Chimeric receptors and methods of use thereof

Abstract

To provide novel and improved therapies for treating CLL-1-associated diseases and disorders.SOLUTION: The present invention provides modified immune cells (such as CARs or TCRs) and antigen-binding molecules (antibodies, scFvs, and the heavy and / or light chains and CDRs of these antigen-binding molecules) that have specificity for CLL-1. The present invention also provides vectors, compositions, and methods of treatment and / or detection using the antigen-binding molecules and modified immune cells.SELECTED DRAWING: None

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 317,068, filed on April 1, 2016, the entire disclosure of which is incorporated herein by reference.

[0002] [Sequence Listing] This application includes a sequence listing that was electronically submitted in ASCII format, the entire disclosure of which is incorporated herein by reference. The name of the ASCII copy created on March 31, 2017 is K - 1029_02_SL.txt, and it is 265829 bytes in size.

Background Art

[0003] C - type lectin - like - 1 (CLL - 1; also known as CLEC - 1, CLEC12A, MICL, dendritic cell - associated lectin - 1 (DCAL - 1) and DCAL - 2) is a glycoprotein receptor and a member of the C - type lectin - like receptor family involved in the regulation of cell proliferation and immune regulation. CLL - 1 is expressed on innate immune cells in hematopoietic cells, mainly including monocytes, granulocytes, dendritic cells and myeloid progenitor cells (Non - Patent Document 1). CLL - 1 has been associated with the regulation of the proliferation and differentiation of myeloid cells (Non - Patent Documents 2, 3), and is present on acute myeloid (myelogenous) leukemia (AML) cells and leukemia stem cells (Non - Patent Document 4).

[0004] Therefore, CLL - 1 is associated with a plurality of diseases including, but not limited to, acute myeloid (myelogenous) leukemia (AML), chronic myeloid (myelogenous) leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monocytic leukemia, acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorders, myeloid neoplasms, myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN) or combinations thereof.

[0005] CLL-1 may additionally play a role in inflammatory or autoimmune diseases such as rheumatoid arthritis, psoriasis, allergy, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

[0006] The human CLL-1 protein comprises a polypeptide of the following amino acid sequence: MSEEVTYADLQFQNSSEMEKIPEIGKFGEKAPPAPSHVWRPAALFLTLLCLLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA (SEQ ID NO: 140).

[0007] Additional sequence information is included in the CLL-1 Uniprot list (http: / / www.uniprot.org / uniprot / Q5QGZ9) and the NCBI reference sequence NP_612210.4 (http: / / www.ncbi.nlm.nih.gov / protein / NP_612210.4).

[0008] When referring to CLL-1, such reference includes related polypeptides including, but not limited to, its fragments, as well as allelic variants, splice variants, induced variants, substitution variants, deletion variants, and / or insertion variants including the addition of an N-terminal methionine It will be understood to encompass fusion polypeptides and interspecies homologs. In certain embodiments, the CLL-1 polypeptide includes terminal residues such as, but not limited to, leader sequence residues, target residues, amino-terminal methionine residues, lysine residues, tag residues, and / or fusion protein residues.

[0009] A certain specific antibody against CLL-1 is described in Patent Document 1 and Patent Document 2.

[0010] Modified immune cells have been shown to have desirable qualities in therapeutic treatment, particularly in oncology. Two major types of modified immune cells are those containing chimeric antigen receptors (referred to as "CAR" or "CAR-T") and T cell receptors ("TCR"). These modified cells are modified to confer antigen specificity while retaining or enhancing the ability to recognize and kill target cells. A chimeric antigen receptor can include, for example, (i) an antigen-specific component ("antigen-binding molecule"), (ii) an extracellular domain, (iii) one or more co-stimulatory domains, and (iv) one or more activation domains. Each domain may be heterologous, i.e., composed of sequences derived from (or corresponding to) different protein chains. Immune cells (such as T cells) expressing chimeric antigen receptors can be used in various therapies including cancer therapy. It will be appreciated that the use of co-stimulating domains can enhance the activation of CAR-expressing cells against the target antigen, thereby increasing the efficacy of adoptive immunotherapy. be enhanced, thereby increasing the efficacy of adoptive immunotherapy.

[0011] A certain CAR against CLL-1 is described in, for example, Patent Document 3.

[0012] T cells can be modified to have specificity for one or more desired targets. For example, DNA or other genetic material encoding one or more antigen-binding molecules such as single-chain variable fragments ("scFv") of an antibody, together with one or more signaling molecules and / or one or more activation domains such as CD3 zeta, can be transduced into T cells.

[0013] In addition to the ability of CAR-T cells to recognize and destroy target cells, successful T cell therapies benefit from the ability of CAR-T cells to sustain and maintain the ability to proliferate in response to the antigen.

[0014] The T cell receptor (TCR) is a molecule found on the surface of T cells that is involved in the recognition of antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains. In approximately 95% of human TCRs, the TCR consists of an alpha (α) chain and a beta (β) chain. In approximately 5% of human T cells, the TCR consists of gamma and delta (γ / δ) chains. Each chain is composed of two extracellular domains of the immunoglobulin superfamily: a variable (V) region and a constant (C) region. Similar to other immunoglobulins, the variable domains of the TCR alpha and beta chains (or gamma and delta (γ / δ) chains) each have three hypervariable regions, namely complementarity-determining regions (CDRs). When the TCR binds to an antigenic peptide and MHC (peptide / MHC), the T cell is activated, enabling the T cell to attack and destroy target cells.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0016]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0017] However, current therapies show a variable level of efficacy with undesirable side effects. Therefore, there is a need to identify new and improved therapies for treating CLL-1 related diseases and disorders.

Means for Solving the Problems

[0018] The present invention relates to modified immune cells (such as CAR or TCR) having specificity for CLL-1, antigen-binding molecules (including antibodies, scFv, heavy and / or light chains of these antigen-binding molecules, and CDRs, but not limited thereto).

[0019] The present invention further relates to a novel CD28 extracellular (hinge) sequence useful as a co-stimulatory domain in these cells.

[0020] The chimeric antigen receptor of the present invention typically comprises (i) a CLL-1 specific antigen-binding molecule, (ii) an extracellular (optionally including a hinge) domain, (iii) one or more co-stimulatory domains, and (iv) one or more activation domains. It will be understood that each domain may be heterologous and thus may be composed of sequences derived from (or corresponding to) different protein chains.

[0021] In some embodiments, a chimeric antigen receptor comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule comprises: a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 51, 73, and 95; b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 52, 74, and 96; c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 53, 75, and 97; d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 56, 78, and 100; e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 57, 79, and 101; and f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102. The chimeric antigen receptor may further comprise at least one co-stimulatory domain. The chimeric antigen receptor according to claim 1 further comprises at least one activation domain.

[0022] In certain embodiments, the invention relates to a chimeric antigen receptor having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the chimeric antigen receptor defined herein.

[0023] Chimeric antigen receptors having eight or fewer amino acid substitutions are also encompassed by the invention.

[0024] In certain embodiments, the co-stimulatory domain is CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD 247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R delta, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 and a ligand that specifically binds thereto, or a signal transduction domain (or other suitable portion) of any combination thereof.

[0025] In some embodiments, the costimulatory domain can include all or a portion of the 4-1BB nucleic acid sequence defined in SEQ ID NO: 141 and the corresponding amino acid sequence defined in SEQ ID NO: 142. In other embodiments, the costimulatory domain can include all or a portion of the amino acid sequence of OX40 defined in SEQ ID NO: 143. Hombach et al., Oncoimmunology. 2012 Jul. 1; See also 1(4): 458 - 466. In further embodiments, the co - stimulatory domain may comprise all or part of the ICOS molecule as described in Guedan et al., August 14, 2014; Blood: 124 (7) and Shen et al., Journal of Hematology & Oncology (2013) 6:33. In still further embodiments, the co - stimulatory domain may comprise all or part of CD27 as described in Song et al., Oncoimmunology. 2012 Jul. 1;1(4): 547 - 549.

[0026] Preferred embodiments include the incorporation of one or more of the following sequences into the CAR of the invention: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. Additional preferred embodiments include the incorporation of the sequence defined in SEQ ID NO: 14 into the CAR of the invention.

[0027] In further embodiments, the activation domain is CD3, preferably CD3 zeta, more preferably CD3 zeta having the sequence defined in SEQ ID NO: 10.

[0028] In other embodiments, the invention relates to a chimeric antigen receptor comprising an antigen - binding molecule that further comprises SEQ ID NO: 2 and further comprises SEQ ID NO: 10.

[0029] The present invention further relates to an isolated polynucleotide encoding a chimeric antigen receptor and a vector comprising the polynucleotide. Any vector known in the art may be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (such as pMSVG1), a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus vector (AAV), a lentiviral vector (such as pGAR), or any combination thereof. The pGAR sequence is as follows: AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC

[0030] The pGAR vector map is shown below:

Chemical formula

[0031] Suitable additional exemplary vectors include, for example, pBABE-puro, pBABE-neo large TcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP and pLXIN-Luc.

[0032] Exemplary immune cells include, but are not limited to, T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR-expressing cells, dendritic cells or NK-T cells. The T cells can be autologous, allogeneic or xenogeneic. In other embodiments, the present invention relates to a pharmaceutical composition comprising the immune cells described herein.

[0033] In certain embodiments, the present invention is (a) a VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VL region comprising the amino acid sequence of SEQ ID NO: 21, (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 55, (c) a VH region comprising the amino acid sequence of SEQ ID NO: 72 and a VL region comprising the amino acid sequence of SEQ ID NO: 77, (d) a VH region comprising the amino acid sequence of SEQ ID NO: 94 and a VL region comprising the amino acid sequence of SEQ ID NO: 99, Relates to antigen-binding molecules (and chimeric antigen receptors comprising these molecules) that comprise at least one of the following, wherein the VH and VL region(s) are linked by at least one linker. Also included in the present invention are chimeric antigen receptors and / or antigen-binding molecules having eight or fewer amino acid substitutions.

[0034] The linker can be a polyGly linker such as GGGGSGGGGSGGGGS (SEQ ID NO: 130) or GGGGSGGGGSGGGGS (SEQ ID NO: 145). and the like.

[0035] In other embodiments, the present invention relates to antigen-binding molecules (and chimeric antigen receptors comprising these molecules) wherein the linker comprises at least one of SEQ ID NO: 130 and SEQ ID NO: 132.

[0036] In certain embodiments, the present invention relates to antigen-binding molecules and / or chimeric antigen receptors that have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the antigen-binding molecules and / or chimeric antigen receptors defined herein.

[0037] In other embodiments, the present invention relates to isolated polynucleotides comprising at least one of SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121 and SEQ ID NO: 125.

[0038] In certain embodiments, the invention relates to an isolated polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the polynucleotides defined herein.

[0039] The invention further relates to vectors containing these polynucleotides and cells transfected with these vectors.

[0040] In other embodiments, the invention relates to an isolated polypeptide comprising an amino acid sequence defined by at least one of SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122 and SEQ ID NO: 126. In other embodiments, the invention relates to vectors encoding these polypeptides and immune cells containing these polypeptides. Preferred immune cells include T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR-expressing cells, dendritic cells or NK-T cells. The T cells can be autologous, allogeneic or xenogeneic. Chimeric antigen receptors having eight or fewer amino acid substitutions thereto are also encompassed by the present invention.

[0041] In other embodiments, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule comprises a variable heavy (V H ) chain CDR3 selected from the group consisting of the amino acid sequences of SEQ ID NO: 19, 53, 75 and 97. Chimeric antigen receptors having eight or fewer amino acid substitutions thereto are also encompassed by the present invention. The polynucleotide may further comprise an activation domain. In a preferred embodiment, the activation domain is CD3, more preferably CD3 zeta, and even more preferably the amino acid sequence defined by SEQ ID NO: 9.

[0042] In other embodiments, the present invention relates to CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD1 37, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signal transduction lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand or fragment, or a costimulatory domain comprising a signal transduction domain (or other suitable portion) of these or a combination thereof. Preferred costimulatory domains are listed below.

[0043] In a further embodiment, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CLL-1, and the antigen-binding molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102 and variable light (V L ) chain CDR3. The polynucleotide may further comprise an activation domain. The polynucleotide may further comprise a co-stimulatory domain.

[0044] In other embodiments, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 18), and CDR3 (SEQ ID NO: 19), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24).

[0045] In certain embodiments, the invention relates to an isolated polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to the above sequences.

[0046] In other embodiments, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 53), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 56), CDR2 (SEQ ID NO: 57), and CDR3 (SEQ ID NO: 58).

[0047] In certain embodiments, the invention relates to an isolated polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the above sequence.

[0048] In other embodiments, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 73), CDR2 (SEQ ID NO: 74) and CDR3 (SEQ ID NO: 75), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79) and CDR3 (SEQ ID NO: 80).

[0049] In certain embodiments, the invention relates to an isolated polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the above sequence.

[0050] In other embodiments, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 95), CDR2 (SEQ ID NO: 96) and CDR3 (SEQ ID NO: 97), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101) and CDR3 (SEQ ID NO: 102).

[0051] In certain embodiments, the invention relates to an isolated polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the above-described sequence.

[0052] In a further embodiment, the invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule is (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence GX2X3X4X5X6X7X8X9 (SEQ ID NO: 134), wherein X2 is G, F or Y, X3 is S or T, X4 is I, F or L, X5 is S or T, X6 is absent or S, X7 is absent or G, X8 is absent or E or G, X9 is F, L or Y), (b) a heavy chain variable region (VH) complementarity determining region (CDR) 2 comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 135), wherein X1 is D, H, S or Y, X2 is H, P or Y, X3 is D, E or S, X4 is D or G, X5 is G or S, X6 is absent or D or E), (c) an amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 DY (SEQ ID NO: 136), wherein X1 is E or L, X2 is R, S or V, X3 is R or Y, X4 is C, G or S, X5 is absent or is G or I, X6 is absent or is G, X7 is absent or is D, X8 is absent or is C, X9 is absent or is W or Y, X 10 is absent or is P or S, X 11 is absent or is G or Y, X 12 is F or R) and comprises a heavy chain variable region (VH) complementarity determining region (CDR) 3, (d) the amino acid sequence X1ASQX5X6X7X8X9LX 11 (SEQ ID NO: 137) wherein, X1 is Q or R, X5 is D or S, X6 is I or V, X7 is N or S, X8 is N or S, X9 is F, L or Y, X 11 is N or T) and comprises a light chain variable region (VL) CDR1, (e) the amino acid sequence X1ASX4X5X6X7 (SEQ ID NO: 138) wherein, X1 is D or G, X4 is N, S or T, X5 is L or R, X6 is A, E or K, X7 is S or T) and comprises a light chain variable region (VL) CDR2, and / or, (f) the amino acid sequence QQX3X4X5X6PX8T (SEQ ID NO: 139) wherein, X3 is S or Y, X4 is D, G or Y, X5 is N, S or T, X6 is L, T or Y, X8 is F or I) and comprises a light chain variable region (VL) CDR3, Relates to an isolated polynucleotide comprising

[0053] The present invention further relates to an antigen-binding molecule against CLL-1 comprising at least one variable heavy chain CDR3 or variable light chain CDR3 sequence as defined herein. The present invention further relates to an antigen-binding molecule against CLL-1 comprising at least one variable heavy chain CDR1, CDR2 and CDR3 sequences described herein. The present invention further relates to an antigen-binding molecule against CLL-1 comprising at least one variable light chain CDR1, CDR2 and CDR3 sequences described herein. The present invention further relates to an antigen-binding molecule against CLL-1 comprising both the variable heavy chain CDR1, CDR2, CDR3 sequences and the variable light chain CDR1, CDR2 and CDR3 sequences described herein.

[0054] The present invention further relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an antigen-binding molecule, CAR, TCR, polynucleotide, vector, cell or composition according to the present invention. Suitable diseases for treatment include, but are not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome ( MDS), myeloproliferative disorders, myeloid neoplasms, myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN) or combinations thereof. Additional diseases include inflammatory and / or autoimmune diseases such as rheumatoid arthritis, psoriasis, allergy, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis and celiac disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0055]

Figure 1

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Figure 6

Figure 7

Figure 8

Figures 9A - 9D

Figure 10

Modes for Carrying Out the Invention

[0056] It will be understood that chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs) are genetically modified receptors. These modified receptors can be readily inserted into and expressed in immune cells, including T cells, according to techniques known in the art. By using a CAR, a single receptor can be programmed to recognize a specific antigen and, when bound to that antigen, activate immune cells to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells.

[0057] A CAR can be modified to bind to an antigen (such as a cell surface antigen) by incorporating an antigen-binding molecule that interacts with the target antigen. Preferably, the antigen-binding molecule is an antibody fragment thereof, more preferably one or more single-chain antibody fragments ("scFv"). An scFv is a single-chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked to each other. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv retains the ability of the parental antibody to specifically interact with the target antigen. Since an scFv can be modified to be expressed as part of a single chain together with other CAR components, it is preferred for use in chimeric antigen receptors (ibid.). See also Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626), Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It will be understood that the antigen-binding molecule is customarily contained in the extracellular portion of the CAR so as to be capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs having specificity for two or more target antigens of interest are contemplated within the scope of the present invention.

[0058] Co-stimulatory domain Chimeric antigen receptors can incorporate co-stimulatory (signaling) domains to increase their potency. See U.S. Pat. Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (supra), Song et al., Blood 119:696 - 706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725 - 33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59 - 83 (2016). For example, CD28 is a co - stimulatory protein naturally found on T cells. Although various co - stimulatory molecules are defined herein, it will be understood that additional co - stimulatory molecules are also included within the scope of the present invention.

[0059] The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0060] A particular CD28 domain was used in the chimeric antigen receptor. According to the present invention, a novel CD28 extracellular (hinge) construct, designated "CD28T", has unexpectedly been found to provide certain benefits when utilized in a CAR construct. This construct exhibits the ability to retain (and sometimes exceed) the properties of CARs containing CD28, despite the cleavage (removal) of multiple amino acids from the extracellular CD28 sequence. These benefits include equivalent or superior cytokine production, equivalent or superior cytolytic activity, and / or equivalent or superior CAR expression levels.

