Methods and compositions for immune cell CRISPR screening

Abstract

Disclosed, in part, are methods and compositions for mutating immune cells (e.g., CAR-T cells) and determining cancer killing efficacy of mutated immune cells in vivo.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 344,951, filed on May 23, 2022, entitled “Methods and Compositions for Immune Cell CRISPR Screen,” and U.S. Provisional Application No. 63 / 464,483, filed on May 5, 2023, entitled “Methods and Compositions for Immune Cell CRISPR Screen,” under 35 U.S.C. §119(e), the entire contents of each of which are incorporated herein by reference. Background Art

[0003] Cancer treatment has been radically changed by reprogramming immune cells to include chimeric antigen receptors (CAR), which guide immune cells to target cancers and induce cancer cell killing after binding. CAR is a transmembrane protein, which generally includes an extracellular portion (such as an antigen binding domain), a transmembrane domain, and an intracellular binding domain. The antigen binding domain is usually but not always an antibody that binds to a cancer-associated antigen. After the combination of the antigen binding domain, the intracellular domain activates immune cells (such as T cells), which can cause immune cells to kill cancer cells. However, many factors can affect the effectiveness of CAR-based treatment in killing cancer, including causing a type of CAR-based treatment to have a higher therapeutic efficacy in some patients compared to other patients. Special patient characteristics. Therefore, it is generally necessary to treat patients with personalized CAR treatment in terms of treating specific diseases. Summary of the invention

[0004] 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 immune cell genotypes that enhance CAR immune cancer efficacy. For example, the present disclosure describes a CRISPR guide RNA polynucleotide library, which is designed to target genes that are considered to be related to CAR-T cell efficacy. Therefore, in some embodiments, the CRISPR guide RNA library can be used to produce a library of mutant CAR-T cells, and the mutant CAR-T cell library includes CAR-T cells with many different genotypes and, for example, includes the same CAR. The present disclosure is partly related to the discovery that its changes can affect the collection of genes of CAR-T cell efficacy (e.g., tumor killing and persistence), and the subsequent development of CRISPR guide RNA libraries to target the gene collection. In some embodiments, one or more of the genotypes of the mutant CAR-T cell library improves CAR-T cell efficacy (e.g., tumor killing and persistence). In addition, these mutations may have different effects in different patients and / or when treating different cancers. In some embodiments, the methods described herein include generating a mutated CAR-immune cell library (e.g., a mutated CAR-T cell library or a mutated CAR-NK cell library); applying 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 polynucleotides of the CAR-T cells to identify which CAR-T gene mutants have the most increased prevalence in the sample, thereby serving as an indicator of CAR-T cell efficacy. In some embodiments, the method also includes administering to the patient a CAR-T cell comprising a mutation that increases the prevalence of CAR-T cells in vivo (as determined using the above method). Therefore, in some embodiments, these methods provide strategies for identifying patient-specific and / or cancer-specific CAR-T cell genomic mutations that enhance cancer killing efficacy in vivo.

[0005] Compared with the previous prior art, the compositions and methods described herein provide advantages. When the experiments are adjusted and repeated in different organisms (e.g., in humans), the results obtained from the experiments conducted in model organisms are generally not reproduced. For example, CD19-28z cytotoxic T cells eradicate B cell tumors in mice, but have little effect on cancer in humans. 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-guided CAR-T cells kill cancer in mice, but have little effect on cancer in humans. 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 humans. The CAR-immune cell genotype (e.g., CAR-T cell genotype or CAR-NK cell genotype) identified as improving cancer killing efficacy in humans does not require adjustment from potential non-similar model organisms, and the observed therapeutic effects can be directly applied to humans, such as those screened in vivo. The present disclosure also provides CAR vectors and CRISPR vectors designed for humans, for example, comprising CAR incorporation markers and CRISPR mutation markers compatible with human use.

[0006] In addition, the present disclosure provides methods and compositions capable of providing conclusive screening results in people.The present disclosure provides strategies for determining the characteristics (e.g., size) of guide RNA libraries, which can be used in people and realize sufficient representation of mutated CAR-immune cell libraries for calculations about the phenotype of interest (e.g., cancer killing efficacy) (for calculation details, see, e.g., 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 holo-genome. It is not desirable to be bound by theory, and the holo-genome gRNA library may be too large and contain too many different gRNA polynucleotides to provide conclusive results in people, for example, because the holo-genome CRISPR library contains so many different mutant immune cells (e.g., mutant CAR-T cells) that they may not be fully represented in samples collected from patients, resulting in poor statistical resolution and the mutation that contributes to phenotype (e.g., CAR-T cell efficacy) cannot be identified.

[0007] The present disclosure provides, in part, selected gene sets targeted by guide RNAs of CRISPR libraries suitable for humans (e.g., taking into account expected limitations on guide RNA library size). In some embodiments, the selected genes include genes expected to affect CAR-T cell cancer killing efficacy or persistence and control genes expected not to affect CAR-T cell cancer killing efficacy.

[0008] In some aspects, the present disclosure describes a composition comprising multiple guide RNA (gRNA) polynucleotides, wherein at least 2 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by a sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0009] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 10 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0010] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 20 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0011] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 50 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0012] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 100 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0013] In some embodiments, the composition comprises a plurality of guide RNA (gRNA) polynucleotides, wherein at least 135 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0014] 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 homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

[0015] In some embodiments, at least 10 gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, at least 20 gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, at least 50 gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, at least 100 gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, at least 135 gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.

[0016] In some embodiments, the composition comprises at least 2 gRNA polynucleotides of each gene sequence, wherein the at least 2 gRNA polynucleotides of each gene sequence comprise different sequences. In some embodiments, the composition comprises 8 gRNA polynucleotides of each gene sequence, wherein the 8 gRNA polynucleotides of each gene sequence comprise different sequences. In some embodiments, at least 2 or at least 8 gRNA polynucleotides of each gene sequence are complementary to the non-overlapping regions of the same gene sequence.

[0017] 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 homology region. In some embodiments, the negative control gRNA polynucleotide comprises a homology region complementary to a gene not known to participate in T cell function. In some embodiments, the negative control gRNA polynucleotide comprises a homology region, and the homology region is a complementary intergenic region.

[0018] In some embodiments, the composition comprises at least one gRNA polynucleotide containing the sequence of any one of SEQ ID NOs: 136 to 1315. In some embodiments, the composition comprises at least 10 gRNA polynucleotides each containing 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 containing 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 containing a different sequence of any one of SEQ ID NOs: 136 to 1315. In some aspects, the present application discloses such a composition comprising a plurality of gRNA polynucleotides containing sequences of SEQ ID NOs: 136 to 1315. In some embodiments, the plurality of guide RNA polynucleotides consists of up to 200,000 gRNA polynucleotides having different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of up to 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.

[0019] In some embodiments, the multiple guide RNA polynucleotides consist of up to 15,000 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 10,000 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 5,000 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 2,000 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 1,080 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 500 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 250 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 100 gRNA polynucleotides with different sequences. In some embodiments, the multiple guide RNA polynucleotides consist of up to 50 gRNA polynucleotides with different sequences. In some embodiments, the plurality of guide RNA polynucleotides consists of up to 10 gRNA polynucleotides having different sequences.

[0020] In some embodiments, at least some of the multiple gRNA polynucleotides do not cross-react with more than one gene. In some embodiments, the composition does not include a gRNA homology region complementary to the sense strand or antisense strand of a gene encoding p53, Rb, PTEN, BRCA1 and / or BRCA2 or its variants. In some embodiments, the composition does not include a gRNA complementary to the sense strand 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 its variants.

[0021] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof.

[0022] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof.

[0023] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof.

[0024] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof.

[0025] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by LAG3 (SEQ ID NO: 76) or a variant thereof.

[0026] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by PDCD1 (SEQ ID NO: 91) or a variant thereof.

[0027] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by TGFBR2 (SEQ ID NO: 121) or a variant thereof.

[0028] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by RARA (SEQ ID NO: 99) or a variant thereof.

[0029] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by SmarcB1 (SEQ ID NO: 113) or a variant thereof.

[0030] In some embodiments, at least 1 gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof.

[0031] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by RunX (SEQ ID NO: 107) or a variant thereof.

[0032] In some embodiments, at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by TCF7 (SEQ ID NO: 118) or a variant thereof.

[0033] In some embodiments, at least 2 gRNA polynucleotides each comprise a homology region complementary to the sense strand 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, at least 5 gRNA polynucleotides each comprise a homology region complementary to the sense strand 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, at least seven gRNA polynucleotides each comprise a homology region complementary to the sense strand 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 10 gRNA polynucleotides comprises a homology region complementary to the sense strand 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, each of the 14 gRNA polynucleotides comprises a homology region complementary to the sense strand 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.

[0038] In some embodiments, the composition comprises: a first gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof; a second gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof; a third gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof; a fourth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof; a fifth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by LAG3 (SEQ ID NO: 46) or a variant thereof. NO:76) or its variants encoded homology region complementary to the sense strand or antisense strand of the gene encoded by PDCD1 (SEQ ID NO:91) or its variants; a seventh gRNA polynucleotide, the seventh gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by TGFBR2 (SEQ ID NO:121) or its variants; an eighth gRNA polynucleotide, the eighth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by RARA (SEQ ID NO:99) or its variants; a ninth gRNA polynucleotide, the ninth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by SmarcB1 (SEQ ID NO:113) or its variants; a tenth gRNA polynucleotide, the tenth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by CDKN1B (SEQ ID NO:121) or its variants. NO: 18) or its variants or a homology region complementary to the sense strand or antisense strand of the gene encoded by RunX (SEQ ID NO: 107) or its variants; and / or a twelfth gRNA polynucleotide, the twelfth gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by TCF7 (SEQ ID NO: 118) or its variants.

[0039] In some embodiments, the composition does not include a gRNA polynucleotide containing a homology region complementary to the sense strand or antisense strand of the gene encoded by RASA2 (SEQ ID NO: 102).

[0040] In some embodiments, the composition does not include a gRNA polynucleotide containing a homology region complementary to the sense strand or antisense strand of such a gene, wherein the gene is 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.

[0041] In some embodiments, the composition does not include a gRNA polynucleotide containing a homology region complementary to the sense strand or antisense strand of such genes, wherein the genes are 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.

[0042] In some embodiments, the composition does not include a gRNA polynucleotide containing a homology region complementary to the sense strand or antisense strand of such genes, wherein the genes are encoded by at least five 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.

[0043] In some embodiments, the multiple gRNA polynucleotides are Cas9 protein gRNA polynucleotides or Cas12 protein gRNA polynucleotides. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 15,000 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 10,000 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 5,000 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 2,500 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 1,000 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 500 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 200 different gene sequences. In some embodiments, the multiple gRNA polynucleotides include sequences complementary to up to 135 different gene sequences. In some embodiments, the composition is used for human CAR-T cell screening.

[0044] In some aspects, the present application discloses a guide RNA polynucleotide comprising the sequence of any one of SEQ ID NOs: 136 to 1315.

[0045] In some aspects, the application discloses a plasmid comprising any of the gRNA polynucleotides of any of the compositions described herein or a guide RNA described herein.

[0046] In some aspects, the present application discloses such a plasmid library, which comprises at least 2 plasmids, any of the gRNA polynucleotides of any composition described herein, or the guide RNA described herein. In some embodiments, at least some of the at least 2 plasmids comprise the gRNA polynucleotides of the composition described in any one of claims 1 to 72 or the guide RNA polynucleotides of claim 74. In some embodiments, each plasmid comprises the gRNA polynucleotides of the composition described herein or the guide RNA polynucleotides described herein.

[0047] In some aspects, the present application discloses a plasmid library comprising each of SEQ ID NOs: 136 to 1315 encoded on different plasmids.

[0048] In some aspects, the present application discloses a gRNA vector comprising: (1) a first gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand encoded by the TCR / CD3 complex of a T cell (SEQ ID NOs: 1383 to 1398); and (2) a second gRNA polynucleotide.

[0049] In some embodiments, the second gRNA polynucleotide comprises any one of the gRNA polynucleotides of the composition described herein or the guide RNA described herein. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in reverse orientation (inverted orientation). In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in tandem orientation (tandem orientation). In some embodiments, each gRNA coding sequence is operably connected 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 U6 and EF1a promoters. In some embodiments, the promoter is a U6 promoter or a H1 promoter, optionally wherein the first gRNA polynucleotide is operably connected to the U6 promoter and the second gRNA polynucleotide is operably connected to the H1 promoter.

[0050] In some embodiments, the gRNA vector further comprises a selectable marker, optionally wherein the selectable marker is compatible with human use. In some embodiments, the selectable marker is selected from LGNFR, EGFR, CD19, CD20, CD34, and truncated forms thereof. In some embodiments, the selectable marker is operably linked to a promoter, which is optionally a weak promoter. In some embodiments, the promoter is selected from PGK, CMV, EF1a, and a tissue-specific promoter.

[0051] 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.

[0052] In some embodiments, the vector comprises a suicide gene. In some embodiments, the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20 and tHer2. In some embodiments, the gRNA vector comprises the nucleic acid sequence of any one of SEQ ID NO: 1318.

[0053] In some embodiments, the gRNA vector further comprises a polynucleotide encoding a CRISPR protein. In some embodiments, the CRISPR protein is Cas9 or Cas12.

[0054] In some aspects, the present application discloses such a gRNA vector library, which comprises at least 2 gRNA vectors described herein. In some embodiments, each vector comprises a gRNA polynucleotide of a composition described herein or a guide RNA described herein. In some embodiments, at least 10 vectors of the library each comprise a different gRNA polynucleotide homology region, and the different gRNA polynucleotide homology regions are complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NO: 1 to 135 or its variants.

[0055] In some embodiments, the gRNA vector library is a current 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.

[0056] In some embodiments, at least 20 vectors of the library each comprise a different gRNA polynucleotide homology 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 variants thereof. In some embodiments, at least 50 vectors of the library each comprise a different gRNA polynucleotide homology 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 variants thereof. In some embodiments, at least one hundred vectors of the library each comprise a different gRNA polynucleotide homology 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 variants thereof. In some embodiments, each of the at least one hundred thirty-five vectors of the library comprises a different gRNA polynucleotide homology 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.

[0057] In some aspects, the present application discloses such a CAR vector, which comprises 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.

[0058] In some embodiments, the CAR is operably linked to a promoter, which is optionally an EF1a promoter or an EF1a-short promoter.

[0059] In some embodiments, the vector comprises a suicide gene. In some embodiments, the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20 and tHer2. In some embodiments, the vector comprises a reporter. In some embodiments, the reporter is selected from truncated CD34, tEGFR, tCD19, tCD20, tCD34 and tHer2. In some embodiments, CAR comprises a polynucleotide encoding an antigen binding domain, which binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and claudin 6, binds to any one of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain.

[0060] 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 α, β or ζ chain of the T cell receptor,

[0061] CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11 a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), 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 CD28, 4-1BB, CD27, TCR-ζ, FcR-γ, FcR-β, CD3-γ, CD3-θ, CD3-σ, CD3-η, CD3-ε, CD3-ζ, CD22, CD79a, CD79b, and CD66d.

[0062] In some embodiments, the 2A ribosome skipping element or internal ribosome entry site (internalribosome entry site, IRES) is downstream of CAR. In some embodiments, the CAR vector comprises a sequence of SEQ ID NO: 1319. In some aspects, the present application discloses such a composition comprising a gRNA vector as described in any one of the claims herein and a CAR vector as described herein.

[0063] In some aspects, the present application discloses such a CAR-T cell, comprising a composition containing a gRNA vector as described in any one of the claims described herein and a CAR vector as described herein. In some aspects, the present application discloses such a CAR-T cell, the CAR-T cell comprising one, two or three of the following: a mutation in a gene corresponding to a gene sequence of any one of SEQ ID NO: 1 to 135 or a variant thereof, a gRNA polynucleotide of a composition as described herein, a plasmid as described herein, or a gRNA vector as described herein. In some embodiments, the CAR-T cell also comprises a CAR vector as described herein.

