Chimeric antigen receptor and cells containing the chimeric antigen receptor

Abstract

The present disclosure provides a chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, the chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a single anti-CLL-1 heavy chain variable domain (VH) and a single anti-CLL-1 light chain variable domain (VL), a transmembrane domain, and an intracellular signaling domain. Also disclosed are immune effector cells and immune effector cell-based pharmaceutical compositions comprising the CAR, as well as therapeutic uses thereof in the treatment of diseases associated with CLL-1.

Description

[Background technology]

[0001] Effector cell activation can involve ligand binding to a membrane-bound receptor that contains an extracellular antigen-binding domain and an intracellular signaling domain. The complex formed between this antigen-binding domain and its corresponding target can induce conformational and / or chemical changes in the receptor itself, which can generate a series of signals that are transduced intracellularly. Attempts have been made to exploit this interaction in the development of immune cell therapies. Summary of the Invention

[0002] Various aspects of the present disclosure provide systems, compositions, and methods for inducing immune cell activation.

[0003] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, the chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL), a transmembrane domain, and an intracellular signaling domain, wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, and the single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0004] In some embodiments, the first amino acid sequence is that of SEQ ID NO: 14. In some embodiments, the second amino acid sequence is that of SEQ ID NO: 15.

[0005] In some embodiments, the single heavy chain variable domain is located N-terminal to the single light chain variable domain. In some embodiments, the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0006] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0007] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker, which in some embodiments comprises 50 or fewer amino acid residues.

[0008] In some embodiments, the transmembrane domain is derived from CD8 or CD28.

[0009] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0010] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0011] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the hinge domain is derived from CD28.

[0012] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD28.

[0013] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, the chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL), a transmembrane domain, and an intracellular signaling domain, wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14, and the single light chain variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15.

[0014] In some embodiments, the single heavy chain variable domain is located N-terminal to the single light chain variable domain. In some embodiments, the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0015] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0016] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker, which in some embodiments comprises 50 or fewer amino acid residues.

[0017] In some embodiments, the transmembrane domain is derived from CD8 or CD28.

[0018] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0019] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0020] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the hinge domain is derived from CD28.

[0021] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD28.

[0022] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, the chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a single anti-CLL-1 heavy chain variable domain (VH) and a single anti-CLL-1 light chain variable domain (VL), a transmembrane domain derived from CD8, CD28, 4-1BB, or a combination thereof, and an intracellular signaling domain derived from CD8, CD28, 4-1BB, OX40, ICOS, or a combination thereof.

[0023] In some embodiments, the single heavy chain variable domain of the anti-CLL-1 comprises CDR1, CDR2 and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

[0024] In some embodiments, the single light chain variable domain of the anti-CLL-1 comprises CDR1, CDR2 and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:19.

[0025] In some embodiments, the single heavy chain variable domain of the anti-CLL-1 antibody comprises CDR1, CDR2 and CDR3 of a VH domain comprising the amino acid sequence of SEQ ID NO:14.

[0026] In some embodiments, the single light chain variable domain of the anti-CLL-1 antibody comprises CDR1, CDR2 and CDR3 of a VL domain comprising the amino acid sequence of SEQ ID NO:15.

[0027] In some embodiments, the single heavy chain variable domain is located N-terminal to the single light chain variable domain, hi some embodiments, the single heavy chain variable domain is located N-terminal to the single light chain variable domain.

[0028] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0029] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker, which in some embodiments comprises 50 or fewer amino acid residues.

[0030] In some embodiments, the transmembrane domain is derived from CD8 or CD28.

[0031] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0032] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0033] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the hinge domain is derived from CD28.

[0034] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD28.

[0035] In another aspect, the disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1), the chimeric antigen receptor comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 26.

[0036] In another aspect, the disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1), the chimeric antigen receptor comprising a polypeptide derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27 to 33. In some embodiments, the polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32 to 33.

[0037] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, the chimeric antigen receptor comprising an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL), a transmembrane domain, and an intracellular signaling domain, wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, or the single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0038] In some embodiments, the first amino acid sequence is that of SEQ ID NO: 14. In some embodiments, the second amino acid sequence is that of SEQ ID NO: 15.

[0039] In some embodiments, the single heavy chain variable domain is located N-terminal to the single light chain variable domain. In some embodiments, the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0040] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0041] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker, which in some embodiments comprises 50 or fewer amino acid residues.

[0042] In some embodiments, the transmembrane domain is derived from CD8 or CD28.

[0043] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0044] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0045] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof. In some embodiments, the hinge domain is derived from CD28.

[0046] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD28.

[0047] In another aspect, the present disclosure provides an immune effector cell comprising a CAR disclosed herein. In some embodiments, the immune effector cell is a T cell.

[0048] In another aspect, the present disclosure provides a pharmaceutical composition comprising the immune effector cells disclosed herein and a pharmaceutically acceptable carrier.

[0049] In another aspect, the present disclosure provides a method for treating cancer in an individual that expresses CLL-1, the method comprising administering to the individual an effective amount of any of the immune effector cells or pharmaceutical compositions disclosed herein.

[0050] In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is refractory or relapsed multiple myeloma.

[0051] In some embodiments, the cancer is myeloid leukemia. In some embodiments, the cancer is refractory or relapsed myeloid leukemia.

[0052] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein 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.

[0053] The features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0054] [Figure 1]

[0023] Figure 1 shows the screening of anti-CLL-1 antibodies disclosed herein by flow cytometry. (A) Representative flow cytometry dot plots. (B) Overlaid histograms. Each scFv-Fc antibody was stained at 0.2 nM on a pair of K562-CLL-1 and K562 cells, followed by staining with a fluorochrome-conjugated secondary antibody for flow cytometry analysis. [Figure 2] 1 shows binding of the disclosed anti-CLL-1 antibodies to AML cell lines. [Figure 3] A to B show the Kd values ​​of the top three anti-CLL-1 scFv-Fc clones screened by flow cytometry. [Figure 4] 1 shows the design and construction of a CLL-1 CAR gene carrying evolved anti-CLL antibody hits. [Figure 5] The CLL-1 (61H08) CAR gene with scFv VH-VL swapped and costimulatory domain exchanged is shown. [Figure 6] A-C show the proliferation evaluation of CLL-1 CAR-T cells derived from donor 25: (A) T cell viability, (B) CAR%, CAR-T cell transduction efficiency was assessed by EGFP expression (EGFP%), and (C) CAR-T cell count. [Figure 7A] Proliferative assessment of CLL-1 CAR-T cells derived from donor 26, showing T cell viability. [Figure 7B] Proliferation evaluation of CLL-1 CAR-T cells derived from donor 26, showing CAR%; CAR-T cell transduction efficiency was assessed by EGFP expression (EGFP%). [Figure 7C] Proliferation assessment of CLL-1 CAR-T cells derived from donor 26, showing the number of CAR-T cells. [Figure 8] A–F show the phenotypic profiles of CLL-1 CAR-T cells from donors 25 and 26. (A, D) CD4 and CD8 populations of CAR-T cell clones were analyzed by flow cytometry on day 6. (B, E) CAR-T cell phenotypes, with T cell subsets defined as Tnaive (CCR7+ CD45RA+ CD95-), Tscm (CCR7+ CD45RA+ CD95+), Tcm (CCR7+ CD45RA-), Tem (CCR7- CD45RA-), and Teff (CCR7- CD45RA+ CD95+). (C, F) Expression of inhibitory markers (PD-1, TIM-3, and LAG-3). [Figure 9]Figures A to B show the CAR expression profiles of CLL-1 CAR-T cells from donors 25 and 26. (A) Shows the CAR expression profile of CLL-1 CAR-T cells from donor 25. (B) Shows the CAR expression profile of CLL-1 CAR-T cells from donor 26. The transduction efficiency of CAR-T cells was evaluated by EGFP expression (EGFP%) and CLL-1-ECD antigen labeling, and the CAR expression level was confirmed by MFI (median fluorescence intensity). [Figure 10] Figures A-B show the cytotoxicity of CLL-1 CAR-T cell clones against CLL-1+ U937 cells. (A) CLL-1 CAR-T cell-mediated cytotoxicity from donor 25 is shown. (B) CLL-1 CAR-T cell-mediated cytotoxicity from donor 26 is shown. Individual CAR-T cell clones were cultured with luciferase-modified AML U937 cells at E / T ratios of 0.25, 0.5, 1, 2, 4, and 8 for 6 hours. After the addition of luciferin solution, luciferase activity in the remaining cell lysates was measured using a Cytation™ 5 Cell Imaging Multimode Reader. Specific lysis was calculated from the data according to the formula: % specific lysis = 100 × (experimental release - spontaneous release) / (maximum release - spontaneous release). [Figure 11A] In vitro cytokine release profile of CLL-1 CAR-T cell clones encountering CLL-1+U937 cells, showing cytokine release of CLL-1 CAR-T cells from donor 25. [Figure 11B] Figure 1 shows the in vitro cytokine release profile of a CLL-1 CAR-T cell clone encountering CLL-1+ U937 cells, showing cytokine release from CLL-1 CAR-T cells from donor 26. In a cytotoxicity assay, CLL-1 CAR-T cell clones were co-cultured with U937 cells at E / T=4 for 6 hours, and released cytokines in the culture supernatant were measured using the LEGENDplex™ Human CD8 / NK Panel (13-plex). [Figure 12A]BLI data for CLL-1 CAR-T cell clones in a U937 xenograft model. Spider plots showing tumor burden in xenograft mice implanted with different CAR-T cell clones derived from donor 25. [Figure 12B] BLI data for CLL-1 CAR-T cell clones in a U937 xenograft model. Spider plots showing tumor burden in xenograft mice implanted with different CAR-T cell clones derived from donor 26. [Figure 12C] BLI data of CLL-1 CAR-T cell clones in a U937 xenograft model. BLI images of U937 xenograft mice transplanted with different CAR-T cell clones are shown. [Figure 13] A to D show Kaplan-Meier survival curves of xenografted mice transplanted with CAR-T cells and the persistence of CLL-1 CAR-T cells in vivo. (A, B) Kaplan-Meier survival curves. (C, D) Dynamic monitoring of CLL-1 CAR-T cells in the peripheral blood of xenografted mice. [Figure 14A] Figure 1 shows the in vivo cytokine release profile of xenografted mice transplanted with CLL-1 CAR-T cell clones, showing the cytokine kinetic profile in plasma samples from mice transplanted with CAR-T cells from donor 25. Levels of various human cytokines in collected mouse plasma samples were measured using the LEGENDplex™ Human CD8 / NK Panel (13-plex). [Figure 14B] Figure 1 shows the in vivo cytokine release profile of xenografted mice transplanted with CLL-1 CAR-T cell clones, showing the cytokine kinetic profile in plasma samples from mice transplanted with CAR-T cells from donor 26. Levels of various human cytokines in collected mouse plasma samples were measured using the LEGENDplex™ Human CD8 / NK Panel (13-plex). [Figure 15]Panels A-B show hematotoxicity evaluation of the disclosed CLL-1 CAR-T cell candidates. Hematotoxicity evaluation was performed using bone marrow- or peripheral blood-derived CD34+ stem cells inoculated with the indicated CAR-T cells by colony formation inhibition assay, with an E / T ratio of 1 in (A) and 4 in (B). The mean colony count from the CD34+ group was set to 100%, and values ​​from other groups were adjusted and normalized using the calculation (mean colony count of treated samples / mean colony count of CD34+ samples) × 100%. [Figure 16] Characterization of treated CLL-1 CAR-T cell candidates is shown. CLL-1 CAR-T cell candidates were generated using T cells from donors A011 and A012. EGFP expression was used to define the percentage of CARs. ARD103 CD4 and CD8 populations, T cell subsets, and expression of inhibitory markers were analyzed by flow cytometry. T cell subsets were defined as Tnaive (CCR7+ CD45RA+ CD95-), Tscm (CCR7+ CD45RA+ CD95+), Tcm (CCR7+ CD45RA-), Tem (CCR7- CD45RA-), and Teff (CCR7- CD45RA+ CD95+). PD-1, TIM-3, and LAG-3 were used as inhibitory markers. [Figure 17]Figures A-B show that CLL-1 CAR-T cell candidate-mediated cytotoxicity correlates with CLL-1 antigen expression levels. (A) CLL-1 antigen expression levels in AML cell lines (U937, THP-1, HL60, MOLM-13, and MOLM-14) and CML cell lines (K562) were detected by flow cytometry using an anti-human CLL-1-APC antibody (clone: ​​REA431). (B) The cytolytic activity of ARD103 against AML or CML cell lines at different effector:target (E:T) ratios was measured using a luciferase-based reporter assay. CAR-T cells were cultured with luciferase-modified AML cell lines at E:T ratios of 1, 2, 4, and 8 for 4 to 6 hours. After the addition of luciferin solution, luciferase activity in the remaining cell lysates was measured using a Cytation™ 5 Cell Imaging Multimode Reader. Specific lysis was calculated from the data according to the formula: % specific lysis = 100 x (experimental release - spontaneous release) / (maximum release - spontaneous release). [Figure 18] Figures A-B show that CLL-1 CAR-T cell candidate-mediated cell killing is CLL-1 antigen-specific. (A) CLL-1 antigen expression levels in MOLM-14wt and MOLM-14CLL-1KO cell pools were detected by flow cytometry using an anti-human CLL-1-APC antibody (clone: ​​REA431). (B) Cytolytic activity of CLL-1 CAR-T cell candidates prepared from donors S032 and S040 against MOLM-14wt and MOLM-14CLL-1KO pools at different effector:target (E:T) ratios was measured using a luciferase-based reporter assay. [Figure 19]A-B show the minimum effective dose (MED) and durable antitumor activity of CLL-1 CAR-T cells. Mice (n=6) were implanted with 3x104 U937_Luc cells per mouse on day 0 and then infused with CLL-1 CAR-T cells at the indicated doses on day 5. Tumor volume was assessed by BLI measurements throughout the study. (A) Tumor burden in xenografted mice implanted with different doses of CLL-1 CAR-T cells derived from donor #S011. Surviving mice were re-challenged with U937-Luc cells at the same initial dose on day 43. (B) Tumor burden in xenografted mice implanted with CAR-T cells derived from donor #S012 and subsequently re-challenged with tumor cells. [Figure 20] Figures A-B show cytotoxicity mediated by CLL-1 CAR-T cells derived from R / R AML patients. CLL-CAR-T cells from R / R patients were cultured with autologous primary AML blasts for 24 hours at E / T ratios of 1, 2, 4, and 8. The absolute number of AML blasts (CD45dim CD34+ CD38+) in each culture was counted by ratio comparison with counting beads by flow cytometry. (A) Cytotoxicity of Pt-S008 CLL-1 CAR-T cells. (B) Cytotoxicity of Pt-S015 CLL-1 CAR-T cells. DETAILED DESCRIPTION OF THE INVENTION

