkat cells and engineered K562 target cells were co-cultured for 48 hours and % specific killing was measured via luciferase signal. The engineered K562 target cells over-express CD33 to model the tumor environment (“tumor”) or both CD33 and CD16B to model normal tissue (“normal”).

[0151] FIG. 32 is a panel of flow cytometry plots showing anti-SPN CAR expression in Jurkat reporter cells. Protein L or soluble recombinant human His-tagged protein (sSPN-His) was used to stain the CARs.

[0152] FIG.33 is a panel of histograms showing SPN staining of JNL SPN knock out (KO) and JNL wild type (WT) cells. JNL SPN KO cells were produced using CRISPR and a guide RNA targeting the SPN gene followed by cell sorting to enrich the SPN KO population. Cells were stained using anti-SPN antibody clone 1G10 followed by analysis by flow cytometry.

[0153] FIG. 34 is a panel of graphs showing Jurkat nuclear factor of activated T cells (NFAT) luciferase (JNL) cell activation in a cell titration assay. JNL cells were transfected with 2 ug of activator anti-SPN CAR DNA per 1E6 cells using the Neon electroporation system. 18-24 hours post-transfection, 10,000 JNL cells were co-cultured with serially titrated Target cells (Jurkat E6.1, K562, HeLa, THP-1, and MV4-11) for 6 hours.

[0154] FIG. 35 is a panel of flow cytometry histogram plots showing Flag-CD16B mRNA titrated in Jurkat WT cells, detected with anti-CD16 antibody 3G8 and quantified by converting flow- cytometry mean fluorescence intensity (MFI) signal to molecules of CD16B per cell using QIFIKIT™ quantitative analysis kit.

[0155] FIG.36 shows a panel of flow cytometry plots of anti-SPN CAR expression “CAR 10” and “CAR 11” with or without anti-CD16 Blocker 1 in JNL SPN KO cells where anti-SPN CAR staining was performed using Protein L and anti-CD16 Blocker 1 staining was performed using an anti-CD16 Blocker 1 hinge-specific antibody. Percentages for gated populations shown on plots. CAR 10: VH SEQ ID NO: 269 and VL SEQ ID NO: 289; CAR 11: Light Chain SEQ ID NO: 260 and Heavy Chain SEQ ID NO: 259.

[0156] FIG.37 is a graph showing activation of Jurkat NFAT luciferase (JNL) cells transfected with an anti-CD16 Blocker 1 scFv fused to CAR domains in an mRNA titration assay. Jurkat WT target cells were transfected with serially diluted Flag-CD16B mRNA and JNL SPN KO cells were transfected with 2 ug of activator anti-SPN CAR DNA per 1E6 cells using the Neon™ electroporation system.18-24 hours post-transfection, 10,000 JNL anti-SPN CAR cells were co-cultured with 5000 transfected Jurkat WT target cells for 6 hours. The functional response (RLU) was assessed using ONE™ step luciferase assay system.Attorney Ref.061250-557001WO

[0157] FIG.38A is a graph showing Jurkat WT target cells transfected with serially diluted FLAG- CD16B mRNA and JNL SPN KO cells transfected with 1 ug of activator anti-SPN CAR “CAR 10” DNA and 2 ug of anti-CD16 Blocker 1 DNA per 1E6 cells using the Neon™ electroporation system. 18-24 hours post-transfection, 10,000 JNL cells were co-cultured with 5000 transfected Jurkat cells for 6 hours. The activation of JNL cells was assessed by measuring the functional response (RLU) using the ONE™ step luciferase assay system.

[0158] FIG.38B is a graph showing Jurkat WT target cells transfected with serially diluted FLAG- CD16B mRNA and JNL SPN KO cells transfected with 1 ug of activator anti-SPN CAR “CAR 11” DNA and 2 ug of anti-CD16 Blocker 1 DNA per 1E6 cells using the Neon™ electroporation system. 18-24 hours post-transfection, 10,000 JNL cells were co-cultured with 5000 transfected Jurkat cells for 6 hours. The activation of JNL cells was assessed by measuring the functional response (RLU) using the ONE™ step luciferase assay system.

[0159] FIG. 39A is a diagram of an illustrative engineered receptor system showing the two receptors that comprise the NOT gate. H: hinge, TM: transmembrane domain, ICD: intracellular domain.

[0160] FIG.39B is a graph showing mRNA expression of CD33 and CD16B (FCRG3B) in healthy adult tissue (RNA-Seq data from GTEx database).

[0161] FIG. 39C is a graph showing CD33 and FCGR3B mRNA expression in Primary AML and healthy blood cells including T cells, neutrophils, monocytes, and hematopoietic stem cells (HSC) (data from Ley lab portal TCGA).

[0162] FIG. 40 shows FCGR3B (CD16B) downregulation is not the consequence of genetic deletion. The graph shows data reproduced from cBioportal.org illustrating low genomic alteration of FCGR3B in different sample data sets.

[0163] FIG. 41A shows mRNA expression Log2 (Transcripts Per Million (TPM)+1) of CD33 in AML cell lines (n=44; DepMap).

[0164] FIG.41B shows mRNA expression Log2 (Transcripts Per Million (TPM)+1) of FCGR3B in AML cell lines (n=44; DepMap).

[0165] FIG.42A shows CD33 reactive CARs recognize low levels of CD33, such as levels found in blood cancer cell lines. In the Jurkat reporter cell line activation assay shown, Jurkat cells were transfected with CAR encoding DNA plasmids and cocultured with target cells that were transfected with a titration of the CD33 mRNA. Firefly luciferase read out was measured 6 hours post coculture start. *Indicates experiments where HeLa cells were used as target cells.

[0166] FIG. 42B is a table showing CD33 molecules / cell on the cell surface of blood cancer cell lines.Attorney Ref.061250-557001WO

[0167] FIG. 43 shows a Jurkat reporter cell assay. Jurkat cells were transfected with two plasmids, one plasmid encoding the CD33 reactive CAR and the other plasmid encoding an inhibitor receptor “blocker” CAR. Jurkat cells were cocultured with target cells that were transfected with a titration of CD16B mRNA.

[0168] FIG.44 shows CD16A expression in AML samples is higher than CD16B expression. RNA- seq expression data from primary AML samples was retrieved from the Ley Lab.

[0169] FIG. 45A shows the mouse immunization schedule for the identification of hybridoma derived CD16B-specific binders.

[0170] FIG. 45B shows CD16B specific binders were identified by indirect ELISA screens using biotinylated CD16A or CD16B soluble protein. Wells with CD16B selective binding (red square) were selected for further analysis.

[0171] FIG. 45C shows multiplexed FACS based CD16A vs CD16B binding screens utilized CMFDA loaded K562 cells overexpressing CD16B mixed with non-colored K562 overexpressing CD16A to confirm selective binding to the membrane bound form CD16B.

[0172] FIG.45D shows dendrogram showing diversity of 32 clones chosen for blocker generation. Hamming distance (y-axis) of .1 represents approximately 10 residue differences.

[0173] FIG. 46A is a panel of histograms showing the analysis of CD16B expression in K562 WT cells. Detection was assayed using anti-CD16 antibody 3G8 and quantified by converting flow- cytometry mean fluorescence intensity (MFI) signal to molecules of CD16B per cell using a QIFIKIT™ quantitative analysis kit.

[0174] FIG. 46B is a panel of histograms showing the analysis of CD16A expression in K562 WT cells. Detection was assayed using anti-CD16 antibody 3G8 and quantified by converting flow- cytometry mean fluorescence intensity (MFI) signal to molecules of CD16B per cell using a QIFIKIT™ quantitative analysis kit.

[0175] FIG. 46C is a visualization of a sequence alignment between CD16A (SEQ ID NO: 1175) and CD16B (SEQ ID NO: 1174).

[0176] FIG. 47 is a panel of flow cytometry plots showing staining of Jurkat cells expressing anti- CD16 scFvs CARs using soluble, recombinant human CD16B-biotin (sCD16B-bio) or goat anti- mouse IgG Fab-AF647 antibody.

[0177] FIG. 48 is a panel of graphs showing Jurkat nuclear factor of activated T cells (NFAT) luciferase (JNL) cell activation in a cell titration assay. JNL cells were transfected with CD16B- CARs in an mRNA titration assay. K562 WT target cells were transfected with serially diluted Flag- CD16B mRNA or Flag-CD16a-GPI (CD16a extracellular domain fused to CD16B C-terminal domain). JNL cells were transfected with 3 ug of activator DNA per 1E6 cells using the Neon electroporation system.18-24 hours post-transfection, 10,000 JNL CAR cells were co-cultured withAttorney Ref.061250-557001WO 5000 transfected K562 WT target cells for 6 hours. The functional response (RLU) was assessed using ONE step luciferase assay system.

[0178] FIG. 49A shows CD16B-selective binders mediate blocker function in Jurkat cells. A diagram of a functional screen in Jurkat reporter cell line cocultured with K562 target cells transfected with different amounts of CD16B mRNA.

[0179] FIG.49B shows a diagram of functional parameters estimated from the Jurkat cell assay data.

[0180] FIG. 49C shows rank-ordered inhibition by blockers using a single CD33 CAR; constructs isolated by mini-prep.

[0181] FIG.49D shows rank-ordered inhibition using the average percent blocking with 4 different CD33 CARs; constructs isolated by maxi-prep.

[0182] FIG. 49E shows functional readout from 8-point mRNA titration curves. Selected blocker- activator combinations in color. Top blocker hits are combined with five C2 domain CARs and one V domain CAR as a positive control.

[0183] FIG. 50A shows that cells expressing a CD33 / CD16B engineered receptor system exhibit potent, selective cytotoxicity in vitro. A diagram of high-throughput construct screening in primary T cells using piggyBAC.

[0184] FIG. 50B shows FACS analysis of stable integrants via staining with labeled recombinant human CD33.

[0185] FIG. 50C shows E:T cytotoxicity curves generated from GFP signal at 48 hour time point with each well normalized to the zero time point. Tumor (CD33(+)CD16B(-)) target cell curves are shown with dashed lines and normal (CD33(+)CD16B(+)) target cell curves with solid lines. Black lines are CAR constructs and colored lines are constructs of engineered receptor systems. Tumor cells are K562 cells engineered with CD33 and normal cells are K562 cells engineered to overexpress CD33 and CD16B.

[0186] FIG. 50D shows potency calculated as ET50 of cells expressing an engineered receptor system cocultured with tumor cells.

[0187] FIG. 50E shows selectivity ratios are calculated as ET50 on normal cells divided by ET50 on tumor cells.

[0188] FIG.50F shows kinetic cytotoxicity analysis of a selective and potent construct as compared to the CAR-T. GFP(+) area was used as proxy for target cell viability.

[0189] FIG.51A shows cells expressing a CD33 / CD16B engineered receptor system selectively kill tumor but not “normal” cells in vivo. A Schema for the in vivo experiment.2e6 MV-4-11 AML cells or MV-4-11 cells that overexpress CD16B were injected into NGS-SGM3 mice and 6 days later 7.5e6 T cells were injected.Attorney Ref.061250-557001WO

[0190] FIG. 51B shows selectivity in vitro using MV-4-11 cells. Surrogate normal cells were generated by overexpression of CD16B in the AML cells.

[0191] FIG.51C shows FACS analysis of construct expression by staining with labeled recombinant human CD16 and CD33.

[0192] FIG.51D shows bioluminescence imaging (BLI) at 20 days post target cell injection.

[0193] FIG. 51E shows FACS analysis of MV-4-11 cells in the bone marrow 27 days post target cell injection.

[0194] FIG.51F shows quantification of data shown in panel FIG.51E.

[0195] FIG. 52 shows CD33 gene expression in MV-4-11 matches AML primary samples. CD33 gene expression in primary AML samples retrieved from the Ley lab data set. The level of CD33 in MV-4-11 (from DepMap) is plotted as a dashed line.

[0196] FIG.53 shows MV411 CD33 mRNA expression is representative of primary AML samples. Flow cytometry-based analysis of CD16B expression in the engineered “normal” (non-cancerous) cell line.

[0197] FIG.54 shows mouse anti-CD33 CAR generation workflow.

[0198] FIG. 55 shows ELISA binding data demonstrating CD33 vs Siglec-8 specificity of murine anti-CD33 IgG to either biotinylated huCD33 / Streptavidin HRP tetramers or biotin Siglec- 8 / Streptavidin HRP tetramers. The boxed mouse IgGs were selected as CD33 specific.

[0199] FIG.56 shows an illustrative CAR construct design.

[0200] FIG.57A is a panel of histograms showing CD33 mRNA titration in K562 CD33 knockout (KO) cells.

[0201] FIG. 57B shows human CD33 (huCD33) detected with mouse anti-CD33 antibody WM53 and quantified by converting mean fluorescence intensity (MFI) signal to molecules of CD33 per cell using QIFIKIT™ quantitative analysis kit.

[0202] FIG. 58 shows a panel of flow cytometry plots showing staining of Jurkat cells expressing hybridoma derived mouse anti-CD33 CARs using Protein L (FIG. 58A) or recombinant human CD33-PE labeled protein (FIG.58B).

[0203] FIG.59 shows activation of Jurkat NFAT Luc cells transfected with mouse anti-CD33-CARs in an mRNA titration assay.

[0204] FIG.60 is a table showing mouse anti-CD33 CAR activation EC50 values for FIG.59. EC50 values were obtained and converted to molecule numbers using QIFIKIT™ quantitative analysis kit.

[0205] FIG.61 shows phage -derived human anti-CD33 CAR generation workflow.

[0206] FIG. 62 shows ELISA binding data demonstrating recombinant huCD33 vs recombinant huSiglec-8 specificity of human anti-CD33 CARs to either biotinylated huCD33 / Streptavidin HRPAttorney Ref.061250-557001WO tetramers or biotin hSiglec 8 / Streptavidin HRP tetramers. The boxed scFv were selected as CD33 specific.

[0207] FIG. 63 shows a panel of flow cytometry plots showing staining of Jurkat cells expressing human anti-CD33 CARs using Protein L (FIG. 63A) or recombinant human CD33-PE labeled protein (FIG. 63B).

[0208] FIG.64 shows activation of Jurkat NFAT Luc cells transfected with human anti-CD33-CARs in an mRNA titration assay.

[0209] FIG.65 is a table showing anti-CD33 CAR activation EC50 values for FIG.64. EC50 values were obtained and converted to molecule numbers using QIFIKIT™ quantitative analysis kit.

[0210] FIG. 66 is a panel of flow cytometry histograms showing surface staining of CD4+ / CD8+ / CD56- human primary T cells stably transfected with anti-CD33-CARs. Stably transfected cells were selected using puromycin and stained with recombinant biotinylated human CD33 molecule combined with PE-conjugated streptavidin or AF647-conjugated goat anti-mouse polyclonal antibody.

[0211] FIG.67 is a panel of flow cytometry plots showing specific cytolytic activity with or without CD33 surface expression by CD4+ / CD8+ / CD56- human primary T cells from 3 normal donors stably transfected with CD33-CARs. Samples were normalized to CAR+ T cells and then co-cultured at varying effector CAR+ T cell:tumor cell (E:T) ratios. Specific killing was calculated by normalization to mock transfected T cells from the same donor.

[0212] FIG. 68 is a table showing ET50 and specific killing ratios for anti-CD33CARs across 3 human donors (d1-d3).

[0213] FIG. 69 shows a panel of flow cytometry histograms showing surface staining of CD4+ / CD8+ / CD56- human primary T cells from 3 donors (FIG. 69A d1; FIG. 69B d2; FIG. 69C d3) stably transfected with anti-CD33 / anti-CD16B constructs. Stably transfected cells were selected using puromycin and stained with recombinant biotinylated huCD33 molecule combined with PE- conjugated streptavidin and recombinant biotinylated huCD16b molecule combined with APC- conjugated streptavidin.

[0214] FIG.70 is a panel of flow cytometry plots showing specific cytolytic activity with or without CD33 surface expression by CD4+ / CD8+ / CD56- human primary T cells from 3 normal donors stably transfected with CD33-CARs. Samples were normalized to CAR+ T cells and then co-cultured at varying effector CAR+ T cell:tumor cell (E:T) ratios. Specific killing was calculated by normalization to mock transfected T cells from the same donor.

[0215] FIG. 71 is a table showing ET50 and specific killing ratios for anti-CD33CARs across 3 human donors (d1-d3).Attorney Ref.061250-557001WO

[0216] FIG.72 is a panel of flow cytometry plots showing specific cytolytic activity with or without CD33 or CD16B surface expression by CD4+ / CD8+ / CD56- human primary T cells from 3 normal donors stably transfected with anti-CD33 / anti-CD16B engineered receptor system and then co-cultured at varying effector CAR+ T cell:target MV4-11 cell (E:T) ratios. Specific killing was calculated by normalization to mock transfected T cells from the same donor. (FIG.72A d1; FIG. 72B d2; FIG.72C d3)

[0217] FIG. 73 is a table showing ET50 and specific killing ratios for anti-CD33CARs across 3 human donors (d1-d3) with MV4-11 target cells.

[0218] FIG.74 is a panel of flow cytometry plots showing specific cytolytic activity with or without CD33 or CD16B surface expression by CD4+ / CD8+ / CD56- human primary T cells from 3 normal donors stably transfected with anti-CD33 / anti-CD16B engineered receptor system and then co-cultured at varying effector CAR+ T cell: target HL-60 cell (E:T) ratios. Specific killing was calculated by normalization to mock transfected T cells from the same donor. (FIG.74A d1; FIG. 74B d2; FIG.74C d3)

[0219] FIG. 75 is a table showing ET50 and specific killing ratios for anti-CD33CARs across 3 human donors (d1-d3) with HL-60 target cells.

[0220] FIG.76 shows mouse anti-SPN CAR generation workflow.

[0221] FIG.77A is a panel of histograms showing SPN mRNA titration in K562 WT cells. Human SPN (huSPN) detected with anti-SPN antibody 1G10 were quantified by converting mean fluorescence intensity (MFI) signal to molecules of SPN per cell using QIFIKIT quantitative analysis kit. *below level of QIFIKit sensitivity.

[0222] FIG.77B is a panel of histograms showing SPN mRNA titration in K562 WT cells. Flag- tagged mouse SPN (Flag-mSPN) detected with anti-Flag antibody clone M2 were quantified by converting mean fluorescence intensity (MFI) signal to molecules of SPN per cell using QIFIKIT (quantitative analysis kit, Agilent). *below level of QIFIKit sensitivity.

[0223] FIG.78 is a panel of histograms showing SPN staining of WT and SPN knock-out (KO) cell lines (Jurkat NFAT Luciferase (JNL), MV4-11, K562, and THP-1). Cells were stained using anti- SPN conjugated antibody 1G10-PE followed by analysis by flow cytometry.

[0224] FIG. 79 is a set of bar charts depicting FACS binding of purified mouse anti-SPN IgGs to various WT and SPN KO cell lines. Binding complexes were detected using a goat anti-mouse IgG, Fc specific BV421 secondary antibody.

[0225] FIG.80 is a panel of flow cytometry plots showing staining of JNL SPN KO cells expressing anti-SPN scFvs CARs or pLenti0 control using Protein L (left panel) or soluble human SPN protein containing only the extracellular domain (right panel).Attorney Ref.061250-557001WO

[0226] FIG. 81 is a panel of plots showing activation of JNL SPN KO cells transfected with SPN- CARs in an mRNA titration assay. JNL SPN KO cells were transfected with SPN-CAR DNA and K562 WT target cells were transfected with serially diluted human SPN (huSPN) or Flag-tagged mouse SPN (Flag-mSPN) mRNA. JNL SPN KO SPN-CAR cells were co-cultured with transfected K562 WT target cells and then the functional response (RLU) was assessed using ONE-Step luciferase assay system.

[0227] FIG.82 is a table showing SPN-CAR activation EC50 values for FIG. 81 co-culture Jurkat assay in RNA ng or molecules per cell calculated using QIFIKIT. EC50 values were obtained and converted to molecule numbers using QIFIKIT quantitative analysis kit.

[0228] FIG. 83 is a panel of flow cytometry plots showing staining of SPN-CAR and / or CD16 blocker expression when transiently co-expressed in JNL SPN KO cells.

[0229] FIG.84 is a panel of plots showing titratable blocking of JNL SPN KO cell activation when CD16 Blockers are co-transfected with SPN-CAR activators. The activation of JNL SPN KO cells was assessed by measuring the functional response (RLU) using the ONE step luciferase assay system.

[0230] FIG. 85 is a panel of histograms showing SPN staining for a panel of AML cell lines. JNL cells were included as a non-AML control. SPN was detected with anti-SPN antibody 1G10 and quantified by converting mean fluorescence intensity (MFI) signal to molecules of SPN per cell using QIFIKIT quantitative analysis kit.

[0231] FIG. 86 is a panel of plots showing activation of JNL SPN KO cells transfected with SPN- CARs in a cell titration assay. The functional response (RLU) of JNL cells was assessed using ONE- Step luciferase assay system.

[0232] FIG. 87 is a panel of flow cytometry plots showing surface staining of CD4+ / CD8+ / CD56- human Primary T cells stably transfected with SPN-CARs using piggyBac™ system and SPN KO using CRISPR. CAR scFv was stained with Protein L and SPN was stained with conjugated anti- SPN antibody 1G10-PE.

[0233] FIG.88A is a panel of lots showing % specific killing of colorized MV4-11 target cells with or without SPN KO by CD4+ / CD8+ / CD56- human Primary T cells stably transfected with SPN- CARs using piggyBac™ system and SPN KO using CRISPR. Primary T cell were co-cultured at various Effector:Target cell ratios with target MV4-11 GFP cells + Fluc and + / - SPN KO.

[0234] FIG.88B is a table showing ET50 and specific killing ratios for SPN-CARs.

[0235] FIG.89 shows mouse anti-ITGA4 CAR generation workflow.

[0236] FIG. 90 shows binding data from ELISA and FACs analysis. Indirect ELISA using biotinylated ITGA4B1 extracellular domain (ECD) protein and direct ELISAs using plate bound ITGA4B1 and ITGA4B7 ECDs were performed using hybridoma derived mouse IgG supernatants.Attorney Ref.061250-557001WO Table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on ITGA4+, ITGA4 KO, Beta1 KO, or Beta1 KO overexpressing Beta7 protein in Jurkat host cells. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0237] FIG. 91A shows flow cytometry plots showing staining of Jurkat cells with an NFAT- luciferase reporter (JNL) ITGA4 KO cells expressing anti-ITGA4 CARs or pLenti control. Transfected JNL cells were stained with a polyclonal goat anti-mouse IgG antibody. ITGA4 CARs consist of anti-ITGA4 scFvs fused to CAR activation and costimulatory domains.

[0238] FIG. 91B shows flow cytometry plots showing staining of Jurkat cells with an NFAT- luciferase reporter (JNL) ITGA4 KO cells expressing anti-ITGA4 CARs or pLenti control. Transfected JNL cells were stained with tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1). ITGA4 CARs consist of anti-ITGA4 scFvs fused to CAR activation and costimulatory domains.

[0239] FIG. 92A is a panel of histograms showing human ITGA4 (huITGA4) mRNA titration in K562 WT cells. huITGA4 was detected with anti-ITGA4 antibody (clone L25) and was quantified by converting median fluorescence intensity (MFI) to molecules of ITGA4 per cell using QIFIKIT (quantitative analysis kit, Agilent).

[0240] FIG. 92B is a panel of histograms showing murine ITGA4 (muITGA4) mRNA titration in K562 WT cells. Relative muITGA4 expression was detected with anti-muITGA4 antibody (clone R1-2) conjugated to Alexa Fluor 647 (AF647) and quantified using MFI.

