Chimeric antigen receptors and cells comprising same

Abstract

The present disclosure provides a chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule-1 (CLL-1) comprising a polypeptide comprising: an extracellular antigen binding domain comprising an anti-CLL-1 single heavy chain variable domain (VH) and an anti-CLL-1 single light chain variable domain (VL); a transmembrane domain; and an intracellular signaling domain. Also disclosed are immune effector cells comprising the CARs and pharmaceutical compositions based on the immune effector cells, as well as their therapeutic uses in the treatment of CLL-1 related conditions.

Description

BACKGROUND

[0001] Effector cell activity can involve the binding of a ligand to a membrane-bound receptor comprising an extracellular antigen binding domain and an intracellular signaling domain. The formation of a complex between the antigen binding domain and its corresponding target can result in a conformational and / or chemical modification of the receptor itself, which in turn can generate a series of signals transduced within the cell. Attempts have been made to exploit this interaction to develop immune cell therapies. SUMMARY

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

[0003] In an aspect, the disclosure provides a chimeric antigen receptor (CAR) directed to human C-type lectin-like molecule-1 (CLL-1), comprising a polypeptide 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 as set forth in 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 wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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 SEQ ID NO: 14. In some embodiments, the second amino acid sequence is SEQ ID NO: 15.

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

[0006] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are directly fused 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 connected to each other via a peptide linker. In some embodiments, the peptide linker comprises no more than 50 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 the immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta.

[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) directed to human C-type lectin-like molecule-1 (CLL-1), comprising a polypeptide 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 a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 14, and wherein the single light chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 15.

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

[0015] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are directly fused 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 connected to each other via a peptide linker. In some embodiments, the peptide linker comprises no more than 50 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. In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta.

[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 comprising: an extracellular antigen binding domain comprising an anti-CLL-1 single heavy chain variable domain (VH) and an anti-CLL-1 single light chain variable domain (VL); a transmembrane domain derived from CD8, CD28, 4-1BB, or combinations thereof; and an intracellular signaling domain derived from CD8, CD28, 4-1BB, OX40, ICOS, or combinations thereof.

[0023] In some embodiments, the anti-CLL-1 single heavy chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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 anti-CLL-1 single light chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in 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 anti-CLL-1 single heavy chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in a VH domain comprising the amino acid sequence of SEQ ID NO: 14.

[0026] In some embodiments, the anti-CLL-1 single light chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in 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 on the N-terminal side of the single light chain variable domain. In some embodiments, the single heavy chain variable domain is located on the C-terminal side of the single light chain variable domain.

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

[0029] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are connected to one another via a peptide linker. In some embodiments, the peptide linker comprises no more than 50 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. In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta.

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

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

[0037] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) directed to human C-type lectin-like molecule-1 (CLL-1), comprising a polypeptide 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 as set forth in 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 wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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 SEQ ID NO: 14. In some embodiments, the second amino acid sequence is 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 directly fused to one another via a peptide bond.

[0041] In some embodiments, the single heavy chain variable domain and the single light chain variable domain are connected to one another via a peptide linker. In some embodiments, the peptide linker comprises no more than 50 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. In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta.

[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 N-terminal to 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 an immune effector cell disclosed herein and a pharmaceutically acceptable carrier.

[0049] In another aspect, the present disclosure provides a method of treating a cancer expressing CLL-1 in an individual, comprising administering to the individual an effective amount of the immune effector cell or pharmaceutical composition 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] incorporated by reference

[0053] 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. BRIEF DESCRIPTION OF DRAWINGS

[0054] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings of which:

[0055] Figure 1 Exemplification of screening of anti-CLL-1 antibodies disclosed herein by flow cytometry: (A) Representative flow cytometry dot plots. (B) Overlay histograms. Each scFv-Fc antibody was stained at 0.2 nM with paired K562-CLL-1 and K562 cells, followed by staining with a 2nd antibody conjugated to a fluorescent dye for flow cytometry analysis;

[0056] Figure 2 Exemplification of binding of disclosed anti-CLL-1 antibodies to AML cell lines;

[0057] Figures 3A-3B Exemplification of Kd values of top 3 anti-CLL-1 scFv-Fc clones from flow cytometry screening;

[0058] Figure 4 Exemplification of design and construction of CLL-1 CAR gene with late- stage anti-CLL antibody hits;

[0059] Figure 5 Exemplification of CLL-1 (61H08) CAR gene with scFv VH-VL swap and costimulatory domain swap;

[0060] Figures 6A-6CProductivity assessment of CLL-1 CAR-T cells derived from donor 25 is exemplified: (A) T cell viability. (B) CAR%. Transduction efficiency of CAR-T cells was determined by EGFP expression (EGFP%). (C) CAR-T cell counts.

[0061] Figures 7A-7C Productivity assessment of CLL-1 CAR-T cells derived from donor 26 is exemplified: (A) T cell viability. (B) CAR%. Transduction efficiency of CAR-T cells was determined by EGFP expression (EGFP%). (C) CAR-T cell counts.

[0062] Figure 8 Phenotypic profile of CLL-1 CAR-T cells from donors 25 and 26 is exemplified by A-8F: (A, D) CD4 and CD8 populations of CAR-T cell clones were analyzed by flow cytometry at day 6. (B, E) CAR-T cell phenotype. T cell subsets were defined as T 幼稚 (CCR7+CD45RA+CD95-), T scm (CCR7+CD45RA+CD95+), T cm (CCR7+CD45RA-), T em (CCR7-CD45RA-), and T eff (CCR7-CD45RA+CD95+). (C, F) Inhibitory marker expression (PD-1, TIM-3, and LAG-3);

[0063] Figure 9 CAR expression profile of CLL-1 CAR-T cells from donors 25 and 26 is exemplified by A-9B: (A) CAR expression profile of CLL-1 CAR-T cells derived from donor 25. (B) CAR expression profile of CLL-1 CAR-T cells derived from donor 26. Transduction efficiency of CAR-T cells was determined by EGFP expression (EGFP%) and CLL-1-ECD antigen labeling, and CAR expression levels were revealed by MFI (median fluorescence intensity);

[0064] Figure 10 Cytotoxicity of CLL-1 CAR-T cell clones against CLL-1+ U937 cells is exemplified by A-10B: (A) CLL-1 CAR-T cells derived from donor 25 mediated cytotoxicity. (B) CLL-1 CAR-T cells derived from donor 26 mediated cytotoxicity. Individual CAR-T cell clones were incubated with luciferase engineered AML U937 cells at E / T ratios of 0.25, 0.5, 1, 2, 4, and 8 for 6 hours. Cytation 5 was used to measure the luciferase activity of U937 cells. The percentage of specific cytotoxicity was calculated as follows: (1 - (test sample - spontaneous release) / (maximum release - spontaneous release)) x 100. TM5Cellular Imaging Multi-Mode Plate Reader measured luciferase activity of the remaining cell lysate after addition of luciferin solution. Specific lysis was calculated from the data according to the following formula: % Specific Lysis = 100 x (Experimental Release - Spontaneous Release) / (Maximum Release - Spontaneous Release);

[0065] Figures 11A-11B In vitro cytokine release profiles of CLL-1 CAR-T cell clones upon encounter with CLL-1+ U937 cells are exemplified: (A) donor 25 derived CLL-1 CAR-T cells release cytokines. (B) donor 26 derived CLL-1 CAR-T cells release cytokines. CLL-1 CAR-T cell clones were co-cultured with U937 cells at E / T = 4 for 6 hours in a cytotoxicity assay and LEGENDplex TM Human CD8 / NK Panel (13-plex) measured cytokines released in culture supernatants;

[0066] Figures 12A-12C BLI data of CLL-1 CAR-T cell clones in U937 xenograft model are exemplified: (A) tumor burden spider plot of xenograft mice receiving different CAR-T cell clones derived from donor 25. (B) tumor burden spider plot of xenograft mice receiving different CAR-T cell clones derived from donor 26. (C) BLI images representing U937 xenograft mice receiving different CAR-T cell clones;

[0067] Figure 13 A-13D exemplifies Kaplan-Meier survival curves and persistence of CLL-1 CAR-T cells in vivo of xenograft mice receiving CAR-T cells: (A, B) Kaplan-Meier survival curves, (C, D) kinetic monitoring of CLL-1 CAR-T cells in peripheral blood of xenograft mice;

[0068] Figures 14A-14B In vivo cytokine release profiles of xenograft mice receiving CLL-1 CAR-T cell clones are exemplified: (A) kinetic cytokine profile in plasma samples of mice receiving donor 25 derived CAR-T cells. (B) kinetic cytokine profile in plasma samples of mice receiving donor 26 derived CAR-T cells. LEGENDplex TM Human CD8 / NK Panel (13-plex) measured levels of various human cytokines in collected mouse plasma samples;

[0069] Figure 15A-15B illustrates the blood toxicity evaluation of the disclosed CLL-1 CAR-T cell candidates: Blood toxicity evaluation was performed by colony formation inhibition assay, in which bone marrow or peripheral blood derived CD34+ stem cells were seeded with the indicated CAR-T cells at 1 (A) and 4 (B) E / T ratios. To normalize, the average colony number of the CD34+ group was set to 100%, and the values of other groups were adjusted using the following calculation: (average colony number of treated sample / average colony number of CD34+ sample) x 100%;

[0070] Figure 16 Characterization of processed CLL-1 CAR-T cell candidates is illustrated. CLL-1 CAR-T cell candidates were produced with T cells from donors A011 and A012. CAR% was defined using EGFP expression. CD4 and CD8 populations, T cell subsets, and suppressor marker expression of ARD103 were analyzed by flow cytometry. T cell subsets were defined as T 幼稚 (CCR7+CD45RA+CD95-), T scm (CCR7+CD45RA+CD95+), T cm (CCR7+CD45RA-), T em (CCR7-CD45RA-), and T eff (CCR7-CD45RA+CD95+). Suppressor markers were PD-1, TIM-3, and LAG-3;

[0071] Figures 17A-17B CLL-1 CAR-T cell candidate mediated cytotoxicity correlates with CLL-1 antigen expression level is illustrated: (A) CLL-1 antigen expression level on AML cell lines (U937, THP-1, HL60, MOLM-13, and MOLM14) and CML cell line (K562) was detected by flow cytometry using anti-human CLL-1-APC antibody (clone: REA431). (B) Cytolytic activity of ARD103 against AML cell lines or CML cell line was measured using a luciferase-based reporter assay at different effector to target (E:T) ratios. CAR-T cells were incubated with luciferase-engineered AML cell lines at 1, 2, 4, and 8 E / T ratios for 4-6 hours. Cytation TM 5Cell Imaging Multi-Mode Reader measured luciferase activity of the remaining cell lysate after addition of luciferin solution. Specific lysis was calculated from the data according to the following formula: % Specific Lysis = 100 x (experimental release - spontaneous release) / (maximal release - spontaneous release);

[0072] Figure 18A-18B illustrates that CLL-1 CAR-T cell candidate mediated cell killing is specific for the CLL-1 antigen: (A) CLL-1 antigen expression levels on MOLM-14 wt and MOLM-14 CLL-1KO pools were detected by flow cytometry using anti-human CLL-1-APC antibody (clone: REA431). (B) Cytolytic activity of CLL-1 CAR-T cell candidates prepared from donor S032 and donor S040 against MOLM-14 wt and MOLM-14 CLL-1KO pools at different effector to target (E:T) ratios were measured using a luciferase-based reporter assay;

[0073] Figure 19 A-19B illustrates the minimal effective dose (MED) and durable anti-tumor activity of CLL-1 CAR-T cells. Mice (n=6) received U937_Luc transplants on day 0 at 3 x 10 4 CAR T cells on day 5. Tumor volume was assessed by in vivo BLI measurements. (A) Tumor burden of xenograft mice that received different doses of CLL-1 CAR-T cells derived from donor #S011. On day 43, surviving mice were re-challenged with the same initial amount of U937-Luc cells. (B) Tumor burden of xenograft mice that received CAR-T cells derived from donor #S012 as well as a subsequent re-challenge of tumor cells;

[0074] Figure 20 A-20B illustrates CLL-1 CAR-T cell mediated cytotoxicity of R / R AML patient derived cells. R / R patient derived CLL-CAR-T cells were incubated with autologous primary AML blast cells at E / T ratios of 1, 2, 4, and 8 for 24 hours. Absolute AML blast cells (CD45dimCD34+CD38+) were counted for each culture by flow cytometry by counting beads ratio comparison. (A) Pt-S008 CLL-1 CAR-T cell cytotoxicity. (B) Pt-S015 CLL-1 CAR-T cell cytotoxicity.