[0055] Sequence Listing Sequence number 01 (GMCSFRss) MLLLVTSLLLCELPHPAFLLIP

[0056] SEQ ID NO: 02 (linker) GGGGSGGGGSGGGGS

[0057] SEQ ID NO: 03 (CD8 Hinge-CD8TM) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0058] SEQ ID NO: 04 (CD28 Hinge-CD28TM) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0059] SEQ ID NO: 05 (4-1BB costimulation) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0060] SEQ ID NO: 06 (CD28 costimulation) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0061] SEQ ID NO: 07 (CD3ζ) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0062] SEQ ID NO: 08 (T2A) EGRGSLLTCGDVEENPGP

[0063] SEQ ID NO: 09 (human IgG1 CH2CH3) AAAPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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[0088] Detailed Description of the Disclosure Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0089] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0090] This disclosure recognizes that adoptive immunotherapy, involving the transfer of ex vivo-generated antigen-specific T cells, is a promising therapeutic strategy for viral infections and cancer. T cells used in adoptive immunotherapy can be generated either by the expansion of antigen-specific T cells or by T cell redirection through genetic engineering (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T cells is a well-established treatment used to treat transplant-associated viral infections and rare virus-associated malignancies. Similarly, the isolation and transfer of tumor-specific T cells has been shown to be successful in the treatment of melanoma.

[0091] This disclosure recognizes that genetic transfer of transgenic chimeric antigen receptors (scCARs) has been successful in generating novel specificities in T cells (Jena, Dotti et al. 2010). scCARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Typically, the binding moiety of scCARs consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the light chain variable fragment of a monoclonal antibody connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of first-generation scCARs are derived from the cytoplasmic / intracellular regions of CD3ζ or Fc receptor γ chains. While first-generation scCARs have been shown to successfully redirect T cell cytotoxicity, they failed to confer long-term proliferation or antitumor activity in vivo. Signaling domains derived from costimulatory molecules, including CD28, OX-40 (CD134), and 4-1BB (CD137), have been added alone (second generation) or in combination (third generation) to enhance the survival and proliferation of scCAR-modified T cells. scCARs have been successfully used to redirect T cells to antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors (Jina, Dotti et al. 2010).

[0092] This disclosure recognizes that induction therapy for acute myeloid leukemia (AML) has remained largely unchanged for many years, and AML remains a disease with a poor prognosis. AML is a disease characterized by the rapid proliferation of immature myeloid cells in the bone marrow, resulting in hematopoietic dysfunction. While standard induction chemotherapy can induce complete remission, many patients ultimately relapse and die, necessitating the development of new AML treatments. Recent advances in immunophenotyping of AML cells have revealed several AML-associated cell surface antigens that could be targeted in future treatments.

[0093] In this disclosure, we identify CLL-1 (C-type lectin-like molecule-1) as an interesting tumor antigen target because it is expressed by leukemic blasts in 85-92% of analyzed AML patients at the time of diagnosis. It belongs to the group V C-type lectin-like receptor family and has a molecular weight of 75 kDa. Group V molecules have a lectin-like domain that binds to non-carbohydrate ligands. CLL-1 is a 265-amino acid type II transmembrane glycoprotein (Uniprot database: Q5QGZ9, human protein encoded by gene number 160364 in the Entrez Gene database) with a 200-aa extracellular domain. CLL-1 is also referred to in literature and databases as MICL, CLEC12, and KLRL1.

[0094] This disclosure recognizes that CLL-1 is a cell surface protein that is specifically expressed on most malignant lymphoid stem cells (AML LSCs) but not on normal HSCs (Van Rhenen et al., 2007). Meanwhile, CLL-1 has been shown to be a diagnostic marker in AML (Larsen et al., 2012). Anti-CLL-1 antibodies distinguish malignant cells from normal stem cells both at diagnosis and in remission, allowing for both AML-specific stem cell detection and potentially antigen targeting (van Rhenen et al., 2007).

[0095] This disclosure recognizes that monoclonal antibodies are often used to treat lymphomas, but their use in leukemias has been more limited. Gemtuzumab ozogamicin (Mylotarg®) is a monoclonal antibody conjugated to a cytotoxin. It was previously approved for treating AML in elderly patients, but was withdrawn from the market after investigations revealed some toxicity associated with the product (PMLIVE "ASH: Pfizer eyes re-launch of Mylotarg" press release, December 10, 2010). Other therapeutic monoclonal antibodies have shown side effects over the past decade (Klastersky, J. (2006) "Adverse effects of the humanized antibodies used as cancer therapeutics," Current Opinion in Oncology, 18(4):316-320).

[0096] This disclosure recognizes that targeting CCL-1 by using immune cells bearing specific chimeric antigen receptors based on anti-CLL-1 monoclonal antibodies to redirect the specificity of immune cells to CLL-1-positive cells is a novel approach, and that the engineered immune cells obtained using this approach have proven effective in eliminating CLL-1-positive malignant cells.

[0097] The present disclosure paves the way for the use of adoptive immunotherapy to treat patients with diseases characterized by an excess of CLL-1-expressing cells. Additionally, the present invention provides engineered allogeneic immune cells that can be used as "off the shelf" allogeneic therapeutic agents.

[0098] The methods disclosed herein, unless otherwise indicated, are performed using conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Current Protocols in Molecular Biology series (F.M.A.usubel, et al. eds.), Methods in Enzymology series (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0099] Specific Definitions As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "antigen-binding domain (singular)" includes multiple antigen-binding domains.

[0100] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise noted, when a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0101] As used herein, "cell" may generally refer to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may be from any organism, so long as it has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, and mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.), and the like. C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. The cells may not be derived from a naturally occurring organism (e.g., the cells may be synthetically produced, sometimes referred to as artificial cells).

[0102] As used herein, the term "activation" and its grammatical equivalents may refer to the process by which a cell transitions from a quiescent state to an activated state. This process may include response to antigen, migration, and / or phenotypic or genetic changes to a functionally active state. For example, the term "activation" may refer to the stepwise process of T cell activation. In some instances, at least two signals may be required for a T cell to become fully activated.

[0103] The term "antigen" as used herein refers to a molecule or fragment thereof that can be bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. In some cases, a receptor may function as the antigen, and a ligand may function as the selective binding agent. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent, such as an immune protein (e.g., an antibody). In some cases, an immune protein may function as the antigen, and an antigenic molecule may function as the selective binding agent. An antigen can also refer to a molecule or fragment thereof that can be used in an animal to generate antibodies that can bind to that antigen.

[0104] As used herein, the term "epitope" and its grammatical equivalents can refer to a portion of an antigen that can be recognized by an antigen-binding domain. An antigen-binding domain can include, for example, a protein (e.g., an antibody, antibody fragment) that is present on a surface, e.g., a cell surface (e.g., a B cell, a T cell, a CAR-T cell, or a modified cell).

[0105] As used herein, the term "antibody" refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (IgG1, IgG2, etc.). Derivatives, variants, or fragments thereof may refer to functional derivatives or fragments that retain (e.g., fully and / or partially) the binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), minibodies, diabodies, and single-domain antibodies ("sdAb" or "nanobodies" or "camelid-derived antibodies"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated. Examples of optimized antibodies include affinity-matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0106] As used herein, the term "antigen-binding domain" refers to a protein or fragment thereof capable of binding to an antigen or epitope. As an example, the antigen-binding domain may be a cellular receptor. As an example, the antigen-binding domain may be a modified cellular receptor. As an example, the antigen-binding domain may be a soluble receptor. In some examples, the antigen-binding domain may be a ligand bound by the cellular receptor, the modified cellular receptor, and / or the soluble receptor.

[0107] As used herein, the term "autologous (self) transplant" and its grammatical equivalents may refer to being from the same individual. For example, an autologous sample (e.g., cells) may refer to a sample that is collected, processed, and then returned to the same subject (e.g., patient). Autologous transplantation in terms of the process may be distinguished from allogeneic processes in which the donor of the sample (e.g., cells) and the recipient of the sample are not the same subject.

[0108] As used herein, the terms "cancer neoantigen," "neoantigen," and "neoepitope," and their grammatical equivalents, can refer to antigens that are not encoded in the normal, unmutated host genome. In some instances, "neoantigen" can refer to either an oncogenic viral protein or an abnormal protein that arises as a result of somatic mutation. For example, neoantigens can arise from the destruction of cellular structures via the activity of viral proteins. As another example, neoantigens can arise from exposure to carcinogenic compounds, which, in some cases, can lead to somatic mutation. This somatic mutation can lead to the formation of tumors / cancers.

[0109] As used herein, the term "cytotoxicity" refers to an unintended or undesired change that occurs in a cell in its normal state. The normal state of a cell may refer to the state seen or present before the cell is exposed to a cytotoxic composition, agent, and / or condition. A cell in its normal state may be in a homeostatic state. The unintended or undesired change in a cell in its normal state may be seen in the form of, for example, cell death (e.g., programmed cell death), reduced replicative capacity, reduced cellular integrity such as membrane integrity, reduced metabolic activity, reduced developmental capacity, or any of the cytotoxic effects disclosed herein.

[0110] As used herein, the phrases "reduced cytotoxicity" and "reducing cytotoxicity" refer to a reduction in the degree or frequency of unintended or undesirable changes in normal cells due to exposure to a cytotoxic composition, agent, and / or condition. The phrases can refer to a reduction in the degree of cytotoxicity in an individual cell exposed to a cytotoxic composition, agent, and / or condition, or a reduction in the number of cytotoxic cells in a population of cells when the population is exposed to a cytotoxic composition, agent, and / or condition.

[0111] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. A transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0112] The terms "derivative," "variant," and "fragment," as used herein with respect to polypeptides, refer to polypeptides that are related to a wild-type polypeptide by, for example, amino acid sequence, structure (e.g., secondary and / or tertiary structure), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, relative to the wild-type polypeptide.

[0113] As used herein, the term "percent identity (%)" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences to achieve the maximum percent identity and introducing gaps as necessary (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The percent identity of two sequences can be calculated by aligning the test sequence and the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides in the same position in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0114] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sports animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0115] As used herein, the terms "treatment" and "treating" refer to an approach to obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit and / or a prophylactic effect. For example, treatment can include administering a system or cell population disclosed herein. A therapeutic benefit can refer to a therapeutic improvement or effect in one or more diseases, disorders, or symptoms being treated. For a prophylactic effect, a composition can be administered to a subject at risk of developing a particular disease, disorder, or symptom, or to a subject who experiences one or more physiological symptoms of a disease, even if the disease, disorder, or symptom has not yet manifested.

[0116] A "therapeutic effect" can occur when there is a change in the disease being treated. This change can be positive or negative. For example, a "positive effect" can correspond to an increase in the number of activated T cells in a subject. In another example, a "negative effect" can correspond to a decrease in tumor burden or size in a subject. A "change" in the disease being treated can refer to at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100% change in disease symptoms. The change can be based on an improvement in the severity of the disease being treated in an individual or the difference in the frequency of disease improvement in a population of individuals with and without the administration of treatment. Similarly, the methods of the present disclosure can include administering a "therapeutically effective" amount of cells to a subject. The term "therapeutically effective" should be understood to have a definition corresponding to "having a therapeutic effect."