[0064] 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 cell also comprises an mRNA encoding a CRISPR protein. In some embodiments, the CAR-T cell is CD34 positive and CD3 negative. In some embodiments, the T cells used to prepare the CAR-T cells are from a mammalian subject. In some embodiments, the T cells used to prepare the CAR-T cells are from a human subject. In some embodiments, the T cells used to produce the CAR-T cells are collected using leukocyte apheresis.

[0065] In some embodiments, CAR-T cells are activated. In some embodiments, CAR-T cells are activated using anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, CAR-T cells are activated using anti-CD3 antibodies. In some embodiments, CAR-T cells are activated using anti-CD3 antibodies and IL-2. In some embodiments, CAR-T cells are activated using anti-CD3 antibodies, IL-2 and PHA. In some embodiments, anti-CD3 antibodies, anti-CD28 antibodies, IL2 and PHA are soluble or plate-bound. In some embodiments, CRISPR protein is Cas9 or Cas12. In some embodiments, cells do not include DNA polynucleotides encoding CRISPR proteins. In some embodiments, CAR-T cells are CD34 positive, CD3 negative and LNGFR positive.

[0066] In some embodiments, the CAR-T cells of the library are selected from the CAR-T cells described herein. In some embodiments, at least 2 CAR-T cells of the library each contain different gRNA polynucleotides, and the different gRNA polynucleotides contain homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 10 CAR-T cells of the library each contain different gRNA polynucleotides, and the different gRNA polynucleotides contain homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 20 CAR-T cells of the library each contain different gRNA polynucleotides, and the different gRNA polynucleotides contain homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 50 CAR-T cells of the library each contain different gRNA polynucleotides, and the different gRNA polynucleotides contain homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 100 CAR-T 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 to 135 or variants thereof. In some embodiments, 135 CAR-T 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 to 135 or variants thereof.

[0067] In some embodiments, SEQ ID NOs: 136 to 1315 are each present in at least one CAR-T cell.

[0068] In some aspects, the present application discloses a method for generating a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting T cells with the gRNA vector library described herein; (c) transfecting T cells with the CAR vector described herein; and (d) introducing mRNA encoding a CRISPR protein or a CRISPR protein into T cells.

[0069] In some aspects, the present application discloses a method for producing a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting T cells with the gRNA vector library described herein; and (c) transfecting T cells with the CAR vector described in any one of claims C1 to C11; wherein the gRNA vector or the CAR vector comprises a polynucleotide encoding a CRISPR protein.

[0070] In some embodiments, the method further includes collecting T cells from the subject. In some embodiments, the method further includes purifying T cells comprising a CAR vector and a CRISPR-induced genetic mutation. In some embodiments, T cells are collected using leukocyte removal. In some embodiments, T cells are collected from human or mouse subjects. In some embodiments, T cells are activated using anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the gRNA vector library is transduced with a multiplicity of infection (MOI) of 0.1 to 1. In some embodiments, the MOI is 0.3 to 0.7. In some embodiments, the MOI is 0.4 to 0.6. In some embodiments, the MOI is 0.5. In some embodiments, the CAR vector is transduced with an MOI of up to 20.

[0071] In some embodiments, the CRISPR protein is Cas9 or Cas12. In some embodiments, CRISPR protein mRNA or CRISPR protein is introduced using electroporation. In some embodiments, CRISPR mRNA is expressed by a gRNA vector or a CAR vector. In some embodiments, electroporation is performed 3 to 7 days after transduction with a gRNA vector and a CAR vector. In some embodiments, the method further includes selecting T cells, which are CD3 negative, CD34 positive, and optionally LNGFR positive. In some embodiments, the selection is performed using magnetic beads and / or flow cytometry sorting. In some aspects, the application discloses a mutant CAR-T cell library produced using the method described herein.

[0072] In some aspects, the present application discloses a method for identifying gRNA polynucleotides associated with in vivo CAR-T cell efficacy, comprising: (a) administering to a subject a mutant CAR-T cell library described in any one of claims E1 to E7 or F15; (b) collecting from the subject one or more samples of a plurality of mutant CAR-T cells of the mutant CAR-T cell library; (c) sequencing the gRNA polynucleotides from the mutant CAR-T cells collected in (b); and (d) evaluating changes in the relative abundance of each gRNA polynucleotide based on the sequencing in (c).

[0073] In some embodiments, the method further comprises: (e) identifying a gene associated with a gRNA polynucleotide with 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 a subject.

[0074] In some embodiments, the mutant CAR-T cell library comprises a full genome CAR-T cell mutant library. In some embodiments, the library comprises a negative control CAR-T cell. In some embodiments, the negative control CAR-T cell comprises a negative control gRNA polynucleotide described herein. In some embodiments, the subject is a human. In some embodiments, the subject suffers from cancer. In some embodiments, the cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, and B cell lymphoma. In some embodiments, CAR comprises an antigen binding domain that binds to an antigen expressed by the cancer.

[0075] In some embodiments, CAR comprises an antigen binding domain selected from CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, and any one of CD19 / CD79b, BCMA / TACI is combined, 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, after step (a), after administering the mutant CAR-T cell library, on the 3rd day, the 7th day, the 10th day, the 14th day, the 21st day, the 1st month, the 2nd month, the 3rd month, the 6th month, the 9th month, the 12th month, the 15th month, the 18th month, the 21st month and the 24th month, one or more, collect samples from the subject. In some embodiments, after step (a), a sample is collected from the subject on day 21 after administration of the mutant CAR-T cell library.

[0076] In some embodiments, the method further comprises extracting mutant 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.

[0077] In some embodiments, the method further includes isolating genomic DNA from mutant CAR-T cells. In some embodiments, the method further includes isolating gRNA polynucleotides from mutant CAR-T cells. In some embodiments, the method further includes isolating RNA from mutant CAR-T cells, and optionally sequencing RNA. In some embodiments, the method further includes amplifying the gRNA polynucleotides of mutant CAR-T cells to produce any of them. In some embodiments, the amplified gRNA polynucleotides are genomic DNA, vector DNA, or RNA. In some embodiments, SEQID NO: 1324 to 1325 primers are used for amplification. In some embodiments, gRNA polynucleotides or their amplicon are sequenced. In some embodiments, next generation sequencing is used to sequence gRNA polynucleotides or their amplicon.

[0078] In some embodiments, the efficacy of each mutant CAR-T cell is evaluated based on in vivo persistence and tumor cell toxicity. In some embodiments, in vivo persistence and tumor cell toxicity are measured using flow cytometry and PCR. In some embodiments, quantification of persistence is performed by calculating the relative change of each gRNA over time.

[0079] In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time compared to the gRNA polynucleotides from negative control CAR-T cells. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time through at least 3 time points and calculating the slope of the relative change. In some embodiments, tumor cell toxicity is quantified using clinical imaging and / or bone marrow evaluation. In some aspects, the present application discloses such CAR natural killer (natural killer, NK) cells, which include compositions described herein.

[0080] In some aspects, the present application discloses such CAR-NK cells, which include one, two or three of the following: a mutation in a gene corresponding to a gene sequence of any one of SEQ ID NO: 1 to 135 or a variant thereof, a gRNA polynucleotide of a composition described herein, a plasmid described herein, or a gRNA vector described herein. In some embodiments, the CAR-NK cell further includes 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 results in a frameshift.

[0081] In some embodiments, CAR-NK also includes mRNA and / or CRISPR proteins encoding CRISPR proteins. In some embodiments, NK cells are CD34 positive and CD3 negative. In some embodiments, the NK cells used to prepare the NK cells are from mammalian subjects. In some embodiments, the NK cells used to prepare the NK cells are from human subjects. In some embodiments, the NK cells used to produce the NK cells are collected from blood, and the blood is optionally umbilical cord blood or peripheral blood. In some embodiments, NK cells are activated. In some embodiments, NK cells are activated using irradiated feeder cells, IL-2, and IL-15. In some embodiments, NK cells are activated using irradiated K562 cells. In some embodiments, NK cells are activated using IL-21. In some embodiments, NK cells are activated using 4-1BBL, IL-2, and irradiated K562 cells expressing membrane-bound IL-15. In some embodiments, CRISPR protein is Cas9 or Cas12. In some embodiments, the NK cells do not comprise a DNA polynucleotide encoding a CRISPR protein. In some embodiments, the NK cells are CD34 positive, CD3 negative, and LNGFR positive.

[0082] In some embodiments, the NK cells of the library are selected from the NK cells described herein. In some embodiments, at least 2 CAR-NK cells of the library each comprise different gRNA polynucleotides, and the different gRNA polynucleotides comprise homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 10 CAR-NK cells of the library each comprise different gRNA polynucleotides, and the different gRNA polynucleotides comprise homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 20 CAR-NK cells of the library each comprise different gRNA polynucleotides, and the different gRNA polynucleotides comprise homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. In some embodiments, at least 50 CAR-NK cells of the library each comprise different gRNA polynucleotides, and the different gRNA polynucleotides comprise homology regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants. 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 to 135 or variants 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 to 135 or variants thereof. In some embodiments, SEQ ID NOs: 136 to 1315 are each present in at least one NK cell.

[0083] In some aspects, the present application discloses a method for generating a mutant CAR-NK cell library, comprising: (a) activating CAR-NK cells; (b) transducing CAR-NK cells with the gRNA vector library described herein; (c) transducing CAR-NK cells with the CAR vector described herein; and (d) introducing mRNA encoding CRISPR protein or CRISPR protein into NK cells.

[0084] In some aspects, the present application discloses a method for generating a mutant CAR-NK cell library, comprising: (a) activating CAR-NK cells; (b) transducing CAR-NK cells with the gRNA vector library described herein; and (c) transducing CAR-NK cells with the CAR vector described herein; wherein the gRNA vector or the CAR vector comprises a polynucleotide encoding a CRISPR protein.

[0085] In some embodiments, the method described in the claim further comprises collecting CAR-NK cells from the subject. In some embodiments, the method further comprises purifying CAR-NK cells comprising a CAR vector and a CRISPR-induced genetic mutation. In some embodiments, CAR-NK cells are collected using leukocyte removal. In some embodiments, CAR-NK cells are collected from human or mouse subjects. In some embodiments, CAR-NK cells are activated using anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the gRNA vector library is transduced with a multiplicity of infection (MOI) of 0.1 to 1. In some embodiments, the MOI is 0.3 to 0.7. In some embodiments, the MOI is 0.4 to 0.6. In some embodiments, the MOI is 0.5. In some embodiments, the CAR vector is transduced with an MOI of up to 20. In some embodiments, the CRISPR protein is Cas9 or Cas12.

[0086] In some embodiments, CRISPR protein mRNA or CRISPR protein is introduced using electroporation. In some embodiments, CRISPR mRNA is expressed by gRNA vector or CAR vector. In some embodiments, electroporation is performed 3 to 7 days after transduction with gRNA vector and CAR vector.

[0087] In some embodiments, the method further comprises selecting NK cells, wherein the NK cells are CD3 negative, CD34 positive. In some embodiments, the method further comprises selecting NK cells, wherein the NK cells are CD3 negative, CD34 positive and LNGFR positive.

[0088] In some embodiments, the selection is performed using magnetic beads and / or flow cytometry sorting. In some aspects, the application discloses a library of mutant CAR-NK cells generated using the methods described herein.

[0089] In some aspects, the present application discloses a method for identifying mutations that improve the efficacy of CAR-NK cells in vivo, comprising: (a) administering a mutant CAR-NK cell library described herein to a subject; (b) collecting one or more samples of a plurality of mutant CAR-NK cells from the mutant NK cell library from the subject; (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).

[0090] In some embodiments, the method further includes (e) identifying a gene associated with a gRNA polynucleotide with 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 a subject.

[0091] In some embodiments, the mutant CAR-NK cell library comprises a full genome NK cell mutant library. In some embodiments, the library comprises a negative control CAR-NK cell. In some embodiments, the negative control CAR-NK cell comprises a negative control gRNA polynucleotide described herein. In some embodiments, the subject is a human.

[0092] In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma and B cell lymphoma. In some embodiments, CAR comprises an antigen binding domain that binds to an antigen expressed by the cancer.

[0093] In some embodiments, CAR comprises an antigen binding domain selected from CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, and any one of CD19 / CD79b, BCMA / TACI is combined, 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, removal or tumor resection. In some embodiments, after step (a), after administering the mutant CAR-T cell library, on the 3rd day, the 7th day, the 10th day, the 14th day, the 21st day, the 1st month, the 2nd month, the 3rd month, the 6th month, the 9th month, the 12th month, the 15th month, the 18th month, the 21st month and the 24th month, one or more, collect samples from the subject. In some embodiments, after step (a), a sample is collected from the subject on day 21 after administration of the mutant CAR-T cell library.

[0094] In some embodiments, the method further comprises extracting mutant NK cells from the sample before sequencing. In some embodiments, the extraction is carried out by isolating NK cells, and the NK cells are CD3 negative, CD34 positive, and optionally LNGFR positive. In some embodiments, the method further comprises isolating genomic DNA from mutant NK cells. In some embodiments, the method further comprises isolating gRNA polynucleotides from mutant NK cells. In some embodiments, the method further comprises isolating RNA from mutant NK cells, and optionally sequencing RNA. In some embodiments, the method further comprises amplifying the gRNA polynucleotides of mutant NK cells to produce any of them. In some embodiments, the amplified gRNA polynucleotides are genomic DNA, vector DNA or RNA. In some embodiments, SEQ ID NO: 1324 to 1325 primers are used for amplification. In some embodiments, gRNA polynucleotides or their amplicon are sequenced. In some embodiments, gRNA polynucleotides or their amplicon are sequenced using next generation sequencing. In some embodiments, the effectiveness of each mutant NK cell is evaluated based on in vivo persistence and tumor cell toxicity. In some embodiments, in vivo persistence and tumor cell toxicity are measured using flow cytometry and PCR. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time compared to the gRNA polynucleotides from negative control NK cells. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time through at least 3 time points and calculating the slope of the relative change. In some embodiments, tumor cell toxicity is quantified using clinical imaging and / or bone marrow evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present disclosure, which aspects may be better understood by reference to one or more of these drawings in combination with the detailed description of some specific embodiments presented herein.

[0096] Figure 1Figures showing identification of mutant CAR-T cells that improve CAR-T cell survival in vivo. Patients undergo leukapheresis, and their T cells are transduced with two lentiviral vectors: one for expressing a chimeric antigen receptor (CAR) and a truncated CD34 (CD34t), and the other for expressing 2 guide RNAs (one is a barcoded gRNA from a library, and the other targets the T cell receptor alpha chain -TRAC) and a truncated low-affinity nerve growth factor receptor (tLNGFR). 2) The transduced T cells are electroporated with Cas9 to initiate genetic mutations, thereby knocking out the genes targeted by the library gRNA and CD3 targeted by the TRAC gRNA. 3) Negative selection of successfully mutated T cells based on the absence of CD3 expression is performed using magnetic-activated cell sorting (MACS). 4) Mutated T cells are then positively selected for CD34t to separate CAR T cells, thereby creating a library of mutant CAR T cells. 5) The CAR T cell library is then infused back into the patient. 6) After 7 to 14 days, tumor and blood samples are collected from the patient. The cells are lysed and the DNA is collected. 7) The DNA is sequenced to identify the barcodes of the gRNAs expressed by CAR T cells surviving in the tumor or blood, which allows the identification of genes that improve the survival of CAR T cells in these compartments after being knocked out.

[0097] Figures 2A to 2B The vector map of CAR vector and gRNA vector is shown. Figure 1 A shows a CAR vector comprising a nucleic acid sequence encoding a CD34 truncated (CD34t) selectable marker. Figure 2B Shown is a gRNA vector comprising 2 guide RNA cassettes, a suicide gene (iCasp9) operably linked to a PGK promoter, and a low-affinity nerve growth factor receptor truncated (LNGFRt) selectable marker.

