Methods and compositions for treating a cancer

Abstract

Provided herein are methods for treatment of acute myeloid leukemia (AML) in a subject comprising administration of a composition comprising engineered immune cells. The engineered immune cells can comprise a first nucleotide sequence encoding an anti-CD7 protein expression blocker and a second nucleotide sequence encoding an anti-CD7 chimeric antigen receptor (CAR). The subject described herein may have previously been administered one or more prior lines of therapy before the administration of the engineered immune cells.

Description

Attorney Docket No. 62190-737601METHODS AND COMPOSITIONS FOR TREATING A CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Italian Patent Application No. 102025000004212, filed February 28, 2025, the entire content of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Chimeric antigen receptor (CAR) technology has been developed to target leukemias, lymphomas, and myeloma. However, novel therapies for certain leukemias, lymphomas and myeloma in patients are needed whose disease has relapsed or is refractory to treatment, and progress to date has been slow. In particular, effective immunotherapeutic options are lacking and treatment of acute myeloid leukemia (AML) relies on intensive chemotherapy and hematopoietic stem cell transplant. Despite aggressive treatment regimens associated with significant morbidity, results with these approaches are far from satisfactory.

[0003] CAR T cells have recently been developed in which the target antigen of the CAR is itself expressed in the CAR T cell. To avoid self-killing (e.g., fratricide), the CAR T cells also express a protein expression blocker (PEBL) protein that serves to reduce the expression of the target antigen on the cell surface of the CAR T. To produce viable CAR T cells, a PEBL can be expressed to bind and sequester the target protein prior to the subsequent expression of the CAR. There is a need for enhanced methods for producing an engineered CAR-T cell and eliminating CAR-mediated self-killing or fratricide of engineered cells, and a needed improvement in targeted therapies and treatments for fast-growing cancers, such as AML.BRIEF SUMMARY

[0004] Recognized herein is a need for improved CAR-T cell therapies. The methods and compositions provided herein can be used for the treatment of acute myeloid leukemia (AML) in a subject in need thereof. The compositions and methods provided herein can further produce engineered CAR-T cells and eliminate CAR-mediated self-killing or fratricide of the T cells.

[0005] In some aspects, the present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject in need thereof, the method comprising: (a) administering to the subject a composition comprising an engineered immune cell, wherein the engineered immune cell comprises: (i) a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and (ii) a secondAttorney Docket No. 62190-737601nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; and wherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

[0006] In some embodiments, the AML is a refractory acute myeloid leukemia. In some embodiments, the AML is a relapsed acute myeloid leukemia. In some embodiments, the subject has previously received one or more prior lines of therapy. In some embodiments, the one or more prior lines of therapy comprises at least two prior lines of therapy. In some embodiments, the one or more prior lines of therapy comprise surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, or radiation therapy, or any combination thereof. In some embodiments, the one or more prior lines of therapy comprise administration of a biologic. In some embodiments, the biologic comprises an antibody, antibody drug conjugate (ADC), cellular therapy, peptide, polypeptide, enzyme, vaccine, oligonucleotide, oncolytic virus, polysaccharide, or gene therapy. In some embodiments, the one or more prior lines of therapy comprises administration of a regimen comprising fludarabine, cytarabine, granulocyte colony stimulating factor, and gemtuzumab ozogamicin (FLAG-GO). In some embodiments, the one or more prior lines of therapy comprises administration of a regimen comprising venetoclax. In some embodiments, the regime comprises FLAG-GO and venetoclax.

[0007] In some embodiments, the subject has a risk of recurrence. In some embodiments, the subject has a prior history of recurrence after a prior therapy. In some embodiments, the subject has a treatment failure after at least two prior lines of induction therapy, a relapse within 12 months after first complete response (CR), a relapse after allogeneic hematopoietic stem cell transplantation, has at least one prior relapse, or any combination thereof. In some embodiments, the subject has been diagnosed with AML. In some embodiments, the method further comprises, prior to administering, identifying the subject as having AML. In some embodiments, identifying comprises measuring CD7 expression on blast cells. In some embodiments, the subject has at least 20% CD7 expression on blast cells. In some embodiments, the subject has at least 98% CD7 expression on blast cells. In some embodiments, the AML is a CD7-expressing AML. In some embodiments, the subject has been identified as having a relapsed or refractory AML. In some embodiments, the method further comprises, prior to administering, identifying the subject as having the relapsed or refractory AML. In some embodiments, the subject has been identified as having the relapsed orAttorney Docket No. 62190-737601refractory AML based on persistence of myeloid blasts in a bone marrow sample from the subject after one or more prior lines of therapy.

[0008] In some embodiments, the method further comprises, prior to administering, subjecting the subject to a pre-conditioning regimen. In some embodiments, the pre-conditioning regimen comprises a lymphodepletion. In some embodiments, the lymphodepletion comprises administration of fludarabine, etoposide, cyclophosphamide, bendamustine, busulfan, cytarabine, or alemtuzumab, or any combination thereof. In some embodiments, the etoposide is administered at a dose from about 50 mg / m2to about 300 mg / m2. In some embodiments, the etoposide is administered at a dose of about 150 mg / m2. In some embodiments, the etoposide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days. In some embodiments, the etoposide is administered at a dose of about 150 mg / m2per day for 3 days. In some embodiments, the etoposide is administered at a dose of about 150 mg / m2per day for 3 consecutive days.

[0009] In some embodiments, the cyclophosphamide is administered at a dose from about 10 mg / kg to about 100 mg / kg. In some embodiments, the cyclophosphamide is administered at a dose of about 60 mg / kg. In some embodiments, the cyclophosphamide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days. In some embodiments, the cyclophosphamide is administered at a dose of about 60 mg / kg per day for 2 days. In some embodiments, the cyclophosphamide is administered at a dose of about 60 mg / kg per day for 2 consecutive days. In some embodiments, the lymphodepletion comprises administration of (i) etoposide at a dose of about 150 mg / m2per day for 3 consecutive days, and (ii) cyclophosphamide at a dose of about 60 mg / kg per day for 2 consecutive days.

[0010] In some embodiments, the cytarabine is administered at a dose from about 1.0 g / m2to about 4.0 g / m2. In some embodiments, the cytarabine is administered at a dose of about 2.0 g / m2. In some embodiments, the cytarabine is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days. In some embodiments, the cytarabine is administered at a dose of 2.0 g / m2per day for 5 days. In some embodiments, the cytarabine is administered at a dose of 2.0 g / m2per day for 5 consecutive days.

[0011] In some embodiments, the fludarabine is administered at a dose from about 10 mg / sqm / day to about 100 mg / sqm / day. In some embodiments, the fludarabine is administered at a dose from about 20 mg / sqm / day to about 50 mg / sqm / day. In some embodiments, the fludarabine is administered at a dose of about 30 mg / sqm / day. In some embodiments, the fludarabine is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days. In some embodiments, the fludarabine is administered at a dose of about 30 mg / sqm / day for 4Attorney Docket No. 62190-737601days. In some embodiments, the fludarabine is administered at a dose of about 30 mg / sqm / day for 4 consecutive days.

[0012] In some embodiments, the cyclophosphamide is administered at a dose from about 500 mg / sqm / day to about 1500 mg / sqm / day. In some embodiments, the cyclophosphamide is administered at a dose from about 800 mg / sqm / day to about 1200 mg / sqm / day. In some embodiments, the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day. In some embodiments, the cyclophosphamide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days. In some embodiments, the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 days. In some embodiments, the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 consecutive days. In some embodiments, the lymphodepletion comprises administration of (i) fludarabine at a dose of about 30 mg / sqm / day for 4 consecutive days, and (ii) cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 consecutive days. In some embodiments, the lymphodepletion comprises administration of (i) fludarabine at a dose of about 30 mg / sqm / day on Day -6, -5, -4, and -3, and (ii) cyclophosphamide is administered at a dose of about 1000 mg / sqm / day on Day -4 and -3, when Day 0 is the day for administering the composition.

[0013] In some embodiments, the composition is administered once per day for at least about 5 days, at most about 30 days, or from about 1 to 30 days following the pre-conditioning regimen. In some embodiments, the composition is not administered immediately after the preconditioning regimen. In some embodiments, the composition is administered at least 1 day, 2 days, 3 days, or more after a last dosing of the pre-conditioning regimen. In some embodiments, the composition is administered once per day for at least about 5 days, at most about 30 days, or from about 1 to 30 days. In some embodiments, the composition is administered at a dose of at least about IxlO5engineered immune cells / kg. In some embodiments, the composition is administered at a dose of at least about 5xl05engineered immune cells / kg. In some embodiments, the composition is administered at a dose of at least about IxlO6engineered immune cells / kg. In some embodiments, the composition is administered at a dose of about 3xl06engineered immune cells / kg. In some embodiments, the composition is administered at a dose of about IxlO7engineered immune cells / kg.

[0014] In some embodiments, the composition is administered as a single dose. In some embodiments, the composition is administered as multiple doses on a single day or over multiple days. In some embodiments, the composition is administered as 2, 3, 4, 5, or more doses. In some embodiments, the method further comprises administering a second dose of the engineered immune cell no sooner than 1 month and no later than 1 year following completionAttorney Docket No. 62190-737601of a first dose. In some embodiments, the composition is administered as multiple doses over a duration of time. In some embodiments, the duration of time is at least about 1 week, at least about 1 month, or greater than about 6 months.

[0015] In some embodiments, the method further comprises subjecting the subject to one or more bridging therapies. In some embodiments, the one or more bridging therapies is administered prior to, concomitantly with, or subsequent to the composition. In some embodiments, the one or more bridging therapies comprise 5-azacytidine, venetoclax, or any combination thereof. In some embodiments, the method does not comprise any bridging therapy.

[0016] In some embodiments, the method further comprises, prior to and / or subsequent to administering, measuring a level of myeloid blasts of a sample from the subject. In some embodiments, the sample is a bone marrow sample. In some embodiments, the myeloid blasts are CD7+CD33+CD34+ myeloid blasts. In some embodiments, measuring the level of myeloid blasts comprises measuring an expression level of one or more markers selected from the group consisting of CD9, CD7, CD18, CD25, CD32, CD33, CD34, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a / c, CD366, CD371, and CX3CR1. In some embodiments, measuring the level of myeloid blasts comprises measuring an expression level of CD7, CD33, and / or CD34. In some embodiments, a percentage of myeloid blasts in the bone marrow sample is at least about 40% prior to administering. In some embodiments, a percentage of myeloid blasts in the bone marrow sample is at least about 80% prior to administering. In some embodiments, a percentage of myeloid blasts in the bone marrow sample is at most about 20% subsequent to administering. In some embodiments, the percentage of myeloid blasts in the bone marrow sample is at most about 10% subsequent to administering. In some embodiments, the bone marrow sample subsequent to administering does not comprise any detectable level of myeloid blasts.

[0017] In some embodiments, the composition is administered into the subject by intravenous infusion, intra-arterial infusion, direct injection into tumor and / or perfusion of tumor bed after surgery, implantation at a tumor site in an artificial scaffold, intrathecal administration, or intraocular administration. In some embodiments, administering the composition to the subject increases a percentage of engineered immune cells expressing the CAR in a peripheral blood sample or a bone marrow sample from the subject. In some embodiments, the percentage of engineered immune cells expressing the CAR, at least 9 days subsequent to the administering, is at least about 70%. In some embodiments, the percentage of engineered immune cells expressing the CAR, at least 14 days subsequent to the administering, is at least about 80%. InAttorney Docket No. 62190-737601some embodiments, the percentage of engineered immune cells expressing the CAR, at least 20 days subsequent to the administering, is at least about 60%. In some embodiments, the percentage of engineered immune cells expressing the CAR, at least 28 days subsequent to the administering, is at least about 40%. In some embodiments, the engineered immune cell persists in the subject for at least 5 days, 10 days, 14 days, 28 days, or more.

[0018] In some embodiments, the method further comprises, subsequent to administering, detecting a minimal residual disease (MRD) of the subject. In some embodiments, detecting the MRD of the subject comprises determining a level of myeloid blasts in a sample from the subject. In some embodiments, the myeloid blasts are positive for one or more markers selected from the group consisting of CD9, CD7, CD18, CD25, CD32, CD33, CD34, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a / c, CD366, CD371, and CX3CR1. In some embodiments, the myeloid blasts are CD7+CD33+CD34+ blasts. In some embodiments, the sample is a blood sample or a bone marrow sample. In some embodiments, the method further comprises administering an additional therapeutic agent or therapy. In some embodiments, the method further comprises obtaining a population of immune cells from the subject prior to administering the composition, and transducing one or more cells from the population of immune cells with a recombinant nucleic acid comprising the first nucleotide sequence encoding the target-binding domain linked to the localizing domain and the second nucleotide sequence encoding the CAR, thereby generating a population of engineered immune cells.

[0019] In some embodiments, the localizing domain comprises an ER retention signal. In some embodiments, the ER retention signal comprises the amino acid sequence of KDEL (SEQ ID NO: 35), KKMP (SEQ ID NO: 36), KKTN (SEQ ID NO: 37), SEKDEL (SEQ ID NO: 40), or AEKDEL (SEQ ID NO: 41). In some embodiments, the ER retention signal comprises the amino acid sequence of KKXX, where X is any amino acid. In some embodiments, the localizing domain comprises a Golgi retention sequence. In some embodiments, the localizing domain comprises a proteasome localizing sequence. In some embodiments, the proteasome localizing sequence comprises a PEST motif. In some embodiments, the PEST motif comprises an amino acid sequence as set forth in SEQ ID NO: 43 or 44. In some embodiments, the localizing domain comprises a transmembrane domain sequence derived from CD8a, CD8P, 4-1BB, CD28, CD34, CD4, FcsRIy, CD16, 0X40, CD3i CD3s, CD3y, CD38, TCRa, CD32, CD64, VEGFR2, FAS, or FGFR2B. In some embodiments, the first nucleotide sequence further encodes a CD8a signal peptide as set forth in SEQ ID NO: 54. In some embodiments,Attorney Docket No. 62190-737601the localizing domain comprises an amino acid sequence as set forth in SEQ ID NO: 31-33 or 45-50.

[0020] In some embodiments, the localizing domain further comprises a linker. In some embodiments, the localizing domain comprises, in N-terminus to C-terminus order, a linker and a retention signal. In some embodiments, the linker comprises a sequence of at least 5 amino acids. In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 53. In some embodiments, the linker comprises a human influenza hemagglutinin (HA) tag or a Myc tag.

[0021] In some embodiments, the first target-binding domain is a first antibody or antigen binding fragment thereof. In some embodiments, the second target-binding domain is a second antibody or antigen binding fragment thereof. In some embodiments, the first antibody or antigen binding fragment thereof is a first single chain variable fragment (scFv). In some embodiments, the second antibody or antigen binding fragment thereof is a second single chain variable fragment (scFv). In some embodiments, the first antibody or antigen binding fragment thereof and the second antibody or antigen binding fragment thereof comprise the same scFv.

[0022] In some embodiments, the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of DEVRGYLDV (SEQ ID NO: 3). In some embodiments, the VH comprises a HC CDR2 comprising the amino acid sequence of SISSGGFTYYPDSVKG (SEQ ID NO: 2) or SSGGF (SEQ ID NO: 5). In some embodiments, the VH comprises a HC CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 1) or GLTFSSY (SEQ ID NO: 4). In some embodiments, the first target-binding domain and / or the second target-binding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QQYSKLPYT (SEQ ID NO: 8). In some embodiments, the VL comprises a LC CDR2 comprising the amino acid sequence of YTSSLHS (SEQ ID NO: 7). In some embodiments, the VL comprises a LC CDR1 comprising the amino acid sequence of SASQGISNYLN (SEQ ID NO: 6). In some embodiments, the first target-binding domain and / or the second target-binding domain comprises: a HC CDR3 sequence of DEVRGYLDV (SEQ ID NO: 3), a HC CDR2 sequence of SISSGGFTYYPDSVKG (SEQ ID NO: 2) or SSGGF (SEQ ID NO: 5), aHC CDR1 sequence of SYAMS (SEQ ID NO: 1) or GLTFSSY (SEQ ID NO: 4), a LC CDR3 sequence of QQYSKLPYT (SEQ ID NO: 8), a LC CDR2 sequence of YTSSLHS (SEQ ID NO: 7), and a LC CDR1 sequence of SASQGISNYLN (SEQ ID NO: 6). In some embodiments, the VHAttomey Docket No. 62190-737601comprises a sequence with at least 80% sequence identity to the sequence EVQLVESGGGLVKPGGSLKLSC AASGLTF S S YAMSWVRQTPEKRLEWVASIS SGGFT YYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTV TVSS (SEQ ID NO: 9). In some embodiments, the VL comprises a sequence with at least 80% sequence identity to the sequence AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSL HSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR (SEQ ID NO: 10). In some embodiments, the VH comprises a sequence of EVQLVESGGGLVKPGGSLKLSC AASGLTF S S YAMSWVRQTPEKRLEWVASIS SGGFT YYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTV TVSS (SEQ ID NO: 9). In some embodiments, the VL comprises a sequence of AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSL HSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR (SEQ ID NO: 10).

[0023] In some embodiments, the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of GGVYYDLYYYALDY (SEQ ID NO: 13). In some embodiments, the VH comprises a HC CDR2 comprising the amino acid sequence of KINPSNGRTNYNEKFKS (SEQ ID NO: 12) or NPSNGR (SEQ ID NO: 15). In some embodiments, the VH comprises a HC CDR1 comprising the amino acid sequence of SYWMH (SEQ ID NO: 11) or GYTFTSY (SEQ ID NO: 14). In some embodiments, the first target-binding domain and / or the second targetbinding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QQSNSWPYT (SEQ ID NO: 18). In some embodiments, the VL comprises a LC CDR2 comprising the amino acid sequence of SASQSIS (SEQ ID NO: 17). In some embodiments, the VL comprises a LC CDR1 comprising the amino acid sequence of RASQSISNNLH (SEQ ID NO: 16). In some embodiments, the first target-binding domain and / or the second target-binding domain comprises: a HC CDR3 sequence of GGVYYDLYYYALDY (SEQ ID NO: 13), a HC CDR2 sequence of KINPSNGRTNYNEKFKS (SEQ ID NO: 12) or NPSNGR (SEQ ID NO: 15), a HC CDR1 sequence of SYWMH (SEQ ID NO: 11) or GYTFTSY (SEQ ID NO: 14), a LC CDR3 sequence of QQSNSWPYT (SEQ ID NO: 18), a LC CDR2 sequence of SASQSIS (SEQ ID NO: 17), and a LC CDR1 sequence of RASQSISNNLH (SEQ ID NO: 16). In some embodiments, theAttorney Docket No. 62190-737601VH comprises a sequence with at least 80% sequence identity to the sequence QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSN GRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALD YWGQGTTVTVSS (SEQ ID NO: 19). In some embodiments, the VL comprises a sequence with at least 80% sequence identity to the sequence DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPS RFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKR (SEQ ID NO: 20). In some embodiments, the VH comprises a sequence of QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSN GRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALD YWGQGTTVTVSS (SEQ ID NO: 19). In some embodiments, the VL comprises a sequence of DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPS RFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKR (SEQ ID NO: 20).

[0024] In some embodiments, the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of WGNYPHYAMDY (SEQ ID NO: 23). In some embodiments, the VH comprises a HC CDR2 comprising the amino acid sequence of YISSGSSTLHYADTVKG (SEQ ID NO: 22) or SSGSST (SEQ ID NO: 25). In some embodiments, the VH comprises aHC CDR1 comprising the amino acid sequence of SFGMH (SEQ ID NO: 21) or GFTFSSF (SEQ ID NO: 24). In some embodiments, the first target-binding domain and / or the second target-binding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QHSRELPYT (SEQ ID NO: 28). In some embodiments, the VL comprises a LC CDR2 comprising the amino acid sequence of LASNLES (SEQ ID NO: 27). In some embodiments, the VL comprises a LC CDR1 comprising the amino acid sequence of RASKSVSASGYSYMH (SEQ ID NO: 26). In some embodiments, the first target-binding domain and / or the second target-binding domain comprises: a HC CDR3 sequence of WGNYPHYAMDY (SEQ ID NO: 23), a HC CDR2 sequence of YISSGSSTLHYADTVKG (SEQ ID NO: 22) or SSGSST (SEQ ID NO: 25), a HC CDR1 sequence of SFGMH (SEQ ID NO: 21) or GFTFSSF (SEQ ID NO: 24), a LC CDR3 sequence of QHSRELPYT (SEQ ID NO: 28), a LC CDR2 sequence of LASNLES (SEQ ID NO: 27), and a LC CDR1 sequence ofAttorney Docket No. 62190-737601RASKSVSASGYSYMH (SEQ ID NO: 26). In some embodiments, the VH comprises a sequence with at least 80% sequence identity to the sequence D VQLVESGGGL VQPGGSRKLSC AASGFTFS SFGMHWVRQAPEKGLEWVAYIS SGS S TLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARWGNYPHYAMDYWG QGTSVTVSS (SEQ ID NO: 29). In some embodiments, the VL comprises a sequence with at least 80% sequence identity to the sequence DIVMTQSPASLAVSLGQRATISCRASKSVSASGYSYMHWYQQKPGQPPKLLIYLASN LESGVPARF SGSGSGTDFTLNIHPVEEED AVT YYCQHSRELP YTFGGGTKLEIK (SEQ ID NO: 30). In some embodiments, the VH comprises a sequence of D VQLVESGGGL VQPGGSRKLSC AASGFTFS SFGMHWVRQAPEKGLEWVAYIS SGS S TLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARWGNYPHYAMDYWG QGTSVTVSS (SEQ ID NO: 29). In some embodiments, the VL comprises a sequence of DIVMTQSPASLAVSLGQRATISCRASKSVSASGYSYMHWYQQKPGQPPKLLIYLASN LESGVPARF SGSGSGTDFTLNIHPVEEED AVT YYCQHSRELP YTFGGGTKLEIK (SEQ ID NO: 30)

[0025] In some embodiments, the CAR further comprises a transmembrane domain, a 4- IBB intracellular signaling domain, and a CD3(^ intracellular signaling domain. In some embodiments, the transmembrane domain comprises an amino acid sequence as set forth in SEQ ID NO: 50. In some embodiments, the 4-1BB intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 51. In some embodiments, the CD3(^ intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 52. In some embodiments, the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell that comprises the second nucleotide sequence but not the first nucleotide sequence.

[0026] In some embodiments, the engineered immune cell is a T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the engineered immune cell is a natural killer (NK) cell.

[0027] In some embodiments, the first target-binding domain linked to the localizing domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 61-64. In some embodiments, the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 65-68.

[0028] In some embodiments, the first nucleotide sequence and the second nucleotide sequence are on different molecules. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are on a same molecule. In some embodiments, the first nucleotideAttorney Docket No. 62190-737601sequence and the second nucleotide sequence are on a bicistronic vector. In some embodiments, the bicistronic vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked by an Internal Ribosome Entry Site (IRES) or a ribosomal codon skipping site. In some embodiments, the IRES is derived from Encephalomyocarditis virus (EMCV) or an Enterovirus. In some embodiments, the IRES comprises a polynucleotide sequence as set forth in SEQ ID NO: 82. In some embodiments, the ribosomal codon skipping site comprises a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of a F2A peptide (foot-and-mouth disease virus 2A peptide), an E2A peptide (equine rhinitis A virus 2 A peptide), a P2A peptide (porcine teschovirus-12 A peptide), and a T2A peptide (thosea asigna virus 2A). In some embodiments, the 2A self-cleaving peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 83-86. In some embodiments, the bicistronic vector further comprises a promoter element. In some embodiments, the promoter element is selected from the group consisting of a CMV promoter, EFla promoter, EFS promoter, MSCV promoter, and PGK promoter. In some embodiments, the promoter element is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NOs: 92-96.

[0029] Use of a composition in the manufacturing of a medicament for the treatment of acute myeloid leukemia (AML) in a subject in need thereof, wherein the composition comprises an engineered immune cell, wherein the engineered immune cell comprises: a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; and wherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

[0030] A pharmaceutical composition for treating acute myeloid leukemia (AML) in a subject in need thereof, wherein the pharmaceutical composition comprises an engineered immune cell and a pharmaceutically acceptable carrier, wherein the engineered immune cell comprises: a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second targetbinding domain that binds to CD7; and wherein the engineered immune cell is from anAttorney Docket No. 62190-737601autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.INCORPORATION BY REFERENCE

[0031] 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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0033] FIG. 1 shows a series of flow cytometry plots depicting CAR positive and CD3 positive cells in peripheral blood of an AML patient over time. The percentage of CAR T cells was determined by staining with a specific CD7-conjugated reagent. From Day 7 to Day 14, the level of CD3+ CAR+ T cells increased following anti-CD7 CAR T treatment.

[0034] FIG. 2 shows a graph depicting the percentage of CD3+ cells for CAR T cells and percentage of blasts in peripheral blood of an AML patient. Following treatment with anti-CD7 CAR T cells (on Day 0), the level of blasts decreased while the level of CAR T cells increased.