[0075] SEQUENCE LISTING

[0076] SEQ ID NO: 01 (GMCSFRss)

[0077] MLLLVTSLLLCELPHPAFLLIP

[0078] SEQ ID NO: 02 (Linker)

[0079] GGGGSGGGGSGGGGS

[0080] SEQ ID NO:03(CD8铰链-CD8TM)

[0081] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0082] SEQ ID NO:04(CD28铰链-CD28TM)

[0083] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0084] SEQ ID NO:05(4-1BB共刺激)

[0085] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0086] SEQ ID NO:06(CD28共刺激)

[0087] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0088] SEQ ID NO:07(CD3ζ)

[0089] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0090] SEQ ID NO:08(T2A)

[0091] EGRGSLLTCGDVEENPGP

[0092] SEQ ID NO:09(人IgG1 CH2CH3)

[0093] AAAPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LS PG

[0094] SEQ ID NO: 10 (anti-CLL-l-59A09_VH; CDRs are underlined)

[0095] EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDYYIH WVRQAPGKGLEWVS MISPSGGGEAYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR AHDFDDFDY WGQGTLVTVSS

[0096] SEQ ID NO: 11 (anti-CLL-l-59A09_VL; CDRs are underlined)

[0097] DIQMTQSPSSLSASVGDRVTITC RASQSIRYSLA WYQQKPGKASKLLIY DASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNNSPLT FGQGTKVEIKR

[0098] SEQ ID NO: 12 (anti-CLL-l-61A07_VH; CDRs are underlined)

[0099] EVQLVESGGGLVQPGGSLRLSCAAS GFTFSNHWMH WVRQAPGKGLEWVS SISGAGGGKSYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DESSYAYDGDY WGQGTLVTVSS

[0100] SEQ ID NO: 13 (anti-CLL-l-61A07_VL; CDRs are underlined)

[0101] DIQMTQSPSSLSASVGDRVTITC RANQSIRNYLNWYQQKPGKAPKLLIY AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNTSSPST FGQGTKVEIKR

[0102] SEQ ID NO: 14 (anti-CLL-l-61H08_VH; CDRs underlined)

[0103] EVQLVESGGGLVQPGGSLRLSCAAS GFTFDDYGMH WVRQAPGKGLEWVS GIWPSGGNKEYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR LLALTYDYFDY WGQGTLVTVSS

[0104] SEQ ID NO: 15 (anti-CLL-l-61H08_VL; CDRs underlined)

[0105] DIQMTQSPSSLSASVGDRVTITC RASQGISSYLA WYQQKPGKAPKLLIY DASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPFT FGQGTKVEIKR

[0106] SEQ ID NO: 16 (anti-CLL-l-65D01_VH; CDRs underlined)

[0107] QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYYWS WIRQPPGKGLEWIG EINHSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAAYYCAR SPLQWLDDAFDI WGQGTMVTVSS

[0108] SEQ ID NO: 17 (anti-CLL-l-65D01_VL; CDRs underlined)

[0109] QSVLTQPPSVSAAPGQRVTISC SGSSSNIGSNYVS WYQQLPGTAPKLLIY DNNERPS GIPDRFSGSKSATSATLDITGLQTGDEADYFC GTWDNSPSTDWV FGGGTKVTVLG

[0110] SEQ ID NO: 18 (anti-CLL-l-72C10_VH)

[0111] EVQLVESGGGLVQPGGSLRLSCAAS GFTFSVNAMH WVRQAPGKGLEWVS TISGSDGHKYYADSVKS RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DVDSEIGDGYHEDI WGQGTLVTVSS

[0112] SEQ ID NO: 19 (anti-CLL-l-72C10_VL)

[0113] DIQMTQSPSSLSASVGDRVTITC RASQSVTNALN WYQQKPGKAPKLLIY AASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQTNNSPAT FGQGTKVEIKR

[0114] SEQ ID NO: 20 (anti-CLL-l-72C10_VH) -61H08VH- 61H08VL-CD8 hinge-CD8TM-4-lBB costimulatory - T2A )

[0115] Amino acid sequence:

[0116]

[0117] SEQ ID NO: 21 (anti-CLL-l-61H08_VL) -61H08VL- 61H08VH-CD8 hinge-CD8TM-4-lBB costimulatory - T2A )

[0118] Amino acid sequence:

[0119]

[0120]

[0121] SEQ ID NO: 22 (anti-CLL-l-65D01_VH) -65D01VH- 65D01VL-CD8 hinge-CD8TM-4-lBB costimulatory - T2A )

[0122] Amino acid sequence:

[0123]

[0124] SEQ ID NO:23 -65D01 VL- 65D01 VH-CD8 hinge-CD8 TM-4-1BB costimulatory - T2A )

[0125] Amino acid sequence:

[0126]

[0127] SEQ ID NO:24 -72C10 VH- 72C10 VL-CD8 hinge-CD8 TM-4-1BB costimulatory - T2A )

[0128] Amino acid sequence:

[0129]

[0130] SEQ ID NO:25 -61H08 VH- 61H08 VL-CD28 hinge-CD28 TM-CD28 costimulatory - T2A )

[0131] Amino acid sequence:

[0132]

[0133] SEQ ID NO:26 -61H08 VL- 61H08 VH-CD28 hinge-CD28 TM-CD28 costimulatory - T2A )

[0134] Amino acid sequence:

[0135]

[0136]

[0137] SEQ ID NO:27 -61H08 VH- 61H08 VL-CD8 hinge-CD8 TM-4-1BB costimulatory - T2A )

[0138] DNA sequence:

[0139]

[0140] SEQ ID NO:28 -61H08 VL- 61H08 VH-CD8 hinge-CD8 TM-4-1BB costimulatory - T2A )

[0141] DNA sequence:

[0142]

[0143] SEQ ID NO:29( -65D01VH- 65D01VL-CD8 Hinge-CD8TM-4-1BB Costimulator- - T2A )

[0144] DNA sequence:

[0145]

[0146] SEQ ID NO:30 -65D01VL- 65D01VH-CD8 hinge-CD8 TM-4-1BB costim- - T2A )

[0147] DNA sequence:

[0148]

[0149] SEQ ID NO:31 -72C10 VH- 72C10 VL-CD8 hinge-CD8 TM-4-1BB costimulatory - T2A )

[0150] DNA sequence:

[0151]

[0152] SEQ ID NO:32( -61H08VH- 61H08VL-CD28 Hinge-CD28TM-CD28 Costimulator- - T2A )

[0153] DNA sequence:

[0154]

[0155] SEQ ID NO:33 -61H08 VL- 61H08 VH-CD28 Hinge-CD28 TM-CD28 costimulatory - T2A )

[0156] DNA sequence:

[0157] DETAILED DESCRIPTION

[0158] Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0159] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, where appropriate, methods and materials are described herein. 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0160] The present disclosure recognizes that adoptive immunotherapy involving the transfer of ex vivo generated antigen-specific T cells is a promising strategy for the treatment of viral infections and cancer. T cells for adoptive immunotherapy can be generated by expansion of antigen-specific T cells or by genetic engineering to redirect T cells (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T cells is a well-established procedure that is used to treat transplant-associated viral infections and rare viral-associated malignancies. Similarly, the isolation and transfer of tumor-specific T cells has been shown to successfully treat melanoma.

[0161] The present disclosure recognizes that novel specificities in T cells can be successfully generated by genetic transfer of transgenic chimeric antigen receptors (scCARs) (Jena, Dotti et al. 2010). scCARs are synthetic receptors composed of a targeting moiety associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of scCARs is composed of an antigen-binding domain of a single-chain antibody (scFv) that includes the light and variable fragments of a monoclonal antibody connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation scCARs is derived from the cytoplasmic / intracellular region of CD3 zeta or Fc receptor gamma chain. First-generation scCARs have been shown to successfully redirect T cell cytotoxicity, however they are unable to provide long-term expansion and anti-tumor activity in vivo. Signaling domains from costimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have been added either individually (second generation) or in combination (third generation) to enhance the survival of scCAR-modified T cells and to increase the proliferation of scCAR-modified T cells. scCARs have successfully redirected T cells against antigens expressed on the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0162] The present disclosure recognizes that induction therapy for acute myeloid leukemia (AML) has remained largely unchanged for many years, and AML remains a disease with poor prognosis. AML is a disease characterized by the rapid proliferation of immature myeloid cells in the bone marrow leading to hematopoietic dysfunction. Although standard induction chemotherapy can induce complete remission, many patients eventually relapse and die from the disease, thus the need to develop new therapeutic agents for AML. Recent advances in immunophenotypic analysis of AML cells have revealed several AML-associated cell surface antigens that can serve as targets for future therapies.

[0163] The present disclosure recognizes CLL-1 (C-type lectin-like molecule-1) as an interesting tumor antigen target, since it is expressed by leukemic blasts at diagnosis in 85-92% of AML patients analyzed. 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-sugar ligands. CLL-1 is a 265 amino acid type II transmembrane glycoprotein containing a 200 AA extracellular domain (Uniprot database: Q5QGZ9, human protein encoded by gene n° 160364 in the "Entrez Gene" database). CLL-1 is also known in the literature and databases as MICL, CLEC12 and KLRL1.

[0164] The present 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). At the same time, CLL-1 was revealed to be a diagnostic marker for AML (Larsen et al., 2012). Anti-CLL-1 antibodies enable AML-specific stem cell detection and possible antigen targeting, thus distinguishing between malignant and normal stem cells at diagnosis and remission (van Rhenen et al., 2007).

[0165] The present disclosure recognizes that monoclonal antibodies are commonly used for the treatment of lymphomas, but their use in leukemias is more limited. Gemtuzumab ozogamicin, ) is a monoclonal antibody that is attached to a cytotoxin. Gemtuzumab ozogamicin was previously approved for the treatment of AML in older patients, but was withdrawn from the market after studies found that the product was associated with some toxicities (PMLIVE press release "ASH: Pfizer eyes re-launch of Mylotarg" December 10, 2010). Over the past decade, other monoclonal therapeutic antibodies have shown adverse effects (Klastersky, J. (2006) "Adverse effects of the humanized antibodies used as cancer therapeutics" Current Opinion in Oncology. 18(4):316-320).

[0166] The present disclosure recognizes a novel approach to target CCL-1 using immune cells with chimeric antigen receptors based on the specificity of anti-CLL-1 monoclonal antibodies that redirect the specificity of immune cells to CLL-1 positive cells. In addition, the present disclosure recognizes that engineered immune cells obtained using this approach have been shown to have efficacy in eliminating CLL-1 positive malignant cells.

[0167] The present disclosure opens the way for methods of using adoptive immunotherapy to treat patients with a condition characterized by an excess of CLL-1 expressing cells. In addition, the present invention provides engineered allogeneic immune cells that can be used as "off-the-shelf" allogeneic therapeutic products.

[0168] The practice of some of the methods disclosed herein employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th ed. (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel et al. eds.); the series Methods in Enzymology (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 Animal Cell Culture: Basic and Applied Aspects, 6th Ed. (R.I. Freshney ed. (2010)).

[0169] Certain Definitions

[0170] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "antigen binding domain" includes multiple antigen binding domains.

[0171] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend 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 than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean ranges approximately 20% above and below a given value, approximately 10%, approximately 5%, or approximately 1%. Alternatively, especially 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. Where particular values are described in the application and claims, unless otherwise stated the term "about" means within an acceptable error range for the particular value.

[0172] As used herein, "cell" can generally refer to a biological cell. A cell can be a basic structural, functional, and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-celled eukaryotic organism, a protozoan cell, a cell from a plant (e.g., from a plant crop, fruit, vegetable, grain, soybean, maize, wheat, seed, tomato, rice, cassava, sugarcane, pumpkin, hay, potato, cotton, hemp, tobacco, flowering plant, conifer, gymnosperm, fern, lycopodium, duckweed, liverwort, moss, algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweed (e.g., kelp), fungal cell (e.g., yeast cell, cell from a mushroom), animal cell, cell from an invertebrate (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), cell from a vertebrate (e.g., fish, amphibian, reptile, bird, mammal), cell from a mammal (e.g., pig, cow, goat, sheep, rodent, rat, mouse, non-human primate, human, etc.), etc. Sometimes a cell is not derived from a natural organism (e.g., a cell can be synthetic, sometimes referred to as an artificial cell).

[0173] As used herein, the term“activate” and grammatical equivalents thereof can refer to the process by which a cell transitions from a quiescent state to an active state. This process can include a response to an antigen, migration, and / or a phenotypic or genetic change to a functionally active state. For example, the term“activate” can refer to the stepwise process of T cell activation. In some cases, T cells require at least two signals to fully activate.

[0174] As used herein, the term“antigen” refers to a molecule or fragment thereof that is capable of being bound by a selective binding agent. As an example, an antigen can be a ligand that is capable of being bound by a selective binding agent, such as a receptor. In some cases, a receptor can act as an antigen, and a ligand can act as a selective binding agent. As another example, an antigen can be an antigenic molecule that is capable of being bound by a selective binding agent, such as an immunological protein (e.g., an antibody). In some cases, an immunological protein can act as an antigen, and an antigenic molecule can act as a selective binding agent. An antigen can also refer to a molecule or fragment thereof that is capable of being used in an animal to generate an antibody that is capable of binding to the antigen.

[0175] As used herein, the term“epitope” and grammatical equivalents thereof can refer to a portion of an antigen that can be recognized by an antigen binding domain. An antigen binding domain can comprise, for example, a protein (e.g., an antibody, an 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 an engineered cell)).

[0176] As used herein, the term“antibody” refers to a protein binding molecule having immunoglobulin-like function. The term antibody includes antibodies (e.g., monoclonal antibodies and polyclonal antibodies) as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of various classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (such as IgGl, IgG2, etc.). A derivative, variant, or fragment thereof can refer to a functional derivative or fragment that retains (e.g., fully and / or partially) the binding specificity of the corresponding antibody. Antigen binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibody, diabody, and single-domain antibody (“sdAb” or“nanobody” or“camelid”). 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 (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0177] As used herein, the term "antigen binding domain" refers to a protein or fragment thereof that is capable of binding an antigen or epitope. As an example, an antigen binding domain can be a cellular receptor. As an example, an antigen binding domain can be an engineered cellular receptor. As an example, an antigen binding domain can be a soluble receptor. In some cases, an antigen binding domain can be a ligand that binds to a cellular receptor, an engineered cellular receptor, and / or a soluble receptor.

[0178] As used herein, the term "autologous" and its grammatical equivalents can refer to something that is derived from the same existence. For example, an autologous sample (e.g., a cell) can refer to a sample that is removed, processed, and then returned to the same subject (e.g., a patient) at a later time. In terms of process, autologous can be distinguished from allogeneic processes in which the donor of a sample (e.g., a cell) and the recipient of the sample are not the same subject.

[0179] As used herein, the terms "cancer neoantigen," "neoantigen," and "neoepitope," and their grammatical equivalents, can refer to an antigen that is not encoded in a normal, non-mutated host genome. In some cases, a "neoantigen" can represent a cancer-causing viral protein or an abnormal protein that results from a somatic mutation. For example, a neoantigen can arise from the disruption of cellular mechanisms by the activity of a viral protein. As another example, a neoantigen can arise from exposure to a cancer-causing compound, which in some cases can result in a somatic mutation. Such somatic mutations can result in the formation of a tumor / cancer.