[0061] The nucleotide sequence of the CD28T molecule, including the extracellular domain and the CD28 transmembrane and intracellular domains, is defined in SEQ ID NO: 1 below: CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC

[0062] The corresponding amino acid sequence is defined by SEQ ID NO: 2 below: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRS

[0063] The nucleotide sequence of the extracellular portion of CD28T is defined by SEQ ID NO: 3 below: CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA

[0064] The corresponding amino acid sequence of the CD28T extracellular domain is defined by SEQ ID NO: 4 below: LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP

[0065] The nucleotide sequence of the CD28 transmembrane domain is defined by SEQ ID NO: 5 below: TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGG TT

[0066] The amino acid sequence of the CD28 transmembrane domain is defined by SEQ ID NO: 6 below: FWVLVVVGGV LACYSLLVTV AFIIFWV

[0067] The nucleotide sequence of the CD28 intracellular signaling domain is defined by SEQ ID NO: 7 below: AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC

[0068] The amino acid sequence of the CD28 intracellular signaling domain is defined by SEQ ID NO: 8 below: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0069] Additional CD28 sequences suitable for use in the present invention include the CD28 nucleotide sequence defined by SEQ ID NO: 11 below: ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGTACCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGGCCATCAAAGCCC

[0070] The corresponding amino acid sequence is defined by SEQ ID NO: 12 below: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0071] It will be understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc. that comprise at least one isolated nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It will be further understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc. wherein the extracellular portion consists of at least one isolated nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Additionally, it will be understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc., wherein the extracellular portion consists essentially of at least one isolated nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0072] It will be understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc. that include the amino acid sequence of at least one of SEQ ID NO: 2 or SEQ ID NO: 4. It will be further understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc. in which the extracellular portion consists of the amino acid sequence of at least one of SEQ ID NO: 2 or SEQ ID NO: 4. It will also be understood that the present invention relates to antigen-binding molecules, CARs, TCRs, etc. in which the extracellular portion consists essentially of the amino acid sequence of at least one of SEQ ID NO: 2 or SEQ ID NO: 4.

[0073] Another suitable source for the extracellular and / or transmembrane domain can be derived from (or corresponding to) a part or all of CD8. The nucleotide sequence of the suitable CD8 extracellular and transmembrane domain is defined in SEQ ID NO: 13 below: GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTTCTTGCCGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTACCATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGGGCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCCCTGGCCGGAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGTAATCACAGGAAC

[0074] The corresponding amino acid sequence is defined in SEQ ID NO: 14 below: AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

[0075] Suitable costimulatory domains within the scope of the present invention can be derived from (or corresponding to), for example, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signal transduction lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand or fragment, or a combination thereof.It will be understood that additional costimulatory molecules or fragments thereof not listed above are included within the scope of the present invention.

[0076] Activation domain CD3 is an element of the T cell receptor on natural T cells and has been shown to be an important intracellular activation element in CARs. In a preferred embodiment, CD3 is CD3 zeta, the nucleotide sequence of which is defined by SEQ ID NO: 9 below: AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG

[0077] The corresponding amino acids of intracellular CD3 zeta are defined by SEQ ID NO: 10 below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0078] Domain orientation with respect to the cell Structurally, it will be understood that the domains described herein correspond to positions relative to immune cells or other cells. For this reason, these domains can be part of (i) a "hinge" or extracellular (EC) domain, (ii) a transmembrane (TM) domain, and / or (iii) an intracellular / cytoplasmic domain (IC). Intracellular components often partially include activation domains such as those of CD3 family members, preferably part of CD3 zeta. This domain is capable of activating T cells when the antigen-binding molecule binds to its target. It will be understood that the intracellular domain typically further includes one or more co-stimulatory molecules described herein.

[0079] As used herein, "activation" or "stimulation" refers to a primary response induced by the binding of an activating molecule to its cognate ligand, which mediates a signal transduction event.

[0080] "Activating molecule" or "stimulatory molecule" refers to a TCR / CD3 complex that specifically binds to a cognate stimulatory ligand present on, for example, an antigen-presenting cell, a molecule on a T cell. Suitable activating molecules are described herein.

[0081] As used herein, "co-stimulatory molecule" refers to a molecule that generates signals that mediate T cell responses, including but not limited to proliferation, activation, differentiation, etc. Co-stimulatory molecules can generate signals in addition to the primary signal generated by the activating molecules described herein.

[0082] Suitable costimulatory molecules include, but are not limited to, all or part of CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signal transduction lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand or fragment, or combinations thereof.The hinge region may contain some or all of the immunoglobulin family members such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragments thereof. It will be understood.

[0083] In some embodiments, the extracellular domain is located between the antigen-binding molecule and the transmembrane domain.

[0084] Exemplary CAR constructs according to the present invention are defined in Table 1.

[0085] [Table 1]

[0086] As described above, the modified T cells of the present invention include an antigen-binding molecule (such as scFv), an extracellular domain (which may include a "hinge" domain), a transmembrane domain, and an intracellular domain. The intracellular domain includes at least partially an activation domain and may preferably be composed of CD3 family members such as CD3 zeta, CD3 epsilon, CD3 gamma or a part thereof.

[0087] It will be further understood that the antigen-binding molecule (e.g., one or more scFv) is modified such that it is located in the extracellular portion of the molecule / construct and can recognize and bind its target(s).

[0088] Extracellular domain The extracellular domains for specific uses in the present invention are CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 and can be derived from (i.e., include) all or part of any combination thereof. The extracellular domain may be derived from either a natural source or a synthetic source.

[0089] The extracellular domain often includes a hinge portion, which may be referred to as the "spacer" region. Various hinges containing a part or derivative of the molecule described herein can be used according to the present invention.

[0090] In certain embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of the extracellular domain defined herein.

[0091] In certain embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of the extracellular nucleotide defined herein.

[0092] Transmembrane domain CARs can be designed using transmembrane domains fused to the extracellular domain of the CAR. This may also be fused to the intracellular domain of the CAR. In some cases, 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 and to minimize interaction with other members of the receptor complex. The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The extracellular regions for particular uses in the present invention are CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4) , derived from a ligand that specifically binds to CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof (i.e., including or corresponding to them).

[0093] Optionally, a short linker may form a connection between any one or several of the extracellular, transmembrane, and intracellular domains of the CAR.

[0094] In other embodiments, the transmembrane domain in the CAR of the present invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the transmembrane portion of the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence contained in SEQ ID NO: 14.

[0095] In certain embodiments, the transmembrane domain in the CAR of the present invention is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 5. In one embodiment, the CD28 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.

[0096] Intracellular (cytoplasmic) domain The intracellular (cytoplasmic) domain of the modified T cell of the present invention can bring about the activation of at least one of the normal effector functions of immune cells. The effector function of T cells may refer to cytolytic activity or helper activity including, for example, the secretion of cytokines.

[0097] Suitable intracellular molecules are those that specifically bind to ligands derived from CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof (or corresponding thereto) and include (i.e., comprise) signal transduction domains, but are not limited thereto. It will be understood that this is not limited thereto.

[0098] In a preferred embodiment, the intracellular / cytoplasmic domain of the CAR is designed to include the CD3 zeta domain itself or can be combined with any other desired intracellular domain(s) useful in the context of the CARs of the present invention. For example, the intracellular domain of the CAR can include a CD3 zeta chain moiety and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences of the intracellular signaling portion of the CARs of the present invention can be linked to each other randomly or in a specified order.

[0099] In another preferred embodiment, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of 4-1BB. In another embodiment, the intracellular domain in the CAR is designed to include CD28 and a portion of CD3 zeta, and the intracellular CD28 includes the nucleic acid sequence defined in SEQ ID NO: 7 and the amino acid sequence defined in SEQ ID NO: 8. The CD3 zeta nucleic acid sequence is defined in SEQ ID NO: 9, and the amino acid sequence is defined in SEQ ID NO: 8.

[0100] It will be appreciated that one preferred orientation of the CARs according to the present invention involves including an antigen-binding molecule (such as scFv) together with an extracellular and / or hinge domain, a co-stimulatory domain, and an activation domain. It will be further appreciated that multiple domains can be utilized together.

[0101] In some embodiments, an isolated nucleic acid is provided that comprises a promoter operably linked to a first polynucleotide encoding an antigen-binding molecule, at least one co-stimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0102] In some embodiments, the modified immune cell is a T cell, a tumor-infiltrating lymphocyte (TIL), a NK cell, a TCR-expressing cell, a dendritic cell, or a NK-T cell. In some embodiments, the cell is obtained or prepared from peripheral blood. In some embodiments, the cell is obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained or prepared from bone marrow. In some embodiments, the cell is obtained or prepared from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the nucleic acid vector is transfected or transduced into the cell using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes.

[0103] In some embodiments, a chimeric antigen receptor is expressed in the modified immune cell comprising the nucleic acid of the present application. These chimeric antigen receptors of the present application may, in some embodiments, comprise (i) an antigen-binding molecule (such as scFv), (ii) a transmembrane region, and (iii) a T cell activation molecule or region.

[0104] Whenever an aspect is described herein in conjunction with the word "comprising", in no case shall "consisting of" and / or "consisting essentially of" Another similar aspect described in terms of "(consisting essentially of)" is also provided. This is further understood.

[0105] Additionally, the terms "about" or "comprising essentially of" refer to values or compositions within an acceptable error range for a particular value or composition specified by those skilled in the art, depending in part on how that value or composition is measured or specified, i.e., on the limitations of the measurement system. For example, "about" or "comprising essentially of" can mean within one standard deviation or greater than one standard deviation per implementation in the relevant technical field. Alternatively, "about" or "comprising essentially of" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any numerical value between 2.7 mg and 3.3 mg (in the case of 10%). Further, especially with respect to biological systems or processes, the above terms may mean up to an order of magnitude or up to five times a particular value. When a particular value or composition is provided in this application and the claims, unless otherwise indicated, the meaning of "about" or "comprising essentially of" should be considered to be within the acceptable error range for that particular value or composition.

[0106] Antigen-binding molecule Antigen-binding molecules are within the scope of the present invention. As used herein, "antigen-binding molecule" means any protein that binds to a designated target antigen. In this application, the designated target antigen is the CLL-1 protein or a fragment thereof. Examples of antigen-binding molecules include, but are not limited to, antibodies and their binding portions, such as immunologically functional fragments. Peptibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are a preferred example of another antigen-binding molecule.

[0107] In certain embodiments, the present invention provides (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence GX2X3X4X5X6X7X8X9 (SEQ ID NO: 134), where X2 is G, F, or Y, X3 is S or T, X4 is I, F, or L, X5 is S or T, X6 is absent or S, X7 is absent or G, X8 is absent or E or G, X9 is F, L, or Y); (b) a heavy chain variable region (VH) complementarity determining region (CDR) 2 comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 135), where X1 is D, H, S, or Y, X2 is H, P, or Y, X3 is D, E, or S, X4 is D or G, X5 is G or S, X6 is absent or D or E); (c) an amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 DY (SEQ ID NO: 136), where X1 is E or L, X2 is R, S, or V, X3 is R or Y, X4 is C, G, or S, X5 is absent or G or I, X6 is absent or G, X7 is absent or D, X8 is absent or C, X9 is absent or W or Y, X 10 is absent or P or S, X 11 is absent or G or Y, X12 a heavy chain variable region (VH) complementarity determining region (CDR) 3 that includes (d) the amino acid sequence X1ASQX5X6X7X8X9LX 11 (SEQ ID NO: 137) where X1 is Q or R, X5 is D or S, X6 is I or V, X7 is N or S, X8 is N or S, X9 is F, L or Y, X 11 is N or T) and a light chain variable region (VL) CDR1, (e) the amino acid sequence X1ASX4X5X6X7 (SEQ ID NO: 138) where X1 is D or G, X4 is N, S or T, X5 is L or R, X6 is A, E or K, X7 is S or T) and / or a light chain variable region (VL) CDR2, and / or (f) the amino acid sequence QQX3X4X5X6PX8T (SEQ ID NO: 139) where X3 is S or Y, X4 is D, G or Y, X5 is N, S or T, X6 is L, T or Y, X8 is F or I) and a light chain variable region (VL) CDR3, relates to an antigen-binding molecule that includes.

[0108] In some embodiments, the invention provides (a) a variable heavy chain CDR1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 51, 73, 95, 5 and 97, (b) a variable heavy chain CDR2 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 52, 74, 96, (c) a variable heavy chain CDR3 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 53, 75 and 97, (d) A variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 56, 78, and 100, (e) A variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 57, 79, and 101, (f) A variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102, relating to an antigen-binding molecule comprising at least one of the foregoing.

[0109] In other embodiments, the invention relates to (a) A VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VL region comprising the amino acid sequence of SEQ ID NO: 21, (b) A VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 55, (c) A VH region comprising the amino acid sequence of SEQ ID NO: 72 and a VL region comprising the amino acid sequence of SEQ ID NO: 77 comprising a VL region, (d) A VH region comprising the amino acid sequence of SEQ ID NO: 94 and a VL region comprising the amino acid sequence of SEQ ID NO: 99, relating to an antigen-binding molecule (and chimeric antigen receptors comprising these molecules) wherein the VH and VL region(s) are linked by at least one linker. In other embodiments, the invention relates to an antigen-binding molecule (and chimeric antigen receptors comprising these molecules) wherein the linker comprises at least one of SEQ ID NOs: 130 and 132.

[0110] In a further embodiment, the invention relates to an antigen-binding molecule comprising a variable light (V L ) chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102.

[0111] In other embodiments, the invention relates to an isolated polynucleotide encoding an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 18) and CDR3 (SEQ ID NO: 19), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 24).

[0112] In other embodiments, the invention relates to an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 56), CDR2 (SEQ ID NO: 57) and CDR3 (SEQ ID NO: 58).

[0113] In other embodiments, the invention relates to an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 73), CDR2 (SEQ ID NO: 74) and CDR3 (SEQ ID NO: 75), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79) and CDR3 (SEQ ID NO: 80).

[0114] In other embodiments, the invention relates to an isolated polynucleotide encoding an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 95), CDR2 (SEQ ID NO: 96) and CDR3 (SEQ ID NO: 97), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101) and CDR3 (SEQ ID NO: 102).

[0115] In certain embodiments, the present invention relates to isolated polynucleotides encoding anti-CLL-1 antigen-binding molecules that cross-compete with one or more of the antibodies or antigen-binding molecules thereof described herein. In one embodiment, the present invention relates to isolated polynucleotides encoding anti-CLL-1 antigen-binding molecules that bind to the same epitope as one or more of the antigen-binding molecules described herein.

[0116] In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen. In further embodiments, the antigen-binding molecule is an antibody or a fragment thereof comprising one or more of its complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv).

[0117] The term "immunely functional fragment" (or "fragment") of an antigen-binding molecule refers to a portion of an antibody (regardless of how that portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain, but is still capable of specifically binding to an antigen. The fragments are antigen-binding molecule species containing fragments of the same or similar structure (e.g., fragments containing a CDR that does not contain a CDR that is not present in the fragment). Such fragments are biologically active in that they can bind to the target antigen and compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments can block or reduce the activity of CLL-1. In one aspect, such fragments retain at least one CDR present in a full-length light or heavy chain, and in some embodiments, comprise a single heavy and / or light chain or a portion thereof. These fragments can be produced by recombinant DNA methods or by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.

[0118] As immune function immunoglobulin fragments, there are scFv fragments, Fab fragments (Fab’, F(ab’)2, etc.), one or more CDRs, diabodies (light chain variable domains and heavy chain variable domains on the same polypeptide connected via a short peptide linker that is too short to allow pairing between two domains on the same chain), domain antibodies, and single-chain antibodies, but are not limited thereto. These fragments can be derived from any mammalian source including, but not limited to, humans, mice, rats, camelids, or rabbits. As will be understood by those skilled in the art, the antigen-binding molecule may contain non-protein components.

[0119] Variants of the antigen-binding molecule, such as variable light chains and / or variable heavy chains each having at least 70% - 80%, 80% - 85%, 85% - 90%, 90% - 95%, 95% - 97%, 97% - 99% or more than 99% identity to the amino acid sequences of the sequences described herein are also within the scope of the present invention. In some cases, such molecules contain at least one heavy chain and one light chain, while in other cases, the variant form contains two identical light chains and two identical heavy chains (or subparts thereof). Those skilled in the art can determine suitable variants of the antigen-binding molecule defined herein using known techniques. In certain embodiments, those skilled in the art can identify suitable sites of the molecule that can be altered without disrupting activity by targeting regions that are not considered important for activity.

[0120] In certain embodiments, the polypeptide structure of the antigen-binding molecule is based on antibodies including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof. In some embodiments, the antigen-binding molecule comprises or consists of avimers.

[0121] In some embodiments, the antigen-binding molecule against CLL-1 is administered as part of a CAR, TCR, or other immune cell. In such immune cells, the antigen-binding molecule against CLL-1 may be under the control of the same promoter region or separate promoters. In certain embodiments, the protein agents and / or the gene encoding the antigen-binding molecule against CLL-1 may be in separate vectors.

[0122] The present invention further provides a pharmaceutical composition comprising an antigen-binding molecule against CLL-1 together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the pharmaceutical composition comprises two or more different antigen-binding molecules against CLL-1. In certain embodiments, the pharmaceutical composition comprises two or more antigen-binding molecules against CLL-1, and the antigen-binding molecule against CLL-1 binds two or more epitopes. In some embodiments, the various antigen-binding molecules do not compete with each other for binding to CLL-1.