[0098] Figures 3A to 3E Shown is the knockout of multiple genes in primary T cells using a dual guide cassette vector (pMGH354-CP1780). Figure 3A Shown is the experimental workflow for generating double knockout human T cells expressing a drug-inducible caspase-9 (iCasp9) safety switch and truncated LNGFR electroporated with Cas9. Figure 3BA schematic diagram of the lentiviral vector introduced into T cells containing sgRNAs targeting the T cell receptor alpha (TRAC) and CD5 genes, followed by the iCasp9 safety switch and truncated LNGFR (for positive selection) is shown. Figure 3C Shown are flow cytometry data demonstrating T cell receptor complex (TCR) knockout. Figure 3D Shown are flow cytometry data demonstrating knockout of CD5 using two different sgRNAs. Figure 3E Shown are flow cytometry data demonstrating surface expression of truncated LNGFR in cells transduced with lentiviral vectors.

[0099] Figures 4A to 4C Shown are guide RNA abundances in the pooled libraries determined by sequencing. Figure 4A A schematic diagram of a lentiviral vector containing two sgRNAs followed by a drug-inducible caspase-9 (iCasp9) safety switch and a truncated LNGFR (for positive selection) introduced into T cells is shown. Figure 4B Shown is the experimental workflow for generating double knockout T cells expressing a drug-inducible caspase-9 (iCasp9) safety switch and truncated LNGFR electroporated with Cas9, followed by treatment with Rimducid to induce iCasp9 activation, leading to cell death. Figure 4C Shown is flow cytometric evaluation of iCasp9-induced cell death by monitoring the loss of CAR+ cells over time.

[0100] Figure 5 A schematic diagram showing CRISPR screening in cancer patients.

[0101] Figure 6 The functional distribution of 135 genes targeted by the sgRNA CRISPR (Mario) library is shown.

[0102] Figures 7A to 7D Construct design and validation of gene knockout in human T cells is shown. Fig. 7A The lentiviral vector design to be used for in vivo screening is shown. The pCAR vector contains the CAR. The pGuide vector contains a dual guide cassette for delivering two sgRNAs. Figure 7B Shown is the expression of the NGFR reporter from the pGuide vector on human T cells. Figure 7C The editing efficiency of an example sgRNA targeting CD5 cloned into sgRNA position 1 is shown. Fig.7D The editing efficiency of the TRAC-targeting sgRNA cloned into position 2 of the pMario construct is shown.

[0103] Figures 8A to 8C Shown is the generation of mesothelin CAR T cells with a CRISPR (Mario) library for in vivo testing. Fig. 8A Construct design for creating CAR-T cells with the CRISPR Mario library is shown. Figure 8B Schematic diagram showing the process of generating mesothelin CAR T cells with CRISPR (Mario) library. Figure 8C The transduction efficiency of the two lentiviral vectors before and after selection of CD3 negative cells is shown. The CAR construct was labeled with mCherry, while the CRISPR library construct was evaluated using knockout of CD3.

[0104] Fig. 9 Schematic diagram showing the experimental design for in vivo CRISPR screening in mice.

[0105] Figures 10A to 10B Shown are the results of an in vivo loss of function (LOF) CRISPR screen in mice bearing APSC1 tumors (pancreatic tumors). Fig. 10A A guide distribution comparison is shown, demonstrating the ability to capture the complete guide library in tumor-engrafted CAR T cells and spleen-isolated CAR T cells. Fig. 10B Hypergeometric tests are shown, indicating the enriched and depleted guides in captured CAR t cells from tumors and spleens. In addition to depleted RASA2 and enriched IL2RA, new gene targets that may be potentially important for CAR T cell function were identified, such as GATA3 and ETS1.

[0106] Figures 11A to 11B Large-scale plasmid preparations and guide distribution in transduced human T cells are shown. Fig.11A Shown are histograms of guide abundance in the original plasmid primary library stock (blue line) and large-scale plasmid preparations (orange line) using PCR products flanking the plasmid guide region. Fig. 11B Guide distribution in human primary T cells transduced with lentiviral vectors containing a CRISPR library is shown. Lentiviral vectors were prepared using either primary plasmid library stocks or large-scale plasmid preparations before being transduced into human normal donor T cells.

[0107] Fig.12 The proposed preparation process for Meso-Mario-CART cells is shown.

[0108] Fig.13 Shown is the timeline for a Phase I study of Meso-Mario-CAR-T cells in human patients.

[0109] Fig.14 The sample collection and analysis plan for the Phase I study of Meso-Mario-CAR-T cells in human patients is shown.

[0110] Fig.15 Construct schematics of the BCMA CAR (4-1BB co-stimulatory domain, CD34t transduction marker) and dual guide cassette (LNGFR marker) used are shown.

[0111] Fig.16 The BCMA CAR-T CRISPR screening experimental design is shown. After 21 days of transplantation with 1E6 MM1.s tumor cells, mice were treated with 2E6 double-transduced BCMA+ library constructs. On day 7, mice were sacrificed and bone marrow (vertebra, femur, tibia) was harvested as part of the collection of BCMA CAR-T CRISPR cells.

[0112] Fig.17 The results of the measurement of the tumor killing activity of BCMA CAR-T cells and BCMACAR-T cell CRISPR libraries performed with normal donor 216T cells until the 7th day are shown. Body luminescence imaging (BLI) in both groups (left) and single mice. BCMACAR and double transduction BCMA+ library constructs showed anti-tumor activity, while tumors alone and libraries alone could not control tumors.

[0113] Fig.18 Shown is expression using the STARS score STARS ranking score, which allows quantification of significantly enriched (top IL2RA) and depleted hits (top IRF4).

[0114] Figures 19A to 19B Enriched and depleted gene knockouts in the BCMA CAR-T CRISPR screen are shown. Fig.19A The enriched per-mouse STARS score ranking assessment is shown. Enriched gene hits that were among the top 10 enriched genes in 6 mice or more were identified based on STARS score. For example, IL2RA was among the top 10 enriched hits in all 15 mice tested. Fig.19B The STARS score ranking evaluation of each mouse depleted is shown. Enriched gene hits that were among the top 10 enriched genes in 6 mice or more were identified based on STARS score. Example: IRF4 and RASA were among the top 10 depletion hits in all 15 mice tested.

[0115] Figures 20A to 20FResults from the BCMA CRISPR CAR-T screening and definitive selection phase are shown. Fig. 20A The BCMA replicate screening experimental design is shown. Fig.16 Similarly, the screening was repeated using T cells from two additional healthy donors. Fig. 20B Tumor activity measurements of T cells from normal donors 106 and 99 are shown as BLI for both groups (left) and individual mice up to day 21. BCMA CAR and dual transduction BCMA+ library constructs showed effective and rapid tumor clearance, while tumor alone and library alone failed to control tumors. Fig. 20C The expression measured using STARS score from day 0 to day 7 is shown. Fig.18 Similarly, STARS ranking scores in T cells from two other normal donors from day 0 to day 7 (in vivo period) are shown. The highest enriched CAR-T cell gene CRISPR target was IL2RA, and the highest depleted was IRF4 - both consistent with the results from the first ND (see Fig.18 ) are consistent. Fig.20D The expression measured using STARS score from day 0 to day 21 is shown. Fig.18 Similarly, STARS scores in T cells from two other normal donors from day 0 to day 21 (in vivo period) are shown. The highest enriched CAR-T cell gene CRISPR target was IL2RA, and the highest depleted was STAT3. Fig.20E The expression measured using STARS score from day -11 to day 21 is shown. Fig.18 Similarly, STARS scores in T cells from two other normal donors from day -11 to day 21 (in vitro & in vivo phase) are shown. The highest enriched CAR-T cell gene CRISPR target was CDKN1B, and the highest depleted was IL2RA. Fig.20F Shown is expression measured using STARS scoring from day -11 to day 7. The most highly enriched gene CRISPR targets were PTPN2 and SOCS1, and the most highly depleted was IRF4.

[0116] Figures 21A to 21C The generation of a current good manufacturing practice cGMP gRNA plasmid library is shown. Fig.21A The first attempt at making a library is shown, and the resulting low yield and low quality DNA that was insufficient to generate a cGMP gRNA plasmid library. Fig. 21B It was shown that increasing the amount of antibiotic used in cGMP library preparation (two-fold or four-fold) increased gRNA plasmid library yield. Fig. 21CIt is shown that increasing the amount of antibiotic produced a gRNA plasmid library with the expected yield and quality as determined by restriction digestion. Specific implementation plan

[0117] A common challenge in cell therapy (e.g., CAR-T cell therapy) is to identify CAR-T cell modifications (e.g., gene mutations) that improve the efficacy of cell therapy. This is challenging for several different reasons. First, although the efficacy of CAR-T cells in vitro and in mice can inform the efficacy in humans, CAR-T cells may have different efficacy in humans than in vitro or in mice. Second, it may be difficult to predict how a given mutation (e.g., a loss-of-function mutation) will affect the efficacy of CAR-T cells in humans, so it may be necessary to test many mutations to identify mutations that improve the efficacy of CAR-T cancer killing. However, it is impractical to test many different modified CAR-T cells in humans alone. Third, different subjects may have different responses to different CAR-T cell modifications. For example, a first modified CAR-T cell may have a higher efficacy in a first subject but a lower efficacy in a second subject compared to a second modified CAR-T cell. 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 libraries of modified CAR-T cells in vivo (e.g., in humans) in a single experiment to identify CAR-T cell modifications that improve CAR-T cell potency (e.g., in vivo persistence).

[0118] General restrictions

[0119] The terms "reduce", "reduced", "reduce" or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce", "reduce" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or medicament), and can include, for example, a reduction of 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. "Complete inhibition" is 100% inhibition compared to a reference level. Where applicable, reduction may preferably be reduced to an acceptable level within the normal range for individuals without a given disease. The term "increased", "improved", "enhanced" or "activated / activated" is all used herein to mean the increase of statistically significant amount. In some embodiments, the term "increased", "improved", "enhanced" or "activated / activated" can mean an increase of at least 10% compared to a 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 and including 100% increase or any increase of 10% to 100% compared to a reference level, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 2 times to 10 times or greater compared to a reference level. In the case of markers or symptoms, "increase" is a statistically significant increase of such a level.

[0120] A "disease" is a state of health in an animal (e.g., a human) in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is not as good as it would be in the absence of the disorder. Without treatment, the disorder does not necessarily lead to a further decline in the animal's health. In some embodiments, the disease is cancer or a tumor.

[0121] 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 thus be used to target cancer cells. Cancer antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are only expressed at certain stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53); and fusion proteins caused by internal deletions or chromosomal translocations. Other cancer antigens may be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many tumor antigens have been defined for a variety of solid tumors: MAGE 1, 2, & 3, defined by immunity; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (HER2), mucins (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, 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.

[0122] The term "chimeric" as used herein refers to a product of fusion of parts of at least two or more different polynucleotide molecules. In some embodiments, the term "chimeric" refers to a gene expression element produced by manipulating known elements or other polynucleotide molecules.

[0123] In some embodiments, "activation" may refer to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation may refer to induced cytokine production. In other embodiments, activation may refer to detectable effector function.

[0124] At a minimum, "activated T cells" as used herein are proliferating T cells.

[0125] The term "specific binding" and its variations used herein refer to a physical interaction between two molecules, compounds, cells and / or particles, wherein the binding of a first entity to a second target entity has greater specificity and affinity than its binding to a third entity as a non-target. In some embodiments, specific binding may refer to an affinity of a first entity to a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity of a first entity to a third non-target entity under the same conditions. An agent that is specific for a given target is an agent that exhibits specific binding to the target under the assay conditions used. Non-limiting examples include antibodies or ligands that recognize and bind to cognate binding partners (e.g., stimulatory and / or co-stimulatory molecules present on T cells) proteins.

[0126] As used herein, "stimulatory ligand" refers to a ligand that, when present on antigen presenting cells (APCs) (e.g., macrophages, dendritic cells, B cells, artificial APCs, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule" or "co-stimulatory molecule") on a T cell, thereby mediating the primary response of 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 particularly also encompass peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0127] The term "stimulatory molecule" as used herein means a molecule on a T cell that specifically binds to a corresponding cognate stimulatory ligand present on an antigen presenting cell. The term "costimulatory ligand" as used herein includes molecules that are on an APC and specifically bind to a corresponding costimulatory molecule on a T cell, thereby providing a signal that (in addition to the primary signal provided by the binding of, for example, a TCR / CD3 complex to an MHC molecule loaded with a peptide) mediates a T cell response, including but not limited to proliferation, activation, differentiation, etc. Co-stimulatory ligands may include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, IL T3, ILT4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands may also include, but are not limited to, antibodies that specifically bind to the following, and ligands that specifically bind to CD83: co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0128] "Co-stimulatory molecules" refer to corresponding binding partners on T cells, which specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as but not limited to proliferation. Co-stimulatory molecules include but are not limited to 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.

[0129] In some embodiments, the term "engineering" and its grammatical equivalents used herein may refer to one or more human-designed changes in nucleic acids (e.g., nucleic acids within the genome of an organism). In another embodiment, engineering may refer to the alteration, addition, and / or deletion of a gene. An "engineered cell" may refer to a cell with an added, deleted, and / or altered gene.

[0130] As used herein, the terms "cell" or "engineered cell" and their grammatical equivalents may refer to cells of human or non-human animal origin.

[0131] The term "operably linked" as used herein refers to a first polynucleotide molecule (e.g., a promoter) being linked to a second transcribable polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged so 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 the promoter regulates or mediates transcription of a target gene in a cell, the promoter is operably linked to the target gene.

[0132] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides are also contemplated. For amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence (which alters a single amino acid or a small portion of amino acids in the encoding sequence) are "conservatively modified variants," wherein the alteration results in replacement of an amino acid with a chemically similar amino acid and retention of the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0133] A given amino acid may be substituted with a residue having similar physicochemical characteristics, for example, replacing one aliphatic residue with another (e.g., replacing Ile, Val, Leu, or Ala with each other), or replacing another with a polar residue (e.g., between Lys and Arg; between Glu and Asp; or between Gin and Asn). Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are well known. Polypeptides comprising conservative amino acid substitutions may be tested in any of the assays described herein to confirm that the desired activity, such as ligand-mediated receptor activity and specificity of a native or reference polypeptide, is retained. Amino acids can be grouped according to the similarity of their side chain properties (ALL Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: 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, naturally occurring residues can be divided into the following groups 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 would require exchanging a member of one of these classes for another. Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; ile to Leu or to Val; Leu to ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0134] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide according to assays known in the art or described herein below. Functional fragments may comprise conservative substitutions of sequences disclosed herein.

[0135] In some embodiments, the polypeptide described herein can be a variant of a polypeptide or molecule as described herein. In some embodiments, the variant is a conservatively modified variant. For example, a conservatively substituted variant can be obtained by a mutation of a native nucleotide sequence. "Variant" mentioned herein is a polypeptide substantially homologous to a natural or reference polypeptide, but it has an amino acid sequence different from a natural or reference polypeptide due to one or more deletions, insertions or substitutions. The DNA sequence encoding the variant polypeptide encompasses sequences that include one or more nucleotide additions, deletions or substitutions but that encode a variant protein or a fragment thereof that retains the activity of a non-variant polypeptide when compared to a natural or reference DNA sequence. A wide variety of site-specific mutagenesis methods based on PCR are known in the art and can be used by those of ordinary skill in the art.

[0136] Variant amino acid or DNA sequence can have 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 identity with native or reference sequence. The degree of homology (percent identity) between native sequence and mutant sequence can be determined, for example, by comparing two sequences using freely available computer programs (e.g., BLASTp or BLASTn with default settings) commonly used for this purpose on the World Wide Web.

