Methods and Compositions for Immune Cell CRISPR Screening

Abstract

This application partially discloses a method and composition for mutating immune cells (e.g., CAR-T cells) and determining the cancer-killing effect of the mutated immune cells in vivo.

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 63 / 344,951, filed May 23, 2022, and U.S. Provisional Application No. 63 / 464,483, filed May 5, 2023, both entitled "Methods and Compositions for Immune Cell CRISPR Screening", the entire contents of each of which are incorporated herein by reference.

Background Art

[0002] Background Cancer treatment has been revolutionized by reprogramming immune cells to incorporate chimeric antigen receptors (CARs). CARs direct immune cells to target cancer and, upon binding, induce cancer cell death. A CAR is typically a transmembrane protein that includes an extracellular portion (e.g., an antigen-binding domain), a transmembrane domain, and an intracellular binding domain. The antigen-binding domain is often an antibody that binds to a cancer-related antigen, but not necessarily. When the antigen-binding domain binds, the intracellular domain activates the immune cell (such as a T cell), and as a result, the immune cell may kill the cancer cell. However, many factors can affect the effectiveness of CAR-based therapies in killing cancer, including specific patient characteristics in which CAR-based therapies of certain types result in higher therapeutic efficacy in some patients than in others. Thus, there is a general need for CAR therapeutics that can be individualized to the patient in the treatment of a particular disease.

Summary of the Invention

[0003] In some aspects, the present disclosure describes methods and compositions for performing CAR immune cell (e.g., CAR-T cell or CAR-NK cell) CRISPR screening (e.g., in humans) to identify genotypes of immune cells that enhance the efficacy of CAR immunotherapy against cancer. For example, the present disclosure describes a CRISPR guide RNA polynucleotide library designed to target genes thought to be involved in the efficacy of CAR-T cells. Thus, in some embodiments, a mutant CAR-T cell library can be created using the CRISPR guide RNA library, the mutant CAR-T cell library comprising CAR-T cells having many different genotypes, e.g., including the same CAR. The present disclosure is directed, in part, to the discovery of a collection of genes whose modification may affect the efficacy of CAR-T cells (e.g., tumor killing and persistence), and the subsequent development of a CRISPR guide RNA library targeting the collection of genes. In some embodiments, one or more of the genotypes of the mutant CAR-T cell library improve the efficacy of CAR-T cells (e.g., tumor killing and persistence). Further, these mutations may have different effects when treating different patients and / or different cancers. In some embodiments, the methods described herein include creating a mutant CAR immune cell library (e.g., a mutant CAR-T cell library or a mutant CAR-NK cell library), administering the library to a patient (e.g., a human patient), collecting one or more samples of the CAR-T cell library from the patient, and sequencing the guide RNA polynucleotide of the CAR-T cells to identify which CAR-T gene variants have the highest prevalence in the sample as an indicator of the efficacy of the CAR-T cells. In some embodiments, the method further includes administering to the patient a CAR-T cell comprising a mutation that increases the prevalence of the CAR-T cells in vivo (determined using the methods described above).Thus, in some embodiments, these methods provide strategies for identifying patient-specific and / or cancer-specific CAR-T cell genomic mutations that enhance the in vivo cancer killing effect.

[0004] The compositions and methods described herein provide advantages over previous existing technologies. Results obtained from experiments performed in model organisms are often not reproduced when the experiments are adapted and repeated in another organism, such as a human. For example, CD19-28z cytotoxic T cells eradicated B cell tumors in mice but had little effect on human cancers. Brentjens et al., Nature medicine 9.3 (2003): 279-286 and Brentjens et al., Blood, The Journal of the American Society of Hematology 118.18 (2011): 4817-4828. In another example, mesothelin-targeted CAR-T cells killed cancer in mice but had little effect on human cancers. Carpenito et al., Proceedings of the National Academy of Sciences 106.9 (2009): 3360-3365 and Beatty et al., Gastroenterology 155.1 (2018): 29-32. The compositions and methods of the present disclosure are designed, in part, for use in humans. Genotypes of CAR immune cells identified to improve the cancer killing effect in humans (e.g., CAR-T cell genotype or CAR-NK cell genotype) potentially do not require adaptation from dissimilar model organisms, and the observed therapeutic effects can be applied directly to humans, such as the humans in whom the screening was performed. The present disclosure also provides CAR and CRISPR vectors designed for use in humans, including, for example, CAR integration markers and CRISPR mutation markers that are compatible with use in humans.

[0005] Furthermore, the present disclosure provides methods and compositions that can provide definitive screening results in humans. The present disclosure provides a strategy for determining the characteristics (e.g., size) of a guide RNA library that can be used in humans and achieve sufficient representation of a mutant CAR immune cell library for calculations regarding a phenotype of interest, such as a cancer killing effect (e.g., for details of the calculations, see Example 2 below). In some embodiments, the number of different gRNA polynucleotides in the gRNA library is determined based on these calculations. In some embodiments, the gRNA library designed for human CAR immune cell CRISPR screening (e.g., CAR-T cell screening or CAR-NK cell screening) is not genome-wide. Without wishing to be bound by theory, genome-wide gRNA libraries may be too large and contain a very large number of different gRNA polynucleotides, and thus may not be able to provide definitive results in humans. For example, genome-wide CRISPR libraries may contain a very large number of different mutant immune cells (such as mutant CAR-T cells), and thus may not be properly represented in samples collected from patients, resulting in a decrease in statistical resolution and the inability to identify mutations contributing to phenotypes such as the efficacy of CAR-T cells.

[0006] The present disclosure partially provides a selected set of genes targeted by guide RNAs of a CRISPR library suitable for human use, taking into account, for example, the expected limitations regarding the size of the guide RNA library. In some embodiments, the genes selected include genes expected to affect the cancer killing efficacy or persistence of CAR-T cells and control genes not expected to affect the cancer killing efficacy of CAR-T cells.

[0007] In some embodiments, the present disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0008] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 10 gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0009] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 20 gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0010] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 50 gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0011] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 100 gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0012] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 135 gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0013] In some aspects, the present application discloses a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.

[0014] In some embodiments, at least 10 gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 20 gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 50 gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 100 gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 135 gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.

[0015] In some embodiments, the composition comprises at least two gRNA polynucleotides per gene sequence, and the at least two gRNA polynucleotides per gene sequence comprise different sequences. In some embodiments, the composition comprises eight gRNA polynucleotides per gene sequence, and the eight gRNA polynucleotides per gene sequence comprise different sequences. In some embodiments, the at least two or at least eight gRNA polynucleotides per gene sequence are complementary to non-overlapping regions of the same gene sequence.

[0016] In some embodiments, the composition comprises one or more negative control gRNA polynucleotides. In some embodiments, the negative control gRNA polynucleotide comprises a randomly generated homologous region. In some embodiments, the negative control gRNA polynucleotide comprises a homologous region that is complementary to a gene not known to be involved in T cell function. In some embodiments, the negative control gRNA polynucleotide comprises a homologous region that is an intergenic complementary region.

[0017] In some embodiments, the composition comprises at least one gRNA polynucleotide comprising any one of the sequences of SEQ ID NOs: 136 to 1315. In some embodiments, the composition comprises at least 10 gRNA polynucleotides, each comprising a different sequence of any one of SEQ ID NOs: 136 to 1315. In some embodiments, the composition comprises at least 100 gRNA polynucleotides, each comprising a different sequence of any one of SEQ ID NOs: 136 to 1315. In some embodiments, the composition comprises at least 1000 gRNA polynucleotides, each comprising a different sequence of any one of SEQ ID NOs: 136 to 1315. In some aspects, the present application discloses a composition comprising a plurality of gRNA polynucleotides comprising the sequences of SEQ ID NOs: 136 to 1315. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 200,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 100,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 50,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 25,000 gRNA polynucleotides having different sequences.

[0018] In some embodiments, the plurality of guide RNA polynucleotides consists of at most 15,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 10,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 5,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 2,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 1,080 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 500 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 250 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 100 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 50 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of at most 10 gRNA polynucleotides having different sequences.

[0019] In some embodiments, at least some of the plurality of gRNA polynucleotides are not cross-reactive with a plurality of genes. In some embodiments, the composition does not contain a gRNA homologous region that is complementary to the sense or antisense strand of a gene encoding p53, Rb, PTEN, BRCA1, and / or BRCA2, or a variant thereof. In some embodiments, the composition does not contain a gRNA that is complementary to the sense or antisense strand of a gene encoding P53, PTEN, APC, P16INK4a, P15INK4b, cadherin-1, RB1, BRCA1, Wilms tumor 1, STK11, Smad4, BRCA2, CHEK2, P14arf, P21, P73, PTCH1, and / or MSH2, or a variant thereof.

[0020] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof.

[0021] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof.

[0022] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof.

[0023] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof.

[0024] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by LAG3 (SEQ ID NO: 76) or a variant thereof.

[0025] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by PDCD1 (SEQ ID NO: 91) or a variant thereof.

[0026] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by TGFBR2 (SEQ ID NO: 121) or a variant thereof.

[0027] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by RARA (SEQ ID NO: 99) or a variant thereof.

[0028] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by SmarcB1 (SEQ ID NO: 113) or a variant thereof.

[0029] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof.

[0030] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by RunX (SEQ ID NO: 107) or a variant thereof.

[0031] In some embodiments, at least one gRNA polynucleotide comprises a homologous region that is complementary to the sense or antisense strand of a gene encoded by TCF7 (SEQ ID NO: 118) or a variant thereof.

[0032] In some embodiments, at least two gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

[0033] In some embodiments, at least five gRNA polynucleotides each comprise a homologous region that is complementary to the sense or antisense strand of a different gene encoded by any one of IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

[0034] In some embodiments, each of at least seven gRNA polynucleotides comprises a homologous region complementary to the sense or antisense strand of a different gene encoded by any one of IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

[0035] In some embodiments, each of the ten gRNA polynucleotides comprises a homologous region complementary to the sense or antisense strand of a different gene encoded by any one of IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

[0036] In some embodiments, each of the 14 gRNA polynucleotides comprises a homologous region complementary to either the sense or antisense strand of a different gene encoded by any one of IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

[0037] In some embodiments, the composition comprises: a first gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof; a second gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof; a third gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof; a fourth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof; a fifth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by LAG3 (SEQ ID NO: 76) or a variant thereof; a sixth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by PDCD1 (SEQ ID NO: 91) or a variant thereof; a seventh gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by TGFBR2 (SEQ ID NO: 121) or a variant thereof; an eighth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by RARA (SEQ ID NO: 99) or a variant thereof; a ninth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by SmarcB1 (SEQ ID NO: 113) or a variant thereof; a tenth gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof; an eleventh gRNA polynucleotide comprising a homology region complementary to the sense or antisense strand of a gene encoded by RunX (SEQ ID NO: 107) or a variant thereof;and / or a 12th gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand of a gene encoded by TCF7 (SEQ ID NO: 118) or a variant thereof; In some embodiments, the composition does not comprise a gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand of a gene encoded by RASA2 (SEQ ID NO: 102).

[0038] In some embodiments, the composition does not comprise a gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand of a gene encoded by one or more of RASA2 (SEQ ID NO: 102) or a variant thereof, TBL1XR1 (SEQ ID NO: 117) or a variant thereof, MEF2D (SEQ ID NO: 80) or a variant thereof, ARIH2 (SEQ ID NO: 6) or a variant thereof, NDUFB10 (SEQ ID NO: 82) or a variant thereof, STAT3 (SEQ ID NO: 115) or a variant thereof, ELOB (SEQ ID NO: 33) or a variant thereof, IRF2 (SEQ ID NO: 67) or a variant thereof, SMARCA4 (SEQ ID NO: 112) or a variant thereof, CYB5R4 (SEQ ID NO: 24) or a variant thereof, and LEF1 (SEQ ID NO: 78) or a variant thereof.

[0039] In some embodiments, the composition does not comprise a gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand of a gene encoded by at least three of RASA2 (SEQ ID NO: 102) or a variant thereof, TBL1XR1 (SEQ ID NO: 117) or a variant thereof, MEF2D (SEQ ID NO: 80) or a variant thereof, ARIH2 (SEQ ID NO: 6) or a variant thereof, NDUFB10 (SEQ ID NO: 82) or a variant thereof, STAT3 (SEQ ID NO: 115) or a variant thereof, ELOB (SEQ ID NO: 33) or a variant thereof, IRF2 (SEQ ID NO: 67) or a variant thereof, SMARCA4 (SEQ ID NO: 112) or a variant thereof, CYB5R4 (SEQ ID NO: 24) or a variant thereof, and LEF1 (SEQ ID NO: 78) or a variant thereof.

[0040] In some embodiments, the composition does not contain a gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand of a gene encoded by at least 5 of RASA2 (SEQ ID NO: 102) or a variant thereof, TBL1XR1 (SEQ ID NO: 117) or a variant thereof, MEF2D (SEQ ID NO: 80) or a variant thereof, ARIH2 (SEQ ID NO: 6) or a variant thereof, NDUFB10 (SEQ ID NO: 82) or a variant thereof, STAT3 (SEQ ID NO: 115) or a variant thereof, ELOB (SEQ ID NO: 33) or a variant thereof, IRF2 (SEQ ID NO: 67) or a variant thereof, SMARCA4 (SEQ ID NO: 112) or a variant thereof, CYB5R4 (SEQ ID NO: 24) or a variant thereof, and LEF1 (SEQ ID NO: 78) or a variant thereof.

[0041] In some embodiments, the plurality of gRNA polynucleotides are Cas9 protein gRNA polynucleotides or Cas12 protein gRNA polynucleotides. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 15,000 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 10,000 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 5,000 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 2,500 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 1,000 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 500 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 200 different gene sequences. In some embodiments, the plurality of gRNA polynucleotides comprise sequences complementary to at most 135 different gene sequences. In some embodiments, the composition is for use in human CAR-T cell screening.

[0042] In some embodiments, the present application discloses a guide RNA polynucleotide comprising any one of the sequences of SEQ ID NOs: 136 to 1315.

[0043] In some embodiments, the present application discloses a plasmid comprising any one of the gRNA polynucleotides of any one of the compositions described herein or any one of the guide RNAs described herein.

[0044] In some embodiments, the present application discloses a plasmid library comprising at least two plasmids, each comprising any one of the gRNA polynucleotides of any one of the compositions described herein or any one of the guide RNAs described herein. In some embodiments, at least some of the at least two plasmids comprise the gRNA polynucleotide of the composition according to any one of claims 1 to 72 or the guide RNA polynucleotide according to claim 74. In some embodiments, each plasmid comprises the gRNA polynucleotide of the composition described herein or the guide RNA polynucleotide described herein.

[0045] In some embodiments, the present application discloses a plasmid library comprising each of SEQ ID NOs: 136 to 1315 encoded on different plasmids.

[0046] In some embodiments, the present application discloses a gRNA vector comprising (1) a first gRNA polynucleotide comprising a homologous region complementary to the sense or antisense strand encoded by the TCR / CD3 complex (SEQ ID NOs: 1383 to 1398) of a T cell, and (2) a second gRNA polynucleotide.

[0047] In some embodiments, the second gRNA polynucleotide comprises any one of the gRNA polynucleotides of the compositions described herein or the guide RNAs described herein. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in reverse orientation. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in tandem orientation. In some embodiments, each gRNA coding sequence is operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter or an inducible promoter. In some embodiments, the promoter is selected from the group consisting of a U6 promoter and an EF1a promoter. In some embodiments, the promoter is a U6 promoter or an H1 promoter, and optionally, the first gRNA polynucleotide is operably linked to the U6 promoter and the second gRNA polynucleotide is operably linked to the H1 promoter.

[0048] In some embodiments, the gRNA vector further comprises a selectable marker, and optionally, the selectable marker is suitable for use in humans. In some embodiments, the selectable marker is selected from the group consisting of LGNFR, EGFR, CD19, CD20, CD34, and truncated forms thereof. In some embodiments, the selectable marker is operably linked to a promoter, optionally a weak promoter. In some embodiments, the promoter is selected from the group consisting of PGK, CMV, EF1a, and tissue-specific promoters.

[0049] In some embodiments, the vector is an adeno-associated vector, a retroviral vector, or a lentiviral vector. In some embodiments, the lentiviral vector is a third-generation self-inactivating (SIN) lentiviral vector.

[0050] In some embodiments, the vector contains a suicide gene. In some embodiments, the suicide gene is selected from the group consisting of icaspase9, tEGFR, tCD29, CD20, and tHer2. In some embodiments, the gRNA vector contains any one of the nucleic acid sequences of SEQ ID NO: 1318.

[0051] In some embodiments, the gRNA vector further contains a polynucleotide encoding a CRISPR protein. In some embodiments, the CRISPR protein is Cas9 or Cas12.

[0052] In some aspects, the present application discloses a gRNA vector library containing at least two gRNA vectors described herein. In some embodiments, each vector contains the gRNA polynucleotide of the composition described herein or the guide RNA described herein. In some embodiments, at least 10 vectors of the library each contain a different gRNA polynucleotide homologous region that is complementary to the sense strand or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0053] In some embodiments, the gRNA vector library is a Good Manufacturing Practice (cGMP) grade gRNA vector library. In some embodiments, the cGMP grade gRNA vector library is a cGMP grade gRNA lentiviral gRNA vector library.

[0054] In some embodiments, at least 20 vectors of the library each contain a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 50 vectors of the library each contain a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 100 vectors of the library each contain a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 135 vectors of the library each contain a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0055] In some aspects, the present application discloses a CAR vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the vector is a self-inactivating vector. In some embodiments, the vector is an adeno-associated vector, a retroviral vector, or a lentiviral vector. In some embodiments, the vector is a third-generation self-inactivating (SIN) lentiviral vector.

[0056] In some embodiments, the CAR is operably linked to a promoter, optionally EF1a or the EF1a short promoter.

[0057] In some embodiments, the vector comprises a suicide gene. In some embodiments, the suicide gene is selected from the group consisting of icaspase9, tEGFR, tCD29, CD20, and tHer2. In some embodiments, the vector comprises a reporter. In some embodiments, the reporter is selected from the group consisting of truncated CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2. In some embodiments, the CAR comprises a polynucleotide encoding an antigen-binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen-binding domain.

[0058] In some embodiments, the CAR comprises a polynucleotide encoding an antigen-binding domain that binds to mesothelin. In some embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the intracellular signaling domain is selected from the group consisting of CD28, 4-1BB, CD27, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3 epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0059] In some embodiments, the 2A ribosome skip element or the internal ribosome entry site (IRES) is downstream of the CAR. In some embodiments, the CAR vector comprises the sequence of SEQ ID NO: 1319. In some aspects, the present application discloses a composition comprising the gRNA vector according to any one of the claims described herein and the CAR vector described herein.

[0060] In some aspects, the present application discloses CAR-T cells comprising a composition comprising the gRNA vector according to any one of the claims described herein and the CAR vector described herein. In some aspects, the present application discloses CAR-T cells comprising a mutation in a gene corresponding to any one of the gene sequences of SEQ ID NOs: 1-135 or a variant thereof, the gRNA polynucleotide of the composition described herein, the plasmid described herein, or one, two, or three of the gRNA vectors described herein. In some embodiments, the CAR-T cells further comprise the CAR vector described herein.

[0061] In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation is an insertion or deletion. In some embodiments, the insertion or deletion mutation causes a frameshift. In some embodiments, the CAR-T cells further comprise mRNA encoding a CRISPR protein. In some embodiments, the CAR-T cells are CD34 positive and CD3 negative. In some embodiments, the T cells used to generate the CAR-T cells are derived from a mammalian subject. In some embodiments, the T cells used to generate the CAR-T cells are derived from a human subject. In some embodiments, the T cells used to generate the CAR-T cells are collected using leukapheresis.

[0062] In some embodiments, the CAR-T cells are activated. In some embodiments, the CAR-T cells are activated using an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the CAR-T cells are activated using an anti-CD3 antibody. In some embodiments, the CAR-T cells are activated using an anti-CD3 antibody and IL-2. In some embodiments, the CAR-T cells are activated using an anti-CD3 antibody, IL-2, and PHA. In some embodiments, the anti-CD3 antibody, anti-CD28 antibody, IL2, and PHA are soluble or plate-bound. In some embodiments, the CRISPR protein is Cas9 or Cas12. In some embodiments, the cell does not contain a DNA polynucleotide encoding the CRISPR protein. In some embodiments, the CAR-T cells are CD34 positive, CD3 negative, and LNGFR positive.

[0063] In some embodiments, the CAR-T cells of the library are selected from the CAR-T cells described herein. In some embodiments, at least two CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 10 CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 20 CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 50 CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 100 CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the 135 CAR-T cells of the library each contain different gRNA polynucleotides containing a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, each of SEQ ID NOs: 136-1315 is present in at least one CAR-T cell.

[0064] In some aspects, the present application discloses a method for creating a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting the T cells with the gRNA vector library described herein; (c) transfecting the T cells with the CAR vector described herein; and (d) introducing into the T cells an mRNA encoding a CRISPR protein or a CRISPR protein.

[0065] In some embodiments, the present application is a method of creating a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting the T cells with the gRNA vector library described herein; and (c) transfecting the T cells with the CAR vector according to any one of claims C1-C11, wherein the transfection comprises transfecting with a gRNA vector or a CAR vector that contains a polynucleotide encoding a CRISPR protein.

[0066] In some embodiments, the method further comprises collecting T cells from a subject. In some embodiments, the method further comprises purifying T cells that contain the CAR vector and contain CRISPR-induced genetic mutations. In some embodiments, the T cells are collected using leukapheresis. In some embodiments, the T cells are collected from a human or mouse subject. In some embodiments, the T cells are activated using anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the gRNA vector library is transduced at a multiplicity of infection (MOI) between 0.1 and 1. In some embodiments, the MOI is between 0.3 and 0.7. In some embodiments, the MOI is between 0.4 and 0.6. In some embodiments, the MOI is 0.5. In some embodiments, the CAR vector is transduced at an MOI of up to 20.