[0123] In other embodiments, the pharmaceutical composition can be selected for parenteral delivery, inhalation, or delivery via the gastrointestinal tract such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the ability of those skilled in the art. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the therapeutic composition can be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired antigen-binding molecule to CLL-1, with or without additional therapeutic agents in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water formulated as a sterile isotonic solution in which the antigen-binding molecule to CLL-1 is properly preserved, with or without at least one additional therapeutic agent. In certain embodiments, the preparation includes the formulation of the desired molecule with a polymeric compound (such as polylactic acid or polyglycolic acid), beads, or liposomes that can result in controlled or sustained release of the product that can be delivered by depot injection later. In certain embodiments, an implantable drug delivery device can be used to introduce the desired molecule.

[0124] In some embodiments, the antigen-binding molecule is used as a diagnostic or verification tool. The antigen-binding molecule can be used to assay the amount of CLL-1 present in a sample and / or a subject. In some embodiments, the diagnostic antigen-binding molecule is not neutralizing. In some embodiments, the antigen-binding molecules disclosed herein are used or provided in an assay kit and / or method for detecting CLL-1 in mammalian tissues or cells to screen / diagnose a disease or disorder associated with a change in CLL-1 levels. The kit may include an antigen-binding molecule that binds to CLL-1, along with means for indicating the binding of the antigen-binding molecule to CLL-1 and optionally the CLL-1 protein level if present.

[0125] The antigen-binding molecule is further understood in view of the following definitions and explanations.

[0126] The "Fc" region comprises two heavy chain fragments that include the CH1 and CH2 domains of the antibody. The two heavy chain fragments are linked by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0127] The "Fab fragment" comprises one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fab' fragment" comprises one light chain and a portion of one heavy chain containing the VH domain and the CH1 domain, and further the region between the CH1 domain and the CH2 domain, with interchain disulfide bonds formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. The "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 domain and the CH2 domain, with interchain disulfide bonds formed between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments linked by a disulfide bond between the two heavy chains.

[0128] The "Fv region" contains variable regions derived from both the heavy and light chains, but lacks the constant regions.

[0129] A "single-chain variable fragment" ("scFv"; also referred to as a "single-chain antibody") refers to an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker so as to form a single polypeptide chain and form an antigen-binding region. See International Publication No. WO 88 / 01649 of the PCT application, as well as U.S. Patent Nos. 4,946,778 and 5,260,203, the entire disclosures of which are incorporated herein by reference.

[0130] A "bivalent antigen-binding molecule" contains two antigen-binding sites. In some cases, the two binding moieties The positions have the same antigen specificity. The bivalent antigen-binding molecule can be bispecific. A "multispecific antigen-binding molecule" targets two or more antigens or epitopes. A "bispecific", "dual-specific" or "bifunctional" antigen-binding molecule is a hybrid antigen-binding molecule or antibody that each has two different antigen-binding sites. The two binding sites of the bispecific antigen-binding molecule bind to two different epitopes that may be present on the same or different protein targets.

[0131] An antigen-binding molecule is said to "specifically bind" to its target antigen when the dissociation constant (K d ) is about 1×10 -7 M. An antigen-binding molecule specifically binds an antigen with "high affinity" when K d is 1×10 -9 M to 5×10 -9 M, and specifically binds an antigen with "very high affinity" when K d is 1×10 -10 M to 5×10 -10 M. In one embodiment, the antigen-binding molecule has a K -9 of 10 d M. In one embodiment, the off-rate is less than 1×10 -5 . In other embodiments, the antigen-binding molecule binds to human CLL-1 with a K -7 of about 10 -13 M to about 10 d M, and in yet another embodiment, the antigen-binding molecule binds with a K -10 of 1.0×10 -10 to 5×10 d .

[0132] In some embodiments, the antibody or antigen-binding molecule of the present invention specifically binds CLL-1 (e.g., hCLL-1). In certain embodiments, the anti-CLL-1 antibody or antigen-binding molecule of the present invention binds human CLL-1 with a K -6 less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10D binds with. In a particular embodiment, the anti-CLL-1 antibody or antigen-binding molecule binds human CLL-1 with a K -7 less than 1×10 D M. In another embodiment, the anti-CLL-1 antibody or antigen-binding molecule binds human CLL-1 with a K -8 less than 1×10 D M. In some embodiments, the anti-CLL-1 antibody or antigen-binding molecule binds human CLL-1 with a K of about 1×10 -7 M, about 2×10 -7 M, about 3×10 -7 M, about 4×10 -7 M, about 5×10 -7 M, about 6×10 -7 M, about 7×10 -7 M, about 8×10 -7 M, about 9×10 -7 M, about 1×10 -8 M, about 2×10 -8 M, about 3×10 -8 M, about 4×10 -8 M, about 5×10 -8 M, about 6×10 -8 M, about 7×10 -8 M, about 8×10 -8 M, about 9×10 -8 M, about 1×10 -9 M, about 2×10 -9 M, about 3×10 -9 M, about 4×10 -9 M, about 5×10 -9 M, about 6×10 -9 M, about 7×10 -9 M, about 8×10 -9 M, about 9×10 -9 M, about 1×10 -10 M or about 5×10 -10 M of K D binds with. In a certain embodiment, K D is calculated from the quotient of k off / k on and k on and k off are determined using a monovalent antibody such as a Fab fragment, measured, for example, by BIAcore™ surface plasmon resonance technology. In other embodiments, K D is koff / k on calculated from the quotient of, k on and k off is identified using a bivalent antibody such as a Fab fragment, measured by, for example, BIAcore™ surface plasmon resonance technology.

[0133] In other embodiments, the anti-CLL-1 antibody or antigen-binding molecule binds with a K of less than 1×10 -9 M, less than 3×10 -9 M, less than 5×10 -9 M, less than 1×10 -10 M, less than 3×10 -10 M, or less than 5×10 -10 [[ID=2,3]]M. In other embodiments, the anti-CLL-1 antibody or antigen-binding molecule binds with a K of less than 1×10 D M, less than 1×10 -5 M, less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, or less than 1×10 -10 M. D

[0134] In some embodiments, the anti-CLL-1 antibody or antigen-binding molecule binds with a human CLL-1 of less than 1×10 -4 M -1 s -1 less than 2×10 -4 M -1 s -1 less than 3×10 -4 M -1 s -1 less than 4×10 -4 M -1 s -1 less than 5×10 -4 M -1 s -1 less than 6×10 -4 M -1 s -1 less than 7×10 -4 M -1 s -1 less than 8×10 -4 M -1 s -1 ​Less than, 9×10 -4 M -1 s -1 Less than, 1×10 -5 M -1 s -1 Less than, 2×10 -5 M -1 s -1 Less than, 3×10 -5 M -1 s -1 Less than, 4×10 -5 M -1 s -1 Less than, 5×10 -5 M -1 s -1 Less than, 6×10 -5 M -1 s -1 Less than, 7×10 -5 M -1 s -1 Less than, 8×10 -5 M -1 s -1 Less than, 9×10 -5 M -1 s -1 Less than or 1×10 -6 M -1 s -1 Less than, 2×10 -6 M -1 s -1 Less than, 3×10 -6 M -1 s -1 Less than, 4×10 -6 M -1 s -1 Less than, 5×10 -6 M -1 s -1 Less than, 6×10 -6 M -1 s -1 Less than, 7×10 -6 M -1 s -1 Less than, 8×10 -6 M -1 s -1 Less than, 9×10 -6 M -1 s -1 Less than or 1×10 -7 M -1 s -1 Less than the meeting rate (k on) is combined. In certain embodiments, k on is identified using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore™ surface plasmon resonance technology. In other embodiments, k on is identified using a bivalent antibody measured, for example, by BIAcore™ surface plasmon resonance technology.

[0135] In some embodiments, the anti-CLL-1 antibody or antigen-binding molecule binds human CLL-1 at 1×10 -2 s -1 less than, 2×10 -2 s -1 less than, 3×10 -2 s -1 less than, 4×10 -2 s -1 less than, 5×10 -2 s -1 less than, 6×10 -2 s -1 less than, 7×10 -2 s -1 less than, 8×10 -2 s -1 less than, 9×10 -2 s -1 less than, 1×10 -3 s -1 less than, 2×10 -3 s -1 less than, 3×10 -3 s -1 less than, 4×10 -3 s -1 less than, 5×10 -3 s -1 less than, 6×10 -3 s -1 less than, 7×10 -3 s -1 less than, 8×10 -3 s -1 less than, 9×10 -3 s -1 less than, 1×10 -4 s -1 less than, 2×10 -4 s -1 less than, 3×10 -4 s -1 less than, 4×10 -4 s -1 less than, 5×10-4 s -1 less than 6×10 -4 s -1 less than 7×10 -4 s -1 less than 8×10 -4 s -1 less than 9×10 -4 s -1 less than 1×10 -4 s -1 less than or 5×10 -4 s -1 dissociation rate (k off ). In certain embodiments, k off is specified using a monovalent antibody such as a Fab fragment, measured, for example, by BIAcore™ surface plasmon resonance technology. In other embodiments, k off is specified using a divalent antibody, measured, for example, by BIAcore™ surface plasmon resonance technology.

[0136] An antigen-binding molecule is said to be "selective" if it binds more tightly to one target than to a second target.

[0137] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for binding to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is an antigen-binding molecular species as defined herein. Intact antibodies generally include at least two full-length heavy chains and two full-length light chains, but may in some cases include fewer chains, such as antibodies that naturally occur in camels, which may contain only heavy chains. An antibody may be derived from a single source only, or may be chimeric, i.e., different parts of the antibody may be derived from two different antibodies, as further explained below. Antigen-binding molecules, antibodies, or binding fragments can be made in hybridomas by recombinant DNA methods, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and mutant proteins thereof, examples of which are described below. Further, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively.

[0138] The variable regions typically exhibit the same overall structure of relatively conserved framework regions (FRs) joined by three hypervariable regions (i.e., "CDRs"). The CDRs from each pair of two chains typically align by the framework regions, enabling binding to a specific epitope. From the N-terminus towards the C-terminus, both the light chain and heavy chain variable regions typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions of the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions of the light chain are typically referred to as LC CDR1, CDR2, and CDR3. The amino acid assignments to each domain typically follow the Kabat, Chothia definitions, or the AbM definition.

[0139] The terms "Kabat numbering" and similar terms are recognized in the art and refer to systems for numbering the amino acid residues in the variable regions of the heavy and light chains of an antibody, or in its antigen-binding portion. In certain embodiments, the CDRs of an antibody can be identified according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). When using the Kabat numbering system the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), which may optionally contain one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). When using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme.

[0140] In certain embodiments, the CDRs of an antibody can be identified according to the Chothia numbering scheme that refers to the positions of the structural loops of immunoglobulins (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917, Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948, Chothia C et al., (1992) J Mol Biol 227: 799-817, Tramontano A et al., (1990) J Mol Biol 215(1): 175-82, and U.S. Patent No. 7,709,226). This is desired). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at amino acids 26-32, 33 or 34 of the heavy chain, the Chothia CDR-H2 loop is present at amino acids 52-56 of the heavy chain, and the Chothia CDR-H3 loop is present at amino acids 95-102 of the heavy chain, while the Chothia CDR-L1 loop is present at amino acids 24-34 of the light chain, the Chothia CDR-L2 loop is present at amino acids 50-56 of the light chain, and the Chothia CDR-L3 loop is present at amino acids 89-97 of the light chain. The ends of the Chothia CDR-HI loop vary between H32-H34 depending on the length of the loop when numbered using the Kabat numbering convention (this is because the Kabat numbering scheme places insertions at H35A and H35B, i.e., the loop ends at 32 if neither 35A nor 35B is present, at 33 if only 35A is present, and at 34 if both 35A and 35B are present).

[0141] In certain embodiments, the CDRs of the antibodies described herein are identified according to the Chothia numbering scheme.

[0142] Several definitions of CDRs, namely Kabat numbering, Chothia numbering, AbM numbering or contact numbering, are commonly used. A The bM definition is one of two compromises used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on an analysis of available complex crystal structures.

[0143]

Table 2

[0144] As used herein, the term "heavy chain" when used in reference to an antibody refers to any different type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), that gives rise to antibodies of the classes IgA, IgD, IgE, IgG, and IgM, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence of the constant domain.

[0145] As used herein, the term "light chain" when used in reference to an antibody may refer to any different type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are known in the art. In certain embodiments, the light chain is a human light chain.

[0146] The term "variable region" or "variable domain" typically refers to a portion of the light chain and / or heavy chain of an antibody that includes approximately the first 120 to 130 amino acids at the amino terminus in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. The variable region of an antibody typically determines the specificity of that particular antibody for its target.

[0147] The variability is not evenly distributed throughout the variable domain of an antibody or antigen-binding molecule but is concentrated in each of the sub-domains of the variable regions of the heavy and light chains. These sub-domains are 、As further described herein, they are referred to as "hypervariable regions" or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRM or FR), which provide a scaffold for the six CDRs in three-dimensional space and form the antigen-binding surface. The variable domains of naturally occurring heavy and light chains each adopt a predominantly β-sheet conformation, connected by three hypervariable regions that form loops connecting β-sheet structures and, in some cases, forming part of them, and four FRM regions (FR1, FR2, FR3, and FR4). The hypervariable regions of each chain are very closely approximated and associated with the hypervariable regions of the other chain by the FRM and contribute to the formation of the antigen-binding site (see Kabat et al., further described herein).

[0148] Typically, CDRs form loop structures that can be classified into canonical structures. The term "canonical structure" refers to the backbone conformation adopted by the antigen-binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsional angles of the polypeptide backbone . Thus, loops between corresponding antibodies can have very similar three-dimensional structures, despite the high amino acid sequence variability of most of the loop (Chothia and Lesk, J. MoI. Biol., 1987, 196: 901, Chothia et al., Nature, 1989, 342: 877, Martin and Thornton, J. MoI. Biol, 1996, 263: 800). Furthermore, a correlation is recognized between the loop structure adopted and the amino acid sequence around it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop and in the conserved framework (i.e., outside the loop). Thus, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0149] The term "canonical structure" can also include considerations regarding the linear sequence of an antibody, as cataloged, for example, by Kabat (Kabat et al. in this specification). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of antibody variable domains in a consistent manner and is the preferred scheme applied to the present invention, as also referred to in other parts of this specification. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, differences not fully reflected in Kabat numbering can be accounted for by the numbering system of Chothia et al. and / or revealed by other techniques, such as crystal structure analysis and two-dimensional or three-dimensional computational modeling. Thus, a given antibody sequence can be classified into canonical classes that, in particular, enable the identification of suitable chassis sequences (e.g., based on a desire to include various canonical structures in a library). The Kabat numbering of antibody amino acid sequences and the structural considerations described by Chothia et al. (in this specification) and their impact on the interpretation of canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional arrangements of various classes of immunoglobulins are known in the art. For an overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0150] The CDR3 of the light chain and especially that of the heavy chain can potentially be the most important determinants of antigen binding in the light and heavy chain variable regions. In some antibody constructs, the heavy chain CDR3 appears to constitute the major contact site between the antigen and the antibody. In vitro selection schemes that vary CDR3 alone can be used to alter the binding properties of the antibody or to determine which residues contribute to antigen binding. Thus, CDR3 is typically the largest source of molecular diversity in the antibody binding site. For example, H3 can be as short as two amino acid residues or larger than 26 amino acids.

[0151] As used herein, the terms "constant region" and "constant domain" are synonymous and have the meaning generally recognized in the art. The constant region is the antibody portion of the light chain and / or heavy chain that does not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions such as interaction with Fc receptors, for example, the carboxyl-terminal portion. The constant regions of immunoglobulin molecules generally have a more conserved amino acid sequence compared to the immunoglobulin variable domains.

[0152] "Binding affinity" generally refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of the binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured and / or represented in a number of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). K D is calculated from the quotient of k off / k on , and K A is calculated from the quotient of k on / k off . k on refers, for example, to the association rate constant of an antibody for an antigen, and koff refers to, for example, the dissociation of an antibody with respect to an antigen. k on and k off can be determined by techniques known to those skilled in the art, such as BIAcore (trademark) or KinExA.

[0153] The term "neutralize" refers to each of an antigen-binding molecule, scFv or antibody that binds to a ligand and blocks or reduces the biological effect of the ligand. This can be done, for example, by directly blocking the binding site on the ligand or by binding to the ligand and changing the binding ability of the ligand by indirect means (such as a structural change or energy change in the ligand). In some embodiments, the term may mean that the protein to which the antigen-binding molecule binds prevents the performance of a biological function.

[0154] The term "target" or "antigen" refers to a molecule or a part of a molecule to which an antigen-binding molecule can bind. In certain embodiments, the target may have one or more epitopes.

[0155] The term "competing" when used in the context of an antigen-binding molecule competing for the same epitope means competition between antigen-binding molecules (e.g., an antibody or an immunologically functional fragment thereof) being tested, as determined by an assay in which the antigen-binding molecule blocks or inhibits (e.g., reduces) the specific binding of a reference antigen-binding molecule to an antigen. A number of types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA using I-125 labeling (Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0156] As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitopes), or an epitope may be, for example, derived together from two or more non-contiguous regions of a polypeptide(s) (conformational, non-linear, discontinuous, or non-contiguous epitopes). In certain embodiments, the epitope to which an antibody binds can be identified, for example, by NMR spectroscopy, X-ray crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallographic studies, crystallization can be performed using any of the methods known in the art (e.g., Giege R. et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350, McPherson A (1990) Eur J Biochem 189: 1-23, Chayen NE (1997) Structure 5: 1269-1274, McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques, and X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. Patent Application Pub It can be refined using computer software such as BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60, Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), and the like. Mutagenesis mapping studies can be performed using any method known to those skilled in the art. This can be done, for example, for descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques, see Champe M et al., (1995) J Biol Chem 270: 1388-1394, and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0157] As used herein, the terms "label" or "labeled" refer to the incorporation of a detectable marker, for example, by the incorporation of a radiolabeled amino acid or by the addition of a biotin moiety to a polypeptide that can be detected by a labeled avidin (e.g., a fluorescent marker or streptavidin having enzymatic activity that can be detected by optical or colorimetric methods). In certain embodiments, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.