[0035] FIG.3 shows CD7+CD33+CD34+ cells in bone marrow (BM) at pre- and post-infusion timepoints. The upper row shows pre-infusion of anti-CD7 CAR T treatment, and the bottom row shows Day 14 post-infusion of anti-CD7 CAR T treatment. Over the treatment period of fourteen days, the level of myeloid blasts decreased from 84.99% (as shown in the first column, top row) to 0.02% (as shown in the first column, bottom row).

[0036] FIG. 4 shows CAR T cells in the bone marrow of an AML patient at Day 14 posttreatment with anti-CD7 CAR T cells. The plot demonstrates that 94% of CD3+ cells were CAR+, indicating that CAR T cells were able to enter the patient’s bone marrow and persist for at least 14 days post-infusion.Attorney Docket No. 62190-737601DETAILED DESCRIPTION

[0037] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed.Overview

[0038] CAR T cells have been developed to target T cell malignancies. Patients with hematological malignancies expressing certain T cell-associated markers, such as acute myeloid leukemia (AML), may present with refractory / relapsed AML following any number of prior treatments. For example, after treatment with chemotherapy, some patients with diagnosed AML may not go into remission. AML is a fast-growing cancer that occurs when the bone marrow of a subject produces too many abnormal white blood cells called myeloblasts. AML represents one of the most common types of acute leukemia, and has also been referred to as acute myelogenous leukemia or acute nonlymphocytic leukemia. In refractory AML or relapsed AML, the myeloblasts may return in the blood and / or bone marrow.

[0039] Engineered chimeric antigen receptor T cells (CAR T cells) provide another therapeutic intervention for inducing remission in these patients prior to stem cell transplantation, and may improve the survival rate. One concern regarding an allogeneic CAR T cell transplant is a condition called graft versus host disease (or GvHD). In GvHD, the donated cells view the recipient’s body as foreign, thereby attacking the recipient’s body, and this can be lifethreatening. GvHD is mediated by receptors on the T cell surface.

[0040] The present disclosure provides compositions comprising engineered immune cells. The engineered immune cells can comprise anti-CD7 CAR that recognize, target, and kill CD7+ target cells, and a target-binding domain that binds to CD7. The anti-CD7 target binding domain may remove surface CD7 expression of the engineered immune cells to hinder or prevent fratricide or self-killing. The combination of these two components - an anti-CD7 CAR and anti-CD7 target-binding domain - can provide for viable CAR-expressing T lymphocytes capable of targeting T cell antigens and reducing fratricide.Attorney Docket No. 62190-737601Definitions

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

[0042] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0043] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0044] The term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value. For example, the amount “about 10” includes amounts from 9 to 11. The term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0045] As used herein, the term “nucleic acid” refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). “Nucleic acid” includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In some embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, “nucleic acid” can refer to either or both strands of the molecule. Nucleic acids and polynucleotides as used herein are interchangeable.

[0046] The term “nucleotide sequence,” in reference to a nucleic acid, refers to a contiguous series of nucleotides that are joined by covalent linkages, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl-phosphonate, phosphorothioate, phosphotriester bonds), and / or non-phosphorus linkages (e.g., peptide and / or sulfamate bonds). In some embodiments, the nucleotide sequence encoding, e.g., a target-binding domain linked to a localizing domain is a heterologous sequence (e.g., a gene that is of a different species or cell type origin).

[0047] The terms “nucleotide” and “nucleotide monomer” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotidesAttorney Docket No. 62190-737601comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0048] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0049] The term “sequence identity” means that two nucleotide sequences or two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least, e.g., 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. For sequence comparison, typically one sequence acts as a reference sequence (e.g., parent sequence), to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0050] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly accessible through the National Institutes of Health NCBI internet server). Typically, defaultAttorney Docket No. 62190-737601program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0051] As will be appreciated by those of skill in the art, in some aspects, the nucleic acid further comprises a plasmid sequence. The plasmid sequence can include, for example, one or more sequences of a promoter sequence, a selection marker sequence, or a locus-targeting sequence.

[0052] The term “protein expression blockers” or “PEBL” as used herein refers to a polypeptide construct containing a target-binding molecule that binds a target (e.g., CD7) to be removed, neutralized or blocked linked to a domain (e.g., a localizing domain, intracellular retention domain, or a intracellular targeting signal, which terms can be used interchangeably herein) that directs the polypeptide to specific cellular compartments, such as the Golgi, endoplasmic reticulum (ER), proteasome, or cellular membrane, depending on the application. In some embodiments, a PEBL further comprises a signal peptide. In some embodiments, a PEBL can further comprise a transmembrane domain. In some embodiments the transmembrane domain contributes to intracellular localization. In some embodiments, the PEBL described herein can be a CD7 PEBL, e.g., a CD7-PEBL comprising a CD7-binding domain, linked to a localizing domain or an intracellular retention domain. In some embodiments, the CD7 binding domain comprises a single-chain variable fragment antibody fragment comprising the VH and VL domains of a CD7 antibody. “CD7 PEBL” and “anti-CD7 PEBL” can be used interchangeably in the present disclosure.

[0053] The term “promoter” or “promoter element” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, that may assist to initiate the specific transcription of a polynucleotide sequence.

[0054] The term “retroviral vector” can refer to a gammaretroviral vector. A retroviral vector may include, e.g., a promoter, a packaging signal, a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and polynucleotides of interest, e.g., a polynucleotide encoding a CAR and a polynucleotide encoding a PEBL. A retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary retroviral (e.g., gammaretroviral) vectors include Murine Embryonic Stem Cell Virus (MESV), Murine Stem Cell Virus (MSCV), Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., inMaetzig et al., Viruses, 2011; 3(6): 677-713.Attomey Docket No. 62190-737601

[0055] The term “lentivirus” refers to a genus of the Retroviridae family that may be used as a gene delivery vector as described herein. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell. HIV, SIV, and FIV are all examples of lentiviruses.

[0056] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX® vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0057] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. In some embodiments, the term is construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0058] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0059] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-Attomey Docket No. 62190-737601associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0060] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Exemplary promoters include the immediate early cytomegalovirus (CMV), EF-la, ubiquitin C, or phosphoglycerokinase (PGK) promoters. A strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto can be used. Other constitutive promoter sequences may be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- 1 Ovian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, and the like. In some embodiments, the promoter is an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0061] As used herein, “antibody” means an intact antibody or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment modified or engineered, or that is a human antibody. Examples of antibodies modified or engineered are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-Attorney Docket No. 62190-737601binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g., scFv), minibodies and diabodies. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. In some embodiments, an antibody comprises at least one heavy chain and one light chain. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region ("LCVR or "VL") and a light chain constant region (CL). An “antibody” includes, but are not limited to, monoclonal, polyclonal, bispecific, multi specific, murine, chimeric, camelid VHH, humanized and human antibodies. In some embodiments, the antibody disclosed herein can be a CD7 antibody.

[0062] The term “specifically (or selectively) binds” or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0063] In some embodiments, the antibody that binds CD7 can be a single-chain variable fragment antibody (“scFv antibody”). scFv refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology Of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, PCT Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. As would beAttorney Docket No. 62190-737601appreciated by those of skill in the art, various suitable linkers can be designed and tested for optimal function, as provided in the art, and as disclosed herein.

[0064] In some embodiments, an antibody can be modified or engineered, e.g., chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies e.g., bispecific antibodies). In some embodiments, an antibody can be a nanobody-based heavy chain antibody engineered and / or modified from an immunoglobulin.

[0065] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The term “antibody heavy chain (VH)” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs. The term “antibody light chain (VL)” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (1) light chains refer to the two major antibody light chain isotypes. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. The VH and VL regions can be further subdivided into regions of hypervariability, termed hypervariable region (HVR) or complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, these CDRs can be distributed between their appropriate framework regions. In some embodiments of the disclosure, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences or may be naturally or artificially modified.

[0066] The term “binding domain” as used herein (e.g., “CD7 binding domain”) refers to a polypeptide having affinity to a target. For example, the binding domain can be an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The binding domain can be antibodies or antibody fragments. In some embodiments, the binding domain can be a portion of a receptor that can bind to a ligandAttorney Docket No. 62190-737601(e.g., a ligand-binding domain of a receptor) or a ligand of a receptor. The term “binding domain” may be used interchangeably with the term “common domain”.

[0067] As used herein, an “engineered” immune cell includes an immune cell that has been genetically modified as compared to a naturally-occurring immune cell. For example, an engineered T cell produced according to the present methods carries a nucleic acid comprising a nucleotide sequence that does not naturally occur in a T cell from which it was derived, such as the nucleic acids exemplified herein. In some embodiments, the engineered immune cell is an engineered T cell, an engineered natural killer (NK) cell, an engineered NK / T cell, an engineered monocyte, an engineered macrophage, or an engineered dendritic cell.

[0068] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro or ex vivo. In other embodiments, the cells are not cultured in vitro or ex vivo.

[0069] In some embodiments, an “immune activating receptor” as used herein refers to a receptor on the surface of an immune cell that activates an immune response upon binding a cancer cell ligand. In some embodiments, the immune activating receptor comprises a molecule that, upon binding (ligation) to a ligand (e.g., peptide or antigen) expressed on a cancer cell, is capable of activating an immune response. In some embodiments, the immune activating receptor is a chimeric antigen receptor (CAR); methods for designing and manipulating a CAR are known in the art. In other embodiments, the immune activating receptor is a target-binding receptor, which is similar to a CAR, but with the scFv replaced with a target-binding molecule (e.g., a molecule that binds to CD7).

[0070] The term “chimeric antigen receptor” or “CAR” as used herein refers to an engineered cell-surface receptor comprising, at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain (maybe referred to as "cytoplasmic signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. The chimeric antigen receptors of the present disclosure are intended primarily for use with lymphocytes such as T cells and natural killer (NK) cells. In some embodiments, the CAR described herein is a CD7 CAR, e.g., comprising a binding domain that binds to an extracellular domain of CD7, a transmembraneAttorney Docket No. 62190-737601domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some embodiments, the CD7 binding domain comprises a single-chain variable fragment antibody fragment comprising the VH and VL domains of a CD7 antibody.

[0071] The portion of the CAR of the disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody.

[0072] The term “intracellular signaling domain,” as used herein, refers to an intracellular (e.g., cytoplasmic) portion of a molecule sufficient to transduce an effector function signal. In embodiments, the intracellular signal domain transduces the effector function signal and directs the cell to perform a specialized function. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain.

[0073] The term “isolated” as used herein, refers to altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment, e.g., a host cell.

[0074] As used herein, a “CD7 CAR+ / CD7-negative” T cell refers to a T cell expressing a chimeric antigen receptor against human CD7 and having low or no surface expression of endogenous CD7. In some instances, a “CD7 CAR+ / CD7-negative” T cell can refer to “PCART7” cells. In some embodiments, the low or no surface expression of endogenous CD7 can be due to expression of a PEBL against human CD7 which prevents or hinders endogenous CD7 protein to translocated to the surface of the T cell. In some instances, surface expression of CD7 can be determined using standard methods known to those in the art such as but not limited to immunocytochemistry, flow cytometry, or FACS.

[0075] As used herein, a “PCART7” T cell refers to a T cell expressing a chimeric antigen receptor against human CD7 and having low or no surface expression of endogenous CD7. In some instances, the “PCART7” T cell can be autologous T cells. For example, T cells obtained from a subject having cancer can be used to engineer the PCART7 T cells, which can be administered to the subject for treating cancer. In some embodiments, the low or no surfaceAttorney Docket No. 62190-737601expression of endogenous CD7 is due to expression of a PEBL against human CD7, which prevents or hinders endogenous CD7 protein, to translocate to the surface of the T cells. In some instances, surface expression of CD7 can be determined using standard methods known to those in the art such as but not limited to immunocytochemistry, flow cytometry, or FACS.

[0076] The term “autologous” and its grammatical equivalents as used herein can refer to as originating from the same being. For example, a sample (e.g., cells) can be removed, processed, and given back to the same subject (e.g., patient) at a later time. An autologous process is distinguished from an allogenic process where the donor and the recipient are different subjects.

[0077] The term “allogeneic”, as used herein, refers to any material derived from an individual that is transplanted into a genetically different recipient of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically. The term can refer to a graft derived from a different animal of the same species.

[0078] As used herein, the terms “treat,” “treating,” or “treatment,” refer to counteracting a medical condition (e.g., a condition related to a hematological malignancy) to the extent that the medical condition is improved according to a clinically-acceptable standard.

[0079] As used herein, “subject” refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In some embodiments, the subject is a human. A “subject in need thereof’ refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented) by inducing T cells to exert specific cytotoxicity against malignant myeloid cells.

[0080] As used herein, a “therapeutic amount” refers to an amount that, when administered to a subject, is sufficient to achieve a desired therapeutic effect (treats a condition related to a myeloid cell malignancy) in the subject under the conditions of administration. An effective amount of the agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art, and is dependent on several factors including, for example, the particular agent chosen, the subject’s age, sensitivity, tolerance to drugs and overall well-being.

[0081] The term “alleviate” as used herein, in context to a disease refers to reducing the severity of one or more symptoms of the disease.

[0082] The term “express” as used herein refers to the transcription and / or translation of a particular nucleotide sequence into a protein. Proteins may be expressed and remainAttorney Docket No. 62190-737601intracellular, become a component of the cell surface membrane, or be secreted into extracellular matrix or medium.

[0083] The term “fratricide” as used herein refers to when one cell in the population kills a second cell in the population wherein the first cell and the second cell are of the same type, e.g., both cells are T cells.

[0084] The term “reducing and / or preventing fratricide” as used herein relates to the decrease in the occurrence of fratricide in a population of cells as compared to a suitable control population of cells (typically, but not necessarily, a population of identical cells with normal expression of the target of a CAR).

[0085] As used herein, a “kill gene” or a “suicide gene” refers to a gene that, upon activation, will induce cell death either by itself (e.g., self-induced apoptosis) or by other mechanism (e.g., as in effector cell mediated immune response, complement-dependent cytotoxicity, antibodydependent cellular cytotoxicity, etc.). The activation of the kill gene can be done using an agent, e.g., antibody or drug, and this results in cell death.

[0086] As used herein, “multiplicity of infection (MOI)” refers to the ratio of the number of agents (e.g., viral particles) to the number of infection targets (e.g., host cells) in an infection medium. A MOI may affect transduction or infection. For a construct, a higher MOI can achieve a higher transduction rate.

[0087] As used herein, “vector copy number (VCN)” refers to the number of agents (e.g., viral particles) within a host cell. For example, a VCN of 1 can refer to a single viral particle transduced and integrated into the genome of a single host cell.

[0088] As used herein, “subject” refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In some embodiments, the subject is a human. A “subject in need thereof’ refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented) by inducing T cells to exert specific cytotoxicity against malignant myeloid cells.

[0089] The term “cancer” as referred herein refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma leukemia, lung cancer and the like. As used herein, the term “cancer” includes premalignant, as well as malignant cancers. In some embodiments, theAttorney Docket No. 62190-737601cancer described herein is a stage I cancer, a stage II cancer, a stage III cancer, or a stage IV cancer.

[0090] The term “myeloid cell disease,” as used herein, refers to a disease caused by myeloid cell defects or disorders. A myeloid cell disease can be a cancer caused by overproduction and overgrowth of malignant and / or immature myeloid cells. A myeloid cell disease can be a myeloid cell mediated disease. In some cases, the cancer is a myeloid cell malignancy. In some cases, a myeloid cell disease is an immune disorder.

[0091] The term “T cell” and its grammatical equivalents as used herein can refer to a T cell from any origin. For example, a T cell can be a primary T cell, e.g., an autologous T cell, an allogeneic T cell, a T cell line, etc. The T cell can also be human or non-human. The term “T cell activation” or “T cell triggering” and its grammatical equivalents as used herein can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation, cytokine production and / or detectable effector function. In some cases, “full T cell activation” can be similar to triggering T cell cytotoxicity. T cell activation can be measured using various assays known in the art. Said assays can be an ELISA to measure cytokine secretion, an ELISPOT, flow cytometry assays to measure intracellular cytokine expression, flow cytometry assays to measure proliferation, and cytotoxicity assays (51Cr release assay or flow cytometry assays to enumerate live target cells) to determine target cell elimination. Said assays typically use controls (non-engineered cells) to compare to engineered cells (CAR T) to determine relative activation of an engineered cell compared to a control. Additionally, said assays can compare engineered cells incubated or put in contact with a target cell not expressing the target antigen. For example, said comparison can be a CD7 CAR T cell incubated with a target cell that does not express CD7.CD7

[0092] CD7 is a 40 kDa type I transmembrane glycoprotein which is the primary marker for T cell malignancies, and which can be highly expressed in cases of T-cell acute lymphoblastic leukemia (T-ALL), including early T cell progenitor acute lymphoblastic leukemia (ETP-ALL). CD7 is also expressed in >30% of patients with AML in their leukemic blasts and malignant progenitor cells. An anti-CD7 CAR can induce T cells to exert specific cytotoxicity against T and myeloid cell malignancies that express CD7 on their blasts and malignant cells. Further, T cell cytotoxicity has been shown to be markedly increased when an anti-CD7 CAR was used in combination with downregulation of CD7 expression on the effector T cells. Downregulation (e.g., elimination, reduction, and / or relocalization) of CD7 in a T cell viaAttorney Docket No. 62190-737601expression of anti-CD7 PEBL can prevent the fratricidal effect exerted by a corresponding antiCD? CAR. This combination may lead to greater T cell recovery after CAR expression as compared to an expression of cells that retain the target antigen (e.g., CD7), and a more effective cytotoxicity against T and myeloid leukemia / lymphoma cells.Methods of Treatment

[0093] Provided herein are methods of treating a cancer in a subject in need thereof. The cancer may be a myeloid cell malignancy. In some embodiments, the cancer to be treated in the subject may be a solid tumor or a hematologic malignancy. The hematologic malignancy may include, but is not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia, myelodysplasia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin and non-Hodgkin lymphoma. In some embodiments, the solid tumor may comprise lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, neuroblastoma, rhabdomyosarcoma, or brain tumor. As an example, the cancer may be AML. The cancer can be refractory or relapsed AML.

[0094] The methods may comprise administering a composition to the subject. The composition can comprise an engineered immune cell comprising a first nucleotide sequence. The first nucleotide sequence may encode a first target-binding domain. The first target-binding domain may comprise a target-binding domain described herein. For example, the first nucleotide sequence may encode a first target-binding domain linked to a localizing domain. The localizing domain can comprise a linker sequence. The localizing domain can comprise a retention signal. In some embodiments, the localizing domain can comprise a linker sequence and a retention signal. In some embodiments, the first target-binding domain can bind to CD7.

[0095] The composition can comprise an engineered immune cell comprising a second nucleotide sequence. The second nucleotide sequence may encode an immune activating receptor. In some embodiments, the immune activating receptor can be a chimeric antigen receptor (CAR). The CAR may comprise a second target-binding domain. In some embodiments, the second target-binding domain may bind to the same target as the first targetbinding domain. In some embodiments, the second target-binding domain may bind to a different target as the first target-binding domain. In some embodiments, the second targetbinding domain may bind to CD7.

[0096] In some embodiments, the engineered immune cell may have reduced CD7 surface expression compared to a corresponding immune cell that comprises the second nucleotideAttorney Docket No. 62190-737601sequence but not the first nucleotide sequence. The engineered immune cell can be a T cell. In some embodiments, the T cell can be a CD4+ T cell or a CD8+ T cell. In some embodiments, the engineered immune cell can be a natural killer (NK) cell.

[0097] As an example, the present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject in need thereof, the method comprising: administering to the subject a composition comprising an engineered immune cell, wherein the engineered immune cell comprises: (i) a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; and wherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered. In some embodiments, the composition may be administered by intravenous infusion, intra-arterial infusion, direct injection into tumor and / or perfusion of tumor bed after surgery, implantation at a tumor site in an artificial scaffold, intrathecal administration, or intraocular administration.

[0098] In some embodiments, the method may further comprise obtaining a population of immune cells from the subject. The population of immune cells may be obtained prior to administering the composition to the subject. One or more cells of the population of immune cells can be transduced with a recombinant nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence described herein. Transduction of the one or more cells of the population of immune cells can thus generate a population of engineered immune cells.

[0099] In some embodiments, the subject may have received one or more prior lines of therapy before being administered the composition described herein. The subject may have been administered at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or greater than about 20 prior lines of therapy. In some embodiments, the subject had been in complete remission following the one or more prior lines of therapy. The subject may have experienced one or more relapse following the prior therapy. The subject may have experienced 1, 2, 3, 4, or more relapses. In some embodiments, the subject may have experienced one or more relapses after a duration of time following the prior therapy and / or following remission after the therapy. The subject may have had a relapse within about 1 month, about 2 months, about 3 months, aboutAttorney Docket No. 62190-7376014 months, about 5 months, about 6 months, about 12 months, about 18 months, 24 months, about 36 months, or about 48 months following the prior therapy and / or following remission after the therapy.

[0100] In some embodiments, the prior therapy may comprise surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, or radiation therapy, hematopoietic stem cell transplant, targeted therapy with small molecule inhibitors, or any combination thereof. The prior line(s) of therapy may comprise administration of a biologic. The biologic may be one or more of an antibody, antibody drug conjugate (ADC), cellular therapy, peptide, polypeptide, enzyme, vaccine, oligonucleotide, oncolytic virus, polysaccharide, or gene therapy. The prior line of therapy may comprise chemotherapy regimen. The hematopoietic stem cell transplant can be allogeneic or autologous. The prior line of therapy (e.g., chemotherapy regimen) can comprise administration of a regimen comprising fludarabine, cytarabine, granulocyte colony stimulating factor, and gemtuzumab ozogamicin (FLAG-GO). In some embodiments, the prior line of therapy may comprise venetoclax. In some embodiments, the subject may have received a prior line of therapy comprising FLAG-GO and venetoclax.

[0101] The subject may have a risk of recurrence. In some embodiments, a subject is known to have recurrence following a prior therapy. A cancer recurrence can occur when cancer returns after a period of remission. It can happen when a small number of cancer cells survive treatment and grow into tumors. For example, recurrence may occur following about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 12 months, about 18 months, 24 months, about 36 months, or about 48 months following the prior therapy and / or following remission after the therapy. A prior history of recurrence can indicate that a subject has experienced a cancer recurrence at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more than 5 times after the prior therapy and / or following remission after the therapy. In some embodiments, a subject may have a relapse following allogeneic hematopoietic stem cell transplantation. Allogeneic hematopoietic stem cell transplantation (HSCT) is a procedure that replaces a patient's unhealthy blood-forming stem cells with healthy stem cells from a donor. In some embodiments, a subject may have a treatment failure after at least two prior lines of induction therapy, a relapse within 12 months after first complete response (CR), a relapse after allogeneic hematopoietic stem cell transplantation, has at least one prior relapse, or any combination thereof.

[0102] A subject may have a treatment failure after at least two prior lines of induction therapy and a relapse within 12 months after first complete response (CR). A subject may have aAttorney Docket No. 62190-737601treatment failure after at least two prior lines of induction therapy and a relapse after allogeneic hematopoietic stem cell transplantation. A subject may have a treatment failure after at least two prior lines of induction therapy and at least one prior relapse. A subject may have a relapse within 12 months after first complete response (CR) and a relapse after allogeneic hematopoietic stem cell transplantation. A subject may have a relapse after allogeneic hematopoietic stem cell transplantation and at least one prior relapse.

[0103] A subject may have been diagnosed with AML. In some embodiments, the method may further comprise identifying a subject as having AML. The AML may be a CD7-expressing AML. The method may comprise measuring a level of CD7 expression on blast cells of the subject. The blast cells can be myeloid blast cells. Myeloid blast cells (e.g., myeloblasts), are immature white blood cells that develop in the bone marrow. They are produced from stem cells and normally mature into white blood cells that help fight infection. In some embodiments, a subject may be evaluated for a level of CD7 expression on blast cells prior to administering. Prior to administering the composition described herein, a subject may have at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% CD7+ expression on blast cells. In some embodiments, the subject may have at least about 95% CD7 expression on blast cells. In some embodiments, the subject may have at least about 96% CD7 expression on blast cells. In some embodiments, the subject may have at least about 97% CD7 expression on blast cells. In some embodiments, the subject may have at least about 98% CD7 expression on blast cells. In some embodiments, the subject may have at least about 99% CD7 expression on blast cells. In some embodiments, the subject may have at least about 100% CD7 expression on blast cells.