[0180] As used herein, the term "cytotoxicity" refers to an unintended or undesirable alteration from a normal state of a cell. The normal state of a cell can refer to a state exhibited or present prior to exposure of the cell to a cytotoxic composition, agent, and / or condition. A cell in a normal state can be in homeostasis. The unintended or undesirable alteration from a normal state of a cell can manifest as, for example, cell death (e.g., programmed cell death), reduced replicative potential, reduced cellular integrity (such as membrane integrity), reduced metabolic activity, reduced developmental capacity, or in the form of any cytotoxic effect disclosed herein.

[0181] As used herein, the phrases "reducing cytotoxicity" and "reduce cytotoxicity" refer to a reduction in the degree or frequency of an unintended or undesirable alteration from a normal state of a cell upon exposure to a cytotoxic composition, agent, and / or condition. The phrases can refer to a reduction in the degree of cytotoxicity of an individual cell exposed to a cytotoxic composition, agent, and / or condition, or to a reduction in the number of cells of a population that exhibit cytotoxicity when a population of cells is exposed to a cytotoxic composition, agent, and / or condition.

[0182] The term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as to an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0183] When the terms "derivative," "variant," and "fragment" are used herein in reference to a polypeptide, they refer to a polypeptide that is related to a wild-type polypeptide, e.g., by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0184] As used herein, the term "percent (%) identity" means the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of a candidate sequence and a reference sequence to achieve the best alignment, and non-identical sequences can be disregarded for comparison purposes). For purposes of determining percent identity, the comparison

[0185] 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, sport animals, and pets. Also encompassed are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0186] As used herein, the terms“treatment” and“treating” refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. For example, treatment can include administration of a system or population of cells disclosed herein. Therapeutic benefit can refer to any therapeutic-related improvement or effect on one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom can not yet be present.

[0187] A“treatment effect” can occur if the condition being treated changes. The 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 the amount or size of a tumor in a subject. A“change” in the condition being treated can refer to a change of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100% in the condition. The change can be based on an improvement in the severity of the condition being treated in an individual, or based on a difference in the frequency of improvement in the condition in a population of individuals administered and not administered the therapy. Similarly, the methods of the disclosure can include administering a“therapeutically effective” amount of cells to a subject. The term“therapeutically effective” is understood to have a definition corresponding to“having a treatment effect”.

[0188] The term“effective amount” or“therapeutically effective amount” refers to the amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), which is sufficient to effect a desired activity after administration to a subject in need thereof. The term“therapeutically effective” can refer to an amount of a composition that is sufficient to delay the manifestation, halt the progression, relieve, or lessen at least one symptom of a disorder being treated by the methods of the disclosure.

[0189] As used herein, the term“TIL” or tumor infiltrating lymphocyte, and grammatical equivalents thereof, can refer to a cell isolated from a tumor. A TIL can be any cell found within a tumor. For example, a TIL can be a cell that has migrated to a tumor. A TIL can be a cell that has infiltrated a tumor. A TIL can be a T cell, a B cell, a monocyte, a natural killer (NK) cell, or any combination thereof. A TIL can be a mixed population of cells. A population of TILs can comprise cells of different phenotypes, cells of different degrees of differentiation, cells of different lineages, or any combination thereof.

[0190] CLL1 single-chain specific chimeric antigen receptor

[0191] The present invention relates to CLL1 specific chimeric antigen receptors comprising an extracellular ligand binding domain specific for a portion of the CLL1 antigen, a transmembrane domain and a signal transduction domain.

[0192] Chimeric antigen receptors (CARs) are molecules that combine an extracellular binding domain directed against a component present on a target cell (e.g., antibody-based specificity directed against a desired antigen (e.g., a tumor antigen)) with immune cell receptor components to produce a chimeric protein that transduces an activation or inhibitory signal to the cellular immune activity.

[0193] The present invention more particularly relates to CLL-1 specific chimeric antigen receptors (anti-CLL-1 CARs) comprising at least: an extracellular anti-CLL-1 antigen binding domain, a transmembrane domain and a cytoplasmic / intracellular / intracellular signaling domain.

[0194] Preferably, the CLL-1 specific chimeric antigen receptors according to the present invention further comprise a costimulatory domain, and more preferably a CD28 or 4-1BB costimulatory domain, as described for example in Jena, B., G. Dotti et al. (2010). It can also comprise a transmembrane domain which can be a Cd8a transmembrane domain and optionally a hinge.

[0195] The signal transduction domain or "cytoplasmic / intracellular signaling domain" of the CAR according to the present invention is responsible for intracellular signaling upon binding of the extracellular ligand binding domain to the target, resulting in the activation or inhibition of the immune cell and the immune response. In other words, the signal transduction domain is responsible for the activation or inactivation of at least one normal effector function of the immune cell expressing the CAR. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "cytoplasmic / intracellular signaling domain" refers to the portion of a protein that transduces an effector signal function signal and directs the cell to perform a specialized function.

[0196] The cytoplasmic / intracellular signaling domain from a human protein involved in a signal transduction pathway determines whether the anti-CLL-1 CAR is a positive CAR (PCAR) or a negative CAR (NCAR) depending on the nature of the signaling. Respectively, a CAR-T is a PCAR when the signaling domain (such as CD3 zeta from human TCR receptor) has the effect of stimulating the cellular immune activity of the immune cell when the extracellular ligand-binding domain binds to CLL-1. Conversely, the anti-CLL-1 CAR is a NCAR or an inhibitory CAR (iCAR) when the signaling domain (such as the signaling domain of human immunoinhibitory receptors CTLA-4 and PD-1) has the effect of reducing the cellular immune activity (Federov et al., Sci Transl Med. 2013 Dec. 11; 5(215): 215ra172). Preferred examples of signal transduction domains for the anti-CLL1 CAR can be the cytoplasmic / intracellular sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence having the same functional ability. The signal transduction domain comprises two different categories of cytoplasmic / intracellular signaling sequences, one that initiates antigen-dependent primary activation and the other that provides a secondary or costimulatory signal in an antigen-independent manner. The primary cytoplasmic / intracellular signaling sequences can comprise a signaling motif known as the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are well-defined signaling motifs that occur in the intracytoplasmic / intracellular tails of various receptors as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention can include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d as non-limiting examples. In a preferred embodiment, the signaling domain of the anti-CLL1 CAR can include a CD3 zeta signaling domain having an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the signaling domain in the amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0197] Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for effective immune responses. A "co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including but not limited to proliferative activation, differentiation, etc., in addition to the primary signal provided by, for example, TCR / CD3 complex binding to MHC molecules loaded with peptide. Co-stimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3. Co-stimulatory ligands also encompass, among others, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. A "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, among others.

[0198] In preferred embodiments, the co-stimulatory domain of the anti-CLL1 CAR of the application comprises a portion of a co-stimulatory signaling molecule selected from the group consisting of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1) fragments. Specifically, the signaling domain of the anti-CLL1 CAR of the application comprises an amino acid sequence comprising at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to a co-stimulatory domain in an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0199] The anti-CLL1 CAR according to the application generally further comprises a transmembrane domain (TM). Notable features of a suitable transmembrane domain include the ability to be expressed on the surface of a cell, preferably an immune cell, in particular a lymphocyte or natural killer (NK) cell, in the present application, and the ability to interact to direct a cellular response of the immune cell against a predetermined target cell. The transmembrane domain can be derived from a natural source or a synthetic source. The transmembrane domain can be derived from any membrane-bound protein or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of a T cell receptor (such as alpha, beta, gamma or zeta), a polypeptide constituting a CD3 complex, IL2 receptor p55 (alpha chain), p75 ((beta chain) or gamma chain, a subunit chain of an Fc receptor, in particular Fc gamma receptor III or a CD protein. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from the human CD8 alpha chain (e.g. NP_001139345.1). The transmembrane domain can further comprise a hinge region between the extracellular ligand binding domain and the transmembrane domain.

[0200] The term "hinge region" as used herein generally means any oligo- or polypeptide that serves to link the transmembrane domain with the extracellular ligand binding domain. In particular, the hinge region serves to provide more flexibility and accessibility to the extracellular ligand binding domain. The hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region can be derived from all or part of a naturally occurring molecule, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. In a preferred embodiment, the hinge domain comprises a hinge polypeptide exhibiting preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with a hinge domain in an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26. According to one embodiment, the hinge can also be a human Ig (immunoglobulin) hinge, e.g. a PD-1 hinge, an IgG4 hinge.

[0201] According to a preferred embodiment, the anti-CLL-1 CAR according to the application comprises a transmembrane domain more particularly selected from CD8 and / or CD28.

[0202] The anti-CLL-1 CAR according to the application generally further comprises a transmembrane domain (TM), more particularly a TM derived from CD8 and / or CD28, and even more particularly exhibiting identity with the polypeptide of SEQ ID NO. 6 or 7.

[0203] In a preferred embodiment, the anti-CLL-1 CAR according to the application further comprises a TM domain showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the TM domain in an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0204] Downregulation or mutation of the target antigen is often observed in cancer cells, resulting in antigen-loss escape variants. Therefore, in order to counteract tumor escape and to make the immune cell more specific for the target, the CLL-1 specific anti-CLL-1 CAR according to the application can comprise another extracellular ligand binding domain to simultaneously bind different elements in the target, thereby enhancing activation and function of the immune cell. In one embodiment, the extracellular ligand binding domains can be placed in tandem on the same transmembrane polypeptide and, optionally, can be separated by a linker. In another embodiment, the different extracellular ligand binding domains can be placed on different transmembrane polypeptides that make up the anti-CLL-1 CAR. In another embodiment, the present application relates to a population of anti-CLL-1 CARs comprising each a different extracellular ligand binding domain. Specifically, the present application relates to a method of engineering an immune cell comprising providing an immune cell and expressing on the surface of said cell a population of anti-CLL-1 CARs, each of said anti-CLL-1 CARs comprising a different extracellular ligand binding domain. In another specific embodiment, the present application relates to a method of engineering an immune cell comprising providing an immune cell and introducing into said cell a polynucleotide encoding a polypeptide that makes up a population of anti-CLL-1 CARs, each of said anti-CLL-1 CARs comprising a different extracellular ligand binding domain. A population of anti-CLL-1 CARs means at least two, three, four, five, six or more anti-CLL-1 CARs, each of said anti-CLL-1 CARs comprising a different extracellular ligand binding domain. The different extracellular ligand binding domains according to the application can preferably simultaneously bind different elements in the target, thereby enhancing activation and function of the immune cell. The present application also relates to an isolated immune cell comprising a population of anti-CLL-1 CARs, each of said anti-CLL-1 CARs comprising a different extracellular ligand binding domain.

[0205] The CLL-1 specific chimeric antigen receptors according to the present application can have different architectures, as they can be expressed, for example, in the form of single chain chimeric proteins (scCARs) or in the form of several polypeptides (multi-chain) comprising at least one such chimeric protein. Such multi-chain CAR architectures are disclosed in WO2014 / 039523, which is incorporated herein by reference.

[0206] The present application discloses several anti-CLL-1 single chain CARs against CLL-1 antigen comprising as non-limiting examples the following amino acid sequences: SEQ ID NOs: 20-26.

[0207] The CLL-1 CAR of the application can also be a "multi-chain CAR" as mentioned previously, which means that the extracellular binding domain and the signaling domain are preferably located on different polypeptide chains, while the costimulatory domain can be located on the same or a third polypeptide. Such multi-chain CARs can be derived from FcεRI (Ravetch et al., 1989) by replacing the high affinity IgE binding domain of the FcεRI alpha chain with an extracellular ligand binding domain such as a scFv, while the N and / or C-terminal tails of the FcεRI beta and / or gamma chains are fused to the signal transduction domain and the costimulatory domain, respectively. The extracellular ligand binding domain has the effect of redirecting T cell specificity to a cellular target, while the signal transduction domain either activates or reduces the immune cell response. Indeed, the different polypeptides derived from the alpha, beta and gamma polypeptides of FcεRI are transmembrane polypeptides located in a juxtamembrane position, which provides a more flexible architecture to the CAR, increases the specificity to the targeted molecule and reduces the background activation of the immune cell, as described in WO2014 / 039523.

[0208] Extracellular antigen binding domain

[0209] As used herein, the term "extracellular antigen binding domain" is defined as an oligo- or polypeptide capable of binding a ligand. Preferably, the domain is capable of interacting with a cell surface molecule. For example, the extracellular ligand binding domain can be selected to recognize a ligand that is a cell surface marker on a target cell associated with a particular disease state. For example, it can be a binding domain derived from a ligand, a receptor, a human or mouse antibody, or an antigen recognition domain derived from a camelid or cartilaginous fish.

[0210] In a preferred embodiment, the extracellular ligand binding domain comprises a single chain antibody fragment (scFv) comprising the light (VL) and heavy (VH) variable fragments of a target antigen specific monoclonal anti-CLL-1 antibody linked by a flexible linker. The VL and / or VH preferably show at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the VH and VL domains of the amino acid sequences of SEQ ID NOs. 20 to 26.

[0211] As used herein, the term "recombinant antibody" means an antibody or antibody fragment produced using recombinant DNA technology, such as, for example, an antibody or antibody fragment expressed by a bacteriophage, a yeast expression system, or a mammalian cell expression system. The term shall also be construed to mean an antibody or antibody fragment produced by a synthetic DNA molecule encoding the antibody or antibody fragment and expressing the antibody or antibody fragment protein, or the amino acid sequence specifying the antibody or antibody fragment, wherein the DNA or amino acid sequence is obtained using recombinant or synthetic DNA or amino acid sequence technology available and well known in the art.

[0212] Disclosed herein are CLL-1 specific single chain chimeric antigen receptors (anti- CLL-1 scCARs) as described above, wherein the extracellular ligand binding domain comprises VH and VL chains, which in some embodiments are humanized.