[0241] FIG. 93 is a panel of graphs showing functional activation of JNL ITGA4 KO cells transfected with ITGA4 CARs co-cultured with K562 target cells expressing ITGA4. JNL ITGA4 KO cells were transfected with ITGA4 CAR DNA and K562 target cells were transfected with serially diluted human ITGA4 mRNA.18-24 hours post-transfection, JNL ITGA4 KO ITGA4 CAR cells were co-cultured with 10,000 transfected K562 target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0242] FIG.94 is a panel of flow plots showing staining of Jurkat E6 cell lines engineered to express different combinations of ITGA4 and its heterodimer binding partners, ITGB1 and ITGB7. KO cell lines were produced using CRISPR and a guide targeting the indicated gene followed by bulk sorting to enrich KO populations. ITGB7 was introduced using piggybac transposase and a transposon containing the ITGB7 gene, followed by bulk sorting to enrich the ITGB7+ population. Cells were stained using anti-ITGA4 (L25), anti ITGB1 (TS2 / 16), or anti-ITGB7 (473207) unconjugated antibody followed by Alexa Fluor 647-conjugated secondary antibody.Attorney Ref.061250-557001WO

[0243] FIG. 95 is a panel of flow plots showing staining of Jurkat cells with an NFAT-luciferase reporter (JNL). ITGA4 KO cells were engineered to express anti-ITGA4 CARs or pLenti control. Expression of each construct was measured using tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) antibody or anti-G4S linker antibody. ITGA4 CARs consist of anti-ITGA4 scFvs fused to CAR activation and costimulatory domains.

[0244] FIG. 96 is a panel of graphs showing functional activation of JNL ITGA4 KO cells transfected with ITGA4 CARs co-cultured with K562 target cells expressing ITGA4. JNL ITGA4 KO cells were transfected with ITGA4 CAR DNA and K562 target cells were transfected with serially diluted human ITGA4 (huITGA4) mRNA or mouse ITGA4 (muITGA4) mRNA . 18-24 hours post-transfection, 10,000 JNL ITGA4 KO ITGA4 CAR cells were co-cultured with 10,000 transfected K562 target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0245] FIG.97 is a table showing ITGA4 CAR activation EC50 values for co-culture assay shown in FIG. 96 in ngs of mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent)

[0246] FIG. 98 is a panel of graphs showing functional activation of JNL WT or JNL ITGA4 KO cells transfected with ITGA4 CARs co-cultured with K562 target cells expressing ITGA4. JNL WT or JNL ITGA4 KO cells were transfected with ITGA4 CAR DNA and K562 target cells were transfected with serially diluted human ITGA4 mRNA. 18-24 hours post-transfection, 10,000 JNL WT or JNL ITGA4 KO ITGA4 CAR cells were co-cultured with 10,000 transfected K562 target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0247] FIG. 99 is a panel of graphs showing functional activation of JNL ITGA4 KO cells transfected with ITGA4 CARs co-cultured with Jurkat E6 target cells engineered with different combinations of ITGA4, ITGB1, and ITGB7 (see FIG. 94). JNL ITGA4 KO cells were transfected with ITGA4 CAR DNA and Jurkat E6 target cells were engineered as described in Fig. 94. 18-24 hours post-JNL transfection, 10,000 JNL ITGA4 KO ITGA4 CAR cells were co-cultured with the indicated number of each target cell type. After 6 hours of co-culture, the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0248] FIG. 100A is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) was used to detect ITGA4 CAR expression.

[0249] FIG. 100B is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs with or without an anti-CD16b blocking receptor (23I12) (co- expressed) or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1)Attorney Ref.061250-557001WO was used to detect ITGA4 CAR expression and tetramerized CD16b was used to detect CD16b blocking receptor expression.

[0250] FIG. 100C is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs with or without an anti-CD16b blocker (3G15) (co-expressed) or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) was used to detect ITGA4 CAR expression and tetramerized CD16b was used to detect CD16b blocker expression.

[0251] FIG.101 is a panel of graphs showing titratable blocking of JNL ITGA4 KO cell activation when CD16b blocking receptors are co-transfected with ITGA4 CARs. K562 target cells were transfected with serially diluted CD16b mRNA and constant ITGA4 mRNA, and JNL ITGA4 KO cells were transfected with ITGA4 CAR and CD16b blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL ITGA4 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0252] FIG. 102A is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) was used to detect ITGA4 CAR expression.

[0253] FIG. 102B is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs with or without an anti-CLEC9A blocking receptor (3D21) (co- expressed) or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) was used to detect ITGA4 CAR expression and CLEC9A monomer was used to detect CLEC9A blocking receptor expression.

[0254] FIG. 102C is a flow cytometry plot showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs with or without an anti-CLEC9A blocking receptor (6B7) (co- expressed) or pLenti control. Tetramerized integrin alpha 4 integrin beta 1 heterodimer (ITGA4B1) was used to detect ITGA4 CAR expression and CLEC9A monomer was used to detect CLEC9A blocking receptor expression.

[0255] FIG.103 is a panel of graphs showing titratable blocking of JNL ITGA4 KO cell activation when CLEC9A blocking receptors are co-transfected with ITGA4 CARs. K562 target cells were transfected with serially diluted CLEC9A mRNA and constant ITGA4 mRNA, and JNL ITGA4 KO cells were transfected with ITGA4 CAR and CLEC9A blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL ITGA4 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.Attorney Ref.061250-557001WO

[0256] FIG. 104 is a panel of flow cytometry plots showing surface staining of CD4 / 8+ CD56- human primary T cells stable transfected with ITGA4 CARs or ITGA4 CARs with CD16b blocking receptor (ITGA4 / CD16b dual receptor system) using the piggybac transposase system and ITGA4 KO using CRISPR. Staining shown here is 23 days after initial T cell thaw and stimulation (21 days post-transfection). CAR and blocker scFvs were stained using polyclonal goat anti-mouse IgG antibody.

[0257] FIG. 105 is a panel of flow cytometry plots showing surface staining of CD4 / 8+ CD56- human primary T cells stable transfected with ITGA4 CARs or ITGA4 CARs with CD16b blocking receptors (ITGA4 / CD16b dual receptor system) using the piggybac transposase system and ITGA4 KO using CRISPR. Staining shown here is 14 days after initial T cell thaw and stimulation (12 days post-transfection / CRISPR KO). ITGA4 KO was confirmed using PE-conjugated anti-ITGA4 antibody (9F10).

[0258] FIG. 106 is a panel of graphs showing the percentage of specific killing of MV4-11 target cells with or without ITGA4 and with or without CD16b blocking receptors by CD4 / 8+ CD56- human primary T cells stably transfected with ITGA4 CARs or ITGA4 / CD16b and ITGA4 KO (as described above). Target cells were engineered to express firefly luciferase using lentiviral transduction followed by FACS enrichment. Primary T effector cells were co-cultured with MV4-11 target cells at indicated effector: target (E:T) cell ratios 23 days after initial T cell thaw / stimulation. Target cell death was assessed after 48-hour co-culture using the ONE-Step luciferase system. The percentage of specific killing of MV4-11 cells was calculated by normalizing CAR alone or ITGA4 / CD16b to UTD ITGA4 KO T cells.

[0259] FIG. 107 is a table showing E:T ratio at 50% specific killing of MV4-11 target cells with indicated ITGA4 / CD16b dual receptor system expression following the co-culture assay described in FIG.106. Specific killing ratios indicate the ET50 of CD16b+ target cells divided by the ET50 of ITGA4+ target cells. Selectivity windows consist of the ET50 of ITGA4+ CD16b+ target cells divided by the ET50 of ITGA4+ target cells, indicating blocking of primary T cell cytotoxicity against ITGA4+ cells when CD16b is also present on the same target cells.

[0260] FIG.108 shows mouse anti-CD162 CAR generation workflow.

[0261] FIG. 109 shows binding data from ELISA and FACs analysis. Indirect ELISAs using biotinylated CD162 extracellular domain (ECD) protein were performed using hybridoma derived mouse IgG supernatants. Table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on WT Jurkat, WT MV411, CD162 KO Jurkat, and CD162 KO MV411 cells. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.Attorney Ref.061250-557001WO

[0262] FIG.110A is a panel of histograms showing human CD162 (huCD162) ) mRNA titration in HeLa WT cells. huCD162 detected with anti-CD162 antibody (clone KPL-1) was quantified by converting median fluorescence intensity (MFI) to molecules of CD162 per cell using QIFIKIT (quantitative analysis kit, Agilent).

[0263] FIG.110B is a panel of histograms showing murine CD162 (muCD162) mRNA titration in HeLa WT cells. muCD162 detected with anti-FLAG (clone M5) was quantified by converting median fluorescence intensity (MFI) to molecules of CD162 per cell using QIFIKIT (quantitative analysis kit, Agilent).

[0264] FIG.111A is a series of flow cytometry plots showing staining of Jurkat cells with an NFAT- luciferase reporter (JNL) CD162 KO cells expressing anti-CD162 CARs or pLenti control using anti- G4S linker antibody (Cell Signaling Technology). CD162 CARs consist of anti-CD162 scFvs fused to CAR activation and costimulatory domains.

[0265] FIG.111B is a series of flow cytometry plots showing staining of Jurkat cells with an NFAT- luciferase reporter (JNL) CD162 KO cells expressing anti-CD162 CARs or pLenti control using recombinant human CD162 (rhCD162 R&D Technology). CD162 CARs consist of anti-CD162 scFvs fused to CAR activation and costimulatory domains.

[0266] FIG.112 is a series of plots showing functional activation of JNL CD162 KO cells transfected with CD162 CARs co-cultured with HeLa target cells expressing CD162. JNL CD162 KO cells were transfected with CD162 CAR DNA and HeLa target cells were transfected with serially diluted human CD162 (huCD162) mRNA or mouse CD162 (muCD162) mRNA . 18-24 hours post- transfection, 10,000 JNL CD162 KO CD162 CAR cells were co-cultured with 10,000 transfected HeLa target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0267] FIG.113 is a table showing CD162 CAR activation EC50 values for co-culture assay shown in FIG. 112 in ngs of mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0268] FIG. 114 is a series of plots showing functional activation of JNL WT or JNL CD162 KO cells transfected with CD162 CARs co-cultured with HeLa target cells expressing CD162. JNL WT or JNL CD162 KO cells were transfected with CD162 CAR DNA and HeLa target cells were transfected with serially diluted human CD162 mRNA. 18-24 hours post-transfection, 10,000 JNL WT or JNL CD162 KO CD162 CAR cells were co-cultured with 10,000 transfected HeLa target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.Attorney Ref.061250-557001WO

[0269] FIG. 115A is a series of flow cytometry plots showing staining of JNL CD162 KO cells transiently expressing anti-CD162 CARs or pLenti control. Recombinant human CD162 (rhCD162 R&D Technology) was used to detect CD162 CAR expression.

[0270] FIG. 115B is a series of flow cytometry plots showing staining of JNL CD162 KO cells transiently expressing anti-CD162 CARs with anti-CD16b blocking receptors or pLenti control. Recombinant human CD162 (rhCD162 R&D Technology) was used to detect CD162 CAR expression and tetramerized CD16b was used to detect CD16b blocking receptor expression.

[0271] FIG. 116 is a series of plots showing titratable blocking of JNL CD162 KO cell activation when CD16b blocking receptors are co-transfected with CD162 CARs. K562 target cells were transfected with serially diluted CD16b mRNA, and JNL CD162 KO cells were transfected with CD162 CAR and CD16b blocking receptor DNA at a 2:1 ratio.18-24 hours post-transfection, 10,000 JNL CD162 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours of co- culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0272] FIG.117 shows mouse anti-CD53 CAR generation workflow.

[0273] FIG. 118 shows binding data from FACs analysis. FACS binding assays using hybridoma derived mouse IgG supernatants were performed on K562 CD50 KO, K562 WT, Jurkat WT and transiently transfected CD53 + HeLa cells. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table. “ND” means no data was collected for this cell type.

[0274] FIG.119A is a panel of histograms showing human CD53 (huCD53) mRNA titration in HeLa cells. huCD53 detected with anti-huCD53 antibody (clone 63-5A3) was quantified by converting median fluorescence intensity (MFI) to molecules of CD53 per cell using QIFIKIT (quantitative analysis kit, Agilent).

[0275] FIG. 119B is a panel of histograms showing murine CD53 (muCD53) mRNA titration in HeLa cells. muCD53 detected with anti-muCD53 antibody (clone OX-70) was quantified by converting median fluorescence intensity (MFI) to molecules of CD53 per cell using QIFIKIT (quantitative analysis kit, Agilent).

[0276] FIG. 120 is a series of flow cytometry plots showing staining of JNL CD53 KO cells expressing anti-CD53 CARs or pLenti control using anti-G4S linker antibody (Cell Signaling Technology). CD53 CARs consist of anti-CD53 scFvs fused to CAR activation and costimulatory domains.

[0277] FIG. 121 is a series of graphs showing functional activation of JNL CD53 KO cells transfected with CD53 CARs co-cultured with HeLa target cells expressing CD53. JNL CD53 KO cells were transfected with CD53 CAR DNA. HeLa target cells were transfected with serially dilutedAttorney Ref.061250-557001WO human CD53 (huCD53) mRNA or mouse CD53 (muCD53) mRNA . 18-24 hours post-transfection, 10,000 JNL CD53 KO CD53 CAR cells were co-cultured with 10,000 transfected HeLa target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0278] FIG. 122 is a table showing CD53 CAR activation EC50 values for the co-culture assay shown in FIG. 121 in ngs of mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0279] FIG. 123 is a series of graphs showing functional activation of JNL WT or JNL CD53 KO cells transfected with CD53 CARs co-cultured with HeLa target cells expressing CD53. JNL WT or JNL CD53 KO cells were transfected with CD53 CAR DNA and HeLa target cells were transfected with serially diluted human CD53 mRNA. 18-24 hours post-transfection, 10,000 JNL WT or JNL CD53 KO CD53 CAR cells were co-cultured with 10,000 transfected HeLa target cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0280] FIG. 124A is a series of flow cytometry plots showing staining of JNL CD53 KO cells transiently expressing anti-CD53 alone or pLenti control using G4S-linker.

[0281] FIG. 124B is a series of flow cytometry plots showing staining of JNL CD53 KO cells transiently expressing anti-CD53 with CD16b blocking receptor (3G15) or pLenti control using G4S- linker.

[0282] FIG. 125 is a series of graphs showing titratable blocking of JNL CD53 KO cell activation when CD16b blocking receptors are co-transfected with CD53 CARs. K562 target cells were transfected with serially diluted CD16b mRNA and constant CD53 mRNA. JNL CD53 KO cells were transfected with CD53 CAR and CD16b blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL CD53 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours of co-culture the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0283] FIG.126 shows mouse anti-CLEC9A blocking receptor generation workflow.

[0284] FIG.127 shows phage derived human anti-CLEC9A blocking receptor generation workflow.

[0285] FIG.128 shows binding data from ELISA and FACs analysis. Indirect ELISA was performed using hybridoma derived mouse IgG and phage derived huscFv supernatants with soluble recombinant biotinylated huCLEC9A-Fc, huCLEC12A, huCLEC7A, and huCD161-Fc extracellular domain (ECD) proteins. Table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on either huCLEC9A+ or huCLEC9A- K562 (Host) cells. Values represent median fluorescence intensity (MFI) after detection withAttorney Ref.061250-557001WO appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table. "ND" denotes data not collected for this binder.

[0286] FIG.129A is a panel of histograms showing CLEC9A mRNA titration in K562 cells. CLEC9A expression was detected by anti-huCLEC9A antibody.

[0287] FIG.129B are calibration curves from the QIFIKIT® to interpolate CLEC9A molecules / cell in FIG. 129A.

[0288] FIG. 130 is a series of flow cytometry plots showing staining of WT JNLs expressing hybridoma-derived anti-CLEC9A CARs or pLenti0 control using Fc-tagged soluble human CLEC9a protein (left) or anti-mouse IgG antibody (right).

[0289] FIG.131 is a series of graphs showing activation curves of wildtype Jurkat NFAT luciferase cells (JNLs) transfected with hybridoma-derived CLEC9A-CARs in a CLEC9A mRNA titration assay. CLEC9A-CAR DNA plasmids were produced by fusing anti-CLEC9A scFvs to CAR activation and costimulatory domains. WT JNL cells were transfected with CLEC9A-CAR DNA. K562 WT target cells were transfected with serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0290] FIG.132 is a series of flow cytometry plots showing staining of WT JNLs expressing phage- derived anti-CLEC9A CARs or pLenti0 control using Fc-tagged soluble human CLEC9A protein.

[0291] FIG.133 is a series of graphs showing activation curves of wildtype Jurkat NFAT luciferase cells (JNLs) transfected with phage-derived CLEC9A-CARs in an CLEC9A mRNA titration assay. CLEC9A-CAR DNA plasmids were produced by fusing anti-CLEC9A scFvs to CAR activation and costimulatory domains. WT JNL cells were transfected with CLEC9A-CAR DNA and K562 WT target cells were transfected with serially diluted human CLEC9A mRNA. 18-24 hours post- transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0292] FIG. 134 is a table showing CLEC9A-CAR activation EC50 values for co-culture assay shown in FIG. 131 and FIG. 133 in molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0293] FIG. 135 is a series of flow cytometry plots of ITGA4 activator expression (hu21.6) on the x-axis and the indicated CLEC9A blocking receptor on the y-axis. Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0294] FIG.136 is a series of graphs showing blocking curves of ITGA4 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with ITGA4(hu21.6)-CAR and CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLsAttorney Ref.061250-557001WO were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0295] FIG. 137 is a series of flow cytometry plots of ITGA4 activator expression (HP1-2) on the x-axis and the indicated CLEC9A blocking receptor on the y-axis. Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0296] FIG.138 is a series of graphs showing blocking curves of ITGA4 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with ITGA4(HP1-2)-CAR and CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0297] FIG. 139 is a series of flow cytometry plots showing expression of the hu21.6 ITGA4 activator (x-axis) and the indicated CLEC9A blocking receptor (y-axis). Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0298] FIG.140 is a series of graphs showing blocking curves of ITGA4 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with ITGA4 CARs with phage-derived CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA.18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0299] FIG. 141 is a series of flow cytometry plots of CD162 activator expression (h15A7) on the x-axis and the indicated CLEC9A blocking receptor on the y-axis. Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0300] FIG.142 is a series of graphs showing blocking curves of CD162 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with CD162(h15A7)-CAR and CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0301] FIG.143 is a series of flow cytometry plots of CD162 activator expression (m43B6) on the x-axis and the indicated CLEC9A blocking receptor on the y-axis. Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0302] FIG.144 is a series of graphs showing blocking curves of CD162 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with CD162(m43B6)-CAR and CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activatorAttorney Ref.061250-557001WO antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0303] FIG. 145 is a series of flow cytometry plots of CD53 activator expression (4-J16) on the x- axis and the indicated CLEC9A blocker on the y-axis. Expression was measured by biotinylated anti- activator antigen and Fc-tagged soluble CLEC9A protein.

[0304] FIG.146 is a series of graphs showing blocking curves of CD53 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with CD53(4-J16)-CAR and CLEC9A blocking receptor in a CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0305] FIG.147 is a series of flow cytometry plots of CD53 activator expression (7-A12) on the x- axis and the indicated CLEC9A blocking receptor on the y-axis. Expression was measured by biotinylated anti-activator antigen and Fc-tagged soluble CLEC9A protein.

[0306] FIG.148 is a series of graphs showing blocking curves of CD53 KO Jurkat NFAT luciferase cells (JNLs) co-transfected with CD53(7-A12)-CAR and CLEC9A blocking receptor in an CLEC9A mRNA titration assay. K562 WT target cells were transfected with a constant amount of activator antigen and serially diluted human CLEC9A mRNA. 18-24 hours post-transfection, 10,000 JNLs were cocultured with 10,000 transfected K562 target cells. After 6 hours, activation was quantified as NFAT activation in JNLs as measured by luminescence.

[0307] FIG.149 shows mouse anti-Thy1 blocking receptor generation workflow.

[0308] FIG. 150 shows binding data from ELISA and FACS analysis. Indirect ELISA using biotinylated Thy1 extracellular domain (ECD) protein was performed using hybridoma derived mouse IgG supernatants. ELISA table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on WT Jurkat and Thy1 KO Jurkat cells. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0309] FIG 151 shows human derived anti-THY1 blocking receptor generation workflow.

[0310] FIG 152 shows binding data from ELISA and FACS analysis. Indirect ELISA using biotinylated Thy1 extracellular domain (ECD) protein were performed using phage derived hu scFv supernatants. ELISA table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on WT Jurkat and Thy1 KO Jurkat cells. Values represent median fluorescence intensity (MFI) after detection with appropriateAttorney Ref.061250-557001WO secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0311] FIG 153 is a series of graphs showing functional activation of JNL Thy1 KO cells transfected with Thy1 CARs co-cultured with K562 target cells expressing Thy1. JNL Thy1 KO cells were transfected with Thy1 CAR DNA and K562 target cells were transfected with serially diluted human Thy1 (huThy1) mRNA or mouse Thy1 (muThy1) mRNA . 18-24 hours post-transfection, 10,000 JNL Thy1 KO Thy1 CAR cells were co-cultured with 10,000 transfected K562 target cells for 6 hours, after which the JNL functional response (luminescence, RLU) was assessed using the ONE- Step luciferase assay system.

[0312] FIG. 154 is a table showing Thy1 CAR activation EC50 values for co-culture assay shown in FIG. 153 in molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0313] FIG.155 is a series of graphs showing functional activation of JNL Thy1 KO cells transfected with Thy1 CARs co-cultured with primary Hematopoietic stem cells (HSCs). JNL Thy1 KO cells were transfected with Thy1 CAR DNA and 18-24 hours post-transfection, HSCs were serially diluted and co-cultured with JNL Thy1 KO Thy1 CAR cells for 6 hours, after which the JNL functional response (luminescence, RLU) was assessed using the ONE-Step luciferase assay system.

[0314] FIG.156A is a series of graphs showing titratable blocking of JNL Thy1 KO cell activation when Thy1 blocking receptors are co-transfected with CD33 (2H20) CAR. K562 target cells were transfected with CD33 mRNA and serially diluted Thy1 mRNA, and JNL Thy1 KO cells were transfected with CD33 (2H20) CAR and Thy1 blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL Thy1 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours, the activation of JNL Thy1 KO cells was assessed by measuring the functional response (Luminescence, RLU) using the ONE step luciferase assay system.

[0315] FIG.156B is a series of graphs showing titratable blocking of JNL Thy1 KO cell activation when Thy1 blocking receptors are co-transfected with CD33 (1C20) CAR. . K562 target cells were transfected with CD33 mRNA and serially diluted Thy1 mRNA, and JNL Thy1 KO cells were transfected with CD33 (1C20) CAR and Thy1 blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL Thy1 KO cells were co-cultured with 10,000 transfected K562 cells. After 6 hours, the activation of JNL Thy1 KO cells was assessed by measuring the functional response (Luminescence, RLU) using the ONE step luciferase assay system.

[0316] FIG.157A is a table showing Thy1 CAR blocking receptor IC50 values, span and blocking percentage for co-culture assay shown in FIG.156A. Thy1 blocking receptor were co-expressed with CD33 (2H20) CAR.Attorney Ref.061250-557001WO

[0317] FIG.157B is a table showing Thy1 CAR blocking receptor IC50 values, Span and Blocking percentage for co-culture assay shown in FIG.156B. Thy1 blocking receptor were co-expressed with CD33 (1C20) CAR.

[0318] FIG. 158A is a series of graphs showing titratable blocking of JNL ITGA4 / Thy1 KO cell activation when Thy1 blocking receptors are co-transfected with ITGA4 (2F5) CAR. K562 target cells were transfected with ITGA4 mRNA and serially diluted Thy1 mRNA, and JNL ITGA4 / Thy1 KO cells were transfected with ITGA4(2F5) CAR and Thy1 blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL ITGA4 / Thy1 KO cells were co-cultured with 10,000 transfected K562 cells. After six hours, the activation of JNL ITGA4 / Thy1 KO cells was assessed by measuring the functional response (Luminescence, RLU) using the ONE step luciferase assay system.

[0319] FIG. 158B is a series of graphs showing titratable blocking of JNL ITGA4 / Thy1 KO cell activation when Thy1 blocking receptors are co-transfected with ITGA4 (17J1) CAR. K562 target cells were transfected with ITGA4 mRNA and serially diluted Thy1 mRNA, and JNL ITGA4 / Thy1 KO cells were transfected with ITGA4(17J1) CAR and Thy1 blocking receptor DNA at a 2:1 ratio. 18-24 hours post-transfection, 10,000 JNL ITGA4 / Thy1 KO cells were co-cultured with 10,000 transfected K562 cells. After six hours, the activation of JNL ITGA4 / Thy1 KO cells was assessed by measuring the functional response (Luminescence, RLU) using the ONE step luciferase assay system.