[0067] In some embodiments, the CRISPR protein is Cas9 or Cas12. In some embodiments, the mRNA of the CRISPR protein or the CRISPR protein is introduced using electroporation. In some embodiments, the CRISPR mRNA is expressed from a gRNA vector or a CAR vector. In some embodiments, electroporation is performed 3 to 7 days after transduction with the gRNA vector and the CAR vector. In some embodiments, the method further comprises selecting T cells that are CD3 negative, CD34 positive, and optionally LNGFR positive. In some embodiments, the selection is performed using magnetic beads and / or fluorescence-activated cell sorting. In some aspects, the present application discloses a mutant CAR-T cell library created using the method described herein.

[0068] In some aspects, the present application is a method for identifying a gRNA polynucleotide associated with the efficacy of CAR-T cells in vivo, comprising: (a) administering to a subject the mutant CAR-T cell library according to any one of claims E1-E7 or F15; (b) collecting from the subject one or more samples comprising a plurality of mutant CAR-T cells of the mutant CAR-T cell library; (c) sequencing the gRNA polynucleotide from the mutant CAR-T cells collected in (b); and (d) evaluating a change in the relative abundance of each gRNA polynucleotide based on the sequencing in (c).

[0069] In some embodiments, the method further comprises: (e) identifying a gene associated with a gRNA polynucleotide having an increased relative abundance; (f) generating a modified CAR-T cell comprising a mutation in the gene identified in (e); and (g) administering the modified CAR-T cell to the subject.

[0070] In some embodiments, the variant CAR-T cell library comprises a genome-wide CAR-T cell variant library. In some embodiments, the library comprises negative control CAR-T cells. In some embodiments, the negative control CAR-T cells comprise the negative control gRNA polynucleotides described herein. In some embodiments, the subject is human. In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, and B cell lymphoma. In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen expressed by the cancer.

[0071] In some embodiments, the CAR comprises an antigen-binding domain that binds to any pair of CD19 / CD79b, BCMA / TACI selected from the group consisting of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, or is TriPRIL. In some embodiments, the sample is collected from the subject's blood. In some embodiments, at least 3 samples are collected from the subject. In some embodiments, at least 5 samples are collected by venipuncture, apheresis, or tumor resection. In some embodiments, the sample is collected from the subject at one or more of day 3, day 7, day 10, day 14, day 21, month 1, month 2, month 3, month 6, month 9, month 12, month 15, month 18, month 21, and month 24 after administration of the variant CAR-T cell library after step (a). In some embodiments, a sample is collected from the subject 21 days after administration of the variant CAR-T cell library after step (a).

[0072] In some embodiments, the method further comprises extracting variant CAR T cells from the sample prior to sequencing. In some embodiments, the extraction is performed by isolating T cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

[0073] In some embodiments, the method further comprises isolating genomic DNA from the mutant CAR-T cells. In some embodiments, the method further comprises isolating gRNA polynucleotides from the mutant CAR-T cells. In some embodiments, the method further comprises isolating RNA from the mutant CAR-T cells and optionally sequencing the RNA. In some embodiments, the method further comprises amplifying the gRNA polynucleotides of the mutant CAR-T cells to generate any of their amplicons. In some embodiments, the gRNA polynucleotides to be amplified are genomic DNA, vector DNA, or RNA. In some embodiments, the primers of SEQ ID NOs: 1324-1325 are used for amplification. In some embodiments, the gRNA polynucleotide or its amplicon is sequenced. In some embodiments, the gRNA polynucleotide or its amplicon is sequenced using next-generation sequencing.

[0074] In some embodiments, the efficacy of each mutant CAR-T cell is evaluated based on in vivo persistence and tumor cell cytotoxicity. In some embodiments, in vivo persistence and tumor cell cytotoxicity are measured using flow cytometry and PCR. In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA.

[0075] In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA compared to the gRNA polynucleotide from negative control CAR-T cells. In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA over at least three time points and calculating the slope of the relative change. In some embodiments, tumor cell cytotoxicity is quantified using clinical imaging and / or bone marrow evaluation. In some aspects, the present application discloses CAR natural killer (NK) cells comprising the compositions described herein.

[0076] In some embodiments, the present application discloses CAR-NK cells comprising one, two, or three of a mutation in a gene corresponding to any one of the gene sequences of SEQ ID NOs: 1-135 or a variant thereof, the gRNA polynucleotide of the composition described herein, the plasmid described herein, or the gRNA vector described herein. In some embodiments, the CAR-NK cells further comprise a CAR vector. In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation is an insertion or deletion. In some embodiments, the insertion or deletion mutation causes a frameshift.

[0077] In some embodiments, CAR-NK further comprises a CRISPR protein and / or mRNA encoding a CRISPR protein. In some embodiments, the NK cells are CD34 positive and CD3 negative. In some embodiments, the NK cells used to generate the NK cells are derived from a mammalian subject. In some embodiments, the NK cells used to generate the NK cells are derived from a human subject. In some embodiments, the NK cells used to generate the NK cells are collected from blood, optionally cord blood or peripheral blood. In some embodiments, the NK cells are activated. In some embodiments, the NK cells are activated using irradiated feeder cells, IL-2, and IL-15. In some embodiments, the NK cells are activated using irradiated K562 cells. In some embodiments, the NK cells are activated using IL-21. In some embodiments, the NK cells are activated using irradiated K562 cells expressing 4-1BBL, IL-2, and membrane-bound IL-15. In some embodiments, the CRISPR protein is Cas9 or Cas12. In some embodiments, the NK cells do not contain a DNA polynucleotide encoding a CRISPR protein. In some embodiments, the NK cells are CD34 positive, CD3 negative, and LNGFR positive.

[0078] In some embodiments, the NK cells of the library are selected from the NK cells described herein. In some embodiments, at least two CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 10 CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 20 CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 50 CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, at least 100 CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, 135 CAR-NK cells of the library each comprise a different gRNA polynucleotide comprising a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, SEQ ID NOs: 136-1315 are each present in at least one NK cell.

[0079] In some aspects, the present application discloses a method of generating a library of mutant CAR-NK cells, the method comprising: (a) activating CAR-NK cells; (b) transducing the CAR-NK cells with a library of gRNA vectors described herein; (c) transducing the CAR-NK cells with a CAR vector described herein; and (d) introducing into the NK cells an mRNA encoding a CRISPR protein or a CRISPR protein.

[0080] In some embodiments, the present application is a method for creating a library of variant CAR-NK cells, comprising: (a) activating CAR-NK cells; (b) transducing the CAR-NK cells with the gRNA vector library described herein; and (c) transducing the CAR-NK cells with the CAR vector described herein, wherein the transducing comprises transducing the gRNA vector or the CAR vector that contains a polynucleotide encoding a CRISPR protein.

[0081] In some embodiments, the method according to the claims further comprises collecting CAR-NK cells from a subject. In some embodiments, the method further comprises purifying CAR-NK cells that contain a CAR vector and contain CRISPR-induced genetic mutations. In some embodiments, the CAR-NK cells are collected using leukapheresis. In some embodiments, the CAR-NK cells are collected from a human or mouse subject. In some embodiments, the CAR-NK cells are activated using anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the gRNA vector library is transduced at a multiplicity of infection (MOI) between 0.1 and 1. In some embodiments, the MOI is between 0.3 and 0.7. In some embodiments, the MOI is between 0.4 and 0.6. In some embodiments, the MOI is 0.5. In some embodiments, the CAR vector is transduced at an MOI of up to 20. In some embodiments, the CRISPR protein is Cas9 or Cas12.

[0082] In some embodiments, the mRNA of the CRISPR protein or the CRISPR protein is introduced using electroporation. In some embodiments, the CRISPR mRNA is expressed from the gRNA vector or the CAR vector. In some embodiments, the electroporation is performed 3 to 7 days after the transduction with the gRNA vector and the CAR vector.

[0083] In some embodiments, the method further comprises selecting NK cells that are CD3 negative and CD34 positive. In some embodiments, the method further comprises selecting NK cells that are CD3 negative, CD34 positive, and LNGFR positive.

[0084] In some embodiments, the selection is performed using magnetic beads and / or fluorescence-activated cell sorting. In some aspects, the present application discloses a mutant CAR-NK cell library created using the methods described herein.

[0085] In some aspects, the present application discloses a method for identifying mutations that enhance the efficacy of CAR-NK cells in vivo, the method comprising: (a) administering to a subject a mutant CAR-NK cell library described herein; (b) collecting from the subject one or more samples comprising a plurality of mutant CAR-NK cells of the mutant NK cell library; (c) sequencing the gRNA polynucleotides from the mutant CAR-NK cells collected in (b); and (d) evaluating changes in the relative abundance of each gRNA polynucleotide based on the sequencing in (c).

[0086] In some embodiments, the method further comprises: (e) identifying a gene associated with a gRNA polynucleotide having an increased relative abundance; (f) generating a modified CAR-T cell comprising a mutation in the gene identified in (e); and (g) administering the modified CAR-T cell to the subject.

[0087] In some embodiments, the mutant CAR-NK cell library comprises a genome-wide NK cell mutant library. In some embodiments, the library comprises negative control CAR-NK cells. In some embodiments, the negative control CAR-NK cells comprise a negative control gRNA polynucleotide described herein. In some embodiments, the subject is human.

[0088] In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, and B cell lymphoma. In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen expressed by the cancer.

[0089] In some embodiments, the CAR comprises an antigen-binding domain that binds to any pair of CD19 / CD79b, BCMA / TACI selected from the group consisting of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, or is TriPRIL. In some embodiments, the sample is collected from the blood of the subject. In some embodiments, at least 3 samples are collected from the subject. In some embodiments, at least 5 samples are collected by venipuncture, apheresis, or tumor resection. In some embodiments, the sample is collected from the subject on one or more of the 3rd, 7th, 10th, 14th, 21st, 1st month, 2nd month, 3rd month, 6th month, 9th month, 12th month, 15th month, 18th month, 21st month, and 24th month after administration of the mutant CAR-T cell library after step (a). In some embodiments, the sample is collected from the subject on the 21st day after administration of the mutant CAR-T cell library after step (a).

[0090] In some embodiments, the method further comprises extracting mutant NK cells from a sample prior to sequencing. In some embodiments, the extraction is performed by isolating NK cells that are CD3 negative, CD34 positive, and optionally LNGFR positive. In some embodiments, the method further comprises isolating genomic DNA from the mutant NK cells. In some embodiments, the method further comprises isolating gRNA polynucleotides from the mutant NK cells. In some embodiments, the method further comprises isolating RNA from the mutant NK cells and optionally sequencing the RNA. In some embodiments, the method further comprises amplifying the gRNA polynucleotides of the mutant NK cells to generate amplicons thereof. In some embodiments, the gRNA polynucleotides to be amplified are genomic DNA, vector DNA, or RNA. In some embodiments, the primers of SEQ ID NOs: 1324-1325 are used for amplification. In some embodiments, the gRNA polynucleotide or its amplicon is sequenced. In some embodiments, the gRNA polynucleotide or its amplicon is sequenced using next-generation sequencing. In some embodiments, the efficacy of each mutant NK cell is evaluated based on in vivo persistence and tumor cell cytotoxicity. In some embodiments, in vivo persistence and tumor cell cytotoxicity are measured using flow cytometry and PCR. In some embodiments, quantifying persistence is performed by calculating the relative change over time of each gRNA. In some embodiments, quantifying persistence is performed by calculating the relative change over time of each gRNA compared to the gRNA polynucleotide from negative control NK cells. In some embodiments, quantifying persistence is performed by calculating the relative change over time of each gRNA over at least three time points and calculating the slope of the relative change. In some embodiments, tumor cell cytotoxicity is quantified using clinical imaging and / or bone marrow evaluation.

[0091] The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present disclosure, and can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0093] A common challenge in cell therapy (e.g., CAR-T cell therapy) is to identify CAR-T cell modifications (e.g., genetic mutations) that improve the effectiveness of the cell therapy. This is difficult for several different reasons. First, although the effectiveness of CAR-T cells in vitro and in mice can provide information about their effectiveness in humans, CAR-T cells may have different effectiveness in humans than in vitro or in mice. Second, it may be difficult to predict how a particular mutation (e.g., a loss-of-function mutation) affects the effectiveness of CAR-T cells in humans, so many mutations may need to be tested to identify mutations that improve CAR-T cancer killing effectiveness. However, it is unrealistic to test many different modified CAR-T cells individually in humans. Third, different subjects may respond differently to different CAR-T cell modifications. For example, a first modified CAR-T cell may have higher effectiveness in a first subject compared to a second modified CAR-T cell, but may have lower effectiveness in a second subject. In some aspects, the present disclosure describes methods and compositions for addressing these challenges. For example, the present disclosure describes methods and compositions for screening a library of CAR-T cells modified in vivo (e.g., in humans) in a single experiment to identify CAR-T cell modifications that increase the effectiveness (e.g., in vivo persistence) of CAR-T cells.

[0094] General definition The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a statistically significant amount of decrease. In some embodiments, "reduce", "reduction", or "decrease" or "inhibit" typically means at least a 10% decrease as compared to a reference level (e.g., the absence of a given treatment or agent), for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 98%, at least about 99%, or more decrease may be included. "Complete inhibition" is 100% inhibition compared to the reference level. Where applicable, the decrease can preferably be reduced to a level that is acceptable as within the normal range for an individual without the given impairment. The terms "increased", "increase", "enhance", or "activate" are all used herein to mean a statistically significant amount of increase. In some embodiments, the terms "increased", "increase", "enhance", or "activate" mean an increase of at least 10% compared to the reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase, or any increase between 10% and 100% compared to the reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to the reference level, or any increase between 2-fold and 10-fold or more. In the context of a marker or symptom, "increase" means such a statistically significant increase in level.

[0095] "Disease" refers to the health state of an animal, such as a human, where the animal is unable to maintain homeostasis and, if the disease is not improved, the animal's health will continue to deteriorate. In contrast, an "impairment" of an animal means that the animal can maintain homeostasis, but the animal's health state is less favorable than it would be in the absence of the impairment. Leaving an impairment untreated does not necessarily cause further deterioration of the animal's health state. In some embodiments, the disease is cancer or a tumor.

[0096] As used herein, the terms "tumor antigen," "tumor-associated antigen," and "cancer antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and can thereby be utilized to target cancer cells. Cancer antigens are antigens that can potentially stimulate a tumor-specific immune response. Some of these antigens are not necessarily expressed, but are encoded by normal cells. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at specific stages of differentiation, and those that are transiently expressed such as embryonic or fetal antigens. Other cancer antigens are encoded by mutant cellular genes such as cancer genes (e.g., activated ras cancer gene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by, for example, viral genes contained in RNA and DNA tumor viruses. Many tumor antigens have been defined from the perspective of multiple solid tumors: MAGE1, 2, and 3 defined by immunity; MART-1 / melan A, gp100, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (HER2), mucin (i.e., MUC-1), prostate-specific antigen (PSA), and prostate acid phosphatase (PAP). Furthermore, viral proteins such as those encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papillomavirus (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively. In some embodiments, the tumor-associated antigen is any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, or any pair of CD19 / CD79b or BCMA / TACI.

[0097] As used herein, the term "chimeric" refers to a fusion product of portions of at least two or more different polynucleotide molecules. In some embodiments, the term "chimeric" refers to a gene expression element generated through the manipulation of known elements or other polynucleotide molecules.

[0098] In some embodiments, "activation" can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation can refer to induced cytokine production. In other embodiments, activation can refer to detectable effector function.

[0099] At a minimum, "activated T cells" as used herein are proliferative T cells.

[0100] As used herein, the terms "specific binding" and "binds specifically" refer to a physical interaction between two molecules, compounds, cells, and / or particles, where the first entity binds to the second target entity with a higher specificity and affinity than it binds to a third non-target entity. In some embodiments, specific binding can refer to the affinity of the first entity for the second target entity being at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more greater than its affinity for a third non-target entity under the same conditions. A reagent specific for a given target is a reagent that exhibits specific binding to that target under the conditions of the assay utilized. Non-limiting examples include antibodies or ligands that recognize and bind to cognate binding partner (e.g., stimulatory and / or co-stimulatory molecules present on T cells) proteins.

[0101] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (APC) (e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule" or "costimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0102] "Stimulatory molecule", as the term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. "Costimulatory ligand", as the term is used herein, means a molecule on an APC that specifically binds to a cognate costimulatory molecule on a T cell and thereby provides a signal that mediates a T cell response, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of the TCR / CD3 complex to, for example, a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, IL T3, IL T4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, e.g., ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0103] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates, without limitation, a costimulatory response by the T cell such as proliferation. Costimulatory molecules include, without limitation, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0104] In some embodiments, the term "engineered" as used herein and its grammatical equivalents can refer to one or more modifications of a nucleic acid, such as a nucleic acid within the genome of an organism, designed by a human. In another embodiment, engineered can refer to the modification, addition, and / or deletion of a gene. An "engineered cell" can refer to a cell in which a gene has been added, deleted, and / or modified.

[0105] The term "cell" or "engineered cell" as used herein and their grammatical equivalents can refer to cells derived from a human or non-human animal.

[0106] The term "operatively linked" as used herein refers to a situation where a first polynucleotide molecule, such as a promoter, is linked to a second transcribable polynucleotide molecule, such as a gene of interest, and the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single continuous polynucleotide molecule and may or may not be adjacent. For example, if a promoter regulates or mediates the transcription of a gene of interest in a cell, the promoter is operatively linked to the gene of interest.

[0107] In various embodiments described herein, it is further contemplated that any variant (naturally occurring or otherwise) of a particular polypeptide described, alleles, homologs, conservatively modified variants, and / or conservative substitution variants are included. With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that change a single amino acid or a small percentage of amino acids in the encoded sequence result in an amino acid substitution by a chemically similar amino acid and are "conservatively modified variants" that retain the desired activity of the polypeptide. Such conservatively modified variants are added to, and do not exclude, polymorphic variants, interspecies homologs, and alleles that are consistent with the disclosure.

[0108] A given amino acid can be replaced with residues having similar physicochemical properties, for example, one aliphatic residue can be replaced with another (e.g., Ile, Val, Leu, Ala with each other), or one polar residue can be replaced with another (e.g., between Lys and Arg, Glu and Asp, or Gln and Asn). Other such conservative substitutions, such as substitution of entire regions having similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, such as ligand-mediated receptor activity and the specificity of the native polypeptide or reference polypeptide, is retained. Amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73 - 75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, the naturally occurring residues can be grouped into classes based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging one member of these classes for another.Certain conservative substitutions include, for example, substitution of Ala with Gly or Ser; Arg with Lys; Asn with Gln or His; Asp with Glu; Cys with Ser; Gln with Asn; Glu with Asp; Gly with Ala or Pro; His with Asn or Gln; Ile with Leu or Val; Leu with Ile or Val; Lys with Arg, Gln, or Glu; Met with Leu, Tyr or Ile; Phe with Met, Leu or Tyr; Ser with Thr; Tyr with Ser; Trp with Tyr; Tyr with Trp; and / or Phe with Val, Ile or Leu.

[0109] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide, as known in the art or according to the assays described hereinbelow. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0110] In some embodiments, the polypeptides described herein can be variants of the polypeptides or molecules described herein. In some embodiments, the variant is a conservatively modified variant. Conservatively substituted variants can be obtained, for example, by mutation of a native nucleotide sequence. As used herein, a "variant" refers to a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes one or more additions, deletions, or substitutions of nucleotides as compared to the native or reference DNA sequence, but encodes a variant protein or fragment thereof that retains the activity of the non-variant polypeptide. A variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0111] Variant amino acid or DNA sequences can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the native or reference sequence. The degree of homology (percent identity) between the native and variant sequences can be determined, for example, by comparing the two sequences using freely available computer programs commonly utilized for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0112] Modification of the native amino acid sequence can be achieved by any of a number of techniques known to those of skill in the art. Mutations can be introduced, for example, at a particular locus by synthesizing an oligonucleotide containing a variant sequence adjacent to a restriction enzyme site that permits ligation of the fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertions, substitutions, deletions. Alternatively, an oligonucleotide-directed site-specific mutagenesis procedure can be used to obtain a modified nucleotide sequence having specific codons modified according to the required substitutions, deletions, or insertions. Techniques for performing such modifications are well established and include, for example, Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and those disclosed in U.S. Patent Nos. 4,518,584 and 4,737,462, which are hereby incorporated by reference in their entirety. Cysteine residues that are not involved in maintaining the appropriate conformation of the polypeptide can also generally be substituted with serine in order to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkages can be added to the polypeptide to improve its stability or promote oligomerization.

[0113] The term "polynucleotide" is used interchangeably herein with "nucleic acid molecule" to refer to a polymer of nucleosides. Typically, a polynucleotide consists of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) found naturally in DNA or RNA, which are linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides or nucleoside analogs that include chemically or biologically modified bases, modified backbones, etc., whether or not such molecules are found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to a polynucleotide, it is understood that both DNA and RNA, and in each case single-stranded and double-stranded forms (and the complementary strands of each single-stranded molecule) are provided. As used herein, the "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information that biochemically characterizes a particular nucleic acid (i.e., a series of letters used as abbreviations for bases). In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein, the term "heterologous nucleic acid molecule" refers to a nucleic acid molecule that is not naturally present in a given cell.

[0114] The polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated.

[0115] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. Polypeptides as used herein typically contain amino acids such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can also be used. One or more amino acids in a polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functional groups, etc. A polypeptide to which a non-polypeptide moiety is covalently or non-covalently attached is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis. As used herein, the term "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide substance itself and / or the sequence information that biochemically characterizes the polypeptide (i.e., a series of letters or three-letter codes used as abbreviations for amino acid names). The polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction unless otherwise specified.