[0104] The subject may have relapsed and / or refractory AML. In some embodiments, the method may further comprise identifying the subject as having relapsed and / or refractory AML. The identifying may occur prior to administering the composition to the subject. Identifying a subject as having relapsed and / or refractory AML may involve measuring a persistence of myeloid blasts. A sample may be obtained from the subject to test for a persistence of myeloid blasts. In some embodiments, the sample may comprise a bone marrow sample. A persistence of myeloid blasts may be measured following one or more prior lines of therapy. For example, the method may further comprise identifying a subject as having relapsedAttorney Docket No. 62190-737601and / or refractory AML based on persistence of myeloid blasts in a bone marrow sample from the subject after one or more prior lines of therapy.

[0105] As another example, a subject may undergo a prior line of therapy comprising FLAG-GO and venetoclax and then be identified as having relapsed and / or refractory AML. The subject may undergo a prior line of therapy comprising FLAG-GO and venetoclax and be tested for a persistence of myeloid blasts in the bone marrow. In some embodiments, a subject may have persistence of at least about 5% myeloid blasts, at least about 10% myeloid blasts, at least about 15% myeloid blasts, at least about 20% myeloid blasts, at least about 25% myeloid blasts, at least about 30% myeloid blasts, at least about 35% myeloid blasts, at least about 40% myeloid blasts, at least about 45% myeloid blasts, at least about 50% myeloid blasts, at least about 55% myeloid blasts, at least about 60% myeloid blasts, at least about 70% myeloid blasts, at least about 80% myeloid blasts, at least about 90% myeloid blasts, or greater than about 90% myeloid blasts following one or more prior lines of therapy. In some embodiments, a subject may have persistence of at most about 90% myeloid blasts, at most about 80% myeloid blasts, at most about 70% myeloid blasts, at most about 60% myeloid blasts, at most about 55% myeloid blasts, at most about 50% myeloid blasts, at most about 45% myeloid blasts, at most about 40% myeloid blasts, at most about 35% myeloid blasts, at most about 30% myeloid blasts, at most about 25% myeloid blasts, at most about 20% myeloid blasts, at most about 15% myeloid blasts, at most about 10% myeloid blasts, at most about 5% myeloid blasts, or less than about 5% myeloid blasts following one or more prior lines of therapy.

[0106] In some embodiments, a subject may have persistence of myeloid blasts in bone marrow comprising from about 5% myeloid blasts to about 75% myeloid blasts following one or more prior lines of therapy. In some embodiments, a subject may have persistence of myeloid blasts in bone marrow comprising from about 5% myeloid blasts to about 10% myeloid blasts, about 5% myeloid blasts to about 15% myeloid blasts, about 5% myeloid blasts to about 20% myeloid blasts, about 5% myeloid blasts to about 25% myeloid blasts, about 5% myeloid blasts to about 30% myeloid blasts, about 5% myeloid blasts to about 35% myeloid blasts, about 5% myeloid blasts to about 40% myeloid blasts, about 5% myeloid blasts to about 45% myeloid blasts, about 5% myeloid blasts to about 50% myeloid blasts, about 5% myeloid blasts to about 60% myeloid blasts, about 5% myeloid blasts to about 75% myeloid blasts, about 10% myeloid blasts to about 15% myeloid blasts, about 10% myeloid blasts to about 20% myeloid blasts, about 10% myeloid blasts to about 25% myeloid blasts, about 10% myeloid blasts to about 30% myeloid blasts, about 10% myeloid blasts to about 35% myeloid blasts, about 10% myeloid blasts to about 40% myeloid blasts, about 10% myeloid blasts to about 45% myeloidAttorney Docket No. 62190-737601blasts, about 10% myeloid blasts to about 50% myeloid blasts, about 10% myeloid blasts to about 60% myeloid blasts, about 10% myeloid blasts to about 75% myeloid blasts, about 15% myeloid blasts to about 20% myeloid blasts, about 15% myeloid blasts to about 25% myeloid blasts, about 15% myeloid blasts to about 30% myeloid blasts, about 15% myeloid blasts to about 35% myeloid blasts, about 15% myeloid blasts to about 40% myeloid blasts, about 15% myeloid blasts to about 45% myeloid blasts, about 15% myeloid blasts to about 50% myeloid blasts, about 15% myeloid blasts to about 60% myeloid blasts, about 15% myeloid blasts to about 75% myeloid blasts, about 20% myeloid blasts to about 25% myeloid blasts, about 20% myeloid blasts to about 30% myeloid blasts, about 20% myeloid blasts to about 35% myeloid blasts, about 20% myeloid blasts to about 40% myeloid blasts, about 20% myeloid blasts to about 45% myeloid blasts, about 20% myeloid blasts to about 50% myeloid blasts, about 20% myeloid blasts to about 60% myeloid blasts, about 20% myeloid blasts to about 75% myeloid blasts, about 25% myeloid blasts to about 30% myeloid blasts, about 25% myeloid blasts to about 35% myeloid blasts, about 25% myeloid blasts to about 40% myeloid blasts, about 25% myeloid blasts to about 45% myeloid blasts, about 25% myeloid blasts to about 50% myeloid blasts, about 25% myeloid blasts to about 60% myeloid blasts, about 25% myeloid blasts to about 75% myeloid blasts, about 30% myeloid blasts to about 35% myeloid blasts, about 30% myeloid blasts to about 40% myeloid blasts, about 30% myeloid blasts to about 45% myeloid blasts, about 30% myeloid blasts to about 50% myeloid blasts, about 30% myeloid blasts to about 60% myeloid blasts, about 30% myeloid blasts to about 75% myeloid blasts, about 35% myeloid blasts to about 40% myeloid blasts, about 35% myeloid blasts to about 45% myeloid blasts, about 35% myeloid blasts to about 50% myeloid blasts, about 35% myeloid blasts to about 60% myeloid blasts, about 35% myeloid blasts to about 75% myeloid blasts, about 40% myeloid blasts to about 45% myeloid blasts, about 40% myeloid blasts to about 50% myeloid blasts, about 40% myeloid blasts to about 60% myeloid blasts, about 40% myeloid blasts to about 75% myeloid blasts, about 45% myeloid blasts to about 50% myeloid blasts, about 45% myeloid blasts to about 60% myeloid blasts, about 45% myeloid blasts to about 75% myeloid blasts, about 50% myeloid blasts to about 60% myeloid blasts, about 50% myeloid blasts to about 75% myeloid blasts, or about 60% myeloid blasts to about 75% myeloid blasts following one or more prior lines of therapy.

[0107] In some embodiments, the method comprising administering the composition comprising the engineered immune cell to the subject in need thereof further comprises administering to the subject a pre-conditioning regimen prior to administration of the composition. The pre-conditioning regimen may comprise a lymphodepleting chemotherapyAttorney Docket No. 62190-737601regimen. The lymphodepletion can comprise administration of various drugs. For example, the lymphodepletion can comprise administration of fludarabine, etoposide, cyclophosphamide, bendamustine, busulfan, cytarabine, or alemtuzumab, or any combination thereof. In some embodiments, fludarabine and cyclophosphamide are administered to the subject. In some embodiments, etoposide and cyclophosphamide are administered to the subject. A lymphodepleting chemotherapy regimen may be tailored for a patient on a clinical basis. Lymphodepletion can comprise modifications of drugs and / or doses. In some embodiments, one or more of the drugs administered as part of the lymphodepleting chemotherapy regiment may be administered intravenously. For dose measurement, the units of milligram per square meter (mg / sqm) and mg / m2may be used interchangeably.

[0108] For example, a lymphodepleting chemotherapy regimen (e.g., lymphodepletion) described herein may comprise administering etoposide. Etoposide may be administered to the subject at a dose of at least about 10 mg / m2, at least about 20 mg / m2, at least about 30 mg / m2, at least about 40 mg / m2, at least about 50 mg / m2, at least about 60 mg / m2, at least about 70 mg / m2, at least about 80 mg / m2, at least about 90 mg / m2, at least about 100 mg / m2, at least about 150 mg / m2, at least about 200 mg / m2, at least about 250 mg / m2, at least about 300 mg / m2, at least about 400 mg / m2, at least about 500 mg / m2, or greater than about 500 mg / m2. Etoposide may be administered to the subject at a dose of at most about 500 mg / m2, at most about 400 mg / m2, at most about 300 mg / m2, at most about 250 mg / m2, at most about 200 mg / m2, at most about 150 mg / m2, at most about 100 mg / m2, at most about 90 mg / m2, at most about 80 mg / m2, at most about 70 mg / m2, at most about 60 mg / m2, at most about 50 mg / m2, at most about 40 mg / m2, at most about 30 mg / m2, at most about 20 mg / m2, at most about 10 mg / m2, or less than about 10 mg / m2. Etoposide may be administered to the subject at a dose from about 10 mg / m2to about 500 mg / m2. Etoposide may be administered to the subject at a dose from about 10 mg / m2to about 20 mg / m2, about 10 mg / m2to about 30 mg / m2, about 10 mg / m2to about 40 mg / m2, about 10 mg / m2to about 50 mg / m2, about 10 mg / m2to about 100 mg / m2, about 10 mg / m2to about 150 mg / m2, about 10 mg / m2to about 200 mg / m2, about 10 mg / m2to about 250 mg / m2, about 10 mg / m2to about 300 mg / m2, about 10 mg / m2to about 400 mg / m2, about 10 mg / m2to about 500 mg / m2, about 20 mg / m2to about 30 mg / m2, about 20 mg / m2to about 40 mg / m2, about 20 mg / m2to about 50 mg / m2, about 20 mg / m2to about 100 mg / m2, about 20 mg / m2to about 150 mg / m2, about 20 mg / m2to about 200 mg / m2, about 20 mg / m2to about 250 mg / m2, about 20 mg / m2to about 300 mg / m2, about 20 mg / m2to about 400 mg / m2, about 20 mg / m2to about 500 mg / m2, about 30 mg / m2to about 40 mg / m2, about 30 mg / m2to about 50 mg / m2, about 30 mg / m2to about 100 mg / m2, about 30 mg / m2to about 150 mg / m2, about 30Attorney Docket No. 62190-737601mg / m2to about 200 mg / m2, about 30 mg / m2to about 250 mg / m2, about 30 mg / m2to about 300 mg / m2, about 30 mg / m2to about 400 mg / m2, about 30 mg / m2to about 500 mg / m2, about 40 mg / m2to about 50 mg / m2, about 40 mg / m2to about 100 mg / m2, about 40 mg / m2to about 150 mg / m2, about 40 mg / m2to about 200 mg / m2, about 40 mg / m2to about 250 mg / m2, about 40 mg / m2to about 300 mg / m2, about 40 mg / m2to about 400 mg / m2, about 40 mg / m2to about 500 mg / m2, about 50 mg / m2to about 100 mg / m2, about 50 mg / m2to about 150 mg / m2, about 50 mg / m2to about 200 mg / m2, about 50 mg / m2to about 250 mg / m2, about 50 mg / m2to about 300 mg / m2, about 50 mg / m2to about 400 mg / m2, about 50 mg / m2to about 500 mg / m2, about 100 mg / m2to about 150 mg / m2, about 100 mg / m2to about 200 mg / m2, about 100 mg / m2to about 250 mg / m2, about 100 mg / m2to about 300 mg / m2, about 100 mg / m2to about 400 mg / m2, about 100 mg / m2to about 500 mg / m2, about 150 mg / m2to about 200 mg / m2, about 150 mg / m2to about 250 mg / m2, about 150 mg / m2to about 300 mg / m2, about 150 mg / m2to about 400 mg / m2, about 150 mg / m2to about 500 mg / m2, about 200 mg / m2to about 250 mg / m2, about 200 mg / m2to about 300 mg / m2, about 200 mg / m2to about 400 mg / m2, about 200 mg / m2to about 500 mg / m2, about 250 mg / m2to about 300 mg / m2, about 250 mg / m2to about 400 mg / m2, about 250 mg / m2to about 500 mg / m2, about 300 mg / m2to about 400 mg / m2, about 300 mg / m2to about 500 mg / m2, or about 400 mg / m2to about 500 mg / m2. In some embodiments, etoposide can be administered to the subject at a dose of about 150 mg / m2.

[0109] Etoposide may be administered for any duration of time prior to administration of the engineered immune cell described herein. For example, etoposide may be administered once per day or more than once per day (e.g., 2x per day, 3x per day, or more). Etoposide may be administered once per day for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than 10 days. Etoposide may be administered once per day for at most about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, etoposide may be administered from about 1 day to about 14 days. In some embodiments, etoposide may be administered from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 14 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about 14 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 daysAttorney Docket No. 62190-737601to about 9 days, about 3 days to about 10 days, about 3 days to about 12 days, about 3 days to about 14 days, about 4 days to about 5 days, about 4 days to about 6 days, about 4 days to about 7 days, about 4 days to about 8 days, about 4 days to about 9 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 14 days, about 5 days to about 6 days, about 5 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, about 5 days to about 10 days, about 5 days to about 12 days, about 5 days to about 14 days, about 6 days to about 7 days, about 6 days to about 8 days, about 6 days to about 9 days, about 6 days to about 10 days, about 6 days to about 12 days, about 6 days to about 14 days, about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 12 days, about 7 days to about 14 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 12 days, about 8 days to about 14 days, about 9 days to about 10 days, about 9 days to about 12 days, about 9 days to about 14 days, about 10 days to about 12 days, about 10 days to about 14 days, or about 12 days to about 14 days. In some embodiments, etoposide may be administered from 1 day to 10 days.

[0110] The lymphodepleting chemotherapy regimen can comprise at least 1 dose of etoposide, at least 2 doses of etoposide, at least 3 doses of etoposide, at least 4 doses of etoposide, at least 5 doses of etoposide, or greater than about 5 doses of etoposide. The lymphodepleting chemotherapy regimen can comprise at most 5 doses of etoposide, at most 4 doses of etoposide, at most 3 doses of etoposide, at most 2 doses of etoposide, or at most 1 dose of etoposide. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of etoposide to about 5 doses of etoposide. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of etoposide to about 2 doses of etoposide, about 1 dose of etoposide to about 3 doses of etoposide, about 1 dose of etoposide to about 4 doses of etoposide, about 1 dose of etoposide to about 5 doses of etoposide, about 2 doses of etoposide to about 3 doses of etoposide, about 2 doses of etoposide to about 4 doses of etoposide, about 2 doses of etoposide to about 5 doses of etoposide, about 3 doses of etoposide to about 4 doses of etoposide, about 3 doses of etoposide to about 5 doses of etoposide, or about 4 doses of etoposide to about 5 doses of etoposide.[oni] In some embodiments, etoposide may be administered on consecutive days prior to administration of the composition comprising the engineered immune cell. For consecutive administration, etoposide may be administered on a subset of consecutive days. For example, etoposide may be administered for 3 days and 2 days be consecutive days of administration (e.g., etoposide may be administered on days -5, -4, and -2 relative to administration of the composition comprising the engineered immune cell). For consecutive administration,Attorney Docket No. 62190-737601etoposide may be administered on all consecutive days. As another example, etoposide may be administered for 3 days and all 3 days may be consecutive days of administration (e.g., etoposide may be administered on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell). In some embodiments, etoposide may be administered on non-consecutive days prior to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered on days -6, -4, and -2 relative to administration of the composition comprising the engineered immune cell.

[0112] In some embodiments, etoposide may be administered to the subject at a dose of about 150 mg / m2per day for about 3 days. In some embodiments, etoposide may be administered to the subject at a dose of about 150 mg / m2per day for about 3 consecutive days. In some embodiments, etoposide may be administered to the subject at a dose of about 150 mg / m2per day for about 3 non-consecutive days. In some embodiments, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on any combination of days -7 through -2 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -6, -5, and -3 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -6, -5, and -2 relative to administration of the composition comprising the engineered immune cell. For example, etoposide may be administered to the subject at a dose of about 150 mg / m2per day on days -5, -3, and -2 relative to administration of the composition comprising the engineered immune cell.

[0113] A lymphodepleting chemotherapy regimen (e.g., lymphodepletion) described herein may comprise administering cytarabine. Cytarabine may be administered to the subject at a dose of at least about 0.01 g / m2, at least about 0.05 g / m2, at least about 0.1 g / m2, at least about 0.5 g / m2, at least about 1.0 g / m2, at least about 1.5 g / m2, at least about 2.0 g / m2, at least about 2.5 g / m2, at least about 3.0 g / m2, at least about 3.5 g / m2, at least about 4.0 g / m2, at least about 5.0 g / m2, or greater than about 5.0 g / m2. Cytarabine may be administered to the subject at aAttorney Docket No. 62190-737601dose of at most about 5.0 g / m2, at most about 4.0 g / m2, at most about 3.5 g / m2, at most about 3.0 g / m2, at most about 2.5 g / m2, at most about 2.0 g / m2, at most about 1.5 g / m2, at most about 1.0 g / m2, at most about 0.5 g / m2, at most about 0.1 g / m2, at most about 0.05 g / m2, at most about 0.01 g / m2, or less than about 0.01 g / m2. Cytarabine may be administered to the subject at a dose from about 0.01 g / m2to about 5 g / m2. Cytarabine may be administered to the subject at a dose from about 0.01 g / m2to about 0.05 g / m2, about 0.01 g / m2to about 0.1 g / m2, about 0.01 g / m2to about 0.5 g / m2, about 0.01 g / m2to about 1 g / m2, about 0.01 g / m2to about 1.5 g / m2, about 0.01 g / m2to about 2 g / m2, about 0.01 g / m2to about 2.5 g / m2, about 0.01 g / m2to about 3 g / m2, about 0.01 g / m2to about 3.5 g / m2, about 0.01 g / m2to about 4 g / m2, about 0.01 g / m2to about 5 g / m2, about 0.05 g / m2to about 0.1 g / m2, about 0.05 g / m2to about 0.5 g / m2, about 0.05 g / m2to about 1 g / m2, about 0.05 g / m2to about 1.5 g / m2, about 0.05 g / m2to about 2 g / m2, about 0.05 g / m2to about 2.5 g / m2, about 0.05 g / m2to about 3 g / m2, about 0.05 g / m2to about 3.5 g / m2, about 0.05 g / m2to about 4 g / m2, about 0.05 g / m2to about 5 g / m2, about 0.1 g / m2to about 0.5 g / m2, about 0.1 g / m2to about 1 g / m2, about 0.1 g / m2to about 1.5 g / m2, about 0.1 g / m2to about 2 g / m2, about 0.1 g / m2to about 2.5 g / m2, about 0.1 g / m2to about 3 g / m2, about 0.1 g / m2to about 3.5 g / m2, about 0.1 g / m2to about 4 g / m2, about 0.1 g / m2to about 5 g / m2, about 0.5 g / m2to about 1 g / m2, about 0.5 g / m2to about 1.5 g / m2, about 0.5 g / m2to about 2 g / m2, about 0.5 g / m2to about 2.5 g / m2, about 0.5 g / m2to about 3 g / m2, about 0.5 g / m2to about 3.5 g / m2, about 0.5 g / m2to about 4 g / m2, about 0.5 g / m2to about 5 g / m2, about 1 g / m2to about 1.5 g / m2, about 1 g / m2to about 2 g / m2, about 1 g / m2to about 2.5 g / m2, about 1 g / m2to about 3 g / m2, about 1 g / m2to about 3.5 g / m2, about 1 g / m2to about 4 g / m2, about 1 g / m2to about 5 g / m2, about 1.5 g / m2to about 2 g / m2, about 1.5 g / m2to about 2.5 g / m2, about 1.5 g / m2to about 3 g / m2, about 1.5 g / m2to about 3.5 g / m2, about 1.5 g / m2to about 4 g / m2, about 1.5 g / m2to about 5 g / m2, about 2 g / m2to about 2.5 g / m2, about 2 g / m2to about 3 g / m2, about 2 g / m2to about 3.5 g / m2, about 2 g / m2to about 4 g / m2, about 2 g / m2to about 5 g / m2, about 2.5 g / m2to about 3 g / m2, about 2.5 g / m2to about 3.5 g / m2, about 2.5 g / m2to about 4 g / m2, about 2.5 g / m2to about 5 g / m2, about 3 g / m2to about 3.5 g / m2, about 3 g / m2to about 4 g / m2, about 3 g / m2to about 5 g / m2, about 3.5 g / m2to about 4 g / m2, about 3.5 g / m2to about 5 g / m2, or about 4 g / m2to about 5 g / m2. In some embodiments, etoposide can be administered to the subject at a dose of about 2 g / m2.

[0114] Cytarabine may be administered for any duration of time prior to administration of the engineered immune cell described herein. For example, cytarabine may be administered once per day or more than once per day (e.g., 2x per day, 3x per day, or more). Cytarabine may be administered once per day for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7Attorney Docket No. 62190-737601days, 8 days, 9 days, 10 days, or more than 10 days. Cytarabine may be administered once per day for at most about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, cytarabine may be administered from about 1 day to about 14 days. In some embodiments, cytarabine may be administered from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 14 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about 14 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 12 days, about 3 days to about 14 days, about 4 days to about 5 days, about 4 days to about 6 days, about 4 days to about 7 days, about 4 days to about 8 days, about 4 days to about 9 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 14 days, about 5 days to about 6 days, about 5 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, about 5 days to about 10 days, about 5 days to about 12 days, about 5 days to about 14 days, about 6 days to about 7 days, about 6 days to about 8 days, about 6 days to about 9 days, about 6 days to about 10 days, about 6 days to about 12 days, about 6 days to about 14 days, about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 12 days, about 7 days to about 14 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 12 days, about 8 days to about 14 days, about 9 days to about 10 days, about 9 days to about 12 days, about 9 days to about 14 days, about 10 days to about 12 days, about 10 days to about 14 days, or about 12 days to about 14 days. In some embodiments, cytarabine may be administered from 1 day to 10 days.

[0115] The lymphodepleting chemotherapy regimen can comprise at least 1 dose of cytarabine, at least 2 doses of cytarabine, at least 3 doses of cytarabine, at least 4 doses of cytarabine, at least 5 doses of cytarabine, or greater than about 5 doses of cytarabine. The lymphodepleting chemotherapy regimen can comprise at most 5 doses of cytarabine, at most 4 doses of cytarabine, at most 3 doses of cytarabine, at most 2 doses of cytarabine, or at most 1 dose of cytarabine. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of cytarabine to about 5 doses of cytarabine. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of cytarabine to about 2 doses of cytarabine, about 1 dose ofAttorney Docket No. 62190-737601cytarabine to about 3 doses of cytarabine, about 1 dose of cytarabine to about 4 doses of cytarabine, about 1 dose of cytarabine to about 5 doses of cytarabine, about 2 doses of cytarabine to about 3 doses of cytarabine, about 2 doses of cytarabine to about 4 doses of cytarabine, about 2 doses of cytarabine to about 5 doses of cytarabine, about 3 doses of cytarabine to about 4 doses of cytarabine, about 3 doses of cytarabine to about 5 doses of cytarabine, or about 4 doses of cytarabine to about 5 doses of cytarabine.

[0116] In some embodiments, cytarabine may be administered on consecutive days prior to administration of the composition comprising the engineered immune cell. For consecutive administration, cytarabine may be administered on a subset of consecutive days. For example, cytarabine may be administered for 3 days and 2 days be consecutive days of administration (e.g., cytarabine may be administered on days -5, -4, and -2 relative to administration of the composition comprising the engineered immune cell). For consecutive administration, cytarabine may be administered on all consecutive days. As another example, cytarabine may be administered for 5 days and all 3 days may be consecutive days of administration (e.g., cytarabine may be administered on days -6, -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell). In some embodiments, cytarabine may be administered on non-consecutive days prior to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered on days -6, -4, and -2 relative to administration of the composition comprising the engineered immune cell.

[0117] In some embodiments, cytarabine may be administered to the subject at a dose of about 2 g / m2per day for about 5 days. In some embodiments, cytarabine may be administered to the subject at a dose of about 2 g / m2per day for about 5 consecutive days. In some embodiments, cytarabine may be administered to the subject at a dose of about 2 g / m2per day for about 5 non-consecutive days. In some embodiments, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on any combination of days -7 through -2 relative to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on days -7, -6, -5, -4, and -3, relative to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on days -7, -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on days -7, -6, -5, -3, and -2 relative to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on days -7, -6, -5, -4, and -2Attorney Docket No. 62190-737601relative to administration of the composition comprising the engineered immune cell. For example, cytarabine may be administered to the subject at a dose of about 2 g / m2per day on days -6, -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell.