[0117] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, including immune cells, such as lymphocytes (e.g., T lymphocytes and / or NK cells), sufficient to produce a desired activity when administered to a subject in need thereof. The term "therapeutically effective" can refer to an amount of a composition sufficient to delay the onset of, arrest the progression of, alleviate, or relieve at least one symptom of a disorder treated by the methods of the present disclosure.

[0118] As used herein, the term "TIL" or tumor-infiltrating lymphocytes and its grammatical equivalents may refer to cells isolated from a tumor. TILs may be any cell found within a tumor. For example, TILs may be cells that have migrated into a tumor. TILs may be cells that have infiltrated a tumor. TILs may be T cells, B cells, monocytes, natural killer (NK) cells, or any combination thereof. TILs may be a mixed population of cells. A population of TILs may include cells with different phenotypes, cells with different degrees of differentiation, cells of different lineages, or any combination thereof.

[0119] CLL1-specific single-chain chimeric antigen receptor The present invention relates to a CLL1-specific chimeric antigen receptor that comprises an extracellular ligand-binding domain specific for a portion of the CLL1 antigen, a transmembrane domain, and a signaling domain.

[0120] A chimeric antigen receptor (CAR) refers to a molecule that combines an extracellular binding domain for a component present on a target cell, e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen), with an immune cell receptor component to generate a chimeric protein that transmits an activating or inhibitory signal, thereby activating cellular immunity.

[0121] More specifically, the present invention relates to a CLL-1-specific chimeric antigen receptor (anti-CLL-1 CAR) comprising at least an extracellular anti-CLL-1 antigen-binding domain, a transmembrane domain, and a cytoplasmic / intracellular / intracellular domain.

[0122] Preferably, the CLL-1-specific chimeric antigen receptor of the present invention further comprises a costimulatory domain, more preferably a CD28 or 4-1BB costimulatory domain, as described, for example, by Jena, B., G. Dotti et al. (2010), and may also comprise a transmembrane domain, which may be a Cd8α transmembrane domain, and optionally a hinge.

[0123] The signaling domain or "cytoplasmic / intracellular signaling domain" of the CAR of the present invention is responsible for intracellular signaling after the extracellular ligand-binding domain binds to a target, resulting in the activation or suppression of immune cells and immune responses. That is, the signaling domain is responsible for activating or inactivating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "cytoplasmic / intracellular signaling domain" refers to the portion of a protein that transmits the functional signal of the effector signal and instructs the cell to perform a specialized function.

[0124] The cytoplasmic / intracellular signaling domain, preferably derived from a human protein involved in signal transduction pathway(s), determines whether the anti-CLL1 CAR is a positive CAR (PCAR) or a negative CAR (NCAR) depending on the nature of the signal transduction. In either case, if the signaling domain, such as CD3ζ from the human TCR receptor, has the effect of stimulating the cellular immune activity of immune cells when the extracellular ligand-binding domain binds to CLL-1, the CAR is a PCAR. Conversely, if the signaling domain has the effect of reducing cellular immune activity, such as the human immune inhibitory receptors CTLA-4 and PD-1, the anti-CLL-1 CAR is an NCAR or an inhibitory CAR (iCAR) (Federov et al., Sci Transl Med. 2013 Dec. 11;5(215):215ral72). Preferred examples of signaling domains for use in anti-CLL1 CARs can be cytoplasmic / intracellular sequences of T cell receptors and coreceptors that act together to initiate signal transduction after antigen receptor engagement, or they can be derivatives or variants of these sequences or synthetic sequences with the same functional capabilities. Signaling domains include two distinct classes of cytoplasmic / intracellular signaling sequences: one that initiates antigen-dependent primary activation and the other that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic / intracellular signaling sequences can contain signaling motifs known as ITAM immunoreceptor activation tyrosine-based motifs. ITAMs are distinct signaling motifs found in the cytoplasmic / intracellular tails of various receptors that serve as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs for use in the present invention can include, by way of non-limiting example, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.In a preferred embodiment, the signaling domain of the anti-CLL1 CAR may include a CD3ζ signaling domain having an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99%, or 100% sequence identity to a signaling domain of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0125] A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is required for an efficient immune response. A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation, in addition to the primary signal provided by, for example, binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind Toll ligand receptors, and ligands that specifically bind B7-H3. Costimulatory ligands include, among others, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated Also encompassed are ligands that specifically bind to lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0126] In a preferred embodiment, the costimulatory domain of an anti-CLL1 CAR of the invention comprises a portion of a costimulatory signal molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). Specifically, the signaling domain of an anti-CLL1 CAR of the invention comprises an amino acid sequence that has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity to a costimulatory domain within an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0127] The anti-CLL1 CARs of the present invention generally further comprise a transmembrane domain (TM). Distinguishing features of a suitable transmembrane domain include its ability to be expressed on the surface of a cell, preferably an immune cell in the present invention, particularly a lymphocyte or natural killer (NK) cell, and to interact with and direct the immune cell's cellular response to a predetermined target cell. The transmembrane domain can be derived from either natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane polypeptides include subunits of the T cell receptor, such as α, β, γ, or ζ; polypeptides constituting the CD3 complex; p55 (α chain), p75 (β chain), or γ chain of the IL2 receptor; Fc receptors, particularly Fcγ receptor III or subunit chains of CD proteins. Alternatively, the transmembrane domain can be synthetic and comprise primarily hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain.

[0128] As used herein, the term "hinge region" generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the hinge region is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The hinge region may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In a preferred embodiment, the hinge domain comprises a hinge polypeptide exhibiting at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to a hinge domain within an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26. According to one embodiment, the hinge may also be a human Ig (immunoglobulin) hinge, such as a PD-1 hinge, an IgG4 hinge.

[0129] According to a preferred embodiment, the anti-CLL-1 CAR according to the invention comprises a transmembrane domain, more particularly selected from CD8 and / or CD28.

[0130] The anti-CLL-1 CARs of the present invention generally further comprise a transmembrane domain (TM), more particularly a TM derived from CD8 and / or CD28, and even more particularly one that exhibits identity to the polypeptide of SEQ ID NO: 6 or 7.

[0131] In a preferred embodiment, the anti-CLL-1 CAR of the present invention further comprises a TM domain that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a TM domain within an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0132] In cancer cells, downregulation or mutation of target antigens commonly leads to the generation of antigen-deficient escape mutants. Therefore, to compensate for tumor escape and make immune cells more specific to their targets, the anti-CLL-1 CARs specific for CLL-1 of the present invention can contain different extracellular ligand-binding domains, which can simultaneously bind different elements within the target and enhance immune cell activation and function. In one embodiment, the extracellular ligand-binding domains can be arranged in tandem on the same transmembrane polypeptide, optionally separated by a linker. In another embodiment, the different extracellular ligand-binding domains can be arranged on different transmembrane polypeptides constituting the anti-CLL-1 CAR. In another embodiment, the present invention relates to a population of anti-CLL-1 CARs, each comprising a different extracellular ligand-binding domain. Specifically, the present invention relates to a method for modifying immune cells, comprising providing immune cells and expressing a population of anti-CLL-1 CARs, each comprising a different extracellular ligand-binding domain, on the surface of the cells. In another specific embodiment, the present invention relates to a method for modifying immune cells, comprising providing immune cells and introducing into the cells polynucleotides encoding polypeptides constituting a population of anti-CLL-1 CARs, each comprising a different extracellular ligand-binding domain. A population of anti-CLL-1 CARs refers to at least two, three, four, five, six, or more anti-CLL-1 CARs, each comprising a different extracellular ligand-binding domain. The different extracellular ligand-binding domains of the present invention preferably simultaneously bind different elements within a target, thereby enhancing the activity and function of the immune cells. The present invention also relates to isolated immune cells comprising a population of anti-CLL-1 CARs, each comprising a different extracellular ligand-binding domain.

[0133] The CLL-1-specific chimeric antigen receptor of the present invention may have different structures, for example, it can be expressed as a single-chain chimeric protein (scCAR) or in the form of several polypeptides (multi-chain) containing at least one such chimeric protein. Such multi-chain CAR structures are disclosed in WO2014 / 039523, which is incorporated herein by reference.

[0134] The present application discloses several anti-CLL-1 single chain CARs against the CLL-1 antigen, including, by way of non-limiting example, the amino acid sequences: SEQ ID NOs: 20-26.

[0135] The CLL-1 CAR of the present invention may be a "multi-chain CAR" as described above, meaning that the extracellular binding domain and signaling domain are preferably located on different polypeptide chains, while the costimulatory domain may be located on the same polypeptide or on a third polypeptide. Such multi-chain CARs can be derived from FcεRI by replacing the high-affinity IgE-binding domain of the FcεRI α chain with an extracellular ligand-binding domain such as an scFv (Ravetch et al., 1989), while the N- and / or C-terminal tails of the FcεRI β and / or γ chains are fused to the signaling and costimulatory domains, respectively. The extracellular ligand-binding domain is responsible for redirecting T cell specificity to cellular targets, while the signaling domain activates or attenuates immune cell responses. The fact that different polypeptides derived from the α, β, and γ polypeptides derived from FcεRI are transmembrane polypeptides located in the vicinity of the membrane makes the structure of the CAR more flexible, improves specificity for target molecules, and reduces background activity of immune cells, as described in WO2014 / 039523.

[0136] Extracellular antigen-binding domain

[0137] As used herein, the term "extracellular antigen-binding domain" is defined as an oligopeptide or polypeptide capable of binding to a ligand. Preferably, this domain is capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. It may also be, for example, a binding domain derived from a ligand, receptor, human or mouse antibody, or an antigen recognition domain derived from camelids or cartilaginous fish.

[0138] In a preferred embodiment, the extracellular ligand-binding domain comprises a single-chain antibody fragment (scFv) comprising variable fragments of the light (VL) and heavy (VH) chains of a monoclonal anti-CLL-1 antibody specific for a target antigen, linked by a flexible linker. Preferably, the VL and / or VH exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH and VL domains of the amino acid sequences of SEQ ID NOs: 20-26.

[0139] As used herein, the term "recombinant antibody" refers to an antibody or antibody fragment produced using recombinant DNA techniques, such as an antibody or antibody fragment expressed by a bacteriophage, a yeast expression system, or a mammalian cell expression system. The term is also intended to refer to an antibody or antibody fragment produced by synthesizing a DNA molecule encoding the antibody or antibody fragment and expressing the antibody or antibody fragment protein, or by synthesizing an amino acid sequence specifying the antibody or antibody fragment, where the DNA or amino acid sequence has previously been obtained using recombinant DNA or amino acid sequence or synthetic techniques available and well known in the art.

[0140] The present invention discloses the above-mentioned CLL-1-specific single-chain chimeric antigen receptors (anti-CLL1 scCARs), in some embodiments, wherein the extracellular ligand-binding domains comprise humanized VH and VL chains.

[0141] As used herein, the term "human / humanized antibody" means that a polypeptide comprises a human / humanized heavy chain variable region and a human / humanized light chain variable region. For example, the polypeptide may comprise the framework (FR) regions of the light and heavy chain variable regions of a human antibody while substantially retaining the antigen-binding specificity of the parent monoclonal antibody. The human / humanized heavy chain variable region and / or the human / humanized light chain variable region, excluding the complementarity-determining regions (CDRs), are at least about 87% human / humanized, at least about 90% human / humanized, at least about 95% human / humanized, at least about 98% human / humanized, or at least about 100% human / humanized. The antigen-binding polypeptide molecule may be derived from a monoclonal antibody donor (e.g., a murine monoclonal antibody donor) and may comprise CDRs from a monoclonal antibody (e.g., murine monoclonal CDRs).

[0142] As used herein, the term "monoclonal antibody" refers to an antibody produced by laboratory-cultured cell clones, either hybridomas or virally transformed lymphocytes, which are more abundant and homogeneous than natural antibodies and can specifically bind to a single site on the CLL1 antigen. These antibodies are monospecific, produced by the same immune cells, all cloned from a single parent cell, unlike polyclonal antibodies, which are produced by several different immune cells. Monoclonal antibodies have a monovalent affinity for binding to the same epitope. The current methodology applied to humanization is that of Lefranc MP et al. (Lefranc, MP, Ehrenmann F, Ginestoux C, Giudicelli V, Duroux P, "Use of IMGT® databases and tools for antibody engineering and humanization," Methods Mol Biol. 2012;907:3-37). Four alignments are shown.

[0143] Humanized antibodies can be produced using various techniques known in the art, including, but not limited to, CDR-grafting (see, e.g., European Patent No. 239,400, International Publication No. WO 91 / 09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering, or resurfacing (see, e.g., European Patent Nos. 592,106 and 519,596, each of which is incorporated herein by reference in its entirety; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1995, Protein Engineering, 7(6):805-814). al., 1994, PNAS, 91:969-973), chain shuffling methods (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), and methods described in, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al. al.,J.Biol.Chem.,272(16):10678-84(1997),Roguska et al.,Protein Eng.,9(10):895-904(1996),Couto et al.,Cancer Res.,55(23 Supp):5973s-5977s(1995),Couto et al.,Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10(1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Framework residues in framework regions are often substituted with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089, and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

[0144] Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within an anti-CLL-1 CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the ability of the modified anti-CLL-1 CAR to bind to CLL-1 can be tested using the functional assays described herein.

