CD33-targeted chimeric antigen receptor-modified T cells for the treatment of CD33-positive malignancies

Abstract

Chimeric antigen receptors that target CD33 and their use for treating various cancers and reducing myeloid-derived suppressor cells in patients are described.

Description

[Technical field]

[0001] The present disclosure relates to leukemia-associated CD33-specific chimeric antigen receptor (CAR) recombinant T cells, methods for their formulation, and methods for their use as anti-cancer agents selective for CD33-positive cells. [Background technology]

[0002] Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has the highest mortality rate (Non-Patent Documents 1-3). CD33 is expressed on myeloblasts in 87-98% of AML cases (Non-Patent Documents 4 and 5). CD33 is also expressed on myeloablative suppressor cells (MDSCs), leukemia stem cells (LSCs), and hematopoietic stem cells (HSCs) (Non-Patent Documents 6 and 7). The current cure rate for primary AML is 35% and decreases with age, making new therapies for AML patients, especially those with relapsed or refractory (R / R) AML, an urgent need. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Budde et al. (2017) Blood 130: 811 [Non-Patent Document 2] Doehner et al. (2015) N Engl J Med 373:1136-52 [Non-Patent Document 3] Schuster et al. (2017) N Engl J Med 377:2545-54 [Non-Patent Document 4] Ehninger et al. (2014) Blood Cancer 4:e218 [Non-Patent Document 5] Andrews RG et al. (1989) J Exp Med 169:1721-31 [Non-Patent Document 6] Elliot LA et al. (2017) Front Immunol 8:86 [Non-Patent Document 7] Walter RB et al. (2012) Blood 119:6198-208 Summary of the Invention

[0004] Described herein are methods of treating various cancers, such as acute myeloid leukemia (AML), using CD33-targeted CAR T cells (also referred to herein as CD33 CAR T cells).

[0005] Described herein are nucleic acid molecules encoding a polypeptide comprising a chimeric antigen receptor (CAR), the CAR comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO: 30, or at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO: 31. Also described are nucleic acid molecules encoding a polypeptide comprising a chimeric antigen receptor (CAR), the CAR comprising the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31, in which no more than five (e.g., one, two, three, four or five) of a single amino acid have been modified. In one embodiment, the amino acid modifications are the following sequences: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1) Or the following sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 32) Or the following sequence: DIVMTQSPDSLAVSLGERATMSCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 33) be completely present within

[0006] Also described herein are expression vectors (e.g., viral or lentiviral vectors) comprising the nucleic acid molecules. Also described are populations of human T cells or NK cells transduced with the vectors or comprising the nucleic acid molecules. In various embodiments, the cells comprise, consist of, or consist essentially of central memory T cells, naive memory T cells, panT cells, NK cells, or peripheral blood mononuclear cells (PBMCs) from which CD25+ and CD14+ cells have been substantially depleted.

[0007] Also described herein is a method of treating leukemia in a patient comprising administering a population of autologous or allogeneic human T cells or NK cells transduced with a vector comprising the nucleic acid molecule, or T cells or NK cells comprising a nucleic acid molecule as described herein, wherein the leukemia comprises cells expressing CD33. In various embodiments, the chimeric antigen receptor is administered locally or systemically; the cells expressing CD33 are cancerous T cells or T regulatory cells; the chimeric antigen receptor is administered once or repeatedly; the patient is administered a methylation inhibitor prior to or simultaneously with administration of the population of autologous or allogeneic human T cells; the methylation inhibitor is a DNA methyltransferase inhibitor; the methylation inhibitor is selected from 5-azacytidine and decitabine; and the hematological cancer is selected from acute myeloid leukemia (AML); myelodysplastic syndrome; myeloproliferative neoplasm; chronic myeloid leukemia (CML); and blastic plasmacytoid dendritic cell neoplasm.

