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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
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  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C12N2510/00—Genetically modified cells

Definitions

• the engineered T cell comprises: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising the amino acid sequence of SEQ ID NO: 104; (ii) a disrupted b2M gene; and a disrupted CD33 gene.
• AAATCCTCATCTGGCACT (SEQ ID NO: 311), AAATCCT, AAACCCTGGCACT (SEQ ID NO: 312), AAATCCTCTGGCACT (SEQ ID NO: 313), AAATCCCCCTGGCACT (SEQ ID NO: 314), AAATCCTCACT (SEQ ID NO: 315), ACATCCCTGGCACT (SEQ ID NO:
• proliferative capacity of engineered T cells of the population is within 10% of proliferative capacity of control cells.
• the CAR comprises the nucleotide sequence of any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 110, 113, 116 or 119.
• FIG. 2B shows the % of cells expressing CAR in TRAC-/p2M- T cells over time. All anti-CD33 CAR-T cells expanded over the two week period.
• CTX-965b and CTX-970 CAR T cells are effective at inducing cell lysis in MV4-l l cells at cell ratios of 0.05:l to 1 : 1 CAR-T cells:MV4-l 1.
• FIG. 6B shows that CTX-965b and CTX-970 CAR T cells are effective at secreting IFNy in the presence of THP-1 cells at cell ratios of 0.05: 1 to 1: 1 CAR-T cells:THP-l.
• FIG. 6C shows that CTX-965b and CTX-970 CAR T cells are effective at secreting IFNy in the presence of MV-411 cells at cell ratios of 0.05: l to 1: 1 CAR-T cells:MV-411.
• FIG. 6D shows that CTX-965b and CTX-970 CAR T cells are effective at secreting IFNy in the presence of KGl cells at cell ratios of 0.05: 1 to 1: 1 CAR-T cells:KGl.
• FIG. 6E shows that CTX-965b CAR T cell growth is cytokine dependent.
• FIG. 8 includes a graph showing that TRAC-/p2M-/anti-CD33 CAR+ T cells (CTX- 965b CAR T cells) are effective at reducing tumor volume of well-established tumors (starting tumor volume of approximately 150 mm 3 ) in a subcutaneous THP-1 AML cancer in vivo mouse model.
• CTX- 965b CAR T cells TRAC-/p2M-/anti-CD33 CAR+ T cells
• gRNA sequences may be designed using the TRAC gene sequence located on chromosome 14 (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734).
• an edited b2M gene comprises at least one nucleotide sequence selected from the following sequences in Table 2:
• ACTCCCCAGTTCATGGTTAC SEQ ID NO: 196
• SEQ ID NO: 196 ACTCCCCAGTTCATGGTTAC
• the edited CD 33 gene may lack a fragment comprising
• ACTACTCACTCCTCGGTGCT SEQ ID NO: 268, or a portion thereof, which may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides.
• Such an edited CD33 gene may be produced using a guide RNA comprising a spacer sequence of SEQ ID NO: 170 (e.g, the gRNA of SEQ ID NO: 148).
• AAATCCCCTGGCACT SEQ ID NO: 307
• ACATCCTCATTCCCTGGCACT SEQ ID NO: 308
• ACATCCTGGCACT SEQ ID NO: 309
• AAATCCTCTCCCTGGCACT SEQ ID NO: 310
• AAATCCTCATCTGGCACT SEQ ID NO: 311
• AAATCCT AAATCCT
• Such an edited CD33 gene may be produced using a guide RNA comprising a spacer sequence of SEQ ID NO: 173 ⁇ e.g., the gRNA of SEQ ID NO: 151).
• ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence- specific manner through one or more zinc fingers.
• ZFBD zinc finger DNA binding domain
• a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
• a designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S.
• a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
• a "transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA.
• TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
• the sgRNA can comprise 5 uracil (UUUUU) at the 3' end of the sgRNA sequence.
• the sgRNA can comprise 6 uracil (UUUUUU) at the 3' end of the sgRNA sequence.