[0320] FIG. 158C is a table showing Thy1 blocking receptor IC50 values, span and blocking percentage for co-culture assay shown in FIG.158A. Thy1 Cars were co-expressed with ITGA4(2F5) CAR.

[0321] FIG.158D is a table showing Thy1 CAR blocking receptor IC50 values, Span and Blocking percentage for co-culture assay shown in FIG. 158B. Thy1 Cars were co-expressed with ITGA4 (17J1) CAR.

[0322] FIG. 159A is a series of Flow cytometry plots showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 CARs with or without anti-THY1 blockers (co-expressed) or pLenti control using tetramerized ITGA4B1 and tetramerized THY1.

[0323] FIG.159B is a series of graphs showing titratable blocking of JNL ITGA4 KO cell activation when THY1 Blockers are co-transfected with ITGA4 CARs.

[0324] FIG.160 shows mouse anti-CEACAM3 blocking receptor generation workflow.

[0325] FIG. 161 shows binding data from ELISA and FACS analysis. Indirect ELISA were performed using hybridoma derived mouse IgG supernatants with soluble recombinant biotinylated CEACAM1, CEACAM3, CEACAM4, CEACAM5, and CEACAM21 extracellular domain (ECD)Attorney Ref.061250-557001WO proteins. Table values represent the optical density (OD) readings after addition of colorimetric substrate.

[0326] FIG. 162 is a series of histograms showing CEACAM3 mRNA titration in K562 WT cells. Human CEACAM3 detected with anti-human CD66d / e (Clone 308 / 3-3)was quantified by converting median fluorescence intensity (MFI) signal to molecules of CEACAM3 per cell using QIFIKIT (quantitative analysis kit, Agilent). *below level of QIFIKit sensitivity.

[0327] FIG.163 is a series of flow cytometry plots showing surface staining of JNL cell transfected to transiently express CEACAM3 CARs.

[0328] FIG. 164 is a table showing CEACAM3-CAR activation EC50 values for co-culture assay shown in FIG. 165 in ng mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0329] FIG. 165 is a series of graphs showing functional activation of JNL cells transfected with CEACAM3-CARs in the mRNA titration assay. CEACAM3-CAR DNA was produced by fusing anti-CEACAM3 scFvs to CAR activation and costimulatory domains. JNL cells were transfected with CARs and K562 WT target cells were transfected with serially diluted human CEACAM3 mRNA. 18-24 hours post-transfection, 10,000 JNL-CAR cells were co-cultured with 10,000 transfected K562 WT target cells for 6 hours and then the functional response (RLU) was assessed using ONE-Step luciferase assay system.

[0330] FIG. 166 is a series of graphs showing functional activation of JNL cells transfected with CEACAM3-CARs in the mRNA titration assay. CEACAM3-CAR DNA was produced by fusing anti-CEACAM3 scFvs to CAR activation and costimulatory domains. JNL cells were transfected with CARs and K562 WT target cells were transfected with serially diluted human CEACAM5 mRNA. 18-24 hours post-transfection, 10,000 JNL-CAR cells were co-cultured with 10,000 transfected K562 WT target cells for 6 hours and then the functional response (RLU) was assessed using ONE-Step luciferase assay system.

[0331] FIG. 167 is a series of graphs showing functional activation of JNL cells transfected with CEACAM3-CARs in the mRNA titration assay. CEACAM3-CAR DNA was produced by fusing anti-CEACAM3 scFvs to CAR activation and costimulatory domains. JNL cells were transfected with CARs and K562 WT target cells were transfected with serially diluted human CEACAM1 mRNA. 18-24 hours post-transfection, 10,000 JNL-CAR cells were co-cultured with 10,000 transfected K562 WT target cells for 6 hours and then the functional response (RLU) was assessed using ONE-Step luciferase assay system.

[0332] FIG. 168 is a series of graphs showing functional activation of JNL cells transfected with CEACAM3-CARs in the mRNA titration assay. JNL cells were transfected with CARs and K562 WT target cells were transfected with serially diluted human CEACAM4 and CEACAM21 mRNA.Attorney Ref.061250-557001WO 18-24 hours post-transfection, 10,000 JNL-CAR cells were co-cultured with 10,000 transfected K562 WT target cells for 6 hours and then the functional response (RLU) was assessed using ONE-Step luciferase assay system.

[0333] FIG.169 is a series of flow cytometry plots showing surface staining of JNL cells transfected to transiently express CD33-CAR and CEACAM3 blocking receptor.

[0334] FIG. 170 is a series of graphs showing titratable blocking of JNL cell activation when CEACAM3 Blockers are co-transfected with CD33-CAR activator. K562 target cells were transfected with serially diluted CEACAM3 mRNA and JNL cells were transfected with CD33 CAR and Blocker DNA at a 1:2 ratio. 18-24 hours post-transfection, 10,000 JNL cells were co-cultured with 10,000 transfected K562 cells for 6 hours. The activation of JNL cells was assessed by measuring the functional response (RLU) using the ONE step luciferase assay system.

[0335] FIG.171 is a series of flow cytometry plots showing surface staining of JNL cells transfected to transiently express SPN-CAR (1G10 or DFT1) and CEACAM3 blocking receptor.

[0336] FIG. 172 is a series of graphs showing titratable blocking of JNL SPN KO cell activation when CEACAM3 Blockers are co-transfected with SPN-CAR activator. K562 target cells were transfected with serially diluted CEACAM3 mRNA and JNL cells were transfected with SPN CARs and Blocker DNA at a 1:2 ratio. 18-24 hours post-transfection, 10,000 JNL cells were co-cultured with 10,000 transfected K562 cells for 6 hours. The activation of JNL cells was assessed by measuring the functional response (RLU) using the ONE step luciferase assay system.

[0337] FIG.173 is a series of flow cytometry plots showing surface staining of JNL cells transfected to transiently express ITGA4-CAR (Natalizumab or HP1-2) and CEACAM3 blocking receptor expression.

[0338] FIG. 174 is a series of graphs showing titratable blocking of JNL cell activation when CEACAM3 Blockers are co-transfected with ITGA4-CAR activator. K562 target cells were transfected with serially diluted CEACAM3 mRNA and 100 ng ITGA4 mRNA and JNL cells were transfected with ITGA4 CAR and Blocker DNA at a 1:2 ratio.18-24 hours post-transfection, 10,000 JNL cells were co-cultured with 10,000 transfected K562 cells for 6 hours. The activation of JNL cells was assessed by measuring the functional response (RLU) using the ONE step luciferase assay system.

[0339] FIG.175 shows mouse anti-CLL1 blocking receptor generation workflow.

[0340] FIG. 176 shows binding data from ELISA and FACS analysis. Indirect ELISA using biotinylated CLL1 or Clec7a extracellular domain (ECD) protein were performed using hybridoma derived mouse IgG supernatants. Table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on K562 WT, K562 overexpressing Cec9a, and K562 overexpressing CLL1. Values represent median fluorescenceAttorney Ref.061250-557001WO intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0341] FIG. 177 shows binding data from ELISA and FACS analysis. Indirect ELISA using biotinylated CLL1 or Clec7a extracellular domain (ECD) protein were performed using hybridoma derived mouse IgG supernatants. Table values represent the optical density (OD) readings after addition of colorimetric substrate. FACS binding assays were performed on K562 WT, K562 overexpressing Cec9a, and K562 overexpressing CLL1. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0342] FIG.178 is a series of plots showing functional activation of JNL WT cells transfected with CLL1 CARs co-cultured with K562 target cells expressing CLL1.

[0343] FIG. 179 is a table showing CLL1 activator receptor activation EC50 values for co-culture assay shown in FIG.178 in ng mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent).

[0344] FIG. 180 is a plot showing the percentage of specific killing of HL-60 target cells with or without CLL1 by CD4 / 8+ CD56- human primary T cells stably transduced with CLL1 CARs.

[0345] FIG.181 is a table showing effector: target ratio at 50% specific killing of HL-60 target cells with indicated CLL1 expression and the specific killing ratio following the co-culture assay described in FIG. 180.

[0346] FIG. 182 is a series of flow cytometry plots showing surface staining of CLL1 activator receptor and CEACAM3 blocker receptor expression when transiently expressed in JNL.

[0347] FIG.183 is a series plots showing titratable blocking of JNL cell activation when CEACAM3 blocking receptors are co-transfected with CLL1-CAR activator receptors.

[0348] FIG.184 is a series of plots showing the percentage of specific killing of HL-60 target cells engineered to express CLL1, CLL1 and CD16b or CD16b alone. HL-60 target cells were co cultured with CD4 / 8+ CD56- human primary T cells engineered to express CLL1 activator receptors (CAR6 or 1075.7)in combination with CD16b blocker receptors.

[0349] FIG.185 is a table showing effector: target ratio at 50% specific killing of HL-60 target cells with indicated CLL1 expression and the specific killing ratio following the co-culture assay described in FIG. 184.

[0350] FIG. 186 is the workflow for humanizing mouse anti-CD16b single chain Fv (scFv) using grafting and structure-guided rational design.

[0351] FIG. 187 is a series of flow cytometry plots showing staining of JNL WT cells expressing anti-CD16 scFv-CARs or pLenti0 control using Goat-anti-mouse Fab2-647 (Jackson Immuno) or soluble human CD16b protein containing only the extracellular domain (Acrobio).Attorney Ref.061250-557001WO

[0352] FIG.188 is a series of plots showing activation of JNL WT transfected with CD16b activator receptors in an mRNA titration assay.

[0353] FIG. 189 is a series of flow cytometry plots showing staining of anti-CD332H20 activator receptor (sCD33-PE) or CD16b-blocker receptor (anti-blocker hinge-APC) expression when transiently co-expressed in JNL WT cells.

[0354] FIG. 190A is a of plot showing titratable blocking of JNL WT cell activation when CD16 blocker receptor 3G15 (SEQ ID NO: 1146), 3G15_Graft_v1-4 (SEQ ID NO: 6073), or 3G15_Graft_v1-2 (SEQ ID NO: 6071) is co-transfected with anti-CD332H20-CAR (SEQ ID NO: 2562) activator.

[0355] FIG. 190B is a of plot showing titratable blocking of JNL WT cell activation when CD16 blocker receptor 23I12 (SEQ ID NO: 1147), 23I12_Graft_v1-2 (SEQ ID NO: 6076), 23I12_Graft_v2-1 (SEQ ID NO: xx), or 23I12_Graft_v2-2 (SEQ ID NO: 6080), is co-transfected with anti-CD332H20-CAR (SEQ ID NO: 2562) activator.

[0356] FIG.190C is a table showing the IC50, the span and the percentage blocking of the coculture experiments described in FIG.190A and FIG.190B.

[0357] FIG. 191A is a flow cytometry plot showing surface staining of JNL CD53 knockout cells transiently expressing anti-CD53 activator receptor, using anti-G4S linker antibody.

[0358] FIG. 191B is a flow cytometry plot showing surface staining of JNL CD53 knockout cells transiently expressing anti-CD53 activator receptors with CD16b 3G15 blocking receptor (SEQ ID NO: 1146), using anti-G4S linker antibody.

[0359] FIG. 191C is a flow cytometry plot showing surface staining of JNL CD53 knockout cells transiently expressing anti-CD53 activator receptors with CD16b(h3G15v1-4) blocking receptor (SEQ ID NO: 6073), using anti-G4S linker antibody.

[0360] FIG. 192 is a series of plots showing titratable blocking of JNL CD53 KO cell activation when humanized CD16b blocker receptors are co-transfected with CD53 CARs.

[0361] FIG.193 is a table describing the humanization of each of the CD16b blocking receptors.

[0362] FIG.194 shows mouse anti-Siglec9 blocking receptor generation workflow.

[0363] FIG.195A shows binding data from FACS and ELISA analysis. FACS binding assays were performed on K562 cells engineered to overexpress Siglec7, Siglec9, Siglec10, and Siglec14. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Indirect ELISA using biotinylated huSiglec9 or huSiglec3 extracellular domain (ECD) protein were performed using hybridoma derived mouse IgG supernatants. Table values represent the optical density (OD) readings after addition of colorimetric substrate. Binders characterized with scFv functional data are denoted with a “yes” in the table.Attorney Ref.061250-557001WO

[0364] FIG.195B shows binding data from FACS and ELISA analysis. FACS binding assays were performed on K562 cells engineered to overexpress Siglec7, Siglec9, Siglec10, and Siglec14. Values represent median fluorescence intensity (MFI) after detection with appropriate secondary antibody. Indirect ELISA using biotinylated huSiglec9 or huSiglec3 extracellular domain (ECD) protein were performed using hybridoma derived mouse IgG supernatants. Table values represent the optical density (OD) readings after addition of colorimetric substrate. Binders characterized with scFv functional data are denoted with a “yes” in the table.

[0365] FIG. 196 is a series of plots showing functional activation of JNL cells transfected with Siglec9 activator receptors co-cultured with K562 target cells expressing Siglec9.

[0366] FIG.197 is a table showing Siglec9 activator receptor activation EC50 values for co-culture assay shown in FIG. 196 in molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent). Span was calculated from maximum activation level subtracted by minimum activation level from titration curve non-linear fit in FIG.196.

[0367] FIG. 198 is a series of flow cytometry plots showing staining of JNL ITGA4 KO cells transiently expressing anti-ITGA4 activator receptor 2F5 (SEQ ID NO: 3037) with or without anti- Siglec9 blocking receptors (co-expressed) or pLenti control using tetramerized ITGA4B1 and tetramerized SIGLEC9.

[0368] FIG. 199 is a series of plots showing titratable blocking of JNL ITGA4 KO cell activation when Siglec9 blocker receptors are co-transfected with an ITGA4(2F5) CAR (SEQ ID NO: 3037).

[0369] FIG. 200 is a table showing Siglec9 blocker receptor IC50 values for co-culture shown in FIG. 199 in ng mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent). Span (Top – Bottom) and % Blocking (Span / Top) are also shown.

[0370] FIG.201A is a flow cytometry plot showing surface staining of JNL ITGA4 knockout cells transiently expressing anti-ITGA4 activator receptor or pLenti control using tetramerized ITGA4B1.

[0371] FIG.201B is a flow cytometry plot showing surface staining of JNL ITGA4 knockout cells transiently expressing anti-ITGA4 activator receptor with or without anti-SIGLEC9 blocker receptor 14M14 (SEQ ID NO: 5201) (co-expressed) or pLenti control using tetramerized ITGA4B1 and tetramerized Siglec9.

[0372] FIG.201C is a flow cytometry plot showing surface staining of JNL ITGA4 knockout cells transiently expressing anti-ITGA4 activator receptor with or without anti-SIGLEC9 blocker receptor 20F11 (SEQ ID NO: 5220) (co-expressed) or pLenti control using tetramerized ITGA4B1 and tetramerized Siglec9.

[0373] FIG. 202 is a series of plots showing titratable blocking of JNL ITGA4 KO cell activation when SIGLEC9 blocker receptors are co-transfected with anti-ITGA4 activator receptor.Attorney Ref.061250-557001WO

[0374] FIG. 203A is a table showing Siglec9 blocker receptor 14M14 (SEQ ID NO: 5201) IC50 values for co-culture shown in FIG.202 in ng mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent). Span (Top – Bottom) and % Blocking (Span / Top) are also shown.

[0375] FIG. 203A is a table showing Siglec9 blocker receptor 20F11 (SEQ ID NO: 5220) IC50 values for co-culture shown in FIG.202 in ng mRNA or molecules per cell calculated using QIFIKIT (quantitative analysis kit, Agilent). Span (Top – Bottom) and % Blocking (Span / Top) are also shown.

[0376] FIG.204 is a series of plots showing blocking in primary monocytes and K562 cell lines of JNL ITGA4 KO cell activation when SIGLEC9 blocker receptors are co-transfected with ITGA4 or CD33 CARs. JNL ITGA4 KO cells were transfected with ITGA4 or CD33 activator receptor DNA and with or without SIGLEC9 blocker receptor DNA at 2:1 ratio. Monocytes were isolated from PBMC (thawed overnight) by negative selection. K562 target cells were transfected with human ITGA4, CD33, SIGLEC9 mRNA as indicated. 18-24 hours post-JNL transfection, 10,000 JNL ITGA4 KO cells were co-cultured with the indicated number of each target cell type for 6hours, after which the JNL functional response (luminescence, RLU) was assessed using ONE-Step luciferase assay system.

[0377] FIG.205 is a table showing the percent activation of JNL cells from FIG.204 on Monocytes and on K562 target cells transfected with human ITGA4 and CD33 with or without SIGLEC9 mRNA following the co-culture assay. DETAILED DESCRIPTION

[0378] The present disclosure relates, in part, to identification of suitable target molecules for logic- gated immune cell therapy. Provided herein are methods to identify suitable sets of activator (A) and blocker (B) antigens for use in the treatment of blood cancers. Further provided are immune cells that integrate, through selective binding, signals provided by sets of A and B antigens. The immune cells of the disclosure operate as a “logic gate” wherein signals from A antigens and B antigens together trigger a desired response by the immune cell.

[0379] Immune cells of the present disclosure are engineered to specifically activate when contacted with blood cancer cells. The immune cells do not activate when contacted with non-cancerous cells, such as hematopoietic stem cells. Accordingly, adverse side effects caused by damage to non- cancerous cells may be reduced. Sets of A and B antigens, where A antigens activate the engineered immune cells and B antigens inhibit them, may be used to selectively target blood cancer cells using the synthetic signal-integration systems of the present disclosure.

[0380] The set of A antigens may comprise a single member A, or the set of A antigens may comprise n antigens denoted A1, A2, …, An. The set of B antigens may comprise a single member B, or the set of B antigens may comprise n antigens denoted B1, B2, …, Bn.Attorney Ref.061250-557001WO

[0381] Logic gates may include OR-NOT, AND-NOT, and other variations. For example, the disclosure contemplates the following, non-limiting, list of logic gates:

[0382] A NOT B

[0383] (A1OR A2) NOT B

[0384] (A1AND A2) NOT B

[0385] (A1 OR A2) NOT (B1 OR B2)

[0386] (A1OR A2) NOT (B1AND B2)

[0387] A NOT (B1 OR B2)

[0388] A NOT (B1 AND B2)

[0389] NOT logic may be provided by, for example and without limitation, use of an inhibitory receptor that specifically binds a B antigen; when activated, the inhibitory receptor provides a signal that blocks the activator receptor.

[0390] OR logic may be provided by pairs of activator receptors or pairs of inhibitor receptors. For example, an (A1 OR A2) signal may be provided by a first activator receptor that specifically binds A1and a second activator receptor that specifically binds A2. Activation of an (A1OR A2) activator receptor occurs upon contact with a cell expressing either antigen A1or antigen A2, or both. Likewise, a NOT (B1OR B2) signal may be provided by a first inhibitor receptor that specifically binds B1and a second inhibitor receptor that specifically binds B2. Activation of a (B1OR B2) inhibitor receptor occurs upon contact with a cell expressing either antigen B1or antigen B2, or both.

[0391] In a variation, OR logic may be provided by a single receptor that specifically binds two antigens. For example, an (A1 OR A2) signal may be provided by an activator receptor that specifically binds A1 and specifically binds A2. Likewise, a NOT (B1 OR B2) signal may be provided by an inhibitor receptor that specifically binds B1 and specifically binds B2.

[0392] AND logic may be provided by two receptors that each individually activate in response to an antigen but provide an activator (or inhibitor) signal that is below a threshold for activation (or inhibition) of the immune cell. For example, an (A1 AND A2) signal may be provided by a first activator receptor that specifically binds A1 and a second activator receptor that specifically binds A2, where either activator signal alone is too weak to activate the immune cell. Likewise, a NOT (B1AND B2) signal may be provided by a first inhibitor receptor that specifically binds B1and a second inhibitor receptor that specifically binds B2, where either inhibitor signal alone is too weak to block the activation of the immune cell.

[0393] In a variation, a single activator (or inhibitor) receptor may be used, where the single receptor is activated only when contacted by both antigens. For example, an (A1AND A2) signal may be provided by an activator receptor that specifically binds A1 and that specifically binds A2, where the activator receptor only activates when it binds both A1 and A2. Likewise, a NOT (B1 AND B2) signalAttorney Ref.061250-557001WO may be provided by an inhibitor receptor that specifically binds B1 and that specifically binds B2, where the inhibitor receptor only activates, to block an activator signal, when it binds both B1and B2.

[0394] Illustrative, non-limiting, examples of A and B antigens in logic gates contemplated by the present disclosure are shown in Table 1. Illustrative, non-limiting, examples of A and B antigen combinations in logic gates contemplated by the present disclosure are shown in Table 2. Table 1: Illustrative A and B antigensTable 2: Illustrative A and B antigen combinationsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0395] The present disclosure describes an algorithm to identify A and B antigens for use in engineered receptor system activator (A) and blocker (B) modules. Unlike other methods that begin with known, specific target antigens, the algorithm described herein (shown in FIG. 5) begins with putative activator antigens that are widely expressed on early hematopoietic lineages, but not on red blood cells (RBCs) and putative inhibitor antigens widely expressed on cell types or lineages that should be protected (e.g., hematopoietic stem cells (HSCs) or macrophages).

[0396] In an embodiment, a blood-tumor targeting cell therapy comprises a synthetic signal- integration system that integrates signals from activator and inhibitor antigens to trigger selective tumor cell killing. The synthetic signal-integration system contains receptor(s) that activate an effector response and receptor(s) that inhibit a response.

[0397] In some embodiments, A1 and A2 specificities are both expressed on a single activator receptor. In some embodiments, A1 and A2 specificities are expressed on individual activator receptors. In some embodiments, B1 and B2 specificities are both expressed on a single activator receptor. In some embodiments, B1 and B2 specificities are expressed on individual activator receptors.

[0398] The present disclosure describes engineered receptors, such as chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), adoptive cell therapies, and methods of use thereof.Attorney Ref.061250-557001WO

[0399] In some embodiments, the present disclosure provides an immune cell that acts as an A NOT B logic gate, where A is SPN and B is CD16B. The applicants have identified SPN and CD16B using the algorithm shown in FIG. 5 and described in Example 1. Moreover, testing immune cells that express an activator receptor that specifically binds SPN and an inhibitor receptor that specifically binds CD16B will demonstrate that SPN and CD16B are effective as A and B antigens for an A NOT B logic gate.

[0400] Accordingly, in some embodiments, an engineered activator receptor may target the SPN antigen and activate immune cells genetically altered to express the SPN targeting receptor. Immune cells expressing the SPN targeting activator receptors can be used in compositions as part of cell adoptive therapies in the treatment, for example, of blood cancers. The immune cells expressing the SPN targeting activator receptor are also contemplated to further comprise an inhibitory receptor targeting CD16B. The inhibitory receptor may prevent or inhibit the activation of an immune cell mediated by the activator receptor. For example, an immune cell engineered to express an activator receptor that specifically binds SPN when the activator contacts a cell expressing SPN and an inhibitory receptor that specifically binds CD16B will not be activated when the inhibitory receptor contacts a cell expressing CD16B; collectively, the receptors generate a NOT logic gate such that the immune cell specifically activates when the immune cell contacts a blood cancer cell that expresses SPN but not CD16B. The immune cell does not activate, or activates at a reduced level, when the immune cell contacts a non-blood cancer cell (e.g., a healthy blood cell or healthy solid tissue cell), expressing both SPN and CD16B.

[0401] Selective activation and inhibition of the immune cell is useful for reducing toxicity in adoptive cell therapies. The inventors have found that this selective activation and inhibition strategy can be employed using the SPN-targeting activator receptors and CD16B-targeting inhibitor receptors described herein.

[0402] In some embodiments, the present disclosure provides an immune cell that acts as an A NOT B logic gate, where A is CD33 and B is CD16B. Testing immune cells that express an activator receptor that specifically binds CD33 and an inhibitor receptor that specifically binds CD16B will demonstrate that CD33 and CD16B are effective as A and B antigens for an A NOT B logic gate.