[0008] Also described herein is a method of reducing myeloid-derived suppressor cells (MDSCs) in a patient suffering from cancer, comprising administering to the patient immune cells comprising a nucleic acid molecule as described herein. In some cases, the MDSCs are lineage negative (LIN-), HLA-DR negative, and CD33 positive. In some cases, the CD33 CAR-expressing cells described herein target MDS blasts and MDSCs. In embodiments, the CD33 CAR-expressing cells described herein are used to treat multiple myeloma, chronic lymphocytic leukemia (CLL), or solid malignancies such as ovarian cancer, colon cancer, or breast cancer.

[0009] Also described herein is a method of preparing CD33 CAR T cells, comprising: providing a population of autologous or allogeneic human T cells; and transducing the T cells with a vector comprising the nucleic acid molecule of claim 1.

[0010] Described herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an scFv targeting CD33, a spacer, a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0011] In various embodiments, the transmembrane domain is selected from a CD4 transmembrane domain or a variant thereof modified by 1-5 amino acids, a CD8 transmembrane domain or a variant thereof modified by 1-5 amino acids, or a CD28 transmembrane domain or a variant thereof modified by 1-5 amino acids; the spacer comprises 20-150 amino acids and is located between the scFv and the transmembrane domain; the transmembrane domain is a CD4 transmembrane domain or a variant thereof modified by 1-5 amino acids; the transmembrane domain is a CD4 transmembrane domain; the chimeric antigen receptor comprises a transmembrane domain selected from a CD4 transmembrane domain or a variant thereof modified by 1-2 amino acids, a CD8 transmembrane domain or a variant thereof modified by 1-2 amino acids, or a CD28 transmembrane domain or a variant thereof modified by 1-2 amino acids; the spacer region is the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof having 1 to 5 amino acid modifications; the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:21; a linker of 3 to 15 amino acids is located between the 4-1BB costimulatory domain and the CD3ζ signaling domain or a variant thereof; the CAR comprises the amino acid sequence of SEQ ID NO:30 or 31 or a variant thereof having 1 to 5 amino acid modifications, e.g. 1 to 5 single amino acid substitutions; and the scFv comprises the amino acid sequence of SEQ ID NO:1.

[0012] Also described herein are viral vectors comprising the nucleic acid molecules described herein; populations of human T cells (e.g., populations that include central memory T cells) transduced with vectors comprising the nucleic acid molecules described herein.

[0013] Also described herein are methods of treating CD33-positive cancer (including, e.g., AML, R / R AML, acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), myeloma, myeloproliferative neoplasms, other CD33+ hematologic malignancies, etc.) in a patient comprising administering a population of autologous or allogeneic human T cells transduced with a vector comprising a nucleic acid molecule described herein, wherein the cancer (or disease or condition) comprises cells expressing CD33. In various embodiments, the chimeric antigen receptor is administered locally or systemically; the cells expressing CD33 are cancerous T cells; and the chimeric antigen receptor is administered once or repeatedly.

[0014] In various embodiments, the chimeric antigen receptor has the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1) wherein up to ten single amino acid substitutions are made in SEQ ID NO:1.

[0015] Also described herein are T cells comprising a vector expressing a CD33 CAR. In various embodiments, at least 20%, 30%, or 40% of the transduced human T cells are central memory T cells; at least 30% of the transduced human T cells are CD4+ and CD62L+ or CD8+ and CD62L+. In various embodiments, the population of human T cells comprises a vector expressing a chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NO: 30 or 31, or a variant (e.g., substitution) thereof in which one to five amino acids have been modified (e.g., one or two); the population of human T cells is central memory T cells (T CM cells), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells are T CM or the population of T cells is a combination of central memory T cells, naive T cells and stem memory cells (T CM / SCM / N cells), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells are T CM / SCM / N In some embodiments, the population of T cells includes both CD4+ and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+, at least 3% of the CD3+ T cells are CD8+, and at least 70, 80, or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells are CD4+, and at least 4%, 5%, 8%, 10%, 20% of the CD3+ cells are CD8+ cells). In some embodiments, the population of human T cells is autologous to the patient. In some embodiments, the population of human T cells is allogeneic to the patient.