• the sgRNA can comprise 7 uracil (UUUUUUU) at the 3 ' end of the sgRNA sequence.
• the sgRNA can comprise 8 uracil (UUUUUUUUU) at the 3' end of the sgRNA sequence.
• a gRNA comprises a spacer sequence.
• a spacer sequence is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target nucleic acid of interest.
• the spacer sequence is 15 to 30 nucleotides.
• the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
• a spacer sequence is 20 nucleotides.
• The“target sequence” is adjacent to a PAM sequence and is the sequence modified by an RNA-guided nuclease ( e.g ., Cas9).
• the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3' (SEQ ID NO: 19).
• the spacer of a gRNA interacts with a target nucleic acid of interest in a sequence- specific manner via hybridization (i.e ., base pairing).
• the nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
• the target nucleic acid comprises the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.
• a chimeric antigen receptor refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by tumor cells.
• a CAR is designed for a T cell and is a chimera of a signaling domain of the T-cell receptor (TCR) complex and an antigen-recognizing domain (e.g . , a single chain fragment (scFv) of an antibody or other antibody fragment) (Enblad et al., Human Gene Therapy. 2015; 26(8):498- 505).
• TCR T-cell receptor
• scFv single chain fragment
• a T cell that expresses a CAR is referred to as a CAR T cell.
• CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner.
• a chimeric antigen receptor is a first generation CAR.
• a chimeric antigen receptor is a second generation CAR.
• a chimeric antigen receptor is a third generation CAR.
• the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence: IYIW APFAGTCGVFFFSFVITFY (SEQ ID NO: 163).
• the term“antibody” encompasses not only intact (i.e ., full-length) monoclonal antibodies, but also antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single chain variable fragment (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, single domain antibodies (e.g ., camel or llama VF1F1 antibodies), multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
• antigen-binding fragments such as Fab, Fab', F(ab')2, Fv
• scFv single chain variable fragment
• mutants thereof fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies,
• an antibody of the present disclosure is a humanized antibody.
• Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
• Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• an antibody of the present disclosure specifically binds a target antigen, such as human CD33.
• a target antigen such as human CD33.
• An antibody that“specifically binds” (used interchangeably herein) to a target or an epitope is a term well understood in the art, and methods to determine such specific binding are also well known in the art.
• a molecule is said to exhibit“specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets.
• An antibody “specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
• the anti-CD33 antibody may comprise a VL CDR1, a VL CDR2, and a VL CDR3, which collectively contains no more than 10 amino acid variations (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation) as compared with the VL CDR1, VL CDR2, and VL CDR3 of the reference antibody.
• the anti-CD33 antibody disclosed herein may comprise a VH CDR1, a VH CDR2, and a VH CDR3, at least one of which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation) as the counterpart VH CDR of a reference antibody such as Antibody B (VH: SEQ ID NO: 77; VL: SEQ ID NO: 78).
• the antibody comprises a VH CDR3, which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation) as the VH CDR3 of a reference antibody such as Antibody B (VH: SEQ ID NO: 77; VL: SEQ ID NO: 78).
• Modes of administration include injection, infusion, instillation, or ingestion.
• the engineered T cell of embodiment 1 further comprising a disrupted T cell receptor alpha chain constant region ( TRAC) gene.
• TRAC T cell receptor alpha chain constant region
• E5 The engineered T cell of any one of embodiments 1-4, wherein the ectodomain of the CAR comprises an anti-CD33 antibody.
• E24 The population of embodiment 22, wherein at least 25% of engineered T cells of the population express the CAR following at least 7 days or at least 14 days of in vitro proliferation.
• E25 The population of any one of embodiments 22-24, wherein at least 50% of engineered T cells of the population do not express a detectable level of T cell receptor (TCR) protein.
• TCR T cell receptor
• E26 The population of embodiment 25, wherein at least 90% of engineered T cells of the population do not express a detectable level of TCR protein.