[0403] Accordingly, in some embodiments, an engineered activator receptor may target the CD33 antigen and activate immune cells genetically altered to express the CD33 targeting receptor. Immune cells expressing the CD33 targeting activator receptors can be used in compositions as part of cell adoptive therapies in the treatment, for example, of blood cancers. The immune cells expressing the CD33 targeting activator receptor are also contemplated to further comprise an inhibitory receptor targeting CD16B. The inhibitory receptor may prevent or inhibit the activation of an immune cell mediated by the activator receptor. For example, an immune cell engineered to express an activatorAttorney Ref.061250-557001WO receptor that specifically binds CD33 when the activator contacts a cell expressing CD33 and an inhibitory receptor that specifically binds CD16B will not be activated when the inhibitory receptor contacts a cell expressing CD16B; collectively, the receptors generate a NOT logic gate such that the immune cell specifically activates when the immune cell contacts a blood cancer cell that expresses CD33 but not CD16B. The immune cell does not activate, or activates at a reduced level, when the immune cell contacts a non-blood cancer cell (e.g., a healthy blood cell or healthy solid tissue cell), expressing both CD33 and CD16B.

[0404] In some embodiments, the present disclosure provides an immune cell that acts as an (A1 OR A2) NOT B logic gate, where A1 is SPN, A2 is CD33, and B is CD16B. Engineered Receptors

[0405] The disclosure provides an activator receptor comprising an extracellular region, the extracellular region comprising a first ligand binding domain capable of specifically binding a first ligand that activates or promotes activation of the receptor, which promotes activation of effector cells expressing the receptor. The disclosure further provides an inhibitor receptor comprising a second ligand binding domain capable of binding a second ligand, wherein binding of the second ligand by the second ligand binding domain inhibits, reduces, or prevents activation of effector cells even when the activator receptor is bound to the first ligand. The first ligand may also be referred to as an activator ligand or antigen. The second ligand may also be referred to as an inhibitor ligand or antigen. The activator and inhibitor receptors that bind to these ligands may also be referred to generally herein as engineered receptors. Engineered receptors can refer to either chimeric antigen receptors (CARs) or T cell receptors (TCRs) described in the disclosure. The term “engineered receptor” may also refer to any receptor designed using the binding domains, hinge regions, transmembrane domains, and / or cytoplasmic domains described herein. Engineered receptors are sometimes referred to herein as fusion proteins. Activator Receptor Antigens CD45

[0406] The activator receptors disclosed can specifically bind to a CD45 antigen.

[0407] The term “CD45” also known as leukocyte common antigen (LCA), protein tyrosine phosphatase receptor type C (PTPRC), and other aliases refers to human CD45 protein and species, isoforms, and other sequence variants thereof. Thus, CD45 can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on leukocytes, and which plays an important role in the function of these cells (reviewed in Altin, JG (1997) Immunol Cell Biol. 75(5):430-45, incorporated herein by reference). For example, the extracellular domain of CD45 is expressed inAttorney Ref.061250-557001WO several different isoforms on T cells, and the particular isoform(s) expressed depends on the particular subpopulation of cell, their state of maturation, and antigen exposure. Expression of CD45 is important for the activation of T cells via the TCR, and different CD45 isoforms display a different ability to support T cell activation.

[0408] In some embodiments, the target antigen is a peptide antigen of CD45. In some embodiments, the CD45 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 1-9 or 327. However, all CD45 isoforms, and sequences and subsequences thereof that act as CD45 antigens are envisaged as within the scope of the disclosure. In some embodiments, A CD45 antigen comprises a protein comprising a sequence any one of SEQ ID NOS: 1-9 or 327, or a peptide fragment of any one of SEQ ID NOS: 1-9 or 327. CD11a / LFA-1 / ITGAL

[0409] The activator receptors disclosed can specifically bind to a CD11a antigen.

[0410] The term “CD11a” also known as integrin alpha L, ITGAL, p180, lymphocyte function- associated antigen 1 - alpha polypeptide, LFA-1, LFA1A, integrin subunit alpha L, and other aliases refers to the human CD11a protein and species, isoforms, and other sequence variants thereof. Thus, CD11a can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein.

[0411] CD11a is expressed on all leukocytes and is involved in leukocyte intercellular adhesion through interactions with its ligands, ICAMs 1-3 (intercellular adhesion molecules 1 through 3), and also functions in lymphocyte costimulatory signaling. CD11a is one of the two components, along with CD18, which form lymphocyte function-associated antigen-1 (LFA-1). LFA-1 plays a role in emigration, which is the process by which leukocytes leave the bloodstream to enter the tissues. Additionally, LFA-1 is involved in the process of cytotoxic T cell mediated killing as well as antibody mediated killing by granulocytes and monocytes.

[0412] In some embodiments, the target antigen is a peptide antigen of CD11a. In some embodiments, the CD11a antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 169-171. However, all CD11a isoforms, and sequences and subsequences thereof that act as CD11a antigens are envisaged as within the scope of the disclosure. In some embodiments, a CD11a antigen comprises a protein comprising a sequence of any one of SEQ ID NOS: 169-171, or a peptide fragment of any one of SEQ ID NOS: 169-171. SPN / CD43

[0413] The activator receptors disclosed can specifically bind to a sialophorin (SPN) antigen.Attorney Ref.061250-557001WO

[0414] The term “sialophorin” also known as SPN, Leukosialin, CD43, GPL115, galactoglycoprotein (GALGP), leukocyte sialoglycoprotein, and other aliases refers to the human SPN protein and species, isoforms, and other sequence variants thereof. Thus, SPN can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein.

[0415] SPN is a large, negatively charged type I transmembrane sialoglycoprotein and is expressed on the surface of human T lymphocytes, monocytes, granulocytes, and some B lymphocytes. SPN functions as an anti-adhesion molecule, mediating repulsion among leukocytes. The SPN extracellular domain has a rod-like structure that is predicted to extend from the cell surface and contains more than 80 serine or threonine residues, most of which are modified with heavily sialylated O-linked glycans. SPN's cytoplasmic tail binds ERM-family proteins. Two major SPN glycoforms (115 kD and 130 kD) have been identified in humans and mice. The 130-kD SPN glycoform, modified with sLex-like structures, is reported to bind E-selectin in activated T cells.

[0416] In some embodiments, the target antigen is a peptide antigen of SPN. In some embodiments, the SPN antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 172-173. However, all SPN isoforms, and sequences and subsequences thereof that act as SPN antigens are envisaged as within the scope of the disclosure. In some embodiments, a SPN antigen comprises a protein comprising a sequence of any one of SEQ ID NOS: 172-173, or a peptide fragment of any one of SEQ ID NOS: 172-173. CD33

[0417] The activator receptors disclosed can specifically bind to a CD33 antigen.

[0418] The term “CD33” also known as siglec-3, sialic acid binding Ig-like lectin 3, gp67, p67, and other aliases refers to the human CD33 protein and species, isoforms, and other sequence variants thereof. Thus, CD33 can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein.

[0419] CD33 is a sialoadhesin molecule and a member of the immunoglobulin supergene family. It is expressed by myeloid stem cells, myeloblasts and monoblasts, monocytes / macrophages, granulocyte precursors (with decreasing expression with maturation), and mast cells.

[0420] In some embodiments, the target antigen is a peptide antigen of CD33. In some embodiments, the CD33 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 174-176. However, all CD33 isoforms, and sequences and subsequences thereof that act as CD33 antigens are envisaged as within the scope of the disclosure. In some embodiments,Attorney Ref.061250-557001WO a CD33 antigen comprises a protein comprising a sequence of any one of SEQ ID NOS: 174-176, or a peptide fragment of any one of SEQ ID NOS: 174-176. FLT3

[0421] The activator receptors disclosed can specifically bind to a FLT3 antigen.

[0422] The term “FLT3” also known as CD135, fms-like tyrosine kinase 3 (FLT-3), receptor-type tyrosine-protein kinase FLT3, fetal liver kinase-2 (Flk2), and other aliases refers to the human FLT3 protein and species, isoforms, and other sequence variants thereof. Thus, FLT3 can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein.

[0423] FLT3 is a class III receptor tyrosine kinase that regulates hematopoiesis. FLT3 is activated by binding of the fms-related tyrosine kinase 3 ligand to the extracellular domain, which induces homodimer formation in the plasma membrane leading to auto-phosphorylation of FLT3. The activated receptor kinase subsequently phosphorylates and activates multiple cytoplasmic effector molecules in pathways involved in apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow.

[0424] In some embodiments, the target antigen is a peptide antigen of FLT3. In some embodiments, the FLT3 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 177-178. However, all FLT3 isoforms, and sequences and subsequences thereof that act as FLT3 antigens are envisaged as within the scope of the disclosure. In some embodiments, a FLT3 antigen comprises a protein comprising a sequence of any one of SEQ ID NOS: 177-178, or a peptide fragment of any one of SEQ ID NOS: 177-178. CD123

[0425] The activator receptors disclosed can specifically bind to a CD123 antigen.

[0426] The IL-3R is a heterodimer composed by alpha and beta chains. The term “CD123” also known as the alpha-chain of the interleukin-3 receptor (IL-3RA) and other aliases refers to a glycoprotein composed of an extracellular domain, a predicted Ig-like domain, two FnIII domains, a transmembrane domain, and an intracellular domain. In some embodiments, the target antigen is a peptide antigen of CD123. All CD123 isoforms, and sequences and subsequences thereof that act as CD123 antigens are envisaged as within the scope of the disclosure.

[0427] In some embodiments, the anti-CD123 VH region comprises any one of SEQ ID NOs: 408, 410, 412, 414, 416, 418, 424-426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, or 450, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD123 VL region comprises any one of SEQ ID NOs: 409, 411, 413, 415, 417, 419, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, or 451 or aAttorney Ref.061250-557001WO sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. ITGA4

[0428] The activator receptors disclosed can specifically bind to ITGA4 antigen.

[0429] The term “ITGA4” also known as Integrin Subunit Alpha 4, CD49d, Alpha 4 Subunit of VLA-4 Receptor, CD49 Antigen-Like Family Member D, VLA-4 Subunit Alpha, Integrin Alpha-IV, Very Late Activation Protein 4 Receptor, 269C Wild Type, IA4, and other aliases refers to human ITGA4, protein and species, isoforms, and other sequence variants thereof. Thus, ITGA4, can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. ITGA4 encodes a member of the integrin alpha chain family of proteins. Integrins may be heterodimeric integral membrane proteins composed of an alpha chain and a beta chain that can function in cell surface adhesion and signaling. ITGA4 can be expressed as a preprotein that can be proteolytically processed to generate light and heavy chains that comprise the alpha 4 subunit. Alternative splicing can result in multiple transcript variants. The alpha 4 subunit can associate with a beta 1 or beta 7 subunit to form an integrin that can play a role in cell motility and migration. Alpha 4 subunit is a therapeutic target for the treatment of multiple sclerosis, Crohn's disease, and inflammatory bowel disease.

[0430] In some embodiments, the target antigen is a peptide antigen of ITGA4. In some embodiments, the ITGA4 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of SEQ ID NO: 3313. However, all ITGA4 isoforms, and sequences and subsequences thereof that act as ITGA4 antigens are envisaged as within the scope of the disclosure. In some embodiments, an ITGA4 antigen comprises a protein comprising a sequence of SEQ ID NO: 3313, or a peptide fragment of SEQ ID NO: 3313. CD162

[0431] The activator receptors disclosed can specifically bind to CD162 antigen.

[0432] The term “CD162” also known as Selectin P Ligand, SELPLG, PSGL-1, P- Selectin Glycoprotein Ligand 1, Cutaneous Lymphocyte-Associated Antigen, CLA and other aliases refers to human CD162, protein and species, isoforms, and other sequence variants thereof. Thus, CD162, can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturallyAttorney Ref.061250-557001WO occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. CD162 encodes a member of the integrin alpha chain family of proteins. CD162 is a glycoprotein that can function as a high affinity counter-receptor for the cell adhesion molecules P-, E- and L- selectin expressed on myeloid cells and stimulated T lymphocytes. CD162 can play a critical role in leukocyte trafficking during inflammation by the tethering of leukocytes to activated platelets or endothelia expressing selectins. CD162 can undergo two post-translational modifications, tyrosine sulfation and the addition of the sialyl Lewis x tetrasaccharide (sLex) to its O-linked glycans. CD162 can undergo alternate splicing that results in multiple transcript variants. The two post- translational modifications can impact the high-affinity binding activity of CD162. Aberrant expression and polymorphisms of CD162 have been associated with defects in the innate and adaptive immune response.

[0433] In some embodiments, the target antigen is a peptide antigen of CD162. In some embodiments, the CD162 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of SEQ ID NO: 3314. However, all CD162 isoforms, and sequences and subsequences thereof that act as CD162 antigens are envisaged as within the scope of the disclosure. In some embodiments, the CD162 antigen comprises a protein comprising a sequence of SEQ ID NO: 3314, or a peptide fragment of SEQ ID NO: 3314. CD53

[0434] The activator receptors disclosed can specifically bind to CD53 antigen.

[0435] The term “CD53” also known as Cell Surface Glycoprotein CD53, Leukocyte Surface Antigen CD53, Tetraspanin-25, Tspan-25, TSPAN25, MOX44, CD53 Tetraspan Antigen, CD53 glycoprotein, and other aliases refers to human CD53, protein and species, isoforms, and other sequence variants thereof. Thus, CD53, can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. CD53 encodes a member of the transmembrane 4 superfamily, also known as the tetraspanin family. CD53 is a cell surface glycoprotein that can complex with integrins. It can contribute to the transduction of CD2-generated signals in T cells and natural killer cells and has been suggested to play a role in growth regulation. Familial deficiency of this gene has been linked to an immunodeficiency associated with recurrent infectious diseases caused by bacteria, fungi, and viruses. CD53 can be a structural component of specialized membrane microdomains known as tetraspanin-enriched microdomains (TERMs), which can act as platforms for receptor clustering and signaling. CD53 can be involved in in diverse biological functions such as cell signal transduction, adhesion, migration, and protein trafficking. CD53 can also play a role in the activation of monocytes and B-cells.Attorney Ref.061250-557001WO

[0436] In some embodiments, the target antigen is a peptide antigen of CD53. In some embodiments, the CD53 antigen comprises a sequence or subsequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence or a subsequence of SEQ ID NO: 3315. However, all CD53 isoforms, and sequences and subsequences thereof that act as CD53 antigens are envisaged as within the scope of the disclosure. In some embodiments, an CD53 antigen comprises a protein comprising a sequence of SEQ ID NO: 3315, or a peptide fragment of SEQ ID NO: 3315. CLL1

[0437] The activator receptors disclosed can specifically bind CLL1.

[0438] The term “CLL1” also known as C-type Lectin Domain Family 12 Member A (CLEC12A), DCAL-2, CLL1, MICL, Myeloid Inhibitory C-Type Lectin-Like receptor, Dendritic Cell-Associated Lectin 2, CD371, C-Type Lectin-Like Molecule-1, C-Type Lectin Protein CLL1 and other aliases refers to human CLL1, protein and species, isoforms, and other sequence variants thereof. Thus, CLL1, can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. CLL1 encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of the C-type lectin family may share a common protein fold and have diverse functions, such as such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. CLL1 may act as a negative regulator of granulocyte and monocyte function. Several alternatively spliced transcript variants of CLL1 have been described. This gene has been closely linked to other CTL / CTLD superfamily members in the natural killer gene complex region on chromosome 12p13. CLL1 may be used as a biomarker for diagnosis and prognosis in acute myeloid leukemia.

[0439] In some embodiments, the target antigen is a peptide antigen of CLL1. CD96

[0440] The activator receptors disclosed can specifically bind CD96.

[0441] The term “CD96” also known as T-cell Surface protein Tactile, T cell Activated Increased Late Expression Protein, TACTILE, and other aliases refer to human CD96, protein and species, isoforms, and other sequence variants thereof. Thus, CD96 can be the native, full-length protein or can be a truncated fragment or a sequence variant (e.g., a naturally occurring isoform, or recombinant variant) that retains at least one biological activity of the native protein. CD96 can be a receptor protein that may be expressed on T cells and NK cells. CD96 belongs to the immunoglobulin superfamily. CD96 may play a role in the adhesion of activated T and NK cells to their target cells during the late phase of the immune response. It may also function in antigen presentation. Alternative splicing occurs at the CD96 locus and two transcript variants encoding distinct isoformsAttorney Ref.061250-557001WO have been identified. CD96 can be a transmembrane glycoprotein that has three extracellular immunoglobulin-like domains and may be expressed by all resting human and mouse NK cells. CD155 is a ligand of CD96.

[0442] In some embodiments, the target antigen is a peptide antigen of CD96. Activator Receptors Activator Ligands

[0443] The disclosure provides an activator ligand and an engineered receptor comprising a ligand binding domain that binds to the activator ligand. In some embodiments, the first engineered receptor is an activator receptor. In some embodiments, the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the first activator ligand is a CD45 antigen. In some embodiments, the first activator ligand is a CD11a antigen. In some embodiments, the first activator ligand is a SPN antigen. In some embodiments, the first activator ligand is a CD33 antigen. In some embodiments, the first activator ligand is a FLT3 antigen. In some embodiments, the CD45 antigen comprises a sequence or subsequence that shares at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence or a subsequence of any one of SEQ ID NOs: 1-9.

[0444] In some embodiments, the activator antigen comprises a peptide of CD45 that lacks amino acids 34-194, as compared with the CD45 peptide of SEQ ID NO: 1. In some embodiments, the activator antigen comprises a peptide of CD45 that lacks amino acids 147-194, as compared with the CD45 peptide of SEQ ID NO: 1. In some embodiments, the activator antigen comprises a peptide of CD45 that lacks amino acids 100-146, as compared with the CD45 peptide of SEQ ID NO: 1. In some embodiments, the activator antigen comprises a peptide of CD45 that lacks amino acids 147- 194, as compared with the CD45 peptide of SEQ ID NO: 1. In some embodiments, the activator antigen comprises a peptide of CD45 that lacks amino acids 34-146, as compared with the CD45 peptide of SEQ ID NO: 1.

[0445] As used herein, an “activator,” “activator antigen,” or “activator ligand” refers to a first ligand that binds to a first, activator ligand binding domain (LBD) of an engineered receptor of the disclosure, such as a CAR or TCR, thereby mediating activation of a T cell or other immune cell expressing the activator receptor. The activator ligand is expressed by target cells, for example cancer cells, and may also be expressed more broadly than just the cancer cells. For example the activator can be expressed on some, or all types of normal, non-cancerous cells such as hematopoietic stem cells (HSCs) or blood cells.Attorney Ref.061250-557001WO

[0446] In some embodiments, the activator ligand is expressed by cancer cells and is not expressed by non-cancerous cells (i.e. normal cells not targeted by the adoptive cell therapy). In some embodiments, the target cells are cancer cells and the normal cells are non-cancerous cells.

[0447] In some embodiments, the activator ligand has high cell surface expression on the cancer cells. In some embodiments, the A antigen is expressed by cancer cells and is not expressed by allogenic donor stem cells. In some embodiments, the target cells are a subject’s cancer cells and / or the subject’s blood cells.

[0448] In some embodiments, the A antigen has high cell surface expression on cancerous and non- cancerous blood cells.

[0449] This high cell surface expression confers the ability to deliver large activation signals. Methods of measuring cell surface expression will be known to the person of ordinary skill in the art and include, but are not limited to, immunohistochemistry using an appropriate antibody against the activator ligand, followed by microscopy, fluorescence activated cell sorting (FACS), and / or flow cytometry.

[0450] The activator ligand is present on cancer cells. In some embodiments, the target cells are cancer cells, such as blood cancer cells.

[0451] In some embodiments, the activator ligand is present on a plurality of cancer cells. In some embodiments, the activator ligand is present on at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of cancer cells. In some embodiments, the activator ligand is present on at least 95% of cancer cells. In some embodiments, the activator ligand is present on at least 99% of cancer cells.

[0452] The A antigen may be present on all blood cells, or on a subset of blood cells. Generally, the subset of blood cells is selected to include cancer cells, although application of the disclosed compositions and methods in a subject not having cancer is also contemplated. For example, the engineered immune cells may be used to condition subjects having aplastic anemia or other non- malignant conditions for stem cell transplant, or to promote engraftment or expansion of stem cells after a subject has received stem cell transplant for cancer or for another non-malignant condition.

[0453] In some embodiments, the A antigen is present on a plurality of patient blood cells. In some embodiments, the A antigen is present on at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of patient blood cells. In some embodiments, the A antigen is present on at least 95% of patient blood cells. In some embodiments, the A antigen is present on at least 99% of patient blood cells.Attorney Ref.061250-557001WO

[0454] In some embodiments, the activator ligand is expressed by a plurality of cancer cells and a plurality of non-cancerous cells. In some embodiments, the plurality of non-cancerous cells expresses both the activator ligand and the inhibitor ligand.

[0455] In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non-cancerous cells at a ratio of about 1:100 to about 100:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and the inhibitor ligand are present on the plurality of non-cancerous cells at a ratio of about 1:50 to about 50:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non-cancerous cells at a ratio of about 1:40 to about 40:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:30 to about 30:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:20 to about 20:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:10 to about 10:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:5 to about 5:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:3 to about 3:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:2 to about 2:1 of the activator ligand to the inhibitor ligand. In some embodiments, the activator ligand and inhibitor ligand are present on the plurality of non- cancerous cells at a ratio of about 1:1 of the activator ligand to the inhibitor ligand.

[0456] In some embodiments, the A antigen is expressed by a plurality of cancer cells and a plurality of allogeneic donor cells. In some embodiments, the plurality of allogeneic donor cells expresses both the A antigen and the inhibitor ligand.

[0457] In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:100 to about 100:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and the inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:50 to about 50:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:40 to about 40:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:30 to about 30:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:20 toAttorney Ref.061250-557001WO about 2:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:10 to about 10:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:5 to about 5:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:3 to about 3:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:2 to about 2:1 of the A antigen to the inhibitor ligand. In some embodiments, the A antigen and inhibitor ligand are present on the plurality of allogeneic donor cells at a ratio of about 1:1 of the A antigen to the inhibitor ligand. Extracellular Ligand Binding Domains

[0458] The disclosure provides chimeric antigen receptors (CARs) 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, antigen-binding domains from antibodies, antibody fragments, scFv, antigen-binding domains derived from T cell receptors, and the like. All forms of antigen-binding domains known in the art are envisaged as within the scope of the disclosure.

[0459] An “extracellular domain,” as used herein, refers to the extracellular portion of a protein. For example, the TCR alpha and beta chains each comprise an extracellular domain, which comprise a constant and a variable region involved in peptide-MHC recognition. The “extracellular domain” can also comprise a fusion domain, for example of fusions between additional domains capable of binding to and targeting a specific antigen and the endogenous extracellular domain of the TCR subunit.

[0460] The term “antibody,” as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0461] The terms “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof that contains the antigen-binding domain, i.e., an antigenic determining variable region of an intact antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. :dMY\XQ_ [R MZ`UN[Pe R^MSYQZ`_ UZOXaPQ& Na` M^Q Z[` XUYU`QP `[& ;MN& ;MNq& ;#MNq$,& MZP ;b R^MSYQZ`_& single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.Attorney Ref.061250-557001WO

[0462] An antibody, antibody fragment, or antibody-binding domain that “does not bind” with an antigen (e.g., CD16A) is intended to refer to an antibody, or the like portion thereof, that exhibits substantially no detectable binding above background to an antigen in a conventional immunoassay, such as ELISA or flow cytometry.

[0463] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0464] “Light chain variable region” or “VL” with regard to an antibody refers to the fragment of the light chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0465] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0466] Unless specified, as used herein a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0467] The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains \^Q_QZ` UZ MZ`UN[Pe Y[XQOaXQ_ UZ `TQU^ ZM`a^MXXe [OOa^^UZS O[ZR[^YM`U[Z_( ?M\\M #jrk$ MZP XMYNPM #jvk$ XUST` OTMUZ_ ^QRQ^ `[ `TQ `c[ YMV[^ MZ`UN[Pe XUST` OTMUZ U_[`e\Q_(

[0468] The term “recombinant antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0469] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CD45, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CD45 antibody is a human anti-CD45 antibody, a humanized anti-CD45 antibody, or a chimeric anti- CD45 antibody. In some embodiments, the anti-CD45 antibody is an anti-CD45 monoclonalAttorney Ref.061250-557001WO antibody. Illustrative anti-CD45 antibodies include antibodies BC8, 4B2, GAP8.3 or 9.4. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CD45 is specific to one CD45 isoform or binds to more than one CD45 isoform, e.g., is a pan-CD45 antibody or antigen- binding fragment.