[0158] Therapeutic methods By using adoptive immunotherapy, natural T cells can be (i) removed from a patient, (ii) genetically modified to express a chimeric antigen receptor (CAR) that binds to at least one tumor antigen, (iii) expanded ex vivo to a larger population of modified T cells, and (iv) reintroduced into the patient. See, e.g., U.S. Patent Nos. 7,741,465 and 6,319,494, Eshhar et al. (Cancer Immunol, supra); Krause et al. (supra); Finney et al. (supra). After reintroducing the modified T cells into the patient, the modified T cells mediate an immune response against cells expressing the tumor antigen. See, e.g., Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626). This immune response includes the secretion of IL-2 and other cytokines by the T cells, clonal expansion of T cells that recognize the tumor antigen, and specific killing of target positive cells mediated by the T cells. See Hombach et al., Journal of Immun. 167: 6123-6131 (2001).

[0159] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that constitutes a major component of the innate immune system. NK cells reject tumor and virus-infected cells. NK cells act through the process of apoptosis, or programmed cell death. NK cells are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (antibody-independent). Their T cell receptor (TCR) differentiates them from other types of lymphocytes. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell memory T cells such as naive cells), and SCM Cell(stem memory T SCM (ii) central memory T cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells; CM cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T EM There are various types of T cells, including T cells (Tregs, suppressor T cells or CD4+CD25+ regulatory T cells, but do not express L-selectin or CCR7, but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. On the other hand, B cells play the most important role in humoral immunity (antibody-mediated immunity). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and, after activation by antigen interaction, become memory B cells. In mammals, immature B cells are formed in the bone marrow, from which they take their name.

[0160] The terms "genetically modified" or "modified" refer to methods of modifying a cell's genome, including, but not limited to, deletions in coding or non-coding regions or portions thereof, or insertions in coding regions or portions thereof. In some embodiments, the cell to be modified is a lymphocyte, such as a T cell that can be obtained from either a patient or a donor. The cell can be modified to express a foreign construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), for example, one that is integrated into the cell's genome.

[0161] "Immune response" refers to the selective targeting, binding to, damaging, destroying, and / or elimination from the vertebrate body of normal human cells or tissues in the case of an invading pathogen, a cell or tissue infected with a pathogen, a cancerous or other abnormal cell, or an autoimmune or pathological inflammation, brought about by the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced by any of these cells or by the liver.

[0162] The term "immunotherapy" refers to the treatment of a subject afflicted with, suffering from, or at risk of recurrence of a disease by methods including inducing, enhancing, suppressing, or otherwise modulating an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, It may include tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. However, those skilled in the art will recognize that the conditioning methods disclosed herein can enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and International Publication No. 2008 / 081035.

[0163] The T cells for immunotherapy can be derived from any source known in the art. For example, the T cells may be differentiated in vitro from a hematopoietic stem cell population, or the T cells may be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue derived from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells may be derived from one or more T cell lines available in the art. Also, the T cells can be obtained from blood units collected from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is hereby incorporated by reference in its entirety to form a part of this specification.

[0164] The term "engineered autologous cell therapy", also known as adoptive cell transfer and abbreviated as "eACT™", is a process in which a patient's own T cells are collected and subsequently genetically engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be modified to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are modified to express an extracellular single-chain variable fragment (scFv) with specificity for a particular tumor antigen, linked to an intracellular signaling moiety that includes at least one co-stimulatory domain and at least one activation domain. The co-stimulatory domain can be derived from (or correspond to), for example, CD28, and the activation domain can be derived from (or correspond to), for example, CD3-zeta. In certain embodiments, the CAR is designed to have two, three, four or more co-stimulatory domains.

[0165] The term "autologous" refers to any substance derived from the same individual that is later re-introduced. For example, the methods of engineered autologous cell therapy (eACT™) described herein include collecting lymphocytes from a patient, subsequently modifying them, for example, to express a CAR construct, and then administering them back to the same patient.

[0166] The term "allogeneic" refers to any substance derived from one individual that is later introduced into another individual of the same species (e.g., allogeneic T cell transfer).

[0167] Accordingly, in some aspects, the invention includes a method of treating or preventing a disorder associated with undesired and / or elevated CLL-1 levels in a patient, the method comprising administering to a patient in need thereof an effective amount of at least one isolated antigen-binding molecule, CAR, or TCR disclosed herein.

[0168] A method of treating a disease or disorder including cancer is provided. In some embodiments, the invention relates to generating a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of the modified immune cells of the present application. In some embodiments, the T cell-mediated immune response is against target cell(s). In some embodiments, the modified immune cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the target cells are tumor cells. In some aspects, the invention includes a method of treating or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding molecule described herein. In some aspects, the invention includes a method of treating or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor, T cell receptor, and / or isolated antigen-binding molecule described herein.

[0169] In some aspects, the invention includes a pharmaceutical composition comprising at least one antigen-binding molecule described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active substance.

[0170] The antigen-binding molecules, CARs, TCRs, immune cells, etc. of the present invention can be used for treating myeloid diseases including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndromes (MDS), myeloproliferative disorders, myeloid neoplasms, myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or combinations thereof. Additional diseases include inflammatory and / or autoimmune diseases such as rheumatoid arthritis, psoriasis, allergy, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

[0171] CAR + / CAR-T+ / TCR + The target dose of the cells is preferably 1×10 6 cells / kg to 2×10 10 cells / kg, more preferably 2×10 6 cells / kg. It will be understood that doses above and below this range may be appropriate for a particular subject, and that the appropriate dose level can be determined by a healthcare provider as needed. Additionally, multiple doses of cells can be provided according to the present invention.

[0172] A method of reducing the size of a tumor in a subject, comprising administering to the subject a modified cell of the present invention, wherein the cell comprises a chimeric antigen receptor, a T cell receptor or a T cell receptor-based chimeric antigen receptor that binds to an antigen on the tumor, is also provided. In some embodiments, the subject has a solid tumor, or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the modified cells are delivered to the tumor bed. In some embodiments the cancer is present in the bone marrow of the subject. In some embodiments, the modified cells are autologous T cells. In some embodiments, the modified cells are allogeneic T cells. In some embodiments, the modified cells are xenogeneic T cells. In some embodiments, the modified cells of the present application are transfected or transduced in vivo. In other embodiments, the modified cells are transfected or transduced ex vivo. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, in vitro cells can include T cells.

[0173] The above method may further comprise administering one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapy It is a pharmaceutical agent. Beneficial preconditioning treatment regimens, along with relevant beneficial biomarkers, are described in U.S. Provisional Patent Application Nos. 62 / 262,143 and 62 / 167,750, which are hereby incorporated by reference in their entirety and made a part of this specification. These include, for example, a method of conditioning a patient in need of T cell therapy, which involves administering to the patient a specified beneficial dose of cyclophosphamide (200 mg / m 2 / day to 2000 mg / m 2 / day) and a specified dose of fludarabine (20 mg / m 2 / day to 900 mg / m 2 / day). A preferred dosing regimen includes administering to the patient approximately 500 mg / m 2 / day of cyclophosphamide and approximately 60 mg / m 2 / day of fludarabine daily for 3 days, including the treatment of the patient, prior to administering to the patient a therapeutically effective amount of modified T cells.

[0174] In other embodiments, an antigen-binding molecule, a transduced (or otherwise modified) cell (such as a CAR or TCR), and a chemotherapeutic agent are each administered to a subject in an amount effective to treat a disease or condition in the subject.

[0175] In certain embodiments, the compositions comprising immune effector cells expressing the CARs disclosed herein can be administered with various chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburemabicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as actinomycin D, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, cardifolin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogues such as fludarabine, 6-mercaptopurine, thioguanine; pyrimidine analogues such as ara-C, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; platinum coordination complexes such as cisplatin, carboplatin, oxaliplatin; vinca alkalides such as vincristine, vinblastine, vinorelbine; taxanes such as pacific taxol, docetaxel; topoisomerase inhibitors such as amsacrine, etoposide, etoposide phosphate, teniposide, topotecone; biological response modifiers such as interferon, interleukin, colony stimulating factor, erythropoietin, granulocyte macrophage colony stimulating factor; and other agents such as hydroxyurea, L-asparaginase, procarbazine, mitotane, aminoglutethimide, leuprolidine, goserelin, tamoxifen, toremifene, aromatase inhibitors such as anastrozole, letrozole, exemestane; antiandrogens such as flutamide, nilutamide, bicalutamide, cyproterone acetate; gonadotropin releasing hormone agonists such as goserelin, leuprolidine, buserelin, triptorelin; and other agents such as Purine analogs such as amiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU; castrone, propionic acid Androgens such as drostanolone, epitioestanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; Folic acid replenishers such as folic acid; aceglatone; aldophosphamide glico Sid; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL (trademark), Bristol-Myers Squibb) and docetaxel (TAXOTERE (trademark), Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopur Methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoid acid derivatives such as Targretin (trademark) (bexarotene), Panretin (trademark) (alitretinoin); ONTAK (trademark) (denileukin diftitox); esperamicin; Capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Anti-hormonal agents that act to modulate or inhibit the hormonal action on tumors, such as anti-estrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above are also included in this definition. Where appropriate, combinations of chemotherapeutic agents including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan (trademark)), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin (trademark)) and prednisone are also administered.

[0176] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week of the administration of the modified cell, polypeptide or nucleic acid. In other embodiments, the chemotherapeutic agent is administered one week to four weeks, i.e., one week to one month, one week to two months, one week to three months, one week to six months, one week to nine months, or one week to twelve months after the administration of the modified cell, polypeptide or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month before administering the cell, polypeptide or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0177] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, useful potential additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo™), pembrolizumab (Keytruda™), pembrolizumab, pidilizumab, and atezolizumab.

[0178] Additional therapeutic agents suitable for combination with the present invention include ibrutinib (Imbruvica™), ofatumumab (Arzerra™), rituximab (Rituxan™), bevacizumab (Avastin™), trastuzumab (Herceptin™), trastuzumab emtansine (KADCYLA™), i matinib (Gleevec™), cetuximab (Erbitux™), panitumumab (Vectibix™), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tibotinib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, luxorutinib, paku ritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as CDK inhibitor (palbociclib), but are not limited thereto.

[0179] In additional embodiments, a composition comprising CAR-bearing immunity can be administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and non-steroidal anti-inflammatory drugs (NSAIDs) including mycophenolate, but are not limited thereto. Exemplary NSAIDs include ibuprofen, naproxen, sodium naproxen, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors (e.g., TNF antagonists (e.g., etanercept (ENBREL (trademark)), adalimumab (HUMIRA (trademark)), and infliximab (REMICADE (trademark))), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0180] In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" means a protein released by one population of cells that acts on another cell as an intercellular mediator. Examples of cytokines are lymphokines, monokines, and conventional polypeptide hormones. Among cytokines, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial cell growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet-derived growth factor; transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; erythropo etin (EPO); osteogenic factor; interferons such as interferon-alpha, beta, and gamma; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). The term cytokine as used herein includes proteins of natural origin or from recombinant cell cultures, and biologically active equivalents of native sequence cytokines.

[0181] In some embodiments, the invention has a K smaller than 100 pM dIt comprises an antigen-binding molecule that binds to CLL-1. In some embodiments, the antigen-binding molecule binds with a K of less than 10 pM. d In other embodiments, the antigen-binding molecule binds with a K of less than 5 pM. d It binds.

[0182] Production method Various known techniques can be used to produce the polynucleotides, polypeptides, vectors, antigen-binding molecules, immune cells, compositions, etc. according to the present invention.

[0183] Before in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a blood unit collected from a subject using various techniques known to those of skill in the art, such as FICOLL™ separation. The cells can preferably be obtained from the individual's circulating blood by apheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS. As will be appreciated, the washing step can be performed by using, for example, a semi-automated flow-through centrifuge such as a Cobe™ 2991 cell processor, a Baxter CytoMate™, etc. After washing, the cells can be resuspended in various biocompatible buffers or other aqueous saline solutions with or without a buffer. In certain embodiments, unwanted components of the apheresis sample can be removed.

[0184] In certain embodiments, T cells are isolated from PBMCs by lysis of red blood cells and depletion of monocytes, for example, by using centrifugation through a PERCOLL™ gradient. + , CD4 + , CD8 + , CD45RA + and CD45RO + Specific subpopulations of T cells, such as T cells from the negatively selected T cells, can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished by a combination of antibodies against surface markers unique to the cells being negatively selected. One method used herein is negative magnetic immunoadherence or flow spectroscopy, which uses a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. Sorting and / or selection of cells by cytometry. For example, negative selection of CD4 + To enrich for cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0185] PBMCs can be transfected with immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, after PBMC isolation, T lymphocytes can be further isolated and sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion, for both cytotoxic and helper T lymphocytes.

[0186] In some embodiments, CD8 + These various CD8 cells +By identifying cell surface antigens associated with cells, they are further sorted into naive cells, central memory cells, and effector cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO + , CD62L + , CD8 + T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4 + T cells are further sorted into subpopulations. For example, CD4 + helper T cells can be sorted into naive cells, central memory cells, and effector cells by identifying a cell population having cell surface antigens.

[0187] Immune cells, such as T cells, can be isolated and then genetically modified using known methods, or the immune cells can be activated and expanded (i.e., differentiated in the case of progenitor cells) in vitro prior to genetic modification. In another embodiment, immune cells, such as T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, and International Publication No. 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and co-stimulatory molecules, such as anti-CD3 antibody and anti-CD28 antibody attached to beads or other surfaces, in a culture medium containing an appropriate cytokine, such as IL-2. The anti-CD3 antibody and anti-CD28 antibody attached to the same beads serve as a "surrogate" antigen-presenting cell (APC). One example is the Dynabeads™ system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, methods as described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and International Publication No. 2012 / 129514 (the contents of which are hereby incorporated by reference in their entirety) can be used to activate, stimulate, and expand T cells with feeder cells, as well as appropriate antibodies and cytokines.

[0188] Certain methods of making the constructs and modified immune cells of the present invention are described in International Application PCT / US15 / 14520, the contents of which are hereby incorporated by reference in their entirety. Additional methods of making the constructs and cells can be found in U.S. Provisional Patent Application No. 62 / 244036, the contents of which are hereby incorporated by reference in their entirety.

[0189] It will be appreciated that the PBMC may further contain other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of the chimeric receptor disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. The successfully transduced T cells carrying the expression vector are sorted using flow cytometry to isolate CD3-positive T cells and then, in addition to cell activation using anti-CD3 antibody and IL-2, or other methods known in the art as described elsewhere herein , they can be further expanded to increase the number of these CAR-expressing T cells. Standard procedures are used for cryopreservation of T cells expressing CARs for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture and / or expansion of T cells is performed in the absence of products derived from non-human animals such as fetal calf serum and fetal bovine serum.

[0190] For cloning of polynucleotides, a vector can be introduced into a host cell (isolated host cell) and the copy of the polynucleotide contained therein can be amplified by enabling the replication of the vector itself. Cloning vectors generally may contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence and a selectable marker. These elements can be selected by those skilled in the art as needed. For example, the origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0191] In certain embodiments, the present disclosure provides an isolated host cell containing the vectors presented herein. The host cell containing the vector can be useful for the expression or cloning of the polynucleotide contained in the vector. Suitable host cells include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae (intestinal bacteria family), for example, Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescens, and Shigella, Bacillus such as B. subtilis ) and Bacillus such as B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces etc. can be mentioned .

[0192] The vector can be introduced into a host cell using any suitable method known in the art, including but not limited to delivery mediated by DEAE-dextran, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, particle bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan, and peptides. Standard methods of transfection and transformation of cells for expression of the vector of interest are known in the art. In a further embodiment, in the genetic modification of a donor population of immune effector cells, a mixture of various expression vectors can be used, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of modified cells, some of which are modified cells expressing two or more different CARs.

[0193] In one embodiment, the invention provides a method of storing genetically modified cells that express a CAR or TCR that targets the CLL-1 protein. This includes cryopreserving immune cells such that the cells remain viable upon thawing. A fraction of immune cells expressing the CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients afflicted with a malignancy. Optionally, the cryopreserved transformed immune cells can be thawed, grown, and expanded into more such cells.

[0194] As used herein, "cryopreserving" refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 Kelvin, i.e., -196 °C (the boiling point of liquid nitrogen). To prevent damage to the cells upon freezing at low temperature or upon warming to room temperature, cryoprotective agents are often used at sub-zero temperatures. Cryopreservative agents and optimal Cell damage can be prevented by the cooling rate. Cryoprotectants that can be used in accordance with the present invention include dimethyl sulfoxide (DMSO) (Lovelock & Bishop, Nature (1959); 183: 1394-1395, Ashwood-Smith, Nature (1961); 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci. (1960); 85: 576) and poly ethylene glycol (Sloviter & Ravdin, Nature (1962); 196: 48), but are not limited thereto. The preferred cooling rate is 1 °C / min to 3 °C / min.

[0195] The term "substantially pure" is used to indicate that a given component is present at a high level. The component is desirably the main component present in the composition. Preferably, the component is present at a level of more than 30%, more than 50%, more than 75%, more than 90% or even more than 95%, and the level is determined on a dry weight / dry weight basis relative to the total composition under consideration. At very high levels (e.g., more than 90%, more than 95% or more than 99%), the component can be considered to be in "pure form". The bioactive substances (CAR, TCR, isolated polypeptide, isolated nucleic acid molecule, antigen-binding molecule, moiety) of the present invention can be provided in a form substantially free of one or more contaminants that might otherwise be associated with the substance. When a composition is substantially free of a given contaminant, the contaminant is at a low level (e.g., less than 10%, less than 5% or less than 1% on the dry weight / dry weight basis described above).