[0137] The change of native amino acid sequence can be realized by any of many technologies known to those skilled in the art.Can introduce sudden change, for example can introduce sudden change by synthesizing such oligonucleotide at specific locus, and described oligonucleotide contains mutant sequence and the restriction site that side joint allows to be connected with the fragment of native sequence.After connecting, the reconstructed sequence encoding obtained has the analogue of desired amino acid insertion, replacement or disappearance.Or, can adopt oligonucleotide directed site-specific mutagenesis program to provide the nucleotide sequence through change with specific codon through change according to required replacement, disappearance or insertion. Techniques for making such changes 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. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine ​​residue that is not involved in maintaining the proper conformation of the polypeptide may also be replaced, usually with serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to a polypeptide to increase its stability or promote oligomerization.

[0138] The term "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to represent the polymer of nucleoside. Generally, polynucleotides are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA connected by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs, which contain chemically modified or biologically modified bases, modified backbones, etc., regardless of whether they are present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application relates to polynucleotides, it should be understood that DNA and RNA are provided, and both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided in each case. "Polynucleotide sequence" used herein may refer to polynucleotide material itself and / or to sequence information (i.e., continuous letters used as base abbreviations) that characterizes specific nucleic acids biochemically. In some embodiments, nucleic acid molecules are heterologous nucleic acid molecules. As used herein, the term "heterologous nucleic acid molecule" refers to a nucleic acid molecule that does not naturally occur in a given cell.

[0139] Unless otherwise indicated, polynucleotide sequences presented herein are presented in the 5' to 3' direction.

[0140] The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide can be a relatively short polypeptide, typically about 2 to 60 amino acids in length. The polypeptides used herein typically contain amino acids, such as the 20 most common L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art can 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; modified by linkers for conjugation, functionalization, etc. A polypeptide having a non-polypeptide portion covalently or non-covalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. A polypeptide can be purified from a natural source, produced using recombinant DNA technology, or synthesized by chemical means (e.g., conventional solid phase peptide synthesis, etc.). The term "polypeptide sequence" or "amino acid sequence" used herein can refer to the polypeptide substance itself and / or to sequence information (i.e., consecutive letters or three-letter codes used as abbreviations of amino acid names) that characterizes a polypeptide biochemically. Unless otherwise indicated, the polypeptide sequences shown herein are shown in the N-terminal to C-terminal direction.

[0141] The term "gene" may refer to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably connected to a suitable regulatory sequence. A gene may include or not include regions before and after the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "tailing" sequence, and intervening sequences (introns) between each coding segment (exon). In some embodiments, a nucleic acid encoding a polypeptide (such as a CAR polypeptide) as described herein is included in a vector (such as a CAR vector). In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein or any module thereof is operably connected to a vector. The term "vector" used herein refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. The vector used herein may be viral or non-viral. The term "vector" encompasses any genetic element that can replicate and can transfer a gene sequence to a cell when associated with a suitable control element. A vector may include, but is not limited to, a cloning vector, an expression vector, a plasmid, a phage, a transposon, a clay, an artificial chromosome, a virus, a virion, etc.

[0142] The term "expression vector" as used herein may refer to a vector that directs RNA or polypeptides to be expressed from a sequence connected to a transcriptional regulatory sequence on the vector. The expressed sequence is usually, but not necessarily, heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems so that it can be maintained in two organisms, such as for expression in human cells and for cloning and amplification in prokaryotic hosts. The term "expression" refers to the cellular process involved in producing RNA and proteins and, where appropriate, secreting proteins, including, but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene.

[0143] The term "viral vector" as used herein may refer to a nucleic acid vector construct that comprises at least one element of viral origin and has the ability to be packaged into viral vector particles. A viral vector may comprise a nucleic acid encoding a polypeptide as described herein in place of a non-essential viral gene. The vector and / or particle may be used for the purpose of transferring nucleic acid into a cell in vitro or in vivo. Many forms of viral vectors are known in the art.

[0144] "Recombinant vector" can be a vector comprising a heterologous nucleic acid sequence or a "transgenic" that can be expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and treatments. In some embodiments, the vector is episomal. The use of suitable episomal vectors provides a method for maintaining the target nucleotide in a subject in a high copy number extrachromosomal DNA to eliminate the potential effects of chromosomal integration.

[0145] "Signal peptide" or "signal sequence" used herein refers to a peptide at the N-terminus of a newly synthesized protein that is used to direct the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 or lgK signal peptide.

[0146] The term "clustered regularly interspaced short palindromic repeats" or "CRISPR" as used herein may refer to a gene editing system comprising a guide RNA component and a CRISPR-related (Cas) protein component. The guide RNA polynucleotide may include a homology region complementary to the target gene and a stem-loop region capable of binding to the Cas protein. The Cas protein may include a guide RNA binding site and nuclease activity. The Cas protein and the guide RNA may form a complex capable of binding to the target gene (based on the homology region) and cutting DNA (using the nuclease activity of the Cas protein). In some embodiments, the Cas protein guides the RNA complex to bind to a sequence adjacent to and downstream of the protospacer adjacent motif (PAM). Cutting causes DNA strand breaks, and the repair of the strand break can introduce mutations (e.g., single nucleotide polymorphisms, insertions, or deletions). In some embodiments, the Cas protein is any suitable Cas protein (herein, the Cas protein may also be referred to as a CRISPR protein) for mutating and / or changing the expression of the target gene. In some embodiments, the Cas protein is selected from a Cas9 protein, a Cas12 protein, or a Cas13 protein. It will be appreciated by the skilled artisan that Cas proteins (e.g., Cas9) may have many different orthologs (e.g., SpyoCas9, spCas9, spyCas9, and geoCas9). In some embodiments, the Cas protein is SpyoCas9. Cas proteins and their 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., selecting homologous region sequences to target specific genes) are also well known in the art, as described in Liu, Guanqing L. et al., Computational and Structural Biotechnology Journal 18 (2020): 35-44, which are incorporated by reference in their 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 incorporated by reference in their entireties.

[0147] In some embodiments, polypeptides, polynucleotides, plasmids and / or vectors as described herein optionally further include reporters, for example to determine whether the vector is correctly expressed in the cell. In some embodiments, the reporter can be a fluorescent protein (e.g., GFP, YFP, RF), an antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2) and a radioactive isotope. In some embodiments, the reporter is hygromycin phosphotransferase (hph), which can be imaged alone or in combination with a substrate or chemical (e.g., 9-[4-[18F] fluoro-3-(hydroxymethyl) butyl] guanine ([18F] FHBG)).

[0148] In some embodiments, GFP and mCherry can be used as fluorescent tags for imaging CAR expressed on T cells (eg, CAR-T cells). It is expected that substantially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, CAR does not need to include a fluorescent tag or fluorescent protein. Therefore, in each example of a specific construct provided herein, any marker present in the construct can be removed. The present invention includes constructs with or without markers. Therefore, when a specific construct is mentioned herein, it can be considered (with or without any marker or tag (including, for example, a histidine tag, such as a histidine tag of HHHHHH (SEQ ID NO: 1370))) included in the present invention.

[0149] The term "statistically significant" or "significant" means statistically significant and generally means a difference of two standard deviations (2SD) or greater.

[0150] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about".

[0151] When used in conjunction with a value, the term "about" or "approximately" may mean that the value or the statement reciting the value covers a range of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±1% to 5%, ±2% to 7%, ±3% to 8%, ±4% to 9%, or ±5% to 10% of the value.

[0152] Unless the context clearly indicates otherwise, expressions without numerical qualifiers include plural references. Similarly, unless the context clearly indicates otherwise, the word "or / or" is intended to include "and". 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 "eg" is derived from the Latin exempli gratia and is used herein to represent non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the term "for example".

[0153] As set forth herein, additional terms are defined throughout the description of various aspects and embodiments of the technology.

[0154] Guide RNA polynucleotide library

[0155] The term "guide RNA (gRNA) polynucleotide" used herein 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 (clustered regularly interspaced short palindromic repeat, CRISPR) protein or CRISPR-related protein, and includes such a sequence that contains another sequence (i.e., homology region) complementary to the target polynucleotide. For example, the guide RNA polynucleotide can be a Cas9 protein guide RNA polynucleotide or a Cas12 protein guide RNA polynucleotide. Cas9 protein guide RNA is compatible with Cas9 CRISPR protein, and is well known in the art, for example, as described in Adli et.al., Nature communications 9.1 (2018): 1-13, which is incorporated by reference in its entirety. Cas12 protein guide RNA polynucleotides are compatible with Cas12 CRISPR proteins and are well known in the art, for example, as described in Zetsche et al., Cell 163.3 (2015): 759-771, which is incorporated by reference in its entirety. In some embodiments, the guide RNA polynucleotide is a base editor guide RNA polynucleotide. In some embodiments, gRNA is a lead editor guide RNA polynucleotide. In some embodiments, the guide RNA polynucleotide encodes a homology region (e.g., a spacer) and a region (e.g., repeated in the same direction) bound to a CRISPR protein. In some embodiments, the guide RNA polynucleotide is a single guide RNA polynucleotide comprising a homology region and a region bound to a CRISPR protein.

[0156] In some embodiments, the homology region comprises a continuous series of about 10 to 30 or about 15 to 25 nucleotides. In some embodiments, the homology region comprises about 20 nucleotides. In some embodiments, the homology region is complementary to a target gene (e.g., a gene associated with immune cell function). In some embodiments, 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.

[0157] In some embodiments, the gRNA polynucleotide has no cross-reactivity or has minimal cross-reactivity. Cross-reactive gRNA polynucleotides refer to such guide RNA polynucleotides, which include homologous regions with sufficient complementarity to more than one target polynucleotide (e.g., gene sequence), so that the gRNA can induce CRISPR mutations in more than one target polynucleotide. Design algorithms can be used to guide RNA polynucleotide design to reduce the chance of cross-reactivity (e.g., Rule Set 3 (RS3) with sequence and target information and Chen 2013 tracr (Chen, Baohui, et al. Cell 155.7 (2013): 1479-1491) can be used to score the on-target activity of gRNA. Tier-agnostic 1 mismatch aggregated Cutting Frequency Determination (CFD) scoring can also be used to score the off-target activity of gRNA). The skilled person will understand that the gRNA polynucleotides designed with such an algorithm may still have a certain degree of cross-reactivity, however, the risk of cross-reactivity is expected to be reduced or can be specified as a selected threshold in the algorithm. In some embodiments, cross-reactivity is a function of complementarity. In some embodiments, the gRNA polynucleotides without cross-reactivity have no more than 80% complementarity with more than one gene. In some embodiments, the gRNA polynucleotides without cross-reactivity have no more than 85% complementarity with more than one gene. In some embodiments, the gRNA polynucleotides without cross-reactivity have no more than 90% complementarity with more than one gene. In some embodiments, the gRNA polynucleotides without cross-reactivity have no more than 95% complementarity with more than one gene.

[0158] The term "complementary" as used herein refers to the degree of Watson-Crick base pairing between two polynucleotides. For example, if 9 / 10 of the nucleotides in each of the polynucleotides form Watson-Crick base pairs, the two polynucleotides may be 90% complementary. In some embodiments, complementarity may refer to at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nucleotides in the first polynucleotide being Watson-Crick base paired with the second polynucleotide. In some embodiments, a homologous region of a gRNA is complementary to a gene sequence when the homologous region is capable of hybridizing to the gene sequence and at least a certain 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 for the gene sequence. In some embodiments, a homologous region is complementary to a gene sequence when the homologous region is capable of hybridizing to the gene sequence and initiating cleavage of the gene sequence by a CRISPR protein and at least a certain 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 for the gene sequence. In some embodiments, when the nucleotides in 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 continuous nucleotides of the target gene sequence, such as 20 continuous nucleotides), the homologous region is complementary to the target gene sequence. 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 is complementary to the antisense strand of the target gene sequence. A homologous region complementary to a gene encoded by SEQ ID NO: 1) may refer to a homologous region complementary to 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 region described herein may be complementary to any one of SEQ ID NOs: 1 to 135 (i.e., the sense strand), or complementary to the reverse complement (i.e., the antisense strand) of any one of SEQ ID NOs: 1 to 135.

[0159] 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 complementary to a region of the target gene sequence adjacent to the protospacer adjacent motif (PAM) and downstream thereof.

[0160] In some embodiments, the disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 2 (e.g., at least 3, at least 5, at least 10, at least 15, at least 25, at least 50, at least 75, at least 100, or at least 135) gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135, or a variant thereof.

[0161] In some embodiments, the disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 2 (e.g., at least 3, at least 5, at least 10, at least 15, at least 25, at least 50, at least 75, at least 100, or at least 135) gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene sequence of any one of SEQ ID NOs: 1 to 135, or a variant thereof.

[0162] In some embodiments, the disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 2 (e.g., at least 3, at least 5, at least 10, at least 15, at least 25, at least 50, at least 75, at least 100, or at least 135) gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof, or wherein at least 2 (e.g., at least 3, at least 5, at least 10, at least 15, at least 25, at least 50, at least 75, at least 100, or at least 135) gRNA polynucleotides each comprise a homology region complementary to a different gene sequence selected from the reverse complement of any one of SEQ ID NOs: 1 to 135.

[0163] In some embodiments, the present disclosure describes a composition comprising a plurality of guide RNA (gRNA) polynucleotides. In some embodiments, at least 2 gRNA polynucleotides, for example, each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. For example, a plurality of guide RNA (gRNA) polynucleotides may comprise a first gRNA polynucleotide and a second gRNA polynucleotide, wherein each of the first gRNA nucleotide and the second gRNA polynucleotide is complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, each homology region of at least 2 gRNA polynucleotides is complementary to the sense strand of a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, each homology region of at least 2 gRNA polynucleotides is complementary to the antisense strand of a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.

[0164] In some embodiments, the composition comprises at least 2 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 3 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 4 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 5 gRNA polynucleotides. In some embodiments, the composition comprises at least 6 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 7 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 8 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 9 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 10 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 11 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 12 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 13 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 14 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the composition comprises at least 15 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 16 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 17 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 18 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 19 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 20 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 21 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 22 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 23 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 24 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 25 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 26 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the composition comprises at least 27 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 28 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 29 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 30 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 40 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 50 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 60 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 70 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 80 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 90 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 100 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 110 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the composition comprises at least 120 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the composition comprises at least 130 gRNA polynucleotides, wherein each gRNA polynucleotide comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or variants thereof.

[0165] In some embodiments, the plurality of guide RNA polynucleotides comprises at least 2 guide RNA polynucleotides having different sequences (e.g., at least 2, at least 5, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 2500, at least 5000, at least 10000, at least 15000, at least 20000, at least 25000, at least 30000, at least 50000, at least 100000, or at least 1000000 guide RNA polynucleotides). In some embodiments, the plurality of gRNA polynucleotides comprises no more than 1000, 2500, 5000, 10,000, 15,000, or 20,000 polynucleotides. In some embodiments, the plurality of guide RNA polynucleotides 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, 5 to 1000, 5 to 2500, 5 to 5000, 5 to 1000, 5 to 15000, 5 to 20000, 5 to 40000, 5 to 5 10,000, 5 to 100,000, 10 to 1,000, 10 to 2,500, 10 to 5,000, 10 to 1,000, 10 to 15,000, 10 to 20,000, 10 to 40,000, 10 to 50,000, 10 to 100,000, 20 to 1,000, 20 to 2,500, 20 to 5,000, 20 to 1,000, 20 to 15,000, 20 to 20,000, 20 to 4 0,000, 20 to 50,000, 20 to 100,000, 50 to 1,000, 50 to 2,500, 50 to 5,000, 50 to 10,000, 50 to 15,000, 50 to 20,000, 50 to 40,000, 50 to 50,000, 50 to 100,000, 100 to 1,000, 100 to 2,500, 100 to 5,000, 100 to 10,000, 100 to 150 135 to 100, 100 to 20,000, 100 to 40,000, 100 to 50,000, 100 to 100,000, 135 to 1000, 135 to 2500, 135 to 5000, 135 to 10000, 135 to 15000, 135 to 20,000, 135 to 40,000, 135 to 50,000, or 135 to 100,000 guide RNA polynucleotides having different sequences. In some embodiments, the different sequences are complementary to one or more of SEQ ID NOs: 1 to 135.In some embodiments, a plurality of guide RNA polynucleotides include 2 to 1300 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 2 to 1200 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 2 to 1100 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 2 to 1080 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 5 to 1300 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 5 to 1200 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 5 to 1100 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 5 to 1080 guide RNA polynucleotides with different sequences. In some embodiments, a plurality of guide RNA polynucleotides include 10 to 1300 guide RNA polynucleotides with 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.