[0116] The term "gene" can refer to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to an appropriate regulatory sequence. A gene may or may not include regions before and after the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons). In some embodiments, the nucleic acid encoding a polypeptide described herein (e.g., a CAR polypeptide) is included in a vector (e.g., a CAR vector). In some aspects described herein, the nucleic acid sequence encoding a given polypeptide described herein, or any module thereof, is operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when combined with appropriate control elements and can transfer a gene sequence into a cell. Vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like.

[0117] As used herein, the term "expression vector" can refer to a vector that directs the expression of RNA or a polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed will often, but not necessarily, be heterologous to the cell. An expression vector can contain additional elements. For example, an expression vector can have two replication systems, thereby allowing it to be maintained in two organisms, e.g., for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the production of RNA and proteins and the intracellular processes appropriately involved in protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0118] As used herein, the term "viral vector" can refer to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. A viral vector can contain a nucleic acid encoding a polypeptide described herein in place of a non-essential viral gene. The vector and / or particle can be utilized for the purpose of transferring the nucleic acid into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0119] A "recombinant vector" can be a vector that contains a heterologous nucleic acid sequence or "transgene" that is expressible in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means for maintaining the nucleotide of interest in high copy number extrachromosomal DNA in a subject, thereby eliminating the potential impact of integration into the chromosome.

[0120] As used herein, "signal peptide" or "signal sequence" means the N-terminal peptide of a newly synthesized protein that serves to direct the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 or IgK signal peptide.

[0121] As used herein, the term "clustered regularly interspaced short palindromic repeats" or "CRISPR" may refer to a gene editing system that includes a guide RNA component and a CRISPR-associated (Cas) protein component. The guide RNA polynucleotide may include a homology region that is complementary to a target gene and a stem-loop region that can bind to a Cas protein. The Cas protein may include a guide RNA binding site and nuclease activity. The Cas protein and the guide RNA can form a complex that can bind to the target gene (based on the homology region) and cleave DNA (using the nuclease activity of the Cas protein). In some embodiments, the Cas protein-guide RNA complex binds to a sequence adjacent to and downstream of a protospacer adjacent motif (PAM). Cleavage results in the cleavage of the DNA strand, and mutations (such as single nucleotide polymorphisms, insertions, or deletions) can be introduced by the repair of the strand break. In some embodiments, the Cas protein is any suitable Cas protein for mutating and / or modifying the expression of the target gene (the Cas protein may also be referred to herein as a CRISPR protein). In some embodiments, the Cas protein is selected from the group consisting of a Cas9 protein, a Cas12 protein, or a Cas13 protein. One of ordinary skill in the art will understand that a Cas protein (e.g., Cas9) can have many different orthologs (e.g., SpyoCas9, spCas9, spyCas9, and geoCas9). In some embodiments, the Cas protein is SpyoCas9. The Cas protein and its orthologs are well known in the art as discussed in Gasiunas, Giedrius, et al, Nature communications 11.1 (2020):1-10; and Fancheng Y et al., Cell Biology and Toxicology 35.6 (2019):489-492, each of which is incorporated by reference in its entirety.Methods for designing guide RNAs (e.g., selection of homologous region sequences for targeting specific genes) are also well known in the art, as described in Guanqing L. et al., Computational and Structural Biotechnology Journal 18(2020):35-44, which is hereby incorporated by reference in its entirety. In some embodiments, the gRNA (encoded by the gRNA polynucleotide) is designed using CRISPick (portals.broadinstitute.org / gppx / crispick / public) as described in Doench et al., Nature Biotechnology, 34(2), 184-191(2016) and Sanson et al., Nature Communications, 9(1), 5416(2018), both of which are hereby incorporated by reference in their entirety.

[0122] In some embodiments, the polypeptides, polynucleotides, plasmids and / or vectors described herein optionally further comprise a reporter molecule, e.g., to determine whether a vector is being properly expressed intracellularly. In some embodiments, the reporter molecule can be a fluorescent protein (e.g., GFP, YFP, RF), an antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), and a radioisotope. In some embodiments, the reporter molecule is hygromycin phosphotransferase (hph) that can be imaged alone or in combination with a substrate or chemical (e.g., 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

[0123] In some embodiments, GFP and mCherry can be used as fluorescent tags to image CARs expressed on T cells (e.g., CAR-T cells). It is contemplated that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR need not include a fluorescent tag or fluorescent protein. Thus, in each example of the specific constructs provided herein, any marker present in the construct can be removed. The invention includes constructs that include a marker or do not include a marker. Thus, when a specific construct is referred to herein, it can be considered to be included in the invention, regardless of the presence or absence of a marker or tag (e.g., a histidine tag such as the HHHHHH (SEQ ID NO: 1370) histidine tag).

[0124] The terms "statistically significant" or "significantly" refer to statistical significance and generally mean a difference of two standard deviations (2SD) or more.

[0125] Except in the Examples or where otherwise indicated, all numerical values representing the amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about".

[0126] The term "about" or "approximately" when used in connection with a value means that the value or the description in which the value is recited can encompass a range of plus or minus 1%, plus or minus 2%, plus or minus 3%, plus or minus 4%, plus or minus 5%, plus or minus 6%, plus or minus 7%, plus or minus 8%, plus or minus 9%, plus or minus 10%, plus or minus 1-5%, plus or minus 2-7%, plus or minus 3-8%, plus or minus 4-9%, or plus or minus 5-10% of that value.

[0127] The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0128] Other terms are defined in the description of the various aspects and embodiments of the technology as described herein.

[0129] Guide RNA polynucleotide library As used herein, the term "guide RNA (gRNA) polynucleotide" refers to a DNA or RNA polynucleotide encoding a guide RNA (gRNA). The guide RNA polynucleotide includes a sequence that binds to a clustered regularly interspaced short palindromic repeat (CRISPR) protein or a CRISPR-associated protein, and a sequence that is complementary to a target polynucleotide (i.e., a homologous region). For example, the guide RNA polynucleotide can be a Cas9 protein guide RNA polynucleotide or a Cas12 protein guide RNA polynucleotide. The Cas9 protein guide RNA is compatible with the Cas9 CRISPR protein and is well known in the art, as described, for example, by Adli et al., Nature communications 9.1 (2018): 1-13, which is incorporated herein by reference in its entirety. The Cas12 protein guide RNA polynucleotide is compatible with the Cas12 CRISPR protein and is well known in the art, as described, for example, by Zetsche et al., Cell 163.3 (2015): 759-771, which is incorporated herein by reference in its entirety. In some embodiments, the guide RNA polynucleotide is a base editing guide RNA polynucleotide. In some embodiments, the gRNA is a prime editing guide RNA polynucleotide. In some embodiments, the guide RNA polynucleotide encodes a homologous region (e.g., a spacer) and a region that binds to a CRISPR protein (e.g., a direct repeat). In some embodiments, the guide RNA polynucleotide is a single guide RNA polynucleotide that includes a homologous region and a region that binds to a CRISPR protein.

[0130] In some embodiments, the homologous region comprises a contiguous sequence of about 10 to 30 or about 15 to 25 nucleotides. In some embodiments, the homologous region comprises about 20 nucleotides. In some embodiments, the homologous region is complementary to a target gene (e.g., a gene related to immune cell function). In some embodiments, the gRNA is designed using an algorithm (e.g., CRISPick). CRISPick is described in Kim et al., Nat Biotechnology 36, 239-241 (2018); Doench et al., Nature Biotechnology, 34(2), 184-191 (2016); and Sanson et al., Nature Communications, 9(1), 5416 (2018), each of which is incorporated by reference in its entirety.

[0131] In some embodiments, the gRNA polynucleotide is non-cross-reactive or has minimal cross-reactivity. A cross-reactive gRNA polynucleotide refers to a guide RNA polynucleotide that contains a homologous region with sufficient complementarity to two or more target polynucleotides (e.g., gene sequences) such that the gRNA can induce CRISPR mutations in two or more target polynucleotides. Design algorithms can be used in the design of guide RNA polynucleotides to reduce the potential for cross-reactivity (e.g., the gRNA can be scored for on-target activity using Rule Set 3 (RS3) which includes sequence and target information and Chen2013 tracr (Chen, Baohui, et al. Cell 155.7 (2013): 1479-1491). The gRNA can also be scored for off-target activity using the Tier-agnostic 1 mismatch aggregated Cutting Frequency Determination (CFD) score. One of ordinary skill in the art will understand that gRNA polynucleotides designed with such algorithms may still have some degree of cross-reactivity, but the risk of cross-reactivity is expected to be reduced or can be specified at a threshold selected in the algorithm. In some embodiments, cross-reactivity is a function of complementarity. In some embodiments, a non-cross-reactive gRNA polynucleotide does not have more than 80% complementarity to more than one gene. In some embodiments, a non-cross-reactive gRNA polynucleotide does not have more than 85% complementarity to more than one gene. In some embodiments, a non-cross-reactive gRNA polynucleotide does not have more than 90% complementarity to more than one gene. In some embodiments, a non-cross-reactive gRNA polynucleotide does not have more than 95% complementarity to more than one gene.

[0132] As used herein, the term "complementary" refers to the degree of Watson-Crick base pairing between two polynucleotides. For example, two polynucleotides can be 90% complementary if 9 / 10 nucleotides of each polynucleotide form Watson-Crick base pairs. In some embodiments, complementary can mean that at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nucleotides of a first polynucleotide form Watson-Crick base pairs with a second polynucleotide. In some embodiments, the homologous region of a gRNA is complementary to a gene sequence if the homologous region can hybridize to the gene sequence and at least a threshold percentage (e.g., at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%)) of the nucleotides are Watson-Crick base pairs to the gene sequence. In some embodiments, the homologous region is complementary to a gene sequence if the homologous region can hybridize to the gene sequence, initiate cleavage of the gene sequence by a CRISPR protein, and at least a threshold percentage (e.g., at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%)) of the nucleotides are Watson-Crick base pairs to the gene sequence. In some embodiments, the homologous region is complementary to a target gene sequence if the nucleotides of the homologous region are 100% complementary to a continuous portion of the target gene sequence (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides of the target gene sequence, e.g., 20 consecutive nucleotides). In some embodiments, the homologous region is complementary to the sense strand of the target gene sequence. In some embodiments, the homologous region is complementary to the antisense strand of the target gene sequence.In some embodiments, the homologous region that is complementary to the gene encoded by the sequence (e.g., SEQ ID NO: 1) can refer to a homologous region that is complementary to either the sense strand of the gene sequence (e.g., SEQ ID NO: 1) or the antisense strand of the gene sequence (e.g., the reverse complement of SEQ ID NO: 1). For example, the homologous regions described herein may be complementary to any one of SEQ ID NOs: 1-135 (i.e., the sense strand), or may be the reverse complement of any one of SEQ ID NOs: 1-135 (i.e., the antisense strand).

[0133] In some embodiments, the homologous region is complementary to a region of the target gene sequence adjacent to the protospacer adjacent motif (PAM). In some embodiments, the homologous region is downstream of the protospacer adjacent motif (PAM) and is complementary to a region of the target gene sequence adjacent to the protospacer adjacent motif.

[0134] In some embodiments, the present disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two (e.g., at least three, at least five, at least ten, at least fifteen, at least twenty-five, at least fifty, at least seventy-five, at least one hundred, or at least one hundred and thirty-five) gRNA polynucleotides each comprise a homologous region that is complementary to the sense strand or the antisense strand of a different gene encoded by any one of the sequences of SEQ ID NOs: 1-135 or a variant thereof.

[0135] In some embodiments, the present disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two (e.g., at least three, at least five, at least ten, at least fifteen, at least twenty-five, at least fifty, at least seventy-five, at least one hundred, or at least one hundred and thirty-five) gRNA polynucleotides each comprise a homologous region that is complementary to the sense strand or the antisense strand of a different gene of any one of the sequences of SEQ ID NOs: 1-135 or a variant thereof.

[0136] In some embodiments, the present disclosure provides a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two (e.g., at least three, at least five, at least ten, at least fifteen, at least twenty-five, at least fifty, at least seventy-five, at least one hundred, or at least one hundred and thirty-five) of the gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof, or at least two (e.g., at least three, at least five, at least ten, at least fifteen, at least twenty-five, at least fifty, at least seventy-five, at least one hundred, or at least one hundred and thirty-five) of the gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence selected from the reverse complement of any one of SEQ ID NOs: 1-135.

[0137] In some embodiments, the present disclosure provides a composition comprising a plurality of guide RNA (gRNA) polynucleotides. In some embodiments, at least two of the gRNA polynucleotides each comprise a homologous region that is complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. For example, the plurality of guide RNA (gRNA) polynucleotides can comprise a first gRNA polynucleotide and a second gRNA polynucleotide, wherein the first gRNA polynucleotide and the second gRNA polynucleotide are each complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, each homologous region of at least two of the gRNA polynucleotides is complementary to the sense strand of a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, each homologous region of at least two of the gRNA polynucleotides is complementary to the antisense strand of a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.

[0138] In some embodiments, the composition comprises at least two gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least three gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least four gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least five gRNA polynucleotides. In some embodiments, the composition comprises at least six gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least seven gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least eight gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least nine gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least ten gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the composition comprises at least 11 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 12 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 13 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 14 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 15 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 16 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 17 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 18 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 19 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the composition comprises at least 20 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 21 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 22 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 23 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 24 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 25 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 26 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 27 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 28 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the composition comprises at least 29 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 30 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 40 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 50 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 60 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 70 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 80 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 90 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 100 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the composition comprises at least 110 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 120 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the composition comprises at least 130 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof.

[0139] In some embodiments, the plurality of guide RNA polynucleotides comprises at least two guide RNA polynucleotides having different sequences (e.g., at least two, at least five, at least twenty-five, at least fifty, at least one hundred, at least two hundred and fifty, at least five hundred, at least one thousand, at least two thousand, at least two thousand five hundred, at least five thousand, at least ten thousand, at least fifteen thousand, at least twenty thousand, at least twenty-five thousand, at least thirty thousand, at least fifty thousand, at least one hundred thousand, or at least one million guide RNA polynucleotides). In some embodiments, the plurality of gRNA polynucleotides comprises 1,000, 2,500, 5,000, 10,000, 15,000, or 20,000 or fewer polynucleotides.In some embodiments, the plurality of guide RNA polynucleotides comprise from 2 to 1000, from 2 to 2500, from 2 to 5000, from 2 to 10000, from 2 to 15000, from 2 to 20000, from 2 to 40000, from 2 to 50000, from 2 to 100000, from 5 to 1000, from 5 to 2500, from 5 to 5000, from 5 to 10000, from 5 to 15000, from 5 to 20000, from 5 to 40000, from 5 to 50000, from 5 to 100000, from 10 to 1000, from 10 to 2500, from 10 to 5000, from 10 to 10000, from 10 to 15000, from 10 to 20000, from 10 to 40000, from 10 to 50000, from 10 to 100000, from 20 to 1000, from 20 to 2500, from 20 to 5000, from 20 to 10000, from 20 to 15000, from 20 to 20000, from 20 to 40000, from 20 to 50000, from 20 to 100000, from 50 to 1000, from 50 to 2500, from 50 to 5000, from 50 to 10000, from 50 to 15000, from 50 to 20000, from 50 to 40000, from 50 to 50000, from 50 to 100000, from 100 to 1000, from 100 to 2500, from 100 to 5000, from 100 to 10000, from 100 to 15000, from 100 to 20000, from 100 to 40000, from 100 to 50000, from 100 to 100000, from 135 to 1000, from 135 to 2500, from 135 to 5000, from 135 to 10000, from 135 to 15000, from 135 to 20000, from 135 to 40000, from 135 to 50000, or from 135 to 100000 guide RNA polynucleotides having different sequences. In some embodiments, the different sequences are complementary to one or more of SEQ ID NOs: 1-135. In some embodiments, the plurality of guide RNA polynucleotides comprise 2 to 1300 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprise 2 to 1200 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprise 2 to 1100 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprise 2 to 1080 guide RNA polynucleotides having different sequences.In some embodiments, the plurality of guide RNA polynucleotides comprises 5 to 1300 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 5 to 1200 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 5 to 1100 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 5 to 1080 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 10 to 1300 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 10 to 1200 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 10 to 1100 guide RNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides comprises 10 to 1080 guide RNA polynucleotides having different sequences.

[0140] In some embodiments, the composition comprises at most 1000 (e.g., at most 2500, at most 5000, at most 7500, at most 10000, at most 15000, at most 20000, at most 25000, at most 30000, at most 35000, at most 40000, at most 45000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000 polynucleotides, at most 250,000, at most 500,000, at most 750,000 or at most 1,000,000) guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 1000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 2500 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 5000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 7500 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 10000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 15000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 20000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 25000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 30000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 35000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 40000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 45000 guide RNA polynucleotides having different sequences.In some embodiments, the composition comprises at most 50,000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 60,000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 70,000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 80,000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 90,000 guide RNA polynucleotides having different sequences. In some embodiments, the composition comprises at most 100,000 guide RNA polynucleotides having different sequences.In some embodiments, the composition comprises 2 to 1000, 2 to 2500, 2 to 5000, 2 to 1000, 2 to 15000, 2 to 20000, 2 to 40000, 2 to 50000, 2 to 100000, 2 to 1000000, 5 to 1000, 5 to 2500, 5 to 5000, 5 to 1000, 5 to 15000, 5 to 20000, 5 to 40000, 5 to 50000, 5 to 100000, 5 to 1000000, 10 to 1000, 10 to 2500, 10 to 5000, 10 to 1000, 10 to 15000, 10 to 20000, 10 to 40000, 10 to 50000, 10 to 100000, 10 to 1000000, 20 to 1000, 20 to 2500, 20 to 5000, 20 to 1000, 20 to 15000, 20 to 20000, 20 to 40000, 20 to 50000, 20 to 100000, 20 to 1000000, 50 to 1000, 50 to 2500, 50 to 5000, 50 to 10000, 50 to 15000, 50 to 20000, 50 to 40000, 50 to 50000, 50 to 100000, 50 to 1000000, 100 to 1000, 100 to 2500, 100 to 5000, 100 to 10000, 100 to 15000, 100 to 20000, 100 to 40000, 100 to 50000, 100 to 100000, 100 to 1000000, 135 to 1000, 135 to 2500, 135 to 5000, 135 to 10000, 135 to 15000, 135 to 20000, 135 to 40000, 135 to 50000, or 135 to 100000, 135 to 1000000, 1080 to 1000, 1080 to 2500, 1080 to 5000, 1080 to 10000, 1080 to 15000, 1080 to 20000, 1080 to 40000, 1080 to 50000, or 1080 to 100000, 1080 to 1000000 guide RNA polynucleotides having different sequences. One of ordinary skill in the art will understand that many copies of any one guide RNA polynucleotide may be present in the composition and that these copies have the same sequence.

[0141] In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 20,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 19,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 18,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 17,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 16,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 15,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 14,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 13,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 12,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 11,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 10,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 9,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 8,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 7,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 6,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 5,000 different gene sequences.In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 4,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 3,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 2,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 1,000 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 900 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 800 different gene sequences.

[0142] In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 700 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 600 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 500 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 400 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 300 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 200 different gene sequences. In some embodiments, the guide RNA polynucleotide of the composition is complementary to at most 135 different gene sequences.

[0143] In some embodiments, the guide RNA polynucleotide of the composition is 135 different complementary gene sequences (e.g., SEQ ID NOs: 1-135).

[0144] In some embodiments, the guide RNA of the composition does not include a guide RNA polynucleotide that is complementary to the sense or antisense strand of a gene encoding a tumor suppressor. Without being bound by theory, mutations (e.g., loss of function) of tumor suppressor genes in cells (e.g., CAR-T cells) can result in the cells becoming tumorigenic or cancerous. In some embodiments, this is undesirable in the guide RNA polynucleotide compositions described herein because these compositions can be used to generate cell therapies (e.g., CAR-T cell therapy) that are administered to a subject (e.g., a human subject having cancer). If a guide RNA polynucleotide library includes guide RNAs that are complementary to tumor suppressors, this can result in the generation of cells (e.g., CAR T cells) that can potentially give a subject cancer or a tumor. Genes encoding tumor suppressors are known in the art, for example, as described in Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Tumor Suppressor Genes (available from ncbi.nlm.nih.gov / books / NBK9894 / ).

[0145] In some embodiments, the compositions described herein (e.g., compositions comprising multiple gRNA polynucleotides) do not include one or more of p53 (e.g., Uniprot:P04637), Rb (e.g., Uniprot:P06400), PTEN (e.g., Uniprot:P60484), BRCA1 (e.g., Uniprot:P38398), and / or BRCA2 (e.g., Uniprot:P51587). In some embodiments, the compositions described herein (e.g., compositions comprising multiple gRNA polynucleotides) do not include one or more of P53 (e.g., Uniprot:P04637), PTEN (e.g., Uniprot:P60484), APC (e.g., Uniprot:P25054), P16INK4a (e.g., Uniprot:P42771), P15INK4b (e.g., Uniprot:P42772), Cadherin-1 (e.g., Uniprot:Q12864), RB1 (e.g., Uniprot:P06400), BRCA1 (e.g., Uniprot:P38398), Wilmstumor1 (e.g., Uniprot:P19544), STK11 (e.g., Uniprot:Q15831), Smad4 (e.g., Uniprot:Q13485), BRCA2 (e.g., Uniprot:P51587), CHEK2 (e.g., Uniprot:O96017), P14arf (e.g., Uniprot:Q8N726), P21 (e.g., Uniprot:Q9H633), P73 (e.g., Uniprot:O15350), PTCH1 (e.g., Uniprot:Q13635), and / or MSH2 (e.g., Uniprot:P43246).