[0213] As used herein, the term "human / humanized antibody" means that the polypeptide comprises a human / humanized heavy chain variable region and a human / humanized light chain variable region. For example, the polypeptide can comprise the framework (FR) regions of a human antibody light and heavy chain variable region 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 is 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, except for the complementarity determining regions (CDRs). The antigen binding polypeptide molecule can be derived from a monoclonal antibody donor (e.g., a mouse monoclonal antibody donor) and can include CDRs from the monoclonal antibody (e.g., mouse monoclonal CDRs).

[0214] As used herein, the term "monoclonal antibody" means an antibody produced by a clone of cells grown in the laboratory, either a hybridoma or a virus-transformed lymphocyte, which is more homogeneous and abundant than natural antibodies and is able to bind specifically to a single site on the CLL1 antigen. They are monospecific antibodies produced by identical immune cells, all clones of a unique parent cell, in contrast to polyclonal antibodies, which are produced by several different immune cells. Monoclonal antibodies have monovalent affinity because they bind to the same epitope. The methods currently applied for humanization are according to the methods of Lefranc M P et al. (Lefranc, MP, Ehrenmann F, Ginestoux C, Giudicelli V, Duroux P "Use of The four alignments have been indicated.

[0215] 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. EP 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. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91 :969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Patent No. 5,565,332, incorporated herein by reference in its entirety), and techniques disclosed, e.g., in U.S. Patent Application Publication No. US 2005 / 0042664, U.S. Patent Application Publication No. US 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. 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., 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 J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Generally, framework residues in the framework regions will be substituted with corresponding residues from the CDR donor antibody to alter (e.g., improve) antigen binding. These framework substitutions can be identified, for example, by modeling of the interactions of CDR and framework residues to identify framework residues contributing to antigen binding, and by sequence comparison to identify unusual framework residues at particular positions.(see, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated by reference herein in their entireties).

[0216] Conservative amino acid substitutions are those substitutions of amino acid residues that have similar side chains. Families of amino acid residues having 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), beta-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 present application can be replaced with other amino acid residues from the same side chain family, and the altered anti-CLL-1 CAR can be tested for its ability to bind CLL-1 using the functional assays described herein.

[0217] In preferred embodiments, the present application discloses an anti-CLL-1 specific single chain chimeric antigen receptor (“anti-CLL-1 scCAR” or “scCAR”) having a polypeptide structure selected from the group consisting of SEQ ID NO. 20-26, or a polypeptide structure showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO. 20 to 26 (SEQ ID NO. 20-26). In some embodiments, the structure comprises an extracellular antigen binding domain comprising a VH and a VL from a monoclonal anti-CLL-1 antibody, a hinge, a transmembrane domain, a cytoplasmic / intracellular domain comprising a signaling domain and a costimulatory domain.

[0218] Polynucleotides and vectors

[0219] The present application also relates to polynucleotides and vectors that allow for the heterologous expression of an anti-CLL-1 CAR according to the present application into a cell, which encode the polypeptide sequences previously detailed in great detail.

[0220] The polynucleotides can be comprised in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or 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).

[0221] In particular embodiments, different nucleic acid sequences can be contained in one polynucleotide or vector, which comprises a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. 2A peptides identified in the foot-and-mouth disease virus subgroup of picornaviruses cause 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 and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). By "codon" is meant the three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, when a polypeptide is separated by an in frame 2A oligopeptide sequence, two polypeptides can be synthesized from a single continuous open reading frame within the mRNA. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0222] To direct a transmembrane polypeptide into the secretory pathway of a host cell, a secretion signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide sequence or vector sequence. The secretion 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. The secretion signal sequence is typically located 5' of the nucleic acid sequence encoding the polypeptide of interest, although certain secretion signal sequences can be located elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). In preferred embodiments, the CAR polypeptide is derived from the nucleic acid sequence 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.

[0223] Those skilled in the art will recognize that there can be considerable sequence variation between these polynucleotide molecules in view of the degeneracy of the genetic code. Preferably, the nucleic acid sequences of the application are codon-optimized for expression in mammalian cells, preferably in human cells. Codon-optimization refers to the exchange of codons in a sequence of interest that are generally rare in highly expressed genes of a given species for codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acid as the exchanged codon.

[0224] Delivery methods

[0225] The present invention encompasses different means of expressing the anti-CLL-1 chimeric antigen receptors (CARs) described herein in immune cells.

[0226] 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 viral-mediated methods.

[0227] The polynucleotide can be introduced into the cell by, for example, a recombinant viral vector (e.g., retrovirus, adenovirus), a liposome, or the like. For example, transient transformation methods include, for example, microinjection, electroporation or particle bombardment, cell fusion. In view of expression in the cell, the polynucleotide can be included in a vector, more particularly a plasmid or virus. The plasmid vector can comprise a selectable marker, which provides for the identification and / or selection of cells that have received the vector.

[0228] Different transgenes can be included in one vector. The vector can comprise a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. The 2A peptide identified in the foot-and-mouth disease virus 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)).

[0229] "Codon" means the three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, when a polypeptide is separated by an in-frame 2A oligopeptide sequence, two polypeptides can be synthesized from a single continuous open reading frame within the mRNA. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for expressing several proteins encoded by a single messenger RNA.

[0230] In a more preferred embodiment of the application, the polynucleotide encoding the polypeptide according to the application can be an mRNA introduced directly into the cell, for example by electroporation. The present inventors determined the optimal conditions for mRNA electroporation in T cells. The present inventors used the cytoPulse technology which allows transient permeabilization of living cells by using pulsed electric fields to deliver material into cells. The technology based on the use of PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, Mass. 01746, USA) electroporation waveforms allows precise control of pulse duration, intensity and pulse interval (US Patent No. 6,010,613 and International PCT Application WO2004083379). All these parameters can be modified to reach optimal conditions, achieving high transfection efficiency and minimal mortality. Basically, the first high electric field pulse allows pore formation, while the subsequent low electric field pulses allow the movement of the polynucleotide into the cell.

[0231] The different methods described above involve the introduction of the scCAR into a cell. As a non-limiting example, the scCAR can be introduced as a transgene encoded by one plasmid vector. The plasmid vector can also contain a selection marker which provides for the identification and / or selection of cells that have received the vector.

[0232] As a result of the introduction of the polynucleotide encoding the polypeptide into the cell, the polypeptide can be synthesized in situ in the cell. Alternatively, the polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art and include, as non-limiting examples, stable transformation methods in which the polynucleotide construct 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. The polynucleotide can be introduced into the cell by, for example, a recombinant viral vector (e.g. retrovirus, adenovirus), a liposome, etc. For example, transient transformation methods include, for example, microinjection, electroporation or particle bombardment. In view of the expression in the cell, the polynucleotide can be included in a vector, more particularly a plasmid or a virus.

[0233] Activation and expansion of T cells

[0234] Whether before or after genetic modification of the T cells, even if the genetically modified immune cells of the present invention are activated and proliferated independently of the antigen binding mechanism, the immune cells of the present invention, particularly T cells, can be further activated and expanded using generally described methods, such as, for example, those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0235] In general, the T cells of the present invention are expanded by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to generate activation signals for the T cells. For example, chemical substances such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or mitogen lectins (such as phytohemagglutinin (PHA)) can be used to generate activation signals for T cells.

[0236] As non-limiting examples, a population of T cells can be stimulated in vitro, such as by contacting anti-CD3 antibodies or antigen-binding fragments thereof or anti-CD2 antibodies immobilized on a surface, or by contacting a protein kinase C activator (e.g., bryostatin) conjugated to a calcium ionophore. To co-stimulate the T cell surface for accessory molecules, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Conditions suitable for T cell culture include appropriate media (e.g., Minimal Essential Media or RPMI media 1640 or X-vivo 5, (Lonza)) which can contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, IL-21, and TNF- or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma salts, and reducing agents such as N-acetyl cysteine and 2-mercaptoethanol. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer with added amino acids, sodium pyruvate, and vitamins, can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokines sufficient for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures to be infused into a subject. Target cells are maintained under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2). T cells exposed to different stimulation times can exhibit different characteristics.

[0237] In another particular embodiment, the cells can be expanded by co-culture with a tissue or cells. The cells can also be expanded in vivo, e.g., in the blood of a subject after administration of the cells to the subject.

[0238] Engineered immune cells

[0239] According to the present application, "cell" generally refers to a cell of hematopoietic origin that functionally participates in initiating and / or executing an innate and / or adaptive immune response. The cell according to the present application is preferably an isolated immune cell, and more preferably a T cell obtained from a donor. The immune cell according to the present application can also be derived from a stem cell. The stem cell can be an adult stem cell, a non-human embryonic stem cell, more particularly a non-human stem cell, a 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 can also be a dendritic cell, a killer dendritic cell, a mast cell, an 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 can 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 application, the cell source can be obtained from a subject by various non-limiting methods. The cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present application, any number of T cell lines available and known to those skilled in the art can be used.

[0240] In another embodiment, the cell can be derived from a healthy donor, a patient diagnosed with cancer or a patient diagnosed with an infection. In another embodiment, the cell is part of a mixed cell population exhibiting different phenotypic characteristics. Cell lines obtained from the transformed T cells according to the methods described previously are also encompassed within the scope of the present application. Modified cells resistant to immunosuppressive therapy and susceptible to be obtained by the previous methods are encompassed within the scope of the present application.

[0241] As a preferred embodiment, the present application provides a T cell or a population of primary T cells having a CLL-1 CAR as described above, which do not express a functional TCR and are reactive against CLL-1 positive cells for their allogeneic transplantation into a patient.

[0242] As a more preferred embodiment, the present application provides T cells or a population of T cells having a CLL-1 scCAR and being reactive against CLL-1 positive cells as described above, which do not express a functional TCR and are resistant to a selected drug, for their allogeneic transplantation into a patient treated with said selected drug. The present application encompasses a method of preparing engineered immune cells for immunotherapy, which comprises introducing a polynucleotide or a vector encoding a CLL-1 CAR into said immune cells ex vivo according to the transformation method as described in WO2014 / 130635, WO2013176916, WO2013176915 and incorporated by reference and into the present document.

[0243] In a preferred embodiment, the polynucleotide is introduced into the immune cell by means of a retroviral vector in view of its stable integration into the genome of the cell.

[0244] Method of engineering immune cells with a CAR

[0245] The present application also aims at generating immune cells with an anti-CLL-1 CAR, which have a lower or no allogeneic reactivity, which can be used for allogeneic treatments (i.e. reduce the risk of inducing a graft versus host reaction) and / or which are resistant to various standard of care treatments.

[0246] As further described in the present specification, the method can further comprise the step of genetically modifying the immune cell by using at least one endonuclease.

[0247] The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Regardless of its sequence, an endonuclease does not cleave a DNA or RNA molecule, but recognizes and cleaves a DNA or RNA molecule at a specific polynucleotide sequence, further referred to as "target sequence" or "target site". An endonuclease can be classified as a rare-cutting endonuclease when it typically has a polynucleotide recognition site of more than 12 base pairs (bp), more preferably of 14-55 bp in length.

[0248] Preferably, the method according to the application involves a rare-cutting endonuclease. Rare-cutting endonucleases can be, for example, homing endonucleases (Paques and Duchateau 2007), chimeric zinc finger nucleases (ZFN) resulting from the fusion of engineered zinc finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus and Carroll 2005), TALE nucleases, Cas9 endonucleases from the CRISPR system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) as described below, or chemical endonucleases (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptidic cleaving agent is conjugated to a nucleic acid polymer or to another DNA recognizing a specific target sequence, thereby targeting the cleaving activity to a specific sequence. Chemical endonucleases also encompass synthetic nucleases such as conjugates of orthophenanthroline, DNA cleaving molecules and triple-stranded forming oligonucleotides (TFOs) known to bind to specific DNA sequences (Kalish and Glazer 2005). Rare-cutting endonucleases can be used to inactivate a gene at a locus or to integrate a transgene by homologous recombination (HR), i.e. by inducing a DNA double-strand break (DSB) at a locus and inserting foreign DNA at this locus by gene repair mechanisms (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007).

[0249] A "TALE-nuclease" (TALEN) is a fusion protein consisting of a nucleic acid binding domain, usually derived from a Transcription Activator-Like Effector (TALE), and a nuclease catalytic domain for cleaving a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain with endonuclease activity, such as e.g. I-Tevl, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease, such as e.g. I-Crel and 1-Onul or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE-nuclease. A monomeric TALE-nuclease is a TALE-nuclease that does not require dimerization for specific recognition and cleavage, such as the fusion of an engineered TAL repeat sequence to the catalytic domain of I-Tevl described in WO2012138927. Transcription Activator-Like Effector (TALE) is a protein from the bacterial species Xanthomonas, comprising multiple repeat sequences, each repeat sequence comprising a double-residue (RVD) at positions 12 and 13 that is specific for each nucleotide base of a nucleic acid target sequence. Binding domains with similar modular base-pair base nucleic acid binding properties (MBBBD) can also be derived from novel modular proteins recently discovered by the present applicant in different bacterial species. The advantage of the new modular proteins is that they exhibit more sequence variability than TAL repeat sequences. Preferably, the RVDs associated with recognition of different nucleotides are HD for recognition of C, NG for recognition of T, NI for recognition of A, NN for recognition of G or A, NS for recognition of A, C, G or T, HG for recognition of T, IG for recognition of T, NK for recognition of G, HA for recognition of C, ND for recognition of C, HI for recognition of C, HN for recognition of G, NA for recognition of G, SN for recognition of G or A and YG for recognition of T, TL for recognition of A, VT for recognition of A or G and SW for recognition of A. In another embodiment, the key amino acids 12 and 13 can be mutated to other amino acid residues to modulate their specificity for the nucleotides A, T, C and G, in particular to enhance this specificity. TALE-nucleases have been described and used to stimulate gene targeting and genetic modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Engineered TAL-nucleases are commercially available under the trade name TALEN TM(Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France) and can be ordered from manufacturers such as Life Technologies (Carlsbad, Calif., USA).