[0016] CD33 targeting CAR The CD33-targeting CAR described herein comprises an scFv that targets CD33. In one embodiment, the CAR has the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1), or the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 32) and the following amino acid sequence linked by a flexible linker: DIVMTQSPDSLAVSLGERATMSCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 33) and wherein the scFv comprises:

[0017] A useful CD33 CAR can consist of or comprise the amino acid sequence of SEQ ID NO: 30 or 31 (signal sequence-depleted mature CAR), or the CD33 CAR can consist of or comprise the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31 preceding the GMCSFRa signal sequence (SEQ ID NO: 34) (GMCSFR signal sequence-containing immature CAR; (MLLVTSLLCHHPAFLLIP; SEQ ID NO: 34)). That is, a CAR can be expressed with additional sequences useful for monitoring expression, such as a T2A skip sequence and a truncated EGFRt. A CAR can be expressed with additional sequences useful for monitoring expression, such as a T2A skip sequence and a truncated CD19t. Thus, a CAR can comprise or consist of the amino acid sequence of SEQ ID NO: 30 or 31, or can comprise or consist of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO: 30 or 31. A CAR can comprise or consist of the amino acid sequence of either SEQ ID NO: 30 or 31 with up to 1, 2, 3, 4 or 5 amino acid modifications (preferably conservative amino modifications). A CAR can comprise an scFv that targets CD33, for example an scFv comprising SEQ ID NO: 32 with up to 1, 2, 3, 4 or 5 amino acid modifications (preferably conservative amino modifications) and an scFv comprising SEQ ID NO: 33 linked by a flexible linker with up to 1, 2, 3, 4 or 5 amino acid modifications (preferably conservative amino modifications).

[0018] In one embodiment, the nucleic acids encoding the amino acid sequences of SEQ ID NOs: 1 to 34 are codon-optimized. In one embodiment, the nucleic acids encoding the amino acid sequences of SEQ ID NOs: 1 to 34 are not codon-optimized.

[0019] Spacer Region The CARs described herein can include a spacer located between the CD33 targeting domain (i.e., the CD33 targeting ScFv or variant thereof) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least a portion of a human Fc region, such as a hinge portion of a human Fc region, or a CH3 domain or variant thereof. Table 1 below provides various spacers that can be used in the CARs described herein. Table 1: Examples of spacers

[0020] [Table 1] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. The immunoglobulin-derived sequence can include one or more amino acid modifications, e.g., 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0021] The hinge / linker region can also include an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3). The hinge / linker region can also include the sequence ESKYGPPCPPCP (SEQ ID NO:3), followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2), followed by the IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:12). Thus, the entire linker / spacer region can comprise the sequence ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11). In some cases, the spacer contains 1, 2, 3, 4 or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 11. In some cases, the IgG4 Fc hinge / linker region is mutated at two positions (L235E; N297Q) to reduce binding by the Fc receptor (FcR).

[0022] Transmembrane domain A variety of transmembrane domains can be used. Examples of suitable transmembrane domains are provided in Table 2. When a spacer region is present, the transmembrane domain (TM) is located carboxy-terminal to the spacer region. Table 2: Examples of transmembrane domains

[0023] [Table 2] Costimulatory domain The costimulatory domain can be any domain suitable for use with a CD3ζ signaling domain. In some cases, the co-signaling domain is a 4-1BB co-signaling domain, including a sequence that is at least 90%, at least 95%, at least 98% identical to KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:24. The costimulatory domain is located between the transmembrane domain and the CD3ζ signaling domain. Table 3 illustrates the sequence of the CD3ζ signaling domain and suitable costimulatory domains. Table 3: Examples of CD3 ζ domains and costimulatory domains

[0024] [Table 3] In various embodiments, the costimulatory domain is selected from the group consisting of a costimulatory domain shown in Table 3 or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, and an OX40 costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, the 4-1BB costimulatory domain or a variant thereof is modified with 1-5 (e.g., 1 or 2) amino acids. In certain embodiments, there are two costimulatory domains, for example, a CD28 costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a 4-1BB costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions. The costimulatory domain is amino-terminal to the CD3 zeta signaling domain, and a short linker of 2-10 amino acids, e.g., 3 amino acids (e.g., GGG), may be located between the costimulatory domain and the CD3 zeta signaling domain.