[0146] In some embodiments, the guide RNA of the composition does not include a guide RNA polynucleotide that is complementary to the sense or antisense strand of a gene essential for T cell function. In some embodiments, the guide RNA of the composition does not include a guide RNA polynucleotide that is complementary to the sense or antisense strand of a gene encoding DAD1 (e.g., Uniprot:P61803), SUPT4H1 (e.g., Uniprot:P63272), ZNF626 (e.g., Uniprot:Q68DY1), RAC2 (e.g., Uniprot:P15153), MARS (e.g., Uniprot:P56192), NAA10 (e.g., Uniprot:P41227), ORAOV1 (e.g., Uniprot:Q8WV07), TRMT112 (e.g., Uniprot:Q9UI30), RPP21 (e.g., Uniprot:Q9H633), VHL (e.g., Uniprot:P40337), NOP14 (e.g., Uniprot:P78316), OGT (e.g., Uniprot:O15294), UTP3 (e.g., Uniprot:Q9NQZ2), VARS (e.g., Uniprot:P26640), CCND3 (e.g., Uniprot:P30281), TAF6 (e.g., Uniprot:P49848), CDK6 (e.g., Uniprot:Q00534), CD247 (e.g., Uniprot:P20963), LCP2 (e.g., Uniprot:Q13094), EXOSC6 (e.g., Uniprot:Q5RKV6), RHOH (e.g., Uniprot:Q15669), VAV1 (e.g., Uniprot:P15498), MYC (e.g., Uniprot:P01106), POLR3H (e.g., Uniprot:Q9Y535), LAT (e.g., Uniprot:O43561), CD3D (e.g., Uniprot:P04234), WDR18 (e.g., Uniprot:Q9BV38), POLR2L (e.g., Uniprot:P62875), TMX1 (e.g., Uniprot:Q9H3N1), PRF1 (e.g., Uniprot:Q42449), STX11 (e.g., Uniprot:O75558), or STXBP2 (e.g., Uniprot:Q15833), or one or more of these variants.

[0147] In some embodiments, the variant refers to a homolog, ortholog, or paralog of a gene (e.g., a gene encoded by any one of SEQ ID NOs: 1 to 135). In some embodiments, variants of the same gene are different alleles. In some embodiments, the variant refers to an amino acid sequence that is at least 60% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 65% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 75% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 99% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 99.5% identical to any one of SEQ ID NOs: 1 to 135. In some embodiments, the variant refers to an amino acid sequence that is at least 99.9% identical to any one of SEQ ID NOs: 1 to 135.

[0148] In some embodiments, the composition comprises at least X gRNA polynucleotides per gene sequence, and the at least X gRNA polynucleotides comprise different sequences complementary to a given gene. For example, in some embodiments, the composition comprises at least 2 gRNA polynucleotides per gene sequence, and the at least 2 gRNA polynucleotides comprise different sequences complementary to a given gene (e.g., the sense or antisense strand of the gene). Thus, in such embodiments, if the composition comprises gRNA polynucleotides that contain homologous regions complementary to Y different genes (e.g., any one of Y different SEQ ID NOs), the composition comprises at least 2Y gRNA polynucleotides, at least 2 of the gRNA polynucleotides contain homologous regions complementary to each of the Y genes, and at least 2 of those gRNA polynucleotides comprise different sequences complementary to a given gene. In some embodiments, the at least X gRNA polynucleotides comprise homologous regions complementary to different regions of the same gene sequence (e.g., different exons or introns, or different regions encoding different domains of a protein encoded by the gene). In some embodiments, the at least X gRNA polynucleotides comprise homologous regions complementary to overlapping regions of the same gene sequence. In some embodiments, the composition comprises at least 2 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences (e.g., SEQ ID NOs: 1-135) complementary to the gene sequence.

[0149] In some embodiments, the composition comprises at least 2 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 3 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 4 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 5 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 6 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 7 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 8 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 9 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135).In some embodiments, the composition comprises at least 10 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 12 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 15 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 18 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135). In some embodiments, the composition comprises at least 20 gRNA polynucleotides per gene sequence, and the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1-135, or the reverse complement of any one of SEQ ID NOs: 1-135).

[0150] In some embodiments, the composition comprises from 1 to 20, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 5, 4 to 20, 4 to 15, 4 to 10, 4 to 8, 3 to 15, 3 to 10, 3 to 8, 3 to 6, 4 to 20, 4 to 15, 4 to 10, 4 to 8, 4 to 6, 5 to 20, 5 to 15, 5 to 10, 5 to 8, 6 to 20, 6 to 15, 6 to 10, 6 to 8, 7 to 20, 7 to 15, 7 to 10, or 7 to 8 polynucleotides per gene sequence, and each gRNA polynucleotide comprises a different sequence complementary to the gene sequence or the reverse complement of the gene sequence. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more gRNA polynucleotides per gene sequence, and each gRNA polynucleotide comprises a different sequence complementary to the gene sequence or the reverse complement of the gene sequence. In some embodiments, the composition comprises 8 gRNA polynucleotides per gene sequence, and each gRNA polynucleotide comprises a different sequence complementary to the gene sequence or the reverse complement of the gene sequence. In some embodiments, the plurality of gRNA polynucleotides (e.g., 8) are complementary to different regions of the same gene sequence or the reverse complement of the gene sequence.

[0151] In some embodiments, the composition comprises one or more negative control guide RNA polynucleotides. In some embodiments, the negative control guide RNA polynucleotide is a guide RNA polynucleotide that is not expected to have an effect on the efficacy of the CAR-T cells. In some embodiments, the negative control guide RNA polynucleotide is a guide RNA polynucleotide whose homology region is not complementary to a gene in the genome of an immune cell (e.g., a CAR-T cell). In some embodiments, the negative control gRNA comprises a homology region that is a complementary intergenic region. In some embodiments, the negative control gRNA polynucleotide comprises a randomly generated homology region. In some embodiments, the negative control guide RNA sequence comprises any one of the nucleotide sequences of SEQ ID NOs: 1216 to 1315.

[0152] In some embodiments, the composition comprises at least 1 gRNA polynucleotide of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 2 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 3 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 4 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 5 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 6 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 7 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 8 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 9 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 10 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 11 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 12 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 13 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof.In some embodiments, the composition comprises at least 14 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 15 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 16 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 17 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 18 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 19 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 20 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 21 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 22 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 23 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 24 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 25 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 26 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof.In some embodiments, the composition comprises at least 27 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 28 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 29 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 30 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 40 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 50 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 60 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof.

[0153] In some embodiments, the composition comprises at least 70 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 80 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 90 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 100 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 200 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 300 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 400 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof.

[0154] In some embodiments, the composition comprises at least 500 gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least 1000 gRNA polynucleotides selected from the group consisting of SEQ ID NOs: 136 to 1215 or any variant thereof.

[0155] In some embodiments, the composition comprises a gRNA polynucleotide comprising the sequence of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises a gRNA polynucleotide comprising the sequence of SEQ ID NOs: 136 to 1215. In some embodiments, the composition comprises a gRNA polynucleotide comprising any one of SEQ ID NOs: 1216 to 1315. In some embodiments, the composition comprises a gRNA polynucleotide comprising SEQ ID NOs: 1216 to 1315.

[0156] In some aspects, the present application discloses guide RNA polynucleotides. In some embodiments, the guide RNA polynucleotide is any one of the guide RNA polynucleotides of any of the compositions described herein. In some embodiments, the guide RNA polynucleotide comprises any one of the sequences of SEQ ID NOs: 136 to 1315, or a variant of any of them.

[0157] Plasmids, vectors, and libraries thereof Plasmid In some aspects, the present application discloses a plasmid comprising any one of the gRNA polynucleotides disclosed herein. As used herein, "plasmid" may refer to a circular portion of DNA that contains sequence elements for the replication and expression of a polynucleotide (e.g., gRNA). In some cases, the replication element is an origin of replication (e.g., a bacterial origin of replication). In some embodiments, the expression sequence elements include a promoter and a terminator. In some embodiments, the plasmid comprises a selectable marker (e.g., an antibiotic marker).

[0158] The "plasmid library" described herein may include at least two plasmids described herein that encode different gRNA polynucleotides. In some embodiments, the plasmid library comprises at least 3 (e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, at least 15000, at least 20000, at least 30000, at least 40000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 1000000) plasmids that encode different gRNA polynucleotides (e.g., gRNA polynucleotides disclosed herein or variants thereof).

[0159] gRNA vector In some embodiments, the present application discloses a gRNA vector comprising (1) a first gRNA polynucleotide comprising a homologous region complementary to a marker protein (e.g., any one of the TCR / CD3 complexes of T cells (SEQ ID NOs: 1383 to 1389)), and (2) a second gRNA polynucleotide.

[0160] In some embodiments, the first gRNA polynucleotide comprises the homologous region of SEQ ID NO: 1316. In some embodiments, the first guide RNA polynucleotide is used as a marker of CRISPR activity based on the loss of TCR / CD3 expression on immune cells (e.g., T cells) when transfected into the immune cells. In some embodiments, the first gRNA polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is a tissue-specific promoter (e.g., an immune cell-specific promoter). In some embodiments, the promoter is an inducible promoter (e.g., the tet promoter or the lac promoter). In some embodiments, the promoter is selected from the group consisting of the CMV promoter, the EF1a promoter, the CAG promoter, the PGK promoter, the H1 promoter, or the U6 promoter. In some embodiments, the promoter is the U6 promoter.

[0161] In some embodiments, the second gRNA polynucleotide of the gRNA vector comprises any one of the guide RNA polynucleotides described herein. In some embodiments, the second gRNA polynucleotide comprises a sequence complementary to the sense or antisense sequence of a gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the second gRNA polynucleotide comprises any one of the sequences of SEQ ID NOs: 136-1315, or a variant thereof. In some embodiments, the second gRNA polynucleotide comprises any one of the sequences of SEQ ID NOs: 136-1315. In some embodiments, the second gRNA polynucleotide comprises a negative control gRNA sequence described herein. In some embodiments, the second gRNA polynucleotide comprises any one of the sequences of SEQ ID NOs: 1216-1315, or a variant thereof. In some embodiments, the second gRNA polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is a tissue-specific promoter (e.g., an immune cell-specific promoter). In some embodiments, the promoter is an inducible promoter (e.g., a tet promoter or a lac promoter). In some embodiments, the promoter is selected from the group consisting of a CMV promoter, an EF1a promoter, a CAG promoter, a PGK promoter, an H1 promoter, or a U6 promoter. In some embodiments, the U6 promoter is derived from a non-human species. In some embodiments, the U6 promoter is derived from a human U6 promoter. In some embodiments, the U6 promoter is derived from bovine, mouse, rat, porcine, yeast, canine, feline, Drosophila, or nematode. In some embodiments, the promoter is an H1 promoter. In some embodiments, the first gRNA polynucleotide is operably linked to a first promoter, and the second gRNA polynucleotide is operably linked to a second promoter.In some embodiments, the first and second promoters are the same promoter (i.e., two different copies of the same promoter). In some embodiments, the first and second promoters are different promoters (i.e., contain different sequences). In some embodiments, the first promoter is constitutive and the second promoter is inducible or tissue-specific. In some embodiments, both the first and second promoters are constitutive and tissue-specific. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are operably linked to a single promoter (e.g., transcribed as a single polynucleotide and, for example, cleaved after transcription).

[0162] In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in reverse orientation within the guide RNA vector. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in tandem orientation within the guide RNA vector. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are encoded within the same CRISPR array. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are single guide RNAs.

[0163] In some embodiments, the gRNA vector comprises a selectable marker. A "selectable marker" can refer to a molecule that enables the detection of a biological event. For example, the selectable marker can provide evidence that a cell is expressing the gRNA vector. In some embodiments, the selectable marker is a guide RNA (e.g., the first guide RNA described above) that can knock out a gene (e.g., a cell surface gene), and the gene knockout can be used as a selectable marker. In some embodiments, the selectable marker is a fluorescent marker. In some embodiments, the selectable marker provides resistance to a toxin (e.g., geneticin, hygromycin B, puromycin, or zeocin). In some embodiments, the selectable marker is suitable for use in humans. In some embodiments, the selectable marker is the expression of a non-endogenous cell surface protein. In some embodiments, the selectable marker is selected from the group consisting of LGNFR (nerve growth factor receptor), EGFR, CD19, CD20, CD34, and cleavage forms thereof. In some embodiments, the selectable marker is LNGFR. In some embodiments, the selectable marker is operably linked to a promoter (e.g., a promoter described herein). In some embodiments, the selectable marker is operably linked to a weak promoter (e.g., PGK or UBC). In some embodiments, the selectable marker is operably linked to a tissue-specific promoter (e.g., the HP1, CD14, CD43, CD45, C68, elastase, endoglin, fibronectin, Flt, GFAP, GPIIb, ICAM-2, mIFN-β, Mb, NphsI, OG-2, SP-B, SYN1, or WASP gene promoter). In some embodiments, the selectable marker is selected against CRISPR activity. In some embodiments, the selectable marker is based on negative selection (e.g., knockout of a target protein). In some embodiments, the selectable marker is a knockout of CD3.

[0164] In some embodiments, the gRNA vector contains a suicide gene. A "suicide gene" refers to a gene that is toxic to a host cell (e.g., a cell that transcribes or translates the suicide gene) when transcribed or translated. In some embodiments, the suicide gene is operably linked to a promoter (e.g., a promoter described herein). In some embodiments, the suicide gene is operably linked to an inducible promoter. In some embodiments, the suicide gene is operably linked to a weak promoter (e.g., the PGK promoter). In some embodiments, the suicide gene is selected from the group consisting of icaspase9, tEGFR, tCD29, CD20, and tHer2. In some embodiments, the Cas protein is icaspase9.

[0165] In some embodiments, the gRNA vector is a viral vector as described above. In some embodiments, the gRNA vector is selected from the group consisting of an adeno-associated vector, a retroviral vector, or a lentiviral vector. In some embodiments, the lentiviral vector is a third-generation self-inactivating (SIN) lentiviral vector.

[0166] In some embodiments, the gRNA vector contains the sequence of SEQ ID NO: 1318 and the sequence of the guide RNA polynucleotide described herein. In some embodiments, the gRNA vector contains the sequence of SEQ ID NO: 1318 and the sequence of the guide RNA polynucleotide homologous region of any one of SEQ ID NOs: 136 to 1215 or a variant thereof. In some embodiments, the gRNA vector contains the sequence of SEQ ID NO: 1318 and the sequence of the guide RNA polynucleotide homologous region of any one of SEQ ID NOs: 136 to 1315 or a variant thereof. In some embodiments, the gRNA vector further contains a polynucleotide encoding a CRISPR protein (e.g., Cas9 or Cas12) as described above.

[0167] gRNA vector library The "gRNA vector library" refers to a composition comprising at least two gRNA vectors encoding different gRNA polynucleotides as described herein. In some embodiments, the gRNA vector library comprises at least three gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least four gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least five gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least six gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least seven gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least eight gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least nine gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least ten gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least eleven gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least twelve gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least thirteen gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least fourteen gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least fifteen gRNA vectors encoding different gRNA polynucleotides.In some embodiments, the gRNA vector library comprises at least 16 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 17 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 18 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 19 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 20 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 21 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 22 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 23 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 24 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 25 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 26 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 27 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 28 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 29 gRNA vectors encoding different gRNA polynucleotides.In some embodiments, the gRNA vector library comprises at least 30 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 40 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 50 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 60 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 70 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 80 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 90 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 100 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 200 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 300 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 400 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 500 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 1000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 5000 gRNA vectors encoding different gRNA polynucleotides.In some embodiments, the gRNA vector library comprises at least 10,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 15,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 20,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 30,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 40,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 50,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 60,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 70,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 80,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 90,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 100,000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 1,000,000 gRNA vectors encoding different gRNA polynucleotides.

[0168] In some embodiments, the gRNA vector library comprises at least two gRNAs, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least three gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least four gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least five gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least six gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least seven gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 8 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 9 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 10 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 11 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 12 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 13 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 14 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 15 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 16 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 17 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 18 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 19 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 20 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 21 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 22 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 23 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 24 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 25 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 26 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 27 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 28 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 29 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 30 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 40 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 50 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 60 gRNA vectors, each of which is SEQ ID NOs: 1-135. Comprising at least 60 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 70 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 80 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 90 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 100 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 200 gRNA vectors, each containing a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.In some embodiments, the gRNA vector library comprises at least 300 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 400 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 500 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 1000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 5000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 10000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0169] In some embodiments, the gRNA vector library comprises at least 15,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 15,000 gRNA vectors. In some embodiments, the gRNA vector library comprises at least 20,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 20,000 gRNA vectors. In some embodiments, the gRNA vector library comprises at least 30,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 30,000 gRNA vectors. In some embodiments, the gRNA vector library comprises at least 40,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 40,000 gRNA vectors. In some embodiments, the gRNA vector library comprises at least 50,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 50,000 gRNA vectors. In some embodiments, the gRNA vector library comprises at least 60,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof, and comprises at least 60,000 gRNA vectors.In some embodiments, the gRNA vector library comprises at least 70,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 80,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 90,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 100,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the gRNA vector library comprises at least 1,000,000 gRNA vectors, each comprising a different gRNA polynucleotide homology region that is complementary to the sense or antisense strand of a different gene encoded by any one of SEQ ID NOs: 1-135 or a variant thereof.

[0170] In some embodiments, the gRNA vector library comprises 135 gRNA vectors, each of the gRNA vectors comprising a first guide RNA polynucleotide targeting a marker protein (e.g., CD3), and a second guide RNA comprising a homologous region complementary to the sense or antisense strand of a gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof. In some embodiments, the gRNA vector library comprises 1080 gRNA vectors, each of the gRNA vectors comprising a first guide RNA polynucleotide targeting a marker protein (e.g., CD3), and a second guide RNA comprising a homologous region complementary to the sense or antisense strand of a gene encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0171] In some embodiments, the gRNA vector library comprises gRNA vectors comprising a gRNA polynucleotide homolog region complementary to at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120 or at least 135) gene sequences of any one of SEQ ID NOs: 1 to 135.

[0172] In some embodiments, the gRNA vector library comprises any one of the guide RNA polynucleotides of any of the compositions described herein. In some embodiments, the gRNA vector library comprises a guide RNA polynucleotide comprising any one of SEQ ID NOs: 136 to 1215 or a variant thereof. In some embodiments, the gRNA vector library comprises a guide RNA polynucleotide comprising any one of SEQ ID NOs: 136 to 1315 or a variant thereof.

[0173] CAR vector In some embodiments, the present application provides a vector comprising a sequence encoding a chimeric antigen receptor (CAR).

[0174] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" or "CAR" or "CAR" refers to an engineered T cell receptor that confers ligand or antigen specificity on a T cell (e.g., naive T cell, central memory T cell, effector memory T cell, or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors.

[0175] A CAR places a chimeric antigen-binding domain that specifically binds to a target, e.g., a polypeptide, expressed on the surface of a cell that is the target of a T cell response, on a construct comprising a transmembrane domain and an intracellular domain of a T cell receptor molecule. In some embodiments, the chimeric antigen-binding domain comprises an antigen domain of an antibody reagent that specifically binds to an antigen expressed on a cell that is the target of a T cell response. In some embodiments, the chimeric antigen-binding domain comprises a ligand that specifically binds to an antigen expressed on a cell that is the target of a T cell response.

[0176] As used herein, "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, the CAR has the ability to utilize the antigen-binding properties of a monoclonal antibody to redirect the specificity and reactivity of the T cell, in an MHC-unrestricted manner, towards a selected target. MHC-unrestricted antigen recognition confers upon CAR-expressing T cells the ability to recognize antigens independently of antigen processing, bypassing a major mechanism of tumor escape.

[0177] As used herein, "CAR-NK cell" or "CAR-NK" refers to a natural killer (NK) cell that expresses a CAR.

[0178] In some embodiments, the CAR excludes the CD8 signal peptide described herein. As can be determined by one of ordinary skill in the art, the various functionally similar or equivalent components of these CARs can be exchanged or substituted for one another, as are known in the art or are other similar or functionally equivalent components listed herein.

[0179] Any cell surface moiety can potentially be targeted by a CAR. In many cases, the target is a cell surface polypeptide that can be differentially or preferentially expressed on the cells that one wishes to target with a T cell response. In some embodiments, the extracellular target binding domain binds to any one of CD19, CD37, CD70, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain, e.g., as described in PCT / US2020 / 065733, PCT / US2020 / 036108, PCT / US2018 / 013215, PCT / US2018 / 013213, PCT / US2018 / 027783, PCT / US2018 / 013221, PCT / US2018 / 022974, PCT / US2019 / 042268, PCT / US2019 / 038518, PCT / US2019 / 066357, PCT / US2019 / 013103, PCT / US2019 / 017727, PCT / US2020 / 051018, and / or PCT / US2018 / 013095.

[0180] Antigen binding domain As used herein, the term "antigen-binding domain" refers to a polypeptide found outside the cell that is sufficient to promote binding to a target. The extracellular target-binding domain specifically binds to its binding partner, i.e., the target. By way of non-limiting example, the antigen-binding domain can include the antigen domain of an antibody or antibody reagent that recognizes and binds a cognate binding partner protein, or a ligand. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. The cognate binding partners of ligands useful in the methods and compositions described herein can generally be found on the surface of cells. Ligand:cognate partner binding can result in a change in the receptor having the ligand, or can activate a physiological response, such as activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a function conserved across at least two species.

[0181] Any cell surface moiety can be targeted by a CAR. In some embodiments, the target is a cell surface polypeptide that can be differentially or preferentially expressed on the cells that one wishes to target for a T cell response. To target Tregs, an antibody reagent can target, for example, the repetitive dominant glycoprotein A (GARP), latent associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB, and LAG-3.

[0182] In some embodiments, the CAR vector comprises a CAR polynucleotide encoding an antigen-binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen-binding domain.