[0118] A lymphodepleting chemotherapy regimen (e.g., lymphodepletion) described herein may comprise administering cyclophosphamide. Cyclophosphamide may be administered to the subject at a dose of at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 70 mg / kg, at least about 80 mg / kg, at least about 90 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 200 mg / kg, or greater than about 200 mg / kg. Cyclophosphamide may be administered to the subject at a dose of at most about 200 mg / kg, at most about 150 mg / kg, at most about 100 mg / kg, at most about 90 mg / kg, at most about 80 mg / kg, at most about 70 mg / kg, at most about 60 mg / kg, at most about 50 mg / kg, at most about 40 mg / kg, at most about 30 mg / kg, at most about 20 mg / kg, at most about 10 mg / kg, at most about 5 mg / kg, at most about 2 mg / kg, or less than about 2 mg / kg. Cyclophosphamide may be administered to the subject at a dose from about 2 mg / kg to about 100 mg / kg. Cyclophosphamide may be administered to the subject at a dose from about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 30 mg / kg, about 2 mg / kg to about 40 mg / kg, about 2 mg / kg to about 50 mg / kg, about 2 mg / kg to about 60 mg / kg, about 2 mg / kg to about 70 mg / kg, about 2 mg / kg to about 80 mg / kg, about 2 mg / kg to about 90 mg / kg, about 2 mg / kg to about 100 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 5 mg / kg to about 40 mg / kg, about 5 mg / kg to about 50 mg / kg, about 5 mg / kg to about 60 mg / kg, about 5 mg / kg to about 70 mg / kg, about 5 mg / kg to about 80 mg / kg, about 5 mg / kg to about 90 mg / kg, about 5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 40 mg / kg, about 10 mg / kg to about 50 mg / kg, about 10 mg / kg to about 60 mg / kg, about 10 mg / kg to about 70 mg / kg, about 10 mg / kg to about 80 mg / kg, about 10 mg / kg to about 90 mg / kg, about 10 mg / kg to about 100 mg / kg, about 20 mg / kg to about 30 mg / kg, about 20 mg / kg to about 40 mg / kg, about 20 mg / kg to about 50 mg / kg, about 20 mg / kg to about 60 mg / kg, about 20 mg / kg to about 70 mg / kg, about 20 mg / kg to about 80 mg / kg, about 20 mg / kg to about 90 mg / kg, about 20 mg / kg to about 100 mg / kg, about 30 mg / kg to about 40 mg / kg, about 30 mg / kg to about 50 mg / kg, about 30 mg / kg to about 60 mg / kg, about 30 mg / kg to about 70 mg / kg, about 30 mg / kg to about 80 mg / kg, about 30 mg / kg to about 90 mg / kg, about 30 mg / kg to about 100 mg / kg, about 40Attorney Docket No. 62190-737601mg / kg to about 50 mg / kg, about 40 mg / kg to about 60 mg / kg, about 40 mg / kg to about 70 mg / kg, about 40 mg / kg to about 80 mg / kg, about 40 mg / kg to about 90 mg / kg, about 40 mg / kg to about 100 mg / kg, about 50 mg / kg to about 60 mg / kg, about 50 mg / kg to about 70 mg / kg, about 50 mg / kg to about 80 mg / kg, about 50 mg / kg to about 90 mg / kg, about 50 mg / kg to about 100 mg / kg, about 60 mg / kg to about 70 mg / kg, about 60 mg / kg to about 80 mg / kg, about 60 mg / kg to about 90 mg / kg, about 60 mg / kg to about 100 mg / kg, about 70 mg / kg to about 80 mg / kg, about 70 mg / kg to about 90 mg / kg, about 70 mg / kg to about 100 mg / kg, about 80 mg / kg to about 90 mg / kg, about 80 mg / kg to about 100 mg / kg, or about 90 mg / kg to about 100 mg / kg. In some embodiments, cyclophosphamide may be administered at a dose of about 60 mg / kg.

[0119] In some embodiments, cyclophosphamide may be administered in units of milligram per square meter (mg / sqm). Cyclophosphamide may be administered to the subject at a dose of at least about 100 mg / sqm, at least about 200 mg / sqm, at least about 300 mg / sqm, at least about 400 mg / sqm, at least about 500 mg / sqm, at least about 600 mg / sqm, at least about 700 mg / sqm, at least about 800 mg / sqm, at least about 900 mg / sqm, at least about 1000 mg / sqm, at least about 1200 mg / sqm, at least about 1400 mg / sqm, at least about 1600 mg / sqm, at least about 1800 mg / sqm, at least about 2000 mg / sqm, or greater than about 2000 mg / sqm. Cyclophosphamide may be administered to the subject at a dose of at most about 2000 mg / sqm, at most about 1800 mg / sqm, at most about 1600 mg / sqm, at most about 1400 mg / sqm, at most about 1200 mg / sqm, at most about 1000 mg / sqm, at most about 900 mg / sqm, at most about 800 mg / sqm, at most about 700 mg / sqm, at most about 600 mg / sqm, at most about 500 mg / sqm, at most about 400 mg / sqm, at most about 300 mg / sqm, at most about 200 mg / sqm, at most about 100 mg / sqm, or less than about 100 mg / sqm. Cyclophosphamide may be administered to the subject at a dose from about 100 mg / sqm to about 1,500 mg / sqm. Cyclophosphamide may be administered to the subject at a dose from about 100 mg / sqm to about 200 mg / sqm, about 100 mg / sqm to about 300 mg / sqm, about 100 mg / sqm to about 400 mg / sqm, about 100 mg / sqm to about 500 mg / sqm, about 100 mg / sqm to about 750 mg / sqm, about 100 mg / sqm to about 1,000 mg / sqm, about 100 mg / sqm to about 1,200 mg / sqm, about 100 mg / sqm to about 1,400 mg / sqm, about 100 mg / sqm to about 1,500 mg / sqm, about 200 mg / sqm to about 300 mg / sqm, about 200 mg / sqm to about 400 mg / sqm, about 200 mg / sqm to about 500 mg / sqm, about 200 mg / sqm to about 750 mg / sqm, about 200 mg / sqm to about 1,000 mg / sqm, about 200 mg / sqm to about 1,200 mg / sqm, about 200 mg / sqm to about 1,400 mg / sqm, about 200 mg / sqm to about 1,500 mg / sqm, about 300 mg / sqm to about 400 mg / sqm, about 300 mg / sqm to about 500 mg / sqm, about 300 mg / sqm to about 750 mg / sqm, about 300 mg / sqm to about 1,000 mg / sqm, about 300 mg / sqm to about 1,200 mg / sqm, about 300 mg / sqm to about 1,400 mg / sqm,Attorney Docket No. 62190-737601about 300 mg / sqm to about 1,500 mg / sqm, about 400 mg / sqm to about 500 mg / sqm, about 400 mg / sqm to about 750 mg / sqm, about 400 mg / sqm to about 1,000 mg / sqm, about 400 mg / sqm to about 1,200 mg / sqm, about 400 mg / sqm to about 1,400 mg / sqm, about 400 mg / sqm to about 1,500 mg / sqm, about 500 mg / sqm to about 750 mg / sqm, about 500 mg / sqm to about 1,000 mg / sqm, about 500 mg / sqm to about 1,200 mg / sqm, about 500 mg / sqm to about 1,400 mg / sqm, about 500 mg / sqm to about 1,500 mg / sqm, about 750 mg / sqm to about 1,000 mg / sqm, about 750 mg / sqm to about 1,200 mg / sqm, about 750 mg / sqm to about 1,400 mg / sqm, about 750 mg / sqm to about 1,500 mg / sqm, about 1,000 mg / sqm to about 1,200 mg / sqm, about 1,000 mg / sqm to about 1,400 mg / sqm, about 1,000 mg / sqm to about 1,500 mg / sqm, about 1,200 mg / sqm to about 1,400 mg / sqm, about 1,200 mg / sqm to about 1,500 mg / sqm, or about 1,400 mg / sqm to about 1,500 mg / sqm.

[0120] Cyclophosphamide may be administered for any duration of time prior to administration of the engineered immune cell described herein. For example, cyclophosphamide may be administered once per day or more than once per day (e.g., 2x per day, 3x per day, or more). Cyclophosphamide may be administered once per day for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than 10 days. Cyclophosphamide may be administered once per day for at most about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, cyclophosphamide may be administered from about 1 day to about 14 days. In some embodiments, cyclophosphamide may be administered from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 14 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about 14 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 12 days, about 3 days to about 14 days, about 4 days to about 5 days, about 4 days to about 6 days, about 4 days to about 7 days, about 4 days to about 8 days, about 4 days to about 9 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 14 days, about 5 days to about 6 days, about 5 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, about 5 days to about 10 days, about 5 days to about 12 days, about 5 days to about 14 days, about 6 days toAttorney Docket No. 62190-737601about 7 days, about 6 days to about 8 days, about 6 days to about 9 days, about 6 days to about 10 days, about 6 days to about 12 days, about 6 days to about 14 days, about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 12 days, about 7 days to about 14 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 12 days, about 8 days to about 14 days, about 9 days to about 10 days, about 9 days to about 12 days, about 9 days to about 14 days, about 10 days to about 12 days, about 10 days to about 14 days, or about 12 days to about 14 days. In some embodiments, cyclophosphamide may be administered from 1 day to 10 days.

[0121] The lymphodepleting chemotherapy regimen can comprise at least 1 dose of cyclophosphamide, at least 2 doses of cyclophosphamide, at least 3 doses of cyclophosphamide, at least 4 doses of cyclophosphamide, at least 5 doses of cyclophosphamide, or greater than about 5 doses of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise at most 5 doses of cyclophosphamide, at most 4 doses of cyclophosphamide, at most 3 doses of cyclophosphamide, at most 2 doses of cyclophosphamide, or at most 1 dose of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of cyclophosphamide to about 5 doses of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of cyclophosphamide to about 2 doses of cyclophosphamide, about 1 dose of cyclophosphamide to about 3 doses of cyclophosphamide, about 1 dose of cyclophosphamide to about 4 doses of cyclophosphamide, about 1 dose of cyclophosphamide to about 5 doses of cyclophosphamide, about 2 doses of cyclophosphamide to about 3 doses of cyclophosphamide, about 2 doses of cyclophosphamide to about 4 doses of cyclophosphamide, about 2 doses of cyclophosphamide to about 5 doses of cyclophosphamide, about 3 doses of cyclophosphamide to about 4 doses of cyclophosphamide, about 3 doses of cyclophosphamide to about 5 doses of cyclophosphamide, or about 4 doses of cyclophosphamide to about 5 doses of cyclophosphamide.

[0122] In some embodiments, cyclophosphamide may be administered on consecutive days prior to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered for 2 days and 2 days be consecutive days of administration (e.g., cyclophosphamide may be administered on days -4 and -3 relative to administration of the composition comprising the engineered immune cell). In some embodiments, cyclophosphamide may be administered on non-consecutive days prior to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered on days -4 and -2 relative to administration of theAttorney Docket No. 62190-737601composition comprising the engineered immune cell. In some embodiments, cyclophosphamide may be administered to the subject on 2 consecutive days prior to administration of the composition comprising the engineered immune cell.

[0123] In some embodiments, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day for about 2 days. In some embodiments, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day for about 2 consecutive days. In some embodiments, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day for about 2 non-consecutive days. In some embodiments, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on any combination of days -7 through -2 relative to administration of the composition comprising the engineered immune cell (e.g., administered on day 0). For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -6 and -4 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -6 and -3 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -6 and -2 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -5 and -3 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -5 and -2 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -4 and -2 relative to administration of the composition comprising the engineered immune cell. For example, cyclophosphamide may be administered to the subject at a dose of about 60 mg / kg per day on days -3 and -2 relative to administration of the composition comprising the engineered immune cell.Attorney Docket No. 62190-737601

[0124] In some embodiments, the lymphodepleting chemotherapy regimen can comprise a combination of agents. For example, the lymphodepleting chemotherapy regimen can comprise administration of etoposide and cyclophosphamide. The etoposide and cyclophosphamide may be administered on at least one same day. The etoposide and cyclophosphamide may be administered on different days. The combination of agents may comprise cyclophosphamide at any dose or administration schedule, as described herein, and etoposide at any dose or administration schedule, as described herein. As an example, the lymphodepletion can comprise administration of (i) etoposide at a dose of about 150 mg / m2per day for 3 consecutive days, and (ii) cyclophosphamide at a dose of about 60 mg / kg per day for 2 consecutive days. In some embodiments, the subject may be administered at least one dose of etoposide and at least one dose of cyclophosphamide prior to administration of the composition comprising the engineered immune cell.

[0125] In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -6, -5, and -4 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -3 and -2 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -5, -4, and -3 relative toAttorney Docket No. 62190-737601administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -3 and -2 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of etoposide at a level of about 150 mg / m2per day on days -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administrationAttorney Docket No. 62190-737601of etoposide at a level of about 150 mg / m2per day on days -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 60 mg / kg per day on days -3 and -2 relative to administration of the composition comprising the engineered immune cell.

[0126] A lymphodepleting chemotherapy regimen (e.g., lymphodepletion) described herein may comprise administering fludarabine. Fludarabine may be administered to the subject at a dose of at least about 1 mg / sqm, at least about 5 mg / sqm, at least about 10 mg / sqm, at least about 20 mg / sqm, at least about 30 mg / sqm, at least about 40 mg / sqm, at least about 50 mg / sqm, at least about 60 mg / sqm, at least about 70 mg / sqm, at least about 80 mg / sqm, at least about 90 mg / sqm, at least about 100 mg / sqm, at least about 150 mg / sqm, at least about 200 mg / sqm, or greater than about 200 mg / sqm. Fludarabine may be administered to the subject at a dose of at most about 200 mg / sqm, at most about 150 mg / sqm, at most about 100 mg / sqm, at most about 90 mg / sqm, at most about 80 mg / sqm, at most about 70 mg / sqm, at most about 60 mg / sqm, at most about 50 mg / sqm, at most about 40 mg / sqm, at most about 30 mg / sqm, at most about 20 mg / sqm, at most about 10 mg / sqm, at most about 5 mg / sqm, at most about 1 mg / sqm, or less than about 1 mg / sqm. Fludarabine may be administered to the subject at a dose from about 1 mg / sqm to about 100 mg / sqm. Fludarabine may be administered to the subject at a dose from about 1 mg / sqm to about 5 mg / sqm, about 1 mg / sqm to about 10 mg / sqm, about 1 mg / sqm to about 20 mg / sqm, about 1 mg / sqm to about 30 mg / sqm, about 1 mg / sqm to about 40 mg / sqm, about 1 mg / sqm to about 50 mg / sqm, about 1 mg / sqm to about 60 mg / sqm, about 1 mg / sqm to about 70 mg / sqm, about 1 mg / sqm to about 80 mg / sqm, about 1 mg / sqm to about 90 mg / sqm, about 1 mg / sqm to about 100 mg / sqm, about 5 mg / sqm to about 10 mg / sqm, about 5 mg / sqm to about 20 mg / sqm, about 5 mg / sqm to about 30 mg / sqm, about 5 mg / sqm to about 40 mg / sqm, about 5 mg / sqm to about 50 mg / sqm, about 5 mg / sqm to about 60 mg / sqm, about 5 mg / sqm to about 70 mg / sqm, about 5 mg / sqm to about 80 mg / sqm, about 5 mg / sqm to about 90 mg / sqm, about 5 mg / sqm to about 100 mg / sqm, about 10 mg / sqm to about 20 mg / sqm, about 10 mg / sqm to about 30 mg / sqm, about 10 mg / sqm to about 40 mg / sqm, about 10 mg / sqm to about 50 mg / sqm, about 10 mg / sqm to about 60 mg / sqm, about 10 mg / sqm to about 70 mg / sqm, about 10 mg / sqm to about 80 mg / sqm, about 10 mg / sqm to about 90 mg / sqm, about 10 mg / sqm to about 100 mg / sqm, about 20 mg / sqm to about 30 mg / sqm, about 20 mg / sqm to about 40 mg / sqm, about 20 mg / sqm to about 50 mg / sqm, about 20 mg / sqm to about 60 mg / sqm, about 20 mg / sqm to about 70 mg / sqm, about 20 mg / sqm to about 80 mg / sqm, about 20 mg / sqm to about 90 mg / sqm, about 20 mg / sqm to about 100 mg / sqm, about 30 mg / sqm to about 40 mg / sqm, about 30 mg / sqm to about 50 mg / sqm, about 30 mg / sqm to about 60 mg / sqm, aboutAttorney Docket No. 62190-73760130 mg / sqm to about 70 mg / sqm, about 30 mg / sqm to about 80 mg / sqm, about 30 mg / sqm to about 90 mg / sqm, about 30 mg / sqm to about 100 mg / sqm, about 40 mg / sqm to about 50 mg / sqm, about 40 mg / sqm to about 60 mg / sqm, about 40 mg / sqm to about 70 mg / sqm, about 40 mg / sqm to about 80 mg / sqm, about 40 mg / sqm to about 90 mg / sqm, about 40 mg / sqm to about 100 mg / sqm, about 50 mg / sqm to about 60 mg / sqm, about 50 mg / sqm to about 70 mg / sqm, about 50 mg / sqm to about 80 mg / sqm, about 50 mg / sqm to about 90 mg / sqm, about 50 mg / sqm to about 100 mg / sqm, about 60 mg / sqm to about 70 mg / sqm, about 60 mg / sqm to about 80 mg / sqm, about 60 mg / sqm to about 90 mg / sqm, about 60 mg / sqm to about 100 mg / sqm, about 70 mg / sqm to about 80 mg / sqm, about 70 mg / sqm to about 90 mg / sqm, about 70 mg / sqm to about 100 mg / sqm, about 80 mg / sqm to about 90 mg / sqm, about 80 mg / sqm to about 100 mg / sqm, or about 90 mg / sqm to about 100 mg / sqm. In some embodiments, fludarabine may be administered at a dose of about 30 mg / sqm.

[0127] Fludarabine may be administered for any duration of time prior to administration of the engineered immune cell described herein. For example, fludarabine may be administered once per day or more than once per day (e.g., 2x per day, 3x per day, or more). Fludarabine may be administered once per day for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than 10 days. Fludarabine may be administered once per day for at most about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, fludarabine may be administered from about 1 day to about 14 days. In some embodiments, fludarabine may be administered from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 14 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about 14 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 12 days, about 3 days to about 14 days, about 4 days to about 5 days, about 4 days to about 6 days, about 4 days to about 7 days, about 4 days to about 8 days, about 4 days to about 9 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 14 days, about 5 days to about 6 days, about 5 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, about 5 days to about 10 days, about 5 days to about 12 days, about 5 days to about 14Attorney Docket No. 62190-737601days, about 6 days to about 7 days, about 6 days to about 8 days, about 6 days to about 9 days, about 6 days to about 10 days, about 6 days to about 12 days, about 6 days to about 14 days, about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 12 days, about 7 days to about 14 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 12 days, about 8 days to about 14 days, about 9 days to about 10 days, about 9 days to about 12 days, about 9 days to about 14 days, about 10 days to about 12 days, about 10 days to about 14 days, or about 12 days to about 14 days. In some embodiments, fludarabine may be administered from 1 day to 10 days prior to administration of the composition comprising the engineered immune cell.

[0128] The lymphodepleting chemotherapy regimen can comprise at least 1 dose of fludarabine, at least 2 doses of fludarabine, at least 3 doses of fludarabine, at least 4 doses of fludarabine, at least 5 doses of fludarabine, or greater than about 5 doses of fludarabine. The lymphodepleting chemotherapy regimen can comprise at most 5 doses of fludarabine, at most 4 doses of fludarabine, at most 3 doses of fludarabine, at most 2 doses of fludarabine, or at most 1 dose of fludarabine. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of fludarabine to about 5 doses of fludarabine. The lymphodepleting chemotherapy regimen can comprise from about 1 dose of fludarabine to about 2 doses of fludarabine, about 1 dose of fludarabine to about 3 doses of fludarabine, about 1 dose of fludarabine to about 4 doses of fludarabine, about 1 dose of fludarabine to about 5 doses of fludarabine, about 2 doses of fludarabine to about 3 doses of fludarabine, about 2 doses of fludarabine to about 4 doses of fludarabine, about 2 doses of fludarabine to about 5 doses of fludarabine, about 3 doses of fludarabine to about 4 doses of fludarabine, about 3 doses of fludarabine to about 5 doses of fludarabine, or about 4 doses of fludarabine to about 5 doses of fludarabine.

[0129] In some embodiments, fludarabine may be administered on consecutive days prior to administration of the composition comprising the engineered immune cell. For consecutive administration, fludarabine may be administered on a subset of consecutive days. For example, fludarabine may be administered for 4 days and 3 days be consecutive days of administration (e.g., fludarabine may be administered on days -6, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell). For consecutive administration, fludarabine may be administered on all consecutive days. As another example, fludarabine may be administered for 4 days and all 4 days may be consecutive days of administration (e.g., fludarabine may be administered on days -6, -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell). In some embodiments, fludarabine mayAttorney Docket No. 62190-737601be administered on non-consecutive days prior to administration of the composition comprising the engineered immune cell. For example, fludarabine may be administered on days -7, -5, -3, and -1 relative to administration of the composition comprising the engineered immune cell.

[0130] In some embodiments, the lymphodepleting chemotherapy regimen can comprise a combination of agents. For example, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine and cyclophosphamide. The fludarabine and cyclophosphamide may be administered on at least one same day. The fludarabine and cyclophosphamide may be administered on different days. The combination of agents may comprise cyclophosphamide at any dose or administration schedule, as described herein, and fludarabine at any dose or administration schedule, as described herein. As an example, the lymphodepletion can comprise administration of (i) fludarabine at a dose of about 30 mg / sqm per day for 4 consecutive days, and (ii) cyclophosphamide at a dose of about 1000 mg / sqm per day for 2 consecutive days. In some embodiments, the subject may be administered at least one dose of fludarabine and at least one dose of cyclophosphamide prior to administration of the composition comprising the engineered immune cell.

[0131] In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -6, -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -6, -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -6, -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -6, -5, -4, and -3 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per dayAttorney Docket No. 62190-737601on days -3 and -2 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -6 and -5 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -5 and -4 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -4 and -3 relative to administration of the composition comprising the engineered immune cell. In some embodiments, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg / sqm per day on days -5, -4, -3, and -2 relative to administration of the composition comprising the engineered immune cell, and further comprise administration of cyclophosphamide at a level of about 1000 mg / sqm per day on days -3 and -2 relative to administration of the composition comprising the engineered immune cell.

[0132] The composition comprising the engineered immune cell comprising the first nucleotide sequence and second nucleotide sequence described herein may be administered at any point following the pre-conditioning regimen (e.g., the lymphodepletion). In some embodiments, the composition can be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, or greater than about 50 days following the pre-conditioning regimen (e.g., the lymphodepletion). In some embodiments, the composition can be administered at most about 50 days, at most about 45 days, at most about 40 days, at most about 35 days, at most about 30 days, at most about 25 days, at most about 20 days, at most about 15 days, at most about 10 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 1 day, orAttorney Docket No. 62190-737601less than about 1 day following the pre-conditioning regimen (e.g., the lymphodepletion). In some embodiments, the composition can be administered from about 1 day to about 50 days following the pre-conditioning regimen (e.g., the lymphodepletion). In some embodiments, the composition can be administered from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 10 days, about 1 day to about 15 days, about 1 day to about 20 days, about 1 day to about 25 days, about 1 day to about 30 days, about 1 day to about 40 days, about 1 day to about 50 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 10 days, about 2 days to about 15 days, about 2 days to about 20 days, about 2 days to about 25 days, about 2 days to about 30 days, about 2 days to about 40 days, about 2 days to about 50 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 10 days, about 3 days to about 15 days, about 3 days to about 20 days, about 3 days to about 25 days, about 3 days to about 30 days, about 3 days to about 40 days, about 3 days to about 50 days, about 4 days to about 5 days, about 4 days to about 10 days, about 4 days to about 15 days, about 4 days to about 20 days, about 4 days to about 25 days, about 4 days to about 30 days, about 4 days to about 40 days, about 4 days to about 50 days, about 5 days to about 10 days, about 5 days to about 15 days, about 5 days to about 20 days, about 5 days to about 25 days, about 5 days to about 30 days, about 5 days to about 40 days, about 5 days to about 50 days, about 10 days to about 15 days, about 10 days to about 20 days, about 10 days to about 25 days, about 10 days to about 30 days, about 10 days to about 40 days, about 10 days to about 50 days, about 15 days to about 20 days, about 15 days to about 25 days, about 15 days to about 30 days, about 15 days to about 40 days, about 15 days to about 50 days, about 20 days to about 25 days, about 20 days to about 30 days, about 20 days to about 40 days, about 20 days to about 50 days, about 25 days to about 30 days, about 25 days to about 40 days, about 25 days to about 50 days, about 30 days to about 40 days, about 30 days to about 50 days, or about 40 days to about 50 days following the pre-conditioning regimen (e.g., the lymphodepletion).

[0133] The composition may not be administered immediately after a pre-conditioning regimen (e.g., the lymphodepletion). For example, a composition as described herein may not be administered about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, or 5 days after the pre-conditioning regimen (e.g., the lymphodepletion). The composition may be administered following a duration of time after the last dosing of the pre-conditioning regimen (e.g., the lymphodepletion). The last dosing of the pre-conditioning regimen (e.g., the lymphodepletion) may be at least about 1, 2, 3, 4, 5, or more days before administration of the composition described herein. For example, the composition may be administered at least aboutAttorney Docket No. 62190-7376011 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, or greater than about 50 days after the last dosing of the pre-conditioning regimen (e.g., the lymphodepletion). In some embodiments, the composition may be administered at most about 50 days, at most about 45 days, at most about 40 days, at most about 35 days, at most about 30 days, at most about 25 days, at most about 20 days, at most about 15 days, at most about 10 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 1 day, or less than about 1 day after the last dosing of the pre-conditioning regimen (e.g., the lymphodepletion).