[0166] 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 with different sequences. In some embodiments, the composition comprises at most 5000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 7500 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 10000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 15000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 20000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 25000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 30000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 35000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 40000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 45000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 50,000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 60,000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 70,000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 80,000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 90,000 guide RNA polynucleotides with different sequences. In some embodiments, the composition comprises at most 100,000 guide RNA polynucleotides with 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 100000, 5 to 100000, 5 to 2500, 5 to 5000, 5 to 10000, 5 to 15000, 5 to 20000, 5 to 40000, 5 to 50000, 5 to 100000, 5 to 100000, 10 to 2500, 10 to 5,000 species, 10 to 1,000 species, 10 to 15,000 species, 10 to 20,000 species, 10 to 40,000 species, 10 to 50,000 species, 10 to 100,000 species, 10 to 1,000,000 species, 20 to 1,000,000 species, 20 to 1,000,000 species, 20 to 2,500 species, 20 to 5,000 species, 20 to 1,000 species, 20 to 15,000 species, 20 to 20,000 species, 20 to 40,000 species, 20 to 50,000 species, 20 to 100,000 species, 20 to 1,000,000 species, 50 to 1,000 species, 50 to 2,500 species, 50 to 5,000 species, 50 to 1,000 0, 50 to 15,000, 50 to 20,000, 50 to 40,000, 50 to 50,000, 50 to 100,000, 50 to 1,000,000, 100 to 1,000, 100 to 2,500, 100 to 5,000, 100 to 10,000, 100 to 15,000, 100 to 20,000, 100 to 40,000, 100 to 50,000, 100 to 100,000, 100 to 1,000,000, 135 to 1,000, 135 to 2,500, 135 to 5,000, 135 to 10 000, 135 to 15000, 135 to 20000, 135 to 40000, 135 to 50000, or 135 to 100000, 135 to 100000, 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 with different sequences. The skilled person will understand that many copies of any one guide RNA polynucleotide may be present in the composition, and that the copies have the same sequence.

[0167] In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 20,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 19,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 18,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 17,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 16,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 15,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 14,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 13,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 12,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 11,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 10,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 9,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 8,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 7,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 6,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 5,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 4,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 3,000 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 2,000 different gene sequences. In some embodiments, the guide RNA polynucleotides in the composition are complementary to at most 1,000 different gene sequences. In some embodiments, the guide RNA polynucleotides in the composition are complementary to at most 900 different gene sequences. In some embodiments, the guide RNA polynucleotides in the composition are complementary to at most 800 different gene sequences.

[0168] In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 700 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 600 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 500 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 400 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 300 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 200 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to at most 135 different gene sequences. In some embodiments, the guide RNA polynucleotide in the composition is complementary to 135 different gene sequences (e.g., SEQ ID NO: 1 to 135).

[0169] In some embodiments, the guide RNA in the composition does not include a guide RNA polynucleotide complementary to the sense strand or antisense strand of the gene encoding the 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 cause cells to become tumorigenic or cancerous. In some embodiments, this is undesirable in the guide RNA polynucleotide compositions described herein, because these compositions can be used to produce cell therapies (e.g., CAR-T cell therapies) administered to subjects (e.g., human subjects with cancer). If the guide RNA polynucleotide library includes a guide RNA complementary to the tumor suppressor, this can produce cells (e.g., CAR T cells) that can be given to the subject cancer or tumor. Genes encoding tumor suppressors are known in the art, such as Cooper GM. The Cell: A Molecule Approach. 2nd Edition. Sunderland (MA): Sinauer Associates; 2000. Tumor Suppressor Genes Described. Can be obtained from ncbi.nlm.nih.gov / books / NBK9894 / .

[0170] In some embodiments, a composition described herein (e.g., a composition comprising multiple gRNA polynucleotides) does not include one or more of the following: 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 the following: 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: P3839 8), Wilms' tumor 1 (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).

[0171] In some embodiments, the guide RNA in the composition does not include a guide RNA polynucleotide complementary to the sense strand or antisense strand of a gene essential for T cell function. In some embodiments, the guide RNA in the composition does not include a guide RNA polynucleotide complementary to the sense strand or antisense strand of a gene encoding one or more of the following: 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), ORAO V1 (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: P302 81), 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: Q13094), ot: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 variants thereof.

[0172] In some embodiments, variants thereof refer to homologs, orthologs, or lateral homologs 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, variants thereof refer to amino acid sequences having at least 60% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 65% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 70% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 75% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 80% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 85% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 90% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 95% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 99% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 99.5% identity with any one of SEQ ID NOs: 1 to 135. In some embodiments, variants thereof refer to amino acid sequences having at least 99.9% identity with any one of SEQ ID NOs: 1 to 135.

[0173] In some embodiments, the composition comprises at least X gRNA polynucleotides / gene sequences, wherein 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 / gene sequences, wherein at least 2 gRNA polynucleotides comprise different sequences complementary to a given gene (e.g., the sense strand or antisense strand of a gene). Therefore, in such embodiments, if the composition comprises a gRNA polynucleotide containing a homology region complementary to Y different genes (e.g., any one of Y different SEQ ID NOs), then the composition will comprise at least 2Y gRNA polynucleotides, wherein at least 2 gRNA polynucleotides comprise a homology region complementary to each of Y genes, and wherein the at least 2 gRNA polynucleotides comprise different sequences complementary to a given gene. In some embodiments, at least X gRNA polynucleotides comprise homology 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 a gene). In some embodiments, at least X gRNA polynucleotides comprise homology regions complementary to overlapping regions of the same gene sequence. In some embodiments, the composition comprises at least 2 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequences (e.g., SEQ ID NO: 1 to 135).

[0174] In some embodiments, the composition comprises at least 2 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 3 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 4 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 5 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complementary sequence of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 6 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complementary sequence of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 7 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complementary sequence of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 8 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 9 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 10 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135).In some embodiments, the composition comprises at least 12 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 15 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 18 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequence (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135). In some embodiments, the composition comprises at least 20 gRNA polynucleotides / gene sequences, wherein the gRNA polynucleotides comprise different sequences complementary to the gene sequences (e.g., SEQ ID NOs: 1 to 135, or the reverse complement of any one of SEQ ID NOs: 1 to 135).

[0175] In some embodiments, the composition comprises 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 / gene sequences, wherein 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 / gene sequences, wherein each gRNA polynucleotide comprises different sequences complementary to the gene sequence or the reverse complementary sequence of the gene sequence. In some embodiments, the composition comprises 8 kinds of gRNA polynucleotides / gene sequences, wherein each gRNA polynucleotide comprises different sequences complementary to the reverse complementary sequence of the gene sequence or the gene sequence. In some embodiments, a variety of gRNA polynucleotides (e.g., 8 kinds) are complementary to different regions of the reverse complementary sequence of the same gene sequence or the gene sequence.

[0176] In some embodiments, the composition includes one or more negative control guide RNA polynucleotides. In some embodiments, the negative control guide RNA polynucleotide is a guide RNA polynucleotide that is expected to have no effect on CAR-T cell efficacy. In some embodiments, the negative control guide RNA polynucleotide is a guide RNA polynucleotide whose homology region is not complementary to the gene in the genome of immune cells (eg, CAR-T cells). In some embodiments, the negative control gRNA includes a homology region for complementary intergenic regions. In some embodiments, the negative control gRNA polynucleotide includes a randomly generated homology region. In some embodiments, the negative control guide RNA sequence includes SEQ ID NO: The nucleotide sequence of any one of 1216 to 1315.

[0177] In some embodiments, the composition comprises at least one gRNA polynucleotide of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least two gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least three gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least four gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least five gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least six gRNA polynucleotides of any one of SEQ ID NOs: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises at least seven 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.

[0178] 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. 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 SEQ ID NOs: 136 to 1215 or any variant thereof.

[0179] In some embodiments, the composition comprises a gRNA polynucleotide comprising a sequence of SEQ ID NO: 136 to 1215 or any variant thereof. In some embodiments, the composition comprises a gRNA polynucleotide comprising a sequence of SEQ ID NO: 136 to 1215. In some embodiments, the composition comprises a gRNA polynucleotide comprising any one of SEQ ID NO: 1216 to 1315. In some embodiments, the composition comprises a gRNA polynucleotide comprising SEQ ID NO: 1216 to 1315.

[0180] In some aspects, the present application discloses guide RNA polynucleotides. In some embodiments, the guide RNA polynucleotide is a guide RNA polynucleotide of any of the compositions described herein. In some embodiments, the guide RNA polynucleotide comprises a sequence of any one of SEQ ID NO: 136 to 1315 or any variant thereof.

[0181] Plasmids, vectors and their libraries

[0182] Plasmids

[0183] In some aspects, the application discloses a plasmid comprising any one of the gRNA polynucleotides disclosed herein. "Plasmid" as described herein may refer to a circular DNA fragment comprising sequence elements for polynucleotide (e.g., gRNA) replication and expression. In some cases, the replication element is a replication origin (e.g., a bacterial replication origin). In some embodiments, the expression sequence element comprises a promoter and a terminator. In some embodiments, the plasmid comprises a selectable marker (e.g., an antibiotic marker).

[0184] The "plasmid library" described herein may include at least two plasmids as described herein encoding different gRNA polynucleotides. In some embodiments, the plasmid library includes at least 3 species, (e.g., at least 4 species, at least 5 species, at least 6 species, at least 7 species, at least 8 species, at least 9 species, at least 10 species, at least 11 species, at least 12 species, at least 13 species, at least 14 species, at least 15 species, at least 16 species, at least 17 species, at least 18 species, at least 19 species, at least 20 species, at least 21 species, at least 22 species, at least 23 species, at least 24 species, at least 25 species, at least 26 species, at least 27 species, at least 28 species, at least 29 species, at least 30 species, at least 40 species, at least 50 species, at least 60 species, at least 70 species, at least 80 species, at least 81 species, at least 82 species, at least 83 species, at least 84 species, at least 85 species, at least 86 species, at least 87 species, at least 88 species, at least 89 species, at least 90 species, at least 91 species, at least 92 species, at least 93 species, at least 94 species, at least 95 species, at least 96 species, at least 97 species, at least 98 species, at least 99 species, at least 100 species, at least 101 species, at least 102 species, at least 103 species, at least 104 species, at least 105 species, at least 106 species, at least 107 species, at least 108 species, at least 109 species, at least 110 species, at least 1110 species, at least 112 species, at least 1 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 encoding different gRNA polynucleotides (e.g., gRNA polynucleotides disclosed herein or variants thereof).

[0185] gRNA vector

[0186] In some embodiments, the present application discloses a gRNA vector comprising: (1) a first gRNA polynucleotide comprising a homology region complementary to a marker protein (e.g., a TCR / CD3 complex of a T cell (any one of SEQ ID NOs: 1383 to 1389)), and (2) a second gRNA polynucleotide.

[0187] In some embodiments, the first gRNA polynucleotide comprises a homology region of SEQ ID NO: 1316. In some embodiments, when transfected into immune cells (e.g., T cells), the first guide RNA polynucleotide is used as a marker of CRISPR activity based on TCR / CD3 expression loss on immune cells. In some embodiments, the first gRNA polynucleotide is operably connected 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., tet or lac promoter). In some embodiments, the promoter is selected from: CMV promoter, EF1a promoter, CAG promoter, PGK promoter, H1 promoter or U6 promoter. In some embodiments, the promoter is a U6 promoter.

[0188] In some embodiments, the second gRNA polynucleotide in 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 sequence or antisense sequence of a gene encoded by any one of SEQ ID NO: 1 to 135 or its variant. In some embodiments, the second gRNA polynucleotide comprises a sequence of any one of SEQ ID NO: 136 to 1315 or its variant. In some embodiments, the second gRNA polynucleotide comprises a sequence of any one of SEQ ID NO: 136 to 1315. In some embodiments, the second gRNA polynucleotide comprises a negative control gRNA sequence as described herein. In some embodiments, the second gRNA polynucleotide comprises a sequence of any one of SEQ ID NO: 1216 to 1315 or its variant. In some embodiments, the second gRNA polynucleotide is operably connected 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 or lac promoter). In some embodiments, the promoter is selected from: CMV promoter, EF1a promoter, CAG promoter, PGK promoter, H1 promoter or U6 promoter. In some embodiments, the U6 promoter is from non-human species. In some embodiments, the U6 promoter is from human U6 promoter. In some embodiments, the U6 promoter is from cattle, mice, rats, pigs, yeast, dogs, cats, fruit flies (drosophila) or Caenorhabditis elegans (C.elegans). In some embodiments, the promoter is an H1 promoter. In some embodiments, the first gRNA polynucleotide is operably connected to the first promoter, and the second gRNA polynucleotide is operably connected to the second promoter. In some embodiments, the first and second promoters are the same promoter (that is, two different copies of the same promoter). In some embodiments, the first and second promoters are different promoters (that is, comprising 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 (eg, transcribed as a single polynucleotide, eg, and cleaved following transcription).

[0189] In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in opposite directions in the guide RNA vector. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are in a tandem direction in the guide RNA vector. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are encoded in the same CRISPR array. In some embodiments, the first gRNA polynucleotide and the second gRNA polynucleotide are single guide RNAs.

[0190] In some embodiments, the gRNA vector includes a selectable marker. "Selectable marker" may refer to a molecule that allows detection of biological events. For example, a selectable marker may provide evidence that a cell has expressed a gRNA vector. In some embodiments, a 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), wherein the knockout of a gene may be used as a selectable marker. In some embodiments, a selectable marker is a fluorescent marker. In some embodiments, a selectable marker provides resistance to toxins (e.g., geneticin, hygromycin B, puromycin, or Zeocin). In some embodiments, a selectable marker is suitable for use in humans. In some embodiments, a selectable marker is the expression of a non-endogenous cell surface protein. In some embodiments, a selectable marker is selected from LGNFR (nerve growth factor receptor), EGFR, CD19, CD20, CD34, and a truncated form thereof. In some embodiments, a selectable marker is LNGFR. In some embodiments, a selectable marker is operably connected to a promoter (e.g., a promoter described herein). In some embodiments, the selectable marker is operably connected to a weak promoter (e.g., PGK or UBC). In some embodiments, the selectable marker is operably connected to a tissue-specific promoter (e.g., HP1, CD14, CD43, CD45, C68, elastase, endothelin, fibronectin, Flt, GFAP, GPIIb, ICAM-2, mIFN-β, Mb, NphsI, OG-2, SP-B, SYN1 or WASP gene promoter). In some embodiments, selectable markers are selected for CRISPR activity. In some embodiments, selectable markers are based on negative selection (e.g., knockout of target protein). In some embodiments, selectable markers are knockouts of CD3.

[0191] In some embodiments, the gRNA vector comprises a suicide gene. "Suicide gene" refers to a gene that is toxic to a host cell (e.g., a cell that transcribes or translates a suicide gene) when transcribed or translated. In some embodiments, the suicide gene is operably connected to a promoter (e.g., a promoter described herein). In some embodiments, the suicide gene is operably connected to an inducible promoter. In some embodiments, the suicide gene is operably connected to a weak promoter (e.g., a PGK promoter). In some embodiments, the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20, and tHer2. In some embodiments, the suicide gene is icaspase9.