[0145] In preferred embodiments, the present invention discloses an anti-CLL-1-specific single-chain chimeric antigen receptor ("anti-CLL-1 scCAR" or "scCAR") having a polypeptide structure selected from SEQ ID NOs: 20-26, or a polypeptide structure exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NOs: 20-26. In some embodiments, the structure comprises an extracellular antigen-binding domain comprising a VH and a VL derived from a monoclonal anti-CLL-1 antibody, and a cytoplasmic / intracellular domain comprising a hinge, a transmembrane domain, a signaling domain, and a costimulatory domain.

[0146] Polynucleotides and Vectors

[0147] The present invention also relates to polynucleotides and vectors that allow the heterologous expression in cells of the anti-CLL-1 CARs of the present invention that encode the polypeptide sequences detailed above.

[0148] The polynucleotide may be contained within an expression cassette or expression vector (e.g., a plasmid for introduction into bacterial host cells, a viral vector such as a baculovirus vector for transfection of insect host cells, or a plasmid or viral vector such as a lentivirus for transfection of mammalian host cells).

[0149] In certain embodiments, different nucleic acid sequences can be included in a single polynucleotide or vector, including a nucleic acid sequence encoding a ribosomal skipping sequence, such as a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to the next, without the formation of a peptide bond between the two amino acids encoded by the codon (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). A "codon" refers to three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated into a single amino acid residue by the ribosome. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA if they are separated by an in-frame 2A oligopeptide sequence. Such ribosomal skipping mechanisms are well known in the art and are used by several vectors to express several proteins encoded by a single messenger RNA.

[0150] To direct the transmembrane polypeptide into the secretory pathway of the host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are usually located 5' to the nucleic acid sequence encoding the polypeptide of interest, although some specific secretory signal sequences may be located elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferred embodiment, the CAR polypeptide is derived from a nucleic acid of SEQ ID NO: 27-33 or from a nucleic acid that exhibits at least 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 27-33.

[0151] Those skilled in the art will recognize that, given the degeneracy of the genetic code, there can be considerable sequence diversity among these polynucleotide molecules. The nucleic acid sequences of the present invention are preferably codon-optimized for expression in mammalian cells, preferably human cells. Codon optimization refers to replacing codons in a sequence of interest that are usually rare in highly expressed genes of a given species with codons that are usually common in highly expressed genes of such species. Such codons encode the amino acid for which they are replaced.

[0152] Delivery method The present invention encompasses various means for expressing the anti-CLL-1 chimeric antigen receptors (CARs) described herein in immune cells.

[0153] Methods for introducing polynucleotide constructs into cells are known in the art and include, by way of non-limiting example, stable transformation methods in which the polynucleotide construct encoding the CAR is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and virus-mediated methods.

[0154] The polynucleotide may be introduced into cells, for example, by a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, etc. Transient transformation methods include, for example, microinjection, electroporation or particle bombardment, and cell fusion. The polynucleotide may be contained in a vector, more specifically, a plasmid or virus, so that it can be expressed in cells. The plasmid vector may contain a selection marker that allows identification and / or selection of cells that have received the vector.

[0155] Different transgenes can be included in one vector. The vector can contain a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes the ribosome to "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codons (see Donnelly et al., J. of General Virology 82:1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78:13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13):4227-4239 (2008); Atkins et al., RNA 13:803-810 (2007)).

[0156] "Codon" refers to three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated into one amino acid residue by a ribosome. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA if the polypeptides are separated by an in-frame 2A oligopeptide sequence. Such ribosomal skipping mechanisms are well known in the art and are known to be used by some vectors to express several proteins encoded by a single messenger RNA.

[0157] In a more preferred embodiment of the present invention, the polynucleotide encoding the polypeptide of the present invention may be mRNA, which is directly introduced into cells, for example, by electroporation. The inventors have identified optimal conditions for mRNA electroporation in T cells. The inventors used the Cytopulse technique, which uses a pulsed electric field to temporarily permeabilize live cells and allow for the delivery of substances into the cells. This technique, based on the use of electroporation waveforms from PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, Mass. 01746, USA), allows precise control of pulse duration, intensity, and pulse intervals (U.S. Patent No. 6,010,613 and International PCT Application WO2004083379). All of these parameters can be adjusted to optimize conditions for high transfection efficiency with minimal lethality. Essentially, an initial high electric field pulse forms pores, and subsequent lower electric field pulses transport the polynucleotide into the cells.

[0158] The various methods described above involve introducing the scCAR into cells. As a non-limiting example, the scCAR can be introduced as a transgene encoded by a plasmid vector. The plasmid vector may contain a selectable marker that allows for identification and / or selection of cells that have received the vector.

[0159] A polypeptide may be synthesized in situ within a cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. Alternatively, the polypeptide may be produced extracellularly and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art, and non-limiting examples include stable transformation, in which the polynucleotide construct is integrated into the cell's genome, transient transformation, in which the polynucleotide construct is not integrated into the cell's genome, and viral-mediated methods. The polynucleotide may be introduced into a cell, for example, via a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, etc. Transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. The polynucleotide may be contained in a vector, more specifically, a plasmid or virus, for expression in the cell.

[0160] T cell activation and proliferation Although the genetically modified immune cells of the present invention, whether before or after genetic modification of the T cells, are activated and proliferate independently of antigen binding mechanisms, the immune cells of the present invention, particularly T cells, are typically engineered to express the antigens described, for example, in U.S. Pat. Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905, T cells can be further activated and expanded using methods such as those described in U.S. Patent Application Publication Nos. 6,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0161] The T cells of the present invention are generally expanded by contact with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cells to generate a T cell activation signal, such as calcium ionophore A23187, chemicals such as phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA).

[0162] As non-limiting examples, a population of T cells may be stimulated in vitro, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 5 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, -10, -2, IL-15, TGFp, IL-21, and TNF-, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include any of RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, or Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or cytokine(s) in amounts sufficient for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0163] In another specific embodiment, the cells can be expanded by co-culture with tissue or cells, or they can be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0164] Engineered immune cells The term "cell" as used herein generally refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The cell of the present invention is preferably an isolated immune cell, more preferably a T cell obtained from a donor. The immune cell of the present invention may also be derived from a stem cell. The stem cell may be an adult stem cell, a non-human embryonic stem cell, more specifically, a non-human stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell. A representative human cell is a CD34+ cell. The isolated cell may also be a dendritic cell, a killer dendritic cell, a mast cell, a NK cell, a B cell, or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cell may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to expansion and genetic modification of the cells of the present invention, a source of cells can be obtained from a subject using a variety of non-limiting methods. Cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art can be used.

[0165] In another embodiment, the cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In another embodiment, the cells are part of a mixed population of cells exhibiting different phenotypic characteristics. Cell lines obtained from T cells transformed by the above-described methods are also within the scope of the present invention. Modified cells that are resistant to immunosuppressive treatment and are easily obtainable by the above-described methods are also within the scope of the present invention.

[0166] In a preferred embodiment, the present invention provides a population of T cells or primary T cells for allogeneic transplantation into patients that have been equipped with the above-described CLL-1 CAR, do not express a functional TCR, and are reactive against CLL-1 positive cells.

[0167] In a more preferred embodiment, the present invention provides a T cell or population of T cells that has been loaded with a CLL-1 scCAR, is reactive to CLL-1 positive cells as described above, does not express a functional TCR, and is resistant to a selected drug, for allogeneic transplantation into a patient to be treated with the selected drug. The present invention encompasses a method for preparing modified immune cells for immunotherapy, comprising introducing a polynucleotide or vector encoding a CLL-1 CAR into said immune cells ex vivo by the transformation methods previously described in WO2014 / 130635, WO2013176916, WO2013176915, which are incorporated herein by reference.

[0168] In a preferred embodiment, the polynucleotide is introduced into immune cells using a retroviral vector, with a view to stable integration into the cellular genome.

[0169] Methods for modifying immune cells using CARs The present invention also aims to generate anti-CLL-1 CAR-loaded immune cells that are low or non-alloreactive, can be used in allogeneic therapy (i.e., have a low risk of eliciting a graft-versus-host reaction), and / or are rendered resistant to various standard therapies.

[0170] As further described herein, the method may further comprise the step of genetically modifying said immune cells by using at least one endonuclease.

[0171] The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within DNA or RNA molecules, preferably within DNA molecules. Endonucleases do not cleave DNA or RNA molecules regardless of their sequence, but rather recognize and cleave DNA or RNA molecules at specific polynucleotide sequences, further referred to as "target sequences" or "target sites." Endonucleases can be classified as rare-cutting endonucleases if they have polynucleotide recognition sites that are typically longer than 12 base pairs (bp), more preferably 14-55 bp.

[0172] Preferably, the method of the present invention involves a rare-cutting endonuclease, such as a homing endonuclease (Paques and Duchateau 2007), a chimeric zinc finger nuclease (ZFN) resulting from the fusion of an engineered zinc finger domain with the catalytic domain of a restriction enzyme such as FokI (Porteus and Carroll 2005), a TALE nuclease, the Cas9 endonuclease from the CRISPR system described below (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013), or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, chemical or peptidic cleaving agents are conjugated to either a nucleic acid polymer or another DNA that recognizes a specific target sequence, thereby targeting that sequence for cleavage activity. Chemical endonucleases also include synthetic nucleases such as conjugates of orthophenanthroline, DNA cleaving molecules, and triplex-forming oligonucleotides (TFOs), which are known to bind to specific DNA sequences (Kalish and Glazer 2005). Rare-cutting endonucleases can be used to inactivate genes at loci or to integrate transgenes by homologous recombination (HR), i.e., by inducing a DNA double-strand break (DSB) at the locus and inserting foreign DNA into the locus via gene repair mechanisms (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007).

[0173] The term "TALE-nuclease" (TALEN) refers to a fusion protein consisting of a nucleic acid-binding domain, typically derived from a transcription activator-like effector (TALE), and a single nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain, more preferably a domain with endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain can be fused to a meganuclease, such as I-CreI and I-OnuI or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE-nuclease that does not require dimerization for specific recognition and cleavage, such as the fusion of a modified TAL repeat with the catalytic domain of I-TevI ​​described in WO2012138927. Transcription activator-like effectors (TALEs) are proteins derived from the bacterial species Xanthomonas that contain multiple repeats, each of which contains two residues (RVDs) at positions 12 and 13 that are specific for each nucleotide base of a nucleic acid target sequence. A binding domain (MBBBD) with similar modular base-per-base nucleic acid binding properties may also be derived from a novel modular protein recently discovered by the applicant in a different bacterial species. This novel modular protein has the advantage of exhibiting higher sequence variability than TAL repeats. Preferably, the RVDs involved in the recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A, YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.In another embodiment, the critical amino acids 12 and 13 can be mutated to other amino acid residues to modulate, and in particular to enhance, their specificity for the nucleotides A, T, C, and G. TALE-nucleases have been described and used to stimulate gene targeting and modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Modified TAL-nucleases are available under the trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France) or can be ordered from manufacturers such as Life Technologies (Carlsbad, Calif., USA).

[0174] Preferred TALE-nucleases that recognize and cleave target sequences are described in PCT / EP2014 / 075317. Specifically, to enhance the ability to inactivate target genes, an additional catalytic domain can be introduced into cells together with the rare-cutting endonuclease to increase mutagenesis. More specifically, the additional catalytic domain is a DNA end-processing enzyme. Non-limiting examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases. Non-limiting examples of such catalytic domains include protein domains or catalytically active derivatives of protein domains selected from the group consisting of hExoI (EXO1_HUMAN), yeast ExoI (EXO1_YEAST), E. coli ExoI, human TREX2, mouse TREX1, human TREX1, bovine TREX1, rat TREX1, TdT (terminal deoxynucleotidyl transferase), human DNA2, and yeast DNA2 (DNA2_YEAST). In a preferred embodiment, the additional catalytic domain has 3'-5'-exonuclease activity. In a more preferred embodiment, the additional catalytic domain is the catalytic domain of TREX, more preferably TREX2 (WO 2012 / 058458). In another preferred embodiment, the catalytic domain is encoded by a single-chain TREX2 polypeptide. The additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein according to the present invention, optionally via a peptide linker.

[0175] "Cas9 endonuclease" refers to any genome engineering tool developed from the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system (see (Sorek, Lawrence et al. 2013) for a review) based on the RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013). CRISPR-associated (Cas) systems were first discovered in bacteria and function as a defense against foreign DNA, either viral or plasmid. CRISPR-mediated genome engineering begins with the selection of a target sequence, which is often flanked by short sequence motifs called protospacer adjacent motifs (PAMs). After the target sequence is selected, a specific crRNA complementary to this target sequence is engineered. The transactivating crRNA (tracrRNA), required for type II CRISPR systems, pairs with the crRNA and binds to the supplied Cas9 protein. Cas9 acts as a molecular anchor, promoting base pairing of the tracrRNA with the cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the tracrRNA:crRNA duplex acts as a guide RNA, directing the endonuclease Cas9 to its cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. DNA targeting requires complementarity between the target sequence and the crRNA as well as the presence of a short motif adjacent to the protospacer (protospacer adjacent motif - PAM). Following pairing between the duplex RNA and the target sequence, Cas9 then introduces a blunt double-strand break three bases upstream of the PAM motif (Garnau, Dupuis et al. 2010). The use of Cas9 in immune cells, particularly T cells, has already been described in WO2014191128.