[0250] Preferred TALE-nucleases recognizing and cleaving a target sequence are described in PCT / EP2014 / 075317. Specifically, an additional catalytic domain can be further introduced into a cell having said rare-cutting endonuclease to increase mutagenesis and thereby enhance its ability to inactivate a target gene. More specifically, said 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 hExol (EXOl_HUMAN), yeast Exol (EXOl_YEAST), E. coli Exol, human TREX2, mouse TREX1, human TREX1, bovine TREX1, rat TREX1, TdT (terminal deoxynucleotidyl transferase), human DNA2, yeast DNA2 (DNA2_YEAST). In a preferred embodiment, said additional catalytic domain has 3'-5'-exonuclease activity and in a more preferred embodiment, said additional catalytic domain is TREX, more preferably a TREX2 catalytic domain (WO2012 / 058458). In another preferred embodiment, said catalytic domain is encoded by a single-chain TREX2 polypeptide. Said additional catalytic domain can optionally be fused to the nuclease fusion protein or chimeric protein according to the application by a peptide linker.

[0251] By "Cas9 endonuclease" is meant any genome engineering tool developed based on the RNA-guided Cas9 nuclease from the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeat) adaptive immune system (see review (Sorek, Lawrence et al. 2013)). The CRISPR-associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA (virus or plasmid). CRISPR-mediated genome engineering first proceeds by selection of a target sequence, usually flanked by short sequence motifs called protospacer adjacent motifs (PAM). After selection of a target sequence, a specific crRNA complementary to this target sequence is engineered. A trans-activating crRNA (tracrRNA) required for the CRISPR type II system pairs with the crRNA and binds to the provided Cas9 protein. Cas9 serves as a molecular anchor, facilitating base pairing of the tracRNA to the cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure serves as a guide RNA to direct the endonuclease Cas9 to a homologous target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence to determine homology between the target sequence and the crRNA. In addition to target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif - PAM) upstream of the PAM motif. After the dual RNA pairs with the target sequence, Cas9 subsequently introduces a blunt double-strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010). The use of Cas9 in immune cells, in particular T cells, has been previously described in WO2014191128.

[0252] In preferred embodiments, the method of further engineering the immune cell involves introducing into the T cell a polynucleotide, in particular an mRNA, encoding a specific rare-cutting endonuclease to selectively inactivate the above-mentioned genes by DNA cleavage. In more preferred embodiments, the rare-cutting endonuclease is a TALE-nuclease or a Cas9 endonuclease. To date, TAL-nucleases have been shown to have higher specificity and cleavage efficiency than other types of rare-cutting endonucleases, making them the nucleases of choice for large-scale production of engineered immune cells and constant turnover.

[0253] Therapeutic applications

[0254] In another embodiment, the isolated cells obtained by the different methods as previously described or cell lines derived from said isolated cells can be used as a medicament.

[0255] In another embodiment, the medicament can be used for the treatment of cancer, in particular for the treatment of leukemia in a patient in need thereof.

[0256] In another embodiment, the isolated cells according to the application or cell lines derived from said isolated cells can be used for the manufacture of a medicament for the treatment of cancer in a patient in need thereof.

[0257] In a particular embodiment, anti-CLL-1 CAR-expressing T cells are provided as a medicament for the treatment of AML, AML subtypes, AML-related complications, AML-related conditions.

[0258] In another embodiment, the medicament can be used for the treatment of a pathological condition mediated by CLL-1 expressing cells or a condition characterized by direct or indirect activity of CLL1 expressing cells.

[0259] In another aspect, the application relies on a method for treating a patient in need thereof, said method comprising at least one of the following steps:

[0260] (a) providing immune cells obtainable by any one of the methods previously described;

[0261] (b) administering said transformed immune cells to said patient,

[0262] In one embodiment, the T cells of the application can undergo robust in vivo T cell expansion and can be sustained for an extended amount of time.

[0263] The treatment can be ameliorative, curative or prophylactic. It can be part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines or cell populations used to treat a patient are derived from said patient or from a donor that is human leukocyte antigen (HLA) compatible. Allogeneic means that the cells or cell populations used to treat a patient are not derived from said patient but from a donor.

[0264] The previous section describes cells that can be used with the disclosed methods. The treatment can be used to treat a patient diagnosed with a pre-malignant or malignant cancer condition characterized by CLL-1 expressing cells, in particular an excess of CLL-1 expressing cells. Such conditions are found in hematological cancers, such as leukemia.

[0265] In one embodiment, the present application provides a composition for use in the treatment of a CLL-1 expressing cell mediated disease, in particular a CLL-1 expressing cell mediated hematologic cancer, comprising the anti-CLL-1 scCAR expressing T cells of the present application.

[0266] Any other CLL-1 mediated or involving CLL-1 malignant lymphoproliferative disorder disclosed herein can be ameliorated with the anti-CLL-1 CAR expressing cells of the present application.

[0267] In preferred embodiments, the cancer that can be treated using the anti-CLL-1 CAR expressing cells of the present application is a leukemia, a disease associated with leukemia or a complication thereof.

[0268] AML

[0269] The leukemia that can be treated using the anti-CLL-1 CAR expressing cells of the present application can be acute myeloid leukemia (AML). The AML or AML subtype that can be treated using the anti-CLL-1 scCAR expressing cells of the present application can in particular be acute myeloblasts leukemia, minimally differentiated acute myeloblasts leukemia, immature acute myeloblasts leukemia, acute myeloblasts leukemia with granulocyte maturation, promyelocytic or acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, myelomonocytic with bone marrow eosinophilia, acute monocytic leukemia (M5a) or acute monocytic leukemia (M5b), acute erythroid leukemia (including erythroleukemia (M6a) and very rare pure erythroid leukemia (M6b)), acute megakaryocytic leukemia, acute basophilic leukemia, acute panmyelosis with myelofibrosis, whether or not involving CLL-1 positive cells.

[0270] Subtypes of AML also include hairy cell leukemia, philadelphia chromosome-positive acute lymphoblastic leukemi. AML can be classified as AML with specific genetic abnormalities. Classification is based on karyotype to predict response to induction therapy, risk of relapse, ability to survive.

[0271] Thus, the AML that can be treated using the anti-CLL-1 CAR expressing cells of the present application can be AML with translocation between chromosomes 8 and 21, AML with translocation or inversion in chromosome 16, AML with translocation between chromosomes 9 and 11, APL (M3) with translocation between chromosomes 15 and 17, AML with translocation between chromosomes 6 and 9, AML with translocation or inversion in chromosome 3, AML with translocation between chromosomes 1 and 22 (megakaryoblastic).

[0272] The present application is particularly useful for treating AML associated with these specific cytogenetic markers.

[0273] The present application also provides an anti-CLL-1 CAR-expressing T cell for use in treating patients with specific cytogenetic subgroups of AML, such as patients identified with t(15;17)(q22;q21) using all-trans retinoic acid (ATRA)16-19, and patients identified with t(8;21)(q22;q22) or inv(16)(p13q22) / t(16;16)(p13;q22) using repeated doses of high-dose cytarabine.

[0274] Preferably, the present application provides an anti-CLL-1 CAR-expressing T cell for use in treating patients with aberrations such as -5 / del(5q), -7, 3q abnormalities, or complex karyotype, which have been shown to have lower complete remission rates and survival.

[0275] As used herein, the term "therapeutic agent", "chemotherapeutic agent" or "drug" or "anti-cancer drug" refers to a drug, preferably a compound or a derivative thereof, which 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, but are not limited to, alkylating agents (e.g., busulfan, carboplatine, chlorambucil, cisplatine, cyclophosphamide, ifosfamide, melphalan, mechlorethamine, oxaliplatine, uramustine, temozolomide, fotemustine), metabolic antagonists (e.g., purine nucleoside antimetabolites such as clofarabine, methotrexate (MTX), 5-fluorouracil or derivatives thereof, thiopurine, capecitabine, cytarabine, floxuridine, fluorouracil, gemcitabine, methotrexate, pemetrexed), antitumor antibiotics (e.g., mitomycin, adriamycin, bleomycine, daunorubicine, doxorubicine, epirubicine, hydroxyurea, idarubicine, mitomycin C, mitoxantrone), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol, vinblastine, vinorelbine, docetaxel, paclitaxel), topoisomerase inhibitors (irinotecan, topotecan, etoposide).

[0276] In preferred embodiments, as used herein, a therapeutic agent, chemotherapeutic drug refers to a compound or derivative thereof that can be used to treat cancer, in particular to treat hematopoietic cancer cells and more specifically AML, thereby reducing the proliferative state of the cancer cells and / or killing the cancer cells. Examples of chemotherapeutic agents include, but are not limited to, aracytine, Cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vepeside, etoposide (VP 16), arsenic trioxide, trans-retinoic acid, nitrogen mustard, procarbazine, chlorambucil, and combinations thereof.

[0277] In other embodiments of the application, the cells of the application are administered to a patient in combination with a drug (or agent) selected from the group consisting of aracytine, Cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vepeside, etoposide (VP 16), arsenic trioxide, trans-retinoic acid, Cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone, and combinations thereof.

[0278] Such agents can further include, but are not limited to, the anticancer agent TRIMETHOTRIXATE TM (TMTX), TEMOZOLOMIDE TM , RALTRITREXED TM , S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanine (6-BG), bischloronitrosourea (BCNU), and CAMPTOTHECIN TM or a therapeutic derivative of any one thereof.

[0279] In more preferred embodiments, the anti-CLL-l scCAR-expressing T cells are administered to a patient in combination with at least one therapeutic agent selected from the group consisting of aracytine, Cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vepeside, etoposide (VP 16), arsenic trioxide, trans-retinoic acid, and combinations thereof.

[0280] As used herein, a cell that is "resistant or tolerant to an agent" means a cell that has been genetically modified such that the cell proliferates in the presence of an amount of an agent that would inhibit or prevent the proliferation of an unmodified cell.

[0281] In one embodiment, the anti-CLL-1 CAR-expressing T cells of the application can be used as induction therapy, post remission therapy for AML or consolidation therapy for AML patients.

[0282] In one embodiment, the anti-CLL-1 CAR-expressing T cells of the application can be used in case of relapse of AML, or in case of refractory or resistant AML, and more preferably in combination with at least one other anti-cancer drug.

[0283] In another preferred embodiment, the at least one anti-CLL-1 CAR-expressing cell of the application is used to prevent the development of cancer cells, in particular occurring after an anti-cancer treatment, during bone marrow depletion or before bone marrow transplantation, after bone marrow destruction.

[0284] AML complications

[0285] In a particular embodiment, the application provides a medicament that improves the health condition of a patient, in particular of a patient experiencing a complication associated with AML. More preferably, said engineered anti-CLL-1 CAR-expressing T cells of the application express at least one anti-CLL-1 CAR of the application and are used as a medicament for treating a complication associated with AML.

[0286] Complications or diseases associated with AML can include a previous period of myelodysplasia, secondary leukemia (in particular secondary AML), high white blood cell count and absence of Auer rods. Among them, leukostasis and central nervous system (CNS) involvement, leukocytosis, residual disease are also considered as complications or diseases associated with AML.

[0287] AML-related diseases

[0288] In one embodiment, the application also provides an anti-CLL-1 CAR-expressing T cell for use in the treatment of a pathological condition associated with AML.

[0289] The application provides a therapy against AML-associated myeloid neoplasms, a therapy against acute myeloid leukemia and myelodysplastic syndromes, a therapy for the treatment of relapsed or refractory acute myeloid leukemia, a therapy for the treatment of adult relapsed or refractory acute promyelocytic leukemia, a therapy for the treatment of acute promyelocytic leukemia, a therapy for the treatment of acute myeloid leukemia in adults over 60 years old.

[0290] According to another aspect, the application provides a composition for use in the treatment of AML-associated diseases, in particular hematologic malignancies associated with AML.

[0291] Hematologic malignancies associated with AML disorders include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), which is a collection of various hematologic disorders that combine ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML.

[0292] Other pathological disorders or genetic syndromes associated with risk of AML that can be improved by appropriate use of the present application include Down syndrome, Trisomy 21, Fanconi anemia, Bloom syndrome, Ataxia-telangiectasia, Diamond-Blackfan anemia, Schwachman-Diamond syndrome, Li-Fraumeni syndrome, neurofibromatosis type 1, severe congenital neutropenia (also known as Kostmann syndrome).

[0293] Pharmaceutical compositions and methods of treatment

[0294] The present disclosure also provides a composition comprising engineered T cells according to the present disclosure for use thereof or a method for treating a disease.

[0295] In one aspect, the disease is a hematologic cancer, particularly a stem cell cancer, including but not limited to leukemia, such as acute myeloid leukemia (AML) or complications thereof.

[0296] The present disclosure also provides a composition for use thereof or a method for inhibiting proliferation or reducing a population or activity of CLL-1 expressing cells in a patient. Exemplary methods include contacting a population of CLL-1 expressing cells with anti-CLL-1 CAR T cells of the present disclosure that bind to CLL-1 expressing cells, and particularly scCAR T cells.

[0297] In a more particular aspect, the present disclosure provides a composition for use thereof or a method for inhibiting proliferation of a population of CLL-1 expressing cancer cells or reducing the population of cancer cells in a patient, the method comprising contacting a population of CLL-1 expressing cancer cells with anti-CLL-1 CAR T cells of the present disclosure that bind to CLL-1 expressing cells, and particularly scCAR T cells, binding of the anti-CLL-1 CAR T cells, and particularly scCAR T cells, of the present disclosure to CLL-1 expressing cancer cells results in CLL-1 expressing cancer cells being destroyed.

[0298] In certain aspects, the anti-CLL-1 CAR T cells, and particularly scCART of the present disclosure reduce the number, count, amount, or percentage of cells and / or cancer cells in a subject or animal model having a myeloid leukemia or another cancer associated with CLL-1 expressing 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 an undetectable level) relative to a negative control.