[0470] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 3. Table 3: CDRs Corresponding to CD45 Antigen Binding Domains

[0471] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of RYWMS (SEQ ID NO: 10), EINPTSSTINFTPSLKD (SEQ ID NO: 11), and GNYYRYGDAMDY (SEQ ID NO: 12); and the VL region comprises one or more CDR sequences selected from the group consisting of RASKSVSTSGYSYLH (SEQ ID NO: 13), LASNLES (SEQ ID NO: 14), and QHSRELPFTFGSGT (SEQ ID NO: 15).

[0472] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 4A. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 4A. In some embodiments, the binding domain comprises SEQ ID NO: 16 and SEQ ID NO: 18. In some embodiments, the binding domain comprises SEQ ID NO: 17 and SEQ ID NO: 18. In some embodiments, the binding domain comprises SEQ ID NO: 20 and SEQ ID NO: 19. In some embodiments, the binding domain comprises SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, the binding domain comprises SEQ ID NO: 23 and SEQ ID NO: 24. In some embodiments, the binding domain comprises SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the binding domain comprises SEQ ID NO: 27 and SEQ ID NO: 28.Attorney Ref.061250-557001WO Table 4A: VH and VL Domains Corresponding to CD45 Antigen Binding Domains

[0473] In some embodiments, the binding domain is an anti-CD45 scFv domain. In some embodiments, the anti-CD45 scFv domain is selected from a sequence listed in Table 4B. In some embodiments, the anti-CD45 scFv domain comprises a polypeptide comprising SEQ ID NO: 29. In some embodiments, the anti-CD45 scFv domain comprises a polypeptide comprising SEQ ID NO: 30. In some embodiments, the anti-CD45 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 31. In some embodiments, the anti-CD45 scFv domain comprises a polypeptide comprising SEQ ID NO: 32. Table 4B: CD45 scFv Binding Domains

[0474] In some embodiments, the anti-CD45 scFv domain comprises a sequence of any one of SEQ ID NOS: 29, 30, 31, or 32, or a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical thereto.

[0475] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CD11a, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CD11a antibody is a human anti-CD11a antibody, a humanized anti-CD11a antibody, or a chimeric anti-CD11a antibody. In some embodiments, the anti-CD11a antibody is an anti-CD11a monoclonal antibody. Illustrative anti-CD11a antibodies include efalizumab. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CD11a is specific to one CD11a isoform or binds to more than one CD11a isoform, e.g., is a pan-CD11a antibody or antigen-binding fragment.Attorney Ref.061250-557001WO

[0476] In some embodiments, the full length VH and VL regions comprise SEQ ID NO: 179 and SEQ ID NO: 180, respectively. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the binding domain comprises SEQ ID NO: 179 and SEQ ID NO: 180. In some embodiments, the anti-CD11a VH domain comprises a sequence of SEQ ID NO: 179 or a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical thereto. In some embodiments, the anti-CD11a VL domain comprises a sequence of SEQ ID NO: 180 or a sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical thereto.

[0477] In some embodiments, the anti-CD11a full length VH and VL regions comprise SEQ ID NO: 247 and SEQ ID NO: 248, respectively. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the binding domain comprises SEQ ID NO: 247 and SEQ ID NO: 248. In some embodiments, the anti-CD11a VH domain comprises a sequence of SEQ ID NO: 247 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD11a VL domain comprises a sequence of SEQ ID NO: 248 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0478] In some embodiments, the extracellular ligand binding domain comprises a binding agent for SPN, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti-SPN antibody is a human anti-SPN antibody, a humanized anti-SPN antibody, or a chimeric anti-SPN antibody. In some embodiments, the anti-SPN antibody is an anti-SPN monoclonal antibody. Illustrative anti-SPN antibodies include MAB2038 and OX-58. In some embodiments, the antibody molecule or antigen-binding fragment that binds to SPN is specific to one SPN isoform or binds to more than one SPN isoform, e.g., is a pan-SPN antibody or antigen-binding fragment.

[0479] In some embodiments, the cancerous antigen comprises SPN. Various single variable domains known in the art or disclosed herein that bind to and recognize SPN are suitable for use in embodiments. Illustrative VH and VL domains for SPN antigen binding domains are shown in Table 5A below.Attorney Ref.061250-557001WO Table 5A: VH and VL Domains Corresponding to SPN Antigen Binding Domains

[0480] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for SPN antigen binding domains are shown in Table 5B below. Table 5B: CDRs Corresponding to SPN antigen binding domainsAttorney Ref.061250-557001WO

[0481] In some embodiments, the anti-SPN VH domain comprises any one of SEQ ID NOs: 249- 252, 257, 261-280 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-SPN VL domain comprises any one of SEQ ID NOs: 253-256, 258, 281-303 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-SPN HC domain comprises a sequence of SEQ ID NO: 259 or a sequence having at least 90%, at least 95%, at leastAttorney Ref.061250-557001WO 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-SPN LC domain comprises a sequence of SEQ ID NO: 260 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0482] In some embodiments, the first antigen binding domain comprises a scFv domain that binds to a SPN antigen. In some embodiments, the scFv comprises the complementarity determined regions (CDRs) as disclosed in Table 5B. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs as disclosed in Table 5B and the light chain of the antibody comprises the light chain CDRs as disclosed in Table 5B. In some embodiments, the first, activator ligand is SPN, and the activator ligand binding domain comprises a SPN antigen binding domain. In some embodiments, the SPN antigen binding domain comprises a scFv domain.

[0483] In some embodiments, the anti-SPN scFv domain comprises a polypeptide sequence as disclosed in Table 5C. Illustrative scFv domains for SPN antigen binding domains are shown in Table 5C below. Table 5C: SPN scFv Binding DomainsAttorney Ref.061250-557001WO

[0484] In some embodiments, the activator receptor comprises a scFv domain that binds to a SPN antigen. In some embodiments, the activator ligand is SPN and the activator ligand binding domain comprises a SPN antigen binding domain. In some embodiments, the activator receptor is a chimeric antigen receptor comprises a polypeptide sequence as disclosed in Table 5D. Table 5D: Full Length Receptor Corresponding to SPN Binding Domains

[0485] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CD33, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CD33 antibody is a human anti-CD33 antibody, a humanized anti-CD33 antibody, or a chimeric anti- CD33 antibody. In some embodiments, the anti-CD33 antibody is an anti-CD33 monoclonal antibody. Illustrative anti-CD33 antibodies include Gemtuzumab ozogamicin and immunotoxin vadastuximab talirine. In some embodiments, the antibody molecule or antigen-binding fragment thatAttorney Ref.061250-557001WO binds to CD33 is specific to one CD33 isoform or binds to more than one CD33 isoform, e.g., is a pan-CD33 antibody or antigen-binding fragment.

[0486] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 181 and SEQ ID NO: 182, respectively. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the binding domain comprises SEQ ID NO: 181 and SEQ ID NO: 182. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 181 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 182 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0487] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 336 and SEQ ID NO: 335, respectively. In some embodiments, the binding domain comprises SEQ ID NO: 336 and SEQ ID NO: 335. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 336 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 335 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0488] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 337 and SEQ ID NO: 338, respectively. In some embodiments, the binding domain comprises SEQ ID NO: 337 and SEQ ID NO: 338. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 337 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 338 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0489] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 339 and SEQ ID NO: 340, respectively. In some embodiments, the binding domain comprises SEQ ID NO: 339 and SEQ ID NO: 340. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 339 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 340 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.Attorney Ref.061250-557001WO

[0490] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 341 and SEQ ID NO: 342, respectively. In some embodiments, the binding domain comprises SEQ ID NO: 341 and SEQ ID NO: 342. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 341 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 342 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0491] In some embodiments, the anti-CD33 full length HC and LC regions comprise SEQ ID NO: 331 and SEQ ID NO: 332, respectively. In some embodiments, the anti-CD33 HC domain comprises a sequence of SEQ ID NO: 331 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 LC domain comprises a sequence of SEQ ID NO: 332 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0492] In some embodiments, the anti-CD33 full length HC and LC regions comprise SEQ ID NO: 333 and SEQ ID NO: 334, respectively. In some embodiments, the anti-CD33 HC domain comprises a sequence of SEQ ID NO: 333 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 LC domain comprises a sequence of SEQ ID NO: 334 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0493] In some embodiments, the binding domain is an anti-CD33 scFv domain. In some embodiments, the anti-CD33 scFv domain comprises a polypeptide comprising any one of SEQ ID NOs: 304-312. In some embodiments, the anti-CD33 scFv domain comprises a sequence of any one of SEQ ID NOS: 304-312, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0494] In some embodiments, the anti-CD33 full length HC and LC regions comprise SEQ ID NO: 683 and SEQ ID NO: 682, respectively. In some embodiments, the anti-CD33 HC domain comprises a sequence of SEQ ID NO: 683 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 LC domain comprises a sequence of SEQ ID NO: 682 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0495] In some embodiments, the anti-CD33 full length VH and VL regions comprise SEQ ID NO: 684 and SEQ ID NO: 685, respectively. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VLAttorney Ref.061250-557001WO region and the full length VH region. In some embodiments, the binding domain comprises SEQ ID NO: 684 and SEQ ID NO: 685. In some embodiments, the anti-CD33 VH domain comprises a sequence of SEQ ID NO: 684 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises a sequence of SEQ ID NO: 685 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0496] In some embodiments, the CD33 ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize CD33 are suitable for use in embodiments.

[0497] In some embodiments, the anti-CD33 VH domain comprises any one of SEQ ID NOs: 1290- 1396, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD33 VL domain comprises any one of SEQ ID NOs: 1397-1511, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0498] In some embodiments, the cancerous antigen comprises CD33. Various single variable domains known in the art or disclosed herein that bind to and recognize CD33 are suitable for use in embodiments. Illustrative VH and VL domains for CD33 antigen binding domains are shown in Table 6A below. Table 6A: VH and VL Domains Corresponding to CD33 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0499] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for CD33 antigen binding domains are shown in Table 6B below.Attorney Ref.061250-557001WO Table 6B: CDRs Corresponding to CD33 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0500] In some embodiments, the first antigen binding domain comprises a scFv domain that binds to a CD33 antigen. In some embodiments, the scFv comprises the complementarity determined regions (CDRs) as disclosed in Table 6B. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs as disclosed in Table 6B and the light chain of the antibody comprises the light chain CDRs as disclosed in Table 6B. In some embodiments, the first, activator ligand is CD33, and the activator ligand binding domain comprises a CD33 antigen binding domain. In some embodiments, the CD33 antigen binding domain comprises a scFv domain.

[0501] In some embodiments, the anti-CD33 scFv domain comprises a polypeptide sequence as disclosed in Table 6C. Illustrative scFv domains for CD33 antigen binding domains are shown in Table 6C below. Table 6C: CD33 scFv Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0502] In some embodiments, the activator receptor comprises a scFv domain that binds to a CD33 antigen. In some embodiments, the activator ligand is CD33 and the activator ligand binding domain comprises a CD33 antigen binding domain. In some embodiments, the activator receptor is a chimeric antigen receptor comprises a polypeptide sequence as disclosed in Table 6D. Table 6D: Full Length Receptor Corresponding to CD33 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0503] In some embodiments, the extracellular ligand binding domain comprises a binding agent for FLT3, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti-FLT3 antibody is a human anti-FLT3 antibody, a humanized anti-FLT3 antibody, or a chimeric anti-FLT3 antibody. In some embodiments, the anti-FLT3 antibody is an anti-FLT3 monoclonal antibody. Illustrative anti-FLT3 antibodies include LY3012218 (IMC-EB10) and 4G8SDIEM. In some embodiments, the antibody molecule or antigen-binding fragment that binds to FLT3 is specific to one FLT3 isoform or binds to more than one FLT3 isoform, e.g., is a pan-FLT3 antibody or antigen- binding fragment.

[0504] In some embodiments, the anti-FLT3 VH domain comprises any one of SEQ ID NOs: 319, 321, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-FLT3 VL domain comprises any one of SEQ ID NOs: 320, 322, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.Attorney Ref.061250-557001WO

[0505] In some embodiments, the anti-FLT3 HC domain comprises any one of SEQ ID NOs: 344, 346, 348, 350, 352, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-FLT3 LC domain comprises a sequence of SEQ ID NOs: 343, 345, 347, 349, 351, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0506] In some embodiments, the extracellular ligand binding domain comprises a binding agent for ITGA4, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- ITGA4 antibody is a human anti-ITGA4 antibody, a humanized anti-ITGA4 antibody, or a chimeric anti-ITGA4 antibody. In some embodiments, the anti-ITGA4 antibody is an anti-ITGA4 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to ITGA4 is specific to one ITGA4 isoform or binds to more than one ITGA4 isoform, e.g., is a pan- ITGA4 antibody or antigen-binding fragment.

[0507] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 17. Table 17 CDRs Corresponding to ITGA4 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0508] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 18. In some embodiments, the binding domain comprises the full length VH region and VLAttorney Ref.061250-557001WO regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 18. Table 18 VH and VL Corresponding to ITGA4 antigen binding domainsAttorney Ref.061250-557001WO_

[0509] In some embodiments, the binding domain is an anti-ITGA4 scFv domain. In some embodiments, the anti-ITGA4 scFv domain is selected from a sequence listed in Table 19. Table 19 ITGA4 scFv Binding DomainsAttorney Ref.061250-557001WO_

[0510] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CD162, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CD162 antibody is a human anti-CD162 antibody, a humanized anti-CD162 antibody, or a chimeric anti-CD162 antibody. In some embodiments, the anti-CD162 antibody is an anti-CD162 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CD162 is specific to one CD162 isoform or binds to more than one CD162 isoform, e.g., is a pan- CD162 antibody or antigen-binding fragment.

[0511] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavyAttorney Ref.061250-557001WO chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 21. Table 21 CDRs Corresponding to CD162 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0512] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 22. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 22. Table 22 VH and VL Corresponding to CD162 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0513] In some embodiments, the binding domain is an anti-CD162 scFv domain. In some embodiments, the anti-CD162 scFv domain is selected from a sequence listed in Table 23. Table 23 CD162 scFv Binding DomainsAttorney Ref.061250-557001WO

[0514] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CD53, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CD53 antibody is a human anti-CD53 antibody, a humanized anti-CD53 antibody, or a chimeric anti- CD53 antibody. In some embodiments, the anti-CD53 antibody is an anti-CD53 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CD53 is specific to one CD53 isoform or binds to more than one CD53 isoform, e.g., is a pan-CD53 antibody or antigen-binding fragment.

[0515] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 25.Attorney Ref.061250-557001WO

[0516] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 26. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 26. Table 26 VH and VL Corresponding to CD53 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0517] In some embodiments, the binding domain is an anti-CD53 scFv domain. In some embodiments, the anti-CD53 scFv domain is selected from a sequence listed in Table 27. Table 27 CD53 scFv Binding Domains

[0518] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CLL1, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CLL1 antibody is a human anti-CLL1 antibody, a humanized anti-CLL1 antibody, or a chimeric anti-CLL1 antibody. In some embodiments, the anti-CLL1 antibody is an anti-CLL1 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CLL1 is specific to one CLL1 isoform or binds to more than one CLL1 isoform, e.g., is a pan- CLL1 antibody or antigen-binding fragment.Attorney Ref.061250-557001WO

[0519] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 45. Table 45: CDRs Corresponding to CLL1 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0520] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 46. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full lengthAttorney Ref.061250-557001WO VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 46. Table 46 VH and VL Corresponding to CLL1 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0521] In some embodiments, the binding domain is an anti-CLL1 scFv domain. In some embodiments, the anti-CLL1 scFv domain is selected from a sequence listed in Table 47. Table 47 CLL1 scFv Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOCytoplasmic Activation Domain

[0522] The cytoplasmic domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the cytoplasmic activation domain ensures that there is T- cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In some QYN[PUYQZ`_& `TQ M` XQM_` [ZQ Oe`[\XM_YUO MO`UbM`U[Z U_ M 89,.1 Y[XQOaXQ #89-u$ MO`UbM`U[Z P[YMUZ& a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX- MO`UbM`UZS \^[`QUZ [R +, W9M #96D+,$ MO`UbM`U[Z P[YMUZ( >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 33).

[0523] >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_Q_ [^ O[Z_U_`_ Q__QZ`UMXXe [R G:E >9 BC4 --( >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ U_ QZO[PQP Ne M ZaOXQ[`UPQ sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGCGTA GAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGACTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID BC4 -.$( >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ U_ QZO[PQP Ne G:E >9 BC4 -.(Attorney Ref.061250-557001WO Chimeric Antigen Receptors (CARs)

[0524] In some embodiments, the either the first or the second engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the first and second engineered receptors are chimeric antigen receptors. All CAR architectures are envisaged as within the scope of the instant disclosure.

[0525] The term “chimeric antigen receptors” or “CARs” as used herein, may refer to artificial T- cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell, such as a helper T cell (CD4+), cytotoxic T cell (CD8+) or NK cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen such as a CD45 antigen. In some embodiments, CARs comprise an extracellular domain comprising an antigen-binding region, a transmembrane domain, and an intracellular signaling domain. In some embodiments, CARs comprise fusions of single-chain variable fragments (scFvs) or scFabs derived from monoclonal antibodies, fused to a transmembrane domain and intracellular signaling domain(s). The fusion may also comprise a hinge. Either heavy-light (H-L) and light-heavy (L-H) scFvs may be used. The specificity of CAR designs may be derived from ligands of receptors (e.g., peptides). Depending on the type of intracellular domain, a CAR can be an activator receptor or an inhibitor receptor. In some embodiments, for example, when the CAR is an activator receptor, the CAR comprises domains for additional co-stimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors. As used herein, characteristics attributed to a chimeric antigen receptor may be understood to refer to the receptor itself or to a host cell comprising the receptor. Extracellular Domains

[0526] In some embodiments, the extracellular domain of the CAR comprises the antigen binding domains described supra. Hinge Region

[0527] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Illustrative hinges can be isolated or derived from IgG, CD28 or CD8, among others, for example IgG1. Illustrative hinge domains are provided in Table 7A.Attorney Ref.061250-557001WO

[0528] >Z _[YQ QYN[PUYQZ`_& `TQ TUZSQ U_ U_[XM`QP [^ PQ^UbQP R^[Y 892n [^ 89,2( >Z _[YQ QYN[PUYQZ`_& `TQ 892n TUZSQ O[Y\^U_Q_ MZ MYUZ[ MOUP _Q]aQZOQ TMbUZS M` XQM_` 2*" UPQZ`U`e& M` least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 35). In some QYN[PUYQZ`_& `TQ 892n TUZSQ O[Y\^U_Q_ G:E >9 BC4 - / ( >Z _[YQ QYN[PUYQZ`_& `TQ 892n TUZSQ O[Z_U_`_ Q__QZ`UMXXe [R G:E >9 BC4 - / ( >Z _[YQ QYN[PUYQZ`_& `TQ 892n TUZSQ U_ QZO[PQP Ne M nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCC TGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG GGCTGGACTTCGCCTGTGAT (SEQ ID NO: 36).

[0529] >Z _[YQ QYN[PUYQZ`_& `TQ 892n TUZSQ U_ QZO[PQP Ne G:E >9 BC4 -0(

[0530] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 37). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 37. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of:

[0531] TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAAT GGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGAC CTTCTAAGCCC (SEQ ID NO: 38).

[0532] In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 38.

[0533] In some embodiments, the activator receptor comprises a hinge sequence isolated or derived from CD8, CD28, IgG1, or IgG4, or a synthetic hinge. Table 7A: Hinge Domain Sequences and their use in Receptors

[0534] In some embodiments, for example those embodiments where the CAR is an activator receptor, the hinge comprises a sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or that is identical thereto. In some embodiments, for example those embodimentsAttorney Ref.061250-557001WO where the CAR is an activator receptor, the hinge comprises a sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. Transmembrane Domain

[0535] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0536] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0537] In some embodiments of the CARs of the disclosure, the CARs comprise a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 49). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 49. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGT GGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 50).

[0538] In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 50.

[0539] In some embodiments of the CARs of the disclosure, the CARs comprise an IL-2Rbeta transmembrane domain. In some embodiments, the IL-2Rbeta transmembrane domain comprises anAttorney Ref.061250-557001WO amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 51). In some embodiments, the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 51. In some embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of ATTCCGTGGCTCGGCCACCTCCTCGTGGGCCTCAGCGGGGCTTTTGGCTTCATCATCTT AGTGTACTTGCTGATC (SEQ ID NO: 52).

[0540] In some embodiments, the IL-2Rbeta transmembrane domain is encoded by SEQ ID NO: 52.

[0541] In some embodiments, the CAR comprises a transmembrane domain isolated or derived from CD8 or CD28. In some embodiments, the CAR comprises a transmembrane domain sequence in Table 7B. In some embodiments, the CAR comprises a transmembrane domain sequence of SEQ ID NO: 49, 51, 53, or 55, or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical thereto. In some embodiments, the CAR comprises a transmembrane domain sequence of SEQ ID NO: 49, 51, 53, or 55. Table 7B: Transmembrane Domain Sequences and their use in CAR ConstructsCytoplasmic Domain

[0542] The disclosure provides an activator receptor comprising an extracellular ligand binding domain specific to an activator antigen. In some embodiments, the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an intracellular domain isolated or derived from CD28, 4-1BB or CD3z, or a combination thereof.

[0543] The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Effector functions of a regulatory T cell, for example, include the suppression or downregulation of induction or proliferation of effector T cells. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multipleAttorney Ref.061250-557001WO intracellular domains can be combined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0544] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0545] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences).

[0546] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL) (SEQ ID NO: 57).

[0547] In some embodiments, the cytoplasmic domain contains 1, 2, or 3 ITAMs. In some embodiments, the cytoplasmic domain contains 1 ITAM. In some embodiments, the cytoplasmic domain contains 2 ITAMs. In some embodiments, the cytoplasmic domain contains 3 ITAMs. In some embodiments, the cytoplasmic domain contains 4 ITAMs. In some embodiments, the cytoplasmic domain contains 5 ITAMs.

[0548] >Z _[YQ QYN[PUYQZ`_& `TQ Oe`[\XM_YUO P[YMUZ U_ M 89-u MO`UbM`U[Z P[YMUZ( >Z _[YQ QYN[PUYQZ`_& 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_Q_ M _UZSXQ >H6A( >Z _[YQ QYN[PUYQZ`_& 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_Q_ `c[ >H6A_( >Z _[YQ QYN[PUYQZ`_& 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_Q_ three ITAMs.

[0549] >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_UZS M _UZSXQ >H6A O[Y\^U_Q_ MZ amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 58). In some QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_Q_ G:E >9 BC4 / 2( >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_UZS M _UZSXQ >H6A O[Z_U_`_ Q__QZ`UMXXe [R MZ MYUZ[ MOUP _Q]aQZOQ of SEQ ID NO: 58.Attorney Ref.061250-557001WO

[0550] >Z _[YQ QYN[PUYQZ`_& `TQ 89-u MO`UbM`U[Z P[YMUZ O[Y\^U_UZS M _UZSXQ >H6A U_ QZO[PQP by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG HHHH<8686H<86<<888H<88888H8<8 #G:E >9 BC4 / 3$( >Z _[YQ QYN[PUYQZ`_& `TQ 89-u activation domain is encoded by SEQ ID NO: 59.

[0551] Further examples of ITAM containing primary cytoplasmic signaling sequences that can be a_QP UZ `TQ 86F_ [R `TQ UZ_`MZ` PU_OX[_a^Q UZOXaPQ `T[_Q PQ^UbQP R^[Y H8Fu& ;OFt& ;OFs& 89-t& 89-o& 89-p& 89-u& 89 / & 89,,& 8913M& 8913N& MZP 8900P( >` U_ \M^`UOaXM^Xe \^QRQ^^QP `TM` cytoplasmic signaling molecule in the CAR of the instant invention comprises a cytoplasmic _USZMXUZS _Q]aQZOQ PQ^UbQP R^[Y 89-u( Co-Stimulatory Domain

[0552] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the 89-u _USZMXUZS P[YMUZ Ne U`_QXR [^ O[YNUZQP cU`T MZe [`TQ^ PQ_U^QP Oe`[\XM_YUO P[YMUZ#_$ a_QRaX in the context of the CAR of the instant disclosure. For example, the cytoplasmic domain of the CAR OMZ O[Y\^U_Q M 89-u OTMUZ \[^`U[Z MZP M O['_`UYaXM`[^e P[YMUZ( HTQ O['_`UYaXM`[^e P[YMUZ ^QRQ^_ to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include `TQ O['_`UYaXM`[^e P[YMUZ U_ _QXQO`QP R^[Y `TQ S^[a\ O[Z_U_`UZS [R >@',Fs& ;O FQOQ\`[^ SMYYM #;OFt$& ;O FQOQ\`[^ NQ`M #;OFs$& 89-S Y[XQOaXQ SMYYM #89-t$& 89-o& 89-p& 89 / Y[XQOaXQ (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

[0553] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.Attorney Ref.061250-557001WO

[0554] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 60).