[0183] In some embodiments, the CAR vector comprises a CAR comprising an antigen-binding domain that binds to mesothelin. In some embodiments, the mesothelin CAR comprises a polynucleotide encoding an extracellular binding domain comprising a mesothelin antibody (e.g., scFv). In some embodiments, the mesothelin scFv comprises the VH domain of SEQ ID NO: 1366 and the VL domain of SEQ ID NO: 1367, or variants thereof. In some embodiments, the mesothelin scFv comprises SEQ ID NO: 1368 or SEQ ID NO: 1369, or variants thereof. TIFF2025518033000002.tif156170

[0184] Hinge and transmembrane domains In some embodiments, the CAR polypeptide further comprises a transmembrane domain, e.g., a hinge / transmembrane domain, that binds the antigen-binding domain to an intracellular signaling domain. In some embodiments, following the binding domain of the CAR, one or more "hinge domains" follow, which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. The CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8 (e.g., CD8 alpha), CD4, CD28, 4-1BB, and CD7, which may be the wild-type hinge regions of these molecules or may be modified. In some embodiments, the CAR comprises a polynucleotide encoding the CD8 alpha hinge / transmembrane domain. In some embodiments, the CAR comprises a polynucleotide encoding the 41BB intracellular domain.

[0185] In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain comprises the CD8a hinge region.

[0186] As used herein, a "transmembrane domain" (TM domain) refers to the portion of a CAR that fuses an extracellular binding portion, in some embodiments via a hinge domain, to an intracellular portion (e.g., a costimulatory domain and an intracellular signaling domain), thereby anchoring the CAR to the plasma membrane of an immune effector cell. The transmembrane domain is generally the hydrophobic region of the CAR that traverses the plasma membrane of the cell. The TM domain can be a transmembrane region or a fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Specific examples are provided herein and used herein, but other transmembrane domains will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the technology. It is preferred that the selected transmembrane region or fragment thereof does not interfere with the intended function of the CAR.

[0187] When used in connection with the transmembrane domain of a protein or polypeptide, "fragment thereof" means a portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0188] In some embodiments, the transmembrane domain of the CAR described herein, or a fragment thereof, comprises a transmembrane domain selected from the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0189] As used herein, "hinge / transmembrane domain" refers to a domain that includes both a hinge domain and a transmembrane domain. For example, the hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of a CAR or a fragment thereof is derived from or includes the hinge / transmembrane domain of CD8 (e.g., any one of SEQ ID NO: 1 or its variants). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and functions as a T cell coreceptor. CD8 consists of alpha (CD8 alpha or CD8a) and beta (CD8 beta or CD8b) chains. CD8a sequences are known for many species, such as the human CD8a (NCBI Gene ID 925) polypeptide (e.g., NCBI Reference Sequence NP_001139345.1) and mRNA (e.g., NCBI Reference Sequence NM_000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any aspect, for example, in veterinary applications, CD8 can refer to CD8 of, for example, dogs, cats, cows, horses, pigs, etc.

[0190] Homologs and / or orthologs of human CD8 can be readily identified by one of ordinary skill in the art for such species, for example, by using the NCBI ortholog search function or by searching available sequence data for a given species for sequences similar to the reference CD8 sequence.

[0191] In some embodiments, the CD8 hinge and transmembrane sequence correspond to the amino acid sequence of SEQ ID NO: 1371 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC); or comprise the sequence of SEQ ID NO: 1; or comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 1371.

[0192] Co-stimulatory domain Each CAR described herein optionally includes the intracellular domain or co-stimulatory domain of one or more co-stimulatory molecules. As used herein, the term "co-stimulatory domain" refers to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4-1BB, CD27, CD28, or OX40. In one example, the 4-1BB intracellular domain (ICD) can be used (see, for example, below and SEQ ID NO: 1372 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or variants thereof). Additional exemplary examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2CSLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is a co-stimulatory molecule induced by activation and is an important regulator of the immune response.

[0193] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. 4-1BB sequences are known for many species, for example, human 4-1BB, which is also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI reference sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any aspect, for example, in veterinary applications, 4-1BB can refer to 4-1BB of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human 4-1BB can be readily identified by those skilled in the art for such species, for example, using the NCBI ortholog search function or by searching available sequence data for a given species for sequences similar to the reference 4-1BB sequence.

[0194] Intracellular signaling domain In some embodiments, the CAR comprises a polynucleotide encoding a CD3-zeta intracellular signaling domain.

[0195] The properties of the intracellular signaling domain of the CAR can vary, as known in the art and as disclosed herein, but when the chimeric target / antigen-binding domain binds to the target / antigen on the surface of the target cell, the chimeric target / antigen-binding domain renders the receptor sensitive to signal transduction activation.

[0196] Regarding intracellular signaling domains, so-called "first-generation" CARs include those that provide only the CD3-zeta signal upon antigen binding. So-called "second-generation" CARs include those that provide both a co-stimulatory (e.g., CD28 or CD137) domain and an activation (CD3-zeta) domain, and so-called "third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3-zeta). In various embodiments, the CAR is selected to have a high affinity or avidity for the target / antigen - for example, the target or antigen-binding domain derived from an antibody generally has a higher affinity and / or avidity for the target antigen than the naturally occurring T cell receptor. This property, combined with the high specificity that can be selected for the antibody, results in highly specific T cell targeting by CAR-T cells.

[0197] The CARs described herein include an intracellular signaling domain. An "intracellular signaling domain" means the portion of the CAR polypeptide that is involved in transmitting the message of effective CAR binding to the target antigen inside the immune effector cell to induce effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, such as the release of cytotoxic factors to the target cells bound by the CAR, or other cellular responses induced after antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is derived from CD3-zeta (see, e.g., below). Further non-limiting examples of intracellular signaling domains that include immunoreceptor activation tyrosine motifs (ITAMs) particularly used in the art include those derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0198] CD3 is a T cell co-receptor that promotes the activation of T lymphocytes when engaged simultaneously with appropriate co-stimulation (e.g., binding of co-stimulatory molecules). The CD3 complex consists of four different chains; mammalian CD3 consists of a CD3-gamma chain, a CD3-delta chain, and two CD3-epsilon chains.

[0199] These chains associate with a molecule known as the T cell receptor (TCR) and CD3-zeta to generate activation signals in T lymphocytes. The complete TCR complex includes the TCR, CD3-zeta, and the complete CD3 complex.

[0200] In some embodiments of any aspect, the CAR polypeptides described herein include an immunoreceptor activation tyrosine motif or an intracellular signaling domain containing an ITAM derived from CD3-zeta, including variants of CD3-zeta such as, for example, ITAM mutant CD3-zeta, CD3-eta, or CD3-theta. In some embodiments of any aspect, the ITAM includes three motifs of the ITAM of CD3-zeta (ITAM3). In some embodiments of any aspect, the three motifs of the ITAM of CD3-zeta are not mutated and thus include a native or wild-type sequence. In some embodiments, the CD3-zeta sequence includes the sequence of CD3-zeta described in the sequences provided herein, for example, SEQ ID NO: 1373 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or a CD3-zeta sequence of variants thereof.

[0201] For example, the CAR polypeptides described herein include the intracellular signaling domain of CD3-zeta. In some embodiments, the CD3-zeta intracellular signaling domain corresponds to the amino acid sequence of SEQ ID NO: 1373; or includes the sequence of SEQ ID NO: 1373; or has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 1373.

[0202] In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. In some embodiments, the 4-1BB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO: 2; or includes a sequence selected from SEQ ID NO: 2; or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 2.

[0203] The individual CARs and other construct components described herein can be used and interchanged with one another in the various constructs described herein, as would be determined by one of ordinary skill in the art. Each of these components can include, or consist of, any of the corresponding sequences described herein, or variants thereof.

[0204] For a more detailed description of CARs and CAR-T cells, see Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012, each of which is incorporated herein by reference in its entirety.

[0205] Signal peptide In some embodiments, the CAR polypeptides described herein include a signal peptide. The signal peptide can have an extracellular domain or can be derived from any protein that is secreted. The CAR polypeptides described herein can include any signal peptide known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, e.g., corresponding to the amino acid sequence of SEQ ID NO: 1374 (MALPVTALLLPLALLLHAARP), or includes the amino acid sequence of SEQ ID NO: 5, or includes an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 1374, and includes a CD8 signal peptide.

[0206] In further embodiments, the CAR polypeptides described herein can optionally exclude one of the signal peptides described herein, e.g., the CD8 signal peptide of SEQ ID NO: 1374 or the IgK signal peptide of SEQ ID NO: 1375 (METDTLLLWVLLLWVPGSTGD).

[0207] Linker domain In some embodiments, the CAR further includes a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region about 2 to 100 amino acids in length that links any of the domains / regions of the CAR described herein. In some embodiments, the linker can include or consist of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Linker sequences useful in the present invention can be 2 to 100 amino acids in length, 5 to 50 amino acids in length, 10 to 15 amino acids in length, 15 to 20 amino acids in length, or 18 to 20 amino acids in length, and include any suitable linker known in the art. For example, linker sequences useful in the present invention include, but are not limited to, glycine / serine linkers such as GGGSGGGSGGGS (SEQ ID NO: 1376) and Gly4Ser (G4S) (SEQ ID NO: 1378) linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO: 1377)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 1378)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1379) described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, the contents of which are incorporated herein by reference in their entirety; the linker sequence of GGSSRSSSSGGGGSGGGG (SEQ ID NO: 1380) described by Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011(9), 2011, the contents of which are incorporated herein by reference in their entirety; and linker sequences with added functionality such as, for example, coding sequences or epitope tags containing Cre-Lox recombination sites described by Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000, the contents of which are incorporated herein by reference in their entirety. Longer linkers can be used if it is desired to prevent the two adjacent domains from sterically interfering with each other.

[0208] Furthermore, the linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 1381, GSGATNFSLLKQAGDVEENPGP) and T2A (SGGGGEGRGSLLTCGDVEENPGPR, SEQ ID NO: 1382), 2A-like linkers or functional equivalents thereof, and combinations thereof).

[0209] For example, the P2A linker sequence can correspond to the amino acid sequence of SEQ ID NO: 1381. In various examples, linkers having the sequences described herein, or variants thereof, are used. It should be understood that the designation of a particular linker in a construct at a particular position does not mean that only that linker can be used there. Rather, different linker sequences (e.g., P2A and T2A) can be exchanged with each other (e.g., in the context of the constructs of the present invention) as can be determined by one of ordinary skill in the art. In some embodiments, the linker region is T2A derived from Theea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. Such linkers can be used in the context of a polyprotein such as those described below. For example, they can be used to separate the CAR component of the polyprotein from the therapeutic agent component of the polyprotein (e.g., an antibody such as an scFv, a single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., TEAM)) (see below).

[0210] Full CAR In some embodiments, the CAR is selected from the group consisting of (1) a CAR that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and Claudin 6; (2) a CAR that binds to any pair of CD19 / CD79b or BCMA / TACI; or (3) a TriPRIL antigen-binding domain. In some embodiments, the CAR polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with any one of the sequences of SEQ ID NOs: 1325-1365. In some embodiments, the CAR comprises any one of the amino acid sequences of SEQ ID NOs: 1325-1365. In some embodiments, the CAR polypeptide consists of any one of the amino acid sequences of SEQ ID NOs: 1325-1365. In some embodiments, the CAR polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 1325-1365.

[0211] In some embodiments, the CAR comprises a polynucleotide encoding a mesothelin scFv, a CD8 alpha hinge / transmembrane, a 4-1BB intracellular domain, and a CD3 zeta signaling domain. In some embodiments, the CAR polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with any one of the sequences of SEQ ID NOs: 1325-1328. In some embodiments, the CAR comprises any one of the amino acid sequences of SEQ ID NOs: 1325-1328. In some embodiments, the CAR polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 1325-1328.

[0212] CAR vector In some embodiments, the gRNA vector is a viral vector as described above. In some embodiments, the CAR vector is an adeno-associated vector, a retroviral vector, or a lentiviral vector. In some embodiments, the CAR vector is a self-inactivating vector (self-inactivating lentiviral vector or self-inactivating retroviral vector). In some embodiments, the CAR vector is a third-generation self-inactivating (SIN) lentiviral vector. In some embodiments, the CAR is operably linked to a promoter. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is a chemically inducible promoter. In some embodiments, the promoter is a weak constitutively active promoter. In some embodiments, the promoter is a promoter described herein. In some embodiments, the promoter is EF1a or the EF1a short promoter.

[0213] In some embodiments, the CAR vector contains a suicide gene. A "suicide gene" can refer to a gene that results in cell death upon transcription or translation. Thus, a suicide gene can be included in the CAR vector to inactivate CAR therapy after treatment is complete or if there are harmful side effects in the subject. In some embodiments, the suicide gene is selected from the group consisting of icaspase9, tEGFR, tCD29, CD20, and tHer2. In some embodiments, the CAR vector contains a selectable marker (e.g., a reporter). In some embodiments, the selectable marker is selected from the group consisting of truncated CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2. In some embodiments, the selected marker is CD34 or LNGFR.

[0214] In some embodiments, the CAR vector contains a 2A ribosome skip element or an internal ribosome entry site (IRES) between the CAR polynucleotide sequence and the selectable marker sequence.

[0215] In some embodiments, the CAR vector further comprises a nucleotide sequence encoding a CRISPR protein described herein. In some embodiments, the CAR vector does not comprise a nucleotide sequence encoding a CRISPR protein.

[0216] In some embodiments, the CAR vector comprises a CAR selected from the group consisting of (1) a CAR that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, (2) a CAR that binds to any pair of CD19 / CD79b, BCMA / TACI, or (3) a TriPRIL antigen-binding domain.

[0217] In some embodiments, the CAR vector comprises a polynucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to SEQ ID NO: 1319. In some embodiments, the CAR vector comprises the polynucleotide sequence of SEQ ID NO: 1319.

[0218] CAR-T cells, NK cells and mutant library cell libraries In some aspects, the present disclosure is directed to immune cells (e.g., CAR-T cells or CAR-NK cells) that contain the guide RNA vectors described herein and / or contain mutations in a gene corresponding to any one of the gene sequences of SEQ ID NOs: 1-135. The present disclosure is, in part, directed to the idea of introducing one or more mutations into immune cells by contacting the immune cells with a guide RNA vector and providing a CRISPR protein (e.g., as part of the guide RNA vector or encoded by a separate vector). Expression of the guide RNA vector and the CRISPR protein can result in mutations in immune cells within or near a sequence complementary to the homologous region of the gRNA polynucleotide encoded by the gRNA vector. Accordingly, the present disclosure is directed to immune cells containing a guide RNA vector, immune cells containing mutations generated by the action of one or more gRNA polynucleotides encoded by the guide RNA vector and a CRISPR protein, and immune cells containing both of them. In some embodiments, the immune cells contain (i) a mutation in a gene corresponding to the guide RNA vector and / or any one of the gene sequences of SEQ ID NOs: 1-135, and (ii) a CAR vector described herein. In some embodiments, the immune cells contain one, two, or three of a mutation in a gene corresponding to any one of the gene sequences of SEQ ID NOs: 1-135 or a variant thereof, a gRNA polynucleotide described herein, a plasmid described herein, or a gRNA vector described herein. In some embodiments, the mammalian cells further contain a CAR vector described herein. In some embodiments, the mammalian cells further contain mRNA encoding a CAR and / or a polypeptide encoding a CAR.

[0219] In some embodiments, the immune cells comprise a mutation in a gene corresponding to any one of the gene sequences of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the mutation is the result of CRISPR cleavage of the gene. In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation is a single nucleotide polymorphism. In some embodiments, the mutation is a transversion. In some embodiments, the mutation is a translocation. In some embodiments, the mutation changes the protein sequence of the target gene. In some embodiments, the mutation introduces a premature stop codon. In some embodiments, the mutation is an insertion or deletion. In some embodiments, the immune cells comprise the gRNA polynucleotide described herein. In some embodiments, the immune cells comprise the gRNA vector described herein. In some embodiments, the immune cells further comprise the CAR vector described herein. In some embodiments, the immune cells comprise the CAR described herein.

[0220] In some embodiments, immune cells comprising the gRNA vector and the CAR vector further comprise a selected marker (e.g., a reporter described herein). In some embodiments, the immune cells are CD34 positive and CD3 negative. In some embodiments, the immune cells are CD34 positive, CD3 negative, and LNGFR positive.

[0221] The immune cells or tissues can be from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs or cats, and any other immune cells can be used. In some embodiments, the immune cells are human.

[0222] In some embodiments, the mammalian cells are immune cells. As used herein, "immune cells" refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include T cells and natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[0223] In some embodiments, the immune cells are obtained from an individual having or diagnosed with cancer, a plasma cell disorder, or an autoimmune disease. T cells can be obtained from a subject using standard techniques known in the art. For example, T cells can be isolated from peripheral blood collected from a donor or patient. T cells can be isolated from a mammal. Preferably, T cells are isolated from a human. In some embodiments, the immune cells are collected using leukapheresis.

[0224] In some embodiments, the immune cells are activated. In some embodiments, the immune cells are activated T cells comprising a gRNA vector and a CAR vector. Methods of activating T cells are well known in the art. In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD3 antibody. In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD28 antibody. In some embodiments, activating the T cells comprises contacting the T cells with interleukin-2 (IL-2). In some embodiments, activating the T cells comprises contacting the T cells with phytohemagglutinin (PHA). In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD28 antibody and an anti-CD3 antibody. In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD3 antibody and interleukin-2 (IL2). In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD3 antibody, interleukin-2 (IL2), and phytohemagglutinin (PHA). In some embodiments, any one of the anti-CD3 antibody, anti-CD28 antibody, interleukin-2 (IL-2), and / or phytohemagglutinin (PHA) is soluble when activating the T cells. In some embodiments, activating the T cells comprises contacting the T cells with an anti-CD3 antibody, interleukin-2 (IL2), and phytohemagglutinin (PHA). In some embodiments, any one of the anti-CD3 antibody, anti-CD28 antibody, interleukin-2 (IL-2), and / or phytohemagglutinin (PHA) is immobilized on a solid medium (e.g., a cell plate) when activating the T cells.

[0225] In some embodiments, the immune cells (e.g., T cells) do not contain a DNA sequence encoding a CRISPR protein (e.g., Cas9).

[0226] In some embodiments, a plurality of immune cells (e.g., T cells or NK cells) are transfected with (1) a CAR vector and (2) a gRNA library described herein to create a mutant CAR-T cell library. Methods of immune cell transfection are well known in the art, as described in Fus-Kujawa et al., "Frontiers in Bioengineering and Biotechnology (2021):634, which is incorporated by reference in its entirety.

[0227] One advantage of using separate vectors to encode the CAR and the gRNA polynucleotide is that the multiplicity of infection (MOI) of the CAR and the gRNA can be controlled separately. The multiplicity of infection can refer to how many of a particular vector enter the cell. For example, if the MOI is 1, it indicates that the average number of a particular vector per T cell in the mutant CAR-T cell library is 1. In some embodiments, the MOI can be a target MOI based on the amount of transfected vector. In some embodiments, the effectiveness of the CAR-T cells can depend on the amount of CAR expressed intracellularly. CAR expression can be controlled by increasing the number of CAR vectors transfected per cell (e.g., increasing the MOI). However, in some embodiments, one of ordinary skill in the art may want to increase the MOI of the CAR vector but not the MOI of the gRNA vector. This is because when multiple gRNA vectors are transfected per cell, multiple different mutations can occur within the same cell, which can result in confounding results. Thus, in some embodiments, the MOI of the gRNA vector is less than 1 and the MOI of the CAR vector is greater than 1. In some embodiments, the gRNA library is transfected into multiple T cells at a multiplicity of infection (MOI) between 0.1 - 1, 0.2 - 1, 0.3 - 1, 0.4 - 1, 0.5 - 1, 0.6 - 1, 0.7 - 1, 0.8 - 1, 0.3 - 1, 0.4 - 0.9, 0.5 - 0.8, or 0.6 - 0.07. In some embodiments, the MOI of the gRNA vector is less than 1. In some embodiments, the MOI of the CAR vector is at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20). In some embodiments, the MOI of the CAR vector is between 1 - 3, 1 - 5, 1 - 10, 1 - 20, 5 - 10, 5 - 15, 5 - 20, or 10 - 20.

[0228] "Variant CAR-T cell library" can refer to two or more CAR-T cells, where at least two of the two or more CAR-T cells contain a CAR (e.g., a CAR vector) and a gRNA vector, and where at least two of the two or more CAR-T cells contain a gRNA vector encoding different guide RNAs. In some embodiments, at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, at least twenty-five, at least fifty, at least one hundred, or at least one hundred and thirty-five) of the CAR-T cells in the library each contain a different gRNA polynucleotide that includes a homologous region complementary to a different gene sequence of any one of SEQ ID NOs: 1-135 or a variant thereof. In some embodiments, the CAR-T cell library includes a gRNA vector as described herein. In some embodiments, the CAR-T cells of the variant CAR-T cell library include any one of SEQ ID NOs: 236-1315. In some embodiments, the variant CAR-T cell library includes any one of the compositions including a gRNA polynucleotide as described herein. In some embodiments, the variant CAR-T cell library includes at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-five, at least fifty, at least seventy-five, at least one hundred, at least two hundred and fifty, at least five hundred, at least seven hundred and fifty, or at least one thousand) of the gRNA homologous region sequences of any one of SEQ ID NOs: 236-1315. In some embodiments, the variant CAR-T cell library includes SEQ ID NOs: 236-1315.

[0229] Method In some aspects, the present disclosure provides methods for creating mutant immune cell libraries. In some embodiments, the immune cell library comprises T cells (mutant CAR-T cell library). In some embodiments, the immune cell library comprises NK cells (mutant CAR-NK cell library). In some embodiments, the cells of the library comprise a CAR or a vector encoding a CAR (e.g., mutant CAR-T cell library or mutant CAR-NK cell library).