• E31 The population of any one of embodiments 22-30, wherein engineered T cells of the population, when co-cultured in vitro with a population of cancer cells that express CD33, induce cell lysis of at least 10%, at least 25%, or at least 50% of the cancer cells of the population.
• E33 The population of embodiment 31 or embodiment 32, wherein engineered T cells of the population, when co-cultured in vitro with a population of cancer cells, secrete IFNy.
• E37 A method comprising administering the population of engineered T cells of any one of embodiments 22-36 to a subject.
• cancer is a leukemia, optionally acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).
• ALL acute lymphoblastic leukemia
• AML acute myeloid leukemia
• CLL chronic lymphocytic leukemia
• CML chronic myeloid leukemia
• a method for producing an engineered T cell comprising
• E44 The method of embodiment 43, wherein the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19, or targets the nucleotide sequence of SEQ ID NO: 40.
• E45 The method of embodiment 43 or 44 wherein the nucleic acid encoding the CAR is flanked by left and right homology arms to the TRAC gene.
• RNA-guided nuclease is a Cas9 nuclease, optionally a S. pyogenes Cas9 nuclease.
• E58 The method of any one of embodiments 43-57, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 109, 112, 115, or 118.
• E60 A method for reducing volume of a tumor in a subject having cancer, the method comprising administering to the subject a population of engineered T cells any one of embodiments 22-36.
• E61 The method of embodiment 60, wherein the volume of the tumor in the subject is reduced by at least 50% relative to a baseline control, optionally wherein lxlO 5 cells to lxlO 7 cells of the population are administered.
• E63 The population of cells of embodiment 62, wherein the CAR comprises (a) an ectodomain that comprises an anti-CD33 antigen-binding fragment, (b) a CD8
• transmembrane domain and (c) an endodomain that comprises a 4 IBB co-stimulatory domain and a CD3z co-stimulatory domain.
• E64. The population of cells of embodiment 62 or embodiment 63, wherein the disrupted TRAC gene comprises the nucleic acid encoding the CAR.
• E65 The population of cells of any one of embodiments 62-64 further comprising a disrupted CD33 gene.
• E66 A population of cells comprising engineered T cells, wherein the engineered T cells comprise:
• disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising the amino acid sequence of SEQ ID NO: 104;
• E75 The engineered T cell of embodiment 74, wherein the CAR comprises (a) an ectodomain that comprises an anti-CD33 antigen-binding fragment, (b) a CD8
• transmembrane domain and (c) an endodomain that comprises a 4 IBB co-stimulatory domain and a CD3z co-stimulatory domain.
• E86 The engineered T cell of any one of embodiments 1-21 and 74-85, wherein the T cell is a human T cell.
• cancer is a leukemia, optionally acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).
• ALL acute lymphoblastic leukemia
• AML acute myeloid leukemia
• CLL chronic lymphocytic leukemia
• CML chronic myeloid leukemia
• E90 An engineered T cell produced by any one of the methods of embodiments 43-59.
• E97 An engineered T cell of any one of embodiments of 1-21, wherein the edited CD 33 gene comprises a nucleotide sequence of ACTCCCCAGTTTCATGGTTAC (SEQ ID NO: 197), ACTCCCCAGTCATGGTTAC (SEQ ID NO: 198), ACTCCCCATGGTTAC (SEQ ID NO: 199), ACTCCCCAGTTAC (SEQ ID NO: 200), ACTCATGGTTAC (SEQ ID NO: 201), ACTCCCCATCATGGTTAC (SEQ ID NO: 202), ACTCCCCATTCATGGTTAC (SEQ ID NO: 203), ACTCCCCAGTGTCATGGTTAC (SEQ ID NO: 204), and/or
• E99 An engineered T cell of any one of embodiments of 1-21, wherein the edited CD 33 gene lacks a fragment, the 3 ' segment of which comprises the nucleotide sequence of ACTCCCCAGTTCATGGTT (SEQ ID NO: 206). E100.