[0176] In a preferred embodiment, the method for further engineering immune cells comprises introducing into the T cells a polynucleotide, specifically an mRNA, encoding a specific rare-cutting endonuclease to selectively inactivate the aforementioned gene by DNA cleavage. In a more preferred embodiment, the rare-cutting endonuclease is a TALE-nuclease or a Cas9 endonuclease. TAL-nucleases have previously been shown to have higher specificity and cleavage efficiency than other types of rare-cutting endonucleases, making them the endonucleases of choice for large-scale production of engineered immune cells with consistent turnover.

[0177] Therapeutic applications In another embodiment, the isolated cells obtained by the various methods described above, and cell lines derived from the isolated cells, can be used as pharmaceuticals.

[0178] In another embodiment, such medicaments can be used to treat cancer, particularly leukemia, in a patient in need thereof.

[0179] In another embodiment, the isolated cells of the present invention or cell lines derived from the isolated cells described above can be used in the manufacture of a medicament for treating cancer in a patient in need thereof.

[0180] In certain embodiments, the anti-CLL-1 CAR-expressing T cells are provided as a medicament for the treatment of AML, AML subtypes, AML-related complications, and AML-related diseases.

[0181] In another embodiment, the medicament can be used to treat a disease mediated by CLL-1 expressing cells or a disorder characterized by the direct or indirect activity of CLL1 expressing cells.

[0182] In another aspect, the invention resides in a method for treating a patient in need thereof, said method comprising at least one of the following steps:

[0183] (a) Providing immune cells obtained by any one of the methods described above.

[0184] (b) administering the transformed immune cells to said patient.

[0185] In one embodiment, the T cells of the present invention are capable of robust T cell proliferation in vivo and long-term survival.

[0186] The treatment may be palliative, curative, or preventative. It may also be part of an autologous or allogeneic immunotherapy treatment. Autologous means that the cells, cell line, or population of cells used to treat a patient are derived from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells, or population of cells used to treat a patient are derived from a donor, not from the patient.

[0187] The cells that can be used in the disclosed methods are described in the previous section. The above treatment can be used to treat patients diagnosed with a pre-cancerous or malignant cancer condition characterized by an excess of CLL-1-expressing cells, particularly CLL-1-expressing cells. Such conditions are found in blood cancers such as leukemia.

[0188] In one embodiment, the present invention provides a composition for use in the treatment of a CLL-1-expressing cell-mediated disease, in particular a CLL-1-expressing cell-mediated hematological cancer, comprising the above-described anti-CLL-1 scCAR expressing T cells of the present invention.

[0189] Any of the other CLL-1 mediated or CLL-1 associated malignant lymphoproliferative diseases disclosed herein may be ameliorated with the anti-CLL-1 CAR expressing cells of the invention.

[0190] In a preferred embodiment, the cancer that may be treated using the anti-CLL-1 CAR-expressing cells of the present invention is leukemia, a disease related to leukemia, or a complication thereof.

[0191] AML

[0192] The leukemia that can be treated using the anti-CLL-1 CAR-expressing cells of the present invention can be acute myeloid leukemia (AML). AML or AML subtypes that can be treated using the anti-CLL-1 CAR-expressing cells of the present invention can specifically be acute myeloblastic leukemia, minimally differentiated acute myeloblastic leukemia, acute myeloblastic leukemia without maturation, acute myeloblastic leukemia with granulocytic maturation, promyelocytic or acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, myelomonocytic leukemia with bone marrow eosinophilia, acute monoblastic leukemia (M5a) or acute monocytic leukemia (M5b), acute erythrocytic leukemia including erythroleukemia (M6a) and the extremely rare pure erythrocytic leukemia (M6b), acute megakaryoblastic leukemia, acute basophilic leukemia, acute panmyelosis with myelofibrosis, regardless of whether CLL-1 positive cells are involved.

[0193] AML subtypes also include hairy cell leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. AML can be classified as AML with specific genetic abnormalities. Classification is based on the ability of the karyotype to predict response to induction therapy, risk of relapse, and survival.

[0194] Thus, AML that can be treated using the anti-CLL-1 CAR-expressing cells of the present invention can be AML with a translocation between chromosomes 8 and 21, AML with a translocation or inversion in chromosome 16, AML with a translocation between chromosomes 9 and 11, APL (M3) with a translocation between chromosomes 15 and 17, AML with a translocation between chromosomes 6 and 9, AML with a translocation or inversion in chromosome 3, AML with a translocation between chromosomes 1 and 22 (megakaryoblastic).

[0195] The present invention is particularly useful in treating AML associated with these particular cytogenetic markers.

[0196] The present invention also provides anti-CLL-1 CAR-expressing T cells for the treatment of patients with specific cytogenetic subsets of AML, such as those with t(15;17)(q22;q21) identified using all-trans retinoic acid (ATRA) 16-19, and for the treatment of patients with t(8;21)(q22;q22) or inv(16)(p13q22) / t(16;16)(p13;q22) identified using repeated high-dose cytarabine.

[0197] Preferably, the present invention provides anti-CLL-1 CAR-expressing T cells for treating patients with abnormalities such as transient abnormalities such as -5 / del(5q), -7, persistent abnormalities of 3q, or complex karyotype, which have been shown to have poor complete remission and survival rates.

[0198] As used herein, the terms "therapeutic agent," "chemotherapeutic agent," or "drug" or "anti-cancer drug" refer to a pharmaceutical compound, preferably a compound or derivative thereof, that can interact with cancer cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of chemotherapeutic agents or "anti-cancer drugs" include alkylating agents (e.g., busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, melphalan, mechlorethamine, oxaliplatin, uramustine, temozolomide, fotemustine), antimetabolites (e.g., clofarabine, methotrexate (MTX), 5-fluorouracil or its derivatives, azathioprine, capecitabine, cytarabine, floxuridine, fluorouracil, gemcitabine, methotrexate), and the like. These include, but are not limited to, purine nucleoside antimetabolites such as serotonin, pemetrexed, etc.), antitumor antibiotics (e.g., mitomycin, adriamycin, bleomycin, daunorubicin, doxorubicin, epirubicin, hydroxyurea, idarubicin, mitomycin C, mitoxantrone), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol, vinblastine, vinorelbine, docetaxel, paclitaxel), and topoisomerase inhibitors (irinotecan, topotecan, etoposide).

[0199] In a preferred embodiment, the term "therapeutic agent" or "chemotherapeutic agent" as used herein refers to a compound or derivative thereof used in the treatment of cancer, particularly hematopoietic cancer cells, more particularly AML, thereby reducing the proliferation state of cancer cells and / or killing cancer cells. Examples of chemotherapeutic agents include, but are not limited to, arachnid, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, bepside, etoposide (VP16), arsenic trioxide, trans-retinoic acid, mechlorethamine, procarbazine, chlorambucil, and combinations thereof.

[0200] In other embodiments of the invention, the cells of the invention are administered to a patient in conjunction with a drug (or agent) selected from arachnid, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, bepside, etoposide (VP16), arsenic trioxide, trans-retinoic acid, cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone, and combinations thereof.

[0201] Such agents may further include, but are not limited to, the anti-cancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU), and CAMPTOTHECIN™, or any therapeutic derivative thereof.

[0202] In a more preferred embodiment, the anti-CLL-1 scCAR-expressing T cells are administered to the patient in combination with at least one therapeutic agent selected from arachnidin, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, pepsid, etoposide (VP16), arsenic trioxide, trans-retinoic acid, and combinations thereof.

[0203] As used herein, a cell that is "resistant or tolerant" to a drug means a cell that has been genetically engineered to grow in the presence of an amount of the drug that would inhibit or prevent growth in unmodified cells.

[0204] In one embodiment, the anti-CLL-1 CAR-expressing T cells of the present invention may be used as induction therapy, post-remission therapy for AML, or consolidation therapy for patients with AML.

[0205] In one embodiment, the anti-CLL-1 CAR-expressing T cells of the present invention may be used in combination with at least one other anti-cancer agent, more preferably when AML relapses or when AML is refractory or resistant.

[0206] In another preferred embodiment, at least one anti-CLL-1 CAR-expressing cell of the present invention is used to prevent the development of cancer cells that occur after myeloablation, especially after anti-cancer treatment, during bone marrow depletion, or before bone marrow transplantation.

[0207] AML complications

[0208] In a specific embodiment, the present invention provides a medicament for improving the health status of patients, particularly patients suffering from complications associated with AML. More preferably, the above-mentioned modified anti-CLL-1 CAR-expressing T cells of the present invention express at least one anti-CLL-1 CAR of the present invention and are used as a medicament for the treatment of complications associated with AML.

[0209] Complications or diseases associated with AML may include a preceding myelodysplastic phase, secondary leukemia, particularly secondary AML, leukocytosis, and the absence of Auer rods. Leukostasis and central nervous system (CNS) involvement, leukocytosis, and residual disease, among others, are also considered complications or diseases associated with AML.

[0210] AML-related diseases

[0211] In one embodiment, the present invention also provides anti-CLL-1 CAR-expressing T cells for the treatment of conditions associated with AML.

[0212] The present invention provides treatments for AML-related myeloid neoplasms, acute myeloid leukemia and myelodysplastic syndromes, treatments for relapsed or refractory acute myeloid leukemia, treatments for relapsed or refractory acute promyelocytic leukemia in adults, treatments for acute promyelocytic leukemia, treatments for acute myeloid leukemia in adults over 60 years of age.

[0213] According to another aspect, the present invention provides compositions for the treatment of AML-related diseases, particularly hematological malignancies associated with AML.

[0214] Malignant hematological disorders associated with AML conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemias"), a collection of diverse hematological conditions united by ineffective production (or dysplasia) of bone marrow blood cells and the risk of transformation to AML.

[0215] Other medical conditions or genetic syndromes associated with the risk of AML may be ameliorated by appropriate use of the present invention, including Down syndrome, trisomy, Fanconi anemia, Bloom's syndrome, ataxia-telangiectasia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, Li-Fraumeni syndrome, neurofibromatosis type 1, and severe congenital neutropenia (also known as Kostmann syndrome).

[0216] Pharmaceutical compositions and methods of treatment

[0217] The present disclosure also provides compositions containing and used modified T cells of the present disclosure or methods for treating diseases.

[0218] In one aspect, the disease is a hematological cancer, particularly a stem cell cancer, including but not limited to leukemias such as acute myeloid leukemia (AML) and their complications.

[0219] The present disclosure also provides compositions or methods for use in inhibiting the proliferation or reducing the activity of a population of CLL-1-expressing cells in a patient. Exemplary methods include contacting a cell population containing CLL-1-expressing cells with anti-CLL-1 CART cells of the present disclosure, particularly CscCART, that bind to the CLL-1-expressing cells.

[0220] In more specific aspects, the present disclosure provides compositions or methods used to inhibit the growth or reduce the number of a population of CLL-1-expressing cancer cells in a patient, the methods comprising contacting the population of CLL-1-expressing cancer cells with anti-CLL-1 CART cells, particularly scCARTs of the present disclosure, that bind to the CLL-1-expressing cells, thereby destroying the CLL-1-expressing cancer cells due to binding of the anti-CLL-1 CAR cells, particularly scCARTs, to the CLL-1-expressing cancer cells.

[0221] In certain aspects, in a subject having myeloid leukemia or other cancer associated with CLL-1-expressing cells or in an animal model thereof, the anti-CLL-1 CART cells of the disclosure, particularly scCART, reduce the content, number, total amount, or percentage of cells and / or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% (to undetectable levels) compared to a negative control.

[0222] The present disclosure also provides compositions or methods for use in preventing, treating, and / or managing diseases or conditions associated with CLL-1-expressing cells (e.g., associated with hematological cancers), comprising administering to a subject in need thereof anti-CLL-1 CART cells, particularly scCART, of the present disclosure that bind to CLL-1-expressing cells. In one embodiment, the subject is a human. Non-limiting examples of disorders associated with CLL-1-expressing cells include inflammatory diseases (e.g., rheumatoid arthritis) and cancer (e.g., hematological cancers, particularly AML or AML complications).

[0223] The present disclosure also provides compositions or methods for use in preventing, treating, and / or managing diseases associated with CLL-1-expressing cells, comprising administering to a subject in need thereof anti-CLL-1 CART cells, particularly scCART, of the present disclosure that bind to CLL-1-expressing cells. In one embodiment, the subject is a human. Non-limiting examples of diseases associated with CLL-1-expressing cells include acute myeloid leukemia (AML), among others.

[0224] The present disclosure also provides compositions or methods for use in treating or preventing recurrence of cancer associated with CLL-1-expressing cells, comprising administering to a subject in need thereof anti-CLL-1 CART cells, particularly scCART, of the present disclosure that bind to CLL-1-expressing cells. In another aspect, the method comprises administering to a subject in need thereof an effective amount of anti-CLL-1 CART cells, particularly scCART, of the present disclosure that bind to CLL-1-expressing cells, in combination with an effective amount of another therapeutic agent.