[0134] The composition may be administered once per day or more than once per day (e.g., 2x per day, 3x per day, or more). The composition may be administered once per day for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, or more than 60 days. The composition may be administered once per day for at most about 60 days, at most about 50 days, at most about 40 days, at most about 30 days, at most about 20 days, at most about 15 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or at most about 1 day. In some embodiments, the composition may be administered once per day from about 1 day to about 60 days. In some embodiments, the composition may be administered once per day from about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 10 days, about 1 day to about 15 days, about 1 day to about 20 days, about 1 day to about 30 days, about 1 day to about 40 days, about 1 day to about 50 days, about 1 day to about 60 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 10 days, about 2 days to about 15 days, about 2 days to about 20 days, about 2 days to about 30 days, about 2 days to about 40 days, about 2 days to about 50 days, about 2 days to about 60 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 10 days, about 3 days to about 15 days, about 3 days to about 20 days, about 3 days to about 30 days, about 3 days to about 40 days, about 3 days to about 50 days, about 3 days to about 60 days, about 4 days to about 5 days, about 4 days to about 10 days, about 4 days to about 15 days, about 4 days to about 20 days, about 4 days to about 30 days, about 4 days toAttorney Docket No. 62190-737601about 40 days, about 4 days to about 50 days, about 4 days to about 60 days, about 5 days to about 10 days, about 5 days to about 15 days, about 5 days to about 20 days, about 5 days to about 30 days, about 5 days to about 40 days, about 5 days to about 50 days, about 5 days to about 60 days, about 10 days to about 15 days, about 10 days to about 20 days, about 10 days to about 30 days, about 10 days to about 40 days, about 10 days to about 50 days, about 10 days to about 60 days, about 15 days to about 20 days, about 15 days to about 30 days, about 15 days to about 40 days, about 15 days to about 50 days, about 15 days to about 60 days, about 20 days to about 30 days, about 20 days to about 40 days, about 20 days to about 50 days, about 20 days to about 60 days, about 30 days to about 40 days, about 30 days to about 50 days, about 30 days to about 60 days, about 40 days to about 50 days, about 40 days to about 60 days, or about 50 days to about 60 days.

[0135] The composition described herein may be administered at a dose to the subject. In some embodiments, the composition may be administered at a dose of at least about IxlO4engineered immune cells / kg, at least about 5xl04engineered immune cells / kg, at least about IxlO5engineered immune cells / kg, at least about 5xl05engineered immune cells / kg, at least about IxlO6engineered immune cells / kg, at least about 5xl06engineered immune cells / kg, at least about IxlO7engineered immune cells / kg, at least about 5xl07engineered immune cells / kg, at least about IxlO8engineered immune cells / kg, or greater than about IxlO8engineered immune cells / kg. In some embodiments, the composition may be administered at a dose of at most IxlO8engineered immune cells / kg, at most about 5xl07engineered immune cells / kg, at most IxlO7engineered immune cells / kg, at most 5x10' engineered immune cells / kg, at most 1x10' engineered immune cells / kg, at most 5x10 engineered immune cells / kg, at most lxl0:engineered immune cells / kg, at most 5x10‘ engineered immune cells / kg, at most 1x10' engineered immune cells / kg, or less than about IxlO4engineered immune cells / kg. In some embodiments, the composition may be administered at a dose of IxlO6engineered immune cells / kg. In some embodiments, the composition may be administered at a dose of 3xl06engineered immune cells / kg.

[0136] In some embodiments, the composition may be administered as a single dose. In some embodiments, the composition may be administered as multiple doses. The composition may be administered as 2 doses, 3 doses, 4 doses, 5 doses, or greater than about 5 doses. A second dose of the composition may be administered after a duration of time following a first dose of the composition. In some embodiments, the method may further comprise administering a second dose of the composition. The second dose may be administered no sooner than 5 days, no sooner than 10 days, no sooner than 1 month, no sooner than 2 months, no sooner than 3Attorney Docket No. 62190-737601months, no sooner than 4 months, no sooner than 5 months, no sooner than 6 months, no sooner than 1 year, or no sooner than 2 years following completion of a first dose. The second dose may be administered no later than 2 years, no later than 1 year, no later than 6 months, no later than 5 months, no later than 4 months, no later than 3 months, no later than 2 months, no later than 1 month, no later than 10 days, or no later than 5 days following completion of a first dose.

[0137] In some embodiments, the composition may be administered multiple doses on a single day (e.g., 2, 3, 4, 5, or more doses on 1 day). The composition may be administered as multiple doses over a duration of time. In some embodiments, the composition may be administered as multiple doses over multiple days. For example, the composition may be administered as 4 doses over 4 days, in which the composition is administered as a single dose on each of the 4 days. As another example, the composition may be administered as 4 doses over 3 days, in which the composition is administered as a single dose on 2 days and as 2 doses on 1 day.

[0138] A bridging therapy can comprise a treatment to help a patient through periods of illness during a transition to another stage of therapy. For example, a bridging therapy may be provided to a subject during a transition to a therapy comprising administering the composition described herein. In some embodiments, the method may further comprise subjecting a subject to one or more bridging therapies. The subject may be subjected to 1, 2, 3, 4, or more bridging therapies. In some embodiments, the bridging therapy may be administered prior to administering the composition comprising the engineered immune cell. The bridging therapy may be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 20 days, at least about 30 days, at least about 60 days, or greater than about 60 days prior to administering the composition comprising the engineered immune cell. In some embodiments, the bridging therapy may be administered concomitantly with administering the composition comprising the engineered immune cell. In some embodiments, the bridging therapy may be administered subsequent to administering the composition comprising the engineered immune cell. The bridging therapy may be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 20 days, at least about 30 days, at least about 60 days, or greater than about 60 days subsequent to administering the composition comprising the engineered immune cell. In some embodiments, the bridging therapy can comprise 5-azacytidine, venetoclax, or any combination thereof. In some embodiments, the method may not comprise administration of any bridging therapy.Attorney Docket No. 62190-737601

[0139] The method may further comprise measuring a level of myeloid blasts of the subject. The level of myeloid blasts may be measured from a sample obtained from the subject. The sample may be a bone marrow sample. The sample may be a blood sample. In some embodiments, the level of myeloid blasts may be measured prior to administering the composition to the subject. In some embodiments, the level of myeloid blasts may be measured subsequent to administering the composition to the subject. In some embodiments, the level of myeloid blasts may be measured both prior to administering the composition to the subject and subsequent to administering the composition to the subject. By measuring at both timepoints, one may determine how a level of myeloid blasts changes following administration of the composition. In some embodiments, the myeloid blasts may be CD7+ , CD33+, CD34+, or any combination thereof (e.g., the myeloid blasts may be CD7+CD33+CD34+ myeloid blasts). Measuring a level of myeloid blasts from a sample may comprise measuring an expression level of CD7, CD33, CD34, or any combination thereof.

[0140] A persistence of myeloid blasts may decrease following administration of the composition. For example, a persistence of myeloid blasts in a bone marrow sample of the subject may be at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than about 1% subsequent to administering the composition described herein. In some embodiments, a bone marrow sample from a subject may not contain any detectable level of myeloid blasts following administration of the composition.

[0141] Administering the composition to the subject may increase a percentage of cells expressing the anti-CD7 CAR described herein. A sample (e.g., a peripheral blood sample and / or a bone marrow sample) obtained from the subject administered the composition may comprise a greater percentage of engineered immune cells expressing the CAR, compared to level of CAR in a sample obtained from an otherwise identical subject administered an engineered immune cell comprising a nucleotide sequence encoding the CAR without a nucleotide sequence encoding the first target-binding domain linked to a localizing domain.

[0142] In some embodiments, subsequent to administration of the composition, a percentage of engineered immune cells expressing the CAR in a sample obtained from the subject may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater than about 95%. In some embodiments, subsequent to administration of the composition, a percentage of engineeredAttorney Docket No. 62190-737601immune cells expressing the CAR in a sample obtained from the subject may be at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20%. A percentage of engineered immune cells expressing CAR may be from about 50% to 90% at least 9 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be about 70% at least 9 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be from about 50% to 100% at least 14 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be about 80% at least 14 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be from about 40% to 80% at least 20 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be about 60% at least 20 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be from about 20% to 60% at least 28 days subsequent to administering the composition to the subject. A percentage of engineered immune cells expressing CAR may be about 40% at least 28 days subsequent to administering the composition to the subject.

[0143] In some embodiments, administration of the composition to the subject may result in expansion of CD4+ and / or CD8+ CAR+ T cells with an effector memory phenotype. This effector memory phenotype may be measured via antibodies for T cell surface markers. For example, effector memory (EM) CD4+CAR+ cells may be CD45RO+ CD4+ CAR+. An effector memory phenotype may comprise cells that are CD45RA negative (CD45RA-), CD45RO positive (CD45RO+), CD 197 negative (CD197-), or any combination thereof. As another example, EM CD8+CAR+ cells may be CD45RO+ CD8+ CAR+. Administration of the composition to the subject may result in expansion of terminal effector memory T reexpressing CD45RA (TEMRA) cells. EM T cells may be measured in peripheral blood samples from the subject, bone marrow samples from the subject, or any combination thereof. Expansion of EM T cells may be detected at any timepoint subsequent to administering the composition. For example, expansion of EM T cells may be detected at about 1 day postadministration, about 2 days post-administration, about 3 days post-administration, about 4 days post-administration, about 5 days post-administration, about 6 days post-administration, about 7 days post-administration, about 14 days post-administration, about 21 days postadministration, about 28 days post-administration, about 30 days post-administration, about 45Attorney Docket No. 62190-737601days post-administration, about 60 days post-administration, or greater than about 60 days postadministration of the composition.

[0144] In some embodiments, a percentage of EM CD4+CAR+ T cells and / or EM CD8+CAR+ T cells subsequent to administration of the composition may be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than about 99% in a bone marrow or peripheral blood sample from the subject. In some embodiments, a percentage of EM CD4+CAR+ T cells and / or EM CD8+CAR+ T cells subsequent to administration of the composition may be at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, or less than about 50% in a bone marrow or peripheral blood sample from the subject.

[0145] In some embodiments, subsequent to administering, one or more tests may be performed to assess a minimal residual disease (MRD) of the subject. In some embodiments, the method may further comprise, subsequent to administering the composition, detecting a minimal residual disease (MRD) of the subject. Detection of MRD can comprise determining a level of myeloid blasts in a sample (e.g., a bone marrow sample) obtained from the subject. The myeloid blasts may be positive for CD9, CD7, CD18, CD25, CD32, CD33, CD34, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a / c, CD366, CD371, and CX3CR1, or any combination thereof. The myeloid blasts may be positive for CD7, CD34, and / or CD33. For example, the myeloid blasts may be CD7+CD33+CD34+ blasts.

[0146] In some embodiments, the method can further comprise administering an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent or therapy may comprise surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, radiation therapy, targeted small molecule therapy or any combinations thereof.

[0147] In some embodiments, the subject may not exhibit any adverse event after administering a dose of the composition described herein. In some embodiments, the subject may exhibit an adverse event after administering a dose of the composition described herein. A second dose of the composition may then be administered after a period of time following the first dose (e.g., at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, or greater than 10 days following the first dose). In some embodiments, the adverse event is selected from the group consisting ofAttorney Docket No. 62190-737601cytokine release syndrome (CRS), neurotoxicity, severe CRS (sCRS), CAR T cell-related encephalopathy syndrome (CRES) or immune effector cell-associated neurotoxicity syndrome (ICANS), Central Nervous System (CNS) toxicity, tumor lysis syndrome (TLS), Immune Effector Cell-Associated Hemophagocytic Lymphohistiocytosis-Like Syndrome (IEC-HS), an infusion reaction, cytopenia, cardiac toxicity, hypogammaglobulinemia, infection, organ toxicity, graft-versus-host-disease, and any combination thereof.

[0148] In some embodiments, CRS (cytokine release syndrome) can be graded in severity from 1-5 as follows. Grades 1-2 are less severe CRS. Grades 3-4 can be considered severe CRS. Grade 5 CRS is defined as death due to CRS. For Grade 1 CRS, symptomatic treatment may only be needed (e.g., nausea, fever, fatigue, myalgias, malaise, headache) and symptoms are not life threatening. For Grade 2 CRS, the symptoms may need moderate intervention and generally can respond to moderate intervention. Subjects having Grade 2 CRS develop hypotension that is responsive to either fluids or one low-dose vasopressor; or they develop grade 2 organ toxicity or mild respiratory symptoms that are responsive to low flow oxygen (<40% oxygen). In Grade 3 CRS subjects, hypotension generally cannot be reversed by fluid therapy or one low-dose vasopressor. These subjects generally require more than low flow oxygen and have grade 3 organ toxicity (e.g., renal or cardiac dysfunction or coagulopathy) and / or grade 4 transaminitis. Grade 3 CRS subjects require more aggressive intervention, e.g., oxygen of 40% or higher, high dose vasopressor(s), and / or multiple vasopressors. Grade 4 CRS subjects suffer from immediately life-threatening symptoms, including grade 4 organ toxicity or a need for mechanical ventilation. Grade 4 CRS subjects generally do not have transaminitis. In Grade 5 CRS subjects, the toxicity causes death. Sets of criteria for grading CRS are provided herein as Table 1, Table 2, and Table 3.Table 1. CRS grading.Attorney Docket No. 62190-737601Table 2. CTCAE v5.0 CRS grading scale.Table 3. NCI CRS grading scale.<>

[0149] In some embodiments, CRS may be graded according to Table 1. In some embodiments, CRS may be graded according to Table 2. In some embodiments, CRS may be graded according to Table 3. In some embodiments, the adverse event may be cytokine release syndrome and / or neurotoxicity. In some embodiments, the adverse event may be > grade 3 cytokine release syndrome and / or > grade 2 neurotoxicity. In some embodiments, the adverse event may be grade 1 cytokine release syndrome. The CRS may only occur for at most about 20 days, at most about 14 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 1 day, or less than about 1 day. In some embodiments, the method does not induce cytokine release syndrome (CRS) above grade 1, above grade 2, or above grade 3.Attorney Docket No. 62190-737601Target Binding Domains to CD7

[0150] In some embodiments, a target-binding domain (e.g., a first target-binding domain and / or a second target-binding domain) that binds to CD7 may be an antibody or antigen binding fragment thereof. In some embodiments, the antibody molecule, can be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, orbispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody). The antibody fragment may be a single-chain variable fragment antibody (“scFv”).

[0151] A target-binding domain may comprise an antibody or antigen binding fragment thereof comprising a heavy chain variable region (VH) and / or a light chain variable region (VL). The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on a TH69 antibody described herein. The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on a 3A1F antibody described herein. The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on a T3-3 Al antibody described herein.

[0152] In some embodiments, the VH may have an amino acid sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 98.5% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 9, 19, or 29. In some embodiments, the VH of the anti-CD7 target-binding domain may have an amino acid sequence as set forth in any one of SEQ ID NOs: 9, 19, or 29. In some embodiments, the heavy chain variable region may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in a sequence as set forth in SEQ ID NO: 9, 19, or 29. In some embodiments, the heavy chain variable region may comprise ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in a sequence as set forth in SEQ ID NO: 9, 19, or 29. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0153] In some embodiments, the VL may have an amino acid sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequenceAttorney Docket No. 62190-737601identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 98.5% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 10, 20, or 30. In some embodiments, the VL of the anti-CD7 target-binding domain may have an amino acid sequence as set forth in any one of SEQ ID NOs: 10, 20, or 30. In some embodiments, the light chain variable region may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in a sequence as set forth in SEQ ID NO: 10, 20, or 30. In some embodiments, the light chain variable region may comprise ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in a sequence as set forth in SEQ ID NO: 10, 20, or 30.

[0154] In some embodiments, the target-binding domain (e.g., the first target-binding domain or the second target-binding domain) can comprise an antibody or antigen binding fragment thereof comprising: (i) a HC CDR1 comprising a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 1, 4, 11, 14, 21, or 24; (ii) a HC CDR2 comprising a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 2, 5, 12, 15, 22, or 25; (iii) a HC CDR3 comprising a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 3, 13, or 23; (iv) a LC CDR1 comprising a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 6, 16, or 26; (v) a LC CDR2 comprising a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 7, 17, or 27; and / or (vi) a LC CDR3 comprising aAttorney Docket No. 62190-737601sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 8, 18, or 28.

[0155] In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on the TH69 antibody. The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) can comprise a heavy chain variable region (VH) and a light chain variable region (VL) having an amino acid sequence that each have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOS: 9 and 10, respectively. The heavy chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH sequence of SEQ ID NO: 9. The light chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 9. In some embodiments, the heavy chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth in SEQ ID NO: 9. In some embodiments, the light chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 10. In some embodiments, the light chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth in SEQ ID NO: 10. Any of the amino acid substitutions described herein can be conservative or nonconservative substitutions.Attorney Docket No. 62190-737601

[0156] ). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7scFv) comprises a HC CDR1 of SEQ ID NO: 1 (SYAMS), a HC CDR2 of SEQ ID NO: 2 (SISSGGFTYYPDSVKG), and a HC CDR3 of SEQ ID NO: 3 (DEVRGYLDV). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises a LC CDR1 of SEQ ID NO: 6 (SASQGISNYLN), a LC CDR2 of SEQ ID NO: 7 (YTSSLHS), and a LC CDR3 of SEQ ID NO: 8 (QQYSKLPYT). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises a LC CDR1 of SEQ ID NO: 6, a LC CDR2 of SEQ ID NO: 7, a LC CDR3 of SEQ ID NO: 8, a HC CDR1 of SEQ ID NO: 1, a HC CDR2 of SEQ ID NO: 2, and a HC CDR3 of SEQ ID NO: 3.

[0157] In embodiments, the VH and / or VL of the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be encoded by one or more nucleotide sequences. In some embodiments, the nucleic acid sequence encoding the VH comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 70. In some embodiments, the nucleic acid sequence encoding the VL comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:71.

[0158] In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on the 3 A1F antibody. The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) can comprise a heavy chain variable region (VH) and a light chain variable region (VL) having an amino acid sequence that each have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOS: 19 and 20, respectively. The heavy chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequenceAttorney Docket No. 62190-737601identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH sequence of SEQ ID NO: 19. The light chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 19. In some embodiments, the heavy chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth in SEQ ID NO: 19. In some embodiments, the light chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 20. In some embodiments, the light chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth in SEQ ID NO: 20. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0159] ). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7scFv) comprises a HC CDR1 of SEQ ID NO: 11 (SYWMH), a HC CDR2 of SEQ ID NO: 12 (KINPSNGRTNYNEKFKS), and a HC CDR3 of SEQ ID NO: 13 (GGVYYDLYYYALDY). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises a LC CDR1 of SEQ ID NO: 16 (RASQSISNNLH), a LC CDR2 of SEQ ID NO: 17 (SASQSIS), and a LC CDR3 of SEQ ID NO: 18 (QQSNSWPYT). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises a LC CDR1 of SEQ ID NO: 16, a LC CDR2 of SEQ ID NO: 17, a LC CDR3 of SEQ ID NO: 18, a HC CDR1 of SEQ ID NO: 11, a HC CDR2 of SEQ ID NO: 12, and a HC CDR3 of SEQ ID NO: 13.

[0160] In embodiments, the VH and / or VL of the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be encoded by one or more nucleotide sequences. In some embodiments, the nucleic acid sequence encoding the VH comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acidAttorney Docket No. 62190-737601sequence set forth in SEQ ID NO: 72. In some embodiments, the nucleic acid sequence encoding the VL comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:73.

[0161] In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be based on the T3-3 Al antibody. The anti-CD7 target-binding domain (e.g., anti-CD7 scFv) can comprise a heavy chain variable region (VH) and a light chain variable region (VL) having an amino acid sequence that each have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOS: 29 and 30, respectively. The heavy chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VH sequence of SEQ ID NO: 29. The light chain variable region can comprise at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 30. In some embodiments, the heavy chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 29. In some embodiments, the heavy chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth in SEQ ID NO: 29. In some embodiments, the light chain variable region comprises at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 30. In some embodiments, the light chain variable region comprises ten or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less) amino acid substitutions in the sequence set forth inAttorney Docket No. 62190-737601SEQ ID NO: 30. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0162] ). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7scFv) comprises a HC CDR1 of SEQ ID NO: 21 (SFGMH), a HC CDR2 of SEQ ID NO: 22 (YISSGSSTLHYADTVKG), and a HC CDR3 of SEQ ID NO: 23 (WGNYPHYAMDY). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises aLC CDR1 of SEQ ID NO: 26 (RASKS VSASGYSYMH), a LC CDR2 of SEQ ID NO: 27 (LASNLES), and a LC CDR3 of SEQ ID NO: 28 (QHSRELPYT). In some embodiments, the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) comprises a LC CDR1 of SEQ ID NO: 26, a LC CDR2 of SEQ ID NO: 27, a LC CDR3 of SEQ ID NO: 28, a HC CDR1 of SEQ ID NO: 21, a HC CDR2 of SEQ ID NO: 22, and a HC CDR3 of SEQ ID NO: 23.

[0163] In embodiments, the VH and / or VL of the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be encoded by one or more nucleotide sequences. In some embodiments, the nucleic acid sequence encoding the VH comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the nucleic acid sequence encoding the VL comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:75.

[0164] In some embodiments, the VH, VL, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, or any combination thereof of the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be an amino acid sequence as set forth in Table 4.Table 4. Amino acid sequences of CDRs, VH regions, and VL regions of exemplary anti-CD7 binders.Atorney Docket No. 62190-737601Attorney Docket No. 62190-737601

[0165] In some embodiments, the VH and / or VL of the anti-CD7 target-binding domain (e.g., anti-CD7 scFv) may be encoded by a nucleotide sequence as set forth in Table 5.Table 5. Nucleotide sequences of VH regions and VL regions of exemplary anti-CD7 binders.Attorney Docket No. 62190-737601Downregulation of Intracellular CD7 via CD7 PEBL

[0166] As described herein, T cell cytotoxicity may be markedly increased when anti-CD7 CAR is used in combination with downregulation of CD7 expression on immune cells. As demonstrated herein, downregulation (e.g., elimination, reduction, and / or relocalization) of surface CD7 can decrease and / or prevent the fratricidal effect exerted by the corresponding anti-CD7 CAR, allowing greater T cell recovery after CAR expression as compared to cells that retain the target antigen (e.g., CD7), and a more effective cytotoxicity against TAttorney Docket No. 62190-737601leukemia / lymphoma cells. Downregulation of CD7 expression on the effector T cells can be achieved according to a variety of known methods including, for example, protein expression blockers (PEBLs) against CD7, RNAi against CD7, or gene editing methods such as, e.g., meganucleases, TALEN, CRISPR / Cas9, and zinc finger nucleases. Gene disruption may also be possible through base editing technologies. Base editors are capable of making single base pair changes at defined genetic loci to alter gene expression. Adenine base editors (ABEs) and cytosine base editors (CBEs) combine a deaminase enzyme with a Cas nickase to mediate gene editing without double-stranded breaks in DNA. Without wishing to be bound by theory, the methods provided herein may provide for an efficient targeted multiplexed editing system. In some embodiments, a base editor system comprises a nucleotide binding domain, a deaminase domain for deaminating nucleobases in a target nucleotide sequence; and one or more guide RNA molecules (gRNAs) targeting Cas to a specific locus. Adenine base editors make A to G (or T to C) point mutations at a target site and cytosine base editors make C to T (or G to A) point mutations at a target site. In some cases, cytosine base editors can be fused with an inhibitor of uracil DNA glycosylase (UGI) to prevent base excision repair. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). In some embodiments, the deaminase may be AID, CDA1, or AP0BEC3G. In some embodiments, ADE or CBE may create 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more simultaneous edits at a genomic target site. In some embodiments, base editors can disrupt gene expression by making point mutations in splicing motifs or start codons. In some embodiments, base editors can disrupt gene expression by making point mutations to create termination codons. In some embodiments, the base editor system can comprise a dual base editor (e.g., a fusion of adenine and cytosine base editing components). A dual base editor (e.g., combinatorial base editor or multifunctional base editor) can comprise an adenosine deaminase domain and a cytidine deaminase domain. A dual base editor may further comprise one or more UGIs.