[0299] The present disclosure also provides a composition for its use or a method for preventing, treating, and / or managing a disorder or condition associated with CLL-1 expressing cells (e.g., associated with a hematologic cancer), the method comprising administering to a subject in need thereof an anti-CLL-1 CAR T cell, and particularly scCART, of the present disclosure that binds to CLL-1 expressing cells. In one aspect, the subject is a human. Non-limiting examples of disorders associated with CLL-1 expressing cells include inflammatory disorders (such as rheumatoid arthritis) and cancers (such as a hematologic cancer, particularly AML or complications of AML).

[0300] The present disclosure also provides a composition for its use or a method for preventing, treating, and / or managing a disorder or condition associated with CLL-1 expressing cells (e.g., associated with a hematologic cancer), the method comprising administering to a subject in need thereof an anti-CLL-1 CAR T cell, and particularly scCART, of the present disclosure that binds to CLL-1 expressing cells. In one aspect, the subject is a human. Non-limiting examples of disorders associated with CLL-1 expressing cells include inflammatory disorders (such as rheumatoid arthritis) and cancers (such as a hematologic cancer, particularly AML or complications of AML).

[0301] The present disclosure provides a composition for its use or a method for treating or preventing relapse of a cancer associated with CLL-1 expressing cells, the method comprising administering to a subject in need thereof an anti-CLL-1 CAR T cell, and particularly scCART, of the present disclosure that binds to CLL-1 expressing cells. In another aspect, the method comprises administering to a subject in need thereof an effective amount of an anti-CLL-1 CAR T cell, and particularly scCART, of the present disclosure that binds to CLL-1 expressing cells in combination with an effective amount of another therapy.

[0302] In one aspect, CLL-1 is considered a "cancer stem cell" marker in AML. Thus, the anti-CLL1 CAR T cells, and particularly scCART, of the present disclosure can prevent relapse of AML, or even treat AML that is primarily CLL-1 negative but has a CLL1+ cell (CLL1 expressing cell) "stem" population.

[0303] In one aspect, the present disclosure provides compositions and methods for treating a subject who has undergone treatment for a disease or disorder associated with elevated levels of CLL-1 expression.

[0304] Treatment with engineered immune cells according to the present disclosure can be combined with one or more therapies for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiation therapy.

[0305] Preferably, treatment with engineered immune cells according to the present disclosure can be administered in combination with (e.g., prior to, concurrently with, or subsequent to) one or more therapies for cancer selected from cytarabine, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vindesine, etoposide (VP 16), arsenic trioxide, trans-retinoic acid, arsenic trioxide, trans-retinoic acid combinations, nitrogen mustard, methylhydrazine, chlorambucil, and combinations thereof.

[0306] According to preferred embodiments of the present disclosure, the treatment can be administered to a patient who is undergoing immunosuppressive therapy. Indeed, the present disclosure preferably relies on cells or cell populations that are resistant to at least one immunosuppressive agent due to inactivation of the gene encoding such immunosuppressive agent receptor. In this aspect, the immunosuppressive therapy should facilitate the selection and expansion of T cells according to the present disclosure within the patient.

[0307] Administration of the cells or cell populations according to the present disclosure can be performed in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0308] Administration of the cells or cell populations can be by administration of 10 4 -10 9 cells / kg body weight, preferably administration of 10 5 to 10 6comprise all integer values of cell numbers within those ranges. The cells or cell population can be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as more than one dose over a period of time. The timing of administration is at the discretion of the attending physician and is dependent upon the clinical condition of the patient. The cells or cell population can be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides therapeutic or prophylactic benefit. The dosage administered will depend on the age, health and weight of the recipient, kind of condition being treated (if any), frequency of treatment, and the nature of the effect desired.

[0309] In another embodiment, the effective amount of cells or compositions comprising those cells is administered parenterally. The administration can be intravenous administration. The administration can be performed directly by intratumoral injection.

[0310] In certain embodiments of the disclosure, the cells are administered (e.g., prior to, concurrently with, or after) to a patient in conjunction with any number of related treatment modalities, including but not limited to treatment with agents such as antiviral therapies, cidofovir and interleukin-2, cytarabine (also known as ARA-C), natalizimab for MS patients, efaliztimab for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the disclosure can be used in combination with chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immune ablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These agents are important because they inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993).

[0311] In further embodiments, the cell compositions of the present disclosure are administered (e.g., prior to, concurrently with, or after) to a patient in conjunction with bone marrow transplantation, T cell ablative therapy with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH.

[0312] In another embodiment, the cell compositions of the present disclosure are administered after B cell ablative therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, a subject can undergo standard therapy of high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present disclosure. In further embodiments, the expanded cells are administered prior to or after surgery.

[0313] In certain embodiments of the present disclosure, the anti-CLL-l scCAR-expressing cells are administered (e.g., prior to, concurrently with, or after) to a patient in conjunction with a drug selected from the group consisting of cytarabine, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vindesine, etoposide (VP 16), arsenic trioxide, transretinoic acid, arsenic trioxide, transretinoic acid, combinations thereof, nitrogen mustard, methyldiazomycin, chlorambucil, and combinations thereof. In these embodiments, the anti-CLL-l scCAR-expressing cells can be resistant to the particular drug or combination of drugs with which the anti-CLL-l scCAR-expressing cells are administered.

[0314] In other embodiments of the present disclosure, the anti-CLL-l scCAR-expressing cells are administered to a patient in conjunction with a drug selected from the group consisting of cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone, and combinations thereof.

[0315] Acute myeloid leukemia (AML) is characterized by the overproduction of immature myeloid cells in the bone marrow and is the most common acute leukemia in adults and the second most common leukemia in children, resulting in an aggressive and heterogeneous cancer that affects normal hematopoiesis. Over the last decades, improvements in the two standard treatments for AML (chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT)) have been limited due to the poor prognosis of refractory / relapsed AML and the low 5-year survival rate of less than 50%. Most patients eventually die from relapsed and / or progressive disease due to chemoresistance or severe and long-term toxic effects on healthy / non-cancerous tissues / organs, indicating an urgent need for new therapeutic strategies including targeted and immunotherapies.

[0316] Recent advances in immunotherapy have brought remarkable breakthroughs in the treatment of hematological malignancies. A special type of engineered autologous T cells that combine the specificity of antibody-targeted recognition with the potent effector mechanism of T cells are called chimeric antigen receptor (CAR) T cells. The therapeutic benefits of CAR-T cells have been demonstrated and approved by the US FDA, with anti-CD19 CAR T cells showing potent and durable anti-tumor activity in acute lymphoblastic leukemia (ALL). CARs are composed of several basic parts, an extracellular antigen-binding domain derived from a single-chain variable fragment (scFv) of a targeting antibody, a transmembrane domain, one or more costimulatory domains such as 4-1BB (CD137), CD28, or ICOS (CD278), and an intracellular signaling domain of CD3-zeta. Target recognition by the scFv domain enables CAR-T cells to mediate tumor cytotoxicity in a major histocompatibility complex (MHC)-independent manner and to avoid immune escape by reducing antigen processing and presentation during tumorigenesis. With MHC-independent antigen recognition, CAR T cells have many advantages, including greater specificity than TCRs, programmable to recognize any tumor antigen, manageable cytotoxic capacity, higher proliferation rate, and longer persistence, making CAR-T cell therapy an excellent therapeutic option for cancer treatment.

[0317] Non-limiting embodiments

[0318] The present disclosure is illustrated herein by the following embodiments, which should not be construed as limiting. Those skilled in the art will understand that the present disclosure can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0319] 1. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising a polypeptide comprising:

[0320] an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL);

[0321] a transmembrane domain; and

[0322] an intracellular signaling domain,

[0323] wherein the single heavy chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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

[0324] wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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.

[0325] 2. The CAR of embodiment 1, wherein the first amino acid sequence is SEQ ID NO: 14.

[0326] 3. The CAR of embodiment 1, wherein the second amino acid sequence is SEQ ID NO: 15.

[0327] 4. The CAR of embodiments 1-3, wherein the single heavy chain variable domain is located on the N-terminal side of the single light chain variable domain.

[0328] 5. The CAR of embodiments 1-3, wherein the single heavy chain variable domain is located on the C-terminal side of the single light chain variable domain.

[0329] 6. The CAR of embodiments 1-5, wherein the single heavy chain variable domain and the single light chain variable domain are directly fused to one another via a peptide bond.

[0330] 7. The CAR of embodiments 1-5, wherein the single heavy chain variable domain and the single light chain variable domain are connected to one another via a peptide linker.

[0331] 8. The CAR of embodiment 7, wherein the peptide linker comprises no more than 50 amino acid residues.

[0332] 9. The CAR of embodiments 1-8, wherein the transmembrane domain is derived from CD8 or CD28.

[0333] 10. The CAR of embodiments 1-9, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0334] 11. The CAR of embodiment 10, wherein the primary intracellular signaling domain is derived from CD3 zeta.

[0335] 12. The CAR of embodiments 1-11, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0336] 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.

[0337] 14. The CAR of embodiment 12, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0338] 15. The CAR of embodiment 12, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0339] 16. The CAR of embodiments 1-15, further comprising a hinge domain.

[0340] 17. The CAR of embodiment 16, wherein the hinge domain is positioned between the C- terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0341] 18. The CAR of embodiments 16-17, wherein the hinge domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, and a combination thereof.

[0342] 19. The CAR of embodiments 16-17, wherein the hinge domain is derived from CD28.

[0343] 20. The CAR of embodiments 1-19, further comprising a signal peptide positioned at the N-terminus of the polypeptide.

[0344] 21. The CAR of embodiment 20, wherein the signal peptide is derived from CD28.

[0345] 22. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising a polypeptide, the polypeptide comprising:

[0346] an extracellular antigen binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL);

[0347] a transmembrane domain; and

[0348] an intracellular signaling domain,

[0349] wherein the single heavy chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 14, and

[0350] wherein the single light chain variable domain comprises a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 15.

[0351] 23. The CAR of embodiment 22, wherein the single heavy chain variable domain is positioned at the N-terminus of the single light chain variable domain.

[0352] 24. The CAR of embodiment 22, wherein the single heavy chain variable domain is located C-terminal to the single light chain variable domain.

[0353] 25. The CAR of embodiments 22-24, wherein the single heavy chain variable domain and the single light chain variable domain are directly fused to one another via a peptide bond.

[0354] 26. The CAR of embodiments 22-24, wherein the single heavy chain variable domain and the single light chain variable domain are connected to one another via a peptide linker.

[0355] 27. The CAR of embodiment 26, wherein the peptide linker comprises no more than 50 amino acid residues.

[0356] 28. The CAR of embodiments 22-27, wherein the transmembrane domain is derived from CD8 or CD28.

[0357] 29. The CAR of embodiments 22-28, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0358] 30. The CAR of embodiment 29, wherein the primary intracellular signaling domain is derived from CD3 zeta.

[0359] 31. The CAR of embodiments 22-30, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0360] 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.

[0361] 33. The CAR of embodiment 31, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof.

[0362] 34. The CAR of embodiment 31, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0363] 35. The CAR of embodiments 22-34, further comprising a hinge domain.

[0364] 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.

[0365] 37. The CAR of 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.

[0366] 38. The CAR of embodiments 35-36, wherein the hinge domain is derived from CD28 or 4- 1BB.

[0367] 39. The CAR of embodiments 22-38, further comprising a signal peptide located at the N- terminus of the polypeptide.

[0368] 40. The CAR of embodiment 39, wherein the signal peptide is derived from CD28 or 4-1BB.

[0369] 41. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising a polypeptide, the polypeptide comprising:

[0370] an extracellular antigen binding domain comprising an anti-CLL-1 single heavy chain variable domain (VH) and an anti-CLL-1 single light chain variable domain (VL);

[0371] a transmembrane domain derived from CD8, CD28, 4-1BB, or combinations thereof; and

[0372] an intracellular signaling domain derived from CD8, CD28, 4-1BB, OX40, ICOS, or combinations thereof.

[0373] 42. The CAR of embodiment 41, wherein the anti-CLL-1 single heavy chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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.

[0374] 43. The CAR of embodiment 41, wherein the anti-CLL-1 single light chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in 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.

[0375] 44. The CAR of embodiment 41, wherein the anti-CLL-1 single heavy chain variable domain comprises CDR1, CDR2, and CDR3 as set forth in a VH domain comprising the amino acid sequence of SEQ ID NO: 14.

[0376] 45. The CAR of embodiment 41, wherein the anti-CLL-1 single light chain variable domain comprises CDR1, CDR2, and CDR3 as shown in the VL domain comprising the amino acid sequence of SEQ ID NO: 15.

[0377] 46. ​​The CAR of embodiments 41-45, wherein the anti-CLL-1 single heavy chain variable domain is located at the N-terminus of the anti-CLL-1 single light chain variable domain.

[0378] 47. The CAR of embodiments 41-45, wherein the anti-CLL-1 single heavy chain variable domain is located at the C-terminus of the anti-CLL-1 single light chain variable domain.

[0379] 48. The CAR of embodiments 41-47, wherein the anti-CLL-1 single heavy chain variable domain and the anti-CLL-1 single light chain variable domain are directly fused to each other via a peptide bond.

[0380] 49. The CAR of embodiments 41-47, wherein the anti-CLL-1 single heavy chain variable domain and the anti-CLL-1 single light chain variable domain are connected to each other via a peptide linker.

[0381] 50. The CAR of embodiment 49, wherein the peptide linker comprises no more than 50 amino acid residues.

[0382] 51. The CAR of embodiments 41-50, wherein the transmembrane domain is derived from CD8 or CD28.

[0383] 52. The CAR of embodiments 41-51, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0384] 53. The CAR of embodiment 52, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0385] 54. The CAR of embodiments 41-53, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain.

[0386] 55. The CAR of embodiment 54, wherein the co-stimulatory signaling domain is derived from CD28, 4-1BB, or a combination thereof.

[0387] 56. The CAR of embodiment 54, wherein the co-stimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0388] 57. The CAR of embodiments 41-56, further comprising a hinge domain.