[0225] In one aspect, CLL-1 is considered a "cancer stem cell" marker in AML. Thus, the anti-CLL1 CART cells of the present disclosure, particularly scCART, can prevent AML recurrence or even treat AML that is largely CLL-1 negative but has a "stem" population of CLL1+ cells (CLL1-expressing cells).

[0226] In one aspect, the disclosure provides compositions and methods for treating a subject who has been treated for a disease or disorder associated with elevated expression levels of CLL-1.

[0227] Treatment using modified immune cells according to the present disclosure may be combined with one or more cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0228] Preferably, treatment with modified immune cells according to the present disclosure may be administered in combination with (e.g., before, simultaneously with, or after) one or more cancer treatments selected from arachnidin, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, pepsid, etoposide (VP16), arsenic trioxide, trans retinoic acid, a combination of arsenic trioxide and trans retinoic acid, mechlorethamine, procarbazine, chlorambucil, and combinations thereof.

[0229] According to a preferred embodiment of the present disclosure, the treatment can be administered to patients undergoing immunosuppressive therapy. Indeed, the present disclosure preferably relies on cells or cell populations that have been made resistant to at least one immunosuppressant drug by inactivating a gene encoding a receptor for the immunosuppressant drug. In this embodiment, the immunosuppressive therapy should aid in the selection and expansion of the T cells of the present invention in the patient.

[0230] Administration of the cells or populations of cells of the present invention may be by any convenient means, including aerosol inhalation, injection, ingestion, transfusion, infusion, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneal routes. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0231] Administration of cells or cell populations is at 10 / kg body weight, including all integer values ​​of cell number within that range. 4 ~10 9 preferably 10 per kg of body weight 5 ~10 6The administration of cells may consist of a single administration of cells or a cell population. The cells or cell population may be administered in a single dose or multiple doses. In another embodiment, the effective amount of cells is administered once. In another embodiment, the effective amount of cells is administered twice or more times over a period of time. The timing of administration is determined by the clinical condition of the patient, within the discretion of the supervising physician. The cells or cell population may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or disorder is within the skill of one of ordinary skill in the art. An effective amount refers to an amount that provides a therapeutic or prophylactic effect. The dosage will depend on the age, health, and weight of the recipient, the type and frequency of concurrent treatment, if any, and the nature of the desired effect.

[0232] In another embodiment, the effective amount of cells or a composition comprising the cells is administered parenterally. This administration can be intravenous. This administration can also be by injection directly into a tumor.

[0233] In one particular embodiment of the present disclosure, the cells are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) relevant treatment modalities, including, but not limited to, drug treatments such as antiviral treatments, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatments for MS patients, or efalizumab treatments for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present disclosure may be used in conjunction with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody treatments, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and radiation treatments. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993).

[0234] In a further embodiment, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation or T cell depletion therapy using either chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

[0235] In another embodiment, the cell composition of the present disclosure is administered after B cell depletion therapy, such as a drug that reacts with CD20, e.g., Rituxan. For example, in one embodiment, a subject may undergo peripheral blood stem cell transplantation after standard treatment with high-dose chemotherapy. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present disclosure. In additional embodiments, the expanded cells are administered before or after surgery.

[0236] In certain embodiments of the present disclosure, anti-CLL-1 scCAR-expressing cells are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) a drug selected from arachnidin, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, bepside, etoposide (VP16), arsenic trioxide, trans retinoic acid, combinations of arsenic trioxide and trans retinoic acid, mechlorethamine, procarbazine, chlorambucil, and combinations thereof. In these embodiments, the anti-CLL-1 scCAR-expressing cells may be resistant to the particular drug or combination of drugs administered in conjunction with the anti-CLL-1 scCAR-expressing cells.

[0237] In other embodiments of the present disclosure, the anti-CLL-1 scCAR-expressing cells are administered to the patient in conjunction with a drug selected from cytarabine, anthracycline, 6-thioguanine, hydroxyurea, prednisone, and combinations thereof.

[0238] Acute myeloid leukemia (AML), characterized by the overproduction of immature myeloid cells in the bone marrow, is the most common acute leukemia in adults and the second most common leukemia in children. It is an aggressive, heterogeneous cancer that affects normal hematopoietic function. Over recent decades, there have been limited improvements in the two standard AML treatments—chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT)—and the prognosis for refractory / relapsed AML remains poor, with a 5-year survival rate of less than 50%. Due to chemotherapy resistance or severe, long-term toxic effects on healthy / non-cancerous tissues / organs, most patients ultimately suffer from recurrent and / or progressive disease, suggesting the urgent need for novel treatment strategies, including targeted therapy and immunotherapy.

[0239] Recent advances in immunotherapy have brought about remarkable breakthroughs in the treatment of malignant hematologic diseases. A special type of engineered autologous T cell, called chimeric antigen receptor (CAR) T cells, combines the specificity of antibody target recognition with the effective effector mechanisms of T cells. The therapeutic efficacy of CAR T cells has been demonstrated with anti-CD19 CAR T cells, which have shown potent and long-lasting antitumor activity in acute lymphoblastic leukemia (ALL) and have been approved by the US FDA. CARs consist of several essential components: an extracellular antigen-binding domain derived from the single-chain variable fragment (scFv) of the targeting antibody; a transmembrane domain; one or more costimulatory domains, such as 4-1BB (CD137), CD28, or ICOS (CD278); and an intracellular signaling domain, CD3-ζ. Target recognition by the scFv domain allows CAR T cells to mediate tumor cytotoxicity in a major histocompatibility complex (MHC)-independent manner, avoiding immune evasion by reducing antigen processing and presentation during tumorigenesis. Due to their MHC-independent antigen recognition, CAR T cells have many advantages, including being more specific than TCRs, being programmable to recognize any tumor antigen, having manageable cytotoxic potential, higher proliferation rate, and longer persistence, making CAR-T cell therapy an excellent therapeutic option for cancer treatment.

[0240] Non-limiting embodiments The present disclosure is illustrated by the following embodiments, which should not be construed as limiting. Those skilled in the art will appreciate that the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0241] 1. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL); a transmembrane domain; an intracellular signaling domain; the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18; The chimeric antigen receptor, wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0242] 2. The CAR of embodiment 1, wherein said first amino acid sequence is that of SEQ ID NO: 14.

[0243] 3. The CAR of embodiment 1, wherein said second amino acid sequence is that of SEQ ID NO: 15.

[0244] 4. The CAR of embodiments 1 to 3, wherein the single heavy chain variable domain is located N-terminal to the single light chain variable domain.

[0245] 5. The CAR of embodiments 1 to 3, wherein the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0246] 6. The CAR of embodiments 1 to 5, wherein the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0247] 7. The CAR of embodiments 1 to 5, wherein the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker.

[0248] 8. The CAR of embodiment 7, wherein the peptide linker comprises 50 or fewer amino acid residues.

[0249] 9. The CAR of embodiments 1 to 8, wherein the transmembrane domain is derived from CD8 or CD28.

[0250] 10. The CAR of embodiments 1-9, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0251] 11. The CAR of embodiment 10, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0252] 12. The CAR of embodiments 1-11, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0253] 13. The CAR of embodiment 12, wherein the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0254] 14. The CAR of embodiment 12, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0255] 15. The CAR of embodiment 12, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0256] 16. The CAR of embodiments 1 to 15, further comprising a hinge domain.

[0257] 17. The CAR of embodiment 16, wherein the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0258] 18. A CAR described in embodiments 16-17, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0259] 19. A CAR according to embodiments 16-17, wherein the hinge domain is derived from CD28.

[0260] 20. The CAR of embodiments 1 to 19, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0261] 21. The CAR of embodiment 20, wherein the signal peptide is derived from CD28.

[0262] 22. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL); a transmembrane domain; an intracellular signaling domain; the single heavy chain variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 14; The chimeric antigen receptor, wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15.

[0263] 23. The CAR of embodiment 22, wherein the single heavy chain variable domain is located N-terminal to the single light chain variable domain.

[0264] 24. The CAR of embodiment 22, wherein the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0265] 25. A CAR according to embodiments 22 to 24, wherein the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0266] 26. A CAR according to embodiments 22 to 24, wherein the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker.

[0267] 27. The CAR of embodiment 26, wherein the peptide linker comprises 50 or fewer amino acid residues.

[0268] 28. A CAR according to embodiments 22 to 27, wherein the transmembrane domain is derived from CD8 or CD28.

[0269] 29. A CAR described in embodiments 22 to 28, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0270] 30. The CAR of embodiment 29, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0271] 31. A CAR described in embodiments 22 to 30, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0272] 32. The CAR of embodiment 31, wherein the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0273] 33. The CAR of embodiment 31, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0274] 34. The CAR of embodiment 31, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0275] 35. A CAR according to embodiments 22 to 34, further comprising a hinge domain.

[0276] 36. The CAR of embodiment 35, wherein the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0277] 37. A CAR described in embodiments 35-36, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0278] 38. A CAR described in embodiments 35-36, wherein the hinge domain is derived from CD28 or 4-1BB.

[0279] 39. A CAR according to embodiments 22 to 38, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0280] 40. The CAR of embodiment 39, wherein the signal peptide is derived from CD28 or 4-1BB.

[0281] 41. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, an extracellular antigen-binding domain comprising a single anti-CLL-1 heavy chain variable domain (VH) and a single anti-CLL-1 light chain variable domain (VL); a transmembrane domain derived from CD8, CD28, 4-1BB, or a combination thereof; and an intracellular signaling domain derived from CD8, CD28, 4-1BB, OX40, ICOS, or a combination thereof.

[0282] 42. The CAR of embodiment 41, wherein the anti-CLL-1 single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18.

[0283] 43. The CAR of embodiment 41, wherein the anti-CLL-1 single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0284] 44. The CAR of embodiment 41, wherein the anti-CLL-1 single heavy chain variable domain comprises CDR1, CDR2 and CDR3 of a VH domain comprising the amino acid sequence of SEQ ID NO: 14.

[0285] 45. The CAR of embodiment 41, wherein the anti-CLL-1 single light chain variable domain comprises CDR1, CDR2 and CDR3 of a VL domain comprising the amino acid sequence of SEQ ID NO: 15.

[0286] 46. ​​The CAR of embodiments 41 to 45, wherein the anti-CLL-1 single heavy chain variable domain is located N-terminal to the anti-CLL-1 single light chain variable domain.

[0287] 47. The CAR of embodiments 41 to 45, wherein the anti-CLL-1 single heavy chain variable domain is located C-terminal to the anti-CLL-1 single light chain variable domain.

[0288] 48. A CAR according to embodiments 41 to 47, wherein the anti-CLL-1 single heavy chain variable domain and the anti-CLL-1 single light chain variable domain are fused directly to each other via a peptide bond.

[0289] 49. A CAR described in embodiments 41 to 47, wherein the anti-CLL-1 single heavy chain variable domain and the anti-CLL-1 single light chain variable domain are linked to each other via a peptide linker.

[0290] 50. The CAR of embodiment 49, wherein the peptide linker comprises 50 or fewer amino acid residues.

[0291] 51. A CAR described in embodiments 41 to 50, wherein the transmembrane domain is derived from CD8 or CD28.

[0292] 52. A CAR described in embodiments 41 to 51, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0293] 53. The CAR of embodiment 52, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0294] 54. A CAR described in embodiments 41 to 53, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0295] 55. The CAR of embodiment 54, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0296] 56. The CAR of embodiment 54, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0297] 57. A CAR according to embodiments 41 to 56, further comprising a hinge domain.

[0298] 58. The CAR of embodiment 57, wherein the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0299] 59. A CAR described in embodiments 57-58, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0300] 60. A CAR according to embodiments 57-58, wherein the hinge domain is derived from CD28.

[0301] 61. A CAR described in embodiments 41 to 60, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0302] 62. The CAR of embodiment 61, wherein the signal peptide is derived from CD28.

[0303] 63. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1), The chimeric antigen receptor, comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26.

[0304] 64. The CAR of embodiment 63, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-26.

[0305] 65. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1), The chimeric antigen receptor, comprising a polypeptide derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27 to 33.

[0306] 66. The CAR of embodiment 65, wherein the polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32-33.

[0307] 67. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL); a transmembrane domain; an intracellular signaling domain; The chimeric antigen receptor, wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, or the single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0308] 68. The CAR of embodiment 67, wherein the first amino acid sequence is that of SEQ ID NO: 14.

[0309] 69. The CAR of embodiments 67-68, wherein the second amino acid sequence is that of SEQ ID NO: 15.

[0310] 70. The CAR of embodiments 67-69, wherein the single heavy chain variable domain is located N-terminal to the single light chain variable domain.

[0311] 71. The CAR of embodiments 67-69, wherein the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0312] 72. A CAR described in embodiments 67 to 71, wherein the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

[0313] 73. A CAR described in embodiments 67 to 71, wherein the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker.

[0314] 74. The CAR of embodiment 73, wherein the peptide linker comprises 50 or fewer amino acid residues.