• E109 An engineered T cell of any one of embodiments of 1-21, wherein the edited CD33 gene lacks a fragment, the 3 ' segment of which comprises the nucleotide sequence of AGGTGAAGTTCG (SEQ ID NO: 256), AGGTGAAGTTCGCTGGAG (SEQ ID NO: 259), AGGTGAAGTTCGCTGG (SEQ ID NO: 260), or AGGTGAAGTT (SEQ ID NO: 261).
• On-target sequence centered on cleavage site, with 10 bp in either direction.
• the portion of the CD33-1 gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.
• Example 5 Stabilization of CAR expression and CD4/CD8 cell populations with CD33 knock-out.
• Activated T cells were first electroporated with 3 distinct Cas9:sgRNA RNP complexes, one containing sgRNAs targeting the TRAC locus (SEQ ID NO: 28), the second containing sgRNAs targeting the b2M locus (SEQ ID NO: 30) and the third containing the sgRNAs targeting CD33 (CD33-10: SEQ ID NO: 151).
• the anti-CD33 CAR construct was comprised of right and left homology arms corresponding to the TRAC locus that flanked a chimeric antigen receptor cassette (-/+ regulatory elements for gene expression).
• the resulting modified T cells are 3X KO (TRAC- ⁇ 2M-/CD33-), anti-CD33 CAR+ T cells.
• the 3X KO anti-CD33 CAR T cells were compared to the 2X KO (TEAO-/b2M-), anti-CD33 CAR T cells generated as described above.
• Anti-CD33 CAR expression and CD4/CD8 cell populations were assessed as described in Example 2.
• TRAC-/B2M-/CAR+ (2X KO, CAR+) edited T cells demonstrated an increase in the percentage of cells that were CAR+ over time (FIG. 2B).
• T cells were edited with six different CAR constructs (CTX-981, CTX-98lb, CTX-982, CTX-982b, CTX-970, CTX-965b) and evaluated for the ability of CD33 KO to stabilize CAR expression.
• FIG. 10 shows that the percentage of cells positive for CAR expression increased between 7 and 14 days for T cells that were edited with 2X KO (TI7LO-/b2M-) and anti-CD33 CAR.
• the T cells were generated from two different human donors and were edited with CAR construct CTX-965b, CTX-970 or CTX-982.
• the T cells were incubated with MV4-11 cells at a ratio of CAR T cell to target cell of 0.05 : 1 1 : 1 and the percentage of cell lysis among MV4-11 cells was determined as described above.
• Both 2X KO CAR+ T cells and 3X KO CAR+ T cells demonstrated efficient killing of MV4-11 cells, even at low CAR T cell: target cell ratios (FIG. 13). This result was consistent for T cells generated from different donors.
• TRAC-/B2M-/anti-CD33 CAR+ T cells e.g.: CTX-965b CAR T cells and CTX-970 CAR T cells
• cytokine secretion in the presence of target cells was evaluated according to the procedure described in Example 2.
• 3X KO CAR+ T cells were co-cultured with CD33 -expressing MV4-11 target cells for 24 hours at a ratio of anti-CD33 CAR-T to MV411 ranging from 0.05: 1 to 1 : 1.
• cytokines e.g., IFNy and IL-2
• cytokine production by 3X KO CAR+ T cells was compared to cytokine production by 2X KO (TRAC-/ 2M-), CAR+ T cells and control T cells (e.g., TRAC+ T cells and TRAC-/ 2M- T cells).
• the anti-CD33 CAR+ T cells generated with CTX-965b CAR produced high levels of IL-2 relative to control T cells, while those generated using the CTX-970 or CTX-982b CAR constructs produced levels of IL-2 that were only slightly higher than control T cells (FIG. 15).
• the low IL-2 production observed for T cells generated with the CTX-970 or CTX-982b CAR constructs was observed for both 2X KO CAR+ T cells and 3X KO CAR+ T cells, indicating that the CD33 knockout did not rescue IL-2 production.
• T cells generated with the CTX-965b CAR T cells that had the 3X KO (TRAC-/p2M-/CD33-) produced higher levels of IL-2 than T cells that had only the 2X KO (TRAC-/ 2M-).