[0196] ​In some embodiments, the cells are first harvested from their culture medium, then washed, and formulated by concentrating them in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic media formulation, typically normal saline solution, Normosol® R (Abbott) or Plasma-Lyte® A (Baxter), but 5% dextrose in water or lactated Ringer's solution can also be used. Human serum albumin may be added to the infusion media.

[0197] The desired therapeutic amount of cells in the composition is generally at least 2 cells (e.g., at least 1 CD8 + central memory T cell and at least 1 CD4 + helper T cell subset), or more generally greater than 10 2 cells and up to a maximum of 10 6 cells, 10 8 cells or up to 10 9 cells, and can be greater than 10 10 cells. The number of cells varies depending on the desired application for which the composition is intended and the type of cells included therein. The desired cell density is typically greater than 10 6 cells / ml, generally 10 7 cells / ml, generally 10 8 cells / ml or greater. The clinically appropriate number of immune cells can be distributed among multiple infusions that cumulatively equal or exceed 10 5 cells, 10 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells or 10 12 cells. In some aspects of the invention, particularly since all of the cells being infused are redirected to a specific target antigen (CLL-1), 10 6 cells / kilogram (10 6 cells to 10 11Fewer cells in the range of (number) can be administered. In CAR therapy, multiple administrations can be performed at dosages within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient receiving the therapy.

[0198] The cell population expressing the CAR of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2 or other cytokines or cell populations. The pharmaceutical composition of the present invention can include a cell population such as a T cell expressing the CAR or TCR described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration. It is preferred.

[0199] The pharmaceutical composition (solution, suspension, etc.) can include one or more of the following: a sterile diluent, such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, synthetic monoglycerides or diglycerides such as non-volatile oils that can act as solvents or suspending agents, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and isotonic agents such as sodium chloride or dextrose. The parenteral preparation can be enclosed in an ampoule made of glass or plastic, a disposable syringe or a multi-dose vial. The injectable pharmaceutical composition is preferably sterile.

[0200] It will be appreciated that adverse events can be minimized by transducing immune cells (including one or more CARs or TCRs) with a suicide gene. Also, it may be desirable to incorporate an inducible "on" or "accelerator" switch into the immune cells There is. As a suitable technique, the use of inducible caspase-9 (U.S. Patent Application Publication No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with the cell being transduced with the CAR construct of the present invention can be mentioned. Additional methods of introducing a suicide gene and / or an "on" switch include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[0201] According to the present invention, additional on-off or other types of control switch technologies can be incorporated into the present invention. These technologies can utilize the use of dimerization domains and the use of any activator of such domain dimerization. Examples of these technologies include those described by Wu et al., Science 2014 350 (6258) which uses the FKBP / Rapalog dimerization system in certain specific cells, the contents of which are hereby incorporated by reference in their entirety to form a part of this specification. Additional dimerization technologies include, for example, those described in U.S. Patent Nos. 5,830,462, 5,834,266, 5,869,337 and 6,165,787 in parallel with Fegan et al. Chem. Rev. 2010, 110, 3315-3336, the contents of which are hereby incorporated by reference in their entirety to form a part of this specification. Additional dimerization pairs can include cyclosporin-A / cyclophilin receptor, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Further examples of dimerization technologies can be found in, for example, International Publication No. 2014 / 127261, International Publication No. 2015 / 090229, U.S. Patent Application Publication No. 2014 / 0286987, U.S. Patent Application Publication No. 2015 / 0266973, U.S. Patent Application Publication No. 2016 / 0046700, U.S. Patent No. 8,486,693, U.S. Patent Application Publication No. 2014 / 0171649 and U.S. Patent Application Publication No. 2012 / 0130076, the contents of which are hereby incorporated by reference in their entirety to further form a part of this specification.

[0202] It is understood that the description in this specification is merely illustrative and explanatory and does not limit the claimed invention. In this application, unless specifically specified otherwise, the use of the singular form includes the plural form.

[0203] The headings of sections used in this specification are for the sole purpose of organization and are not to be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are hereby incorporated by reference in their entirety for all purposes as if fully set forth herein. When used in accordance with the present disclosure, the following terms shall be understood to have the following meanings unless otherwise specified.

[0204] In this application, the use of "or" means "and / or" unless otherwise specified. Further, the term "including" and the use of other forms such as "includes" and "included" are not limiting. Also, the terms "element" or "component" etc. include both elements and components that include one unit and elements and components that include two or more subunits, unless otherwise specifically specified.

[0205] The term "CLL-1 activity" includes any biological effect of CLL-1. In certain embodiments, CLL-1 activity includes the ability of CLL-1 to interact with or bind to a substrate or receptor.

[0206] The terms "polynucleotide", "nucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. This preferably includes the isolated polynucleotides, nucleotides or nucleic acids defined herein. The nucleotides that make up the polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoramidate.

[0207] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. The oligonucleotide can be single-stranded or double-stranded, for example, used in the construction of mutant genes. The oligonucleotide can be a sense oligonucleotide or an antisense oligonucleotide. The oligonucleotide may contain a label including a radiolabel, a fluorescent label, a hapten or an antigen label for a detection assay. The oligonucleotide can be used, for example, as a PCR primer, a cloning primer or a hybridization probe.

[0208] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence to which it ligates. The nature of such control sequences may vary depending on the host organism. In certain embodiments, the control sequences for prokaryotes may include a promoter, a ribosome binding site and a transcription termination sequence. For example, the control sequences for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence and a transcription termination sequence. The "control sequence" may include a leader sequence (signal peptide) and / or a fusion partner sequence.

[0209] In some embodiments, the polynucleotide of the invention encoding a CAR or TCR may further include a leader sequence or peptide (also referred to herein as "signal peptide"). In certain embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 144). In some embodiments, the leader peptide comprises the amino acid sequence of SEQ ID NO: 144.

[0210] As used herein, "operatively linked" means that the components to which the term applies are in a relationship such that each can perform its unique function under suitable conditions.

[0211] The term "vector" is used to transfer protein coding information into a host cell and means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus). The term "expression vector" or "expression construct" refers to a vector containing nucleic acid sequences suitable for transformation of a host cell and directing and / or controlling the expression of one or more heterologous coding regions operatively linked thereto ( with the host cell). An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of a coding region operatively linked thereto.

[0212] The term "host cell" refers to a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thereby expressing a target gene. The term includes progeny of the parental cell as long as the target gene is present, regardless of whether the progeny's form or genetic makeup is identical to that of the original parental cell.

[0213] The term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed if it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its native state by introducing new genetic material by transfection, transduction or other techniques. After transfection or transduction, the transformed DNA can recombine with the cell's DNA by physically integrating into the cell's chromosome, or can be maintained transiently as an episomal element without replication, or can replicate independently as a plasmid. A cell is considered to be "stably transformed" when the transformed DNA is replicated by cell division.

[0214] The term "transfection" refers to the uptake of exogenous or foreign DNA by cells. A number of transfection methods are known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (supra), Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, Chu et al., 1981, Gene 13:197.

[0215] The term "transduction" refers to the process by which foreign DNA is introduced into cells by a viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

[0216] The term "polypeptide" or "protein" refers to a macromolecule having an amino acid sequence of a protein that includes deletions, additions and / or substitutions of one or more amino acids of a native sequence, preferably having eight or fewer amino acid substitutions thereto. The polypeptide or protein is preferably isolated as defined herein. The terms "polypeptide" and "protein" particularly include a CLL-1 antigen-binding molecule, an antibody or a sequence having deletions, additions and / or substitutions of one or more amino acids of an antigen-binding protein, preferably having eight or fewer amino acid substitutions thereto. The term "polypeptide fragment" refers to an isolated polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion and / or an internal deletion as compared to a full-length native protein. Such a fragment may contain modified amino acids as compared to the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules. Useful fragments include, but are not limited to, one or more CDR regions, variable domains of heavy and / or light chains, portions of other parts of antibody chains, etc.

[0217] The term "isolated" means (i) not containing at least some of the other proteins that are usually present together, (ii) essentially not containing other proteins from the same source, e.g., from the same species, (iii) being separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates or other substances that are naturally associated, (iv) operatively associating (by covalent or non-covalent interactions) a polypeptide that is not naturally associated, or (v) not occurring naturally.

[0218] A "variant" of a polypeptide (e.g., an antigen-binding molecule or an antibody) includes an amino acid sequence in which one or more amino acid residues are inserted, deleted and / or substituted in the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0219] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by aligning and comparing the sequences. "Percent identity" means the percentage of residues that are identical between the amino acids or nucleotides in the molecules being compared, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an "algorithm").

[0220] To calculate percent identity, the sequences being compared are usually aligned in a way that gives the greatest match between the sequences. An example of a computer program that can be used to determine percent identity is the GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, University of Wisconsin, Madison, WI, Genetics Computer Group) program included in the GCG program package. Using the computer algorithm GAP, two polypeptides or polynucleotides for which percent sequence identity is to be determined are aligned. The sequences are aligned for their optimal matching of each of their amino acids or nucleotides (a "matched span" as determined by the algorithm). In certain embodiments, a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix, Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0221] ​​As used herein, the 20 common (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, Golub and Gren, Eds., Sinauer Assoc., Sunderland, Mass. (1991)) (incorporated herein by reference for any purpose). Stereoisomers of the 20 common amino acids (e.g., D - amino acids), unnatural amino acids such as alpha, alpha - disubstituted amino acids, N - alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include 4 - hydroxyproline, gamma - carboxyglutamic acid, epsilon - N,N,N - trimethyllysine, e - N - acetyllysine, O - phosphoserine, N - acetylserine, N - formylmethionine, 3 - methylhistidine, 5 - hydroxylysine, sigma - N - methylarginine, and other similar amino acids and imino acids (e.g., 4 - hydroxyproline). In the polypeptide notations used herein, in accordance with standard usage and convention, the left - hand direction is the amino - terminal direction and the right - hand direction is the carboxy - terminal direction.

[0222] Conservative amino acid substitutions can include non - naturally occurring amino acid residues incorporated by chemical peptide synthesis rather than synthesis in a biological system. These include peptidomimetics and other reverse - or inverted - type amino acid moieties. Naturally occurring residues can be divided into the following classes based on common side - chain characteristics: a) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, b) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln , c) Acidic: Asp, Glu, d) Basic: His, Lys, Arg, e) Residues that affect chain orientation: Gly, Pro, and, f) Aromatic: Trp, Tyr, Phe.

[0223] For example, non-conservative substitutions can involve exchanging a member of one of these classes with a member of another class. Such substituted residues can be introduced, for example, into regions of a human antibody that are homologous to a non-human antibody, or into non-homologous regions of the molecule. Exemplary amino acid substitutions are shown in Table 3.

[0224] [Table 3]

[0225] The term "derivative" refers to a molecule that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, derivatives include covalent modifications including, but not limited to, chemical bonds with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified antigen-binding molecule can have a longer circulating half-life than an unmodified antigen-binding molecule. In some embodiments, the derivative antigen-binding molecule is covalently modified to include the attachment of one or more water-soluble polymers including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol thereof.

[0226] Peptidomimetics are commonly used in the pharmaceutical industry as non-peptide drugs having properties similar to the template peptide. These types of non-peptide compounds are referred to as "peptide mimetics" or "peptidomimetics". Fauchere, J., Adv. Drug Res., 15:29 (1986), Veber & Freidinger, TINS, p.392 (1985) and Evans et al., J. Med. Chem., 30:1229 (1987) (which are hereby incorporated by reference for any purpose as part of this specification).

[0227] A therapeutically effective amount, effective dosage, effective amount, or therapeutically effective dosage of a therapeutic agent, such as a modified CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease, or promotes regression of a disease as demonstrated by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of a dysfunction or physical impairment resulting from the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated using various methods known to a skilled physician, for example, in human subjects during a clinical trial, in an animal model system that predicts efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

[0228] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with a formally diagnosed disorder, subjects without a formally recognized disorder, subjects undergoing treatment, subjects at risk of developing a disorder, etc.

[0229] The terms "treat" and "treatment" include therapeutic procedures, prophylactic treatment, and applications in which the risk of a subject developing a disorder or other risk factor is reduced. Treatment does not require complete cure of the disorder and includes embodiments in which symptoms or potential risk factors are reduced. The term "prevent" does not require elimination of the possibility of an event 100%. Rather, it means that the likelihood of the event occurring is reduced in the presence of the compound or method.

[0230] Standard techniques can be used in recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification methods can be performed according to the manufacturer's specifications, or as generally accomplished in the art, or as described herein. The above-mentioned techniques and procedures generally can be performed according to conventional methods known in the art, as described in various general and more specific reference documents cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for any purpose).

[0231] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference as part of this specification. However, the citation of reference documents in this specification should not be construed as an admission that such reference documents are prior art to the present invention. In the event that any definition or term provided in a reference document incorporated by reference herein differs from the terms and considerations provided herein, the terms and definitions of the present invention shall control.

[0232] The foregoing specification is considered to be sufficient to enable one skilled in the art to practice the invention. The above description and examples detail certain preferred embodiments of the invention and illustrate the best mode contemplated by the inventors. However, even though the foregoing has been described in great detail herein, it is to be understood that the invention can be practiced in many ways and that the invention is to be construed in accordance with the appended claims and any equivalents thereof.

[0233] The following examples, which include the experiments conducted and the results achieved, are presented for illustrative purposes only and are not to be construed as limiting the present invention.

Example

[0234] Example 1 Determination of CLL-1 CAR activity by mRNA electroporation in human PBMC. A plasmid encoding the T7 promoter, the CAR construct, and the beta-globin stabilizing sequence was linearized by digesting 10 μg of DNA overnight with EcoRI and BamHI (NEB). The DNA was then digested with proteinase K (Thermo Fisher (trademark), 600 U / ml) at 50 °C for 2 hours, purified with phenol / chloroform, and precipitated by adding sodium acetate and 2 volumes of ethanol. The pellet was then dried, resuspended in RNase / DNase-free water, and quantified. Then, 1 μg of linear DNA was used to generate in vitro transcription using mMESSAGE mMACHINE T7 Ultra (Thermo Fisher (trademark)) according to the manufacturer's instructions. The RNA was further purified using the MEGAClear kit (Thermo Fisher (trademark)) according to the manufacturer's instructions and quantified using NanoDrop (trademark). The integrity of the mRNA was evaluated by agarose gel electrophoresis. Various cancer cell lines were evaluated for CLL-1 expression. Namalwa (ATCC), U937 (ATCC), HL-60 (ATCC), EoL-1 (Sigma), KG1a (AT CC), and MV4;11 (ATCC) cells were stained with an anti-CLL-1 antibody conjugated to PE (BD Pharmingen (trademark)) in staining buffer (BD Pharmingen (trademark)).

[0235] Various cancer cell lines were evaluated for CLL-1 expression. Namalwa (ATCC), U937 (ATCC), HL-60 (ATCC), EoL-1 (Sigma), KG1a(AT CC) and MV4;11(ATCC) cells were stained with an anti-CLL-1 antibody conjugated to PE(BD Pharmingen( trademark)) in staining buffer(BD Pharmingen( trademark)). ) with 4 It was stained at 30 minutes at ℃. Subsequently, the cells were washed before data acquisition and resuspended in a staining buffer containing propidium iodide (BD Pharmingen (trademark)). Subsequently, the samples were obtained by flow cytometry and the data were analyzed and plotted on a histogram using FlowJo (trademark). The results of CLL-1 expression can be seen in Figure 1.

[0236] PBMCs were isolated from leukopaks (Hemacare (trademark)) of healthy donors using ficoll-paque density centrifugation according to the manufacturer's instructions and. PBMCs were stimulated using OKT3 (50 ng / ml, Miltenyi Biotec (trademark)) in R10 medium supplemented with IL-2 (300 IU / ml, Proleukin (trademark), Prometheus (trademark) Therapeutics and Diagnostics). Seven days after stimulation the T cells were washed twice with Opti-MEM (trademark) (Thermo Fisher Scientific (trademark)) and resuspended in Opti-MEM at a final concentration of 2.5×10 7 cells / ml. 10 μg of mRNA was used per electroporation. Electroporation of the cells was performed using a Gemini X2 system (Harvard Apparatus BTX (trademark)) set to deliver a single pulse of 400 V for 0.5 ms in a 2 mm cuvette (Harvard Apparatus BTX (trademark)). The cells were immediately transferred to R10+IL-2 medium and cultured. Before using the cells for the activity assay, 0.5×10 6 cells / ml to 2.0×10 6 cells / ml.

[0237] Six hours after mRNA electroporation, T cells were stained with biotinylated Protein L (Thermo Scientific™) in staining buffer (BD Pharmingen™) at 4°C for 30 minutes. Cells were then washed and stained with PE streptavidin (BD Pharmingen™) in staining buffer at 4°C for 30 minutes. Cells were then washed prior to data acquisition and resuspended in staining buffer containing propidium iodide (BD Pharmingen™). The results of CAR detection are shown in Figure 2. Cells were washed and resuspended in staining buffer containing propidium iodide (BD Pharmingen™). The results of CAR detection are shown in Figure 2.

[0238] Effector cells were cultured with target cells at a 1:1 E:T ratio in R10 medium 6 hours after mRNA electroporation. The cell lines tested included Namalwa, U937, HL-60, EoL-1, KG1a, and MV4;11. Sixteen hours after co-culture, supernatants were analyzed by Luminex (EMD Millipore) according to the manufacturer's instructions, and the viability of target cells was evaluated by flow cytometric analysis of propidium iodide (PI) uptake. Results corresponding to the cytokine release assay can be seen in Figure 3. Results of the cytotoxicity assay can be seen in Figures 4 and 5. Results corresponding to the cytokine release assay can be seen in Figure 3. Results of the cytotoxicity assay can be seen in Figures 4 and 5.