[0192] In some embodiments, the gRNA vector is a viral vector as described above. In some embodiments, the gRNA vector is selected from 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.

[0193] In some embodiments, the gRNA vector comprises a sequence of SEQ ID NO: 1318 and a sequence of a guide RNA polynucleotide described herein. In some embodiments, the gRNA vector comprises a sequence of SEQ ID NO: 1318 and a sequence of a guide RNA polynucleotide homology region of any one of SEQ ID NO: 136 to 1215 or a variant thereof. In some embodiments, the gRNA vector comprises a sequence of SEQ ID NO: 1318 and a sequence of a guide RNA polynucleotide homology region of any one of SEQ ID NO: 136 to 1315 or a variant thereof. In some embodiments, the gRNA vector further comprises a polynucleotide encoding a CRISPR protein (e.g., Cas9 or Cas12) as described above.

[0194] gRNA vector library

[0195] "gRNA vector library" refers to a composition comprising at least 2 gRNA vectors encoding different gRNA polynucleotides as described herein. In some embodiments, the gRNA vector library comprises at least 3 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 4 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 5 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 6 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 7 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 8 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 9 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 10 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 11 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 12 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 13 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 14 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 15 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 10000 gRNA vectors encoding different gRNA polynucleotides. In some embodiments, the gRNA vector library comprises at least 15000 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.

[0196] In some embodiments, the gRNA vector library comprises at least 2 gRNAs each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology regions are complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 3 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 4 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 5 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 6 gRNAs each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 7 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 8 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 9 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 10 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the gRNA vector library comprises at least 11 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 12 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 13 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 14 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 15 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 16 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 17 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 18 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 19 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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.In some embodiments, the gRNA vector library comprises at least 20 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 21 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 22 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 23 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 24 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 25 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 26 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 27 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 28 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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.In some embodiments, the gRNA vector library comprises at least 29 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 30 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 40 gRNA vectors each comprising a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region 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. In some embodiments, the gRNA vector library comprises at least 50 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 60 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 70 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 80 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 90 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the gRNA vector library comprises at least 100 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 200 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 300 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 400 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 500 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 1000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 5000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 10000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the gRNA vector library comprises at least 15,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 20,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 30,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 40,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 50,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 60,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 70,000 gRNA vectors encoding different gRNA polynucleotides, each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology regions are complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.In some embodiments, the gRNA vector library comprises at least 80,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 90,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 100,000 gRNA vectors encoding different gRNA polynucleotides each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology region is complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof. In some embodiments, the gRNA vector library comprises at least 1,000,000 gRNA vectors encoding different gRNA polynucleotides, each containing a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology regions are complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.

[0197] In some embodiments, the gRNA vector library comprises 135 gRNA vectors, each gRNA vector comprising a first guide RNA polynucleotide targeting a marker protein (e.g., CD3) and a second guide RNA containing a homology region complementary to the sense strand or antisense strand of a gene encoded by any one of SEQ ID NOs: 1 to 135 or its variants. In some embodiments, the gRNA vector library comprises 1080 gRNA vectors, each gRNA vector comprising a first guide RNA polynucleotide targeting a marker protein (e.g., CD3) and a second guide RNA containing a homology region complementary to the sense strand or antisense strand of a gene encoded by any one of SEQ ID NOs: 1 to 135 or its variants.

[0198] In some embodiments, the gRNA vector library comprises gRNA vectors comprising gRNA polynucleotide homology regions 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.

[0199] In some embodiments, the gRNA vector library comprises any one of the guide RNA polynucleotides in 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.

[0200] CAR vector

[0201] In some embodiments, the present application provides a vector comprising a sequence encoding a chimeric antigen receptor (CAR).

[0202] Chimeric Antigen Receptor (CAR)

[0203] The term "chimeric antigen receptor" or "CAR" or "CARs" used herein refers to a modified T cell receptor that specifically implants a ligand or antigen into a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor.

[0204] CAR places a chimeric antigen binding domain on a construct comprising a transmembrane domain and an intracellular domain of a T cell receptor molecule, and the chimeric antigen binding domain specifically binds to a target (e.g., a polypeptide) expressed on the surface of a cell targeted by a T cell response. In some embodiments, the chimeric antigen binding domain comprises an antigen domain of an antibody reagent specifically bound to an antigen expressed on a cell targeted by a T cell response. In some embodiments, the chimeric antigen binding domain comprises a ligand specifically bound to an antigen expressed on a cell targeted by a T cell response.

[0205] "CAR-T cells" or "CAR-T" used herein refer to T cells expressing CAR. When expressed in T cells, CAR has the ability to redirect T cell specificity and reactivity to selected targets in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby avoiding the main mechanism of tumor escape.

[0206] As used herein, "CAR-NK cell" or "CAR-NK" refers to a natural killer (NK) cell expressing CAR.

[0207] In some embodiments, CAR does not include the CD8 signal peptide described herein. As can be determined by those skilled in the art, a variety of functionally similar or equivalent components of these CARs and other similar or functionally equivalent components known in the art or listed herein can be interchanged or replaced.

[0208] CARs can target any cell surface moiety. Typically, the target will be a cell surface polypeptide that is differentially or preferentially expressed on cells to which one wishes to target a T cell response. In some embodiments, the extracellular target binding domain binds to any of CD19, CD37, CD70, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin 6, binds to any of the pairs CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain, e.g.

[0209] PCT / US2020 / 065733, PCT / US2020 / 036108, PCT / US2018 / 013215, PCT / US2018 / 013213, PCT / US2018 / 027783, PCT / US2018 / 013221, PCT / US2018 / 022974, P CT / US2019 / 042268, PCT / US2019 / 038518, PCT / US2019 / 066357, PCT / US2019 / 013103, PCT / US2019 / 017727, PCT / US2020 / 051018. and / or PCT / US2018 / 013095

[0210] As described in .

[0211] Antigen binding domain

[0212] The term "antigen binding domain" as used herein refers to a polypeptide present outside the cell that is sufficient to promote binding to a target. The extracellular target binding domain will specifically bind to its binding partner (i.e., target). As a non-limiting example, the antigen binding domain may include an antibody or an antibody reagent or an antigenic domain of a ligand that recognizes and binds to a cognate binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or a receptor. The cognate binding partner of a ligand that can be used for the methods and compositions described herein may generally be present on the cell surface. Ligand: cognate partner binding may result in changes in receptors with ligands, or activation of physiological responses, such as activation of signal transduction pathways. In some embodiments, the ligand may be non-natural to the genome. In some embodiments, the ligand has a conserved function in at least two species.

[0213] CAR can target any cell surface part. In some embodiments, the target will be a cell surface polypeptide that can be differentially expressed or preferentially expressed on the cells targeted by the T cell response. In order to target Treg, antibody reagents can be targeted to, for example, glycoprotein A dominant repeat sequence (Glycoprotein A Repetitions Predominant, GARP), latency-associated peptide (latency-associated peptide, LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB and LAG-3.

[0214] 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, and claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain.

[0215] 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 containing a mesothelin antibody (e.g., scFv). In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 1366 and a VL domain of SEQ ID NO: 1367, or a variant thereof. In some embodiments, the mesothelin scFv comprises SEQ ID NO: 1368 or SEQ ID NO: 1369, or a variant thereof.

[0216] Table 1: Mesothelin antibody sequences

[0217]

[0218] Hinge and transmembrane domains

[0219] In some embodiments, the CAR polypeptide further comprises a transmembrane domain, such as a hinge / transmembrane domain connecting the antigen binding domain to the intracellular signaling domain. In some embodiments, the binding domain of CAR is followed by one or more "hinge domains", which play a role in positioning the antigen binding domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation. CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence. Exemplary hinge domains suitable for use in CAR described herein include hinge regions derived from type 1 membrane proteins (e.g., CD8 (e.g., CD8α), CD4, CD28, 4-1BB and CD7) extracellular regions, and the hinge region may be a wild-type hinge region from these molecules or may be an altered hinge region. In some embodiments, CAR comprises a polynucleotide encoding a CD8α hinge / transmembrane domain. In some embodiments, CAR comprises a polynucleotide encoding a 41BB intracellular domain.

[0220] In some embodiments, the hinge region is derived from an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG or IgM), a hinge region of CD28 or CD8. In some embodiments, the hinge domain comprises a CD8a hinge region.

[0221] "Transmembrane domain" (TM domain) used herein refers to a part of such CAR, which fuses the extracellular binding portion (in some embodiments via the hinge domain) with the intracellular portion (e.g., costimulatory domain and intracellular signaling domain), and anchors CAR to the plasma membrane of immune effector cells. Transmembrane domains are generally hydrophobic regions of CARs that pass through the plasma membrane of the cell. TM domains can be transmembrane proteins (e.g., type I transmembrane proteins or other transmembrane proteins), artificial hydrophobic sequences, or transmembrane regions or fragments thereof of a combination thereof. Although specific examples are provided herein and used herein, other transmembrane domains will be obvious to those skilled in the art, and can be used in combination with alternative embodiments of the present technology. The selected transmembrane region or its fragment preferably does not interfere with the intended function of CAR.

[0222] "Fragment thereof" used in relation to a transmembrane domain of a protein or polypeptide refers to a portion of the transmembrane domain that is sufficient to anchor or link the protein to the cell surface.

[0223] In some embodiments, the transmembrane domain of the CAR described herein or a fragment thereof includes a transmembrane domain selected from the group consisting of: α, β or ζ chain of a T cell receptor, CD28, CD3ε, 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, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C transmembrane domain.

[0224] "Hinge / transmembrane domain" used herein refers to a domain comprising both a hinge domain and a transmembrane domain. For example, a hinge / transmembrane domain may be derived from the hinge / transmembrane domain of CD8, CD28, CD7 or 4-1BB. In some embodiments, the hinge / transmembrane domain of CAR or its fragment is derived from the hinge / transmembrane domain of CD8 or comprises the hinge / transmembrane domain of CD8 (for example, SEQ ID NO: 1 or any one of its variants). CD8 is an antigen preferentially present on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a T cell co-receptor. CD8 consists of alpha chains (CD8 alpha or CD8a) and beta chains (CD813 or CD8b). CD8a sequences of many species are known, for example, human CD8a (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 may refer to human CD8, including naturally occurring variants, molecules and alleles thereof. In some embodiments of any aspect, for example, in veterinary applications, CD8 may refer to CD8 of, for example, dogs, cats, cattle, horses, pigs, etc.

[0225] Homologs and / or orthologs of human CD8 for such species are readily identified by one skilled in the art, for example, using the NCBI ortholog search function or searching available sequence data for sequences similar to a reference CD8 sequence for a given species.

[0226] In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO: 1371 ( ), or comprises a sequence of SEQ ID NO: 1; or comprises 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.

[0227] Costimulatory domain

[0228] Each CAR described herein optionally includes an intracellular domain or a costimulatory domain of one or more costimulatory molecules. The term "costimulatory domain" used herein refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal required for the effective activation and function of T lymphocytes when bound to an antigen. The costimulatory domain can be, for example, a costimulatory domain of 4-1BB, CD27, CD28 or OX40. In one example, a 4-1BB intracellular domain (intracellulardomain, ICD) (see, e.g., below and SEQ ID NO: 1372 ( ), or variants thereof). Additional illustrative examples of such costimulatory 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, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-inducible costimulatory molecule and an important regulator in immune response.

[0229] 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. The 4-1BB sequences of many species are known, such as human 4-1BB, also known as TNFRSF9 (NCBI gene 25ID: 3604), and its mRNA (NCBI reference sequence: NM_001561.5). 4-1BB may refer to human 4-1BB, including its naturally occurring variants, molecules and alleles. In some embodiments of either aspect, for example, in veterinary applications, 4-1BB may refer to 4-1BB such as dogs, cats, cattle, horses, pigs, etc. For such species, homologs and / or orthologs of human 4-1BB are easily identified by those skilled in the art, for example, using the NCBI ortholog search function or searching for sequences similar to the reference 4-1BB sequence of a given species for available sequence data to identify.

[0230] Intracellular signaling domain

[0231] In some embodiments, the CAR comprises a polynucleotide encoding a CD3 zeta intracellular signaling domain.

[0232] The properties of the intracellular signaling domain of the CAR can vary as known in the art and disclosed herein, but the chimeric target / antigen binding domain sensitizes the receptor to signaling activation when the chimeric target / antigen binding domain binds to the target / antigen on the surface of the target cell.

[0233] Regarding the intracellular signaling domain, the so-called "first generation" CAR includes CARs that only provide CD3-ζ signals when antigen binds. The so-called "second generation" CAR includes CARs that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3-ζ;) domains, and the so-called "third generation" CAR includes CARs that provide multiple co-stimulation (e.g., CD28 and CD137) domains and activation domains (e.g., CD3-ζ). In multiple embodiments, a CAR with high affinity or avidity for the target / antigen is selected-for example, an antibody-derived target or antigen binding domain typically has a higher affinity and / or avidity for the target antigen compared to a naturally occurring T cell receptor. This property, combined with the high specificity of antibodies that can be selected by people, provides high specificity T cell targeting of CAR-T cells.

[0234] CAR described herein includes an intracellular signaling domain. "Intracellular signaling domain" refers to a part of such a CAR polypeptide, which participates in the effective CAR information transduction combined with the target antigen to the inside of the immune effector cell, to induce effector cell function, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the target cell bound by CAR, or antigens and extracellular CAR domains are combined to induce other cell responses. In multiple examples, the intracellular signaling domain is from CD3-ζ; (for example, see below). Other non-limiting examples of intracellular signaling domains containing an activation motif (immunoreceptor tyrosine-based activation motif, ITAM) particularly useful in the present technology include intracellular signaling domains derived from TCR-ζ, FcR-γ, FcR-β, CD3-γ, CD3-θ, CD3-σ, CD3-η, CD3-ε, CD3-ζ, CD22, CD79a, CD79b and CD66d.

[0235] CD3 is a T cell co-receptor that promotes T lymphocyte activation when simultaneously engaged with an appropriate co-stimulatory agent (e.g., binding to a co-stimulatory molecule). The CD3 complex consists of four different chains; mammalian CD3 consists of one CD3-γ chain, one CD3δ chain, and two CD3-ε chains.

[0236] These chains associate with CD3-ζ and a molecule known as the T cell receptor (TCR), thereby generating an activation signal in T lymphocytes. A complete TCR complex contains the TCR, CD3-ζ, and the complete CD3 complex.

[0237] In some embodiments of any aspect, the CAR polypeptide described herein comprises an intracellular signaling domain containing an immunoreceptor tyrosine-based activation motif or an ITAM from CD3-ζ, wherein the CD3-ζ includes a variant of CD3-ζ, such as CD3-ζ, CD3-η or CD3-θ mutated by ITAM. In some embodiments of any aspect, ITAM comprises three motifs (ITAM3) of the ITAM of CD3-ζ. In some embodiments of any aspect, the three motifs of the ITAM of CD3-ζ are not mutated, and thus include natural or wild-type sequences. In some embodiments, the CD3-ζ sequence comprises a CD3-ζ sequence listed in the sequence provided herein, such as a CD3-ζ sequence of SEQ ID NO: 1373 ( ), or variations thereof.

[0238] For example, the CAR polypeptide described herein comprises an intracellular signaling domain of CD3-ζ. In some embodiments, the CD3-ζ intracellular signaling domain corresponds to the amino acid sequence of SEQ ID NO: 1373 or comprises a sequence of SEQ ID NO: 1373; or comprises 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: 1373.

[0239] 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 comprises a sequence selected from SEQ ID NO: 2; or comprises 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 with a sequence selected from SEQ ID NO: 2.

[0240] As can be determined by those skilled in the art, the individual CARs and other construct components described herein can be used together with each other and swapped in and out of the various constructs described herein. Each of these components can all comprise or consist of any corresponding sequence listed herein or a variant thereof.