[0389] 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.

[0390] 59. The CAR of 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.

[0391] 60. The CAR of embodiments 57-58, wherein the hinge domain is derived from CD28.

[0392] 61. The CAR of embodiments 41-60, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0393] 62. The CAR of embodiment 61, wherein the signal peptide is derived from CD28.

[0394] 63. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising:

[0395] a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-26.

[0396] 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.

[0397] 65. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising:

[0398] a polypeptide derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27-33.

[0399] 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.

[0400] 67. A chimeric antigen receptor (CAR) directed against human C-type lectin-like molecule- 1 (CLL-1), comprising a polypeptide comprising:

[0401] an extracellular antigen-binding domain comprising a single heavy chain variable domain (VH) and a single light chain variable domain (VL);

[0402] a transmembrane domain; and

[0403] an intracellular signaling domain,

[0404] wherein the single heavy chain variable domain comprises CDR1, CDR2 and CDR3 as shown in 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 wherein the single light chain variable domain comprises CDR1, CDR2 and CDR3 as shown in 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.

[0405] 68. The CAR of embodiment 67, wherein the first amino acid sequence is SEQ ID NO: 14.

[0406] 69. The CAR of embodiments 67-68, wherein the second amino acid sequence is SEQ ID NO: 15.

[0407] 70. The CAR of embodiments 67-69, wherein the single heavy chain variable domain is located N-terminus of the single light chain variable domain.

[0408] 71. The CAR of embodiments 67-69, wherein the single heavy chain variable domain is located at the C-terminus of the single light chain variable domain.

[0409] 72. The CAR of embodiments 67-71, wherein the single heavy chain variable domain and the single light chain variable domain are directly fused to each other via a peptide bond.

[0410] 73. The CAR of embodiments 67-71, wherein the single heavy chain variable domain and the single light chain variable domain are connected to each other via a peptide linker.

[0411] 74. The CAR of embodiment 73, wherein the peptide linker comprises no more than 50 amino acid residues.

[0412] 75. The CAR of embodiments 67-74, wherein the transmembrane domain is derived from CD8 or CD28.

[0413] 76. The CAR of embodiments 67-75, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0414] 77. The CAR of embodiment 76, wherein the primary intracellular signaling domain is derived from CD3ζ.

[0415] 78. The CAR of embodiments 67-77, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

[0416] 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.

[0417] 80. The CAR of embodiment 78, wherein the costimulatory signaling domain is derived from CD28, 4-1BB, or combinations thereof.

[0418] 81. The CAR of embodiment 78, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28.

[0419] 82. The CAR of embodiments 67-81, further comprising a hinge domain.

[0420] 83. The CAR of embodiment 82, wherein the hinge domain is positioned between the C- terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0421] 84. The CAR of 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.

[0422] 85. The CAR of embodiments 82-83, wherein the hinge domain is derived from CD28.

[0423] 86. The CAR of embodiments 67-85, further comprising a signal peptide positioned at the N-terminus of the polypeptide.

[0424] 87. The CAR of embodiment 86, wherein the signal peptide is derived from CD28.

[0425] 88. An immune effector cell comprising the CAR of embodiments 1-87.

[0426] 89. The immune effector cell of embodiment 88, wherein the immune effector cell is a T cell.

[0427] 90. A pharmaceutical composition comprising the immune effector cell of embodiments 88- 89 and a pharmaceutically acceptable carrier.

[0428] 91. A method of treating a cancer expressing CLL-1 in an individual, comprising administering to the individual an effective amount of the immune effector cell of embodiments 88- 89 or the pharmaceutical composition of embodiment 90.

[0429] 92. The method of embodiment 91, wherein the cancer is multiple myeloma.

[0430] 93. The method of embodiment 91, wherein the cancer is refractory or relapsed multiple myeloma.

[0431] 94. The method of embodiment 91, wherein the cancer is myeloid leukemia.

[0432] 95. The method of embodiment 91, wherein the cancer is refractory or relapsed myeloid leukemia.

[0433] Non-limiting examples

[0434] The present disclosure is illustrated by the experiments described by the following examples, which are not to be construed as limiting. It will be understood by those skilled in the art that the present disclosure can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0435] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments have been presented by way of example only, as will be apparent to those skilled in the art. Numerous changes, variations and alternatives to the embodiments described herein will be apparent to those skilled in the art. It will be understood that various alternatives to the embodiments of the present disclosure described herein can be employed in practicing the present disclosure. This means 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 are intended to be covered thereby.

[0436] In some embodiments, the present disclosure relates to the development of CAR-T cells against CLL-1 antigen expressed on AML blast cells and leukemia stem cells. Through CAR-T cell hit selection process, through the exchange of antibody variable region and the exchange of costimulatory domain (4-1BB or CD28), the lead optimization selected out the disclosed CAR-T cell candidate 61H08 HL_2828z and 61H08 LH_2828z with high CAR%, normal CD4 / CD8 ratio, high cell expansion fold, more T scm and T cm memory-like phenotype (>80%) and better cytotoxicity to AML cells in vitro and in vivo. In AML U937 xenograft animal model, CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were able to eradicate 3x 10 6Significant anti-tumor activity was revealed at a dose of 1 CAR-T cell / mouse and was essentially sustained in the peripheral blood of xenografted mice. In addition, the disclosed CLL-1 CAR-T candidates 61H08 HL_2828z and LH_2828z were tolerable in that they exerted no or limited inhibition of colony formation by CD34+ cells derived from BM or PB at an E / T ratio of 1 or 4 and incubation for 6 hours or 24 hours. In addition, ex vivo studies with the disclosed CLL-1 (61H08 HL_2828z) CAR-T cells prepared with T cells from r / r AML patients S-008 and S015 could mediate significant cytotoxicity against their paired primary AML blast cells.

[0437] Example I - Screening and characterization of anti-CLL-1 antibodies

[0438] Bio-panning with phage-displayed human scFv library

[0439] Antibodies against the CLL-1 antigen were screened by CRO services using human antibody phage libraries (DSyn2, SLE1, SLE2, SLE3, SLE3-1, SLE3-2, SS1-Mix and SS2). Biopanning was performed on eight antibody libraries, including a fully synthetic human antibody library (DSyn2) with a diversity of 1 x 1010and seven human autoimmunity disease-derived antibody libraries with a diversity of 1 x 1010to 1 x 1010. A total of 67 scFv antibodies with unique CDR sequences were obtained, each unique DNA sequence was then amplified into a mammalian expression vector by recombinant DNA technology for scFv-Fc format protein expression. Among them, 58 scFv-Fc clones were successfully expressed, and then their cell-based CLL-1 antigen binding was determined by flow cytometry, six exemplary clones are shown in Table 1. 10 9 10 Biopanning was performed on eight antibody libraries, including a fully synthetic human antibody library (DSyn2) with a diversity of 1 x 1010and seven human autoimmunity disease-derived antibody libraries with a diversity of 1 x 1010to 1 x 1010. A total of 67 scFv antibodies with unique CDR sequences were obtained, each unique DNA sequence was then amplified into a mammalian expression vector by recombinant DNA technology for scFv-Fc format protein expression. Among them, 58 scFv-Fc clones were successfully expressed, and then their cell-based CLL-1 antigen binding was determined by flow cytometry, six exemplary clones are shown in Table 1.

[0440] Table 1. Exemplary clones screened by phage display

[0441]

[0442] Screening of late anti-CLL-1 scFv-Fc hits by flow cytometry

[0443] As Figure 1 ​​A-1B and 2, purified scFv-Fc antibodies were further evaluated for binding to CLL-1 antigen on K562-Luc-CLL-1 transfectants and CLL-1+ AML cell lines, U937, MOLM-14 and THP-1 by flow cytometry. K562 cells (a human erytholeukemia 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 with the six cell lines, including K562, K562-CLL-1, U937, MOLM-14, THP-1 and Raji. Although the binding properties of each clone varied depending on the cell line tested, the top 3 clones with stronger binding patterns were selected, and the percentage of binding ranged from 52% to 99% for K562-Luc-CLL-1, 25% to 98% for U937, 46% to 37% for MLOM-14, and 1% to 36% for THP-1.

[0444] Determination of interaction between CLL-1 antigen and late anti-CLL-1 scFv-Fc hits by AlphaLISA

[0445] To characterize the antigen binding ability of anti-CLL-1 scFv-Fc antibodies, an AlphaLISA sandwich assay was established with anti-6xHis acceptor beads, Protein A donor beads and CLL-1-ECD. The Kd values of each anti-CLL-1 scFv-Fc antibody were determined by the AlphaLISA assay. The Kd values ranged from 1.1 x 10 d M to 4.9 x 10 d M. The ranking from high to low based on the maximum value of AlphaLISA signal fold was 61H08, 56D01 and 72C10, as exemplified in Figure 3. -9 -9

[0446] Example II - CAR gene design and lentivirus production

[0447] Design and construction of CLL-1 CAR gene with late anti-CLL antibody hits

[0448] CAR lentivirus plasmid is a key material for CAR-T cell production, and includes a 2ndgeneration CAR gene format with essential elements in the order of signal peptide GMCSFRss, scFv, hinge region and transmembrane region of CD8 molecule, cytoplasmic portion of 4-1BB, cytoplasmic component of CD3 zeta, T2A and enhanced GFP will be applied to the construction of CAR LV transfer plasmid, i.e. GMCSFRss-scFv-hinge-TM-co-stimulatory domain-CD3z-T2A-EGFP. Late anti-CLL antibody hits 61H08, 65D01 and 72C10 were applied to the construction of CLL-1 CAR lentivirus vector, as exemplified in Figure 4. Figure 4 ​​Exemplified.

[0449] Each CLL-1 CAR gene was first generated in pMK cloning vector by circular PCR, i.e., each anti-CLL-1 scFv fragment was amplified in frame with CD8 hinge / TM-4-1BB-CD3 zeta-T2A enhanced GFP backbone in pMK cloning vector, and then each promoterless CLL-1 CAR gene in cloning vector was subcloned into lentiviral plasmid by In-Fusion technology.

[0450] Productivity assessment of CLL-1 CAR lentiviral vector

[0451] Although CAR LV titers varied with CAR clones and their binding domain formats (i.e., scFv VH-VL or VL-VH orientation), all CLL-1 CAR LVs were successfully produced with high yields. These CLL-1 CAR LVs were then used to transduce MACSbead-isolated primary T cells from PBMCs of healthy donors, as shown in Table 2.

[0452] Table 2. Productivity evaluation of CLL-1 CAR lentivirus

[0453]

[0454] Optimization of CLL-1 (61H08) CAR gene format by swapping antibody variable regions and swapping costimulatory domains Design

[0455] By in vitro functional assays, CLL-1 (61H08 HL_8BBz and 61H08 LH_8BBz) CAR-T cells were selected as late hits, and their CAR gene formats were further optimized by scFv VH-VL exchange and costimulatory domain exchange to maximize their in vitro and in vivo CAR-T cell potency. In addition, two more CLL-1 CAR-T cells, 65D01 LH_8BBz, and one reference 24C8 HL_8BBz, as well as one control CD19 (FMC63) CAR-T cell were included to lead the potency evaluation with CLL-1 CAR-T cells.

[0456] CLL-1 (61H08 HL_8BBz and 61H08 LH_8BBz) CAR genes were further optimized by exchange with CD28 hinge, CD28 transmembrane domain, and CD28 costimulatory domain, as Figure 5 Exemplified.

[0457] Example III - Generation of CLL-1 CAR-T cell clones

[0458] CLL-1 CAR-T cell bioprocessing

[0459] Peripheral blood mononuclear cells (PBMC) from healthy donors were collected by centrifugation, and then CD3+ T cells isolated with CD3 microbeads were stored in liquid nitrogen tanks prior to the CAR-T cell process. The thawed T cells were stimulated with anti-CD3 / anti-CD28 antibody-coated human T activator beads and cultured. The 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 then the CAR-T cell cultures were re-cultured and expanded. Then, the CAR-T cells were harvested and analyzed for CAR% and phenotype, and then cryopreserved in liquid nitrogen gas phase.

[0460] Productivity assessment of CLL-1 CAR-T cell clones

[0461] Different CLL-1 CAR-T cells (different CLL-1 clones) were manufactured to observe bioprocessing attributes such as CAR%. Two batches of production were performed with donor 25 and donor 26.

[0462] As Figures 6A-6C illustrated, for CLL-1 CAR-T cells derived from donor 25, most CLL-1 CAR-T clones had a viability higher than 95% throughout the culture period. 61H08 HL and 61H08 LH had a slight decrease in viability at day 6, but the viability was still about 90%. For CAR%, 61H08 HL_28 and 61H08 LH_28 had the highest CAR% of about 95%, while 65D01 LH had the lowest CAR% of about 88%. Regarding cell expansion, it is notable that 61H08 HL_28 and 61H08 LH_28 had a significant growth advantage compared to their 4-1BB counterparts. In addition, 65D01 LH also had a large growth advantage.

[0463] As Figures 7A-7C illustrated, for CLL-1 CAR-T cells derived from donor 26, all CLL-1 CAR-T clones had a viability higher than 90% throughout the culture period. For CAR%, 61H08 HL_28 and 61H08 LH_28 had the highest CAR% of about 95%, while 24C8 HL had the lowest CAR% of about 90%. Regarding cell expansion, 61H08 HL_28 and 61H08 LH_28 had a significant growth advantage compared to their 4-1BB counterparts. In addition, 65D01 LH also had a large growth advantage.

[0464] Briefly, both lots of CLL-1 CAR-T production had similar bioprocessing attributes. 61H08 HL_28 and 61H08 LH_28 had higher viability, %CAR, and expansion fold compared to their 4-1BB counterparts. In addition, 61H08 HL_28 and 61H08 LH_28 had the best bioprocessing characteristics among all CLL-1 CAR-T clones.