[0239] Example 2 Determination of CLL-1 CAR activity by lentiviral transduction of human PBMCs. ViraPower™ Lentiviral Packaging Mix (Life Technologies™) Third-generation lentiviral transfer vectors containing various CLL-1 CAR constructs together with [[ID=]] were used to generate lentiviral supernatants. Briefly, a transfection mix was generated by mixing 15 μg of DNA and 22.5 μl of polyethyleneimine (Polysciences®, 1 mg / ml) in 600 μl of OptiMEM® medium. The mix was incubated at room temperature for 5 minutes. At the same time, 293T cells (ATCC) were trypsinized and counted, and a total of 10×10 6 cells were seeded into a T75 flask containing the transfection mix. Three days after transfection, the supernatant was collected, filtered through a 0.45 μm filter, and stored at -80 °C until use.

[0240] PBMCs were isolated from healthy donor leukopak (Hemacare®) using ficoll-paque density centrifugation according to the manufacturer's instructions. PBMCs were stimulated with OKT3 (50 ng / ml, Miltenyi Biotec®) in R10 medium supplemented with IL-2 (300 IU / ml, Proleukin®, Prometheus® Therapeutics and Diagnostics). Forty-eight hours after stimulation, the cells were transduced with lentivirus at an MOI = 10. Cells were maintained at 0.5×10 6 cells / ml to 2.0×10 6 cells / ml before use in the viability assay.

[0241] Twelve days after stimulation, the T cells were stained with biotinylated protein L (Thermo Scientific®) in staining buffer (BD Pharmingen®) for 30 minutes at 4 °C. Then the cells were washed and incubated with PE streptavidin (BD Pharmingen®) in staining buffer for 30 minutes at 4 °C. ) was used to stain at 4 °C for 30 minutes. Subsequently, the cells were washed before data acquisition and resuspended in staining buffer containing propidium iodide (BD Pharmingen™). The results of CAR detection are shown in Fig. 6. The results are shown in Fig. 6.

[0242] Effector cells were cultured with target cells in R10 medium at a 1:1 E:T ratio 12 days after T cell stimulation. The cell lines tested included Namalwa, U937, HL-60, EoL-1, KG1a, and MV4;11. Sixteen hours after co-culture, the supernatant was analyzed by Luminex (EMD Millipore™) according to the manufacturer's instructions, and the viability of target cells was evaluated by flow cytometric analysis of propidium iodide (PI) uptake. The results corresponding to the cytokine release assay can be seen in Fig. 7. The results of the cytotoxicity assay can be seen in Fig. 8. The results corresponding to the cytokine release assay can be seen in Fig. 7. The results of the cytotoxicity assay can be seen in Fig. 8.

[0243] Example 3 Five- to six-week-old female Jackson NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) were used in this study. Irradiated Harlan 2918.15 Rodent Diet and water were freely provided to the mice. The mice were housed in an Innovive™ disposable ventilated cage with corn cob bedding in a Biobubble™ Clean Room supplied with HEPA-filtered air at 100 complete air changes per hour in a bubble environment. All procedures, body weight determinations, and tumor measurements were performed within the bubble environment. The environment was controlled within a temperature range of 70° ± 2°F and a humidity range of 30% - 70%. All procedures were conducted in compliance with all laws, regulations, and guidelines of the National Institutes of Health (NIH) and with approval from the Animal Experimentation Committee of Molecular Imaging, Inc. The mice were housed in an Innovive™ disposable ventilated cage with corn cob bedding in a Biobubble™ Clean Room supplied with HEPA-filtered air at 100 complete air changes per hour in a bubble environment. All procedures, body weight determinations, and tumor measurements were performed within the bubble environment. The environment was controlled within a temperature range of 70° ± 2°F and a humidity range of 30% - 70%. All procedures were conducted in compliance with all laws, regulations, and guidelines of the National Institutes of Health (NIH) and with approval from the Animal Experimentation Committee of Molecular Imaging, Inc. environment) supplied with HEPA-filtered air at 100 complete air changes per hour in a bubble environment. All procedures, body weight determinations, and tumor measurements were performed within the bubble environment. The environment was controlled within a temperature range of 70° ± 2°F and a humidity range of 30% - 70%. All procedures were conducted in compliance with all laws, regulations, and guidelines of the National Institutes of Health (NIH) and with approval from the Animal Experimentation Committee of Molecular Imaging, Inc. All procedures were conducted in compliance with all laws, regulations, and guidelines of the National Institutes of Health (NIH) and with approval from the Animal Experimentation Committee of Molecular Imaging, Inc.

[0244] Tumor cell preparation U937-luc cells were obtained in Lifor™ Preservation Solution. The cells were centrifuged at 200 rcf at 4 °C for 8 minutes, the supernatant was aspirated, and the pellet was resuspended in cold Dulbecco's phosphate buffered saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension was diluted in trypan blue solution and counted using a Luna™ automated cell counter. The cell suspension was centrifuged at 200 rcf at 4 °C for 8 minutes. The supernatant was aspirated, and the cell pellet was resuspended in cold serum-free medium to a final concentration of trypan-excluding cells / ml. The cell suspension was maintained on wet ice during transplantation. On day 0, 1.00E+06 cells in 0.2 ml were transplanted intravenously via the lateral tail vein of the test animals using a 27-gauge needle and syringe. into the test animals via the lateral tail vein using a 27-gauge needle and syringe.

[0245] CAR T Cell Preparation T cells according to the present invention were obtained, frozen on dry ice, and stored in liquid nitrogen. On the treatment day, the prepared cryovials were taken out from cryostorage and thawed in a 37 °C water bath. For each group, the prepared T cells were combined in a single 50-ml conical tube containing warm RPMI 1640 supplemented with 10% FBS. The cryovial tubes were rinsed with warm RPMI 1640 containing 10% FBS in each conical tube to minimize cell loss to achieve a total volume of 50 ml. Each 50-ml conical tube was centrifuged at 200 rcf at 4 °C for 8 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 10 ml of room temperature DPBS. An aliquot of the homogeneous cell suspension was diluted in trypan blue solution and counted manually using a hemocytometer. The cell suspension was centrifuged again at 200 rcf at 4 °C for 8 minutes. The supernatant was aspirated, and the cell pellet was resuspended in room temperature DPBS to the required final concentration. The cell suspension was maintained on wet ice at the time of treatment administration.

[0246] Bioluminescence Imaging In vivo bioluminescence imaging (BLI) was performed using an IVIS Spectrum (Perkin Elmer, Hopkinton, Massachusetts). Animals were imaged up to 5 times at a time under approximately 1% - 2% isoflurane gas anesthesia. Each mouse was injected intraperitoneally (IP) with 150 mg / kg (15 mg / ml) of D-luciferin, and imaged in the ventral recumbent position and then the supine position 10 minutes after injection. Binning of the large and small CCD chips was used, and the exposure time was adjusted so that at least several hundred counts were obtained per image and saturation of the CCD chip was avoided (2 seconds to 2 minutes). BLI images were collected on days 3, 11, 18, and 25. The images were analyzed using Living Image version 4.5 (Perkin Elmer, Hopkinton, Massachusetts) software. Whole-body constant volume ROIs were placed on the ventral and supine position images for each individual animal and labeled based on animal identification. The total luminance expressed in photons / second (p / s) was calculated and exported for all ROIs to facilitate analysis between groups. The ventral and supine position ROIs were summed to estimate the total body tumor burden.

[0247] Treatment All mice were sorted into study groups based on the BLI-estimated total body tumor burden. Mice were allocated so that the average tumor burden of all groups was within 10% of the average tumor burden of the entire study population. Treatment with CAR T cells was initiated on day 3. A fixed volume of 0.2 mL was administered to all mice. The results are shown in Figure 10.

[0248] Evaluation of side effects All animals were observed for clinical signs at least once a day. The animals were weighed on each treatment day. Individual body weights were recorded three times a week.

[0249] The present invention is further illustrated by the following sequences.

[0250] CD28T DNA extracellular, transmembrane, intracellular CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC(SEQ ID NO:1)

[0251] CD28 extracellular, transmembrane, intracellular AA LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:2)

[0252] CD28T DNA - extracellular CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA(SEQ ID NO:3)

[0253] CD28T AA - extracellular LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP(SEQ ID NO:4)

[0254] CD28 DNA transmembrane domain TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTT(SEQ ID NO:5)

[0255] CD28 AA transmembrane domain FWVLVVVGGV LACYSLLVTV AFIIFWV(SEQ ID NO:6)

[0256] CD28 DNA intracellular domain AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ ID NO: 7)

[0257] CD28 AA intracellular domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 8)

[0258] CD3 zeta DNA AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 9)

[0259] CD3 zeta AA RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10)

[0260] CD3 zeta mutant AA RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 146)

[0261] CD28 DNA ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGTACCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGGCCATCAAAGCCC (SEQ ID NO: 11)

[0262] CD28 AA IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 12)

[0263] CD8 DNA extracellular & transmembrane domain GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTTCTTGCCGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTACCATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGGGCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCCCTGGCCGGAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGTAATCACAGGAAC (SEQ ID NO: 13)

[0264] CD8 AA extracellular & transmembrane domain AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 14)

[0265] Clone 24C1 HC DNA CAGGTGCAGCTGCAGGAATCCGGACCGGGGCTGGTGAAGCCCAGCGAGACTCTGAGTCTCACGTGTACAGTTTCTGGAGGTAGCATTAGCTCCTACTATTGGTCATGGATAAGGCAGCCCCCCGGGAAGGGATTGGAATGGATCGGCTATATTTACTACAGTGGGAGCACCAATTACAACCCCTCACTGAAGTCTAGAGTTACAATCAGCGTTGACACCTCAAAGAATCAGTTCAGTTTGAAATTGTCTAGCGTCACAGCAGCTGATACAGCCGTCTATTATTGTGTTTCTCTGGTCTATTGCGGTGGGGATTGTTACAGTGGCTTTGACTATTGGGGGCAGGGTACTCTGGTTACAGTTTCTTCC (SEQ ID NO: 15)

[0266] Clone 24C1 HC AA (CDRs are underlined) QVQLQESGPGLVKPSETLSLTCTVS GGSISSY YWSWIRQPPGKGLEWIGY I YYSGS TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVS LVYCGGDCYSGFDY WGQGTLVTVSS (SEQ ID NO: 16)

[0267] Clone 24C1 HC AA CDR1: GGSISSY (SEQ ID NO: 17)

[0268] Clone 24C1 HC AA CDR2: YYSGS (SEQ ID NO: 18)

[0269] Clone 24C1 HC AA CDR3: LVYCGGDCYS GFDY (SEQ ID NO: 19)

[0270] Clone 24C1 LC DNA GACATCCAGTTGACACAGAGCCCGAGTTCCTTGTCCGCCTCCGTCGGGGATAGAGTGTCATTTACCTGTCAGGCCTCTCAGGATATTAATAACTTTCTGAATTGGTATCAGCAAAAGCCCGGAAAGGCACCCAAGCTGTTGATTTACGACGCCAGTAACCTGGAGACAGGCGTGCCCTCCCGGTTTAGTGGTAGCGGAAGCGGTACGGATTTTACCTTTACTATCAGCTCTCTCCAACCCGAAGACATTGCAACCTACTATTGTCAACAATATGGAAACCTGCCTTTTACATTTGGCGGCGGCACCAAGGTGGAGATTAAGCGG (SEQ ID NO: 20)

[0271] Clone 24C1 LC AA (CDR underlined) DIQLTQSPSSLSASVGDRVSFTC QASQDINNFLN WYQQKPGKAPKLLIY DASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYGNLPFT FGGGTKVEIKR (SEQ ID NO: 21)

[0272] Clone 24C1 LC CDR1 AA: QASQDINNFLN (SEQ ID NO: 22)

[0273] Clone 24C1 LC CDR2 AA: DASNLET (SEQ ID NO: 23)

[0274] Clone 24C1 LC CDR3 AA: QQYGNLPFT (SEQ ID NO: 24)

[0275] Clone 24C1 CD28T CD3 zeta CAR DNA heavy chain & light chain

[0276] TIFF2025111619000005.tif47170

[0277] Clone 24C1 CD28T CD3 zeta CAR DNA heavy chain & light chain CAGGTCCAACTGCAAGAAAGCGGACCCGGACTGGTGAAGCCTTCTGAGACACTTAGTCTGACGTGCACGGTCAGTGGCGGCTCCATCTCCTCCTATTATTGGTCATGGATACGACAACCCCCAGGTAAGGGCCTGGAATGGATTGGCTATATCTACTATTCAGGAAGCACGAACTACAATCCCAGCCTGAAGTCCCGAGTGACAATTTCAGTAGATACCAGTAAAAACCAGTTCAGTCTTAAACTGTCAAGCGTGACAGCTGCCGACACCGCTGTGTATTACTGCGTCTCACTGGTGTATTGTGGAGGGGATTGTTATAGCGGGTTCGATTATTGGGGACAGGGAACCCTGGTGACTGTATCTTCCGGCGGCGGCGGCTCAGGGGGTGGCGGTAGTGGCGGTGGGGGTTCCGATATTCAACTGACACAATCCCCCAGCTCACTCAGCGCCAGCGTGGGGGACAGGGTTAGCTTTACCTGTCAAGCCTCTCAGGATATAAATAACTTTCTGAACTGGTATCAACAGAAGCCTGGGAAGGCGCCCAAACTCCTGATCTATGATGCGTCCAACCTGGAAACTGGCGTGCCTTCACGCTTTAGCGGCTCTGGCAGTGGTACAGACTTCACTTTTACCATCTCTTCACTTCAGCCGGAGGACATCGCCACATATTACTGTCAACAGTACGGAAACTTGCCCTTTACTTTTGGAGGCGGCACCAAAGTTGAAATCAAAAGGGCCGCTGCCCTGGATAACGAAAAGAGCAATGGGACTATAATACATGTTAAAGGAAAACACCTGTGTCCATCTCCCCTGTTCCCTGGACCGTCAAAGCCATTTTGGGTGCTCGTGGTTGTCGGTGGCGTTCTCGCCTGTTATAGCTTGCTGGTGACAGTAGCCTTCATTATCTTTTGGGTGAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATG ACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG(SEQ ID NO: 27)

[0278] Clone 24C1 CD28T CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 28)

[0279] Clone 24C1 CD28 CD3 zeta CAR DNA heavy & light chains

[0280] TIFF2025111619000006.tif47169

[0281] Clone 24C1 CD28 CD3 zeta CAR DNA heavy chain & light chain CAGGTGCAGCTGCAGGAATCCGGACCGGGGCTGGTGAAGCCCAGCGAGACTCTGAGTCTCACGTGTACAGTTTCTGGAGGTAGCATTAGCTCCTACTATTGGTCATGGATAAGGCAGCCCCCCGGGAAGGGATTGGAATGGATCGGCTATATTTACTACAGTGGGAGCACCAATTACAACCCCTCACTGAAGTCTAGAGTTACAATCAGCGTTGACACCTCAAAGAATCAGTTCAGTTTG

[0282] Clone 24C1 CD28 CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO: 32)

[0283] Clone 24C1 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0284] TIFF2025111619000007.tif47169

[0285] Clone 24C1 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0286] Clone 24C1 CD8 CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (SEQ ID NO: 36)

[0287] Clone 24C1 CD28T CD3 zeta CAR DNA heavy chain & light chain ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCGGATATCCAGCTCACGCAATCCCCCTCAAGCTTGAGTGCCTCCGTGGGCGACCGGGTGTCCTTCACATGTCAGGCAAGCCAAGACATAAATAATTTCCTGAATTGGTACCAACAAAAACCCGGCAAGGCTCCCAAACTCCTGATTTATGATGCCTCCAATCTGGAGACCGGGGTCCCTTCTAGATTCAGCGGAAGTGGCAGCGGCACAGACTTTACATTTACTATCTCTTCTCTGCAACCAGAGGACATCGCCACATACTATTGCCAGCAATACGGCAATCTGCCCTTCACCTTCGGAGGCGGAACCAAGGTAGAAATTAAAAGGGGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCCCAAGTACAATTGCAGGAGTCAGGGCCTGGACTCGTGAAGCCTTCAGAAACTTTGTCACTGACATGTACAGTGTCCGGCGGAAGCATTTCCAGTTACTATTGGTCCTGGATTAGACAGCCACCCGGCAAAGGACTGGAATGGATTGGATATATCTACTACTCTGGATCTACAAACTATAATCCCAGCCTCAAATCCAGGGTCACTATTA GTGTGGATACATCAAAGAATCAGTTCTCCTTGAAGCTGAGCTCAGTCACTGCTGCCGACACCGCAGTGTACTATTGTGTGAGCCTGGTCTACTGCGGCGGAGATTGCTACAGCGGTTTCGATTACTGGGGCCAGGGCACCCTGGTTACCGTTAGTTCCGCGGCTGCTCTTGATAACGAGAAGTCCAACGGTACGATTATCCACGTTAAGGGTAAGCACCTTTGCCCTAGCCCGCTGTTCCCAGGCCCCAGTAAGCCCTTTTGGGTCCTCGTTGTGGTAGGTGGGGTACTCGCCTGCTACTCCCTGCTCGTCACTGTCGCATTCATCATCTTCTGGGTCAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGTAA(SEQ ID NO: 37)

[0288] TIFF2025111619000008.tif47169

[0289] Clone 24C1 CD28 T CD3 zeta CAR DNA heavy chain & light chain

[0290] Clone 24C1 CD28T CD3 zeta CAR AA heavy chain & light chain DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 40)

[0291] Clone 24C1 CD28 CD3 zeta CAR DNA AA heavy chain & light chain

[0292] TIFF2025111619000009.tif47169

[0293] Clone 24C1 CD28 CD3 zeta CAR DNA heavy chain & light chain

[0294] Clone 24C1 CD28 CD3 zeta CAR AA heavy chain & light chain DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 4 4)

[0295] Clone 24C1 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0296] TIFF2025111619000010.tif47169