[0315] 75. A CAR described in embodiments 67 to 74, wherein the transmembrane domain is derived from CD8 or CD28.

[0316] 76. A CAR described in embodiments 67 to 75, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0317] 77. The CAR of embodiment 76, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0318] 78. A CAR described in embodiments 66 to 77, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0319] 79. The CAR of embodiment 78, wherein the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0320] 80. The CAR of embodiment 78, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0321] 81. The CAR of embodiment 78, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0322] 82. A CAR according to embodiments 67 to 81, further comprising a hinge domain.

[0323] 83. The CAR of embodiment 82, wherein the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0324] 84. A CAR described in embodiments 82-83, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and combinations thereof.

[0325] 85. A CAR according to embodiments 82-83, wherein the hinge domain is derived from CD28.

[0326] 86. A CAR described in embodiments 67 to 85, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0327] 87. A CAR according to embodiment 86, wherein the signal peptide is derived from CD28.

[0328] 88. An immune effector cell comprising a CAR according to embodiments 1 to 87.

[0329] 89. The immune effector cell of embodiment 88, wherein the immune effector cell is a T cell.

[0330] 90. A pharmaceutical composition comprising the immune effector cells of embodiments 88-89 and a pharmaceutically acceptable carrier.

[0331] 91. A method for treating cancer in an individual expressing CLL-1, comprising administering to the individual an effective amount of any of the immune effector cells described in embodiments 88-89 or the pharmaceutical composition described in embodiment 90.

[0332] 92. The method of embodiment 91, wherein the cancer is multiple myeloma.

[0333] 93. The method of embodiment 91, wherein the cancer is refractory or relapsed multiple myeloma.

[0334] 94. The method of embodiment 91, wherein the cancer is myeloid leukemia.

[0335] 95. The method of embodiment 91, wherein the cancer is refractory or relapsed myeloid leukemia.

[0336] Non-limiting Examples The present disclosure will now be described based on the experiments described in the following examples, but should not be construed as being limited thereto. Those skilled in the art will appreciate that the present disclosure may be embodied in many different forms and should not be construed as being limited to the examples set forth herein.

[0337] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0338] In some embodiments, the present disclosure relates to the development of CAR-T cells against the CLL-1 antigen, which is expressed on AML blasts and leukemia stem cells. Through a process of CAR-T cell hit selection, lead optimization by swapping antibody variable regions and exchanging the costimulatory domains 4-1BB or CD28 with the disclosed CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z resulted in high CAR%, normal CD4 / CD8 ratios, high fold cell expansion, and T cell proliferation with a memory-like phenotype. scm and Tcm In an AML U937 xenograft animal model, CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were selected from those with high affinity (>80%) and good cytotoxicity against AML cells both in vitro and in vivo. In an AML U937 xenograft animal model, CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were selected from those with high affinity (>80%) and good cytotoxicity against AML cells both in vitro and in vivo. 6The CAR-T cells significantly demonstrated excellent antitumor activity at a dose of 100 mg / mL and substantially survived in the peripheral blood of xenografted mice. In contrast, the disclosed CLL-1 CAR-T candidates, 61H08 HL_2828z and LH_2828z, exhibited acceptable, limited or no inhibition of colony formation of BM- or PB-derived CD34+ cells when cultured for 6 or 24 hours at an E / T ratio of 1 or 4. Furthermore, ex vivo testing of the disclosed CLL-1 (61H08 HL_2828z) CAR-T cells prepared with T cells from r / rAML patients S-008 and S015 mediated significant cytotoxicity against their paired primary AML blasts.

[0339] Example I - Screening and characterization of anti-CLL-1 antibodies Biopanning using a phage-displayed human scFv library

[0340] A CRO service screened for antibodies against the CLL-1 antigen using a human antibody phage library (DSyn2, SLE1, SLE2, SLE3, SLE3-1, SLE3-2, SS1-Mix, and SS2). 10 A fully synthetic human antibody library (DSyn2) with a diversity of 1 × 10 9 ~1×10 10 Biopanning was performed on eight antibody libraries, including antibody libraries derived from seven human autoimmune diseases with a diversity of 1000-150 ...

[0341] TIFF2025532633000025.tif122165

[0342] Screening evolved anti-CLL-1 scFv-Fc hits using flow cytometry

[0343] As shown in Figures 1A-B and 2, the purified scFv-Fc antibodies were further evaluated by flow cytometry for their binding to the CLL-1 antigen in K562-Luc-CLL-1 transfectants and to CLL-1+ AML cell lines, U937, MOLM-14, and THP-1. K562 cells, a human erythroleukemia cell line, and Raji-Luc cells, a human B lymphoblastoid cell line, were used as CLL-1 negative control cell lines. Each scFv-Fc antibody was screened on six cell lines, including K562, K562-CLL-1, U937, MOLM-14, THP-1, and Raji. The binding profile of each clone varied depending on the cell line tested. The top three clones with the strongest binding patterns were selected, and the binding rates ranged from 52% to 99% for K562-Luc-CLL-1, 25% to 98% for U937, 6% to 37% for MLOM-14, and 1% to 36% for THP-1.

[0344] Measurement of the interaction between CLL-1 antigen and evolved anti-CLL-1 scFv-Fc hits by AlphaLISA

[0345] To characterize the antigen-binding ability of anti-CLL-1 scFv-Fc antibodies, an AlphaLISA sandwich assay was set up using anti-6xHis acceptor beads, Protein A donor beads, and CLL-1-ECD. The AlphaLISA assay determined the K d The value was measured. d The value range is 1.1×10 -9 M to 4.9 x 10 -9 The rank order based on the maximum fold increase in AlphaLISA signal was 61H08, 56D01, and 72C10, as shown in Figure 3, from highest to lowest.

[0346] Implementation II - CAR gene design and lentivirus production Design and construction of CLL-1 CAR genes carrying evolved anti-CLL-1 antibody hits

[0347] CAR lentiviral plasmids are essential materials for generating CAR T cells. A second-generation CAR gene containing the essential elements (GMCSFRss signal peptide, scFv, hinge and transmembrane domain of CD8, cytoplasmic portion of 4-1BB, cytoplasmic component of CD3ζ, T2A, and enhanced GFP) was used to construct a CAR LV transfer plasmid (GMCSFRss-scFv-hinge-TM-costimulatory domain-CD3z-T2A-EGFP). The evolved anti-CLL antibody hits 61H08, 65D01, and 72C10 were used to construct CLL-1 CAR lentiviral vectors, as shown in Figure 4.

[0348] First, each CLL-1 CAR gene was generated in the pMK cloning vector by circular PCR to amplify each anti-CLL-1 scFv fragment in frame with the CD8-hinge / TM-4-1BB-CD3ζ-T2A-enhanced GFP backbone, which was then subcloned into a lentiviral plasmid by In-Fusion technology for each of the promoterless CLL-1 CAR genes in the cloning vector.

[0349] Productivity evaluation of CLL-1 CAR lentiviral vector

[0350] Although the titers of CAR LVs varied depending on the CAR clone and the format of its binding domain, i.e., whether the scFv was in the VH-VL or VL-VH orientation, all CLL-1 CAR LVs were successfully produced at high yields and were used to transduce MACS-bead-isolated primary T cells obtained from PBMCs of healthy donors, as shown in Table 2.

[0351] TIFF2025532633000026.tif52165

[0352] Optimized design of CLL-1 (61H08) CAR gene format by antibody variable region swapping and costimulatory domain exchange

[0353] Based on in vitro functional assays, CLL-1 (61H08 HL_8BBz and 61H08 LH_8BBz) CAR-T cells were selected as evolutionary hits, and their CAR gene formats were further optimized by scFv VH-VL swapping and costimulatory domain exchange to maximize CAR-T cell potency in vitro and in vivo. Two additional 65D01 LH_8BBz CLL-1 CAR-T cells, one 24C8 HL_8BBz as a reference, and one control CD19 (FMC63) CAR-T cell were also included for efficacy evaluation in lead CLL-1 CAR-T cells.

[0354] The CLL-1 (61H08HL_8BBz and 61H08LH_8BBz) CAR genes were further optimized by exchanging the costimulatory domain with the CD28 hinge, CD28 transmembrane domain, and CD28 costimulatory domain, as shown in Figure 5.

[0355] Example III - Generation of CLL-1 CAR-T cell clones CLL-1 CAR-T cell bioprocess

[0356] Peripheral blood mononuclear cells (PBMCs) from healthy donors were collected by centrifugation, followed by CD3 microbead isolation of CD3+ T cells, which were then stored in a liquid nitrogen tank prior to CAR-T cell treatment. Thawed T cells were stimulated with human T-activation beads coated with anti-CD3 / anti-CD28 Abs and cultured. Activated T cells were transduced with lentiviral vectors encoding CD19 or CLL-1 CAR constructs. The transduction efficiency of each CAR T cell was analyzed by flow cytometry, and CAR-T cell cultures were expanded by subculture. CAR-T cells were then harvested, analyzed for CAR% and phenotype, and cryopreserved in the vapor phase of liquid nitrogen.

[0357] Productivity evaluation of CLL-1 CAR-T cell clones

[0358] Different CLL-1 CAR-T cells (different CLL-1 clones) were produced and their bioprocessing characteristics, such as CAR%, were observed. Two batches were produced using donors 25 and 26.

[0359] As shown in Figures 6A-C, for CLL-1 CAR-T cells derived from donor 25, the viability of most CLL-1 CAR-T clones exceeded 95% throughout the culture period. The viability of 61H08 HL and 61H08 LH decreased on day 6 but remained approximately 90%. Regarding CAR%, 61H08 HL_28 and 61H08 LH_28 had the highest CAR% at approximately 95%, while 65D01 LH had the lowest CAR% at approximately 88%. Regarding cell proliferation, 61H08 HL_28 and 61H08 LH_28 exhibited a significant growth advantage over their 4-1BB counterparts. 65D01 LH also exhibited a significant growth advantage.

[0360] As shown in Figures 7A-C, for CLL-1 CAR-T cells derived from donor 26, the viability of all CLL-1 CAR-T clones exceeded 90% throughout the culture period. Regarding CAR%, 61H08 HL_28 and 61H08 LH_28 had the highest CAR% at approximately 95%, while 24C8 HL had the lowest CAR% at approximately 90%. Regarding cell proliferation, 61H08 HL_28 and 61H08 LH_28 exhibited a significant growth advantage compared to their 4-1BB counterparts. 65D01 LH also exhibited a significant growth advantage.

[0361] Thus, the two batches of CLL-1 CAR-T production had similar bioprocessing characteristics. 61H08 HL_28 and 61H08 LH_28 had higher viability, CAR%, and fold expansion compared to their 4-1BB counterparts. Furthermore, among all CLL-1 CAR-T clones, 61H08 HL_28 and 61H08 LH_28 had the best bioprocessing characteristics.

[0362] Phenotypic profiling of CLL-1 CAR-T cell clones

[0363] The phenotype and CAR expression level of CLL-1 CAR-T cell clones were continuously monitored. The memory phenotype profile and expression of inhibitory markers were analyzed. CAR functional binding or CAR expression was detected by antigen labeling. Looking at the CD4 / CD8 ratio, 61H08 HL_28 and 61H08 LH_28 had a larger CD8 population compared to their 4-1BB counterparts. 61H08 HL and 61H08 LH had a larger CD4 population, while the others had similar CD4 / CD8 ratios. Regarding the memory phenotype, 61H08 HL_28 and 61H08 LH_28 had a larger T population compared to their 4-1BB counterparts. eff and T em The majority of the population in 61H08 HL_BB and 61H08 LH_BB was T scm and T cm While other CLL-1 CAR-T cell clones had similar memory phenotype profiles, regarding the expression of inhibitory markers, triple-positive cells for PD-1, TIM-3, and LAG3 are likely to be exhausted cells. All of our CLL-1 CAR-T cell clones had a low percentage of triple-positive cells, less than 10% in the batch from donor 25 and less than 5% in the batch from donor 26. Overall, all of our CLL-1 CAR-T cell clones had a predominantly memory phenotype and a high percentage of CD4, while 61H08 HL_28 and 61H08 LH_28 had a higher percentage of T em and T effA high percentage of cells (T scm and T cm The CD8 population was large (although CD16 was still the predominant population). The phenotypic profiles of CLL-1 CAR-T cells from donors 25 and 26 are shown in Figures 8A-F.

[0364] Antigen labeling was used to detect CAR expression and expression intensity in our CLL-1 CAR-T cell clones. Results showed that 65D01 LH_BB had low CAR expression and expression intensity. Conversion of 61H08 HL_8BBz to 61H08 HL_2828z upregulated CAR expression, whereas conversion of 61H08 LH_8BBz to 61H08 LH_2828z reduced CAR expression. CAR expression intensity was highest in 61H08 HL_28 and 61H08 HL_BB, followed by 61H08 LH_BB and 61H08 LH_28. All four of the disclosed CLL-1 CAR-T cell clones showed better CAR expression than the reference 24C8 HL_BB. The CAR phenotypic profiles of CLL-1 CAR-T cells from donors 25 and 26 are shown in Figures 9A-B.