[0555] In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 60. In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGG CCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCT ACCGGAGC (SEQ ID NO: 61). In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 61.

[0556] In some embodiments, the intracellular domain of the CARs of the instant disclosure comprises an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2Rbeta domain is truncated. In some embodiments, the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs. In some embodiments, the CAR comprises one or more STAT5-recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

[0557] In some embodiments, the IL-2-Rbeta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEV LERDKVTQLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 62). In some embodiments, the IL2R-beta intracellular domain comprises or consists essentially of SEQ ID NO: 62. In some embodiments, the IL-2R-beta intracellular domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical to a sequence of AACTGCAGGAACACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGAC CCCTCGAAGTTCTTTTCCCAGCTGAGCTCAGAGCATGGAGGCGACGTCCAGAAGTGGC TCTCTTCGCCCTTCCCCTCATCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCG CCACTAGAAGTGCTGGAGAGGGACAAGGTGACGCAGCTGCTCCCCCTGAACACTGATG CCTACTTGTCTCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTG (SEQ ID NO: 63). In some embodiments, the IL-2R-beta intracellular domain is encoded by SEQ ID NO: 63.Attorney Ref.061250-557001WO

[0558] In some embodiments, the IL-2R-beta cytoplasmic domain comprises one or more STAT5- recruitment motifs. Illustrative STAT5-recruitment motifs are provided by Passerini et al. (International Immunology, Vol. 20, No. 3, pp. 421–431, 2008) and by Kagoya et al. (Nature Medicine doi:10.1038 / nm.4478, 2018).

[0559] In some embodiments, the STAT5-recruitment motif(s) consists of the sequence Tyr-Leu- Ser-Leu (SEQ ID NO: 64).

[0560] In some embodiments, the CAR comprises an intracellular domain isolated or derived from 89,2& .'+77 MZP)[^ 89-u& [^ M O[YNUZM`U[Z `TQ^Q[R( >XXa_`^M`UbQ P[YMUZ_ PQ^UbQP R^[Y 89,2& .' +77 MZP)[^ 89-u M^Q _T[cZ UZ Table 7C below. In some embodiments, the CAR comprises an intracellular domain comprising a sequence of SEQ ID NOS: 65, 67, or 69, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or that is identical thereto. In some embodiments, the CAR comprises an intracellular domain comprising a sequence of SEQ ID NO: 65 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or that is identical thereto. In some embodiments, the CAR comprises an intracellular domain comprising a sequence of SEQ ID NOS: 65, 67, or 69. In some embodiments, the CAR comprises an intracellular domain comprising CD28-4-1BB-CD3z and having a sequence of SEQ ID NO: 65. In some embodiments, the CAR intracellular domain is encoded by the sequence set forth in any one of SEQ ID NOs: 66, 68, or 70. In some embodiments, the CAR intracellular domain is encoded by the sequence set forth in SEQ ID NO: 66. Table 7C: Intracellular Domain Sequences and their use in CAR ConstructsCD45 Targeted CAR

[0561] The CD45 targeted CAR (also called “anti-CD45 CAR”) or CD45 targeted polypeptide described herein may include a CD45 targeting scFv.

[0562] In some embodiments, the first receptor is an anti-CD45 CAR comprising any one of SEQ ID NOS: 133-140, 142, 144, 146, 148, 151, 153, 155, 156-168, 328, 329, 330 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0563] In some embodiments, nucleic acids (e.g., isolated nucleic acids) encoding one or more chimeric antigen receptors are provided. In some embodiments, the nucleic acid is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the nucleic acid is a DNA construct.Attorney Ref.061250-557001WO

[0564] In some embodiments, the first receptor is an anti-CD45 CAR encoded by a polynucleotide comprising SEQ ID NO: 141, 143145, 147, 149, 150, 152, 154, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. CD33 Targeted CAR

[0565] The CD33 targeted CAR (also called “anti-CD33 CAR”) or CD33 targeted polypeptide described herein may include a CD33 targeting scFv.

[0566] In some embodiments, the first receptor is an anti-CD33 CAR comprising any one of SEQ ID NOS: 313-318 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. CD123 Targeted CAR

[0567] The CD123 targeted CAR (also called “anti-CD123 CAR”) or CD123 targeted polypeptide described herein may include a CD123 targeting scFv.

[0568] In some embodiments, the first receptor is an anti-CD123 CAR comprising any one of SEQ ID NOS: 420-423 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. SPN Targeted CAR

[0569] The SPN targeted CAR (also called “anti-SPN CAR”) or SPN targeted polypeptide described herein may include an SPN targeting scFv.

[0570] In some embodiments, the first receptor is an anti-SPN CAR comprising an SPN targeting scFv comprising a VH comprising any one of SEQ ID NOS: 249-252, 257, 259, 261-280 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 253-256, 258, 260, 281-303 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. ITGA4 CAR

[0571] The ITGA4 targeted CAR (also called “anti-ITGA4 CAR”) or ITGA4 targeted polypeptide described herein may include an ITGA4 targeting scFv.

[0572] In some embodiments, the first receptor is an anti-ITGA4 CAR comprising an ITGA4 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 2910-2971 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 2972-3033 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.Attorney Ref.061250-557001WO Table 20 Full Length Receptor Corresponding to ITGA4 Antigen Binding DomainsAttorney Ref.061250-557001WOCD162 CAR

[0573] The CD162 targeted CAR (also called “anti-CD162 CAR”) or CD162 targeted polypeptide described herein may include an CD162 targeting scFv.

[0574] In some embodiments, the first receptor is an anti-CD162 CAR comprising an CD162 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 3243-3293 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 3294-3344 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. Table 24 Full Length Receptor Corresponding to CD162 Antigen Binding DomainsAttorney Ref.061250-557001WOCD53 CAR

[0575] The CD53 targeted CAR (also called “anti-CD53 CAR”) or CD53 targeted polypeptide described herein may include an CD53 targeting scFv.

[0576] In some embodiments, the first receptor is an anti-CD53 CAR comprising an CD53 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 3524-3540 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 3541-3557 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.Attorney Ref.061250-557001WO Table 28 Full Length Receptor Corresponding to CD53 Antigen Binding DomainsCLL1 CAR

[0577] The CLL1 targeted CAR (also called “anti-CLL1 CAR”) or CLL1 targeted polypeptide described herein may include an CLL1 targeting scFv.

[0578] In some embodiments, the first receptor is an anti-CLL1 CAR comprising a CLL1 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 5587-5658 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 5659-5730 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0579] In some embodiments, the first receptor is an anti-CLL1 CAR comprising a CLL1 targeting scFv comprising a combination of CDRs from any row of Table 45. In some embodiments, the first receptor is an anti-CLL1 CAR comprising a CLL1 targeting scFv comprising a combination of VH and VL from any row of table 46. Table 48: Full Length Receptor Corresponding to CLL1 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0580] In some embodiments, the first receptor is an anti_CLL1 CAR comprising a CLL1 targeting scFv comprising a combination of CDRs from any row of Table 45. T Cell Receptors (TCRs)

[0581] The disclosure provides a first activator receptor and immune cells comprising same. In some embodiments, the first receptor is a T cell receptor (TCR). Illustrative TCRs comprising cytoplasmic domains for use in the instant disclosure are described in PCT / US2020 / 045250, the contents of which are incorporated herein by reference.

[0582] As used herein, a “TCR,” sometimes also called a “TCR complex” or “TCR / CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha / beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an illustrative 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 two CD3 zeta form a homodimer.

[0583] Any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the TCRs described herein. For example, the ligand binding domain can be an MZ`USQZ NUZPUZS P[YMUZ [R MZ MZ`UN[Pe [^ H8F& [^ O[Y\^U_Q MZ MZ`UN[Pe R^MSYQZ`& M Js [ZXe P[YMUZ& a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).Attorney Ref.061250-557001WO

[0584] In some embodiments, the ligand binding domain is fused to one or more extracellular domains or transmembrane domains of one or more TCR subunits. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma or CD3 zeta. For example, the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.

[0585] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.

[0586] TCR transmembrane domains isolated or derived from any source are envisaged as within the scope of the disclosure. The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein.

[0587] In some embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target. A transmembrane domain of particular use in this disclosure may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0588] In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identical to a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 71). In some embodiments, the TCR alpha transmembrane domain comprises, or consists essentially of, SEQ ID NO: 71. In some embodiments, the TCR alpha transmembrane domain is encoded by a sequence of GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCT GCGGCTGTGG (SEQ ID NO: 72).

[0589] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identical to a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 73). In some embodiments, the TCR beta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 73. In some embodiments, the TCR beta transmembrane domain is encoded by a sequence ofAttorney Ref.061250-557001WO ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGC CCTCGTGCTG (SEQ ID NO: 74).

[0590] TCRs of the disclosure can comprise one or more intracellular domains. In some embodiments, the intracellular domain comprises one or more domains capable of providing a stimulatory signal to a transmembrane domain. In some embodiments, the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain comprises a first, second and third intracellular domain capable of providing a stimulatory signal. The intracellular domains capable of providing a stimulatory signal are selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

[0591] In some embodiments, an intracellular domain comprises at least one intracellular signaling domain. An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T-cell. In some embodiments, the intracellular domain of the first receptor of the disclosure includes at least one intracellular signaling domain. For example, the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.

[0592] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.

[0593] Examples of intracellular domains for use in activator receptors of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0594] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Illustrative primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.Attorney Ref.061250-557001WO

[0595] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, a CD3 zeta intracellular domain, a TCR alpha intracellular domain or a TCR beta intracellular domain.

[0596] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, a TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, a TCR alpha intracellular domain is encoded by a sequence of TCCAGC (SEQ ID NO: 75).

[0597] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity,, or 100% identical to a sequence of: MAMVKRKDSR (SEQ ID NO: 76). In some embodiments, the TCR beta intracellular domain comprises, or consists essentially of SEQ ID NO: 76. In some embodiments, the TCR beta intracellular domain is encoded by a sequence of ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 77).

[0598] In some embodiments, the intracellular signaling domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0599] Illustrative co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecule (CD28). A co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin- like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like. The co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional variant thereof.

[0600] In some embodiments, the stimulatory domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a CD28 or 4-1BB co-stimulatory domain. CD28 and 4-1BB are well characterized co-stimulatory molecules required for full T cell activation andAttorney Ref.061250-557001WO known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain. Likewise, inclusion of co- stimulatory domains, for example CD28 and 4-1BB domains, in TCRs can increase T cell effector function and specifically allow co-stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells. In some embodiments, the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain. Inhibitor Receptors

[0601] The disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to a non-cancerous antigen that has been lost in a cancer cell. The antigen can be lost in the cancer cell through any mechanism, such as, without limitation, epigenetic changes that effect antigen expression, mutations to the gene encoding the non-cancerous antigen, disruption of cellular signaling that regulates expression of the non-cancerous antigen, chromosome loss, partial or complete deletion of the genomic locus, gene silencing through modification of nucleic acids or heterochromatin, or loss of expression through other mechanisms. In variations of the compositions and methods disclosed herein, the cells or subject treated may exhibit a loss of expression of the non- cancerous antigen because of non-genetic changes. Accordingly, the disclosure provides compositions and methods for killing cells lacking expression of the non-cancerous antigen from any cause.

[0602] Illustrative inhibitory receptors are described in PCT / US2020 / 045228, PCT / US2020 / 064607, PCT / US2021 / 029907, and PCT / US2020 / 059856, the contents of each of which are incorporated herein by reference.

[0603] The term “inhibitor chimeric antigen receptor” or “inhibitor receptor” as used herein refers to an antigen-binding domain that is fused to an intracellular signaling domain capable of transducing an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitor receptors have immune cell inhibitory potential, and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential. For example, CARs are activating receptors as they include intracellular stimulatory and / or co-stimulatory domains. Inhibitor receptors are inhibiting receptors that contain intracellular inhibitory domains.

[0604] As used herein “inhibitory signal” refers to signal transduction or changes in protein expression in an immune cell resulting in suppression of an immune response (e.g., decrease in cytokine production or reduction of immune cell activation). Inhibition or suppression of an immune cell can be selective and / or reversible, or not selective and / or reversible. Inhibitor receptors are responsive to non-cancerous or allogeneic donor cell antigens expressed on normal or allogeneic donor cells (e.g. CD16B or HLA-A*02). For example, when a non-cancerous or allogeneic donorAttorney Ref.061250-557001WO cell antigen (e.g. CD16B or HLA-A*02) binds to or contacts the inhibitor receptor, the inhibitor receptor is responsive and initiates an inhibitory signal in the immune cell expressing the inhibitor receptor. Upon binding of the non-cancerous or allogeneic donor cell antigen by the extracellular ligand binding domain of the inhibitor receptor, the inhibitor receptor inhibits activation of an immune cell expressing the engineered inhibitor receptor.

[0605] Inhibitory receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure. Inhibitory receptors are responsive to allogeneic donor cell antigens (e.g. HLA-A*02). For example, when an allogeneic donor cell antigen (e.g. HLA-A*02) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in the immune cell expressing the inhibitory receptor upon binding of the allogeneic donor cell antigen by the extracellular ligand binding domain of the inhibitory receptor.

[0606] A “non-cancerous antigen” or “non-cancerous ligand” as used herein refers to a gene whose expression is reduced or eliminated in a cancer cell, but is expressed or detectably expressed in a normal or healthy cell (e.g., CD16B). For example, a subject may have a cancer in which the cancer cells express SPN and / or CD33. In such a subject, because the inhibitor receptor is designed to be specific to CD16B (or other non-cancerous antigen), the inhibitor receptor of immune cells encountering the cancer cell does not release an inhibitory signal. In normal non-cancerous cells, CD16B (or other non-cancerous antigen) is present and inhibits activation of the engineered immune cell. By this mechanism, the immune cell is selectively activated, and selectively kills, cancer cells expressing SPN and / or CD33 but having lost CD16B (or another non-cancerous antigen). In some embodiments, the non-cancerous antigen is not expressed in a cancer cell of the subject. In some embodiments, the non-cancerous antigen is not expressed in a fraction of the cells in a tumor in the subject. In some embodiments, the non-cancerous antigen is expressed by a cancer cell at a lower expression level relative to the level of expression in a non-cancerous cell.

[0607] In some embodiments, an activator ligand is an antigen expressed by a cancer cell, or a cell capable of becoming a cancer cell. In some embodiments, an activator ligand is an antigen expressed by a blood cancer cell. In some embodiments, an activator ligand is an antigen expressed by an AML cell.

[0608] In some embodiments, a cancer cell in a subject has lost expression of the non-cancerous antigen. Loss of expression or lack of expression of the non-cancerous antigen in a cell can be by any mechanism, such as, without limitation, epigenetic changes that effect non-cancerous gene expression, mutations to the gene encoding the non-cancerous antigen, genetic deletion, or disruption of cellular signaling that regulates expression of the non-cancerous gene.Attorney Ref.061250-557001WO

[0609] An “allogeneic donor cell antigen” or “allogeneic donor cell ligand” as used herein refers to a gene whose expression is reduced or eliminated in an allogeneic donor cell, but is expressed or detectably expressed in a subject’s cell. For example, a subject may have a cancer in which the cancer cells express SPN and / or CD33 and non-cancerous blood cells which express SPN and / or CD33. In such a subject, because the inhibitor receptor is designed to be specific to HLA-A*02 (or other allogeneic donor cell antigen), the inhibitor receptor of immune cells encountering the subject’s cell does not release an inhibitory signal. In allogeneic donor cells, HLA-A*02 (or other allogeneic donor cell antigen) is present and inhibits activation of the engineered immune cell. By this mechanism, the immune cell is selectively activated, and selectively kills, a subject’s cells expressing SPN and / or CD33 but not expressing HLA-A*02 (or another allogeneic donor cell antigen). The allogeneic donor cell may be a stem cell. The stem cell may be a HSC.

[0610] In some embodiments, the second receptor is an inhibitor chimeric antigen receptor (i.e. inhibitor receptor). In some embodiments, the second receptor is an inhibitor receptor. In some embodiments, the second receptor is humanized.

[0611] The disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding domain that can discriminate between different levels of expression of a non-cancerous or allogeneic donor cell antigen. This allows the second receptor to inhibit activation of immune cells comprising the second receptor when contacted by non-cancerous or allogeneic donor cells that express the ligand for the second receptor, but to allow activation of immune cells that are contacted by cancer or the subject’s cells that express low levels, or have no expression, of the ligand for the second receptor. Inhibitor Ligands

[0612] The disclosure provides a second ligand and a second inhibitor receptor comprising a second ligand binding domain that binds to the second ligand. In some embodiments, the second ligand is an inhibitor ligand. In some embodiments, the second ligand is an antigen, and the second inhibitor receptor comprises an antigen binding domain that specifically recognizes the second ligand antigen.

[0613] The disclosure provides a second inhibitor receptor comprising an extracellular region, the extracellular region comprising a second ligand binding domain capable of specifically binding to a second ligand that inhibits activation of effector cells expressing the first and second receptors, wherein the effector cells are activated by binding only the first ligand to the first activator receptor.

[0614] As used herein an “inhibitor” or “inhibitor ligand” or “inhibitor antigen” or “blocker antigen” refers to a second ligand that binds to a second ligand binding domain (inhibitor LBD) of an engineered receptor of the disclosure, but inhibits activation of an immune cell expressing the engineered inhibitor receptor. The inhibitor is not expressed by the cancerous cells. The inhibitor ligand is expressed in a plurality of normal, non-cancerous cells, including normal, non-cancerousAttorney Ref.061250-557001WO cells that express the activator ligand, thereby protecting these cells from the cytotoxic effects of the adoptive cell therapy. Without wishing to be bound by theory, inhibitor ligands can block activation of the effector cells through a variety of mechanisms. For example, binding of the inhibitor ligand to the inhibitor LBD can block transmission of a signal that occurs upon binding of the activator ligand to the activator LBD that would, in the absence of the inhibitor, lead to activation of the immune cell expressing the engineered receptors described herein.

[0615] In some embodiments, the inhibitor is not expressed by the subject’s cells. The inhibitor ligand is expressed in a plurality of allogeneic donor cells, including allogeneic donor cells that express the A antigen, thereby protecting these cells from the cytotoxic effects of the adoptive cell therapy. Without wishing to be bound by theory, inhibitor ligands can block activation of the effector cells through a variety of mechanisms. For example, binding of the inhibitor ligand to the inhibitor LBD can block transmission of a signal that occurs upon binding of the A antigen to the activator LBD that would, in the absence of the inhibitor, lead to activation of the immune cell expressing the engineered receptors described herein.

[0616] Alternatively, or in addition, binding of the inhibitor ligand to the second engineered receptor can cause loss of cell surface expression the first, activator receptor from the surface of the immune cells comprising the two receptor system described herein. Without wishing to be bound by theory, it is thought that immune cell engagement of activator and inhibitor ligands on normal cells acts in part by causing the inhibitory receptor to cause removal of nearby activator receptor molecules from the immune cell surface. This process locally desensitizes the immune cell, reversibly raising its activation threshold. Immune cells that engage only the activator ligand on a cancer cell cause local activation signals which are unimpeded by signals from the second, inhibitory receptor. This local activation increases until release of cytotoxic granules leads to cancer cell selective cell death. However, modulation of surface receptor expression levels may not be the only mechanism by which inhibitory receptors inhibit activation of immune cells by the first activator receptor. Without wishing to be bound by theory, other mechanisms may come into play, including, but not limited to, cross- talk between activator and inhibitory receptor signaling pathways.

[0617] In some embodiments, the second ligand is not expressed by the cancer cells, and is expressed by the non-cancerous cells. In some embodiments, the non-cancerous antigen is not expressed by the cancer cells, and is expressed by non-cancerous cells. In some embodiments, the non-cancerous antigen is expressed by healthy cells, i.e. cells that are not cancer cells. In some embodiments, the cancer cells have lost expression of the non-cancerous antigen. In some embodiments, the non- cancerous cells are a plurality of healthy cells (i.e., normal, or healthy cells), that express both the cancer antigen and the non-cancerous antigen.Attorney Ref.061250-557001WO

[0618] In some embodiments, the inhibitor ligand is an antigen and the second ligand binding domain comprises a scFv domain.

[0619] >Z _[YQ QYN[PUYQZ`_& `TQ UZTUNU`[^ XUSMZP NUZPUZS P[YMUZ O[Y\^U_Q_ M Js'[ZXe XUSMZP binding domain.

[0620] In some embodiments, the inhibitor ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR). For example, the inhibitor ligand binding P[YMUZ O[Y\^U_Q_ H8F n MZP s OTMUZ bM^UMNXQ P[YMUZ_(

[0621] In some embodiments, the inhibitor ligand binding domain is an antigen binding domain. Suitable antigen-binding domains include, but are not limited to, antigen-binding domains from antibodies, antibody fragments, scFv, antigen-binding domains derived from T cell receptors, and the like.

[0622] Any cell surface molecule expressed by the non-cancerous cells that is not expressed by cancer cells may be a suitable non-cancerous antigen for the second receptor extracellular ligand binding domain. For example, a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane domain, a receptor for a neurotransmitter, or a voltage gated ion channel can be used as a non-cancerous antigen.

[0623] Alternatively, or in addition, expression of activator and inhibitor antigens may be correlated, i.e. the two are expressed at similar levels on non-cancerous cells.

[0624] In some embodiments, the inhibitor ligand is a peptide ligand. In some embodiments, the inhibitor ligand is a peptide antigen.

[0625] In some embodiments, the non-cancerous antigen is lost in the cancer cells. In some embodiments, the inhibitor ligand is encoded by a gene that is absent or polymorphic in blood cancer cells.

[0626] Methods of distinguishing the differential expression of inhibitor ligands between cancerous and non-cancerous cells will be readily apparent to the person or ordinary skill in the art. For example, the presence or absence of inhibitor ligands in non-cancerous and cancerous cells can be assayed by immunohistochemistry with an antibody that binds to the inhibitor ligand, followed by microscopy or FACS, RNA expression profiling of cancer cells and non-cancerous cells, or DNA sequencing of non-cancerous and cancerous cells to determine if the genomic locus of the inhibitor ligand comprises mutations or deletions in either the cancerous or non-cancerous cells. CDCP1

[0627] In some embodiments, the non-cancerous antigen is CUB domain-containing protein 1 (CDCP1). In some embodiments, the non-cancerous antigen comprises a peptide of CDCP1. In some embodiments, the CDCP1 non-cancerous cell antigen is expressed by healthy cells of a subject.Attorney Ref.061250-557001WO

[0628] In some embodiments, the inhibitor ligand is CDPC1.

[0629] The term “CDCP1” (also known as SIMA135, TRASK, CD318 and gp140) refers to a 140 kD transmembrane glycoprotein with a large extracellular domain containing three CUB domains, and a smaller intracellular domain. CDCP1 may be cleaved by serine proteases at the extracellular domain to generate a truncated molecule of 80 kDa size. Reference to CDCP1 is also intended to include a further isoform (UniProt number Q9H5V8-3) having an NK to SE substitution at 342 and 343 and amino acids 344-836 missing.

[0630] In vivo, CDCP1 is not cleaved during normal physiological circumstances, but its cleavage may be induced during tumorigenesis or tissue injury.

[0631] Western blot analysis has demonstrated that CDCP1 is a phosphotyrosine protein, consistent with the presence of 5 intracellular tyrosine residues. In addition, the inhibitor PP2 has been used to demonstrate that a Src kinase family member acts to phosphorylate the tyrosines of CDCP1. CDCP1 may contribute to metastasis, in part, by allowing cancer cells to survive and metastasize in the absence of attachment. In the MDA-MB-468 breast cancer cell line, enforced CDCP1 expression induced cell detachment and growth in suspension even in the presence of a suitable adhesive substrate. However, CDCP1-mediated cell detachment is not observed universally, and how CDCP1 induces suspension growth in specific circumstances is unknown.