[0297] Clone 24C1 CD8 CD3 zeta CAR DNA heavy chain & light chain GACATTCAATTGACCCAGTCCCCTAGCAGTCTCTCAGCAAGTGTGGGAGATAGGGTGTCATTCACCTGTCAGGCTTCACAGGACATCAACAACTTCCTCAATTGGTATCAGCAGAAGCCAGGGAAGGCACCAAAGCTGCTCATATATGACGCTTCAAACCTTGAAACCGGAGTACCTAGCCGCTTCAGCGGAAGCGGATCAGGGACTGACTTCACTTTTACCATCTCTTCACTGCAGCCCGAAGACATCGCCACATACTACTGCCAGCAGTACGGAAACTTGCCTTTTACATTTGGGGGCGGCACCAAAGTGGAGATTAAGCGAGGGGGAGGCGGCTCAGGAGGCGGTGGCTCCGGAGGCGGGGGTTCCCAGGTCCAGCTCCAGGAATCCGGCCCAGGTCTGGTTAAGCCCAGTGAAACTTTGTCCCTCACGTGTACTGTGAGCGGTGGTTCAATCTCCTCATACTATTGGTCTTGGATACGGCAACCTCCTGGAAAGGGCCTCGAGTGGATCGGCTATATCTACTATAGTGGCTCCACTAATTACAACCCTTCCCTCAA GTCCAGAGTCACCATTTCCGTGGACACATCTAAGAACCAGTTCAGTCTGAAGTTGTCCAGCGTTACAGCCGCAGACACAGCCGTTTATTACTGTGTGTCTCTTGTTTACTGCGGGGGAGACTGTTATAGCGGCTTCGATTACTGGGGCCAGGGCACCTTGGTCACAGTCTCTTCCGCGGCCGCCCTCTCTAACAGTATTATGTACTTTTCTCATTTTGTACCCGTGTTCCTTCCCGCTAAGCCAACTACTACCCCGGCCCCACGGCCGCCTACCCCTGCACCCACAATAGCCAGTCAGCCTTTGAGCCTGAGACCTGAGGCTTGTCGGCCGGCTGCTGGGGGTGCAGTGCACACACGAGGTCTTGATTTTGCTTGCGACATATACATCTGGGCCCCTCTGGCCGGGACCTGTGGGGTGCTGCTTCTGAGCTTGGTCATCACGCTCTATTGCAACCATCGCAACAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 47)

[0298] Clone 24C1 CD8 CD3 zeta CAR AA heavy chain & light chain DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQA LPPR (SEQ ID NO: 48)

[0299] Clone 24C8 heavy chain (HC) DNA CAGGTACAGCTGCAGGAATCTGGGCCCGGACTTGTCAAGCCAAGTCAGACACTTTCTCTTACATGTACCGTGAGCGGCGGAAGTATAAGCAGTGGAGGCTTTTACTGGTCTTGGATACGGCAGCACCCAGGCAAAGGCTTGGAGTGGATTGGATACATTCATCATTCAGGATCTACACACTATAATCCATCCCTTAAGTCCCGGGTCACCATTAGCATTGATACGTCTAAGAATCTGTTCAGTCTCAGGCTGTCCTCCGTCACTGCTGCCGACACAGCCGTGTACTACTGCGCCTCCTTGGTTTACTGCGGAGGCGACTGTTATAGCGGCTTTGATTATTGGGGGCAGGGGACCCTCGTAACCGTGAGCTCT (SEQ ID NO: 48)

[0300] Clone 24C8 AA HC (CDRs are underlined) QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGGF YWSWIRQHPGKGLEWIGYI HHSGS THYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCAS LVYCGGDCYSGFDY WGQGTLVTVSS (SEQ ID NO: 50)

[0301] Clone 24C8 HC CDR1 AA: GGSISSGGF (SEQ ID NO: 51)

[0302] Clone 24C8 HC CDR2 AA: HHSGS (SEQ ID NO: 52)

[0303] Clone 24C8 HC CDR3 AA: LVYCGGDCYS GFDY (SEQ ID NO: 53)

[0304] Clone 24C8 light chain (LC) DNA GATATCCAGCTCACTCAAAGCCCCTCTAGTCTCTCTGCCTCAGTGGGGGATCGGGTCAGTTTTACTTGTCAAGCTTCACAGGATATCAACAACTTCCTTAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGCTGCTCATCTATGATGCCTCAAATTTGGAGACGGGTGTTCCCAGTCGATTCTCTGGGTCAGGGTCCGGGACCGACTTTACGTTTACGATCTCCTCTCTGCAGCCCGAAGACATCGCCACATACTATTGTCAACAGTACGGCAACTTGCCTTTCACATTTGGGGGCGGGACTAAGGTTGAAATCAAGAGG (SEQ ID NO: 54)

[0305] Clone 24C8 LC AA (CDRs are underlined) DIQLTQSPSSLSASVGDRVSFTC QASQDINNFLN WYQQKPGKAPKLLIY DASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYGNLPFTFGGGTKVEIKR (SEQ ID NO: 55)

[0306] Clone 24C8 LC CDR1 AA: QASQDINNFLN (SEQ ID NO: 56)

[0307] Clone 24C8 LC CDR2 AA: DASNLET (SEQ ID NO: 57)

[0308] Clone 24C8 LC CDR3 AA: QQYGNLPFT (SEQ ID NO: 58)

[0309] Clone 24C8 CD28T CD3 zeta CAR DNA heavy chain & light chain

[0310] TIFF2025111619000011.tif47169

[0311] Clone 24C8 CD28T CD3 zeta CAR DNA heavy chain & light chain CAGGTACAGCTGCAGGAATCTGGGCCCGGACTTGTCAAGCCAAGTCAGACACTTTCTCTTACATGTACCGTGAGCGGCGGAAGTATAAGCAGTGGAGGCTTTTACTGGTCTTGGATACGGCAGCACCCAGGCAAAGGCTTGGAGTGGATTGGATACATTCATCATTCAGGATCTACACACTATAATCCATCCCTTAAGTCCCGGGTCACCATTAGCATTGATACGTCTAAGAATCTGTTCAGTCTCAGGCTGTCCTCCGTCACTGCTGCCGACACAGCCGTGTACTACTGCGCCTCCTTGGTTTACTGCGGAGGCGACTGTTATAGCGGCTTTGATTATTGGGGGCAGGGGACCCTCGTAACCGTGAGCTCTGGAGGGGGTGGGAGCGGGGGAGGAGGTTCAGGGGGGGGCGGCTCCGATATCCAGCTCACTCAAAGCCCCTCTAGTCTCTCTGCCTCAGTGGGGGATCGGGTCAGTTTTACTTGTCAAGCTTCACAGGATATCAACAACTTCCTTAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGCTGCTCATCTATGATGCCTCAAATTTGGAGACGGGTGTTCCCAGTCGATTCTCTGGGTCAGGGTCCGGGACCGACTTTACGTTTACGATCTCCTCTCTGCAGCCCGAAGACATCGCCACATACTATTGTCAACAGTACGGCAACTTGCCTTTCACATTTGGGGGCGGGACTAAGGTTGAAATCAAGAGGGCCGCTGCACTGGACAATGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCTAGTCCTCTGTTCCCAGGCCCATCCAAACCTTTTTGGGTTCTTGTTGTGGTCGGGGGGGTGCTGGCCTGCTATTCTCTGCTGGTCACGGTGGCCTTCATAATTTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATG AATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG(SEQ ID NO: 61)

[0312] Clone 24C8 CD28T CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 62)

[0313] Clone 24C8 CD28 CD3 zeta CAR DNA heavy chain & light chain

[0314] TIFF2025111619000012.tif46169

[0315] Clone 24C8 CD28 CD3 zeta CAR DNA heavy chain & light chain CAGGTGCAGCTGCAGGAAAGCGGTCCGGGACTTGTCAAGCCGTCCCAAACGCTGAGTCTGACGTGTACTGTCTCTGGTGGCTCTATTTCTTCCGGGGGCTTTTATTGGTCTTGGATCAGACAACACCCTGGCAAAGGGCTGGAGTGGATAGGGTATATTCACCACTCTGGGTCCACTCACTACAACCCATCATTGAAATCCAGAGTGACTATCTCAATCGACACATCCAAGAACCTTTTC

[0316] Clone 24C8 CD28 CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO 66)

[0317] Clone 24C8 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0318] TIFF2025111619000013.tif47169

[0319] Clone 24C8 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0320] Clone 24C8 CD8 CD3 zeta CAR AA heavy chain & light chain QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHM QALPPR (SEQ ID NO: 70)

[0321] Clone 20C5.1 HC DNA CAGGTCCAACTGGTGCAGTCCGGAGCCGAAGTCAAGAAACCAGGTGCCTCCGTTAAAGTGAGTTGCAAAGTCTCTGGATACACTCTGACCGAGCTCTCTATGCACTGGGTCCGGCAGGCCCCCGGCAAGGGATTGGAATGGATGGGCGGGTTCGATCCTGAGGACGGAGAGACTATCTACGCTCAAAAATTCCAGGGACGAGTGACTGTGACCGAAGACACTAGTACCGACACTGCCTACATGGAACTTTCCTCTCTGCGATCAGAAGATACCGCAGTGTACTACTGTGCTACTGAATCTAGGGGCATTGGATGGCCCTACTTCGATTACTGGGGTCAGGGAACTCTGGTGACTGTCTCCAGC (SEQ ID NO: 71)

[0322] Clone 20C5.1 AA HC (CDRs are underlined) QVQLVQSGAEVKKPGASVKVSCKVS GYTLTEL SMHWVRQAPGKGLEWMGGF DPEDGE TIYAQKFQGRVTVTEDTSTDTAY MELSSLRSEDTAVYYCAT ESRGIGWPYFDY WGQGTLVTVSS (SEQ ID NO: 72)

[0323] Clone 20C5.1 HC AA CDR1: GYTLTEL (SEQ ID NO: 73)

[0324] Clone 20C5.1 HC AA CDR2: DPEDGE (SEQ ID NO: 74)

[0325] Clone 20C5.1 HC AA CDR3: ESRGIGWPYFDY (SEQ ID NO: 75)

[0326] Clone 20C5.1 LC DNA GATATTCAGATGACTCAATCTCCTTCTTCTCTGTCCGCTTCCGTGGGCGATAGAGTGACCATTACTTGTAGGGCGTCCCAGTCAATCTCCAGTTATTTGAATTGGTATCAGCAGAAGCCCGGGAAAGCACCTAAGCTGTTGATCAGCGGGGCTTCTAGCCTGAAGAGTGGGGTACCTTCACGGTTCAGCGGAAGCGGAAGCGGAACCGATTTCACCCTGACTATCAGCAGCCTGCCACCTGAGGACTTTGCAACTTACTACTGCCAACAGTCATACAGCACTCCGATCACTTTCGGCCAGGGCACCCGGCTCGAAATCAAGCGC (SEQ ID NO: 76)

[0327] Clone 20C5.1 AA LC (CDR is underlined) DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIS GASSLKS GVPSRFSGSGSGTDFTLTISSLPPEDFATYYC QQSYSTPIT FGQGTRLEIKR (SEQ ID NO: 77)

[0328] Clone 20C5.1 AA LC CDR1: RASQSISSYLN (SEQ ID NO: 78)

[0329] Clone 20C5.1 AA LC CDR2: GASSLKS (SEQ ID NO: 79)

[0330] Clone 20C5.1 AA LC CDR3: QQSYSTPIT (SEQ ID NO: 80)

[0331] Clone 20C5.1 CD28T CD3 zeta CAR DNA heavy chain & light chain Array number 81)

[0332] TIFF2025111619000014.tif46169

[0333] Clone 20C5.1 CD28T CD3 zeta CAR DNA heavy chain & light chain

[0334] Clone 20C5.1 CD28T CD3 zeta CAR AA heavy chain & light chain QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 84)

[0335] Clone 20C5.1 CD28 CD3 zeta CAR DNA heavy chain & light chain ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCGCAGGTGCAGCTTGTGCAGAGCGGGGCCGAGGTGAAGAAGCCCGGGGCCAGCGTCAAAGTGTCCTGTAAGGTCAGCGGTTACACCCTCACCGAGCTGAGCATGCACTGGGTACGGCAGGCTCCCGGCAAAGGTCTTGAGTGGATGGGTGGATTTGATCCAGAAGATGGAGAGACTATCTACGCCCAGAAGTTCCAGGGCCGGGTCACCGTAACAGAAGACACCTCAACTGACACCGCTTACATGGAGCTGAGTTCACTGCGGTCCGAGGACACGGCCGTGTATTATTGTGCCACCGAGAGCCGCGGAATCGGATGGCCTTACTTCGACTACTGGGGACAGGGTACACTTGTTACAGTATCATCCGGGGGTGGCGGCTCTGGTGGGGGCGGCTCCGGAGGGGGTGGATCAGATATCCAAATGACTCAAAGTCCAAGTTCCCTGTCTGCCTCAGTCGGAGATAGAGTCACCATAACCTGCAGGGCAAGTCAGTCCATCTCCTCCTATCTGAACTGGTACCAACAGAAACCTGGAAAGGCGCCTAAGCTCCTGATCTCCGGAGCCTCATCTTTGAAATCCGGTGTCCCATCTCGCTTCAGTGGCTCTGGAAGCGGTACAGATTTTACTTTGACCATTAGCAGCCTCCCACCGGAAGACTTTGCTACATATTACTGCCAGCAGTCTTACTCAACCCCAATCACCTTCGGGCAAGGCACCAGACTCGAAATAAAAAGAGCAGC TGCTATCGAGGTTATGTACCCACCGCCGTACTTGGATAACGAAAAAAGCAATGGGACCATCATTCATGTGAAGGGTAAGCACCTTTGCCCTAGCCCACTGTTTCCTGGCCCGAGTAAACCCTTTTGGGTACTTGTGGTCGTCGGCGGCGTGCTGGCCTGCTACTCACTCCTGGTTACCGTCGCATTCATCATCTTTTGGGTGAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGTAA (SEQ ID NO: 85)

[0336] TIFF2025111619000015.tif48169

[0337] Clone 20C5.1 CD28 CD3 zeta CAR DNA heavy chain & light chain

[0338] Clone 20C5.1 CD28 CD3 zeta CAR AA heavy chain & light chain QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR(SEQ ID NO: 8 8)

[0339] Clone 20C5.1 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0340] TIFF2025111619000016.tif48169

[0341] Clone 20C5.1 CD8 CD3 zeta CAR DNA heavy & light chains TGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 91)

[0342] Clone 20C5.1 CD8 CD3 zeta CAR AA Heavy Chain & Light Chain QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 92)

[0343] Clone 20C5.2 HC DNA CAGGTCCAGTTGGTCGAAAGTGGCGGTGGTGTAGTGCAGCCGGGCCGCAGTTTGAGGCTTTCCTGTGCGGCTTCAGGCTTTACTTTTTCCAGCTATGGAATGCACTGGGTGCGGCAGGCCCCCGGCAAAGGACTTGAGTGGGTGGCCGTCATTTCTTATGACGGATCAGATAAGTACTACGTGGACAGCGTCAAGGGCAGATTCACCATCTCTAGGGACAACAGTAAAAATAGACTCTACCTCCAGATGAATAGCCTCAGAGCTGAAGACACGGCCGTCTACTATTGTGCTCGGGAGCGGTATAGTGGCAGAGACTACTGGGGGCAGGGCACACTCGTTACAGTGAGTAGC (SEQ ID NO: 93)

[0344] Clone 20C5.2 AA HC (CDRs are underlined) QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSY GMHWVRQAPGKGLEWVAVI SYDGSD KYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCAR ERYSGRDY WGQGTLVTVSS (SEQ ID NO: 94)

[0345] Clone 20C5.2 HC AA CDR1: GFTFSSY (SEQ ID NO: 95)

[0346] Clone 20C5.2 HC AA CDR2: SYDGSD (SEQ ID NO: 96)

[0347] Clone 20C5.2 HC AA CDR3: ERYSGRDY (SEQ ID NO: 97)

[0348] Clone 20C5.2 LC DNA GAGATTGTTATGACCCAGAGTCCTGCGACCCTCTCAGTCAGCCCCGGGGAGCGCGCAACTTTGTCTTGCAGAGCTAGTCAGTCCGTGTCCTCTCTTCTGACATGGTACCAGCAAAAGCCCGGGCAGGCTCCGCGCCTTTTGATCTTTGGGGCTTCAACAAGAGCCACTGGGATTCCCGCACGATTCTCTGGCTCCGGGAGCGGTACTGGTTTCACCCTGACGATTAGCAGTCTCCAGAGCGAGGACTTCGCCGTATACTACTGCCAGCAGTACGATACGTGGCCATTCACTTTTGGACCAGGGACTAAAGTGGATTTTAAGCGC (SEQ ID NO: 98)

[0349] Clone 20C5.2 AA LC (CDRs are underlined) EIVMTQSPATLSVSPGERATLSC RASQSVSSLLT WYQQKPGQAPRLLIF GASTRAT GIPARFSGSGSGTGFTLTISSLQSEDFAVYYC QQYDTWPFT FGPGTKVDFKR (SEQ ID NO: 99)

[0350] Clone 20C5.2 AA LC CDR1: RASQSVSSLLT (SEQ ID NO: 100)

[0351] Clone 20C5.2 AA LC CDR2: GASTRAT (SEQ ID NO: 101)

[0352] Clone 20C5.2 AA LC CDR3: QQYDTWPFT (SEQ ID NO: 102)

[0353] Clone 20C5.2 CD28T CD3 zeta CAR DNA heavy & light chains ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCGCAGGTCCAGTTGGTCGAAAGTGGCGGTGGTGTAGTGCAGCCGGGCCGCAGTTTGAGGCTTTCCTGTGCGGCTTCAGGCTTTACTTTTTCCAGCTATG 3)

[0354] TIFF2025111619000017.tif47169

[0355] Clone 20C5.2 CD28T CD3 Zeta CAR DNA heavy chain & light chain (Accession No. 105)