[0241] A more detailed description of CAR and CAR-T cells can be found in:

[0242] 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. 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;

[0243] Each of which is incorporated herein by reference in its entirety.

[0244] Signal peptide

[0245] In some embodiments, the CAR polypeptide described herein comprises a signal peptide. The signal peptide may be derived from any protein having an extracellular domain or a secreted protein. The CAR polypeptide described herein may comprise any signal peptide known in the art. In some embodiments, the CAR polypeptide comprises a CD8 signal peptide, for example, an amino acid sequence corresponding to SEQ ID NO: 1374 ( ), or a CD8 signal peptide comprising the amino acid sequence of SEQ ID NO: 5, or a CD8 signal peptide comprising 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.

[0246] In additional embodiments, the CAR polypeptide described herein may optionally not include one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO: 1374 or the IgK signal peptide of SEQ ID NO: 1375 ( ).

[0247] Linker domain

[0248] In some embodiments, CAR also includes a linker domain. As used herein, a "linker domain" refers to an oligopeptide or polypeptide region of about 2 to 100 amino acids in length that connects any domain / region of the CAR described herein. In some embodiments, the linker may include or consist of flexible residues (e.g., glycine and serine) so that adjacent protein domains move freely relative to each other. The length of the linker sequence that can be used in the present invention can be 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids, and includes any suitable linker known in the art. For example, linker sequences that can be used in the present invention include, but are not limited to, glycine / serine linkers, such as and Gly4Ser (G4S) (SEQ ID NO: 1378) linker, e.g. and ; as described in Whitlow et al., Protein Eng. 6(8):989-95,1993 Linker sequence, the contents of which are incorporated herein by reference in their entirety; as described in Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011 (9), 2011 Linker sequences, the contents of which are incorporated herein by reference in their entirety; and linker sequences with additional functions, for example, epitope tags or coding sequences comprising Cre-Lox recombination sites, as described in 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 when it is desired to ensure that two adjacent domains do not interfere with each other spatially.

[0249] In addition, the linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 1381, ) and T2A( , SEQ ID NO: 1382)), 2A-like linker, or its functional equivalent, and combination thereof.

[0250] For example, the P2A linker sequence may correspond to the amino acid sequence of SEQ ID NO: 1381. In multiple examples, a linker having a sequence listed herein or a variant thereof is used. It should be understood that the marking of a specific linker in a construct at a specific position does not mean that only the linker can be used here. More specifically, as can be determined by those skilled in the art, different linker sequences (e.g., P2A and T2A) can be interchanged (e.g., in the case of a construct of the present invention). In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in the present technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. For example, these linkers can be used in the case of polyproteins, such as the following linkers. For example, they can be used to separate the CAR component of a polyprotein from the therapeutic agent (e.g., antibody, such as scFv, single domain antibody (e.g., camelid antibody) or bispecific antibody (e.g., TEAM)) component in the polyprotein (see below).

[0251] Complete CAR

[0252] In some embodiments, the CAR is selected from: (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, 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 to the sequence of any one of SEQ ID NOs: 1325 to 1365. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1325 to 1365. In some embodiments, the CAR polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 1325 to 1365. In some embodiments, the CAR polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1325 to 1365.

[0253] In some embodiments, the CAR comprises a polynucleotide encoding a mesothelin scFv, a CD8α hinge / transmembrane, a 41BB intracellular domain, and a CD3ζ 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 higher sequence identity to the sequence of any one of SEQ ID NOs: 1325 to 1328. 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 higher sequence identity to the sequence of any one of SEQ ID NOs: 1325 to 1328. In some embodiments, the CAR polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1325 to 1328.

[0254] CAR vector

[0255] In some embodiments, the CAR 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, CAR is operably connected 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 EF1a short promoter.

[0256] In some embodiments, the CAR vector comprises a suicide gene. "Suicide gene" may refer to a gene that causes cell death when transcribed or translated. Therefore, the suicide gene may be included in the CAR vector to inactivate the CAR treatment after the treatment is completed or if the subject has adverse side effects. In some embodiments, the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20 and tHer2. In some embodiments, the CAR vector comprises a selectable marker (eg, a reporter). In some embodiments, the selectable marker is selected from truncated CD34, tEGFR, tCD19, tCD20, tCD34 and tHer2. In some embodiments, the selected marker is CD34 or LNGFR.

[0257] In some embodiments, the CAR vector comprises a 2A ribosomal skipping element or an internal ribosome entry site (IRES) between the CAR polynucleotide sequence and the selectable marker sequence.

[0258] In some embodiments, the CAR vector further comprises a nucleotide sequence encoding a CRISPR protein as described herein. In some embodiments, the CAR vector does not comprise a nucleotide sequence encoding a CRISPR protein.

[0259] In some embodiments, the CAR vector comprises a CAR selected from the following: (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, BCMA / TACI, or (3) a TriPRIL antigen binding domain.

[0260] 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 a polynucleotide sequence of SEQ ID NO: 1319.

[0261] CAR-T cells, NK cells and mutant libraries

[0262] In some aspects, the present disclosure relates to such immune cells (e.g., CAR-T cells or CAR-NK cells), which contain guide RNA vectors described herein and / or mutations in genes corresponding to the gene sequences of any one of SEQ ID NO: 1 to 135. The present disclosure relates in part to the concept of introducing one or more mutations into immune cells by contacting immune cells with guide RNA vectors and providing CRISPR proteins (e.g., as part of guide RNA vectors or encoded on separate vectors). The expression of guide RNA vectors and CRISPR proteins can lead to mutations in or near sequences complementary to the homologous regions of gRNA polynucleotides encoded by gRNA vectors in immune cells. Therefore, the present disclosure relates to immune cells comprising guide RNA vectors, immune cells comprising mutations produced by CRISPR proteins and one or more gRNA polynucleotides encoded by guide RNA vectors, and immune cells comprising both of them. In some embodiments, immune cells comprise (i) guide RNA vectors and / or mutations in genes corresponding to the gene sequences of any one of SEQ ID NO: 1 to 135; and (ii) CAR vectors described herein. In some embodiments, the immune cell comprises one, two or three of the following: a mutation in a gene corresponding to a gene sequence of any one of SEQ ID NO: 1 to 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 cell further comprises a CAR vector described herein. In some embodiments, the mammalian cell further comprises an mRNA encoding a CAR and / or a polypeptide encoding a CAR.

[0263] In some embodiments, the immune cell comprises a mutation in a gene corresponding to a gene sequence or a variant thereof of any one of SEQ ID NO: 1 to 135. In some embodiments, the mutation is the result of CRISPR cutting a 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 conversion. In some embodiments, the mutation changes the protein sequence of the target gene. In some embodiments, the mutation introduces an early stop codon. In some embodiments, the mutation is an insertion or deletion. In some embodiments, the immune cell comprises a gRNA polynucleotide as described herein. In some embodiments, the immune cell comprises a gRNA vector as described herein. In some embodiments, the immune cell also comprises a CAR vector as described herein. In some embodiments, the immune cell comprises a CAR as described herein.

[0264] In some embodiments, the immune cells comprising gRNA vectors and CAR vectors also include selected markers (e.g., reporters as described herein). In some embodiments, immune cells are CD34 positive and CD3 negative. In some embodiments, immune cells are CD34 positive, CD3 negative and LNGFR positive.

[0265] The immune cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, goat, canine or feline origin, and any other immune cells can be used. In some embodiments, the immune cells are human.

[0266] In some embodiments, mammalian cells are immune cells. "Immune cells" as used herein refer to cells that play a role in immune responses. Immune cells are of hematopoietic origin and include T cells and natural killer (NK) cells. In some embodiments, immune cells are T cells. In some embodiments, immune cells are NK cells.

[0267] In some embodiments, immune cells are obtained from individuals suffering from or diagnosed with cancer, plasma cell disorders, or autoimmune diseases. 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 taken from a donor or patient. T cells can be isolated from mammals. Preferably, T cells are isolated from humans. In some embodiments, immune cells are collected from a subject using leukapheresis.

[0268] In some embodiments, immune cells are activated. In some embodiments, immune cells are activated T cells comprising gRNA vectors and CAR vectors. Methods for activating T cells are well known in the art. In some embodiments, activating T cells includes contacting T cells with anti-CD3 antibodies. In some embodiments, activating T cells includes contacting T cells with anti-CD28 antibodies. In some embodiments, activating T cells includes contacting T cells with interleukin-2 (IL-2). In some embodiments, activating T cells includes contacting T cells with phytohemagglutinin (PHA). In some embodiments, activating T cells includes contacting T cells with anti-CD28 antibodies and anti-CD3 antibodies. In some embodiments, activating T cells includes contacting T cells with anti-CD3 antibodies and interleukin-2 (IL2). In some embodiments, activating T cells includes contacting T cells with anti-CD3 antibodies, interleukin-2 (IL2) and phytohemagglutinin (PHA). In some embodiments, when activating T cells, any one of anti-CD3 antibodies, anti-CD28 antibodies, interleukin-2 (IL-2) and / or phytohemagglutinin (PHA) is soluble. In some embodiments, activating T cells includes contacting T cells with anti-CD3 antibodies, interleukin-2 (IL2) and phytohemagglutinin (PHA). In some embodiments, when activating T cells, any one of anti-CD3 antibodies, anti-CD28 antibodies, interleukin-2 (IL-2) and / or phytohemagglutinin (PHA) is fixed to a solid culture medium (e.g., a cell plate).

[0269] In some embodiments, the immune cell (e.g., T cell) does not comprise a DNA sequence encoding a CRISPR protein (e.g., Cas9).

[0270] In some embodiments, multiple immune cells (e.g., T cells or NK cells) are transfected with (1) CAR vectors and (2) gRNA libraries described herein to generate mutant CAR-T cell libraries. Methods for 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.

[0271] One advantage of using separate vectors to encode CAR and gRNA polynucleotides is that the multiplicity of infection (MOI) of CAR and gRNA can be controlled respectively. The multiplicity of infection can refer to how many of the given vectors enter the cell. For example, and MOI of 1 indicates that the average number of given vectors for each T cell in the mutant CAR-T cell library is 1. In some embodiments, MOI can be a target MOI based on the amount of transfected vectors. In some embodiments, CAR-T cell efficacy may depend on the amount of CAR expressed in the cell. CAR expression can be controlled by increasing the number of CAR vectors transfected by each cell (e.g., increasing MOI). However, in some embodiments, technicians may wish to increase the MOI of the CAR vector, but not the MOI of the gRNA vector, because when each cell is transfected with more than one gRNA vector, it is possible to return mixed results from a variety of different mutations produced by the same cell. Therefore, 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) of 0.1 to 1, 0.2 to 1, 0.3 to 1, 0.4 to 1, 0.5 to 1, 0.6 to 1, 0.7 to 1, 0.8 to 1, 0.3 to 1, 0.4 to 0.9, 0.5 to 0.8 or 0.6 to .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 1 to 3, 1 to 5, 1 to 10, 1 to 20, 5 to 10, 5 to 15, 5 to 20 or 10 to 20.

[0272] "Mutation CAR-T cell library" may refer to two or more CAR-T cells, wherein at least two of the two or more CAR-T cells include CAR (e.g., CAR vector) and gRNA vectors, wherein at least two of the two or more CAR-T cells include gRNA vectors encoding different guide RNAs. 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 15, at least 20, at least 25, at least 50, at least 100 or at least 135) CAR-T cells in the library each include different gRNA polynucleotides, which include homology regions complementary to different gene sequences of any one of SEQ ID NO:1 to 135 or its variants. In some embodiments, the CAR-T cell library includes a gRNA vector as described herein. In some embodiments, the CAR-T cells of the mutant CAR-T cell library include SEQ ID NO:236 to 1315. In some embodiments, the mutant CAR-T cell library includes any one of the compositions comprising gRNA polynucleotides described herein. In some embodiments, the mutant CAR-T cell library includes 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 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750 or at least 1000) SEQ ID NO: 236 to 1315 of any one of the gRNA homology region sequences. In some embodiments, the mutant CAR-T cell library includes SEQ ID NO: 236 to 1315.

[0273] method

[0274] In some aspects, the disclosure provides a method for producing a mutant immune cell library. In some embodiments, the immune cell library comprises T cells (mutated CAR-T cell library). In some embodiments, the immune cell library comprises NK cells (mutated CAR-NK cell library). In some embodiments, the cells in the library comprise CAR or a vector encoding CAR (e.g., a mutant CAR-T cell library or a mutant CAR-NK cell library).

[0275] Generation of mutant CAR-T cell libraries

[0276] In some aspects, the disclosure provides a method for producing a mutant CAR-T cell library. In some embodiments, the method includes: (a) obtaining CAR-T cells (e.g., previously prepared CAR-T cells), and (b) transfecting T cells with a gRNA vector library (e.g., as described herein). In some embodiments, the obtained CAR-T cells have been previously activated. In some embodiments, the method includes activating CAR-T cells. In some embodiments, CAR-T cells express CRISPR proteins. In some embodiments, the method also includes introducing mRNA or CRISPR proteins encoding CRISPR proteins into CAR-T cells (e.g., by electroporation).

[0277] In some embodiments, the method comprises: (a) activating T cells (e.g., as described above (e.g., using anti-CD3 and anti-CD28 antibodies)), (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 mRNA encoding a CRISPR protein or a CRISPR protein into the T cells (e.g., by electroporation).

[0278] In some embodiments, CRISPR protein is any suitable CRISPR protein for mutating DNA in CAR-T cells. In some embodiments, CRISPR protein is any CRISPR protein described herein. In some embodiments, the introduction of mRNA or CRISPR protein encoding CRISPR protein into T cells is performed 1 to 10 days after gRNA vector and / or CAR vector transfection. In some embodiments, the introduction of mRNA or CRISPR protein encoding CRISPR protein into T cells is performed 3 to 7 days after gRNA vector and / or CAR vector transfection. In some embodiments, the introduction of mRNA or CRISPR protein encoding 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 gRNA vector and / or CAR vector transfection. In some embodiments, the introduced CRISPR protein is CRISPR protein. In some embodiments, the introduced CRISPR protein is introduced as ribonucleoprotein. In some embodiments, the CRISPR protein is encoded on a gRNA vector or a CAR vector.

[0279] In some embodiments, the method further comprises collecting T cells from the object, for example, and then the T cells can be modified using, for example, one or more (e.g., all) of the above steps (a) to (d). "Object" as used herein means a person or an animal. Usually the animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include, for example, chimpanzees, crab-eating monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include, for example, cattle, 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 trout, catfish, and salmon. In some embodiments, the object is a mammal, such as a primate, such as a person. In some embodiments, the object is a person. In some embodiments, the object is a laboratory model organism (e.g., mouse). The terms "individual", "patient" and "subject" are used interchangeably herein. Preferably, the object is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cattle, but is not limited to these examples. Mammals other than humans can be advantageously used as objects representing disease (eg, cancer) animal models. The object may be male or female. The object may be previously diagnosed or identified as having or having a condition requiring treatment (eg, pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer or mesothelioma or another type of cancer expressing an antigen target through the antigen binding domain of CAR) or one or more of the complications associated with such a condition, and optionally, the object has undergone treatment for the condition or one or more complications associated with the condition. Alternatively, the object may also be an object that has not been previously diagnosed as having such a condition or related complications. For example, the object may be an object that exhibits one or more risk factors for the condition or one or more complications associated with the condition, or an object that does not exhibit risk factors. A "subject in need thereof" for use in the treatment of a particular disorder may be a subject suffering from the disorder, diagnosed as suffering from the disorder, or at risk of developing the disorder.