[0230] Generation of Mutant CAR-T Cell Library In some aspects, the present disclosure provides methods for creating mutant CAR-T cell libraries. In some embodiments, the method comprises (a) obtaining CAR-T cells (e.g., pre-generated CAR-T cells), and (b) transfecting the T cells with a gRNA vector library (e.g., as described herein). In some embodiments, the obtained CAR-T cells are pre-activated. In some embodiments, the method comprises activating the CAR-T cells. In some embodiments, the CAR-T cells express a CRISPR protein. In some embodiments, the method further comprises introducing into the CAR-T cells an mRNA encoding the CRISPR protein or the CRISPR protein (e.g., by electroporation).

[0231] In some embodiments, the method comprises (a) activating T cells (e.g., as described above (e.g., using anti-CD3 antibody and anti-CD28 antibody)), (b) transfecting the T cells with a gRNA vector library (e.g., as described herein), (c) transfecting the T cells with a CAR vector (e.g., as described herein), and (d) introducing into the T cells an mRNA encoding the CRISPR protein or the CRISPR protein (e.g., by electroporation).

[0232] In some embodiments, the CRISPR protein is any suitable CRISPR protein for mutating DNA in CAR-T cells. In some embodiments, the CRISPR protein is any CRISPR protein described herein. In some embodiments, introducing the mRNA encoding the CRISPR protein or the CRISPR protein into T cells is performed 1 to 10 days after transfection of the gRNA vector and / or the CAR vector. In some embodiments, introducing the mRNA encoding the CRISPR protein or the CRISPR protein into T cells is performed 3 to 7 days after transfection of the gRNA vector and / or the CAR vector. In some embodiments, introducing the mRNA encoding the CRISPR protein or the CRISPR protein into T cells is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 16 days after transfection of the gRNA vector and / or the CAR vector. In some embodiments, the introduced CRISPR protein is a CRISPR protein. In some embodiments, the introduced CRISPR protein is introduced as a ribonucleoprotein. In some embodiments, the CRISPR protein is encoded on the gRNA vector or the CAR vector.

[0233] In some embodiments, the method includes collecting T cells from a subject, which can then be modified, for example, using one or more (e.g., all) of the above steps (a)-(d). As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus macaque. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. In some embodiments, the subject is a mammal, such as a primate, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a laboratory model organism (e.g., a mouse). The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of diseases such as cancer. The subject can be male or female. The subject has been previously diagnosed with a condition that requires treatment (e.g., pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, or mesothelioma, or another type of cancer that expresses an antigen target by the antigen-binding domain of the CAR) or one or more complications associated with such a condition, or is determined to have or be suffering from such a condition, and in some cases, may have already received treatment for such a condition or one or more complications associated with such a condition. Alternatively, the subject can also be one that has never been diagnosed in the past with having such a condition or associated complications. For example, the subject can be one that exhibits one or more risk factors for such a condition or one or more complications associated with such a condition or does not exhibit risk factors.A "subject in need of treatment" for a particular condition can be a subject who has the condition, is diagnosed as having the condition, or is at risk of developing the condition.

[0234] In some embodiments, the subject is diagnosed with cancer. As used herein, "cancer" can refer to the uncontrolled growth of cells that, due to their unique properties and loss of normal cell control, results in disorganized growth, lack of differentiation, local tissue invasion, and metastasis. Exemplary cancers include, but are not limited to, glioblastoma, prostate cancer, glioma, leukemia, lymphoma, multiple myeloma, or solid tumors such as lung cancer and pancreatic cancer. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is ALL or CLL. Non-limiting examples of lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, hairy cell leukemia (HCL), and T-cell lymphoma (e.g., peripheral T-cell lymphoma (PTCL) including cutaneous T-cell lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)). In some embodiments, the cancer is DLBCL or follicular lymphoma. Non-limiting examples of solid tumors include adrenocortical tumor, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, endodermal sinus tumor, epitheloid hemangioendothelioma, Ewing sarcoma, germ cell tumor (solid tumor), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms tumor. In some embodiments, the cancer expresses mesothelin. In some embodiments, the cancer is uterine cancer. Solid tumors can be found in bone, muscle, or organs and can be sarcomas or carcinomas. It is contemplated that any aspect of the techniques described herein can be used to treat all types of cancer, including cancers not listed in this application. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, for example, of a malignant or benign type.

[0235] In some embodiments, T cells are collected using any suitable method. In some embodiments, T cells are collected from a subject. In some embodiments, T cells are collected from a healthy subject. In some embodiments, T cells are collected by in vitro production (e.g., from induced pluripotent stem cells or embryonic stem cells). In some embodiments, T cells are collected from a subject using leukapheresis.

[0236] In some embodiments, the method further comprises purifying T cells comprising a CAR vector and T cells comprising a gRNA vector. In some embodiments, the purification method can be performed based on a selectable molecule (e.g., a reporter) as described above. For example, the CAR vector can express a selection marker (e.g., CD34t), and the gRNA vector can express a selected marker (e.g., knockout of CD3). In some embodiments, purification can be performed using antibody-based magnetic selection. In some embodiments, purification can be performed using flow cytometry. In some embodiments, purification can be performed using CliniMACS. In some embodiments, CAR-T cells having CRISPR activity are purified based on knockout of a gene (e.g., CD3) for negative selection. In some embodiments, CAR-T cells expressing a CAR are selected based on positive selection (e.g., expression of CD34). In some embodiments, the success of transduction can be determined using flow cytometry based on the number of CD34+CD-LNGF+ cells.

[0237] In some embodiments, the gRNA vector library is transfected into CAR-T cells at the MOI described herein. In some embodiments, the CAR vector is transfected at the MOI described herein.

[0238] In some embodiments, the present application discloses a library of mutant CAR-T cells generated using any of the methods described herein.

[0239] Identifying the impact of CRISPR on the function of CAR-T cells. In some embodiments, the disclosure provides a method for identifying mutant (e.g., CRISPR-generated) CAR-T cells with increased efficacy.

[0240] In some aspects, the present application is a method for identifying gRNA polynucleotides associated with the efficacy of CAR-T cells in vivo, comprising: (a) administering to a subject a library of mutant CAR-T cells (e.g., a library of mutant CAR-T cells disclosed herein, or a library of mutant CAR-T cells generated by the methods described herein); (b) collecting from the subject one or more samples comprising a plurality of mutant CAR-T cells (e.g., via leukapheresis); (c) sequencing the gRNA polynucleotides from the mutant CAR-T cells collected in (b) (e.g., using any reasonable method, such as Illumina sequencing); and (d) evaluating a change in the relative abundance of each gRNA polynucleotide based on the sequencing in (c). For example, a gRNA associated with the efficacy of CAR-T cells is a gRNA that induces mutations that increase the proliferation and / or tumor toxicity of the CAR-T cells. One of ordinary skill in the art will understand that an increase in the relative abundance of a given gRNA after administration of a library of mutant CAR-T cells to a subject indicates that the given gRNA causes mutations that increase the efficacy (e.g., persistence) of the CAR-T cells.

[0241] One of ordinary skill in the art will understand that mutant CAR-T cells (e.g., cells of a library of mutant CAR-T cells) can replicate within a subject. Thus, the plurality of mutant CAR-T cells collected in step b above can include the mutant CAR-T cells administered to the subject and / or the progeny of the mutant CAR-T cells administered to the subject.

[0242] In some embodiments, the methods described herein relate to treating a subject having or diagnosed with cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder with an immune cell comprising any of the CAR polypeptides (and any antibody reagent or cytokine) described herein, or with a nucleic acid encoding any of the CAR polypeptides (and any antibody reagent or cytokine) described herein. The CAR-T cells described herein comprise an immune cell comprising any of the CAR polypeptides (and any antibody reagent or cytokine) described herein, or a nucleic acid encoding any of the CAR polypeptides (and any antibody reagent or cytokine) described herein.

[0243] The compositions described herein can be administered to a subject having or diagnosed with a condition (e.g., cancer). As used herein, "condition" refers to cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder. A subject having a condition can be identified by a physician using methods for diagnosing the current condition. Symptoms and / or complications of the condition that characterize and aid in the diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that may be useful in diagnosing a condition include, but are not limited to, blood screening and bone marrow examination, which are known in the art for a given condition. A family history of a condition, or exposure to risk factors for a condition, can also be useful in determining whether a subject may have that condition or in diagnosing the condition.

[0244] In some embodiments, the methods described herein include administering to a subject an effective amount of the activated CAR-T cells (e.g., a library of mutant CAR-T cells) described herein to identify mutant CAR-T cells with increased efficacy. In some embodiments, the methods described herein include administering to a subject an effective amount of the activated CAR-T cells (e.g., a library of mutant CAR-T cells) described herein to alleviate the symptoms of a condition. As used herein, "alleviating the symptoms of a condition" means improving any condition or symptom associated with the condition. Such a decrease, as compared to an equivalent untreated control, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered to the site of a tumor.

[0245] As used herein, the term "effective amount" can refer to the number of immune cells (e.g., activated CAR-T cells or CAR-NK cells) necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to an amount of a cell preparation or composition sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" can refer to an amount of activated immune cells (e.g., CAR-T cells or NK cells) sufficient to have an effect against a particular condition when administered to a typical subject. Effective amounts, as used herein in various contexts, would also include amounts sufficient to slow the progression of the symptoms of a disease, alter the course of the symptoms of a disease (e.g., but not limited to, slowing the progression of the symptoms), or reverse the symptoms of a condition. Thus, it is generally not practical to specify an exact "effective amount." However, in any case, the appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0246] An effective amount, toxicity, and therapeutic effect can be evaluated by standard pharmaceutical procedures in cell culture or experimental animals. The dosage can vary depending on the dosage form used and the route of administration utilized. The dosage ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. A therapeutically effective dosage can first be estimated from cell culture assays. Also, the dosage can be formulated in an animal model to achieve a concentration range in the circulating plasma that includes the IC50 (i.e., the concentration of activated CAR-T cells that achieves half-maximal inhibition of the symptoms) determined in cell culture or an appropriate animal model. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays, such as assays for bone marrow examination, among others. The dosage is determined by the physician and can be adjusted as needed in accordance with the observed therapeutic effect.

[0247] Modes of administration can include, for example, intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient arterially, intratumorally, intranodally, intraperitoneally, or intramedullarily. In some embodiments, the composition of T cells can be directly injected into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0248] In certain exemplary embodiments, the subject may undergo leukapheresis, and leukocytes are collected, concentrated, or removed ex vivo to select and / or isolate the cells of interest, such as T cells. These T cell isolates can be expanded by contact with artificial APCs, such as aAPCs expressing anti-CD28 and anti-CD3 CDRs, and processed to allow introduction of one or more CAR constructs of the present technology, thereby generating CAR-T cells. These methods can further include transfecting the CAR-T cells with the gRNA libraries described herein.

[0249] In some embodiments, a single treatment regimen is required. In other cases, administration of one or more subsequent doses or treatment regimens may be carried out. For example, after treating for 3 months every other week, the treatment can be repeated once a month for 6 months or more than one year. In some embodiments, no additional treatment is performed after the initial treatment.

[0250] The dosage of the compositions described herein is determined by a physician and can be adjusted as needed to fit the observed treatment effect. Regarding the duration and frequency of treatment, a skilled clinician generally monitors the subject to determine when the treatment will produce a therapeutic effect and further decides whether to administer additional cells, discontinue the treatment, resume the treatment, or make other changes to the treatment regimen. The dosage should not be so high as to cause harmful side effects such as cytokine release syndrome. Generally, the dosage varies depending on the age, condition, and gender of the patient and can be determined by those skilled in the art. The dosage can also be adjusted by an individual physician if any complications occur.

[0251] In some embodiments, the mutant CAR-T cell library administered to the subject comprises a genome-wide CAR-T cell mutant library. A genome-wide CAR-T cell mutant library can refer to a plurality of CAR-T cells transfected with a gRNA library containing at least one guide RNA targeting each gene in the genome of the CAR-T cells. In some embodiments, the genome-wide CAR-T cell mutant library comprises a plurality of CAR-T cells transfected with a gRNA library containing at least one guide RNA targeting each protein-coding gene in the genome of the CAR-T cells. The genome-wide T cell CRISPR library and methods of use in in vitro cell lines are described in Shifrut et al., Cell 175.7(2018):1958-1971.

[0252] In some embodiments, the variant CAR-T cell library includes negative control CAR-T cells. In some embodiments, the negative control CAR-T cells include the CAR vector described herein and the negative control gRNA described herein. In some embodiments, the negative control CAR-T cells include an empty CAR vector that does not encode a CAR. In some embodiments, the negative control CAR-T cells include a CAR that does not include an antigen-binding domain. In some embodiments, the negative control CAR-T cells include a CAR that includes an antigen-binding domain that does not bind to a protein expressed by the subject.

[0253] In some embodiments, the subject is any suitable subject including the subjects described herein. In some embodiments, the subject is a human (e.g., a human subject described herein). In some embodiments, the subject has cancer (e.g., cancer described herein). In some embodiments, the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, or B cell lymphoma.

[0254] In some embodiments, the CAR includes an antigen-binding domain (e.g., the antigen-binding domain described herein) that binds to an antigen expressed by cancer. In some embodiments, the CAR includes an antigen-binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen-binding domain. In some embodiments, the antigen-binding domain binds to mesothelin.

[0255] In some embodiments, one or more samples collected from a subject are collected for any reasonable tissue or body fluid using any reasonable means. In some embodiments, one or more samples are collected from a body fluid (e.g., cerebrospinal fluid, blood or blood product). In some embodiments, the blood product is serum or plasma. In some embodiments, one or more samples are collected from bone marrow. In some embodiments, one or more samples are collected from tissue (e.g., heart, lung, liver, pancreas, brain, skin, intestine, kidney, spleen, lymph node, thyroid, muscle, fat, gallbladder, or tonsil).

[0256] In some embodiments, sample collection is performed using any reasonable means including, but not limited to, venipuncture, apheresis, tissue biopsy or tumor resection.

[0257] In some embodiments, samples are collected from a subject. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more samples (e.g., blood samples) are collected from a subject. In some embodiments, at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 samples (e.g., blood samples) are collected from a subject. In some embodiments, 1-2, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 2-3, 2-5, 2-7, 2-10, 2-12, 2-15, 3-5, 3-7, 3-10, 3-12, or 3-15 samples are collected from a subject. In some embodiments, at least 5 samples are collected by venipuncture, apheresis, or resection of a tumor.

[0258] In some embodiments, the sample is collected from the subject on one or more of the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day, 14th day, 15th day, 16th day, 17th day, 18th day, 19th day, 20th day, 21st day, 22nd day, 23rd day, 24th day, 25th day, 26th day, 27th day, 28th day, 29th day, 30th day, or 31st day after step (a). In some embodiments, the sample is collected from the subject on one or more of the 1st week, 2nd week, 3rd week, 4th week, 5th week, 6th week, 7th week, 8th week, 9th week, 10th week, 11th week, 12th week, 13th week, 14th week, 15th week, 16th week, 17th week, 18th week, 19th week, 20th week, 21st week, 22nd week, 23rd week, 24th week, 25th week, 26th week, 27th week, 28th week, 29th week, or 30th week after step (a). In some embodiments, the sample is collected from the subject on one or more of the 1st month, 2nd month, 3rd month, 4th month, 5th month, 6th month, 7th month, 8th month, 9th month, 10th month, 11th month, 12th month after step (a). In some embodiments, the sample is collected from the subject on the 3rd day, 7th day, 10th day, 14th day, 21st day, 28th day, 2nd month, 3rd month, and 6th month after step (a).

[0259] In some embodiments, the method further comprises extracting variant CAR T cells from a sample prior to sequencing. Methods for isolating CAR-T cells from a sample (e.g., a body fluid) are well known in the art. For example, leukapheresis is a known method for extracting CAR-T cells from a subject. Other methods include immunoprecipitation, immunomagnetic selection, microfluidics, and fluorescence-activated cell sorting. These methods are described in Chiu et al., Scientific Reports 9.1(2019):1-10, which is incorporated by reference in its entirety. In some embodiments, the extraction of variant CAR-T cells from a sample is performed based on cell surface markers expressed by the variant CAR-T cells. In some embodiments, the extraction is performed by isolating T cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

[0260] In some embodiments, the method further comprises extracting genomic DNA from the variant CAR T cells. In some embodiments, the genomic DNA is extracted from a single variant CAR-T cell or a clonal population of variant CAR-T cells. In some embodiments, the method comprises sequencing the extracted genomic DNA to determine mutations induced by a CRISPR guide RNA in a given CAR-T cell. Alternatively, in some embodiments, the method comprises amplifying a region of the genome expected to contain a CRISPR-induced mutation (e.g., based on the sequence of the gRNA homology region) using primers and sequencing that region.

[0261] In some embodiments, the method further comprises isolating RNA from the variant CAR T cells. In some embodiments, the RNA is isolated from a single variant CAR-T cell (e.g., for single cell RNA sequencing). In some embodiments, the RNA isolated from the variant CAR-T cells is sequenced (e.g., using Illumina sequencing).

[0262] In some embodiments, the method includes sequencing the gRNA polynucleotide in the variant CAR-T cells extracted from the subject. In some embodiments, the method includes extracting DNA from the variant CAR-T cells. One of ordinary skill in the art will understand that DNA can be extracted from a pool of variant CAR-T cells collected from the subject. In other words, in some embodiments, it is not necessary to separately isolate the DNA from each variant CAR-T cell collected from the subject in order to determine the effect of the CRISPR mutation on the efficacy of the CAR-T cells. Rather, because the guide RNA homology sequence can have a sequence that is complementary to a particular region of a particular gene, the guide RNA homology sequence can be used to identify which gene is expected to be mutated by CRISPR and the expected location of the mutation in that gene.

[0263] Thus, in some embodiments, the method further comprises amplifying the gRNA polynucleotide of the variant CAR-T cells using a primer to generate any of these amplicons, which are used for sequencing. In some embodiments, the gRNA polynucleotide to be amplified is genomic DNA, vector DNA, or RNA. In some embodiments, universal primers can be used to amplify all of the guide RNA polynucleotides in the variant CAR-T cell library. In some embodiments, the universal primers include a universal forward primer and a universal reverse primer. In some embodiments, the forward primer and the reverse primer are designed to bind to the same region of the gRNA vector and / or gRNA polynucleotide. For example, in some embodiments, the universal forward primer binds to the gRNA polynucleotide upstream of the homologous region. In some embodiments, the universal reverse primer binds to the gRNA polynucleotide downstream of the homologous region. In some embodiments, the universal forward primer comprises the sequence of SEQ ID NO: 1324. In some embodiments, the forward primer and the reverse primer include phosphorothioate modifications. In some embodiments, the universal forward primer includes a stagger sequence (e.g., a variable-length sequence within the primer to improve the resolution of next-generation sequencing). Stagger sequences are also known in the art and are described, for example, in Wohlhieter et al., STAR Protocols 2.2 (2021): 100390. In some embodiments, the stagger sequence includes any one of C, GC, AGC, CAAC, TGCACC, ACGCAAC, and TGAAGACCC. In some embodiments, the universal reverse primer includes a stagger sequence.

[0264] In some embodiments, the universal reverse primer comprises the sequence of SEQ ID NO: 1325.

[0265] In some embodiments, the gRNA polynucleotide or its amplicon is sequenced. Sequencing of the gRNA polynucleotide, its amplicon, or other sequencing described herein can be performed using any suitable method or apparatus including, but not limited to, next-generation sequencing, IlluminaTM sequencing, Ion TorrentTM sequencing, PacBioTM sequencing, nanopore sequencing, 454 sequencing, Sanger sequencing, and SoLidTM sequencing.

[0266] In some embodiments, the method includes assessing changes in the relative abundance of each gRNA polynucleotide based on sequencing. In some embodiments, the relative abundance of each gRNA polynucleotide is based on the number of times a given gRNA polynucleotide is observed in sequencing data before the mutant CAR-T cell library is administered to a patient and after the mutant CAR-T cell library is extracted from the patient. In some embodiments, the relative abundance of a given guide RNA in the sequencing data polynucleotide is determined over time (e.g., in each sample collected). The relative abundance of a given guide RNA can indicate the efficacy of the CAR-T cells.

[0267] In some embodiments, the method includes determining a fold change value by comparing the average gRNA polynucleotide abundance for any given gene in a post-injection sample (extracted from a subject) to its abundance in a pre-injection sample (before administration to the subject). In some embodiments, the threshold for identifying a significant fold change in gRNA polynucleotide abundance is based on z-score normalization of the distribution of fold change values. In some embodiments, a change greater than two standard deviations from the mean of the z-score distribution is considered significant.

[0268] In some embodiments, the efficacy of each variant CAR-T cell is evaluated based on in vivo persistence and tumor cell cytotoxicity. As used herein, the term "persistence" or "persistence of CAR-T cells" refers to the ability of CAR-T cells to remain activated, continue to proliferate, and / or not die. Since the proliferation and death of variant CAR-T cells are expected to change the relative abundance of variant CAR-T cells, persistence can be measured based on the relative abundance of variant CAR-T cells over time. Thus, variant CAR-T cells with an increased relative abundance may have increased persistence.

[0269] In some embodiments, in vivo persistence and tumor cell cytotoxicity are measured using flow cytometry and PCR. In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA. In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA compared to the gRNA polynucleotide from negative control CAR-T cells. In some embodiments, quantifying persistence is done by calculating the relative change over time of each gRNA over at least three time points and calculating the slope of the relative change. In some embodiments, tumor cell cytotoxicity is quantified using clinical imaging and / or bone marrow evaluation.

[0270] In some embodiments, the persistence of variant CAR-T cells is determined based on relative abundance, and then variant CAR-T cells with a high relative abundance are tested for cancer killing effects (e.g., in vitro or in vivo).

[0271] In some embodiments, efficacy is measured for the entire library of variant CAR-T cells administered to a patient. For example, if a patient shows signs of improvement (e.g., reduction in tumor or cancer burden, extension of lifespan, etc.) after administration of a variant CAR-T cell library, that library may be effective as a whole.