[0356] Clone 20C5.2 CD28T CD3 zeta CAR AA heavy chain & light chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLY LQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Accession No. 106)

[0357] Clone 20C5.2 CD28 CD3 zeta CAR DNA heavy chain & light chain

[0358] TIFF2025111619000018.tif47169

[0359] Clone 20C5.2 CD28 CD3 zeta CAR DNA heavy chain & light chain CAGGTGCAGCTCGTGGAGTCTGGCGGCGGCGTGGTCCAGCCCGGCCGGTCCCTGCGCCTGTCCTGCGCCGCCAGCGGGTTTACTTTTTCCTCCTACGGCATGCACTGGGTGCGCCAGGCTCCCGGCAAGGGCCTCGAGTGGGTCGCCGTGATCTCATACGATGGGTCAGACAAATACTATGTCGATTCTGTTAAAGGGCGGTTTACCATTTCAAGAGATAACTCTAAGAATAGGCTGTATTTGCAGATGAACAGCCTGAGGGCTGAAGATACCGCAGTGTACTATTGCGCTAGGGAGCGGTATAGTGGCCGCGATTACTGGGGACAGGGTACACTGGTGACCGTGAGCTCTGGGGGTGGCGGAAGCGGGGGTGGCGGAAGCGGCGGAGGGGGTAGTGAAATTGTGATGACCCAGTCTCCGGCTACACTTTCAGTCTCCCCTGGGGAGAGAGCTACACTGTCATGCAGAGCGTCCCAGTCCGTCTCTTCTCTCCTTACCTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGACTGCTGATCTTCGGTGCCTCCACAAGGGCGACCGGGATTCCAGCCCGCTTCTCAGGTTCTGGGAGCGGAACTGGTTTCACTTTGACAATCAGTTCACTGCAGTCAGAGGATTTCGCCGTGTACTACTGCCAGCAATACGACACATGGCCATTCACTTTCGGACCCGGTACCAAAGTCGATTTCAAGAGAGCCGCGGCCATCGAGGTTATGTACCCACCACCATATCTGGACAATGAAAAAAGCAATGGAACCATTATCCATGTGAAGGGTAAACACCTCTGCCCTAGCCCACTTTTCCCTGGCCCATCAAAGCCCTTCTGGGTCTTGGTGGTCGTGGGGGGTGTGCTGGCCTGTTACAGCCTTCTGGTGACGGTTGCTTTCATTATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGAT TACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG(SEQ ID NO: 109)

[0360] Clone 20C5.2 CD28 CD3 zeta CAR AA heavy chain & light chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 110)

[0361] Clone 20C5.2 CD8 CD3 zeta CAR DNA heavy & light chains

[0362] TIFF2025111619000019.tif48169

[0363] Clone 20C5.2 CD8 CD3 zeta CAR DNA heavy chain & light chain CAGGTGCAGTTGGTTGAATCAGGAGGGGGTGTGGTGCAACCCGGTCGGTCACTGCGCCTCAGTTGTGCTGCTTCCGGGTT

[0364] Clone 20C5.2 CD8 CD3 zeta CAR AA heavy chain & light chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 114)

[0365] Clone 20C5.2 CD28T CD3 zeta CAR DNA heavy chain & light chain 5)

[0366] TIFF2025111619000020.tif47169

[0367] Clone 20C5.2 CD28T CD3 Zeta CAR DNA heavy chain & light chain (Array number 117)

[0368] Clone 20C5.2 CD28T CD3 zeta CAR AA heavy chain & light chain EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Array number 118)

[0369] Clone 20C5.2 CD28 CD3 zeta CAR DNA heavy chain & light chain ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCGGAGATCGTCATGACACAGAGTCCAGCTACCCTGAGCGTGTCCCCTGGAGAGAGAGCCACCCTGTCCTGTAGGGCTAGTCAGAGTGTGTCCAGCCTCCTCACCTGGTATCAACAGAAGCCTGGTCAAGCTCCCCGGCTGCTTATCTTCGGGGCCAGCACGCGAGCCACAGGCATCCCGGCCAGATTCTCTGGCTCTGGCAGTGGCACCGGGTTCACTCTCACGATCTCATCCCTGCAGTCAGAGGATTTCGCTGTGTATTACTGTCAGCAGTACGATACATGGCCCTTCACCTTCGGCCCGGGCACAAAAGTAGATTTCAAGCGCGGCGGCGGGGGTAGTGGGGGCGGGGGATCAGGAGGAGGGGGCTCCCAAGTACAGCTGGTTGAGAGCGGCGGCGGGGTGGTTCAGCCCGGGCGCAGCCTCAGGCTGAGTTGCGCAGCATCAGGATTCACATTCAGTTCTTATGGAATGCATTGGGTCAGACAGGCTCCCGGGAAGGGCCTTGAATGGGTGGCAGTCATTAGCTACGACGGAAGCGATAAGTACTATGTGGACTCAGTTAAAGGGAGATTTACTATCAGCCGCGACAATTCCAAAAACAGATTGTATTTGCAGATGAACTCCCTCAGGGCGGAGGACACTGCTGTATATTACTGCGCACGAGAGAGATACTCCGGCCGAGACTATTGGGGCCAAGGAACATTGGTAACTGTGAGCTCCGCCGCAGCTATTGAGGT CATGTACCCCCCACCTTATCTCGATAATGAGAAGAGTAATGGGACTATAATTCACGTAAAGGGCAAACACCTGTGCCCTTCCCCGCTGTTTCCAGGTCCAAGTAAGCCGTTCTGGGTCCTGGTTGTGGTGGGAGGGGTGCTGGCCTGCTATTCTCTGTTGGTTACCGTGGCCTTTATCATTTTCTGGGTGAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCAGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGGTAA (SEQ ID NO: 119)

[0370] TIFF2025111619000021.tif47169

[0371] Clone 20C5.2 CD28 CD3 zeta CAR DNA heavy chain & light chain

[0372] Clone 20C5.2 CD28 CD3 zeta CAR AA heavy chain & light chain EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 122)

[0373] Clone 20C5.2 CD8 CD3 zeta CAR DNA heavy chain & light chain ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCGGAAATAGTGATGACTCA

[0374] TIFF2025111619000022.tif47169

[0375] Clone 20C5.2 CD8 CD3 zeta CAR DNA heavy chain & light chain

[0376] Clone 20C5.2 CD8 CD3 zeta CAR AA heavy chain & light chain EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 126)

[0377] CAR signal peptide DNA ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCG (SEQ ID NO: 127 )

[0378] CAR signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 128)

[0379] scFv G4S linker DNA GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO: 129)

[0380] scFv G4S linker: GGGGSGGGGSGGGGS (SEQ ID NO: 130)

[0381] Additional G4S linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 145)

[0382] scFv Whitlow linker DNA GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAGGGG (SEQ ID NO: 131)

[0383] scFv Whitlow linker: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 132)

[0384] CD28 AA extracellular domain MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 133)

[0385] GX2X3X4X5X6X7X8X9 (SEQ ID NO: 134)

[0386] X1X2X3X4X5X6 (SEQ ID NO: 135)

[0387] X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 DY (SEQ ID NO: 136)

[0388] X1ASQX5X6X7XδX9LX 11 (SEQ ID NO: 137)

[0389] X1ASX4X5X6X7 (SEQ ID NO: 138)

[0390] QQX3X4X5X6PX8T (SEQ ID NO: 139)

[0391] CLL-1 AA (also known as CLEC12A) MSEEVTYADLQFQNSSEMEKIPEIGKFGEKAPPAPSHVWRPAALFLTLLCLLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA (SEQ ID NO: 140)

[0392] 4-1BB nucleic acid sequence (intracellular domain) AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 141)

[0393] 4-1BB AA (intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 142)

[0394] OX40 AA RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 143)

[0395] Leader sequence AA MALPVTALLLPLALLLHAARP (SEQ ID NO: 144)

Claims

**Claim 1** A chimeric antigen receptor comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 51, 73, and 95, b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 52, 74, and 96, c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 53, 75, and 97, d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 56, 78, and 100, e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 57, 79, and 101, f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102, and comprising at least one of the above, the chimeric antigen receptor. **Claim 2** The chimeric antigen receptor according to claim 1, having eight or fewer amino acid substitutions. **Claim 3** The chimeric antigen receptor according to claim 1, further comprising at least one co-stimulatory domain. **Claim 4** The chimeric antigen receptor according to claim 1, further comprising at least one activation domain. **Claim 5** The co-stimulatory domain is a signal transduction region (or other suitable portion) of a ligand that specifically binds to CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof, the chimeric antigen receptor according to claim 3.

6. The chimeric antigen receptor according to claim 5, wherein the co-stimulatory domain comprises CD28.

7. The chimeric antigen receptor of claim 6, wherein the CD28 costimulatory domain comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:

8.

8. The chimeric antigen receptor of claim 5, wherein the CD8 costimulatory domain comprises SEQ ID NO:

14.

9. The chimeric antigen receptor of claim 4, wherein the activation domain comprises CD3.

10. The chimeric antigen receptor of claim 9, wherein the CD3 comprises CD3 zeta.

11. The chimeric antigen receptor of claim 10, wherein the CD3 zeta comprises SEQ ID NO:

10.

12. The chimeric antigen receptor of claim 1, further comprising SEQ ID NO: 2 and SEQ ID NO:

10.

13. An isolated polynucleotide encoding the chimeric antigen receptor of claim 1.

14. A vector comprising the polynucleotide of claim 13.

15. 15. The vector of claim 14, which is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or any combination thereof.

16. An immune cell comprising the vector of claim 14.

17. The immune cell of claim 16, which is a T cell, a tumor-infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

18. The immune cell of claim 17, which is an autologous T cell.

19. The immune cell of claim 17, which is an allogeneic T cell.

20. A chimeric antigen receptor having at least 90% identity to the sequence of the antigen-binding molecule of claim 1.

21. A chimeric antigen receptor having at least 95% identity to the sequence of the antigen-binding molecule of claim 1.

22. 20. A pharmaceutical composition comprising the T cells of claim 17, 18 or 19.

23. A chimeric antigen receptor, (a) a VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VL region comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 55; (c) a VH region comprising the amino acid sequence of SEQ ID NO: 72 and a VL region comprising the amino acid sequence of SEQ ID NO: 77; (d) a VH region comprising the amino acid sequence of SEQ ID NO: 94 and a VL region comprising the amino acid sequence of SEQ ID NO: 99; A chimeric antigen receptor comprising at least one of the following, wherein the VH region and the VL region are linked by at least one linker.

24. The chimeric antigen receptor according to claim 23, having eight or fewer amino acid substitutions.

25. The chimeric antigen receptor according to claim 23, wherein the linker comprises at least one of SEQ ID NO: 130 and SEQ ID NO:

132.

26. The chimeric antigen receptor according to claim 23, further comprising at least one co-stimulatory domain.

27. The chimeric antigen receptor according to claim 23, further comprising at least one activation domain.

28. The co-stimulatory domain is a signaling domain that specifically binds to a ligand of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof, the chimeric antigen receptor according to claim 26.

29. An immune cell comprising the chimeric antigen receptor according to claim 23.

30. The immune cell according to claim 29, which is a T cell, tumor-infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell or NK-T cell.

31. The T cell according to claim 30, which is an autologous T cell.

32. The T cell according to claim 30, which is an allogeneic T cell.

33. A pharmaceutical composition comprising the immune cell according to claim 29.

34. An isolated polynucleotide comprising at least one of SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121 and SEQ ID NO:

125.

35. A vector comprising the polynucleotide according to claim 34.

36. An immune cell comprising the vector according to claim 33.

37. The immune cell according to claim 36, which is a T cell, tumor-infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell or NK-T cell.

38. The T cell according to claim 37, which is an autologous T cell.

39. The T cell according to claim 37, which is an allogeneic T cell.

40. An isolated polypeptide comprising an amino acid sequence defined by at least one of SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122 and SEQ ID NO:

126.

41. The isolated polypeptide according to claim 40, which has eight or fewer amino acid substitutions.

42. A vector encoding the polypeptide according to claim 40.

43. An immune cell comprising the vector according to claim 42.

44. The immune cell according to claim 43, which is a T cell, tumor-infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell or NK-T cell.

45. The T cell according to claim 44, which is an autologous T cell or an allogeneic T cell.

46. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule comprises a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 53, 75, and 97.

47. The polynucleotide according to claim 46, further comprising an activation domain.

48. The polynucleotide according to claim 47, wherein the activation domain is CD3.

49. The polynucleotide according to claim 48, wherein the CD3 is CD3 zeta.

50. The polynucleotide according to claim 49, wherein the CD3 zeta comprises the amino acid sequence defined in SEQ ID NO:

9.

51. The polynucleotide according to claim 46, further comprising a co-stimulatory domain.

52. The co-stimulatory domain is CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a) , DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, and a ligand that specifically binds to any combination thereof, or a polynucleotide according to claim 51 which is a signal transduction region of any combination thereof.

53. The polynucleotide according to claim 52, wherein the CD28 co-stimulatory domain encodes the amino acid sequence defined in SEQ ID NO:

2.

54. A vector comprising the polynucleotide according to claim 46.

55. An immune cell comprising the vector according to claim 54.

56. The immune cell according to claim 50, wherein the immune cell is a T cell, tumor infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell or NK-T cell.

57. The T cell according to claim 51, which is an autologous T cell or an allogeneic T cell.

58. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CLL-1, and the antigen-binding molecule comprises a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 58, 80, and 102.

59. The polynucleotide according to claim 58, further comprising an activation domain.

60. The polynucleotide according to claim 59, wherein the activation domain is CD3.

61. The polynucleotide according to claim 60, wherein the CD3 is CD3 zeta.

62. The polynucleotide according to claim 61, wherein the CD3 zeta comprises the amino acid sequence defined by SEQ ID NO:

9.

63. The polynucleotide according to claim 58, further comprising a co-stimulatory domain.

64. The co-stimulatory domain is CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell A ligand that specifically binds to costimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or a signaling region of any combination thereof, the polynucleotide according to claim 63.

65. The polynucleotide according to claim 64, wherein the CD28 co-stimulatory domain comprises the nucleotide sequence defined in SEQ ID NO: 3 or SEQ ID NO:

1.

66. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 18) and CDR3 (SEQ ID NO: 19), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 24).

67. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 56), CDR2 (SEQ ID NO: 57) and CDR3 (SEQ ID NO: 58).

68. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 73), CDR2 (SEQ ID NO: 74) and CDR3 (SEQ ID NO: 75), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79) and CDR3 (SEQ ID NO: 80).

69. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the heavy chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 95), CDR2 (SEQ ID NO: 96) and CDR3 (SEQ ID NO: 97), and the light chain of the antigen-binding molecule comprises CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101) and CDR3 (SEQ ID NO: 102).

70. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CLL-1, wherein the antigen-binding molecule comprises (a) Amino acid sequence GX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ (SEQ ID NO: 134) (where X ２ is G, F, or Y, X ３ is S or T, X ４ is I, F or L, X ５ is S or T, X ６ does not exist or is S, X ７ does not exist or is G, X ８ does not exist, or is E or G, X ９ a heavy chain variable region (VH) complementarity determining region (CDR) 1 containing (where X is F, L or Y), (b) Amino acid sequence X １ X ２ X ３ X ４ X ５ X ６ (SEQ ID NO: 135) (where X １ is D, H, S or Y, X ２ is H, P or Y, X ３ is D, E, or S, X ４ is D or G, X ５ is G or S, X ６ is absent or is D or E), and a heavy chain variable region (VH) complementarity determining region (CDR) 2 (c) Amino acid sequence X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ DY (SEQ ID NO: 136) (where X １ is E or L, X ２ is R, S or V, X ３ is R or Y, X ４ is C, G, or S, X ５ does not exist, or is G or I, X ６ does not exist, or is G, X ７ either does not exist or is D, X ８ either does not exist or is C, X ９ does not exist, or is W or Y, X １０ either does not exist, or is P or S, X １１ does not exist, or is G or Y, X １２ a heavy chain variable region (VH) complementarity determining region (CDR) 3 comprising (where X is F or R), (d) Amino acid sequence X １ ASQX ５ X ６ X ７ X ８ X ９ LX １１ (SEQ ID NO: 137) (where X １ is Q or R, X ５ is D or S, X ６ is I or V, X ７ is N or S, X ８ is N or S, X ９ is F, L or Y, X １１ a light chain variable region (VL) CDR1 containing (where X is N or T), (e) Amino acid sequence X １ ASX ４ X ５ X ６ X ７ (SEQ ID NO: 138) (where X １ is D or G, X ４ is N, S, or T, X ５ is L or R, X ６ is A, E or K, X ７ a light chain variable region (VL) CDR2 comprising (where X is S or T), and / or (f) Amino acid sequence QQX ３ X ４ X ５ X ６ PX ８ T (SEQ ID NO: 139) (where X ３ is S or Y, X ４ is D, G or Y, X ５ is N, S or T, X ６ is L, T, or Y, X ８ a light chain variable region (VL) CDR3 comprising (where X is F or I), An isolated polynucleotide comprising.

71. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the polynucleotide according to claim 46, 58, 66, 67, 68, 69 or 70.

72. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the polypeptide according to claim 40.

73. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the chimeric antigen receptor according to any one of claims 1, 20, 21 and 23.

74. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the cell according to any one of claims 16, 29, 36, 43 and 55.

75. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the composition according to claim 22 or 33.

76. The method according to any one of claims 71 to 75, wherein the disease or disorder is cancer.

77. The method according to claim 76, wherein the cancer is leukemia, lymphoma or myeloma.

78. The method according to any one of claims 71 to 75, wherein the disease or disorder is at least one of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN) and inflammatory / autoimmune disease.

79. The method according to claim 78, wherein the inflammatory / autoimmune disease is at least one of rheumatoid arthritis, psoriasis, allergy, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis and celiac disease.

80. The lentiviral vector according to claim 15, comprising pGAR or a derivative thereof.

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