[0025] CD3ζ Signaling Domain The CD3 zeta signaling domain can be any domain suitable for use with the CD3 zeta signaling domain. In some cases, the CD3 zeta signaling domain has the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21) The present invention includes sequences that are at least 90%, at least 95%, or at least 98% identical or identical to In some cases, the CD3ζ signaling has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21.

[0026] Cleaved EGFR and Cleaved CD19 The CD3 zeta signaling domain is followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and the following sequence: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 28) In some cases, the truncated EGFR has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 28.

[0027] Alternatively, the CD3 zeta signaling domain may be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and the following sequence: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 26) A truncated CD19R (referred to as CD19t) may be located in the sequence, which is identical or at least 90%, at least 95%, at least 98% identical to:

[0028] By "amino acid modification" is meant an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. "Amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. Such substitutions can change the amino acid in the resulting protein in a non-conservative manner (i.e., changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristics to an amino acid belonging to another group) or in a conservative manner (i.e., changing a codon from an amino acid belonging to a group of amino acids of a particular size or characteristics to an amino acid belonging to the same group). Such conservative changes generally result in less alteration of the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with non-polar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) Amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (positively charged at pH 6.0). The other group is made up of amino acids containing phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0029] In some cases, CD33 CARs can be produced using vectors in which the CAR open reading frame is followed by a T2A ribosomal skip sequence and a truncated EGFR (EGFRt) with a deleted cytoplasmic signaling tail. In this arrangement, co-expression of EGFRt provides an inert, non-immunogenic surface marker by which genetically modified cells can be accurately measured, positively selected for genetically modified cells, and efficiently tracked cells in vivo for therapeutic T cells after adoptive transfer. Efficient control of proliferation to avoid cytokine storm and off-target toxicity is a key hurdle for successful T cell immunotherapy. EGFRt incorporated into the CD33 CAR lentiviral vector can act as a suicide gene and eliminate CAR+ T cells in the event of treatment-related toxicity.

[0030] The CAR described herein is preferably produced using recombinant DNA technology, but can be produced by any means known in the art. The nucleic acid encoding several regions of the chimeric receptor can be conveniently prepared and the complete coding sequence constructed by standard techniques of molecular cloning known in the art (genomic library screening, overlap PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably T lymphocytes, most preferably autologous T lymphocytes.

[0031] Various T cell subsets isolated from patients may be transduced with vectors for CAR expression. Central memory T cells are one of the useful T cell subsets. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by selecting CD45RO+ / CD62L+ cells, for example, using a CliniMACS® device, and immunomagnetically selecting cells expressing the desired receptor. Cells enriched for central memory T cells can be activated with anti-CD3 / CD28 and transduced with, for example, lentiviral vectors directing the expression of CD33 CAR, as well as non-immunogenic surface markers for in vivo detection, ablation, and potential ex vivo selection. Activated / genetically modified CD33 central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Further methods for preparing CAR T cells are described in PCT / US2016 / 043392.