[0167] The protein expression blocker (PEBL) described herein can comprise a target-binding domain linked to a localizing domain. The present disclosure describes PEBLs that bind target antigens and sequester the target antigens to the cytoplasm of a cell. The present disclosure describes PEBLs that bind target antigens and sequester the target antigens to intracellular compartments of a cell. The first nucleotide sequence as described herein can encode an anti-CD7 PEBL, wherein the PEBL comprises the first antigen-binding domain and a localizing domain.Attorney Docket No. 62190-737601

[0168] In some embodiments, provided herein is a polynucleotide comprising a nucleotide sequence comprising a target-binding molecule (e.g., a CD7 antigen binding domain) linked to a localizing domain. In some instances, the PEBL comprises from the N-terminus to the C-terminus: a CD7 antigen binding domain, an optional domain linker, and an intracellular localizing domain. In some embodiments, the PEBL further comprises a signal peptide fused N-terminal to the CD7 antigen binding domain. In some embodiments, the CD7 antigen binding domain comprises a VL domain, a domain linker, and a VH domain.

[0169] As used herein, “linked” in the context of the protein expression blocker refers to a gene encoding a target-binding molecule connected directly in frame to one or more genes encoding one or more localizing domains. In some cases, the gene encoding a target-binding molecule may be connected to one or more genes encoding one or more localizing domains through a linker sequence. Various linker sequences can be used. For example, the linker sequence can be (GGGGS)n, where n is any integer from 1 to 12 (SEQ ID NO: 53). As another example, the linker may comprise a human influenza hemagglutinin (HA) tag or a Myc tag (SEQ ID NO: 34). The linker sequence may comprise a myc tag and GS-linker, as set forth in EQKLISEEDLGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 110).

[0170] In some embodiments, the localizing domain of the PEBL comprises an endoplasmic reticulum (ER) retention signal or Golgi retention sequence. In some embodiments, the localizing domain of the PEBL may comprise a proteosome localizing sequence. In some embodiments, the localizing domain comprises an endoplasmic reticulum (ER) retention signal as set forth in Table 6. In some embodiments, the localizing domain comprises a proteasome localizing sequence set forth in Table 6. The localizing domain can direct the PEBL to a specific cellular compartment, such as the Golgi or endoplasmic reticulum, the proteasome, or the cell membrane, depending on the application.

[0171] In some embodiments, proteasome localization is achieved by linking the scFv sequence to a tripartite motif containing 21 (TRIM21) targeting domain sequence and coexpressing the sequence encoding the human TRIM21 E3 ubiquitin ligase protein. TRIM21 binds with high affinity to the Fc domains of antibodies and can recruit the ubiquitin-proteosome complex to degrade molecules (e.g., proteins and peptides) bound to the antibodies. The TRIM21 targeting domain sequence encodes amino acid sequences selected from the group of human immunoglobulin G (IgG) constant regions (Fc) genes such as IgGl, IgG2, or IgG4 and is used to form a fusion protein comprising scFv and Fc domains. In this embodiment, the exogenously expressed TRIM21 protein binds the scFv-Fc fusion protein bound to the target protein (e.g., CD7) and directs the complex to the proteasome for degradation.Attorney Docket No. 62190-737601

[0172] In some embodiments, the PEBL may comprise a hinge domain and transmembrane domain sequence derived from CD8a, CD8P, 4- IBB, CD28, CD34, CD4, FcsRIy, CD 16, 0X40, CD3< CD3s, CD3y, CD38, TCRa, CD32, CD64, VEGFR2, FAS, orFGFR2B. In some embodiments, the PEBL comprises a hinge and transmembrane domain selected from the group consisting of a hinge and transmembrane domain of CD8a, a hinge and transmembrane domain of CD8P, a hinge and transmembrane domain of 4- IBB, a hinge and transmembrane domain of CD28, a hinge and transmembrane domain of CD34, a hinge and transmembrane domain of CD4, a hinge and transmembrane domain of FcsRIy, a hinge domain and transmembrane domain of CD 16, a hinge and transmembrane domain of 0X40, a hinge and transmembrane domain of CD3Q, a hinge and transmembrane domain of CD3s, a hinge and transmembrane domain of CD3y, a hinge and transmembrane domain of CD36, a hinge and transmembrane domain of TCRa, a hinge and transmembrane domain of CD32, a hinge and transmembrane domain of CD64, a hinge and transmembrane domain of VEGFR2, a hinge and transmembrane domain of FAS, and a hinge and transmembrane domain of FGFR2B.

[0173] In some embodiments, the PEBL comprises one or more of the components set forth in Table 6Table 6. Amino acid sequences of components of an exemplary CD7 PEBL.Attorney Docket No. 62190-737601

[0174] In some embodiments, the engineered immune cell described herein comprises a localizing domain. In some embodiments, the localizing domain comprises an ER retention signal (e.g., ER retention sequence), a Golgi retention sequence, and / or a proteasome localizing sequence. In some embodiments, the ER retention signal comprises an amino acid sequence of KDEL or KKXX, where X can be any amino acid. In some embodiments, the localizing domain can comprise a Myc tag and / or a linker sequence. In some embodiments, the Myc tag comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 34). In some embodiments, the linker sequence can be (GGGGS)n, where n is any integer from 1 to 12 (SEQ ID NO: 53).

[0175] In some embodiments, the localizing domain may comprise a sequence as set forth in SEQ ID NO: 31, 32, 33, 45, 46, 47, 48, 49, or 50. The localizing domain may comprise an ER retention signal, wherein the ER retention signal is set forth in SEQ ID NO: 35, 36, 37, 38, 39, 40, or 41. The localizing domain may comprise a Golgi retention signal as set forth in SEQ ID NO: 42. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 35. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 36. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 37. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 38. In some embodiments, aAttorney Docket No. 62190-737601localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 39. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 40. In some embodiments, a localizing domain comprises a linker sequence and an ER retention signal as set forth in SEQ ID NO: 41. In some embodiments, a localizing domain comprises a linker sequence and an Golgi retention signal as set forth in SEQ ID NO: 42. The linker sequence may comprise a linker as set forth in SEQ ID NO: 52 or 53. In some embodiments, the localizing domain may comprise a PEST motif. The PEST motif may comprise a sequence as set forth in SEQ ID NO: 43 or 44.

[0176] In some embodiments, a PEBL can comprise a first target-binding domain. In some embodiments, the PEBL comprises, from N-terminus to C-terminus, the first target-binding domain and a localizing domain. The first target-binding domain can comprise a target-binding domain as described herein. In some embodiments, the CD7 PEBL can comprise a CD7 antigen-binding domain comprising an amino acid sequence of SEQ ID NO: 9, an amino acid sequence of SEQ ID NO: 10, and a VH-VL linker. The VH-VL linker can be a (G4S)n linker where n can range from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the CD7 PEBL can comprise an amino acid sequence of SEQ ID NO: 9, an amino acid sequence of SEQ ID NO: 10, and an amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence of SEQ ID NO: 9, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 10, and an amino acid sequence of SEQ ID NO: 52 or 53. In other embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 9, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 10, and an amino acid sequence of SEQ ID NO: 52 or 53.

[0177] In some embodiments, the CD7 PEBL contains CD7 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 19, an amino acid sequence of SEQ ID NO: 20, and a VH-VL linker. The VH-VL linker can be a (G4S)n linker where n can range from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the CD7 PEBL can comprise an amino acid sequence of SEQ ID NO: 19, an amino acid sequence of SEQ ID NO: 20, and an amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least about 90% sequence identity or at least about 95%Attorney Docket No. 62190-737601sequence identity to SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 20, and the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence of SEQ ID NO: 19, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 20, and an amino acid sequence of SEQ ID NO: 52 or 53. In other embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 19, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 20, and an amino acid sequence of SEQ ID NO: 52 or 53.

[0178] In some embodiments, the CD7 PEBL contains CD7 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 29, an amino acid sequence of SEQ ID NO: 30, and a VH-VL linker. The VH-VL linker can be a (G4S)n linker where n can range from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the CD7 PEBL can comprise an amino acid sequence of SEQ ID NO: 29, an amino acid sequence of SEQ ID NO: 30, and an amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 29, the amino acid sequence of SEQ ID NO: 30, and the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the CD7 PEBL comprises an amino acid sequence of SEQ ID NO: 29, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 30, and an amino acid sequence of SEQ ID NO: 52 or 53. In other embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 29, an amino acid sequence having at least about 90% sequence identity or at least about 95% sequence identity to SEQ ID NO: 30, and an amino acid sequence of SEQ ID NO: 52 or 53.

[0179] In some cases, the CD7 PEBL also comprises a CD8a signal peptide comprising the amino acid sequence as set forth in SEQ ID NO: 54. In other cases, the anti-CD7 protein expression blocker also comprises CD8a hinge and transmembrane domains such as but not limited to the CD8a hinge and transmembrane domains encoded by the nucleotide sequence as set forth in SEQ ID NO: 79.

[0180] In some embodiments, a CD7 PEBL described herein may comprise an amino acid sequence as set forth in Table 7.Table 7. Amino acid sequences for exemplary PEBL constructsAttorney Docket No. 62190-737601

[0181] In some embodiments, the CD7 PEBL described herein comprises the sequence of SEQ ID NO: 61 and a proline at the N-terminus. In some embodiments, the CD7 PEBL comprises the sequence of SEQ ID NO: 62. The N-terminal proline residue can arise from the 2A cleavage. In some embodiments, the CD7 PEBL described herein comprises the sequence of SEQ ID NO: 63 and a proline at the N-terminus. In some embodiments, the CD7 PEBL comprises the sequence of SEQ ID NO: 64. In some embodiments, a PEBL described herein may comprise an amino acid sequence having at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 98.5% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or at least about 100% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 61-64.

[0182] In some embodiments, an engineered immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 61. In some embodiments, an engineeredAttorney Docket No. 62190-737601immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 61 and a proline at the N-terminus. In some embodiments, an engineered immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 62. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 61. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 61 and a proline at the N-terminus. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 62. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 61. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 61 and a proline at the N-terminus. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 62.

[0183] In some embodiments, an engineered immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 63. In some embodiments, an engineered immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 63 and a proline at the N-terminus. In some embodiments, an engineered immune cell provided herein comprises a CD7 PEBL comprising the sequence of SEQ ID NO: 64. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 63. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 63 and a proline at the N-terminus. In some embodiments, the engineered immune cell can be a CD4+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 64. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 63. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 63 and a proline at the N-terminus. In some embodiments, the engineered immune cell can be a CD8+ T cell comprising a CD7 PEBL comprising the sequence of SEQ ID NO: 64.

[0184] In some embodiments, the polynucleotide encoding the CD7 PEBL comprises one or more nucleic acid sequences set forth in Table 16.

[0185] In some embodiments, the VH domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence of SEQ ID NO: 70 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotideAttorney Docket No. 62190-737601sequence of SEQ ID NO: 71. In some embodiments, the VH domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 70 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 71. In some embodiments, the VH domain of the first targetbinding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 70 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 71, or a codon optimized variant thereof.

[0186] In some embodiments, the VH domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence of SEQ ID NO: 72 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence of SEQ ID NO: 73. In some embodiments, the VH domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 72 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 73. In some embodiments, the VH domain of the first targetbinding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 72 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 73, or a codon optimized variant thereof.Attorney Docket No. 62190-737601

[0187] In some embodiments, the VH domain of the first target-binding domain (e.g., the antiCD? antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence of SEQ ID NO: 74 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence of SEQ ID NO: 75. In some embodiments, the VH domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 74 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 75. In some embodiments, the VH domain of the first targetbinding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 74 and the VL domain of the first target-binding domain (e.g., the anti-CD7 antibody or antigen binding fragment thereof) of the PEBL comprises the nucleotide sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 75, or a codon optimized variant thereof.

[0188] In some embodiments, the PEBL comprises a component encoded by the nucleotide sequence of any sequence set forth in Table 8.Table 8. Nucleic acid sequence information for components of an exemplary CD7 PEBL.

[0189] In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, or more) sequence identity to SEQ ID NO: 88 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acidAttorney Docket No. 62190-737601sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 88 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 88.

[0190] In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, or more) sequence identity to SEQ ID NO: 89 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 89 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 89.

[0191] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 88. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 PEBL encoded by the bicistronic vector construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 88. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 88. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 88.

[0192] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 89. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 PEBL encoded by the bicistronic vector construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 89. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 89. In some embodiments, the engineered immune cell is a CD3+ T cell comprising aAttorney Docket No. 62190-737601CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90% sequence identity to SEQ ID NO: 89.Anti-CD7 Chimeric Antigen Receptors

[0193] In some embodiments, a CAR described herein comprises a target binding domain (e.g., a second target-binding domain) that binds to CD7. In some embodiments, a first target-binding domain (e.g., binding domain of a PEBL) and a second target-binding domain (e.g., binding domain of a CAR) may comprise a first antibody or antigen binding domain and a second antibody or antigen binding domain, respectively. In some embodiments, an amino acid sequence of the first target-binding domain (e.g., binding domain of a PEBL) and an amino acid sequence of the second target-binding domain (e.g., binding domain of a CAR) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, identical. In some embodiments, an amino acid sequence of the first target-binding domain (e.g., binding domain of a PEBL) and an amino acid sequence of the second target-binding domain (e.g., binding domain of a CAR) can be at least about 90% identical. In some embodiments, an amino acid sequence of the first targetbinding domain (e.g., binding domain of a PEBL) and an amino acid sequence of the second target-binding domain (e.g., binding domain of a CAR) can be identical.

[0194] In some embodiments, the CAR of the present disclosure comprises intracellular signaling domains of 4- IBB and CD3(^, and a second target-binding domain (e.g., an antibody or antigen binding fragment thereof) that specifically binds CD7. The second target binding domain can be a scFv. The CD7 CAR of the present disclosure may be referred to herein as “anti-CD7-41BB-CD3^”. In some embodiments, the CAR may comprise a CD8a hinge domain and transmembrane domain. The CD8a hinge domain and transmembrane domain can comprise an amino acid sequence as set forth in SEQ ID NO: 51.

[0195] In some embodiments, any of the amino acid sequences of the various components disclosed herein (e.g., scFv, intracellular signaling domain, linker, and combinations thereof) can have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the specific corresponding sequences disclosed herein. For example, in some embodiments, the intracellular signaling domain 4-1BB can have at leastAttorney Docket No. 62190-737601about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to SEQ ID NO:55, as long as it possesses the desired function. In some embodiments, the intracellular signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the intracellular signaling domain of 4-1BB may be encoded by a nucleic acid sequence having at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 80.

[0196] As another example, in some embodiments, the intracellular signaling domain 4- IBB can be replaced by another intracellular signaling domain from a co-stimulatory molecule such as CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD30, CD84, CRTAM, DR3, SLAMF1, or CD2. In some embodiments, the intracellular signaling domain of the CAR can have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the intracellular signaling domain of CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2.

[0197] As another example, in certain instances, the intracellular signaling domain of 4-1BB can also comprise another intracellular signaling domain (or a portion thereof) from a costimulatory molecule such as CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD30, CD84, CRTAM, DR3, SLAMF1, or CD2. In some embodiments, the additional intracellular signaling domain can have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the intracellular signaling domain of CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD30, CD84, CRTAM, DR3, SLAMF1, or CD2. In other embodiments, the additional intracellular signaling domainAttorney Docket No. 62190-737601comprises at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to one or more intracellular signaling domain fragment(s) of CD28, 0X40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD30, CD84, CRTAM, DR3, SLAMF1, or CD2.

[0198] As another example, in some embodiments, the intracellular signaling domain CD3(^ can have at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to SEQ ID NO: 56, as long as it possesses the desired function. In some embodiments, the intracellular signaling domain of CD3(^ comprises the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the intracellular signaling domain CD3(^ may be encoded by a nucleic acid sequence having at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 81.

[0199] In some instances, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (IT AM) or a portion thereof, as long as it possesses the desired function. The intracellular signaling domain of the CAR can comprise a sequence having at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to an IT AM. In some instances, the intracellular signaling domain can have at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to FcsRIy, CD4, CD7, CD8, CD28, 0X40, CD79a, CD79b, DAP 12, or H2-Kb, as long as it possesses the desired function.Attorney Docket No. 62190-737601

[0200] In some embodiments, the anti-CD7 CAR further comprises a hinge domain and / or a transmembrane domain. In some embodiments, the hinge and transmembrane domains of the anti-CD7 CAR comprises a signaling domain (e.g., hinge and transmembrane domains) from CD8P, 4-1BB, CD28, CD34, CD4, FcsRIy, CD16, 0X40, CD3< CD3s, CD3y, CD38, TCRa, CD32, CD64, VEGFR2, FAS, FGFR2B, or another transmembrane protein. In some embodiments, the anti-CD7 CAR further comprises a CD8a signal peptide.

[0201] In some embodiments, the chimeric antigen receptor (CAR) can bind to a molecule that is expressed on the surface of a cell including, but not limited to members of the CD1 family of glycoproteins, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

[0202] In some embodiments, the polypeptide comprises a amino acid sequence that encodes a component of the CAR according to Table 9.Table 9. Amino acid sequence information for select components of a CD7 CAR.

[0203] In some embodiments, the CD7 CAR comprises a CD7 antigen binding domain, a 4-1BB intracellular signaling domain, a CD3(^ intracellular signaling domain, and CD8 hinge and transmembrane domain. In some embodiments, the CD7 antigen binding domain comprises a VH domain and a VL domain, and a VH-VL linker, such as but not limited to a (G4S)n linker where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In some embodiments, the CD7 CAR comprises from N-terminus to C-terminus: a CD8 signal peptide, a CD7 antigen binding domain, aCD8 hinge and transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3(^ intracellular signaling domain. In some embodiments, an isolated polypeptide of the present disclosure comprises a amino acid sequence that encodes a CAR according to Table 10Attorney Docket No. 62190-737601Table 10. Amino acid sequences for exemplary CAR constructsAttorney Docket No. 62190-737601

[0204] In some embodiments, the CD7 CAR encoded by a bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the CD7 CAR encoded by a bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the CD7 CAR encoded by a bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the CD7 CAR encoded by a bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 68.

[0205] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65 and additional amino acid residues at the N-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 66. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65 and additional amino acid residues at the C-terminus produced by cleavage of the 2 A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 66. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65 and additional amino acid residues at the C-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 66. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65 and additional amino acid residues at the C-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 66. Also, provided herein are populations comprising such cells.

[0206] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67 and additional amino acid residues at the C-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 68. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67 and additional amino acid residues at the C-terminus produced by cleavage of the 2 A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 68. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 CAR encodedAttorney Docket No. 62190-737601by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67 and additional amino acid residues at the C-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 68. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67 and additional amino acid residues at the C-terminus produced by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 68. Also, provided herein are populations of such cells.

[0207] In some embodiments, the CD7 CAR described herein comprises a sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequence identity to the amino sequence as set forth in SEQ ID NO: 65. In some embodiments, the CD7 CAR described herein comprises a sequence having the amino sequence as set forth in SEQ ID NO: 65. In some embodiments, the CD7 CAR described herein comprises a sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequence identity to the amino sequence as set forth in SEQ ID NO: 66. In some embodiments, the CD7 CAR described herein comprises a sequence having the amino sequence as set forth in SEQ ID NO: 66.

[0208] In some embodiments, the CD7 CAR described herein comprises a sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequenceAttorney Docket No. 62190-737601identity to the amino sequence as set forth in SEQ ID NO: 67. In some embodiments, the CD7 CAR described herein comprises a sequence having the amino sequence as set forth in SEQ ID NO: 67. In some embodiments, the CD7 CAR described herein comprises a sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or greater than about 99.9% sequence identity to the amino sequence as set forth in SEQ ID NO: 68. In some embodiments, the CD7 CAR described herein comprises a sequence having the amino sequence as set forth in SEQ ID NO: 68.

[0209] In some embodiments, the CD7 CAR encoded by the dual promoter vector described herein comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 65. In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 CAR encoded by a dual promoter vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 67. Also, provided herein are populations of such cells.Attorney Docket No. 62190-737601

[0210] In some embodiments, an isolated polynucleotide of a CD7 CAR of the present disclosure comprises one or more nucleic acid sequences of Table 11. In some embodiments, the nucleic acid sequence comprises a sequence encoding one or more components of the CAR as set forth in Table 11.Table 11. Nucleic acid sequence information for select components of a CD7 CAR.<

[0211] In some embodiments, the polynucleotide encoding the CD7 CAR comprises a nucleic acid sequence for an antigen binding domain that binds CD7, a nucleic acid sequence for a CD8a hinge and transmembrane domain, a nucleic acid sequence for an intracellular signaling domain of 4-1BB, and a nucleic acid sequence for an intracellular signaling domain of CD3(^. In some embodiments, the polynucleotide also comprises a nucleic acid sequence for a CD8 signal peptide.

[0212] In some embodiments, the antigen binding domain is an anti-CD7 scFv. In some embodiments, the VH sequence of the scFv comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence of SEQ ID NO: 70 and the VL sequence comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence of SEQ ID NO: 71.

[0213] In some embodiments, the VH sequence of the scFv comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence of SEQ ID NO: 72 and the VL sequence comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to theAttorney Docket No. 62190-737601sequence of SEQ ID NO: 73. In some embodiments, the VH sequence of the scFv comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence of SEQ ID NO: 74 and the VL sequence comprises a nucleic acid sequence having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence of SEQ ID NO: 75.

[0214] In some embodiments, the polynucleotide encoding the CD7 CAR comprises from the 5’ end to the 3’ end: a nucleic acid sequence for an antigen binding domain that binds CD7, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81. In some embodiments, the polynucleotide encoding the CD7 CAR comprises from the 5’ end to the 3’ end: SEQ ID NO:76, a nucleic acid sequence for an antigen binding domain that binds CD7, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81.

[0215] In some embodiments, the CD7 CAR comprises a nucleic acid sequence having at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, or more) sequence identity to SEQ ID NO: 90 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 90 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence having at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, or more) sequence identity to SEQ ID NO: 91 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 91 and binds to CD7.

[0216] In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 88. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having a sequence as set forth in SEQ ID NO: 88. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 89. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having a sequence as set forth in SEQ ID NO: 89.

[0217] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a bicistronic construct or a dual promoter construct comprising a nucleic acid sequence of the CD7 CAR having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 90. In someAttorney Docket No. 62190-737601embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 PEBL encoded by the bicistronic vector construct or a dual promoter construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. Also, provided herein is a population comprising such cells.

[0218] In some embodiments, an engineered immune cell of the present disclosure comprises a CD7 CAR encoded by a bicistronic construct or a dual promoter construct comprising a nucleic acid sequence of the CD7 CAR having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 91. In some embodiments, the engineered immune cell is a CD4+ T cell comprising a CD7 CAR encoded by the bicistronic vector construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. In some embodiments, the engineered immune cell is a CD8+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 PEBL encoded by the bicistronic construct comprising a nucleic acid sequence of the CD7 PEBL having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least aboutAttorney Docket No. 62190-73760199%, at least about 99.5%, or 100% sequence identity to SEQ ID NO: 88 or 89. Also, provided herein is a population comprising such cells.Bicistronic Constructs

[0219] Provided herein are vectors that allow for simultaneous expression of a fratricideinducing chimeric antigen receptor (e.g., a CAR) and a fratricide-preventing protein (e.g., a PEBL) in immune cells (e.g., autologous immune cells) that result in viable CAR-expressing cytotoxic T lymphocytes (CAR T) that target T cell antigens.

[0220] Viral vectors have been produced in which two or more genes can be expressed from a single construct. Typically, these vectors employ either a bicistronic element or a two-promoter configuration. In the case of bicistronic vectors, a sequence element is introduced between two genes that enables the translation of two proteins from a single messenger RNA. Examples include, but are not limited to, the internal ribosome entry site sequences (IRES) and the virally derived "codon skipping" peptide sequences such as P2A, T2A, F2A, E2A, and the like. In the case of two-promoter designed vectors, separate promoter elements are configured upstream of each gene such that each promoter transcribes the mRNA for its proximally linked gene. In some embodiments, an expression vector (e.g., construct) contains a first promoter operably linked to a CAR and a second promoter operably linked to a PEBL.

[0221] Described herein are fratricide-resistant CAR T cells expressing a CAR directed against CD7 and such CAR T cell has reduced or no surface expression of CD7. The present disclosure is based, in part, on co-expression of a chimeric antigen receptor (CAR) directed against surface CD7 and a protein expression blocker (PEBL) directed against intracellular CD7 in immune cells (e.g., T cells) using a bicistronic construct, such as a bicistronic viral vector. In some aspects, the present disclosure relates to an engineered immune cell (e.g., an engineered T cell) comprising a bicistronic construct comprising a polynucleotide sequence encoding an anti-CD7 CAR and a polynucleotide sequence encoding an anti-CD7 PEBL. In some embodiments, the CAR comprises intracellular signaling domains of 4-1BB and CD3(^ and an antibody (e.g., a single chain variable fragment, scFv) that specifically binds CD7. The CD7 CAR of the present disclosure may be referred to herein as “anti-CD7-41BB-CD3^”. In some embodiments, the CAR may comprise a CD8a hinge and transmembrane domain. In some embodiments, the anti-CD7 PEBL may comprise a target-binding domain (e.g., an antibody or fragment thereof) that specifically binds CD7 and a localizing domain. In some embodiments, the anti-CD7 PEBL can comprise an antibody or fragment thereof (e.g., a scFv) that specifically binds CD7, CD8a hinge and transmembrane domains, and a localizing domain.Attorney Docket No. 62190-737601

[0222] The term “bicistronic expression” is typically achieved by operably linking the polynucleotides described herein to a promoter, and incorporating the bicistronic construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors can comprise expression vectors, replication vectors, probe generation vectors, or sequencing vectors.Table 12. Amino acid sequences of ribosomal codon skipping sites.Table 13. Nucleotide sequences of ribosomal codon skipping peptides and IRES.