[0465] Phenotypic profiling of CLL-1 CAR-T cell clones

[0466] The phenotype and CAR expression levels of CLL-1 CAR-T cell clones were continuously monitored. Memory phenotype profile and expression of inhibitory markers were analyzed. Functional binding of CAR or CAR expression was detected via antigen labeling. 61H08 HL_28 and 61H08 LH_28 had more CD8 population compared to their 4-1BB counterparts in terms of CD4 / CD8 ratio. 61H08 HL and 61H08 LH had higher CD4 population, while other cells had similar CD4 / CD8 ratio. 61H08 HL_28 and 61H08 LH_28 had higher T eff and T em cell population compared to their 4-1BB counterparts in terms of memory phenotype. The majority of 61H08 HL_BB and 61H08 LH_BB population were T scm and T cm , while other CLL-1 CAR-T cell clones had similar memory phenotype profile. In terms of inhibitory marker expression, PD-1, TIM-3, LAG3 triple positive cells were more likely to be exhausted cells. The triple positive cell percentage of all our CLL-1 CAR-T cell clones were low, less than 10% in donor 25 lot and less than 5% in donor 26 lot. Overall, the majority of our CLL-1 CAR-T cell clones population were memory phenotype and had higher CD4 percentage, while 61H08 HL_28 and 61H08 LH_28 had higher T em and T eff cell percentage (T scm and T cm were still the major population) and higher CD8 population. The phenotype profile of CLL-1 CAR-T cells from donor 25 and 26 are exemplified in Figure 8 A-8F.

[0467] Using antigen labeling, we can detect CAR expression and expression strength of our CLL-1 CAR-T cell clones. The results show that 65D01 LH_BB has lower CAR expression and CAR expression strength. The conversion of 61H08 HL_8BBz to 61H08 HL_2828z results in upregulation of CAR expression, while the conversion of 61H08 LH_8BBz to 61H08 LH_2828z results in lower CAR expression. 61H08 HL_28 and 61H08 HL_BB have the highest CAR expression strength, followed by 61H08 LH_BB and 61H08 LH_28. All four disclosed CLL-1 CAR-T cell clones have better CAR expression than the reference 24C8 HL_BB. The CAR expression profile of CLL-1 CAR-T cells from donors 25 and 26 is illustrated in Figure 9 A-9B.

[0468] Example IV - Characterization of CLL-1 CAR-T cell clones

[0469] For in vitro cytotoxicity, it is notable that the non-specific killing of Pan T and CD19 CAR-T cells is almost non-existent, indicating that all of the cytotoxicity observed is exerted by the CLL-1 CAR-T cells. 61H08 HL_28 and 61H08 LH_28 have better cytotoxicity against CLL-1+ U937 cells than their 4-1BB counterparts. 61H08 HL_28 and 61H08 LH_28 also exhibit the best cytotoxicity against target cells compared to all other CAR-T cell clones (24C8 HL_BB, 65D01 LH_BB). The cytotoxicity of CLL-1 CAR-T cell clones against CLL-1+ U937 cells is illustrated in Figure 10 A-10B.

[0470] For in vitro cytokine release profiles, the cytokine profiles of CLL-1 CAR-T cell clones in the cytotoxicity assay were analyzed via the LegendPlex multiplex Cytokine Detection Kit. The pattern is similar for both batches, with 61H08 HL_28 and 61H08 LH_28 having higher secretion of effector cytokines (IFN-γ, TNF-α, Granzyme B), stimulatory cytokines (IL-2), regulatory cytokines (IL-4, IL-10), and inflammatory cytokines (IL-17A). The cytokine release profile corresponds to the cytotoxicity results, indicating that 61H08 HL_28 and 61H08 LH_28 have the best tumor killing capacity compared to the other CLL-1 CAR-T cell clones. The in vitro cytokine release profile of CLL-1 CAR-T cell clones upon encounter with CLL-1+ U937 cells is illustrated in Figures 11A-11Bmiddle.

[0471] CLL-1 CAR-T cell-mediated anti-AML tumor activity in vivo in U937 xenograft model

[0472] A xenograft U937-Luc AML model was established to test the anti-AML activity of CLL-1 CAR-T cells in vivo. AML cell expansion was measured weekly using bioluminescence imaging (BLI). 4 Mice that received U937 transplants died approximately 25 days after tumor inoculation. It was demonstrated that 61H08 (HL or LH) CAR-T cells with a CD28 co-stimulatory domain exhibited better anti-AML activity than CAR-T cells with a 4-1BB domain and other CLL-1CAR-T cell clones (ie, clones 24C8_8BBz and 65D01 LH_8BBz). Notably, 61H08 HL_2828z showed a more uniform tumor suppressive effect in both donors. BLI images showed that 61H08 HL_2828z and 61H08LH_2828z had significant tumor suppressive effects compared with the 24C8 HL_8BBz reference clone and the control CD19 CAR-T cell clone. BLI data of CLL-1CAR-T cell clones in the U937 xenograft model are shown in Figures 12A-12C middle.

[0473] For the survival of xenografted mice, mice treated with CLL-1CAR T cells showed slightly improved survival compared to groups treated with saline (PBS) or control CD19 CAR-T cells. Specifically, CLL-1 (61H08 HL or LH) CAR-T cells with a CD28 costimulatory domain prolonged the superior survival of U937 xenografted mice compared to other CAR-T cell clones, of which only one mouse died in the 61H08 HL_2828z group from donor 25. For in vivo CAR-T cell persistence, 65D01 LH_8BBz from donor 26 had an extremely high amount of CAR-T cells in the blood of one mouse, which may eventually die from xGVHD. 61H08 HL_2828z and 61H08 LH_2828z had the best in vivo persistence compared to other CLL-1CAR-T cell clones. There was no significant difference in in vivo persistence between 61H08HL_2828z and 61H08 LH_2828z. Kaplan-Meier survival curves of xenografted mice receiving CAR-T cells and in vivo persistence of CLL-1 CAR-T cells are shown in Figure 13 A-13D.

[0474] In vivo cytokine release profile

[0475] Cytokine release profiles were determined for plasma samples from U937-bearing mice at days 20, 27, and 34 post-CAR-T cell infusion. Responsive effector cytokines such as IFN-γ, granzyme A, perforin, and granulysin were found to be detected in plasma in response to CAR-T cell engagement of target cells, and the levels of the responsive effector cytokines increased over time. Although these cytokine profiles varied in kinetics with CLL-1 CAR-T cell clones, CLL-1 (61H08 HL or LH) CAR-T cells with CD28 costimulatory domains in surviving mice at day 34 still released substantial amounts of these effector cytokines, implicating an active process of eradicating engrafted AML tumor cells. IL-2 (stimulatory) and IL-10 (regulatory) were occasionally detected in the following plasma samples in terms of releasing stimulatory, regulatory, or inflammatory cytokines. In vivo cytokine release profiles of xenograft mice receiving CLL-1 CAR-T cell clones are illustrated in Figures 14A-14B .

[0476] Assessment of hematotoxicity by stem cell (CD34+) colony formation assay

[0477] Blood toxicity evaluation of the disclosed CLL-1 CAR-T cell clones was performed in colony formation assays with CD34+ stem cells derived from normal bone marrow (BM) or peripheral blood (PB). Notably, the disclosed CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z exerted limited or no inhibition of colony formation of CD34+ cells derived from BM or PB at E / T ratios of 1 or 4 and with incubation for 6 hours or 24 hours. Blood toxicity evaluation of the disclosed CLL-1 CAR-T cell candidates is illustrated in Figure 15 A-15B.

[0478] Generation of CLL-1 CAR-T cell candidate (ARD103)

[0479] The CLL-1 CAR-T cell candidate 61H08 HL_2828z was produced through a short bioprocessing procedure. The CLL-1 CAR-T cells retained high cell expansion fold and had a high CAR% (>80%), normal CD4 / CD8 ratio, high T scm and T cm memory-like phenotype percentage (>70%), and relatively low levels of expression of triple positive inhibitory marker (PD-1+TIM-3+LAG-3+). Characteristics of the processed CLL-1 CAR-T cell candidate are illustrated in Figure 16 .

[0480] CLL-1 CAR-T cell candidate-mediated target-specific immune response in vitro

[0481] CLL-1 CAR-T cell candidates mediate cytotoxicity that is specific for CLL-1 antigen and correlates with CLL-1 antigen expression level. Luciferase-based reporter AML cell lines were generated by transduction with lentiviral vectors encoding a luciferase reporter gene and were evaluated with CLL-1 antigen expression levels comparable to their parental cells. Notably, AML cell lines expressing higher CLL-1 antigen, such as U937, THP-1, HL-60, and MOLM-14, were more susceptible to ARD103-mediated cytotoxicity than AML cell lines expressing lower CLL-1 antigen or no CLL-1 antigen, such as MOLM-13 and K562 CML cell lines, respectively. Knocking out the CLL-1 gene in MOLM-14 cell pools by CRISPR-Cas9 rendered them unresponsive to ARD103-mediated cytotoxicity, further confirming the specificity of ARD103 for CLL-1 antigen. CLL-1 CAR-T cell candidate-mediated cytotoxicity correlates with CLL-1 antigen expression level, as Figures 17A-1 7C. CLL-1 CAR-T cell candidate-mediated cell killing is specific for CLL-1 antigen, as Figure 18 A-18B.

[0482] CLL-1 CAR-T cell candidate exhibits anti-tumor activity at minimum effective dose

[0483] In a xenograft U937-Luc AML model, mice were infused with decreasing doses of CLL-1 CAR cell candidate 61H08 HL_2828z, prepared from two donors by a transient bioprocessing procedure. 61H08 HL_2828z rapidly produced from either donor exerted dose-dependent tumor growth inhibition effects, and the minimum effective dose was titrated down to as low as 3 x 10 5 / mouse. Treatment of xenograft mice with 61H08 HL_2828z prolonged the survival of mice, and CAR-T cells persisting in the peripheral blood elicited an effective recall response upon a second challenge with tumor cells. The MED and the durable anti-tumor activity of CLL-1 CAR-T cells are exemplified in Figure 19 A-19B.

[0484] r / r AML patient-derived Ex vivo study of CLL-1 (61H08 HL_2828z) CAR-T cell-mediated cytotoxicity against primary AML blast cells

[0485] Isolated BMMC from patients S-008 and S015 were incubated with their corresponding r / r AML patient CLL-1 (61H08 HL_2828z) CAR-T cells at E / T ratios of 1, 2, 4, and 8 for 24 hours. Absolute AML blast (CD45dimCD34+CD38+) counts were enumerated for each culture by flow cytometry by counting beads. CLL-1 CAR-T cell candidates were shown to mediate cytotoxicity against autologous primary AML blasts compared to corresponding untransduced T cells. R / R AML patient derived CLL-1 CAR-T cell mediated cytotoxicity is exemplified in Figure 20 A-20B.

[0486] As shown in the foregoing examples, CAR-T cells against CLL-1 antigen were designed with a second generation CAR format and optimized by antibody variable region selection and domain (VH-VL) exchange as well as co-stimulatory domain (CD28 or 4-1BB) exchange. CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were selected for high CAR% (>80%), normal CD4 / CD8 ratio, high cell expansion fold, high Tscm and Tcm memory-like phenotype percentage (>70%), significant effector cytokine release, and superior cytotoxicity against AML tumor cells in vitro and in vivo. In the AML U937-Luc xenograft animal model, CLL-1 (61H08 HL or LH) CAR-T cells with CD28 co-stimulatory domain exhibited better anti-tumor activity than CAR-T cells with 4-1BB domain and prolonged survival time and CAR-T cell persistence beyond 43 days after CAR-T cell infusion. Cytokine release profile of xenograft mice receiving CAR-T cells exhibited significant effector cytokine release including IFN-g, granzyme A, perforin, and granulysin. In addition, xenograft mice receiving 3x10 5Xenograft mice of 1 CAR-T cell / mouse were able to elicit potent recall responses upon re-challenge with tumor cells. In terms of on-target de-differentiation toxicity of CAR-T cells, blood toxicity assessment revealed that CLL-1 CAR-T cell candidates 61H08 HL_2828z and 61H08 LH_2828z were tolerable as they exerted 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 made from r / r AML patients and demonstrated their ability to mediate cytotoxicity against paired AML blast cells isolated from bone marrow. Thus, CLL-1 CAR-T cell candidates, i.e., 61H08 HL_2828z and 61H08 HL_2828z, have demonstrated anti-AML potency and minimal safety concerns and will be further developed for the treatment of relapsed or refractory AML patients.

[0487] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments have been presented by way of example only, as will be apparent to those skilled in the art. Various changes in form and detail will present themselves to those skilled in the art having the benefit of this disclosure. It is to be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. This means that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents are intended to be covered.

Claims

1. A chimeric antigen receptor (CAR) against human C-type lectin-like molecule-1 (CLL-1), comprising a polypeptide comprising: an extracellular antigen-binding domain comprising a single heavy-chain variable domain (VH) and a single light-chain variable domain (VL); transmembrane domain; and Intracellular signaling domain, wherein the single heavy chain variable domain comprises CDR1, CDR2 and CDR3 as shown in 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 wherein the single light chain variable domain comprises CDR1, CDR2 and CDR3 as set forth in 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 SEQ ID NO:

14.

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

15.

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

5. The CAR of claim 1, wherein the single heavy chain variable domain is located on the C-terminal side of 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 directly fused 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 connected to each other via a peptide linker.

8. The CAR of claim 7, wherein the peptide linker comprises no more than 50 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 co-stimulatory signaling domain.

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

15. The CAR of claim 12, wherein the co-stimulatory 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 of 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 of claims 1-19, 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 as shown in the amino acid sequence of SEQ ID NO: 14, and wherein the single light chain variable domain comprises CDR1, CDR2, and CDR3 as shown in 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 major intracellular signaling domain of an immune effector cell, and wherein the major intracellular signaling domain is derived from CD3ζ.

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

26. The CAR of 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. A pharmaceutical composition comprising the immune effector cell according to claim 27 and a pharmaceutically acceptable carrier.

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

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

Images