[0632] In some embodiments, the inhibitor ligand comprises CDCP1. There are several CDCP1 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. ABCG2

[0633] In some embodiments, the inhibitor ligand is ABCG2.

[0634] The term “ABCG2” (also known as ATP-binding cassette super-family G member 2, CD338, and CDw338) refers to an ATP-binding cassette (ABC) transporter protein. ABCG2 actively pumps drugs and other compounds against their concentration gradient using the bonding and hydrolysis of ATP as the energy source. The expression of this transport protein is highly conserved throughout the animal kingdom. ABCG2 forms into a homodimer to assume its active transport conformation.

[0635] Substrate binding with compounds occurs in the large central cavity. ABCG2 can bind to a broad range of compounds but binds strongest to flat, polycyclic chemicals with lots of hydrophobic character. In some embodiments, the inhibitor ligand comprises ABCG2. There are several ABCG2 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. CD16A

[0636] In some embodiments, the inhibitor ligand is FCGR3A.

[0637] The term “CD16A” (also known as FCGR3A, also known as low affinity immunoglobulin SMYYM ;O ^QSU[Z ^QOQ\`[^ >>>'6& ;OtF>>>6$ ^QRQ^_ `[ M ^QOQ\`[^ R[^ `TQ UZbM^UMNXQ ;O R^MSYQZ` [R immunoglobulin gamma (IgG). CD16a is optimally activated upon binding of clustered antigen-IgGAttorney Ref.061250-557001WO complexes displayed on cell surfaces, and triggers lysis of antibody-coated cells, a process known as antibody-dependent cellular cytotoxicity (ADCC). CD16A does not bind free monomeric IgG, thus avoiding inappropriate effector cell activation. CD16A binds antigen-IgG complexes generated upon infection and triggers NK cell-dependent cytokine production and degranulation to limit viral load and propagation. CD16A is involved in the generation of memory-like adaptive NK cells capable to produce high amounts of IFNG and to efficiently eliminate virus-infected cells via ADCC. CD16A is expressed on NK cells, monocytes, and macrophages. The major polymorphism in CD16A is a point mutation (T>G) at nucleotide 559 (rs396991), which results in either a valine (V158) or phenylalanine (F158) at amino acid position 158. These alleles are also co-dominantly expressed. In some embodiments, the inhibitor ligand comprises CD16A. There are several CD16A variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. CD16B

[0638] In some embodiments, the inhibitor ligand is CD16B.

[0639] The term “CD16B” (also known as FCGR3B (Fc fragment of IgG, low affinity IIIb, receptor), ;8<-7& ;OtF>>>7& MZP 8Xa_`Q^ [R 9URRQ^QZ`UM`U[Z +07$ ^QRQ^_ `[ M X[c MRRUZU`e ^QOQ\`[^ R[^ `TQ ;O region of IgG gamma. CD16B binds complexed or aggregated IgG and also monomeric IgG and plays a role in phagocytosis and the clearance of immune complexes.

[0640] In some embodiments, the inhibitor ligand comprises CD16B. There are several CD16B variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. Human CD16A and CD16B are 97% identical by polypeptide sequence alignment (FIG.46C) with 6 amino acid differences. PECAM-1

[0641] In some embodiments, the inhibitor ligand is PECAM-1.

[0642] The term “PECAM-1” (also known as Platelet endothelial cell adhesion molecule and CD31) refers to a hemophilic cell adhesion molecule. PECAM-1 is a type I transmembrane protein with six C2-set Ig domains. Homophilic interactions between leukocyte PECAM-1 and endothelial PECAM- 1 are required for the trans-endothelial migration (TEM) of leukocytes. Leukocyte PECAM-1 engages endothelial PECAM-1 upon reaching the apical side of the endothelial border, and when this interaction is disrupted through genetic ablation or function-blocking reagents, leukocytes are arrested on the apical surface of the endothelial cell, above the junction. There are several PECAM- 1 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. HLA-G

[0643] In some embodiments, the inhibitor ligand is HLA-G.

[0644] The term “HLA-G” (also known as HLA-G histocompatibility antigen class I G and human leukocyte antigen G) refers to a non-classical HLA class I heavy chain paralogue. HLA-G sharesAttorney Ref.061250-557001WO 86% sequence similarity with classical HLA class I genes; however, a stop codon in exon 6 produces a mature HLA-G molecule that is shorter than its classical class I counterparts. Alternative splicing of the primary transcript produces seven different isoforms of HLA-G. All isoforms are equally `[XQ^[SQZUO( He\UOMXXe& =@6'< R[^Y_ M A=8 OXM__ > O[Y\XQd `[SQ`TQ^ cU`T s, YUO^[SX[NaXUZ #7,A [^ s,Y$( >Z [ZQ QYN[PUYQZ`& =@6'< ^QRQ^_ `[ `TQ A=8 OXM__ > O[Y\XQd [R =@6'< MZP s, microglobulin. There are several HLA-G variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. HLA-G Targeted CAR

[0645] The HLA-G targeted CAR (also called “anti-HLA-G CAR”) or HLA-G targeted polypeptide described herein may include a HLA-G targeting scFv.

[0646] In some embodiments, the second receptor is an anti-HLA-G CAR comprising any one of SEQ ID NOS: 391-393 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. SIGLEC5

[0647] In some embodiments, the inhibitor ligand is SIGLEC5.

[0648] The term “SIGLEC5” (also known as Sialic acid-binding Ig-like lectin 5 and CD170) refers to a cell surface lectin characterized by structural motifs in the immunoglobulin (Ig)-like domains and sialic acid recognition sites in the first Ig V set domain. The sialic acid-binding immunoglobulin- like lectins (SIGLECs), such as SIGLEC5, are a subgroup of the immunoglobulin superfamily that mediate protein-carbohydrate interactions. They specifically interact with sialic acids in glycoproteins and glycolipids, with each SIGLEC having a particular preference for both the nature of the sialic acid and its glycosidic linkage to adjacent sugars. There are SIGLEC5 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. PDZK1IP1

[0649] In some embodiments, the inhibitor ligand is PDZK1IP1.

[0650] The term “PDZK1IP1” (also known as PDZK1 Interacting Protein 1, MAP17, DD96, and SPAP) refers to a protein which interacts with several PDZ domain–containing molecules, including sodium-hydrogen antiporter regulators. PDZK1IP1 contributes to the internalization of sodium- dependent phosphate transport protein 2b the trans-Golgi network and participates in enhancement of the endogenous uphill transport system in the kidney. There are PDZK1IP1 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. MME

[0651] In some embodiments, the inhibitor ligand is membrane metallo-endopeptidase (MME). The term “MME” (also known as neutral endopeptidase (NEP), CD10, common acute lymphoblastic leukemia antigen (CALLA), and Neprilysin) refers to a zinc-dependent metalloprotease that cleavesAttorney Ref.061250-557001WO peptides at the amino side of hydrophobic residues and inactivates several peptide hormones. There are MME variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure.

[0652] In some embodiments, the anti-MME VH region comprises any one of SEQ ID NOs: 394, 397, 400-402, 404, or 406 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-MME VL region comprises any one of SEQ ID NOs: 395, 396, 398, 399, 403, 405, or 407 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. CLEC9A

[0653] In some embodiments, the non-cancerous antigen is C-type Lectin Domain Containing 9A (CLEC9A). In some embodiments, the non-cancerous antigen comprises a peptide of CLEC9A. In some embodiments, the CLEC9A non-cancerous cell antigen is expressed by healthy human cells of a subject.

[0654] In some embodiments, the inhibitor antigen is CLEC9A.

[0655] The term CLEC9A (also known as HEEE9341, UNQ9342, DNGR-1, and CD370) refers to a 27 kD endocytic receptor that functions on a subset of myeloid cells. CLEC9A can act as a receptor for the uptake and processing of material from dead cells. CLEC9A can recognize filamentous forms of actin in association with actin-binding domains of cytoskeletal proteins, including spectrin, exposed when cell membranes are damaged, and mediate the cross-presentation of dead-cell associated antigens in a Syk-dependent manner.

[0656] In some embodiments, the inhibitor ligand comprises CLEC9A. There are several CLEC9A variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. THY1

[0657] In some embodiments, the non-cancerous antigen is THY1. In some embodiments, the non- cancerous antigen comprises a peptide of THY1. In some embodiments, the THY1non-cancerous cell antigen is expressed by healthy human cells of a subject.

[0658] In some embodiments, the inhibitor ligand is THY1.

[0659] The term THY1 (also known as Thy-1 cell Surface Antigen, Thy-1 Membrane Glycoportein, CD90, CDw90, and Thy-1 T-cell Antigen) refers to a 17 kD cell surface glycoprotein and member of the immunoglobulin superfamily of proteins. THY1can be involved in cell adhesion and cellAttorney Ref.061250-557001WO communication in numerous cell types, but particularly in cells of the immune and nervous systems. THY1has been widely used as a marker for hematopoietic stem cells.

[0660] In some embodiments, the inhibitor ligand comprises THY1. There are several THY1variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. CEACAM3

[0661] In some embodiments, the non-cancerous antigen is Carcinoembryonic Antigen-Related Cell Adhesion Molecule 3 (CEACAM3). In some embodiments, the non-cancerous antigen comprises a peptide of CEACAM3. In some embodiments, the CEACAM3 non-cancerous cell antigen is expressed by healthy human cells of a subject.

[0662] In some embodiments, the inhibitor antigen is CEACAM3.

[0663] The term CEACAM3 (also known as CEA Cell Adhesion Molecule 3, CD66d, CGM1,CGM1a, W264, W282, and CEA) refers to a 27 kD member of the carcinoembryonic antigen (CEA) gene family. CEACAM3 can be a major granulocyte receptor involved in mediating recognition and efficient opsonin-independent phagocytosis of CEACAM-binding microorganisms, including Neissiria, Moxarella and Haemophilus species. CEACAM3 can contain a transmembrane domain.

[0664] In some embodiments, the inhibitor ligand comprises CEACAM3. There are several CEACAM3 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. Siglec9

[0665] In some embodiments, the non-cancerous antigen is Sialic Acid Binding Ig Like Lectin 9 (Siglec9). In some embodiments, the non-cancerous antigen comprises a peptide of Siglec9. In some embodiments, the Siglec9 non-cancerous cell antigen is expressed by healthy human cells of a subject.

[0666] In some embodiments, the inhibitor antigen is Siglec9.

[0667] The term Siglec9 (also known as CD329, Protein FOAP-9, CDw329, OBBP-LIKE, and FOAP-9) refers to a 50 kD protein that can be involved in immune cell signaling. Siglec9 may enable monosaccharide and sialic acid binding activity. Siglec9 may also be involved in cell adhesion and negative regulation of phagocytosis. Recently, it was found that Siglec9 may act as an immune cell checkpoint molecule.

[0668] In some embodiments, the inhibitor ligand comprises Siglec9. There are several Siglec9 variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure.Attorney Ref.061250-557001WO CLDN10b

[0669] In some embodiments, the non-cancerous antigen is Claudin 10b (CLDN10b). In some embodiments, the non-cancerous antigen comprises a peptide of CLDN10b. In some embodiments, the CLDN10b non-cancerous cell antigen is expressed by the healthy human cells of a subject.

[0670] In some embodiments, the inhibitor antigen is CLDN10b.

[0671] The term CLDN10b refers to a 24 kD member of the claudin protein family. Claudins can be integral membrane proteins and components of tight junction strands. Tight junctions can serve as a physical barrier to prevent solutes and water from passing freely through the paracellular space between epithelial or endothelial cell sheets. CLDN10b may also play critical roles in maintaining cell polarity and signal transductions.

[0672] In some embodiments, the inhibitor ligand comprises CLDN10b. There are several CLDN10b variants encoding distinct isoforms, all of which fall within the scope of the instant disclosure. Extracellular Ligand Binding Domains

[0673] Inhibitor receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.

[0674] In some embodiments, the ligand binding domain is an antigen binding domain. Illustrative MZ`USQZ NUZPUZS P[YMUZ_ UZOXaPQ& UZ`Q^ MXUM& _O;b& GP6N& Js'[ZXe P[YMUZ_& MZP H8F MZ`USQZ NUZPUZS P[YMUZ_ PQ^UbQP R^[Y `TQ H8F n MZP s OTMUZ bM^UMNXQ P[YMUZ_(

[0675] Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0676] Illustrative anti-CDCP1 antibodies include CUB1, REA194, OTI2B2, OTI2C1, OTI2B8, OTI4G5, OTI5B3, CSTEM26, 309116, or 309121.

[0677] In some embodiments, the CDCP1 ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize CDCP1 are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind CDCP1 in a peptide- independent way shown in Table 8 below (complementarity determining regions underlined): Table 8: VH and VL Domains Corresponding to CDCP1 Antigen Binding Domains

[0678] In some embodiments, the second antigen binding domain comprises a scFv domain that binds to a CDCP1 antigen.Attorney Ref.061250-557001WO

[0679] In some embodiments, the non-cancerous antigen comprises CDCP1. Various single variable domains known in the art or disclosed herein that bind to and recognize CDCP1 are suitable for use in embodiments.

[0680] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for CDCP1 antigen binding domains are shown in Table 9 below. Table 9: CDRs Corresponding to CDCP1 Antigen Binding Domains

[0681] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 82-87. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOS: 82-87. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 78-87. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 82-84, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 85-87. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 78 or 80, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 79 or 81. In some embodiments, the heavy chain of the antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID NO: 131 and the light chain of the antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID NO: 132.

[0682] In some embodiments, the second, inhibitory ligand is CDCP1, and the inhibitory ligand binding domain comprises a CDCP1 antigen binding domain. In some embodiments, the CDCP1 antigen binding domain comprises a scFv domain. In some embodiments, the CDCP1 antigen binding domain comprises a sequence of any one of SEQ ID NOs: 78, 79, 80, or 81.

[0683] In some embodiments, the ABCG2 ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize ABCG2 are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind ABCG2 in a peptide- independent way shown in Table 10A below: Table 10A: VH and VL Domains Corresponding to ABCG2 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0684] In some embodiments, the second antigen binding domain comprises a scFv domain that binds to an ABCG2 antigen.

[0685] In some embodiments, the non-cancerous antigen comprises ABCG2. Various single variable domains known in the art or disclosed herein that bind to and recognize ABCG2 are suitable for use in embodiments.

[0686] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for ABCG2 antigen binding domains are shown in Table 10B below. Table 10B: CDRs Corresponding to ABCG2 Antigen Binding Domains

[0687] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 184-186, 191-193, 196-198, 200-202, or 204-206. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 184- 186, 191-193, or 200-202, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 196-198 or 204-206. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 187-190 or 199, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 194, 195, or 203.

[0688] In some embodiments, the second, inhibitory ligand is ABCG2, and the inhibitory ligand binding domain comprises an ABCG2 antigen binding domain. In some embodiments, the ABCG2 antigen binding domain comprises a scFv domain.

[0689] In some embodiments, the FCGR3A ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize FCGR3A are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind FCGR3A in a peptide- independent way. In some embodiments, the second antigen binding domain comprises a scFv domain that binds to a FCGR3A antigen.Attorney Ref.061250-557001WO

[0690] In some embodiments, the non-cancerous antigen comprises FCGR3A. Various single variable domains known in the art or disclosed herein that bind to and recognize FCGR3A are suitable for use in embodiments.

[0691] In some embodiments, the anti-FCGR3A scFv comprises a sequence at least 95% identical to any one of SEQ ID NOS: 207-208. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 207-208.

[0692] In some embodiments, the anti-FCG3A VH domain comprises any one of SEQ ID NOs: 235, 237, 245 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-FCGR3A VL domain comprises any one of SEQ ID NOs: 236, 238-244, 246 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0693] In some embodiments, the second, inhibitory ligand is PECAM-1, and the inhibitory ligand binding domain comprises a PECAM-1 antigen binding domain. In some embodiments, the PECAM- 1 antigen binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize PECAM-1 are suitable for use in embodiments. In some embodiments, the non-cancerous antigen comprises PECAM-1. In some embodiments, the second, inhibitory ligand is PECAM-1, and the inhibitory ligand binding domain comprises a PECAM-1 antigen binding domain.

[0694] In some embodiments, the HLA-G ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-G are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind HLA-G in a peptide- independent way shown in Table 11A below (complementarity determining regions underlined): Table 11A: VH and VL Domains Corresponding to HLA-G Antigen Binding Domains

[0695] In some embodiments, the anti-HLA-G VH domain comprises any one of SEQ ID NOs: 221, 223, 231, 323, 325, 379, 381, 383, 385, 387, 388, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-HLA-G VL domain comprises any one of SEQ ID NOs: 222, 224, 232, 324, 326, 380, 382, 384, 386, 389, orAttorney Ref.061250-557001WO a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0696] In some embodiments, the second antigen binding domain comprises a scFv domain that binds to a HLA-G antigen. In some embodiments, the anti-HLA-G scFv domain comprises SEQ ID NO: 390 or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0697] In some embodiments, the non-cancerous antigen comprises HLA-G. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-G are suitable for use in embodiments.

[0698] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for HLA-G antigen binding domains are shown in Table 11B below. Table 11B: CDRs Corresponding to HLA-G Antigen Binding Domains

[0699] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 209-220 or 225-230. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 209-211, 215-217, or 225- 227, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 212-214, 218-220, or 228-230. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 221, 223, or 231, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 222, 224, or 232. In some embodiments, the second, inhibitory ligand is HLA-G, and the inhibitory ligand binding domain comprises a HLA-G antigen binding domain. In some embodiments, the HLA-G antigen binding domain comprises a scFv domain.

[0700] In some embodiments, the CD16B ligand binding domain comprises a scFv domain. Various single variable domains known in the art or disclosed herein that bind to and recognize CD16B are suitable for use in embodiments.

[0701] In some embodiments, the anti-CD16B VH domain comprises any one of SEQ ID NOs: 688- 741, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto. In some embodiments, the anti-CD16B VL domain comprises any one of SEQ IDAttorney Ref.061250-557001WO NOs: 742-795, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% identity, or 100% identical thereto.

[0702] In some embodiments, the non-cancerous antigen comprises CD16B. Various single variable domains known in the art or disclosed herein that bind to and recognize CD16B are suitable for use in embodiments. Illustrative VH and VL domains for CD16B antigen binding domains are shown in Table 12A below. Table 12A: VH and VL Domains Corresponding to CD16B Antigen Binding DomainsAttorney Ref.061250-557001WO

[0703] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR- L1, CDR-L2 and CDR-L3, respectively) for CD16B antigen binding domains are shown in Table 12B below. Table 12B: CDRs Corresponding to CD16B Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0704] In some embodiments, the second antigen binding domain comprises a scFv domain that binds to a CD16B antigen. In some embodiments, the scFv comprises the complementarity determined regions (CDRs) as disclosed in Table 12B. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs as disclosed in Table 12B and the light chain of the antibody comprises the light chain CDRs as disclosed in Table 12B. In some embodiments, the second, inhibitory ligand is CD16B, and the inhibitory ligand binding domain comprises a CD16B antigen binding domain. In some embodiments, the CD16B antigen binding domain comprises a scFv domain.

[0705] In some embodiments, the anti-CD16B scFv domain comprises a polypeptide sequence as disclosed in Table 12C. Illustrative scFv domains for CD16B antigen binding domains are shown in Table 12C below. Table 12C: CD16B scFv Binding DomainsAttorney Ref.061250-557001WO

[0706] In some embodiments, the anti-CD16B scFv domain comprises a polynucleotide sequence as disclosed in Table 12D. Illustrative scFv domains for CD16B antigen binding domains are shown in Table 12D below.Attorney Ref.061250-557001WO Table 12D: CD16B scFv Binding Domains Polynucleotide SequencesAttorney Ref.061250-557001WO

[0707] In some embodiments, the inhibitor receptor comprises a scFv domain that binds to a CD16B antigen. In some embodiments, the inhibitory ligand is CD16B and the inhibitory ligand binding domain comprises a CD16B antigen binding domain. In some embodiments, the inhibitor receptor is a chimeric antigen receptor comprises a polypeptide sequence as disclosed in Table 12E. Table 12E: Full Length Receptor Corresponding to CD16B Antigen Binding DomainsAttorney Ref.061250-557001WO

[0708] In further embodiments of any of the ligand binding domains, each CDR sequence may have 1, 2, 3 or more substitutions, insertions, or deletions. CDR sequences may tolerate substitutions, deletions, or insertions. Using sequence alignment tools, routine experimentation, and known assays, those of skill in the art may generate and test variant sequences having 1, 2, 3, or more substitutions, insertions, or deletions in CDR sequences without undue experimentation.

[0709] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CLEC9A, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- CLEC9A antibody is a human anti-CLEC9A antibody, a humanized anti-CLEC9A antibody, or a chimeric anti-CLEC9A antibody. In some embodiments, the anti-CLEC9A antibody is an anti- CLEC9A monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CLEC9A is specific to one CLEC9A isoform or binds to more than one CLEC9A isoform, e.g., is a pan-CLEC9A antibody or antigen-binding fragment.

[0710] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 29. Table 29 CDRs Corresponding to CLEC9A Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0711] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 30. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 30.Attorney Ref.061250-557001WO Table 30: VH and VL Corresponding to CLEC9A Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0712] In some embodiments, the binding domain is an anti-CLEC9A scFv domain. In some embodiments, the anti-CLEC9A scFv domain is selected from a sequence listed in Table 31.

[0713] In some embodiments, the second receptor is an anti-CLEC9A CAR comprising an CLEC9A targeting scFv comprising a VH comprising any one of SEQ ID NOS: 3967-4071 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 4072-4176 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. Table 31 CLEC9A scFv Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOTable 32 Full Length Receptor Corresponding to CLEC9A Antigen Binding Domains_Attorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0714] In some embodiments, the extracellular ligand binding domain comprises a binding agent for THY1, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- THY1 antibody is a human anti-THY1 antibody, a humanized anti-THY1 antibody, or a chimeric anti-THY1 antibody. In some embodiments, the anti-THY1 antibody is an anti-THY1 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to THY1 is specific to one THY1 isoform or binds to more than one THY1 isoform, e.g., is a pan-THY1 antibody or antigen-binding fragment.

[0715] In some embodiments, the extracellular ligand binding domain of the activator receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 33.Attorney Ref.061250-557001WO Table 33 CDRs Corresponding to THY1 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0716] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 34. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 34. Table 34 VH and VL Corresponding to THY1 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0717] In some embodiments, the binding domain is an anti-THY1 scFv domain. In some embodiments, the anti-THY1 scFv domain is selected from a sequence listed in Table 35.

[0718] In some embodiments, the second receptor is an anti-THY1 CAR comprising an THY1 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 4574-4591 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 4592-4636 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. Table 35 THY1 scFv Binding DomainsAttorney Ref.061250-557001WOTable 36 Full Length Receptor Corresponding to THY1 Antigen Binding DomainsAttorney Ref.061250-557001WO

[0719] In some embodiments, the extracellular ligand binding domain comprises a binding agent for CEACAM3, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti-CEACAM3 antibody is a human anti-CEACAM3 antibody, a humanized anti-CEACAM3 antibody, or a chimeric anti-CEACAM3 antibody. In some embodiments, the anti-CEACAM3 antibody is an anti-CEACAM3 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to CEACAM3 is specific to one CEACAM3 isoform or binds to more than one CEACAM3 isoform, e.g., is a pan-CEACAM3 antibody or antigen-binding fragment.

[0720] In some embodiments, the extracellular ligand binding domain of the inhibitor receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VHAttorney Ref.061250-557001WO and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 37. Table 37 CDRs Corresponding to CEACAM3 Antigen Binding Domains

[0721] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 38. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 38. Table 38 VH and VL Corresponding to CEACAM3 Antigen Binding Domains

[0722] In some embodiments, the binding domain is an anti-CEACAM3 scFv domain. In some embodiments, the anti-CEACAM3 scFv domain is selected from a sequence listed in Table 39.