[0223] Provided herein are recombinant bicistronic viral constructs or vectors that contain a polynucleotide encoding a CAR (e.g., a CAR comprising a binding domain specific for CD7) and a polynucleotide encoding a PEBL (e.g., a PEBL comprising a binding domain specific for CD7), as described herein. In some embodiments, the recombinant bicistronic viral construct comprises an internal ribosomal entry site (IRES) sequence between the nucleic acid sequence of the CAR and the nucleic acid sequence of the PEBL. In some embodiments, the recombinantAttorney Docket No. 62190-737601bicistronic viral construct comprises a ribosomal codon skipping site sequence (also referred to as a sequence encoding a 2A self-cleaving peptide) between the nucleic acid sequence of the CAR and the nucleic acid sequence of the PEBL. In some embodiments of a bicistronic construct, a polynucleotide encoding a CAR is located upstream (at the 5’ end) of an IRES sequence, and a polynucleotide encoding a PEBL is located downstream (at the 3’ end) of the IRES. In some cases, a nucleic acid sequence encoding a CAR is operably linked to an IRES sequence and an IRES sequence is operably linked to a nucleic acid sequence encoding a PEBL. In some cases, a nucleic acid sequence encoding a PEBL is operably linked to an IRES sequence and an IRES sequence is operably linked to a nucleic acid sequence encoding a CAR.

[0224] In some embodiments of a bicistronic construct, a polynucleotide encoding a CAR is located upstream (at the 5’ end) of a polynucleotide encoding 2A self-cleaving peptide, and a polynucleotide encoding a PEBL is located downstream (at the 3’ end) of the polynucleotide encoding 2A self-cleaving peptide. In some cases, a nucleic acid sequence encoding a CAR is operably linked to a nucleic acid sequence encoding a 2A self-cleaving peptide, which is operably linked to a nucleic acid sequence encoding a PEBL. In some cases, a nucleic acid sequence encoding a PEBL is operably linked to a nucleic acid sequence encoding a 2A selfcleaving peptide, which is operably linked to a nucleic acid sequence encoding a CAR.

[0225] The mechanism of ribosomal codon skipping via a 2A peptide sequence is useful for generating two proteins from one transcript; a normal peptide bond is impaired at the 2A sequence, resulting in two discontinuous protein fragments from one translation event. Selfcleaving 2A peptides (e.g., 2A cleavage sites) are described in Kim et al., PLoS One, 2011, 6(4):el8556.

[0226] In some embodiments, the IRES is from an Encephalomyocarditis virus (EMCV). In some embodiments, the IRES is from an Enterovirus. In some embodiments, the nucleic acid sequence of the IRES sequence is set forth in SEQ ID NO: 82 (see, e.g., Table 13).

[0227] In some embodiments, the ribosomal codon skipping site is based on a 2A self-cleaving peptide (see, e.g., Table 12). In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A (porcine teschovirus-1 2A peptide), E2A (equine rhinitis A virus 2A peptide), F2A (foot-and-mouth disease virus 2A peptide), and T2A (thosea asigna virus 2A). In some instances, the P2A peptide comprises a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, or greater than aboutAttorney Docket No. 62190-73760199.9% sequence identity to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the P2A peptide comprises an amino acid sequence as set forth in SEQ ID NO 57.

[0228] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A, and T2A. In some instances, the E2A peptide comprises a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, or greater than about 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the E2A peptide comprises an amino acid sequence as set forth in SEQ ID NO 58.

[0229] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A, and T2A. In some instances, the F2A peptide comprises a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, or greater than about 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the F2A peptide comprises an amino acid sequence as set forth in SEQ ID NO 59.

[0230] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A, and T2A. In some instances, the T2A peptide comprises a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, at least about 90%, or greater than about 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the T2A peptide comprises an amino acid sequence as set forth in SEQ ID NO 60.

[0231] In some embodiments, the viral construct (e.g., retroviral construct) comprises a nucleic acid sequence encoding a 2A self-cleaving peptide (e.g., 2A peptide cleavage site) selected from the group consisting of P2A, E2A, F2A, and T2A, wherein the polynucleotide encoding 2A self-cleaving peptide links the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the PEBL. The polynucleotide encoding 2A self-cleaving peptide may be between the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the PEBL. As described above, in some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding a CAR, a nucleic acidAttorney Docket No. 62190-737601sequence encoding a P2A self-cleaving peptide, and a nucleic acid sequence encoding a PEBL. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a P2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding an E2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding an F2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a T2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein.

[0232] In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding a PEBL, a nucleic acid sequence encoding a P2A self-cleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding a PEBL, a nucleic acid sequence encoding an E2A self-cleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding a PEBL, a nucleic acid sequence encoding an F2A self-cleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from 5’ end to 3’ end: a nucleic acid sequence encoding a PEBL, a nucleic acid sequence encoding a T2A self-cleaving peptide, and a nucleic acid sequence encoding a CAR.

[0233] In some embodiments, the nucleic acid sequence encoding the P2A comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 83. In some embodiments, the nucleic acid sequence encoding the P2A comprises or consisting of a nucleic acid of SEQ ID NO: 83. In some embodiments, the nucleic acid sequence encoding the E2A comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequenceAttorney Docket No. 62190-737601identity to SEQ ID NO: 84. In some embodiments, the nucleic acid sequence encoding the E2A comprises or consisting of a nucleic acid of SEQ ID NO: 84. In some embodiments, the nucleic acid sequence encoding the F2A comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 85. In some embodiments, the nucleic acid sequence encoding the F2A comprises or consisting of a nucleic acid of SEQ ID NO: 85. In some embodiments, the nucleic acid sequence encoding the T2A comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 86. In some embodiments, the nucleic acid sequence encoding the T2A comprises or consisting of a nucleic acid of SEQ ID NO: 86.

[0234] In some embodiments, the nucleic acid sequence encoding the PEBL is disposed (e.g., located) 5’ to the nucleic acid sequence encoding the CAR. In some embodiments, the nucleic acid sequence encoding the CAR is disposed 5’ to the nucleic acid sequence encoding the PEBL. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: (al) SEQ ID NO: 90, SEQ ID NO: 83, and SEQ ID NO: 88; (a2) SEQ ID NO: 90, SEQ ID NO: 83, and SEQ ID NO: 89; (a3) SEQ ID NO: 91, SEQ ID NO: 83, and SEQ ID NO: 88; (a4) SEQ ID NO: 91, SEQ ID NO: 83, and SEQ ID NO: 89; (bl) SEQ ID NO: 90, SEQ ID NO: 84, and SEQ ID NO: 88; (b2) SEQ ID NO: 90, SEQ ID NO: 84, and SEQ ID NO: 89; (b3) SEQ ID NO: 91, SEQ ID NO: 84, and SEQ ID NO: 88; (b4) SEQ ID NO: 91, SEQ ID NO: 84, and SEQ ID NO: 89; (cl) SEQ ID NO: 90, SEQ ID NO: 85, and SEQ ID NO: 88; (c2) SEQ ID NO: 90, SEQ ID NO: 85, and SEQ ID NO: 89; (c3) SEQ ID NO: 91, SEQ ID NO: 85, and SEQ ID NO: 88; (c4) SEQ ID NO: 91, SEQ ID NO: 85, and SEQ ID NO: 89; (dl) SEQ ID NO: 90, SEQ ID NO: 86, and SEQ ID NO: 88; (d2) SEQ ID NO: 90, SEQ ID NO: 86, and SEQ ID NO: 89; (d3) SEQ ID NO: 91, SEQ ID NO: 86, and SEQ ID NO: 88; (d4) SEQ ID NO: 91, SEQ ID NO: 86, and SEQ ID NO: 89; (el) SEQ ID NO: 88, SEQ ID NO: 83, and SEQ ID NO: 90; (e2) SEQ ID NO: 88, SEQ ID NO: 83, and SEQ ID NO: 91; (e3) SEQ ID NO: 89, SEQ ID NO: 83, and SEQ ID NO: 90; (e4) SEQ ID NO: 89, SEQ ID NO: 83, and SEQ ID NO: 91; (fl) SEQ ID NO: 88, SEQ ID NO: 84, and SEQ ID NO: 90; (f2) SEQ ID NO: 88, SEQ ID NO: 84, and SEQ ID NO: 91; (f3) SEQ ID NO: 89, SEQ ID NO: 84, and SEQ ID NO: 90; (f4) SEQ ID NO: 89, SEQ ID NO: 84, and SEQ ID NO: 91; (gl) SEQ ID NO: 88, SEQ ID NO: 85, and SEQ ID NO: 90; (g2) SEQ ID NO: 88, SEQ ID NO: 85, and SEQ ID NO: 91; (g3) SEQ ID NO: 89, SEQ IDAttorney Docket No. 62190-737601NO: 85, and SEQ ID NO: 90; (g4) SEQ ID NO: 89, SEQ ID NO: 85, and SEQ ID NO: 91; (hl) SEQ ID NO: 88, SEQ ID NO: 86, and SEQ ID NO: 90; (h2) SEQ ID NO: 88, SEQ ID NO: 86, and SEQ ID NO: 91; (h3) SEQ ID NO: 89, SEQ ID NO: 86, and SEQ ID NO: 90; or (h4) SEQ ID NO: 89, SEQ ID NO: 86, and SEQ ID NO: 91.

[0235] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0236] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a P2A peptide of SEQ ID NO: 57, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0237] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consistsAttorney Docket No. 62190-737601of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0238] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a E2A peptide of SEQ ID NO: 58, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0239] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69)Attorney Docket No. 62190-737601CAR described herein, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0240] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a F2A peptide of SEQ ID NO: 59, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0241] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR of SEQ ID NO: 65, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (TH69) CAR described herein, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0242] In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (TH69) PEBL of SEQ ID NO: 61. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding aAttorney Docket No. 62190-737601CD7 (TH69) PEBL described herein. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR of SEQ ID NO: 67, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (3A1F) PEBL of SEQ ID NO: 63. In some embodiments, a bicistronic construct comprises or consists of from 5’ to 3’ end: a polynucleotide encoding a CD7 (3A1F) CAR described herein, a polynucleotide encoding a T2A peptide of SEQ ID NO: 60, and a polynucleotide encoding a CD7 (3 A1F) PEBL described herein.

[0243] In some embodiments, the polynucleotide sequence encoding the PEBL is disposed 5’ (upstream) of an IRES site and the IRES site is disposed 5’ to the polynucleotide sequence encoding the CAR. In some embodiments, the polynucleotide sequence encoding the CAR is disposed 5’ of an IRES site and the IRES site is disposed 5’ to the polynucleotide sequence encoding the PEBL.

[0244] In some embodiments, the polynucleotide sequence encoding the PEBL is disposed 5’ (upstream) of the ribosomal codon skipping site and the ribosomal codon skipping site is disposed 5’ to the polynucleotide sequence encoding the CAR. In some embodiments, the polynucleotide sequence encoding the CAR is disposed 5’ of the ribosomal codon skipping site and the ribosomal codon skipping site is disposed 5’ to the polynucleotide sequence encoding the PEBL. In some embodiments, the polynucleotide sequence encoding the PEBL is not disposed 5’ (upstream) to the polynucleotide sequence encoding the CAR. For example, the polynucleotide sequence encoding the PEBL may not be disposed 5’ (upstream) of the ribosomal codon skipping site, which is in turn disposed 5’ to the polynucleotide sequence encoding the CAR.

[0245] In some aspects, provided herein is a recombinant bicistronic construct comprising at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or greater than about 95% sequence identity to a nucleic acid sequence of one or more selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86. In some embodiments, the recombinant bicistronic construct comprises at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, the recombinant bicistronic construct comprises at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 84. In some embodiments, the recombinant bicistronic construct comprises at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 85. In some embodiments, the recombinant bicistronic construct comprises at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the recombinant bicistronic construct comprises a nucleic acidAttorney Docket No. 62190-737601sequence of one selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86.

[0246] The present disclosure provides vectors such as expression vectors in which any of the polynucleotides described herein is inserted. In some embodiments, the vector is derived from retroviruses such as lentiviruses. Such vectors are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of an exogenous polynucleotide (e.g., transgene) and its propagation in daughter cells. Unlike vectors derived from onco-retroviruses such as murine leukemia viruses, lentiviral vectors can transduce non-proliferating cells. Lentiviral vectors also have low immunogenicity. In other embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a plasmid.

[0247] In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the CMV promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 92. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 92. In some embodiments, any of the constructs described herein comprises or consists of a CMV promoter of SEQ ID NO: 92.

[0248] In some embodiments, the promoter comprises an EFla promoter. In some embodiments, the EFla promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 93. In some embodiments, the EFla promoter comprises the sequence of SEQ ID NO: 93. In some embodiments, any of the constructs described herein comprises or consists of a EFla promoter of SEQ ID NO: 93.

[0249] In some embodiments, the promoter comprises an EFS promoter. In some embodiments, the EFS promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 94. In some embodiments, the EFS promoter comprises the sequence of SEQ ID NO: 94. In some embodiments, any of the constructs described herein comprises or consists of a EFS promoter of SEQ ID NO: 94.Attorney Docket No. 62190-737601

[0250] In some embodiments, the promoter comprises a MSCV promoter. In some embodiments, the MSCV promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 95. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO: 95. In some embodiments, any of the constructs described herein comprises or consists of a MSCV promoter of SEQ ID NO: 95.

[0251] In some embodiments, the promoter comprises a PGK promoter. In some embodiments, the PGK promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 96. In some embodiments, the PGK promoter comprises the sequence of SEQ ID NO: 96. In some embodiments, any of the constructs described herein comprises or consists of a PGK promoter of SEQ ID NO: 96.

[0252] In some embodiments, the bicistronic vector comprises or consists of the nucleic acid sequence as set forth in SEQ ID NO: 97. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 97. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a nucleic acid sequence encoding a CD7 PEBL, an IRES sequence, and a nucleic acid sequence encoding a CD7 CAR, and optionally at the 5’ end, a promoter selected from the group consisting of a CMV promoter (e.g., SEQ ID NO: 92), EFla promoter (e.g., SEQ ID NO: 93), EFS promoter (e.g., SEQ ID NO: 94), MSCV promoter (e.g., SEQ ID NO: 95), and PGK promoter (e.g., SEQ ID NO: 96).

[0253] In some embodiments, the bicistronic vector comprises or consists of the nucleic acid sequence as set forth in SEQ ID NO: 98. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 98. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a nucleic acid sequence encoding aAttorney Docket No. 62190-737601CD7 CAR, an IRES sequence, and a nucleic acid sequence encoding a CD7 PEBL, and optionally at the 5’ end, a promoter selected from the group consisting of a CMV promoter (e.g., SEQ ID NO: 92), EFla promoter (e.g., SEQ ID NO: 93), EFS promoter (e.g., SEQ ID NO: 94), MSCV promoter (e.g., SEQ ID NO: 95), and PGK promoter (e.g., SEQ ID NO: 96).

[0254] In some embodiments, the bicistronic vector comprises or consists of the nucleic acid sequence as set forth in SEQ ID NO: 99. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 99. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a nucleic acid sequence encoding a CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL, and optionally at the 5’ end, a promoter selected from the group consisting of a CMV promoter (e.g., SEQ ID NO: 92), EFla promoter (e.g., SEQ ID NO: 93), EFS promoter (e.g., SEQ ID NO: 94), MSCV promoter (e.g., SEQ ID NO: 95), and PGK promoter (e.g., SEQ ID NO: 96).

[0255] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 100. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 100. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a promoter, a nucleic acid sequence encoding a CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL. In some instances the bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a MSCV promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL.

[0256] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 101. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a promoter, a nucleic acid sequence encoding a CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL. In some instances theAttorney Docket No. 62190-737601bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a EFla promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL.

[0257] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 102. In some embodiments, the bicistronic vector comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 102. The bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a promoter, a nucleic acid sequence encoding a CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL. In some instances the bicistronic vector comprises a nucleic acid sequence comprising from 5’ end to 3’ end: a EFS promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL.Dual Promoter Constructs of a CAR and PEBL

[0258] Provided herein are recombinant retroviral constructs (or vectors) for simultaneous expression of a CAR and a PEBL in a cell such as a T cell. In some embodiments, the retroviral constructs comprise a promoter operably linked to a polynucleotide encoding any of the CARs described herein and a promoter operably linked to a polynucleotide encoding any of the PEBLs described herein. In some embodiments, the promoter for the CAR and the promoter for the PEBL share less than 90% sequence identity, e.g., less than 90% identity, less than 80% identity, less than 75% sequence identity, less 70% sequence identity, less than 65% sequence identity, less than 60% sequence identity, less than 55% sequence identity, and the like. In some embodiments, the promoter for the CAR and the promoter for the PEBL share 80% sequence identity or less, e.g., 80% identity, 75% sequence identity, 70% sequence identity, 65% sequence identity, 60% sequence identity, 55% sequence identity, and the like. In some embodiments, the promoter for the CAR and the promoter for the PEBL share at least about 50% sequence identity, e.g., 50% sequence identity, 55% sequence identity, 60% sequence identity, 65% sequence identity, 70% sequence identity, 75% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, or more sequence identity.

[0259] In some embodiments, the promoter for the CAR (referred to as the first promoter) is different than the promoter for the PEBL (referred to as the second promoter). The firstAttorney Docket No. 62190-737601promoter and the second promoter can have the same sequence. In other instances, the first promoter and the second promoter have different sequences. In some embodiments, the first target-binding domain can be preceded by a promoter and the second target-binding domain can be preceded by a promoter.

[0260] In some embodiments, the first promoter and / or second promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 92. In some embodiments, the first promoter and / or second promoter comprises a CMV promoter. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 92.

[0261] In some embodiments, the first promoter and / or second promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 93. In some embodiments, the first promoter and / or second promoter comprises a EFla promoter. In some embodiments, the EFla promoter comprises the sequence of SEQ ID NO: 93.

[0262] In some embodiments, the first promoter and / or second promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 94. In some embodiments, the first promoter and / or second promoter comprises a EFS promoter. In some embodiments, the EFS promoter comprises the sequence of SEQ ID NO: 94.

[0263] In some embodiments, the first promoter and / or second promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 95. In some embodiments, the first promoter and / or second promoter comprises a murine stem cell virus (MSCV) promoter. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO: 95.

[0264] In some embodiments, the first promoter and / or second promoter comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, atAttorney Docket No. 62190-737601least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 96. In some embodiments, the first promoter and / or second promoter comprises a murine stem cell virus (PGK) promoter. In some embodiments, the PGK promoter comprises the sequence of SEQ ID NO: 96.

[0265] In some embodiments, the retroviral constructs from 5 ’ to 3 ’ comprise the first promoter operably linked to the polynucleotide encoding the CAR and the second promoter operably linked to the polynucleotide encoding the PEBL. In various embodiments, the retroviral constructs from 5’ to 3’ comprise the second promoter operably linked to the polynucleotide encoding the PEBL and the first promoter operably linked to the polynucleotide encoding the CAR.

[0266] In some embodiments, the first promoter is located upstream of the second promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is an EFla promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is a MSCV promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is a MSCV promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is a MSCV promoter and the second promoter is an EFla promoter. In some embodiments, the first promoter is a MSCV promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is a MSCV promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is a MSCV promoter and the second promoter is a MSCV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is an EFla promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is a MSCV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EFla promoter and the second promoter is a MSCV promoter. In some embodiments, the first promoter is an EFla promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EFla promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is anAttorney Docket No. 62190-737601EFla promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an EFla promoter and the second promoter is an EFla promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is a MSCV promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is an EFla promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is an EFS promoter.

[0267] In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 103. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 103. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 104. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 104. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 105. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 105. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 106. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 106. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acidAttorney Docket No. 62190-737601sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 107. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 107. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 108. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 108. In some embodiments, the retroviral construct of the present disclosure comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.9%, or greater than about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 109. In some embodiments, the retroviral construct of the present disclosure comprises the nucleic acid sequence of SEQ ID NO: 109.Pharmaceutical Compositions

[0268] In some aspects, provided herein are pharmaceutical compositions for treating acute myeloid leukemia (AML) in a subject in need thereof. The pharmaceutical composition can comprise an engineered immune cell and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition may comprise a population of engineered immune cells and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation can be dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the art. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione); and preservativesAttorney Docket No. 62190-737601(e.g., dimethylsulfoxide, DMSO). Compositions of the present disclosure can be formulated for intravenous administration.

[0269] The engineered immune cell can comprise a first nucleotide sequence. The first nucleotide sequence may encode a first target-binding domain. The first target-binding domain may comprise a target-binding domain described herein. For example, the first nucleotide sequence may encode a first target-binding domain linked to a localizing domain. The localizing domain can comprise a linker sequence. The localizing domain can comprise a retention signal. In some embodiments, the localizing domain can comprise a linker sequence and retention signal. In some embodiments, the first target-binding domain can bind to CD7.

[0270] The engineered immune cell can comprise a second nucleotide sequence. The second nucleotide sequence may encode an immune activating receptor. In some embodiments, the immune activating receptor can be a chimeric antigen receptor (CAR). The CAR may comprise a second target-binding domain. In some embodiments, the second target-binding domain may bind to the same target as the first target-binding domain. In some embodiments, the second target-binding domain may bind to a different target as the first target-binding domain. In some embodiments, the second target-binding domain may bind to CD7.

[0271] The engineered immune cell can be an autologous T cell. For example, immune cells obtained from the subject having cancer (e.g., AML) can be engineered to generated PCART7 T cells. T cells can be engineered from apheresis material. T cells can be engineered from blood draws of from 10 cc to 400 cc. T cells can be engineered from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0272] As an example, provided herein is a pharmaceutical composition for treating acute myeloid leukemia (AML) in a subject in need thereof, wherein the pharmaceutical composition comprises an engineered immune cell and a pharmaceutically acceptable carrier, wherein the engineered immune cell comprises: (i) a first nucleotide sequence encoding a first targetbinding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; and wherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

[0273] The administration of the subject compositions may be carried out in any convenient manner, including by injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, intratumorally, intranodally, intramedullary, by intravenous (i.v.) injection, or intraperitoneally. The T cell compositions ofAttorney Docket No. 62190-737601the present disclosure can be administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node. In some embodiments, described herein are compositions for paren...

Claims

1. Attorney Docket No. 62190-737601CLAIMS WHAT IS CLAIMED IS:

1. A method of treating acute myeloid leukemia (AML) in a subject in need thereof, the method comprising:administering to the subject a composition comprising an engineered immune cell, wherein the engineered immune cell comprises:(i) a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and(ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; andwherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

2. The method of claim 1, wherein the AML is a refractory acute myeloid leukemia.

3. The method of claim 1 or 2, wherein the AML is a relapsed acute myeloid leukemia.

4. The method of any one of claims 1-3, wherein the subject has previously received one or more prior lines of therapy.

5. The method of claim 4, wherein the one or more prior lines of therapy comprises at least two prior lines of therapy.

6. The method of claim 4, wherein the one or more prior lines of therapy comprise surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, or radiation therapy, or any combination thereof.

7. The method of claim 4, wherein the one or more prior lines of therapy comprise administration of a biologic.

8. The method of claim 7, wherein the biologic comprises an antibody, antibody drug conjugate (ADC), cellular therapy, peptide, polypeptide, enzyme, vaccine, oligonucleotide, oncolytic virus, polysaccharide, or gene therapy.

9. The method of any one of claims 4-8, wherein the one or more prior lines of therapy comprises administration of a regimen comprising fludarabine, cytarabine, granulocyte colony stimulating factor, and gemtuzumab ozogamicin (FLAG-GO).

10. The method of any one of claims 4-9, wherein the one or more prior lines of therapy comprises administration of a regimen comprising venetoclax.Attorney Docket No. 62190-73760111. The method of claim 9 or 10, wherein the regime comprises FLAG-GO and venetoclax.

12. The method of any one of claims 1-11, wherein the subject has a risk of recurrence.

13. The method of any one of claims 1-12, wherein the subject has a prior history of recurrence after a prior therapy.

14. The method of any one of claims 1-13, wherein the subject has a treatment failure after at least two prior lines of induction therapy, a relapse within 12 months after first complete response (CR), a relapse after allogeneic hematopoietic stem cell transplantation, has at least one prior relapse, or any combination thereof.

15. The method of any one of claims 1-14, wherein the subject has been diagnosed with the AML.

16. The method of any one of claims 1-15, further comprising, prior to administering, identifying the subject as having the AML.