[0032] In other embodiments, the CD33 CAR-expressing cell is an immune effector cell, e.g., a T cell or an NK cell, that is harboring an exogenous RNA molecule, e.g., an ex vivo transcribed or synthetic RNA comprising a nucleic acid encoding a CAR molecule described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database listings, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the definitions herein will take precedence. Other features and advantages of the invention will be apparent from the following description and drawings, and from the claims. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram showing a representative CD33 CAR construct. [Diagram 2] Graph showing the results of an experiment using CD33 (CD8) CAR T cells with stable expression over a 13-day culture period, using EGFRt as a tracking marker. Mock (non-transduced: 2A) and CD33 CAR T cells (2B) were assessed for EGFR expression by flow cytometry to detect CAR expression. Both Mock and CD33 CAR T cells expanded stably in ex vivo culture (2C). [Diagram 3] 1 shows the expression of CD33 in human AML cell lines.10 is a graph showing the results of an experiment using the human Burkitt's lymphoma cell line RAJI as a negative control. [Figure 4A] Graph showing effector function and killing activity of CD33(CD8) CAR T cells against CD33 expressing AML cell lines. Degranulation assay results with E:T ratio of Mock or CD33 CAR T cells to AML cells at 2:1 are shown. [Figure 4B] Graph showing effector function and killing activity of CD33(CD8) CAR T cells against CD33 expressing AML cell lines.Intracellular IFN-γ staining assay results with 1:1 E:T ratio of Mock or CD33 CAR T cells to AML cells are shown. [Figure 4C] Graph showing effector function and killing activity of CD33 (CD8) CAR T cells against CD33 expressing AML cell lines. Results of re-administration assays with E:T ratios of Mock or CD33 CAR T cells to AML cells of 1:5, 1:10, and 1:20 are shown. RAJI cell line was used as a negative control. [Diagram 5] FIG. 1 shows a representative in vivo study using a NOD-SCID-IL2Rgnull (NSG) xenograft model with the MOLM-14-ffluc cell line, an AML cell line with high CD33 expression levels. [Figure 6] Figures 6A-6B show the ability of CD33 (CD8) CAR T cells to kill CD33-expressing tumor lines in vivo. Figure 6A is a photograph showing that 3x106 CD33 CAR T cells significantly reduced leukemia burden in vivo compared to 3x106 Mock T cells or untreated. Figure 6B is a graph showing that 3x106 CD33 CAR T cells significantly increased overall survival in vivo compared to 3x106 Mock T cells or untreated. [Figure 7] Figure 1 shows the expression of CD33 in AML cell lines on two consecutive days in the presence of 100 nM decitabine or DMSO. The RAJI cell line was used as a negative control. [Figure 8] FIG. 1 is a schematic diagram showing the killing activity of decitabine in combination with CD33 (CD8) CAR T cells in vitro. AML cell lines were treated with 10 nM decitabine or DMSO for two consecutive days, followed by treatment with CD33 CAR or Mock T cells at an E:T ratio of 1:50. After another 48 hours, the killing activity of each group was measured by flow cytometry. RAJI cell line was used as a negative control. [Figure 9] 9A-9B show the mean fluorescence intensity (MFI) of programmed cell death-ligand 1 (PD-L1) and c-type lectin-like molecule-1 (CLL-1) on remaining AML cells after combination of decitabine or DMSO with CD33 (CD8) CAR or Mock T cells. RAJI cell line was used as a negative control. [Figure 10] A representative in vivo study is shown using an NSG xenograft model with MOLM-14-ffluc administered decitabine in combination with CD33 CAR T cell therapy. [Figure 11]11A-11B show the results of the in vivo combination efficacy of decitabine and CD33 (CD8) CAR T cells. A is a graph showing that administration of 1×106 CD33 CAR T cells after administration of 0.5 mg / kg / day×5 days of decitabine significantly reduced leukemia burden in vivo. B is a graph showing that combination therapy of 0.5 mg / kg / d×5 days of decitabine and 1×106 CD33 CAR T cells significantly extended overall survival in vivo. [Figure 12] FIG. 11 shows that treatment with 10 nM decitabine and CD33 (CD8) CAR T cells at a 1:50 E:T ratio significantly increased PD-L1 expression levels by the remaining surviving AML cells. [Figure 13] Graph showing that anti-PD-1 antibody (10 μg / mL) significantly enhanced the anti-AML effect of CD33 (CD8) CAR T cells at an E:T ratio of 1:75. [Figure 14] The annotated amino acid sequences of CD33 CD8 CAR (A; SEQ ID NO: 30) and CD33 EQ CAR (B; SEQ ID NO: 31) are shown. Underlining indicates various components. [Figure 15] 1 shows the annotated nucleic acid and corresponding amino acid sequences of (A) CD33 CD8 CAR preceded by a GMCSR signal sequence and followed by a T2A skip sequence and EGFRt (SEQ ID NO: 35 (nucleic acid) and SEQ ID NO: 36 (amino acid)); and (B) CD33 EQ CAR preceded by a GMCSR signal sequence and followed by a T2A skip sequence and EGFRt (SEQ ID NO: 35 (nucleic acid) and SEQ ID NO: 36 (amino acid)). The codon-optimized portions of each nucleic acid sequence are shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] This disclosure describes the generation and antitumor efficacy of CARs containing a humanized anti-human CD33 scFv antigen-binding domain and, in certain embodiments, a 4-1BB intracellular costimulatory signaling domain. CD33 CAR T cells exhibited potent antigen-dependent cytotoxicity against CD33-expressing human cancer lines. Intraperitoneal or intravenous in vivo administration of CD33 CAR T cells in human leukemia / lymphoma mouse tumor models eliminated antigen-positive disease and extended overall survival. EXAMPLES