[0272] Generation of a Variant NK Cell Library In some embodiments, the methods described herein for generating a variant CAR-NK cell library can be adapted to generate a variant natural killer cell library. In some embodiments, the method includes transfecting a library of gRNA vectors into a plurality of NK cells (e.g., CAR-NK cells) isolated from a subject (e.g., using the methods described herein). In some embodiments, the NK cells are also transfected with a CAR vector as described herein.

[0273] In some aspects, the disclosure provides a method for generating a variant CAR-NK cell library. In some embodiments, the method includes (a) obtaining CAR-NK cells (e.g., pre-generated CAR-NK cells), and (b) transfecting the CAR-NK cells with a library of gRNA vectors (e.g., as described herein). In some embodiments, the obtained CAR-NK cells are pre-activated. In some embodiments, the method includes activating the CAR-NK cells. In some embodiments, the CAR-NK cells express a CRISPR protein. In some embodiments, the method further includes introducing an mRNA encoding the CRISPR protein or the CRISPR protein into the CAR-NK cells (e.g., by electroporation).

[0274] In some embodiments, the present application discloses a method for generating a variant CAR-NK cell library, the method comprising: (a) activating NK cells (e.g., as described above (e.g., using anti-CD3 antibody and anti-CD28 antibody)); (b) transfecting the NK cells with a library of gRNA vectors as described above; (c) transfecting the NK cells with a CAR vector according to any one of the above claims; and (d) introducing an mRNA encoding a CRISPR protein or the CRISPR protein into the NK cells (e.g., by electroporation).

[0275] In some embodiments, the CRISPR protein is any suitable CRISPR CAR for mutating DNA in CAR-NK cells. In some embodiments, the CRISPR protein is any CRISPR protein described herein. In some embodiments, introducing the mRNA encoding the CRISPR protein or the CRISPR protein into NK cells is performed 1 to 10 days after transfection of the gRNA vector and / or the CAR vector. In some embodiments, introducing the mRNA encoding the CRISPR protein or the CRISPR protein into NK cells is performed 3 to 7 days after transfection of the gRNA vector and / or the CAR vector. In some embodiments, the introduced CRISPR protein is the CRISPR Cas9 protein. In some embodiments, the introduced CRISPR protein is introduced as a ribonucleoprotein. In some embodiments, the mRNA of the CRISPR protein is expressed from the gRNA vector or the CAR vector.

[0276] In some embodiments, the method includes collecting NK cells from a subject (e.g., a subject as described above), which can then be modified, for example, using one or more (e.g., all) of steps (a)-(d) above.

[0277] In some embodiments, the NK cells are collected using any suitable method (e.g., including any suitable method described above).

[0278] In some embodiments, the method further includes purifying the NK cells containing the CAR vector and the CRISPR-induced genetic mutation (e.g., including any suitable purification method described above).

[0279] In some embodiments, the gRNA vector library is transduced into CAR-NK cells at an MOI as described above. In some embodiments, the CAR vector is transduced at an MOI as described above.

[0280] In some embodiments, the present application discloses a library of variant CAR-NK cells generated using any of the methods described herein.

[0281] Identification of the impact of CRISPR on the function of CAR-NK cells. In some embodiments, the present disclosure provides methods for identifying variant (e.g., CRISPR-generated) CAR-NK cells with increased efficacy.

[0282] In some aspects, the present application is a method for identifying gRNA polynucleotides associated with the efficacy of CAR-T cells in vivo, comprising: (a) administering to a subject a library of variant CAR-NK cells (e.g., a library of variant CAR-NK cells disclosed herein, or a library of variant CAR-NK cells generated by the methods described herein); (b) collecting from the subject one or more samples comprising a plurality of variant CAR-NK cells (e.g., via leukapheresis); (c) sequencing the gRNA polynucleotides from the variant CAR-NK cells collected in (b) (e.g., using any reasonable method, such as Illumina sequencing); and (d) evaluating a change in the relative abundance of each gRNA polynucleotide based on the sequencing in (c). For example, a gRNA associated with the efficacy of CAR-NK cells is a gRNA that induces mutations that increase the proliferation and / or tumor toxicity of CAR-NK cells. One of ordinary skill in the art will understand that an increase in the relative abundance of a given gRNA after administration of a library of variant CAR-NK cells to a subject indicates that the given gRNA causes a mutation that increases the efficacy (e.g., persistence) of the CAR-NK cells.

[0283] One of ordinary skill in the art will understand that the mutant CAR-NK cells (e.g., cells of a mutant CAR-NK cell library) may replicate within the subject. Thus, the plurality of mutant CAR-NK cells collected in step b above may include the mutant CAR-NK cells administered to the subject and / or the progeny of the mutant CAR-NK cells administered to the subject.

[0284] The CAR-NK cells described herein include immune cells comprising any of the CAR polypeptides (and any antibody reagents or cytokines) described herein, or nucleic acids encoding any of the CAR polypeptides (and any antibody reagents or cytokines) described herein.

[0285] In some embodiments, the methods described herein include administering to a subject an effective amount of the activated CAR-NK cells (e.g., a mutant CAR-NK cell library) described herein to identify mutant CAR-NK cells with increased efficacy. In some embodiments, the methods described herein include administering to a subject an effective amount of the activated CAR-NK cells (e.g., a mutant CAR-NK cell library) described herein to alleviate the symptoms of a condition.

[0286] In some embodiments, the composition of NK cells can be administered to a subject as described above (e.g., in the same or a similar manner as performed using mutant CAR-T cells).

[0287] In some embodiments, the mutant CAR-NK cell library administered to the subject includes a genome-wide CAR-NK cell mutant library (e.g., a genome-wide library generated using the genome-wide gRNA vector library described above for mutant CAR-T cells).

[0288] In some embodiments, the CAR of the CAR-NK cell comprises an antigen-binding domain that binds to an antigen expressed by cancer (e.g., the antigen-binding domains described herein). In some embodiments, the CAR is an antigen-binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen-binding domain. In some embodiments, the antigen-binding domain binds to mesothelin.

[0289] In some embodiments, one or more samples collected from a subject are collected for any reasonable tissue or body fluid using any reasonable means as described above.

[0290] In some embodiments, the methods for collecting variant CAR-NK cells from a subject, extracting genomic DNA and RNA from CAR-NK cells, amplifying and / or sequencing gRNA polynucleotides, determining relative abundance, and determining the efficacy of CAR-NK cells have been described above in the context of CAR-T cells and can be used similarly with CAR-NK cells.

[0291] All patents and other publications, including reference documents, issued patents, published patent applications, and co-pending patent applications cited throughout this application, are hereby expressly incorporated by reference herein for the purpose of explaining and disclosing the methodologies described in such publications, which may be used, for example, in connection with the techniques described herein. These publications are provided only for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventors have no right to antedate such disclosure by virtue of prior art or for any other reason. All statements as to the date or representation of the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the date or content of these documents.

[0292] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the present disclosure, as will be recognized by those of ordinary skill in the relevant art. For example, although method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as needed to provide further embodiments using the compositions, functions, and concepts of the above references and applications. Further, in view of the equivalence of biological functions, some changes can be made to the protein structure without affecting the biological or chemical action in terms of type or amount. In light of the detailed description, these and other changes can be made to the present disclosure. All such changes are intended to be included within the scope of the appended claims.

[0293] Any particular element of any of the foregoing embodiments can be combined with or replaced by elements of other embodiments. Further, although the advantages associated with specific embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0294] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limitations.

Examples

[0295] Examples Example 1: Vector Design of gRNA Vector and CAR Vector Two different vectors are designed that together provide for the expression of a CAR and positive selection using a clinically feasible magnetic bead system, and the expression of two guides, a suicide gene, and a selectable marker that enable specific CRISPR / Cas9 knockout of two genes, one of which is used for negative selection using said clinically feasible magnetic bead system.

[0296] The CAR vector was composed of the backbone of a third-generation self-inactivating (SIN) lentiviral vector (Figure 1A). A promoter (e.g., EF1a) was used to drive the expression of the CAR. A 2A ribosome skip element followed the CAR sequence, followed by a reporter gene (truncated CD34 lacking the intracellular domain) that was detectable / selectable by staining for surface expression.

[0297] The dual guide vector backbone (e.g., gRNA vector) enabled the use of a library of guides. Two guides were encoded in the vector backbone; one was static (TRAC), which enabled knockout of the TCR / CD3 complex on the gene-edited T cells; the absence of CD3 enabled clinically feasible negative selection, and the effector population that had been successfully transduced and gene-edited by CRISPR / Cas9 was selected. In this exemplary embodiment, the guide RNAs on the vector were inverted; in another embodiment, the two guides were configured in tandem (Figure 1B).

[0298] EF1a and EF1a-short (EFS) were used for CAR and reporter expression. The expression of the reporter gene LNGFR and the suicide gene was driven by a weaker promoter (PGK) to avoid leaky and potentially apoptosis-causing overexpression of the suicide gene, which could lead to apoptosis of cell products.

[0299] The CAR vector and the dual guide vector are co-introduced into the same T cell population. The final "active" investigational product of interest is CAR+CD34+LNGFR+CD3- T cells. To enhance safety, a suicide gene (e.g., icaspase9) can also be included in the dual guide vector.

[0300] Example 2: Design and Generation of the gRNA Library Gene Selection Using genomic, transcriptional, and functional data from human and mouse T cells, an initial list of 135 genes that are hypothesized to be CRISPR / Cas9 knockout - capable and improve CAR - T cell function was selected (Table 2). These genes included checkpoint proteins (e.g., PD - 1 encoded by PDCD1), as well as genes in which mutations were observed in subjects with lymphoproliferative diseases or subjects showing abnormal responses to CAR - T cells (e.g., TET2 and cbl - b).

[0301] Determination of the sgRNA Library Size The size of the library was calculated based on manufacturing feasibility, the dose of cells to be administered (usually 100x106), and the number of cells that can be reasonably recovered from the patient after infusion (usually 50x106 - 100x106). Targeting the entire genome results in sub - optimal depth and a low signal - to - noise ratio. On the other hand, targeting less than 10 genes would provide less information about which candidate gene knockout is optimal. To increase the signal - to - noise ratio, redundancy was built into the guides to target 100 - 500 genes, providing depth of coverage, reducing randomness due to the number of T cells recovered after infusion, and presenting a competitive system within and between different patients to rigorously evaluate the effects of various candidate genes. This library also includes controls targeting genes predicted to have some impact on T cell biology.

[0302] sgRNA Library Design For the 135 selected genes, a library of 8 sgRNAs per gene was designed using the dominant transcript IDs in human T cells for each gene (e.g., encoded by SEQ ID NOs: 1 - 135). These transcripts were used with CRISPick to design all possible guides. A total of 34,318 guides were designed. These guides were scored for on - target activity using Rule Set 3 (RS3) and Chen2013 tracr (PMID: 24360272) with sequence and target information. The guides were also scored for off - target activity using the Tier - agnostic 1 mismatch aggregated CFD score. 2009 guides were excluded based on cloning incompatibilities (e.g., BsmBI sites) and poly - T. Five guides had over 10,000 off - target sites with one mismatch. 277 guides with off - targets in 58 tumor suppressor genes were excluded according to the definition of Lenoir et al. Nature communications 12.1(2021):1 - 15. 118 guides flagged for targeting genomic regions with high variability within the human population were also excluded. 5561 guides targeting outside the 2 - 85% protein window were excluded. To avoid spurious guides, 4771 guides with a total Cutting Frequency Determination (CFD) score of less than 0.27 were excluded.

[0303] After all the selections were applied, the top 8 guides per gene were selected by the RS3 score. This library has a total of 1080 gene - targeting guides (Table 4). 100 validated gene - to - gene controls were also included, and the total number of guides was 1180.

[0304] sgRNA library construction To create the sgRNA library, the oligos for each sgRNA are synthesized on-chip and amplified using PCR primers containing BsmBI-compatible cloning sequences. The amplified oligo pool is ligated into the above sgRNA vector, and the ligation product is transformed into electrocompetent E. coli. The transformed E. coli is then plated onto solid agar at a density that achieves 1000-fold expression of each sgRNA in the library and grown overnight. Colonies are scraped from the plate and the pooled sgRNA plasmids are extracted. After sequencing the plasmid library to confirm sgRNA expression, it is re-transformed into electrocompetent E. coli and stored frozen in glycerol medium. An aliquot of the E. coli stored in glycerol is then cultured overnight in liquid culture, and the plasmid is extracted from the grown E. coli. The pooled sgRNA library lentivirus is prepared using standard third-generation lentiviral vector production methods.

[0305] Example 3: Generation and Administration of Mutant CAR-T Cell Library T cells are collected from human subjects by standard leukapheresis. Peripheral blood mononuclear cells are isolated and T cells are purified. The T cells are then activated using anti-CD3 / anti-CD28 antibodies (optionally beads coated with antibodies) and transduced with both vectors. The MOI of the dual guide vector has an upper limit of 1, optionally 0.5, or approaching 0.5. The MOI of the CAR vector is as high as 20.

[0306] Cas9 is introduced into CAR-T cells in the form of mRNA encoding Cas9 or in the form of ribonucleoprotein (RNP), where the soluble Cas9 protein is complexed with a non-targeting guide RNA that does not have a predicted target site within the genome. Regardless of whether mRNA or RNP is used, Cas9 is electroporated into T cells 3 to 7 days after lentiviral transduction.

[0307] T cells are further expanded and selected through a series of magnetic bead treatments. First, the product is negatively selected against CD3 expression using a system such as CliniMACS that removes T cells. The CD3-negative fraction is then positively selected against CD34 expression using a system such as CliniMACS. CD34+CD3− cells are considered investigational products for infusion.

[0308] Flow cytometry analysis is used to determine the number of CAR+ cells based on CD34+ cells, and the number of cells that have been successfully transduced and gene edited based on CD3 negativity and LNGFR+.

[0309] CAR T cells are administered in the range of 10x10 6 (perhaps suboptimal dose levels) to 100x10 6 (typical dose levels) to 300x10 6 (typical maximum allowable dose of CAR T cells). The expression of the library is measured as a baseline to assess manufacturing variability, but is not itself part of dose selection.

[0310] Example 4: Determination of the Efficacy of Mutant CAR-T Cells Samples of mutated T cells are collected from the subject by venipuncture, apheresis, or tumor resection according to the event schedule of the clinical protocol (usually on days 3, 7, 10, 14, 21, 28, 2 months, 3 months, and 6 months). T cells are isolated by standard procedures. CAR+ T cells are purified based on the expression of CD34 and analyzed for the expression of other T cell markers such as CD3 as well as CD4 and CD8. Genomic DNA is isolated from the purified CAR+ T cells for downstream analysis. The sgRNA region from CAR+ T cells is amplified using PCR primers specific to the sequence adjacent to the sgRNA cassette containing the library. The PCR amplicons are then sequenced using Nextgen sequencing to quantify the abundance of each sgRNA in the library and compare the abundance of each guide in CAR+ T cells after injection with the value of its abundance in CAR+ T cells before injection.

[0311] In this method, it is also possible to capture sgRNA and perform single-cell transcriptome profiling of CAR-T cells isolated from patients, enabling the association of gene deletions with transcriptional phenotypes. A standard droplet-based single-cell whole-transcriptome analysis platform such as 10xGenomics is used in combination with the direct capture of sgRNA sequences from cellular RNA using a primer set specific to the sgRNA region.

[0312] Example 5: Knockout of Multiple Genes in Primary T Cells Using a Dual Guide Cassette Vector The CD5 and TRAC genes were knocked out in T cells using a gRNA vector containing two guide RNAs. Naive T cells were activated using anti-CD3 / CD28 beads (Figure 3A). On day 1, activated T cells were transduced with a guide RNA vector containing sgRNAs targeting CD5 and TRAC, important components of the T cell receptor complex (TCR) (Figure 3B). The next day, the transduced cells were bead-depleted (removal of anti-CD3 / CD28 beads), electroporated with Cas9, and fresh medium containing IL-2 was given every two days. On day 10, the transduced T cells were analyzed for the expression of LNGFR, and the knockout of TCR and CD5. (Figures 3C - 3E). Naive T cells and transduced T cells were stained for cell surface expression of TCR, CD5, and LNGFR and then analyzed by flow cytometry. The results show that untransduced naive T cells maintain the expression of TCR (Figure 1C). However, guide RNA vector-transduced cells show knockout of the T cell receptor complex as a result of the expression of TRAC-targeting sgRNA and electroporation with Cas9. Two versions of the dual-guide cassette vector (pMGH354-CP1780) were tested (Figure 3D). Each version contained a different sgRNA targeting CD5 (CD5 sgRNA #1 or CD5 sgRNA #2). Transduced T cells were stained for CD5 using a fluorescent antibody that detects CD5 and analyzed by flow cytometry. As a negative control, an isotype control, a similar fluorescent antibody lacking specificity for CD5, was used. The results show that CD5 surface expression is lost in T cells transduced with CD5 sgRNA #2, but CD5 expression is maintained in T cells transduced with CD5 sgRNA #1. Overall, the strong CD5 knockout seen with CD5 sgRNA #2 indicates proper functioning of the guide RNA vector. The maintained CD5 expression seen with CD5 sgRNA #1 suggests reduced function of the sgRNA and does not reflect the guide RNA vector itself.Naive T cells that were not transduced and T cells transduced with the gRNA vector were stained for the expression of the LNGFR cell surface marker. The results show LNGFR expression only in T cells transduced with the gRNA vector (Figure 3E).

[0313] Example 6: Abundance of guide RNA polynucleotides in a pooled library as determined by sequencing Naive T cells were activated using anti-CD3 / CD28 beads. On day 1, activated T cells were transduced with a dual-guide cassette vector (pMGH354-CP1780) containing two sgRNAs, a drug-inducible caspase-9 (iCasp9) safety switch, and a truncated LNGFR selection marker (Figures 4A - 4B). On day 5, the transduced cells had the beads removed (anti-CD3 / CD28 beads removed), were electroporated with Cas9, and replenished with fresh medium containing IL-2. On day 8, T cells transduced with pMGH354-CP1780 were left untreated (0 nM rimiducid), treated with vehicle only (DMSO control), or treated with various concentrations of rimiducid to activate the iCasp9 safety switch. Each experimental condition was performed 3 times.

[0314] The response to rimiducid was evaluated by measuring the change in the ratio of guide RNA vector (CAR+ve) cells after treatment (Figure 4C). CountBright TM Absolute counting beads and live / dead cell staining solution were mixed with the cell samples and assayed by flow cytometry 24 and 48 hours after rimiducid treatment. By comparing the ratio of bead events to live cell events, the absolute number and ratio of CAR+ve live cells in the sample were calculated. The results show that the ratio of CAR+ve cells is maintained in the untreated and vehicle-treated control groups. However, in the rimiducid-treated group, a decrease in CAR+ve cells over time is shown. This indicates proper expression and function of the iCasp9 safety switch in T cells transduced with pMGH354-CP1780.

[0315] Example 7: Use of CAR-T cell CRISPR libraries in humans with solid tumors Chimeric antigen receptor (CAR) T cells have achieved durable complete remission in approximately 40% of patients with refractory lymphoma, revolutionizing the treatment landscape for patients with hematologic malignancies. However, some patients ultimately relapse, and CAR T cells have thus far been relatively unsuccessful in patients with progressive solid tumors. While some of this acquired resistance is explained by loss of the CAR target antigen, the major factor in treatment failure in both liquid and solid tumors is the inability of CAR T cells to maintain durable effector function in the face of persistent antigen expression, which is often referred to as “exhaustion.” This evolutionary program limits damage to the host during infection and autoimmune inflammation but creates a major barrier to the efficacy of CAR T cells. In solid tumors, this program is accentuated by the suppressive tumor microenvironment (1, 2). Thus, it is highly important to define genes and pathways that can enhance the efficacy of CAR T cells in both solid and liquid tumors in vivo where factors that cause T cell dysfunction are operative.

[0316] There are various genes and pathways that can potentially be regulated theoretically to improve CAR-T cell responses. However, due to the very large number of candidate genes, in this field, it is substantially impossible to conduct individual investigational new drug (IND) and Phase 1 clinical trials for CAR T cells and verify each potential candidate individually. Data obtained from in vitro and in vivo studies provide valuable insights into the factors governing CAR T cell functionality, while parallel in vitro and in vivo gene screening in solid and liquid cancer models shows that the targets identified by in vitro screening do not necessarily correlate with in vivo screening (3, 4). Some of the target genes identified as regulators of T cell function in in vitro gene screening, such as RASA2, have not been identified as top targets in the in vivo screening disclosed herein (e.g., Figure 10B) (5). Furthermore, preclinical in vivo models regarding the efficacy of CAR T cells do not necessarily predict clinical performance. Thus, gene screening of genes that regulate CAR T cell function in human patients may be the only way to identify genes that improve clinical outcomes. To meet this requirement, in this example, screening of selected target genes in CAR T cells administered to patients is performed, and studies are conducted to enable an IND to initiate this process. The inventors believe that by using large-scale and systematic gene screening in cancer patients, regulators of CAR T cell function in solid tumors can be identified. As described herein, a limited set of loss-of-function (LOF) CRISPR screening is used to identify major regulators of T cell performance in human patients.

[0317] The therapeutic product has autologous T lymphocytes transduced with two lentiviral vectors, one encoding a CRISPR guide library (Mario library) containing sgRNAs targeting 135 genes related to T cell function, and the other encoding a chimeric antigen receptor (CAR) targeting mesothelin using an SS1scFv binder (Meso-Mario-CART cells). The manufacture of the product is designed such that each CAR T cell targeting mesothelin expresses a single variable sgRNA for a specific gene and a single fixed sgRNA for the TCRα chain gene (TRAC). A limited set of libraries of knockout Meso-Mario-CAR T cells are delivered to each patient (Figure 5). The diseases to be treated are highly advanced cancers expressing mesothelin, including ovarian cancer and pancreatic cancer. A specific mesothelin CAR construct has been previously tested in both forms of T cells, those electroporated with RNA and those transduced with lentivirus, in a phase 1 clinical trial targeting these patient populations (SS1-BBz) (6 - 12).