• MV4-11 cells were electroporated with Cas9:sgRNA targeting CD33 (CD33-10; SEQ ID NO: 151). Cells were plated at ⁇ l cell per well and allowed to expand for three weeks. Several clonal lines were then tested for CD33 surface expression using flow cytometry and an anti-CD33 antibody (PE-anti-human-CD33, Biolegend catalog #366608) Wild type MV4-11 (WT MV4-1 1) had high level of surface CD33, while one particular clonal line demonstrated no CD33 staining above background. This clonal line (referred to as CD33 KO MV4-11 cells) was subsequently used to evaluate CD33 -selective killing by anti-CD33 CAR+ T cells.
• CD33 KO MV4-11 cells was subsequently used to evaluate CD33 -selective killing by anti-CD33 CAR+ T cells.
• Selective -killing by anti-CD33 CAR+ T cells was evaluated by measuring target cell lysis and cytokine production in the presence of WT MV4-11 or CD33 KO MV4-11.
• CAR+ T cells that were defined as selective were those that induced cell lysis and cytokine production in the presence of CD33 -expressing WT MV4-11 cells, but exhibited no response in the presence of CD33 KO MV4-1 1. This result indicates that the anti-CD33 CAR+ T cell requires recognition of a CD33 antigen on the surface of the target cell to mediate cell killing.
• Selective killing was evaluated by measuring target cell lysis and CAR+ T cell cytokine production as described in example 2.
• both 2X KO CAR+ and 3X KO CAR+ T cells were generated with a CTX-982b CAR induced killing of CD33 KO MV4-11 cells at levels higher than 60%.
• 2X KO or 3X KO anti-CD33 CAR+ T cells generated with a CTX-965b or CTX- 970 CAR are selective for killing CD33 -expressing target cells, while T cells generated with a CAR-982b are non-selective and induce undesirable killing of cells deficient in CD33 expression (/. ⁇ ?., off-target cells).
• cytokine production by CAR+ T cells in the presence of CD33 KO MV4-11 cells was evaluated. Given that CAR+ T cells produce cytokines upon recognition of an antigen on a target cell, selective CAR+ T cells are expected to only produce cytokines when a target cell expressing a CAR-specific antigen is present. Given that CAR+ T cells with a CTX-965 CAR or a CTX-970 CAR were selective for inducing lysis of CD33-expressing target cells (FIG. 16), it was expected that likewise, they would only produce cytokines in the presence of CD33 -expressing target cells and not in the presence of CD33- deficient cells.
• CART T cells were co-cultured with CD33 KO MV4-11 cells and IL-2 and IFNy in the supernatant were measured by ELISA as described in example 2.
• CAR+ T cells produced IL-2 when co-cultured with CD33 KO MV4-1 1 cells (FIG. 18, left panel).
• CTX-982b CAR T cells that were identified as non-selective killers as described above.
• CTX-982b CAR T cells induced significant levels of IFNy when incubated with CD33 KO MV4-11 cells (FIG. 18, right panel).
• This example describes studies performed to evaluate therapeutic effects of anti-CD33 CAR+ T cells in an animal model of acute myeloid leukemia (AML)(MV-4-l 1 NSG model).
• AML acute myeloid leukemia
• the MV-4-11 human AML derived cell line was made to express both luciferase and mCherry (MV-4-11 -Luc-mCh-Puro) genes for in vivo imaging studies.
• Bioluminescence imaging (BLI) correlates with the amount of tumor burden, and therefore BLI was quantitated to assess tumor burden.
• BLI Bioluminescence imaging
• mice were injected with luciferin prior to measuring luciferase using IVIS S5 Lumina (Perkin Elmer).
• IVIS S5 Lumina Perkin Elmer
• MV-4-11 -Luc-mCh-Puro cells were injected into 5-6 week old female NSG mice (The Jackson Laboratory) (2x10 6 cells/mouse).
• CTX-965b CAR+ T cells with or without CD33 knockout appeared to be more efficacious in terms of tumor burden control, as indicated by the lower luminescence

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