[0035] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1 Construction of CD33 CAR T cells containing different linkers The studies described below show that the CD33 CAR can be stably expressed on primary T cells. Several CD33-targeted CAR constructs were designed. All constructs expressed the same codon-optimized humanized CD33 single chain variable fragment. In one embodiment, the CAR construct also contained a CD4 transmembrane domain (TM), a 4-1BB costimulatory domain, and a CD3 zeta domain. In one embodiment, the CAR construct also contained a CD8 transmembrane domain (TM), a 4-1BB costimulatory domain, and a CD3 zeta domain. A representative schematic of a CD33 CAR is shown in FIG. 1. The CAR was co-expressed with a truncated EGFR, which served as a marker for successful transduction of cells with the CAR construct. In one embodiment, the CD33 CAR constructs differ in their linker domain and transmembrane domain. Without being bound by theory, the different lengths of the extracellular portion of the constructs may result in different functions of the CAR to bind antigen and transmit activation signals after antigen binding. These differences may also result in different killing of CD33-expressing tumor cells.

[0036] Figure 14A shows the amino acid sequence of a CD33 CAR comprising a CD33 targeting scFv, a CD8 hinge (spacer), a CD8 transmembrane domain, a 4-1BB costimulatory domain linked by a GGG linker to a CD3 zeta stimulatory domain (SEQ ID NO: 30). Figure 14B shows the amino acid sequence of a CD33 CAR comprising a CD33 targeting scFv, an IgG4 spacer (IgG4 (S228P, L235E, N297Q)), a CD4 transmembrane domain, a 4-1BB costimulatory domain linked by a GGG linker to a CD3 zeta stimulatory domain (SEQ ID NO: 31).

[0037] As previously reported (Priceman SJ, Gerdts EA, Tilakawardane D, Kennewick KT, Murad JP, Park AK, Jeang B, Yamaguchi Y, Yang X, Urak R, Weng L, Chang WC, Wright S, Pal S, Reiter RE, Wu AM, Brown CE, Forman SJ. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+metastatic prostate cancer. Oncoimmunology. 2018;7(2):e1380764), CD33 CAR lentivirus was used to transduce other cell types, including peripheral blood mononuclear cells, T(n / mem) cells, Pan T cells, and enriched T cells from human healthy donors depleted of CD14+ and CD25+ cells (dPBMCs) (EasySep Human T cell isolation Kit. StemCell Technologies). CAR expression was demonstrated by flow cytometry as described above, using EGFRt as a tracking marker. Representative CD33 CAR constructs were stably expressed in T cells (Figures 2A-2C). Seven days after transduction, cells were stained with anti-EGFR to indicate successful uptake of the CD33 CAR construct. CD33 (CD8) CAR was stably expressed 15 days after transduction (Figure 2C).

[0038] Example 2 Expression of CD33 in AML cell lines The studies described below examined the expression of CD33 in various AML cell lines. As shown in FIG. 3, CD33 expression was highest in MOLM-14 and THP-1 cell lines. CD33 expression was moderate in the KG-1A cell line. CD33 expression was lowest in the Raji cell line.