[0318] Mesothelin is highly expressed in ovarian cancer cells compared to normal cells and is an attractive antigen to target with CAR T cells. Ovarian cancer is the fifth leading cause of cancer death in women in the United States (American Cancer Society, 2022). Ninety percent of ovarian cancers are epithelial, and the most common is serous carcinoma. Most serous carcinomas are diagnosed at stage III (51%) or IV (29%), and the 5-year survival rates by cause are 42% and 26% respectively (13). Current treatment options are a combination of debulking surgery, drug therapy, and radiation therapy. However, 70% of ovarian cancer patients relapse after primary tumor debulking surgery and standard first-line chemotherapy (14). The median survival of patients with platinum-resistant or refractory high-grade epithelial ovarian cancer is 9 - 12 months (15). Due to such poor prognosis, this indication represents an unmet medical need. Mesothelin is expressed in 75% of high-grade serous ovarian cancers and is an appropriate target for immunotherapy (16).

[0319] Other malignant tumors that highly express mesothelin include pancreatic adenocarcinoma (16). Pancreatic cancer is one of the most lethal solid tumor malignancies. The 5-year survival rate is 11%, and it is predicted that approximately 50,000 people will die in 2022 (17). Only 10 - 15% of patients have resectable disease at the time of diagnosis (18). In the case of patients with locally advanced and unresectable disease, the treatment regimen usually consists of combination chemotherapy, but 80% of patients do not achieve a response sufficient to undergo surgery (18). Finally, the treatment of metastatic pancreatic cancer consists of a cytotoxic regimen with a median overall response of 6 - 11 months.

[0320] In previous clinical trials of mesothelin-targeted CAR T cells using the single-chain variable fragment derived from SS1 as the binding domain of the CAR, safety was demonstrated in patients with advanced ovarian cancer or pancreatic cancer, but clinically meaningful efficacy was generally lacking (6 - 12). This is thought to be due, in part, to a decrease in the persistence of CAR T cells and the progression of T cell dysfunction in the tumor microenvironment. There are hundreds of candidate genes and pathways that can alter T cell biology, and it is not clear whether it will be possible to conduct a single first-in-human study or randomized study to test the modification of each of these candidate pathways in individual clinical trials or drug development programs. When the therapeutic product is a genetically engineered cell, it is possible to use gene marking itself to test a pool of products containing different genetic modifications and identify the cell product with the highest engraftment rate after injection. In this example, each patient provides information about which genetically modified CAR T cell provides the greatest benefit to that individual. It is also possible to identify the CAR T product with the highest engraftment rate and / or the highest persistence across multiple patients and two diseases expressing the same target antigen (mesothelin in this case).

[0321] CRISPR screening is based on generating a pool of cells with one gene knocked out from a library using a lentiviral library of guide RNAs in combination with transient expression of Cas9 nuclease. Thus, CRISPR screening in human patients can be used to rapidly identify, from a selected library of genes involved in T cell biology, which gene knockouts result in increased engraftment, persistence, and response in patients with progressive solid tumors expressing mesothelin. The inventors believe that Meso-Mario CAR T cells may provide future benefits to individual patients by improving effector function and having a defined specificity (based on the SS1-BBz CAR construct) that has previously been shown to be safe by allowing the therapeutic product to take "multiple shots at the goal" (6, 7). Using this approach, single gene knockouts that result in higher engraftment, persistence, and response across patients or two diseases can be rapidly identified beyond future benefits to individual human subjects; if so, in the context of Meso-CAR-T, the most common drug development pathway of incorporating that specific single gene knockout can be individually pursued to maximize benefits to the most patients.

[0322] Product Description and Design The therapeutic medicinal product is autologous T lymphocytes transduced with two lentiviral vectors - one encoding a CRISPR sgRNA library (Mario library) targeting 135 genes related to T cell function (Table 1) and the TCRα chain (TRAC), and the other encoding a chimeric antigen receptor (CAR) targeting mesothelin (Meso-Mario-CART cells). Example 6 Table 1: Genes included in the sgRNA library (Mario library) of the Meso-Mario-CART cell product. TIFF2025518033000003.tif108170

[0323] Design of CRISPR sgRNA Library The CRISPR library was designed to optimally deconvolve data obtained from each treatment target to maximize safety, the promise of benefit, and learning in the field. 135 genes were selected based on literature search and tested genes (i.e., IFNG, ITK, PDCD1, HAVCR2) (Figure 6). Subsequently, the major transcript of each gene was selected and 34,318 guides were designed using CRISPick (30). To minimize the potential off-target effects of sgRNA guides, guides predicted to have off-target cleavage in tumor suppressor genes were removed (31). Guide RNAs targeting regions of high variability in the human population were removed. The Cutting Frequency Determination (CFD) score was used to reduce the risk of off-target activity of any guide (30). Guides with a total CFD off-target score of 0.27 or less were removed. For each gene, a rule set 3 score incorporating multiple sequence features contributing to sgRNA expression and the generation of on-target gene editing was used to select 8 sgRNAs per gene (32). Finally, 100 validated intergenic control guides were included for a total of 1180 guides in the library. When combined with Cas9 protein (delivered in the form of mRNA by electroporation), cells transduced with the Mario library are expected to have two genes, the TCRα chain and a single gene from the library, knocked out.

[0324] Design of Lentiviral Vector Two lentiviral vector constructs were designed to safely deliver a CRISPR sgRNA library (pMario) and a chimeric antigen receptor targeting mesothelin (pCAR, Figure 7A). For the construct containing the CRISPR library (pMario), an additional layer of safety was added by incorporating an inducible caspase 9 (iCasp9)-based safety switch that can remove CAR T cells in the event of severe adverse events or expansion of CAR T cell clones. To optimize the expression of the two sgRNAs, a dual sgRNA cassette was designed in which the sgRNA library was cloned at position 1 and a sgRNA against the T cell receptor α constant gene (TRAC) was included at position 2. Since an intact TCRα chain is required for the assembly of the native T cell receptor / CD3 complex, knockout of TRAC results in loss of expression of the entire native TCR / CD3 complex. Thus, the TRAC-targeting guide enables negative selection of the edited cells during manufacturing (using CliniMAC for CD3-based magnetic selection of investigational products). Clinically, this selection for CD3-negative T cells also minimizes the risk of enhancing autoreactive T cells (based on TCRs with retained undefined specificities). Finally, a truncated low-affinity nerve growth factor receptor (tLNGFR) reporter was included to enable flow cytometry-based aggregation, recovery, and enrichment of the cells after injection, and to facilitate further analysis of the enriched and depleted sgRNAs. The pCAR vector (Figure 7A) was designed to contain a CAR construct consisting of an SS1 scFv that binds to mesothelin and a 4-1BB intracellular co-stimulatory domain (6 - 11). This construct also contains a CD34t reporter that enables dosing and post-injection monitoring of CAR+ T cells.

[0325] Verification of the sgRNA double cassette and reporter system To determine the reporter expression and editing efficiency of the dual sgRNA cassette, bead-activated T cells (CD3 / CD28 beads at a bead:T cell ratio of 3:1) were transduced with a dual guide vector encoding sgRNAs targeting CD5 and TRAC. After removing the beads, the cells were electroporated with Cas9 mRNA. Next, naive T cells and transduced T cells were analyzed by flow cytometry for cell surface expression of tLNGFR, CD3, and CD5 (Figures 7B - 7D). Isotype controls, which are similar fluorescent antibodies lacking specificity for the targets being analyzed, were used as negative controls. The results show that LNGFR was expressed in dual guide vector-transduced T cells (Figure 7B). Untransduced naive T cells maintain the expression of TCR (CD3), while dual guide vector-transduced cells lack CD3 expression, which is a result of successful targeting of TRAC by the sgRNA after Cas9 electroporation (Figure 7C). LNFGR+ cells also lack CD5 expression, indicating the proper function of the dual guide cassette vector.

[0326] Generation of Meso-Mario-CART cells for in vivo testing A protocol for generating Meso-Mario-CART cells for in vivo testing was determined. On day 0, T cells were activated using CD3 / CD28 beads. The cells were then transduced with a lentivirus carrying an anti-mesothelin CAR. On day 2, the cells were transduced with a lentiviral vector containing a CRISPR (Mario) library. Next, on day 5, the cells were transfected with Cas9 mRNA via electroporation. The cells were grown until day 10, and CD3-negative cells were enriched by depleting CD3-positive cells using an APC-CD3 selection kit. After two rounds of selection, this protocol was able to generate a final product containing up to 88% dual-transduced cells (mCherry+CD3-) (Figures 8A - 8C).

[0327] In vivo pilot study of CRISPR screening As a preparation for human CRISPR screening, an in vivo pilot CRISPR screening was performed in NSG mice (Figure 9). The described Mario sgRNA library targeting 135 selected relevant T cell regulators was generated with 8 sgRNAs per gene. This library and the mesothelin CAR were transduced into T cells, and Cas9 mRNA was electroporated into the T cells as described. T cells transduced with the library were transplanted into mice bearing mesothelin+ASPC1 tumors. After 14 days, following digestion and positive selection for human CD45, CAR T cells were isolated from the tumors. Data were generated by PCR amplification of sgRNAs from the gDNA of the isolated T cells followed by next-generation sequencing (NGS).

[0328] CAR T cells recovered from the tumors and spleens of mice bearing ASPC1 tumors demonstrated the ability to engraft a large number of tumor-infiltrating mesothelin CAR T cells and efficiently capture T cells from the tumors and spleens, enabling a technically robust in vivo screening experiment even in a small number of animals (Figure 10A). The distribution of sgRNAs in the recovered CAR T cells indicated that the complete CRISPR library could be captured, as well represented by the cell pool from which the guides were recovered.

[0329] These results identified that the top hits during in vivo screening were actually the exact opposite of the top predicted hits from the published genome-wide CRISPR screens performed in vitro (5). In these results, knockout of IL2RA was enriched in vivo, while knockout of RASA2 was depleted (Figure 10B). In previous in vitro screening, RASA2 was one of the most enriched gene knockouts in T cells. Furthermore, these data indicate that using in vitro models to predict the best gene knockout targets to enhance CAR T cell function may not actually be able to predict in vivo function or activity. Similar results were observed using BCMA CAR, indicating that the effect of gene knockout is not CAR-specific (see Example 8).

[0330] Generation of a large-scale sgRNA plasmid library Based on the results of preclinical data, the feasibility of creating a plasmid library (Mario library) in a large scale and in a methodology compliant with investigational new drug (IND) in a quality sufficient to enable the production of lentiviral vectors of pharmaceutical good manufacturing practice (cGMP) grade was evaluated. To evaluate the quality of the large-scale guide library, the guide distribution of the original plasmid library stock was compared with the large-scale IND-compliant plasmid preparation. In large-scale preparation, first, the guide abundance in the plasmid was analyzed using next-generation sequencing (NGS) of the PCR products adjacent to the guide region. Next, primary human T cells were transduced with lentivirus prepared together using either the original plasmid primary library stock or the large-scale plasmid preparation.

[0331] The results indicate that the large-scale plasmid preparation process did not significantly alter the distribution of plasmids in the library (Figure 11A). Furthermore, since the distribution of the guides in the transduced T cells did not significantly change (Figure 11B), this plasmid preparation can be used to generate lentivirus and cell products suitable for clinical-grade screening studies. Figure 12 outlines the cGMP-compliant manufacturing process for the Meso-Mario-CAR T cell product (Figure 12).

[0332] Treatment plan Human patients can receive three days of lymphodepleting chemotherapy starting approximately five days before the infusion of Meso-Mario-CART cells on day 0 (Figure 13). Lymphodepletion can be performed either outpatient or inpatient, at the discretion of the healthcare provider. After CAR T cell infusion, patients are monitored for adverse events, clinical status, and laboratory parameters. Research samples are collected for up to 24 months at the indicated time points (Figure 13 and Figure 14) for correlative studies. Research samples are analyzed using the methods described herein (e.g., Examples 4-6).

[0333] References 1. Larson RC, Kann MC, Bailey SR, Haradhvala NJ, Llopis PM, Bouffard AA, et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature [Internet]. April 21, 2022 [cited March 2, 2023];604(7906):563-70. Available from: https: / / www.nature.com / articles / s41586-022-04585-5 2. Bailey SR, Vatsa S, Larson RC, Bouffard AA, Scarfo I, Kann MC, et al. Blockade or Deletion of IFNγ Reduces Macrophage Activation without Compromising CAR T cell Function in Hematologic Malignancies. Blood Cancer Discov [Internet]. March 1, 2022 [cited March 2, 2023]; 3(2):136 - 53. Available from: https: / / aacrjournals.org / bloodcancerdiscov / article / 3 / 2 / 136 / 681793 / Blockade-or-Deletion-of-IFN-Reduces-Macrophage 3. Lin S, Larrue C, Scheidegger NK, Seong BKA, Dharia NV, Kuljanin M, et al. An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML. Cancer Discov [Internet]. February 1, 2022 [cited March 2, 2023]; 12(2):432 - 49. Available from: https: / / aacrjournals.org / cancerdiscovery / article / 12 / 2 / 432 / 678482 / An-In-Vivo-CRISPR-Screening-Platform-for 4. Michels BE, Mosa MH, Streibl BI, Zhan T, Menche C, Abou-El-Ardat K, et al. Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids. Cell Stem Cell [Internet]. May 2020 [cited Mar 2, 2023];26(5):782-792.e7. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S1934590920301429 5. Carnevale J, Shifrut E, Kale N, Nyberg WA, Blaeschke F, Chen YY, et al. RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature [Internet]. Sep 1, 2022 [cited Mar 2, 2023];609(7925):174-82. Available from: https: / / www.nature.com / articles / s41586-022-05126-w 6. Haas AR, Tanyi JL, O’Hara MH, Gladney WL, Lacey SF, Torigian DA, et al. Phase I Study of Lentiviral-Transduced Chimeric Antigen Receptor-Modified T Cells Recognizing Mesothelin in Advanced Solid Cancers. Mol Ther [Internet]. Nov 2019 [cited Mar 15, 2023];27(11):1919-29. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S1525001619303284 7. Beatty GL, O’Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al. Activity of Mesothelin-Specific Chimeric Antigen Receptor T Cells Against Pancreatic Carcinoma Metastases in a Phase 1 Trial. Gastroenterology [Internet]. July 2018 [cited March 15, 2023];155(1):29-32. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S0016508518303238 8. Tanyi JL, Stashwick C, Plesa G, Morgan MA, Porter D, Maus MV, et al. Possible Compartmental Cytokine Release Syndrome in a Patient With Recurrent Ovarian Cancer After Treatment With Mesothelin-targeted CAR-T Cells. J Immunother [Internet]. April 2017 [cited April 3, 2023];40(3):104-7. Available from: https: / / journals.lww.com / 00002371-201704000-00003 9. Ko AH, Jordan AC, Tooker E, Lacey SF, Chang RB, Li Y, et al. Dual Targeting of Mesothelin and CD19 with Chimeric Antigen Receptor-Modified T Cells in Patients with Metastatic Pancreatic Cancer. Mol Ther [Internet]. November 2020 [cited April 3, 2023];28(11):2367-78. Available from: https: / / linkinghub.elsevier.com / retrieve / pii / S1525001620303683 10. Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, et al. T Cells Expressing Chimeric Antigen Receptors Can Cause Anaphylaxis in Humans. Cancer Immunol Res [Internet]. July 1, 2013 [cited April 3, 2023]; 1(1):26 - 31. Available from: https: / / aacrjournals.org / cancerimmunolres / article / 1 / 1 / 26 / 466725 / T-Cells-Expressing-Chimeric-Antigen-Receptors-Can 11. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, et al. Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies. Cancer Immunol Res [Internet]. February 1, 2014 [cited April 3, 2023]; 2(2):112 - 20. Available from: https: / / aacrjournals.org / cancerimmunolres / article / 2 / 2 / 112 / 467025 / Mesothelin-Specific-Chimeric-Antigen-Receptor-mRNA 12. Adusumilli PS, Zauderer MG, Riviere I, Solomon SB, Rusch VW, O’Cearbhaill RE, et al. A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease, in Combination with the Anti-PD-1 Agent Pembrolizumab. Cancer Discov [Internet]. November 1, 2021 [cited April 4, 2023];11(11):2748-63. Available from: https: / / aacrjournals.org / cancerdiscovery / article / 11 / 11 / 2748 / 666400 / A-Phase-I-Trial-of-Regional-Mesothelin-Targeted 13. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, et al. Ovarian cancer statistics, 2018: Ovarian Ca...

Claims

1. A composition comprising multiple guide RNA (gRNA) polynucleotides, wherein at least two gRNA polynucleotides each contain homologous regions complementary to the sense strand or antisense strand of different genes encoded by any one sequence or variant thereof of sequence numbers 1 to 135.

2. The composition according to claim 1, comprising at least eight gRNA polynucleotides per gene sequence, wherein at least eight gRNA polynucleotides per gene sequence comprise different sequences.

3. The composition according to claim 2, wherein at least eight gRNA polynucleotides per gene sequence are complementary to non-overlapping regions of the same gene sequence.

4. The composition according to claim 1, wherein the composition comprises at least one gRNA polynucleotide having one of the sequences of sequence numbers 136 to 1315.

5. The composition according to claim 1, wherein the composition comprises at least 10 gRNA polynucleotides, each containing one different sequence from sequence numbers 136 to 1315.

6. A composition comprising multiple gRNA polynucleotides containing sequences 136 to 1315.

7. The composition according to claim 1, wherein the multiple guide RNA polynucleotides consist of at most 5,000 gRNA polynucleotides having different sequences.

8. The composition according to claim 1, wherein at least one gRNA polynucleotide each comprises a homologous region that is complementary to the sense strand or antisense strand of a different gene encoded by any one of the following: IL2RA (SEQ ID NO: 65) or a variant thereof, GATA3 (SEQ ID NO: 46) or a variant thereof, AGPS (SEQ ID NO: 3) or a variant thereof, PTPN2 (SEQ ID NO: 98) or a variant thereof, LAG3 (SEQ ID NO: 76) or a variant thereof, PDCD1 (SEQ ID NO: 91) or a variant thereof, TGFBR2 (SEQ ID NO: 121) or a variant thereof, RARA (SEQ ID NO: 99) or a variant thereof, SmarcB1 (SEQ ID NO: 113) or a variant thereof, CDKN1B (SEQ ID NO: 18) or a variant thereof, RunX (SEQ ID NO: 107) or a variant thereof, and TCF7 (SEQ ID NO: 118) or a variant thereof.

9. A first gRNA polynucleotide containing a homologous region complementary to the sense strand or antisense strand of a gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof; A second gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof; A third gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof; A fourth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of the gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof; A fifth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of the gene encoded by LAG3 (SEQ ID NO: 76) or its variant; A sixth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by PDCD1 (SEQ ID NO: 91) or a variant thereof; A seventh gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by TGFBR2 (SEQ ID NO: 121) or a variant thereof; An eighth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of the gene encoded by RARA (SEQ ID NO: 99) or its variant; A ninth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by SmarcB1 (SEQ ID NO: 113) or a variant thereof; A tenth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof; An eleventh gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of a gene encoded by RunX (SEQ ID NO: 107) or a variant thereof; and / or, A twelfth gRNA polynucleotide containing a homologous region complementary to the sense or antisense strand of the gene encoded by TCF7 (SEQ ID NO: 118) or its variants. The composition according to claim 1, comprising:

10. A guide RNA polynucleotide containing one of the sequences from sequence numbers 136 to 1315.

11. A plurality of plasmids comprising one or more gRNA polynucleotides of the composition described in claim 1.

12. A CAR vector comprising the sequence of sequence number 1319.

13. A composition comprising one of the gRNA polynucleotides of the composition described in claim 1, and a CAR vector containing the sequence of SEQ ID NO: 1319.

14. A plurality of CAR-T cells, Multiple CAR-T cells, each comprising at least two CAR-T cells, each containing a gRNA polynucleotide that includes a homologous region complementary to the sense or antisense strand of a different gene encoded by one of sequence numbers 1 to 135 or a variant thereof.

15. A method for creating a mutant CAR-T cell library, (a) Activating T cells, (b) Transfecting T cells with the gRNA polynucleotide described in claim 6, (c) Transfecting T cells with a CAR vector containing the sequence of Sequence ID No. 1319, and (d) Introducing mRNA encoding the CRISPR protein or the CRISPR protein into T cells. Methods that include...

16. A method for identifying gRNA polynucleotides associated with the efficacy of CAR-T cells in vivo, (a) Sequence gRNA polynucleotides from multiple mutant CAR-T cells collected from the subject, and (b) Includes evaluating the change in the relative abundance of each gRNA polynucleotide based on the sequencing in (a), The method comprises the plurality of mutant CAR-T cells described in claim 14.

17. A plurality of CAR natural killer (CAR-NK) cells, wherein at least two of the CAR-NK cells contain a gRNA polynucleotide that comprises a homologous region complementary to the sense strand or antisense strand of a different gene encoded by any one sequence or variant thereof of sequence numbers 1 to 135.

18. A method for creating a mutant CAR-NK cell library, (a) Activating CAR-NK cells, (b) Transduction of the gRNA polynucleotide described in claim 6 into CAR-NK cells, (c) Transduction of a CAR vector containing the sequence of Sequence ID No. 1319 into CAR-NK cells, and (d) A method comprising introducing mRNA encoding the CRISPR protein or the CRISPR protein into CAR-NK cells.

19. A mutant CAR-T cell library for administration to human subjects, The mutant CAR-T cell library comprises at least two CAR-T cells, each containing a different gRNA polynucleotide that includes a homologous region complementary to a different gene sequence of any one of sequence numbers 1 to 135 or a variant thereof.