17. The method of claim 16, wherein identifying comprises measuring CD7 expression on blast cells.

18. The method of claim 17, wherein the subject has at least 20% CD7 expression on blast cells.

19. The method of claim 17 or 18, wherein the subject has at least 98% CD7 expression on blast cells.

20. The method of any one of claims 1-19, wherein the AML is a CD7-expressing AML.

21. The method of any one of claims 1-20, wherein the subject has been identified as having a relapsed or refractory AML.

22. The method of any one of claims 1-21, further comprising, prior to administering, identifying the subject as having the relapsed or refractory AML.

23. The method of any one of claims 1-22, wherein the subject has been identified as having the relapsed or refractory AML based on persistence of myeloid blasts in a bone marrow sample from the subject after one or more prior lines of therapy.

24. The method of any one of claims 1-23, further comprising, prior to administering, subjecting the subject to a pre-conditioning regimen.

25. The method of claim 24, wherein the pre-conditioning regimen comprises a lymphodepletion.

26. The method of claim 25, wherein the lymphodepletion comprises administration of fludarabine, etoposide, cyclophosphamide, bendamustine, busulfan, cytarabine, or alemtuzumab, or any combination thereof.Attorney Docket No. 62190-73760127. The method of claim 26, wherein the etoposide is administered at a dose from about 50 mg / m2to about 300 mg / m2.

28. The method of claim 26, wherein the etoposide is administered at a dose of about 150 mg / m2.

29. The method of any one of claims 26-28, wherein the etoposide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days.

30. The method of any one of claims 26-29, wherein the etoposide is administered at a dose of about 150 mg / m2per day for 3 days.

31. The method of claim 30, wherein the etoposide is administered at a dose of about 150 mg / m2per day for 3 consecutive days.

32. The method of any one of claims 26-31, wherein the cyclophosphamide is administered at a dose from about 10 mg / kg to about 100 mg / kg.

33. The method of any one of claims 26-32, wherein the cyclophosphamide is administered at a dose of about 60 mg / kg.

34. The method of any one of claims 26-33, wherein the cyclophosphamide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days.

35. The method of any one of claims 26-34, wherein the cyclophosphamide is administered at a dose of about 60 mg / kg per day for 2 days.

36. The method of claim 35, wherein the cyclophosphamide is administered at a dose of about 60 mg / kg per day for 2 consecutive days.

37. The method of any one of claims 25-35, wherein the lymphodepletion comprises administration of (i) etoposide at a dose of about 150 mg / m2per day for 3 consecutive days, and (ii) cyclophosphamide at a dose of about 60 mg / kg per day for 2 consecutive days.

38. The method of claim 26, wherein the cytarabine is administered at a dose from about 1.0 g / m2to about 4.0 g / m2.

39. The method of claim 26, wherein the cytarabine is administered at a dose of about 2.0 g / m2.

40. The method of claim 38 or 39, wherein the cytarabine is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days.

41. The method of claim 40, wherein the cytarabine is administered at a dose of 2.0 g / m2per day for 5 days.

42. The method of claim 41, wherein the cytarabine is administered at a dose of 2.0 g / m2per day for 5 consecutive days.Attorney Docket No. 62190-73760143. The method of claim 26, wherein the fludarabine is administered at a dose from about 10 mg / sqm / day to about 100 mg / sqm / day.

44. The method of claim 43, wherein the fludarabine is administered at a dose from about 20 mg / sqm / day to about 50 mg / sqm / day.

45. The method of claim 44, wherein the fludarabine is administered at a dose of about 30 mg / sqm / day.

46. The method of any one of claims 43-45, wherein the fludarabine is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days.

47. The method of claim 46, wherein the fludarabine is administered at a dose of about 30 mg / sqm / day for 4 days.

48. The method of claim 47, wherein the fludarabine is administered at a dose of about 30 mg / sqm / day for 4 consecutive days.

49. The method of claim 43, wherein the cyclophosphamide is administered at a dose from about 500 mg / sqm / day to about 1500 mg / sqm / day.

50. The method of claim 49, wherein the cyclophosphamide is administered at a dose from about 800 mg / sqm / day to about 1200 mg / sqm / day.

51. The method of claim 50, wherein the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day.

52. The method of any one of claims 26-33, wherein the cyclophosphamide is administered once per day for at least 1 day, at most 5 days, or from 1 to 10 days.

53. The method of any one of claims 26-34, wherein the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 days.

54. The method of claim 35, wherein the cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 consecutive days.

55. The method of any one of claims 43-54, wherein the lymphodepletion comprises administration of (i) fludarabine at a dose of about 30 mg / sqm / day for 4 consecutive days, and (ii) cyclophosphamide is administered at a dose of about 1000 mg / sqm / day for 2 consecutive days.

56. The method of claim 55, wherein the lymphodepletion comprises administration of (i) fludarabine at a dose of about 30 mg / sqm / day on Day -6, -5, -4, and -3, and (ii) cyclophosphamide is administered at a dose of about 1000 mg / sqm / day on Day -4 and -3, when Day 0 is the day for administering the composition.Attorney Docket No. 62190-73760157. The method of any one of claims 24-37, wherein the composition is administered once per day for at least about 5 days, at most about 30 days, or from about 1 to 30 days following the pre-conditioning regimen.

58. The method of claim 57, wherein the composition is not administered immediately after the pre-conditioning regimen.

59. The method of claim 58, wherein the composition is administered at least 1 day, 2 days, 3 days, or more after a last dosing of the pre-conditioning regimen.

60. The method of any one of claims 1-59, wherein the composition is administered once per day for at least about 5 days, at most about 30 days, or from about 1 to 30 days.

61. The method of any one of claims 1-60, wherein the composition is administered at a dose of at least about 1x105engineered immune cells / kg.

62. The method of any one of claims 1-60, wherein the composition is administered at a dose of at least about 5x105engineered immune cells / kg.

63. The method of any one of claims 1-60, wherein the composition is administered at a dose of at least about 1x106engineered immune cells / kg.

64. The method of any one of claims 1-60, wherein the composition is administered at a dose of about 3xl06engineered immune cells / kg.

65. The method of any one of claims 1-60, wherein the composition is administered at a dose of about IxlO7engineered immune cells / kg.

66. The method of any one of claims 1-65, wherein the composition is administered as a single dose.

67. The method of any one of claims 1-65, wherein the composition is administered as multiple doses on a single day or over multiple days.

68. The method of claim 67, wherein the composition is administered as 2, 3, 4, 5, or more doses.

69. The method of any one of claims 1-68, further comprising administering a second dose of the engineered immune cell no sooner than 1 month and no later than 1 year following completion of a first dose.

70. The method of any one of claims 1-65, wherein the composition is administered as multiple doses over a duration of time.

71. The method of claim 70, wherein the duration of time is at least about 1 week, at least about 1 month, or greater than about 6 months.

72. The method of any one of claims 1-71, further comprising subjecting the subject to one or more bridging therapies.Attorney Docket No. 62190-73760173. The method of claim 72, wherein the one or more bridging therapies is administered prior to, concomitantly with, or subsequent to the composition.

74. The method of claim 72 or 73, wherein the one or more bridging therapies comprise 5-azacytidine, venetoclax, or any combination thereof.

75. The method of any one of claims 1-71, wherein the method does not comprise any bridging therapy.

76. The method of any one of claims 1-75, further comprising, prior to and / or subsequent to administering, measuring a level of myeloid blasts of a sample from the subject.

77. The method of claim 76, wherein the sample is a bone marrow sample.

78. The method of claim 76 or 77, wherein the myeloid blasts are CD7+CD33+CD34+ myeloid blasts.

79. The method of any one of claims 76-78, wherein measuring the level of myeloid blasts comprises measuring an expression level of one or more markers selected from the group consisting of CD9, CD7, CD18, CD25, CD32, CD33, CD34, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD 123, CD200, CD300a / c, CD366, CD371, and CX3CR1.

80. The method of any one of claims 76-79, wherein measuring the level of myeloid blasts comprises measuring an expression level of CD7, CD33, and / or CD34.

81. The method of any one of claims 76-80, wherein a percentage of myeloid blasts in the bone marrow sample is at least about 40% prior to administering.

82. The method of any one of claims 76-81, wherein a percentage of myeloid blasts in the bone marrow sample is at least about 80% prior to administering.

83. The method of any one of claims 76-82, wherein a percentage of myeloid blasts in the bone marrow sample is at most about 20% subsequent to administering.

84. The method of any one of claims 76-83, wherein the percentage of myeloid blasts in the bone marrow sample is at most about 10% subsequent to administering.

85. The method of any one of claims 76-84, wherein the bone marrow sample subsequent to administering does not comprise any detectable level of myeloid blasts.

86. The method of any one of claims 1-85, wherein the composition is administered into the subject by intravenous infusion, intra-arterial infusion, direct injection into tumor and / or perfusion of tumor bed after surgery, implantation at a tumor site in an artificial scaffold, intrathecal administration, or intraocular administration.Attorney Docket No. 62190-73760187. The method of any one of claims 1-86, wherein administering the composition to the subject increases a percentage of engineered immune cells expressing the CAR in a peripheral blood sample or a bone marrow sample from the subject.

88. The method of claim 87, wherein the percentage of engineered immune cells expressing the CAR, at least 9 days subsequent to the administering, is at least about 70%.

89. The method of claim 87, wherein the percentage of engineered immune cells expressing the CAR, at least 14 days subsequent to the administering, is at least about 80%.

90. The method of claim 87, wherein the percentage of engineered immune cells expressing the CAR, at least 20 days subsequent to the administering, is at least about 60%.

91. The method of claim 87, wherein the percentage of engineered immune cells expressing the CAR, at least 28 days subsequent to the administering, is at least about 40%.

92. The method of any one of claims 1-91, wherein the engineered immune cell persists in the subject for at least 5 days, 10 days, 14 days, 28 days, or more.

93. The method of any one of claims 1-92, further comprising, subsequent to administering, detecting a minimal residual disease (MRD) of the subject.

94. The method of claim 93, wherein detecting the MRD of the subject comprises determining a level of myeloid blasts in a sample from the subject.

95. The method of claim 94, wherein the myeloid blasts are positive for one or more markers selected from the group consisting of CD9, CD7, CD 18, CD25, CD32, CD33, CD34, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a / c, CD366, CD371, and CX3CR1.

96. The method of claim 94, wherein the myeloid blasts are CD7+CD33+CD34+ blasts.

97. The method of any one of claims 94-96, wherein the sample is a blood sample or a bone marrow sample.

98. The method of any one of claims 1-97, further comprising administering an additional therapeutic agent or therapy.

99. The method of any one of claims 1-98, further comprising obtaining a population of immune cells from the subject prior to administering the composition, and transducing one or more cells from the population of immune cells with a recombinant nucleic acid comprising the first nucleotide sequence encoding the target-binding domainAttorney Docket No. 62190-737601linked to the localizing domain and the second nucleotide sequence encoding the CAR, thereby generating a population of engineered immune cells.

100. The method of any one of claims 1-99, wherein the localizing domain comprises an ER retention signal.

101. The method of claim 100, wherein the ER retention signal comprises the amino acid sequence of KDEL (SEQ ID NO: 35), KKMP (SEQ ID NO: 36), KKTN (SEQ ID NO: 37), SEKDEL (SEQ ID NO: 40), or AEKDEL (SEQ ID NO: 41).

102. The method of claim 100, wherein the ER retention signal comprises the amino acid sequence of KKXX, where X is any amino acid.

103. The method of any one of claims 1-99, wherein the localizing domain comprises a Golgi retention sequence.

104. The method of any one of claims 1-99, wherein the localizing domain comprises a proteasome localizing sequence.

105. The method of claim 104, wherein the proteasome localizing sequence comprises a PEST motif.

106. The method of claim 105, wherein the PEST motif comprises an amino acid sequence as set forth in SEQ ID NO: 43 or 44.

107. The method of any one of claims 1-99, wherein the localizing domain comprises a transmembrane domain sequence derived from CD8a, CD8P, 4- IBB, CD28, CD34, CD4, FcsRIy, CD16, 0X40, CD3< CD3s, CD3y, CD38, TCRa, CD32, CD64, VEGFR2, FAS, orFGFR2B.

108. The method of claim 107, wherein the first nucleotide sequence further encodes a CD8a signal peptide as set forth in SEQ ID NO: 54.

109. The method of any one of claims 1-99, wherein the localizing domain comprises an amino acid sequence as set forth in SEQ ID NO: 31-33 or 45-50.

110. The method of any one of claims 1-108, wherein the localizing domain further comprises a linker.

111. The method of any one of claims 1-110, wherein the localizing domain comprises, in N-terminus to C-terminus order, a linker and a retention signal.

112. The method of claim 110 or 111, wherein the linker comprises a sequence of at least 5 amino acids.

113. The method of any one of claims 110-112, wherein the linker comprises an amino acid sequence as set forth in SEQ ID NO: 53.Attorney Docket No. 62190-737601114. The method of any one of claims 110-112, wherein the linker comprises a human influenza hemagglutinin (HA) tag or a Myc tag.

115. The method of any one of claims 1-114, wherein the first target-binding domain is a first antibody or antigen binding fragment thereof.

116. The method of any one of claims 1-115, wherein the second target-binding domain is a second antibody or antigen binding fragment thereof.

117. The method of claim 115, wherein the first antibody or antigen binding fragment thereof is a first single chain variable fragment (scFv).

118. The method of claim 116, wherein the second antibody or antigen binding fragment thereof is a second single chain variable fragment (scFv).

119. The method of any one of claims 116-118, wherein the first antibody or antigen binding fragment thereof and the second antibody or antigen binding fragment thereof comprise the same scFv.

120. The method of any one of claims 1-119, wherein the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of DEVRGYLDV (SEQ ID NO: 3).

121. The method of claim 120, wherein the VH comprises a HC CDR2 comprising the amino acid sequence of SISSGGFTYYPDSVKG (SEQ ID NO: 2) or SSGGF (SEQ ID NO: 5).

122. The method of claim 120 or 121, wherein the VH comprises a HC CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 1) or GLTFSSY (SEQ ID NO: 4).

123. The method of any one of claims 120-122, wherein the first target-binding domain and / or the second target-binding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QQYSKLPYT (SEQ ID NO: 8).

124. The method of claim 123, wherein the VL comprises a LC CDR2 comprising the amino acid sequence of YTSSLHS (SEQ ID NO: 7).

125. The method of claim 123 or 124, wherein the VL comprises a LC CDR1 comprising the amino acid sequence of SASQGISNYLN (SEQ ID NO: 6).

126. The method of any one of claims 120-125, wherein the first target-binding domain and / or the second target-binding domain comprises:Attorney Docket No. 62190-737601a HC CDR3 sequence of DEVRGYLDV (SEQ ID NO: 3),a HC CDR2 sequence of SISSGGFTYYPDSVKG (SEQ ID NO: 2) or SSGGF (SEQ ID NO: 5),a HC CDR1 sequence of SYAMS (SEQ ID NO: 1) or GLTFSSY (SEQ ID NO: 4), a LC CDR3 sequence of QQYSKLPYT (SEQ ID NO: 8),a LC CDR2 sequence of YTSSLHS (SEQ ID NO: 7), anda LC CDR1 sequence of SASQGISNYLN (SEQ ID NO: 6).

127. The method of any one of claims 120-126, wherein the VH comprises a sequence with at least 80% sequence identity to the sequence EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASIS SGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYL DVWGAGTTVTVSS (SEQ ID NO: 9).

128. The method of any one of claims 123-127, wherein the VL comprises a sequence with at least 80% sequence identity to the sequence AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIY YTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTK LEIKR (SEQ ID NO: 10).

129. The method of any one of claims 120-128, wherein the VH comprises a sequence of EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASIS SGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYL DVWGAGTTVTVSS (SEQ ID NO: 9).

130. The method of any one of claims 123-129, wherein the VL comprises a sequence of AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIY YTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTK LEIKR (SEQ ID NO: 10).

131. The method of any one of claims 1-130, wherein the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of GGVYYDLYYYALDY (SEQ ID NO: 13).

132. The method of claim 131, wherein the VH comprises a HC CDR2 comprising the amino acid sequence of KINPSNGRTNYNEKFKS (SEQ ID NO: 12) or NPSNGR (SEQ ID NO: 15).Attorney Docket No. 62190-737601133. The method of claim 131 or 132, wherein the VH comprises a HC CDR1 comprising the amino acid sequence of SYWMH (SEQ ID NO: 11) or GYTFTSY (SEQ ID NO: 14).

134. The method of any one of claims 131-133, wherein the first target-binding domain and / or the second target-binding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QQSNSWPYT (SEQ ID NO: 18).

135. The method of claim 134, wherein the VL comprises a LC CDR2 comprising the amino acid sequence of SASQSIS (SEQ ID NO: 17).

136. The method of claim 134 or 135, wherein the VL comprises a LC CDR1 comprising the amino acid sequence of RASQSISNNLH (SEQ ID NO: 16).

137. The method of any one of claims 131-136, wherein the first target-binding domain and / or the second target-binding domain comprises:a HC CDR3 sequence of GGVYYDL YYYALD Y (SEQ ID NO: 13),a HC CDR2 sequence of KINPSNGRTNYNEKFKS (SEQ ID NO: 12) or NPSNGR (SEQ ID NO: 15),a HC CDR1 sequence of SYWMH (SEQ ID NO: 11) or GYTFTSY (SEQ ID NO: 14),a LC CDR3 sequence of QQSNSWPYT (SEQ ID NO: 18),a LC CDR2 sequence of SASQSIS (SEQ ID NO: 17), anda LC CDR1 sequence of RASQSISNNLH (SEQ ID NO: 16).

138. The method of any one of claims 131-137, wherein the VH comprises a sequence with at least 80% sequence identity to the sequence QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKI NPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYY DL YYYALD YWGQGTTVT VS S (SEQ ID NO: 19).

139. The method of any one of claims 134-136, wherein the VL comprises a sequence with at least 80% sequence identity to the sequence DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSI SGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKR(SEQ ID NO: 20).

140. The method of any one of claims 131-139, wherein the VH comprises a sequence of QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKIAttorney Docket No. 62190-737601NPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYY DLYYYALDYWGQGTTVTVSS (SEQ ID NO: 19).

141. The method of any one of claims 134-140, wherein the VL comprises a sequence of DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSI SGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKR(SEQ ID NO: 20).

142. The method of any one of claims 1-141, wherein the first target-binding domain and / or the second target-binding domain comprises a heavy chain variable region (VH), and wherein the VH comprises a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of WGNYPHYAMDY (SEQ ID NO: 23).

143. The method of claim 142, wherein the VH comprises a HC CDR2 comprising the amino acid sequence of YISSGSSTLHYADTVKG (SEQ ID NO: 22) or SSGSST (SEQ ID NO: 25).

144. The method of claim 142 or 143, wherein the VH comprises a HC CDR1 comprising the amino acid sequence of SFGMH (SEQ ID NO: 21) or GFTFSSF (SEQ ID NO: 24).

145. The method of any one of claims 142-144, wherein the first target-binding domain and / or the second target-binding domain further comprises a light chain variable region (VL), and wherein the VL comprises a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of QHSRELPYT (SEQ ID NO: 28).

146. The method of claim 145, wherein the VL comprises a LC CDR2 comprising the amino acid sequence of LASNLES (SEQ ID NO: 27).

147. The method of claim 145 or 146, wherein the VL comprises a LC CDR1 comprising the amino acid sequence of RASKS VS ASGYSYMH (SEQ ID NO: 26).

148. The method of any one of claims 142-147, wherein the first target-binding domain and / or the second target-binding domain comprises:a HC CDR3 sequence of WGNYPHYAMDY (SEQ ID NO: 23),a HC CDR2 sequence of YISSGSSTLHYADTVKG (SEQ ID NO: 22) or SSGSST (SEQ ID NO: 25),a HC CDR1 sequence of SFGMH (SEQ ID NO: 21) or GFTFSSF (SEQ ID NO: 24), a LC CDR3 sequence of QHSRELPYT (SEQ ID NO: 28),a LC CDR2 sequence of LASNLES (SEQ ID NO: 27), andAttorney Docket No. 62190-737601a LC CDR1 sequence of RASKS VS ASGYSYMH (SEQ ID NO: 26).

149. The method of any one of claims 142-148, wherein the VH comprises a sequence with at least 80% sequence identity to the sequence DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYI S SGS STLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTA YYC ARWGNYP HYAMDYWGQGTSVTVSS (SEQ ID NO: 29).

150. The method of any one of claims 145-147, wherein the VL comprises a sequence with at least 80% sequence identity to the sequence DIVMTQSPASLAVSLGQRATISCRASKSVSASGYSYMHWYQQKPGQPPKLLI YLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAVTYYCQHSRELPYTFGGG TKLEIK (SEQ ID NO: 30).

151. The method of any one of claims 142-150, wherein the VH comprises a sequence of DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYI S SGS STLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC ARWGNYP HYAMDYWGQGTSVTVSS (SEQ ID NO: 29).

152. The method of any one of claims 145-151, wherein the VL comprises a sequence of DIVMTQSPASLAVSLGQRATISCRASKSVSASGYSYMHWYQQKPGQPPKLLI YLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAVTYYCQHSRELPYTFGGG TKLEIK (SEQ ID NO: 30).

153. The method of any one of claims 1-152, wherein the CAR further comprises a transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3(^ intracellular signaling domain.

154. The method of any one of claims 1-153, wherein the transmembrane domain comprises an amino acid sequence as set forth in SEQ ID NO: 51.

155. The method of claim 153 or 154, wherein the 4-1BB intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 55.

156. The method of any one of claims 153-155, wherein the CD3(^ intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 56.

157. The method of any one of claims 1-156, wherein the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell that comprises the second nucleotide sequence but not the first nucleotide sequence.

158. The method of any one of claims 1-157, wherein the engineered immune cell is a T cell.

159. The method of claim 158, wherein the T cell is a CD4+ T cell or a CD8+ T cell.Attorney Docket No. 62190-737601160. The method of any one of claims 1-159, wherein the engineered immune cell is a natural killer (NK) cell.

161. The method of any one of claims 1-160, wherein the first target-binding domain linked to the localizing domain comprises an amino acid sequence as set forth in any one of SEQ IDNOs: 61-64.

162. The method of any one of claims 1-161, wherein the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 65-68.

163. The method of any one of claims 1-162, wherein the first nucleotide sequence and the second nucleotide sequence are on different molecules.

164. The method of any one of claims 1-162, wherein the first nucleotide sequence and the second nucleotide sequence are on a same molecule.

165. The method of claim 164, wherein the first nucleotide sequence and the second nucleotide sequence are on a bicistronic vector.

166. The method of claim 165, wherein the bicistronic vector is a viral vector.

167. The method of claim 166, wherein the viral vector is a retroviral vector.

168. The method of claim 166, wherein the viral vector is a lentiviral vector.

169. The method of any one of claims 165-168, wherein the first nucleotide sequence and the second nucleotide sequence are operably linked by an Internal Ribosome Entry Site (IRES) or a ribosomal codon skipping site.

170. The method of claim 169, wherein the IRES is derived from Encephalomyocarditis virus (EMCV) or an Enterovirus .

171. The method of claim 169 or 170, wherein the IRES comprises a polynucleotide sequence as set forth in SEQ ID NO: 82.

172. The method of claim 169, wherein the ribosomal codon skipping site comprises a 2A self-cleaving peptide.

173. The method of claim 172, wherein the 2 A self-cleaving peptide is selected from the group consisting of a F2A peptide (foot-and-mouth disease virus 2A peptide), an E2A peptide (equine rhinitis A virus 2A peptide), a P2A peptide (porcine teschovirus-1 2A peptide), and a T2A peptide (thosea asigna virus 2A).

174. The method of claim 172 or 173, wherein the 2A self-cleaving peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 83-86.

175. The method of any one of claims 165-174, wherein the bicistronic vector further comprises a promoter element.Attorney Docket No. 62190-737601176. The method of claim 175, wherein the promoter element is selected from the group consisting of a CMV promoter, EFla promoter, EFS promoter, MSCV promoter, and PGK promoter.

177. The method of claim 175 or 176, wherein the promoter element is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NOs: 92-96.

178. Use of a composition in the manufacturing of a medicament for the treatment of acute myeloid leukemia (AML) in a subject in need thereof, wherein the composition comprises an engineered immune cell, wherein the engineered immune cell comprises:(i) a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and(ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; andwherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

179. A pharmaceutical composition for treating acute myeloid leukemia (AML) in a subject in need thereof, wherein the pharmaceutical composition comprises an engineered immune cell and a pharmaceutically acceptable carrier, wherein the engineered immune cell comprises:(i) a first nucleotide sequence encoding a first target-binding domain linked to a localizing domain, wherein the first target-binding domain binds to CD7; and(ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second target-binding domain that binds to CD7; andwherein the engineered immune cell is from an autologous immune cell of the subject or an immune cell obtained from the subject to which the composition is to be administered.

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