[0039] Example 3: CD33 CAR T cells exhibit potent and specific cell killing and effector functions in vitro To determine whether CD33 CAR T cells exhibit selective effector function and killing activity against CD33-positive cancer cells, CD33 CAR T cells were expanded in the presence of either CD33-positive or CD33-negative cancer cells. CD107a degranulation levels, intracellular IFN-γ staining, and the percentage of killed cancer cells were then quantified. A schematic diagram of the assay used is shown in Figure 4. For CD107a degranulation assay, Mock or CD33 CAR T cells were co-cultured with target cells at an E:T ratio of 2:1 for 6 h in complete X-VIVO medium supplemented with Golgi stop and CD107a antibody, followed by flow cytometry. The CD33-positive AML cell lines were MOLM-14, THP-1, and KG-1A. The CD33-negative cell line used was RAJI (Figure 4A). For intracellular IFN-γ staining assay, Mock or CD33 CAR T cells were co-cultured with target cells at an E:T ratio of 1:1 for 4–6 h in complete X-VIVO medium. Golgi plug was then added and the cultures were incubated overnight. Cells were then fixed, permeabilized, stained with IFN-γ antibody, and subjected to flow cytometry (Figure 4B). For rechallenge assays, Mock CAR T cells or CD33 CAR T cells were co-cultured with target cells at E:T ratios of 1:5, 1:10, and 1:20. After 48 hours, additional tumor cells were added to the co-culture wells at E:T of 1:5, 1:10, or 1:20, the 48 hour incubation was repeated, and additional tumor cells were added to the co-culture wells at E:T of 1:5, 1:10, or 1:20. After another 48 hours, the % specific lysis of tumor cells was measured using flow cytometry (Figure 4C). CD33(CD8) CAR T cells displayed potent and specific CD107a degranulation levels, intracellular IFN-γ staining, and killing activity against CD33-positive AML cell lines compared to the CD33-negative RAJI cell line (Figures 4A-4C).

[0040] Example 5: Demonstration that delivered CD33 CAR T cells exhibit potent antitumor activity and provide survival benefits in mice in vivo in a mouse model To evaluate the in vivo efficacy of CD33 CAR T cells to selectively target CD33-positive cells in the MOLM-14 model, CD33 CAR T cells were delivered and tumor size and survival were assessed over time. MOLM-14 cells were transduced with lentivirus to express firefly luciferase (ffluc) to track tumor growth by non-invasive optical imaging. Six days after intravenous tumor injection, mice were treated with Mock or CD33 CAR T cells (3 × 10 6 ) (Figure 5). In mice treated with CD33(CD8) CAR T cells via intravenous delivery, a rapid antitumor effect was observed, with the antitumor response peaking 1–2 weeks after treatment (Figure 6A). The antitumor response in mice was sustained for 3–4 weeks. Delivery of CD33(CD8) CAR T cells significantly prolonged mouse survival (Figure 6B).

[0041] Example 6 Decitabine enhances CD33 CAR T cell-mediated AML killing in vitro Decitabine is a hemoglobin methylation inhibitor (HMA) that is often used in the treatment of AML. First, AML cell lines were treated with 100 nM decitabine or DMSO for two consecutive days. RAJI cell line was used as a negative control. Decitabine treatment was observed to upregulate CD33 expression on AML cells (Figure 7). We hypothesized that decitabine may enhance CD33 CAR T cell-mediated AML killing. In an in vitro combination study, AML cell lines were treated with 10 nM decitabine or DMSO for two consecutive days, followed by treatment with CD33 (CD8) CAR or Mock T cells at an E:T ratio of 1:50. After an additional 48 hours, the killing ratio (%) of each group was quantified using flow cytometry. In an in vitro experiment using AML cells as targets, potent killing was observed in the group treated with decitabine followed by CD33 CAR T cells (Figure 8). Furthermore, we detected MFI expression of PD-L1 and CLL-1 in the residual AML cells that survived treatment with decitabine in combination with CD33 CAR T cells. The expression levels of PD-L1 (Figure 9A) and CLL-1 (Figure 9B) were significantly increased by the ex vivo combination treatment.

[0042] Example 7: Decitabine improves the anti-leukemia efficacy of CD33 CAR T cells in vivo 1 × 10 in NSG mice 6 MOLM-14-ffluc cells were administered intravenously on day 0. From days 1 to 5, decitabine 0.5 mg / kg / day or PBS was administered by intraperitoneal (ip) injection for 5 consecutive days. On day 6, mice were administered 1 × 10 6 CD33 (CD8) CAR (SEQ ID NO: 30) or Mock T cells were administered intravenously (Figure 10). Consistent with the in vitro findings, the combination of decitabine with CD33 CAR T cells significantly reduced leukemic burden (Figure 11A) and improved overall survival in vivo (Figure 11B).