[0723] In some embodiments, the second receptor is an anti-CEACAM3 CAR comprising an CEACAM3 targeting scFv comprising a VH comprising any one of SEQ ID NOS: 4773-4780 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto and a VL comprising any one of SEQ ID NOS: 4781-4788 or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.Attorney Ref.061250-557001WO Table 39 CEACAM3 scFv Binding DomainsTable 40 Full Length Receptor corresponding to CEACAM3 antigen binding domains

[0724] In some embodiments, the extracellular ligand binding domain comprises a binding agent for SIGLEC9, such as an antibody or antigen-binding fragment thereof. In some embodiments, the anti- SIGLEC9 antibody is a human anti-SIGLEC9 antibody, a humanized anti-SIGLEC9 antibody, or a chimeric anti-SIGLEC9 antibody. In some embodiments, the anti-SIGLEC9 antibody is an anti- SIGLEC9 monoclonal antibody. In some embodiments, the antibody molecule or antigen-binding fragment that binds to SIGLEC9 is specific to one Siglec9 isoform or binds to more than one Siglec9 isoform, e.g., is a pan-Siglec9 antibody or antigen-binding fragment.

[0725] In some embodiments, the extracellular ligand binding domain of the inhibitor receptor O[Y\^U_Q_ MZ MZ`UN[Pe R^MSYQZ`& M _UZSXQ OTMUZ ;b MZ`UN[Pe R^MSYQZ` #_O;b$& M s OTMUZ bM^UMNXQ P[YMUZ #Js$& [^ M H8F n OTMUZ bM^UMNXQ P[YMUZ MZP M H8F s OTMUZ bM^UMNXQ P[YMUZ( >Z _[YQ embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 41. Table 41 CDRs Corresponding to Siglec9 antigen binding domainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0726] In some embodiments, the first receptor is an anti-Siglec9 inhibitor receptor comprises a Siglec9 targeting scFv comprising a combination of CDRs from any row of Table 41.

[0727] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 42. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 42. Table 42 VH and VL Corresponding to Siglec9 Antigen Binding DomainsAttorney Ref.061250-557001WOAttorney Ref.061250-557001WO

[0728] In some embodiments, the binding...

Claims

What is claimed is:

1. An immune cell for treating a blood cancer, the immune cell comprising an inhibitor receptorcomprising an extracellular ligand binding domain specific to an inhibitor antigen expressedby a non-cancerous blood cell.

2. The immune cell of claim 1, wherein binding the extracellular ligand binding domain to theinhibitor antigen initiates an inhibitory signal in the immune cell which promotes sparing ofthenon-cancerous blood cell from killing, injuring, or distressing by the immune cell.

3. The immune cell of claims 1-2, wherein the non-cancerous blood cell is a myeloid cell, ahematopoietic stem cell, a myeloid stem cell, or a myeloblast.

4. The immune cell of any one of claims 1-3, wherein the inhibitor receptor is a chimeric antigenreceptor (CAR) or is a T cell receptor (TCR).

5. The immune cell of any one of claims 1-4, wherein the immune cell is a CD8+ T cell (e.g., a CD8+CD4- T cell), a CD4+T cell (e.g., a CD8- CD4+ T cell), a gamma delta (y8) T cell, an invariantT cell, a natural killer (NK) cell, an invariant natural killer cell (iNK cell), an invariant naturalkiller T cell (iNKT cell), or a macrophage.

6. The immune cell of any one of claims 1-5, wherein the inhibitory antigen is any B antigenidentified in Table 1.

7. The immune cell of any one of claims 1-6, wherein the inhibitor receptor comprises a bindingdomain as disclosed in Table 12C, Table 31, Table 39, Table 35, Table 43 and Table 51.

8. The immune cell of any one of claims 1-7, wherein the inhibitor antigen expressed by the noncancerous blood cell is THY1.

9. The immune cell of claim 8, wherein the extracellular ligand binding domain of the inhibitorreceptor comprises a heavy chain variable (VH) region and a light chain variable (VL) regioncomprising a set of complement determining region (CDR), wherein:the VH region comprises:(a) a complementarity-determining region VH-1 (CDR-H1) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4387-4410and 5876-5882;(b) a CDR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4411-4434 and 5883-5890; and(c) a CDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4435-4469 and 5891-5897, andthe VL region comprises:350(d) a CDR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4470-4495 and 5898-5902;(e) a CDR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4496-4519 and 5905; and(f) a CDR3-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4520-4546 and 5907-5914.

10. The immune cell of any one of claims 8-9, wherein the extracellular ligand binding domain ofthe inhibitor receptor comprises a set of CDR sequences as disclosed in a row of Table 33.

11. The immune cell of claim 10, wherein the row of Table 33 is identified as 13P13, 3K22, 3G19,or 10H1.

12. The immune cell of any one of claims 8-11, wherein the VH region comprises the sequence ofany one of SEQ ID NOs: 4547-4591 and 5915-5922, or a sequence having at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100%identical thereto; and wherein the VL region comprises the sequence of any one of SEQ IDNOs: 4592-4636 and 5923-5930, or a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

13. The immune cell of any one of claims 8-12, wherein the extracellular ligand binding domain ofthe inhibitor receptor comprises a VH region and a VL region comprising a set of sequencesas disclosed in a row of Table 34, or sequences having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

14. The immune cell of claim 13, wherein the row of Table 34 is identified as 13P13, 3K22, 3G19,or 10H1.

15. The immune cell of any one of claims 8-14, wherein the extracellular ligand binding domain ofthe inhibitor receptor comprises an antibody fragment or a single chain Fv antibody fragment(scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequence selectedfrom the group consisting of SEQ ID NOs: 4637-4681 and 5931-5938.

16. The immune cell of claim 15, wherein the scFv comprises a sequence as disclosed in Table 35identified as 13P13, 3K22, 3G19, or 10H1.

17. The immune cell of any one of claims 8-16, wherein the inhibitor receptor comprises asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identityto, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos:4682-4726 and 5939-5946.

18. The immune cell of any one of claims 8-17, wherein the inhibitor receptor comprises asequence as disclosed in Table 36 identified as 13P13, 3K22, 3G19, or 10H1.35119. The immune cell of any one of claims 1-7, wherein the inhibitor antigen expressed by the noncancerous blood cell is Siglec9.

20. The immune cell of claim 19, wherein the extracellular ligand binding domain of the inhibitorreceptor comprises a heavy chain variable (VH) region and a light chain variable (VL) regioncomprising a set of complement determining region (CDR), wherein:the VH region comprises:(a) a complementarity-determining region VH-1 (CDR-H1) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4805-4836and 5947-5948;(b) a CDR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4837-4886 and 5949-5953; and(c) a CDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4887-4926 and 5955-5960, andthe VL region comprises:(d) a CDR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4927-4958 and 5961-5966;(e) a CDR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4959-4984 and 5967-5971; and(f) a CDR3-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4985-5026 and 5972-5978.

21. The immune cell of any one of claims 19-20, wherein the extracellular ligand binding domainof the inhibitor receptor comprises a set of CDR sequences as disclosed in a row of Table 41.

22. The immune cell of claim 21, wherein the row of Table 41 is identified as 14EE8 or 19M17.

23. The immune cell of any one of claims 19-22, wherein the VH region comprises the sequence ofany one of SEQ ID NOs: 5027-5091 and 5979-5985, or a sequence having at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100%identical thereto; and wherein the VL region comprises the sequence of any one of SEQ IDNOs: 5092-5156 and 5986-5992, or a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

24. The immune cell of any one of claims 19-23, wherein the extracellular ligand binding domainof the inhibitor receptor comprises a VH region and a VL region comprising a set ofsequences as disclosed in a row of Table 42, or sequences having at least 85%, at least 90%,at least 95%, at least 97%, or at least 99% identity thereto, or that are 100% identical thereto.

25. The immune cell of claim 24, wherein the row of Table 42 is identified as 14EE8 or 19M17.35226. The immune cell of any one of claims 19-25, wherein the extracellular ligand binding domainof the inhibitor receptor comprises an antibody fragment or a single chain Fv antibodyfragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequenceselected from the group consisting of SEQ ID NOs: 5157-5221 and 5993-5999.

27. The immune cell of claim 15, wherein the scFv comprises a sequence as disclosed in Table 43identified as 14EE8 or 19M17.

28. The immune cell of any one of claims 19-27, wherein the inhibitor receptor comprises asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identityto, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos:5222-5286 and 6000-6006.

29. The immune cell of any one of claims 19-28, wherein the inhibitor receptor comprises asequence as disclosed in Table 44 identified as 14EE8 or 19M17.

30. The immune cell of any one of claims 1-29, wherein the inhibitor receptor comprises a LILRB1intracellular domain or a functional variant thereof.

31. The immune cell of claim 30, wherein the LILRB1 intracellular domain comprises the sequenceof SEQ ID NO: 113, or a sequence having at least 85%, at least 90%, at least 95%, at least97%, or at least 99% identity thereto, or that is 100% identical thereto.

32. The immune cell of any one of claims 1-31, wherein the inhibitor receptor comprises LILRB1hinge and transmembrane domains, or functional variants thereof.

33. The immune cell of claim 32, wherein the LILRB1 transmembrane domain comprises thesequence of SEQ ID NO: 102, or a sequence having at least 85%, at least 90%, at least 95%,at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

34. The immune cell of claims 32-33, wherein the LILRB1 hinge domain comprises the sequenceof any one of SEQ ID NOs: 103, 104, 115, 116, 117, 118, 119, 120, or a sequence having atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is100% identical thereto.

35. The immune cell of any one of claims 30-34, wherein the LILRB1 hinge, LILRB1transmembrane, and LILRB1 intracellular domains comprise the sequence of SEQ ID NO:108, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least99% identity thereto, or that is 100% identical thereto.

36. The immune cell of any one of claims 1-35, wherein the immune cell further comprises anactivator receptor comprising an extracellular ligand binding domain specific to an activatorantigen expressed by a blood cancer cell.35337. The immune cell of claim 36, wherein binding the extracellular ligand binding domain to theactivator antigen initiates an activating signal in the immune cell which promotes the killing,injuring, or distressing of the cancerous blood cell by the immune cell.

38. The immune cell of claims 36-37, wherein the blood cancer cell is a hematopoietic cancer cell ora hematologic cancer cell.

39. The immune cell of any one of claims 36-38, wherein the blood cancer cell is a lymphoma cell,a leukemia cell (e.g., an acute myeloid leukemia (AML) cell), a myeloma cell, a Reed-Sternbergcell, a myeloproliferative neoplasm cell, or a Waldenstrom macroglobulinemia cell.

40. The immune cell of any one of claims 36-39, wherein the activator receptor is a chimeric antigenreceptor (CAR) or is a T cell receptor (TCR).

41. The immune cell of any one of claims 36-40, wherein the activator antigen is any A antigenidentified in Table 1.

42. The immune cell of any one of claims 36-41, wherein the activator receptor comprises a bindingdomain as disclosed in Table 4B, Table 5C, Table 6C, Table 19, Table 23, Table 27 andTable 47.

43. The immune cell of any one of claims 36-42, wherein the activator antigen expressed by thecancerous blood cell is ITGA4.

44. The immune cell of claim 43, wherein the extracellular ligand binding domain of the activatorreceptor comprises a heavy chain variable (VH) region and a light chain variable (VL) regioncomprising a set of complement determining region (CDR), wherein:the VH region comprises:(a) a complementarity-determining region VH-1 (CDR-H1) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2704-2731;(b) a CDR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2732-2776; and(c) a CDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2777-2812, andthe VL region comprises:(d) a CDR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2813-2846;(e) a CDR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2847-2874; and(f) a CDR3-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2875-2909.35445. The immune cell of any one of claims 43-44, wherein the extracellular ligand binding domain47.of the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 17.

46. The immune cell of claim 45, wherein the row of Table 17 is identified as 16E9, 17J1, or 2F5.The immune cell of any one of claims 43-46, wherein the VH region comprises the sequence ofany one of SEQ ID NOs: 2910-2971, or a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; andwherein the VL region comprises the sequence of any one of SEQ ID NOs: 2972-3033, or asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identitythereto, or that is 100% identical thereto.

48. The immune cell of any one of claims 43-47, wherein the extracellular ligand binding domainof the activator receptor comprises a VH region and a VL region comprising a set ofsequences as disclosed in a row of Table 18, or sequences having at least 85%, at least 90%,at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

49. The immune cell of claim 48, wherein the row of Table 18 is identified as 16E9, 17J1, or 2F5.

50. The immune cell of any one of claims 43-49, wherein the extracellular ligand binding domainof the activator receptor comprises an antibody fragment or a single chain Fv antibodyfragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequenceselected from the group consisting of SEQ ID NOs: 3034-3095.

51. The immune cell of claim 50, wherein the scFv comprises a sequence as disclosed in Table 19identified as 16E9, 17J1, or 2F5.

52. The immune cell of any one of claims 43-51, wherein the activator receptor comprises asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identityto, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos:3096-3157.

53. The immune cell of any one of claims 43-52, wherein the activator receptor comprises asequence as disclosed in Table 20 identified as 16E9, 17J1, or 2F5.

54. The immune cell any one of claims 36-42, wherein the activator antigen expressed by thecancerous blood cell is CLL1.

55. The immune cell of claim 54, wherein the extracellular ligand binding domain of the activatorreceptor comprises a heavy chain variable (VH) region and a light chain variable (VL) regioncomprising a set of complement determining region (CDR), wherein:the VH region comprises:(a) a complementarity-determining region VH-1 (CDR-H1) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 5287-5326;355(b) a CDR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5327-5389; and(c) a CDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5390-5452, andthe VL region comprises:(d) a CDR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5453-5507;(e) a CDR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5508-5513; and(f) a CDR3-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5545-5551.

56. The immune cell of any one of claims 54-55, wherein the extracellular ligand binding domainof the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 45.

57. The immune cell of claim 56, wherein the row of Table 45 is identified as 2N24, 14L11, 7F2,7G3, 9011, 20121, 3F10, or 7F2.

58. The immune cell of any one of claims 54-57, wherein the VH region comprises the sequence ofany one of SEQ ID NOs: 5587-5658, or a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto; andwherein the VL region comprises the sequence of any one of SEQ ID NOs: 5659-5730, or asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identitythereto, or that is 100% identical thereto.

59. The immune cell of any one of claims 54-58, wherein the extracellular ligand binding domainof the activator receptor comprises a VH region and a VL region comprising a set ofsequences as disclosed in a row of Table 46, or sequences having at least 85%, at least 90%,at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

60. The immune cell of claim 59, wherein the row of Table 46 is identified as 2N24, 14L11, 7F2,7G3, 9011, 20121, 3F10, or 7F2.

61. The immune cell of any one of claims 54-60, wherein the extracellular ligand binding domainof the activator receptor comprises an antibody fragment or a single chain Fv antibodyfragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequenceselected from the group consisting of SEQ ID NOs: 5731-5802.

62. The immune cell of claim 61, wherein the scFv comprises a sequence as disclosed in Table 47identified as 2N24, 14L11, 7F2, 7G3, 9011, 20121, 3F10, or 7F2.35663. The immune cell of any one of claims 54-62, wherein the activator receptor comprises asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identityto, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos:5803-5874.

64. The immune cell of any one of claims 54-63, wherein the activator receptor comprises asequence as disclosed in Table 48 identified as 2N24, 14L11, 7F2, 7G3, 9011, 20121, 3F10,or 7F2.

65. The immune cell any one of claims 36-42, wherein the activator antigen expressed by thecancerous blood cell is CD33.

66. The immune cell of claim 65, wherein the extracellular ligand binding domain of the activatorreceptor comprises a heavy chain variable (VH) region and a light chain variable (VL) regioncomprising a set of complement determining region (CDR), wherein:the VH region comprises:(a) a complementarity-determining region VH-1 (CDR-H1) comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1512-1618;(b) a CDR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1619-1725; and(c) a CDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1726-1832, andthe VL region comprises:(d) a CDR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1833-1947;(e) a CDR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1948-2062; and(f) a CDR3-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2063-2177.

67. The immune cell of any one of claims 65-66, wherein the extracellular ligand binding domainof the activator receptor comprises a set of CDR sequences as disclosed in a row of Table 6В.

68. The immune cell of claim 67, wherein the row of Table 6B is identified as Activator 6,Activator 37, or Activator 100.

69. The immune cell of any one of claims 65-68, wherein the VH region comprises the sequence ofany one of SEQ ID NOs: 1290-1396 and 2442-2449, or a sequence having at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100%identical thereto; and wherein the VL region comprises the sequence of any one of SEQ ID357NOs: 1397-1511 and 2450-2457, or a sequence having at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

70. The immune cell of any one of claims 65-69, wherein the extracellular ligand binding domainof the activator receptor comprises a VH region and a VL region comprising a set ofsequences as disclosed in a row of Table 6A, or sequences having at least 85%, at least 90%,at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

71. The immune cell of claim 70, wherein the row of Table 6A is identified as Activator 6,Activator 37, or Activator 100.72.73.The immune cell of any one of claims 65-71, wherein the extracellular ligand binding domainof the activator receptor comprises an antibody fragment or a single chain Fv antibodyfragment (scFv), wherein the scFv comprises a sequence having at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% identity to, or that is 100% identical to a sequenceselected from the group consisting of SEQ ID NOs: 2458-2580.The immune cell of claim 72, wherein the scFv comprises a sequence as disclosed in Table 6Cidentified as Activator 6, Activator 37, or Activator 100.

74. The immune cell of any one of claims 65-73, wherein the activator receptor comprises asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identityto, or that is 100% identical to a sequence selected from the group consisting of SEQ ID Nos:2581-2703.

75. The immune cell of any one of claims 65-74, wherein the activator receptor comprises asequence as disclosed in Table 6D identified as Activator 6, Activator 37, or Activator 100.

76. The immune cell of any one of claims 36-35, wherein the immune cell is modified to reduce oreliminate expression of the B2M gene product.

77. The immune cell of claim 76, wherein expression of the B2M gene product is reduced oreliminated using a short hairpin RNA (shRNA).

78. The immune cell of claim 77, wherein the shRNA comprises a first sequence, having from 5' to3' end a sequence complementary to the B2M mRNA; and a second sequence, having from 5'to 3' end a sequence complementary to the first sequence, wherein the first sequence andsecond sequence form the shRNA.

79. An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressedby cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is ITGA4 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising a358sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID NOs: 3034-3095 or comprising the CDR sequences as disclosed in a rowof Table 17; andwherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 andwherein the inhibitor receptor comprises an extracellular ligand binding domaincomprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDRsequences as disclosed in a row of Table 33.

80. The immune cell of claim 79, wherein the activator receptor comprises a sequence as disclosedin Table 17 identified as 16E9, 17J1, or 2F5 and wherein the inhibitor receptor comprises asequence as disclosed in Table 33 identified as 13P13, 3K22, 3G19, or 10H1.81.82.An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressedby cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is ITGA4 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID NOs: 3034-3095 or comprising the CDR sequences as disclosed in a rowof Table 17: andwherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 andwherein the inhibitor receptor comprises an extracellular ligand binding domaincomprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 5157-5221 and 5993-5999 or comprising the CDRsequences as disclosed in a row of Table 41.The immune cell of claim 81, wherein the activator receptor comprises a sequence as disclosedin Table 17 identified as 16E9, 17J1, or 2F5 and wherein the inhibitor receptor comprises asequence as disclosed in Table 41 identified as 14EE8 or 19M17.

83. An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressed35984.by cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is CLL1 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID NOs: 5731-5802 or comprising the CDR sequences as disclosed in a rowof Table 45; andwherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 andwherein the inhibitor receptor comprises an extracellular ligand binding domaincomprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDRsequences as disclosed in a row of Table 33.The immune cell of claim 83, wherein the activator receptor comprises a sequence as disclosedin Table 45 identified as 2N24, 14L11, 7F2, 7G3, 9011, 20121, 3F10, or 7F2 and wherein theinhibitor receptor comprises a sequence as disclosed in Table 33 identified as 13P13, 3K22,3G19, or 10H1.

85. An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressedby cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is CLL1 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID Nos: 5731-5802 or comprising the CDR sequences as disclosed in a row ofTable 45; andwherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 andwherein the inhibitor receptor comprises an extracellular ligand binding domaincomprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID Nos: 5157-5221 and 5993-5999 or comprising the CDRsequences as disclosed in a row of Table 41.36086. The immune cell of claim 85, wherein the activator receptor comprises a sequence as disclosedin Table 45 identified as 2N24, 14L11, 7F2, 7G3, 9011, 20121, 3F10, or 7F2 and wherein theinhibitor receptor comprises a sequence as disclosed in Table 41 identified as 14EE8 or19M17.

87. An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressedby cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is CD33 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID NOs: 2458-2580 or comprising the CDR sequences as disclosed in a rowof Table 6B: andwherein the inhibitor antigen expressed by the non-cancerous blood cell is THY1 andwherein the inhibitor receptor comprises an extracellular ligand binding domaincomprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 4682-4726 and 5939-5946 or comprising the CDRsequences as disclosed in a row of Table 33.

88. The immune cell of claim 87, wherein the activator receptor comprises a sequence as disclosedin Table 6B identified as 1A4, 2H20, or 1C20 and wherein the inhibitor receptor comprises asequence as disclosed in Table 33 identified as 13P13, 3K22, 3G19, or 10Н1.

89. An immune cell for treating a blood cancer, the immune cell comprising an activator receptorcomprising an extracellular ligand binding domain specific to an activator antigen expressedby cancerous blood cells and an inhibitor receptor comprising an extracellular ligand bindingdomain specific to an inhibitor antigen expressed by non-cancerous blood cells,wherein the activator antigen expressed by the cancerous blood cell is CD33 and whereinthe activator receptor comprises an extracellular ligand binding domain comprising asequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%identity to, or that is 100% identical to a sequence selected from the group consistingof SEQ ID NOs: 2458-2580 or comprising the CDR sequences as disclosed in a rowof Table 6B; andwherein the inhibitor antigen expressed by the non-cancerous blood cell is Siglec9 andwherein the inhibitor receptor comprises an extracellular ligand binding domain36190.comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, orat least 99% identity to, or that is 100% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 5157-5221 and 5993-5999 or comprising the CDRsequences as disclosed in a row of Table 41.The immune cell of claim 89, wherein the activator receptor comprises a sequence as disclosedin Table 6B identified as 1A4, 2H20, or 1C20 and wherein the inhibitor receptor comprises asequence as disclosed in Table 41 identified as 14EE8 or 19M17.

91. The immune cell of any one of claims 1-90, wherein the cancer is leukemia, lymphoma,myeloma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), myeloid leukemia,Non-Hodgkin lymphoma, Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiplemyeloma, myeloproliferative neoplasms (MPNs), juvenile myelomonocytic leukaemia(JMML), Juvenile Chronic Myeloid Leukaemia (JCML), or Chronic MyelomonocyticLeukaemia of Infancy.

92. An activator receptor expressed by an immune cell of any one of claims 36-91.

93. A polynucleotide or polynucleotide system, comprising one or more polynucleotidescomprising one or more polynucleotide sequences encoding the activator receptor of claim92.

94. A method of making an immune cell therapy, comprising transforming immune cells with thepolynucleotide or polynucleotide system of claim 93.

95. An inhibitor receptor expressed by an immune cell of any one of claims 1-91.

96. A polynucleotide or polynucleotide system, comprising one or more polynucleotidescomprising one or more polynucleotide sequences encoding the inhibitor receptor of claim 95.

97. A method of making an immune cell therapy, comprising transforming immune cells with thepolynucleotides or polynucleotide systems of claim 93 and 99.

98. A pharmaceutical composition comprising a therapeutically effective amount of the immunecells of any one of claims 1-91 and a pharmaceutically acceptable carrier, diluent or excipient.

99. A method for treating a blood cancer in a subject, the method comprising administering to thesubject a therapeutically amount of the pharmaceutical composition of claim 98.

100. The method of claim 99, wherein the immune cell is autologous to the subject or is allogeneicto the subject.

101. A method for treating a blood cancer in a subject, the method comprising administering to thesubject a therapeutically amount of an immune cell according to any one of claims 1-91.

102. The method of claim 101, wherein the immune cell is autologous to the subject or isallogeneic to the subject.362103. Use of the immune cells of any one of claims 1-91 in a method for manufacturing amedicament for treating a blood cancer.

104. A kit comprising the immune cell of any one of claims 1-91 or the pharmaceuticalcomposition of claim 94.

Images