[0365] Example IV - Characterization of CLL-1 CAR-T cell clones In vitro cytotoxicity revealed little or no nonspecific killing by Pan T cells and CD19 CAR-T cells, indicating that all observed cytotoxicity was exerted by CLL-1 CAR-T cells. 61H08 HL_28 and 61H08 LH_28 demonstrated superior cytotoxicity against CLL-1+ U937 cells compared with their 4-1BB counterparts. 61H08 HL_28 and 61H08 LH_28 also demonstrated the best cytotoxicity against target cells compared with all other CAR-T cell clones (24C8 HL_BB, 65D01 LH_BB). Cytotoxicity of the CLL-1 CAR-T cell clones against CLL-1+ U937 cells is shown in Figure 10A-B.

[0366] The cytokine profiles of the CLL-1 CAR-T cell clones were analyzed in a cytotoxicity assay using the LegendPlex multiplex cytokine detection kit. The patterns of both batches were similar, with 61H08 HL_28 and 61H08 HL_28 secreting high levels of effector cytokines (IFN-γ, TNF-α, granzyme B), stimulatory cytokines (IL-2), regulatory cytokines (IL-4, IL-28), and proinflammatory cytokines (IL-17A). This cytokine release profile corresponds to the cytotoxicity results, demonstrating that 61H08 HL_28 and 61H08 LH_28 have superior tumor-killing ability compared to other CLL-1 CAR-T cell clones. The in vitro cytokine release profiles of the CLL-1 CAR-T cell clones encountering CLL-1 U937 cells are shown in Figures 11A-B.

[0367] In vivo CLL-1 CAR-T cell-mediated anti-AML tumor activity in a U937-xenograft model

[0368] A xenogeneic U937-Luc AML model was established to test the in vivo anti-AML activity of CLL-1 CAR-T cells. AML cell proliferation was measured weekly using bioluminescence imaging (BLI). In this setting, 1 × 10 cells per mouse were used. 4Mice transplanted with U937 tumors died approximately 25 days after tumor inoculation. We demonstrated that 61H08 (HL or LH) CAR-T cells carrying the CD28 costimulatory domain exhibited superior anti-AML activity compared with those carrying the 4-1BB domain and other CLL-1 CAR-T cell clones, namely, 24C8_8BBz and 65D01 LH_8BBz clones. Furthermore, 61H08 HL_2828z exhibited more consistent tumor-suppressing activity across donors. BLI images revealed that 61H08 HL_2828z and 61H08 LH_2828z had significant tumor-suppressing effects compared with the reference 24C8 HL_8BBz clone and the control CD19 CAR-T cell clone. BLI data for the CLL-1 CAR-T cell clones in the U937 xenograft model are shown in Figures 12A-C.

[0369] Regarding the survival of xenografted mice, mice receiving CLL-1 CAR-T cells showed a slight improvement in survival compared with groups treated with saline (PBS) or control CD19 CAR-T cells. Specifically, CLL-1 (61H08HL or LH) CAR-T cells containing the CD28 costimulatory domain extended the survival of U937 xenografted mice compared with other CAR-T cell clones, with only one mouse dying in the 61H08 HL_2828z group from donor 25. Regarding the persistence of CAR-T cells in vivo, one mouse from donor 26, 65D01 LH_8BB, had a very high amount of CAR-T cells in its blood, and this mouse ultimately died due to xGVHD. 61H08 HL_2828z and 61H08 LH_2828z demonstrated the best in vivo persistence compared to other CLL-1 CAR-T cell clones. There was no significant difference in in vivo persistence between 61H08 HL_2828z and 61H08 LH_2828z. Kaplan-Meier survival curves of xenografted mice transplanted with CAR-T cells and the persistence of CLL-1 CAR-T cells in vivo are shown in Figures 13A-D.

[0370] In vivo cytokine release profile

[0371] Plasma samples from U937-bearing mice were analyzed for cytokine release profiles on days 20, 27, and 34 after CAR-T cell infusion. Target-cell-binding effector cytokines such as IFN-γ, granzyme A, perforin, and granulysin were detected in plasma, and their levels increased over time. Although the dynamics of these cytokine profiles varied depending on the CLL-1 CAR-T cell clone, in mice that survived at day 34, CLL-1 (61H08 HL or LH) CAR-T cells bearing the CD28 costimulatory domain still released significant amounts of these effector cytokines, indicating that the process of eliminating the transplanted AML tumor cells was active. Regarding the release of stimulatory, regulatory, or proinflammatory cytokines, IL-2 (stimulatory) and IL-10 (regulatory) were occasionally detected in the following plasma samples: The in vivo cytokine release profile of xenograft mice transplanted with CLL-1 CAR-T cell clones is shown in Figures 14A-B.

[0372] Evaluation of hematotoxicity by stem cell (CD34+) colony formation assay

[0373] Hematotoxicity assessment of the disclosed CLL-1 CAR-T cell clones was performed in colony formation assays using CD34+ stem cells derived from normal bone marrow (BM) or peripheral blood (PB). The disclosed CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z showed no or limited inhibition of colony formation of CD34+ cells derived from BM or PB when cultured for 6 or 24 hours at an E / T ratio of 1 or 4. Hematotoxicity assessment of the disclosed CLL-1 CAR-T cell candidates is shown in Figures 15A-B.

[0374] Generation of CLL-1 CAR-T cell candidate (ARD103)

[0375] A CLL-1 CAR-T cell candidate, 61H08 HL_2828z, was produced through a short bioprocessing step. CLL-1 CAR-T cells maintained high cell proliferation rates, exhibited a high CAR% (>80%), normal CD4 / CD8 ratios, and exhibited a memory-like phenotype. scm and T cm The treated CLL-1 CAR-T cell candidates were characterized by a high percentage of PD-1+ TIM-3+ LAG-3+ and relatively low expression levels of triple-positive inhibitory markers (>70%). The characteristics of the treated CLL-1 CAR-T cell candidates are shown in Figure 16.

[0376] CLL-1 CAR-T cell candidate-mediated in vitro target-specific immune responses

[0377] The cytotoxicity mediated by CLL-1 CAR-T cell candidates was CLL-1 antigen-specific and correlated with CLL-1 antigen expression levels. Luciferase-based reporter AML cell lines were generated by transduction with a lentiviral vector encoding a luciferase reporter gene and evaluated for CLL-1 antigen expression levels comparable to those of their parental cells. It should be noted that AML cell lines with high CLL-1 antigen expression, such as U937, THP-1, HL-60, and MOLM-14, were more susceptible to ARD103-mediated cytotoxicity than those with low or no CLL-1 antigen expression, such as MOLM-13 and K562 CML cell lines, respectively. Knockout of the CLL-1 gene in a MOLM-14 cell pool using CRISPR-Cas9 resulted in the cell pool becoming unresponsive to ARD103-mediated cytotoxicity, further supporting the specificity of ARD103 for the CLL-1 antigen. As shown in Figures 17A-C, CLL-1 CAR-T cell candidate-mediated cytotoxicity correlates with CLL-1 antigen expression levels, and as shown in Figures 18A-B, CLL-1 CAR-T cell candidate-mediated cell killing is CLL-1 antigen-specific.

[0378] CLL-1 CAR-T cell candidate demonstrated antitumor activity at the lowest effective dose

[0379] In a xenogeneic U937-Luc AML model, mice were infused with decreasing doses of the CLL-1 CAR cell candidate 61H08 HL_2828z, prepared from two donors through a short bioprocessing step. Rapid production of 61H08 HL_2828z from either donor exerted a dose-dependent tumor growth suppression effect, with a minimally effective dose of 3 × 10 per mouse. 5 The dose was titrated at a low dose of 61H08 HL_2828z. Treatment of xenografted mice with 61H08 HL_2828z improved mouse survival, and surviving CAR-T cells in the peripheral blood elicited effective recall responses after rechallenge with tumor cells. The MED and sustained antitumor activity of CLL-1 CAR-T cells are shown in Figures 19A-B.

[0380] Ex vivo testing of r / rAML patient-derived CLL-1 (61H08 HL_2828z) CAR-T cell-mediated cytotoxicity against primary AML blasts

[0381] Isolated BMMCs from patients S-008 and S015 were cultured with corresponding CLL-1 (61H08HL_2828z) CAR-T cells from r / r AML patients at E / T ratios of 1, 2, 4, and 8 for 24 hours. The absolute number of AML blasts (CD45dim CD34+ CD38+) in each culture was counted by ratio comparison using counting beads by flow cytometry. The CLL-1 CAR-T cell candidates were shown to mediate cytotoxicity against primary autologous AML blasts compared to corresponding untransduced T cells. Cytotoxicity mediated by CLL-1 CAR-T cells from R / R AML patients is shown in Figures 20A-B.

[0382] As shown in the previous examples, CAR-T cells against the CLL-1 antigen were engineered in a second-generation CAR format and optimized by antibody variable region selection and domain (VH-VL) swapping, as well as costimulatory domain (CD28 or 4-1BB) exchange. The CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were selected for their high CAR% (CAR%), normal CD4 / CD8 ratios, high fold cell expansion, a high percentage of memory-like Tscm and Tcm (>70%), high release of effector cytokines, and excellent cytotoxicity against AML tumor cells both in vitro and in vivo. In an AML U937-Luc xenograft animal model, CLL-1 (61H08 HL or LH) CAR-T cells bearing the CD28 costimulatory domain demonstrated superior antitumor activity compared with those bearing the 4-1BB domain, prolonging survival and T cell persistence beyond 43 days after CAR-T cell infusion. Cytokine release profiles of xenografted mice implanted with CAR-T cells showed a significant release of effector cytokines, including IFN-γ, granzyme A, perforin, and ranulysin. Furthermore, 3 × 10 per mouse were successfully treated with CAR-T cells. 5 Xenograft mice implanted with CAR-T cells were able to elicit effective recall responses after rechallenge with tumor cells. Regarding on-target / off-tumor toxicity of CAR-T cells, hematologic toxicity assessment revealed that the CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z exhibited tolerable effects, with no or limited inhibition of colony formation of CD34+ cells derived from bone marrow (BM) or peripheral blood (PB). Furthermore, autologous CLL-1 (61H08 HL_2828z) CAR-T cells were successfully generated from patients with relapsed / relapsed AML and demonstrated cytotoxicity against matched AML blasts isolated from bone marrow. Thus, the CLL-1 CAR-T cell candidates, i.e., 61H08 HL_2828z and 61H08 HL_2828z, have demonstrated anti-AML efficacy with minimal safety concerns and will be further developed for the treatment of patients with relapsed or refractory AML.

[0383] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A chimeric antigen receptor (CAR) for human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide, an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL); a transmembrane domain; an intracellular signaling domain; the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of a first amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18; the chimeric antigen receptor, wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 of a second amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:

19.

2. The CAR of claim 1, wherein the first amino acid sequence is that of SEQ ID NO:

14.

3. The CAR of claim 1, wherein the second amino acid sequence is that of SEQ ID NO:

15.

4. The CAR of claim 1 , wherein the single heavy chain variable domain is located N-terminally to the single light chain variable domain.

5. The CAR of claim 1, wherein the single heavy chain variable domain is located C-terminally to the single light chain variable domain.

6. The CAR of claim 1, wherein the single heavy chain variable domain and the single light chain variable domain are fused directly to each other via a peptide bond.

7. The CAR of claim 1, wherein the single heavy chain variable domain and the single light chain variable domain are linked to each other via a peptide linker.

8. The CAR of claim 7, wherein the peptide linker comprises 50 or fewer amino acid residues.

9. The CAR of claim 1, wherein the transmembrane domain is derived from CD8 or CD28.

10. The CAR of claim 1, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

11. The CAR of claim 10, wherein the primary intracellular signaling domain is derived from CD3ζ.

12. The CAR of claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

13. The CAR of claim 12, wherein the costimulatory signaling domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and a combination thereof.

14. The CAR of claim 12, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

15. The CAR of claim 12, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

16. The CAR of claim 1, further comprising a hinge domain.

17. The CAR of claim 16, wherein the hinge domain is located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

18. The CAR according to claim 16, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and a combination thereof.

19. The CAR of claim 16, wherein the hinge domain is derived from CD28.

20. The CAR according to claims 1 to 19, further comprising a signal peptide located at the N-terminus of the polypeptide.

21. The CAR of claim 20, wherein the signal peptide is derived from CD28.

22. The CAR of claim 1, wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14, and the single light chain variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:

15.

23. The CAR of claim 22, wherein the transmembrane domain is derived from CD8 or CD28.

24. The CAR of Claim 23, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, and the primary intracellular signaling domain is derived from CD3ζ.

25. The CAR of claim 24, wherein the intracellular signaling domain comprises a costimulatory signaling domain, and the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

26. The CAR according to claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 26.

27. An immune effector cell comprising the CAR of claim 26.

28. 28. A pharmaceutical composition comprising the immune effector cells of claim 27 and a pharmaceutically acceptable carrier.

29. 28. A method of treating cancer in an individual that expresses CLL-1, comprising administering to said individual an effective amount of the immune effector cells of claim 27.

30. 30. The method of claim 29, wherein the cancer is AML.

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