[0043] Example 8: PD-1 / PD-L1 blockade enhances the efficacy of CD33 CAR T cells Checkpoint blockade targeting programmed cell death-1 (PD-1) / PD-L1 is involved in resistance mechanisms to immune system therapy. Residual AML cells surviving treatment with 10 nM decitabine and CD33 (CD8) CAR (SEQ ID NO: 30) T cells at a 1:50 E:T ratio significantly increased PD-L1 expression (Figure 12). Anti-PD-1 antibody (10 μg / mL) was found to significantly enhance the anti-AML efficacy of CD33 (CD8) CAR (SEQ ID NO: 30) T cells at an E:T ratio of 1:75 (Figure 13).

[0044] Other embodiments Although the present invention has been described in conjunction with its detailed description, it will be understood that the above description is intended to be illustrative and not limiting of the scope of the invention as defined in the appended claims. Other aspects, advantages, and modifications are within the scope of the claims. All references are incorporated herein in their entirety for all purposes.

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence represented by SEQ ID NO: 30 or SEQ ID NO:

31.

2. The nucleic acid molecule of claim 1 , wherein the CAR comprises an amino acid sequence identical to the amino acid sequence represented by SEQ ID NO:

30.

3. The nucleic acid molecule of claim 1 , wherein the CAR comprises an amino acid sequence identical to the amino acid sequence represented by SEQ ID NO:

31.

4. An expression vector comprising the nucleic acid molecule of claim 1.

5. A viral vector comprising the nucleic acid molecule of claim 1.

6. A population of human T cells transduced with a vector comprising the nucleic acid molecule of claim 1.

7. A population of human T cells comprising a nucleic acid molecule according to any one of claims 1 to 3.

8. 8. The population of claim 6 or 7, comprising central memory T cells, naive memory T cells, pan T cells, NK cells, or peripheral blood mononuclear cells (PBMCs) from which CD25+ cells and CD14+ cells have been depleted.

9. 1. A population of human T cells comprising a nucleic acid molecule encoding a CAR, wherein the CAR comprises an amino acid sequence comprising SEQ ID NO: 30 or 31.

10. 13. A population of human T cells comprising the nucleic acid molecule of claim 1 for use in a method for treating leukemia or cancer in a patient.

11. The population of human T cells of claim 10, which is autologous or allogeneic.

12. The population of human T cells of claim 10, which is administered locally or systemically.

13. The population of human T cells of claim 10, wherein the leukemia comprises cells expressing CD33.

14. The population of human T cells described in claim 13, wherein the cells expressing CD33 are cancer T cells or T regulatory cells.

15. The population of human T cells of claim 10, wherein the chimeric antigen receptor is administered once or repeatedly.

16. 11. The population of human T cells of claim 10, wherein the patient is administered a methylation inhibitor prior to or simultaneously with administration of the population of autologous or allogeneic human T cells.

17. The population of human T cells of claim 16, wherein the methylation inhibitor is a DNA methyltransferase inhibitor.

18. The population of human T cells of claim 16, wherein the methylation inhibitor is selected from 5-azacytidine and decitabine.

19. 11. The population of human T cells of claim 10, wherein the cancer is a hematological cancer selected from acute myeloid leukemia (AML); myelodysplastic syndrome; myeloproliferative neoplasm; chronic myeloid leukemia (CML); and blastic plasmacytoid dendritic cell neoplasm.

20. A population of immune cells comprising the nucleic acid molecule of claim 1 for use in a method for reducing myeloid-derived suppressor cells (MDSCs) in a patient suffering from cancer.

21. 21. The population of claim 20, wherein the cancer is a solid tumor.

22. An in vitro method for preparing CD33 CAR T cells, comprising: Providing a population of autologous or allogeneic human T cells; and transducing T cells in vitro with a vector comprising the nucleic acid molecule of claim 1; A method comprising:

Images