[0280] In some embodiments, the subject has been diagnosed with cancer. "Cancer" used herein can refer to the excessive proliferation of cells, and its unique characteristics (loss of normal cell control) lead to uncontrolled 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 (acute myeloid leukemia, AML), chronic myeloid leukemia (chronic myeloid leukemia, CML), acute lymphocytic leukemia (acute lymphocytic leukemia, ALL) and chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL). In some embodiments, cancer is ALL or CLL. Non-limiting examples of lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's 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 tumors, alveolar soft tissue sarcomas, epithelial carcinomas, chondrosarcomas, colorectal cancers, desmoid tumors, desmoplastic small round cell tumors, endocrine tumors, endodermal sinus tumors, epithelioid hemangioendothelioma, Ewing sarcoma, germ cell tumors (solid tumors), giant cell tumors 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 may be present in bones, muscles, or organs, and may be sarcomas or epithelial cancers. It is contemplated that any aspect of the technology described herein may be used to treat all types of cancers, including cancers not listed in this application. The term "tumor" as used herein refers to an abnormal growth of cells or tissues (e.g., malignant or benign types).

[0281] 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., by induced pluripotent stem cells or embryonic stem cells). In some embodiments, T cells are collected from a subject using leukapheresis.

[0282] In some embodiments, the method also includes purifying T cells comprising a CAR vector and a gRNA vector. In some embodiments, the purification method can be performed based on selectable molecules (e.g., reporters) as described above. For example, a CAR vector can express a selectable marker (e.g., CD34t), and a gRNA vector can express a selectable marker (e.g., CD3 knockout). In some embodiments, antibody-based magnetic selection can be used for purification. In some embodiments, flow cytometry can be used for purification. In some embodiments, CliniMACS can be used for purification. In some embodiments, CAR-T cells with CRISPR activity are purified based on knockout of genes (e.g., CD3) for negative selection. In some embodiments, CAR-T cells expressing CAR are selected based on positive selection (e.g., expressing CD34). In some embodiments, flow cytometry can be used to determine the success of transduction based on the number of CD34+CD-LNGF+ cells.

[0283] In some embodiments, the gRNA vector library is transfected into the CAR-T cells at an MOI described herein. In some embodiments, the CAR vector is transfected at an MOI described herein.

[0284] In some embodiments, the present application discloses a mutant CAR-T cell library generated using any of the methods described herein.

[0285] Identify the effects of CRISPR on CAR-T cell function.

[0286] In some embodiments, the present disclosure provides methods for identifying mutated (e.g., by CRISPR) CAR-T cells with improved potency.

[0287] In some aspects, the present application discloses a method for identifying gRNA polynucleotides associated with CAR-T cell potency in vivo, the method comprising: (a) administering a mutant CAR-T cell library (e.g., a mutant CAR-T cell library disclosed herein or a mutant CAR-T cell library produced by the method described herein) to a subject, (b) collecting one or more samples containing multiple mutant CAR-T cells from the subject (e.g., by leukocyte apheresis), (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 the changes in the relative abundance of each gRNA polynucleotide based on the sequencing in (c). For example, a gRNA associated with CAR-T cell potency is a gRNA that induces such a mutation: the mutation increases CAR-T cell proliferation and / or tumor toxicity. The technician will understand that after the mutant CAR-T cell library is applied to the subject, the increase in the relative abundance of a given gRNA indicates that a given gRNA causes a mutation that increases the potency of CAR-T cells (e.g., increases persistence).

[0288] The skilled person will appreciate that mutant CAR-T cells (e.g., mutant CAR-T cells in a mutant CAR-T cell library) can replicate in a subject. Therefore, the multiple mutant CAR-T cells collected in step b above may include mutant CAR-T cells administered to a subject and / or offspring of mutant CAR-T cells administered to a subject.

[0289] In some embodiments, the methods described herein relate to treating an object suffering from or diagnosed with cancer, plasma cell disease or disorder, or autoimmune disease or disorder with immune cells, the immune cells comprising any CAR polypeptides described herein (and optional antibody reagents or cytokines), or encoding any CAR polypeptides described herein (and optional antibody reagents or cytokines) nucleic acid. The CAR-T cells described herein include such immune cells, which include any CAR polypeptides described herein (and optional antibody reagents or cytokines), or encoding any CAR polypeptides described herein (and optional antibody reagents or cytokines) nucleic acids.

[0290] The compositions described herein can be applied to a subject suffering from or diagnosed with a disease (e.g., cancer). "Disease" as used herein refers to cancer, plasma cell disease or disease, or autoimmune disease or disease. The subject suffering from the disease can be identified by a physician using a current method for diagnosing the disease. The symptoms and / or complications of the disease (which characterize these diseases and help diagnose) are well known in the art, and include but are not limited to fatigue, persistent infection, and persistent bleeding. Tests that can help diagnose, for example, diseases, but are not limited to blood screening and bone marrow testing, are known in the art for a given disease. The family history of the disease or exposure to the risk factors for the disease also helps determine whether the subject may suffer from the disease or helps diagnose the disease.

[0291] In some embodiments, the method described herein includes administering to a subject an effective amount of activated CAR-T cells described herein (e.g., mutant CAR-T cell libraries) to identify mutant CAR-T cells with improved efficacy. In some embodiments, the method described herein includes administering to a subject an effective amount of activated CAR-T cells described herein (e.g., mutant CAR-T cell libraries) to alleviate the symptoms of the disease. "Relieve symptoms of disease" used herein is to improve any disease or symptoms associated with the disease. Measured by any standard technique, such a reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more compared to an equivalent untreated control. Various ways of administering the compositions described herein to a subject are known to those skilled in the art. In some embodiments, the compositions described herein are systemically or topically administered. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the tumor site.

[0292] The term "effective amount" used herein may refer to the number of immune cells (e.g., activated CAR-T cells or CAR-NK cells) required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of cell preparations or compositions to provide the desired effect. Therefore, the term "therapeutically effective amount" may refer to the amount of activated immune cells (e.g., CAR-T cells or NK cells) sufficient to provide a specific anti-disease effect when applied to a general subject. The effective amount used herein in various cases also includes an amount sufficient to delay the occurrence of disease symptoms, change the course of symptomatic diseases (e.g., but not limited to slowing down the progression of the disease) or reverse the symptoms of the disease. Therefore, it is generally not feasible to specify an accurate "effective amount". However, for any given situation, a suitable "effective amount" can be determined by a person of ordinary skill in the art using only routine experiments.

[0293] The effective amount, toxicity and therapeutic efficacy can be evaluated by standard drug procedures in cell culture or experimental animals. The dosage can vary according to the dosage form used and the route of administration used. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. The therapeutically effective dose can be preliminarily estimated by cell culture assays. In addition, the dose can be formulated in an animal model to reach a circulating plasma concentration range including IC50 (i.e., the concentration of activated CAR-T cells that achieves half-maximal inhibition of symptoms), as determined in cell culture or in a suitable animal model. The level in plasma can be measured by, for example, high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay, such as a bone marrow test assay, etc. The dosage can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effect.

[0294] Modes of administration may include, for example, intravenous (iv) injection or infusion. The compositions described herein may be administered to the patient via an artery, intratumor, intranodal, intraperitoneal, or intramedullary. In some embodiments, the compositions of T cells may be injected directly 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).

[0295] In a specific exemplary aspect, the subject may undergo leukapheresis, wherein leukocytes are collected, enriched or depleted in vitro, thereby selecting and / or separating cells of interest, such as T cells. These T cell isolates can be amplified by contact with artificial APCs (e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs), and processed so that one or more CAR constructs of the technology can be introduced, thereby producing CAR-T cells. These methods may also include transfecting CAR-T cells with the gRNA library described herein.

[0296] In some embodiments, a single treatment regimen is required. In other embodiments, one or more subsequent doses or treatment regimens may be administered. For example, after a biweekly treatment for three months, the treatment may be repeated monthly for six months or a year or longer. In some embodiments, no additional treatment is administered after the initial treatment.

[0297] The dosage of the compositions described herein can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effects. Regarding the duration and frequency of treatment, for ordinary clinicians, it is usually to monitor the subject to determine when treatment provides therapeutic benefits, and to determine whether to administer additional cells, stop treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be too large to avoid causing adverse side effects, such as cytokine release syndrome. Typically, the dosage will vary with the patient's age, illness, and sex, and can be determined by those skilled in the art. In the event of any complications, the dosage can also be adjusted by an individual physician.

[0298] In some embodiments, the mutant CAR-T cell library administered to the subject includes a whole genome CAR-T cell mutant library. A whole genome CAR-T cell mutant library may refer to a variety of CAR-T cells transfected with a gRNA library, wherein the gRNA library includes at least one guide RNA targeting each gene in the CAR-T cell genome. In some embodiments, a whole genome CAR-T cell mutant library includes a variety of CAR-T cells transfected with a gRNA library, wherein the gRNA library includes at least one guide RNA targeting each protein-encoding gene in the CAR-T cell genome. Shifrut et al., Cell 175.7 (2018): A whole genome T cell CRISPR library and a method using an in vitro cell system are described in 1958-1971.

[0299] In some embodiments, the mutant CAR-T cell library comprises a negative control CAR-T cell. In some embodiments, the negative control CAR-T cell comprises a CAR vector described herein and a negative control gRNA described herein. In some embodiments, the negative control CAR-T cell comprises an empty CAR vector that does not encode CAR. In some embodiments, the negative control CAR-T cell comprises a CAR without an antigen binding domain. In some embodiments, the negative control CAR-T cell comprises a CAR containing an antigen binding domain that is not bound to a protein expressed by the object.

[0300] 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 as described herein). In some embodiments, the subject suffers from cancer (e.g., a cancer described herein). In some embodiments, the cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, or B-cell lymphoma).

[0301] In some embodiments, CAR comprises an antigen binding domain (e.g., an antigen binding domain as described herein) that binds to an antigen expressed by cancer. In some embodiments, CAR comprises an antigen binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and claudin 6, an antigen binding domain that binds to any one of CD19 / CD79b, BCMA / TACI, or a TriPRIL antigen binding domain. In some embodiments, the antigen binding domain binds to mesothelin.

[0302] In some embodiments, one or more samples collected from an object are collected using any reasonable means for any reasonable tissue or body fluid. In some embodiments, one or more samples are collected from a body fluid (e.g., cerebrospinal fluid, blood, or blood products). 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 tissues (e.g., heart, lung, liver, pancreas, brain, skin, intestine, kidney, spleen, lymph node, thyroid, muscle, fat, gall bladder (gullbladder) or tonsils).

[0303] In some embodiments, sample collection is performed using any reasonable means including, but not limited to, venipuncture, apheresis, tissue biopsy, or tumor resection.

[0304] In some embodiments, one sample is 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 to 2, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 2 to 3, 2 to 5, 2 to 7, 2 to 10, 2 to 12, 2 to 15, 3 to 5, 3 to 7, 3 to 10, 3 to 12, or 3 to 15 samples are collected from a subject. In some embodiments, at least 5 samples are collected by venipuncture, apheresis, or tumor resection.

[0305] In some embodiments, the sample is collected from the subject at one or more of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or day 31 after step (a). In some embodiments, the sample is collected from the subject at one or more of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 after step (a). In some embodiments, samples are collected from subjects at one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months after step (a). In some embodiments, samples are collected from subjects at 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, and 6 months after step (a).

[0306] In some embodiments, the method also includes extracting mutant CAR-T cells from the sample before sequencing. The method of separating CAR-T cells from a sample (e.g., body fluid) is well known in the art. For example, leukocyte removal is a known method for extracting CAR-T cells from an object. Other methods may include immunoprecipitation, immunomagnetic selection, microfluidics, and flow cytometry 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, extracting mutant CAR-T cells from a sample is based on cell surface markers expressed by mutant CAR-T cells. In some embodiments, extraction is performed by separating CD3 negative, CD34 positive, and optionally LNGFR positive T cells.

[0307] In some embodiments, the method further includes extracting genomic DNA from mutant CAR-T cells. In some embodiments, genomic DNA is extracted from a single mutant CAR-T cell or a clonal population of mutant CAR-T cells. In some embodiments, the method includes sequencing the extracted genomic DNA to determine the mutations caused by CRISPR guide RNA in a given CAR-T cell. Alternatively, in some embodiments, the method includes amplifying a genomic region (e.g., based on a sequence of a gRNA homologous region) expected to contain a CRISPR-induced mutation using a primer, and sequencing the region.

[0308] In some embodiments, the method further comprises isolating RNA from the mutant CAR-T cells. In some embodiments, RNA is isolated from a single mutant CAR-T cell (e.g., for single cell RNA sequencing). In some embodiments, the isolated RNA from the mutant CAR-T cells is sequenced (e.g., using illumine sequencing).

[0309] In some embodiments, the method includes sequencing the gRNA polynucleotides in the mutant CAR-T cells extracted from the subject. In some embodiments, the method involves extracting DNA from the mutant CAR-T cells. The technician will understand that DNA can be extracted from the mutant CAR-T cell pool collected from the subject. In other words, in some embodiments, DNA from each CAR-T cell does not need to be separated separately from each mutant CAR-T cell collected from the subject to determine the effect of CRISPR mutations on CAR-T cell efficacy. More specifically, the guide RNA homology region sequence can be used to identify which gene is expected to be mutated by CRISPR and the expected position of the mutation in the gene, because the guide RNA homology region can have a sequence complementary to a specific region of a specific gene.

[0310] Therefore, in some embodiments, the method also includes amplifying the gRNA polynucleotides in the mutant CAR-T cells using primers to produce any of them, and these amplicons are used for sequencing. In some embodiments, the gRNA polynucleotides amplified are genomic DNA, vector DNA or RNA. In some embodiments, universal primers can be used to amplify all guide RNA polynucleotides in the mutant CAR-T cell library. In some embodiments, universal primers include universal forward primers and universal reverse primers. 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 polynucleotides upstream of the homology region. In some embodiments, the universal reverse primer binds to the gRNA polynucleotides downstream of the homology region. In some embodiments, the universal forward primer includes the sequence of SEQ ID NO: 1324. In some embodiments, the forward primer and the reverse primer include thiophosphate modification. In some embodiments, the universal forward primer includes a staggered sequence (e.g., a variable length sequence in the primer that improves the resolution of next-generation sequencing). Staggered sequences are also known in the art, such as described in Wohlfieter et al., STAR Protocols 2.2 (2021): 100390. In some embodiments, the staggered sequence includes In some embodiments, the universal reverse primer comprises a staggered sequence.

[0311] In some embodiments, the universal reverse primer comprises the sequence of SEQ ID NO:1325.

[0312] In some embodiments, the gRNA polynucleotide or its amplicon is sequenced. The sequencing of the gRNA polynucleotide, its amplicon, or any other sequencing described herein can be performed using any suitable method or device, including but not limited to next generation sequencing, Illumina™ sequencing, Ion Torrent™ sequencing, PacBio™ sequencing, nanopore sequencing, 454 sequencing, sanger sequencing, and SoLid™ sequencing.

[0313] In some embodiments, the method includes evaluating the change of 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 the sequencing data before applying a mutant CAR-T cell library to the patient and after extracting a mutant CAR-T cell library from the patient. In some embodiments, 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 effectiveness of CAR-T cells.

[0314] In some embodiments, the method includes determining a fold change value by comparing the average gRNA polynucleotide abundance of any given gene in a post-infusion sample (extracted from the subject) to the gRNA polynucleotide abundance in a pre-infusion sample (before administration to the subject). In some embodiments, the threshold for identifying significant fold changes in gRNA polynucleotide abundance is based on the z-score normalization of the distribution of fold change values. In some embodiments, a change greater than 2 standard deviations from the mean of the z-score distribution is considered significant.

[0315] In some embodiments, the effectiveness of each mutant CAR-T cell is evaluated based on in vivo persistence and tumor cell toxicity. The term "persistence" or "CAR-T cell persistence" used herein refers to the ability of CAR-T cells to remain activated, continue to proliferate and / or not die. Persistence can be measured based on the relative abundance of mutant CAR-T cells over time, because the proliferation and death of mutant CAR-T cells will be expected to change the relative abundance of mutant CAR-T cells. Therefore, mutant CAR-T cells with increased relative abundance can also have increased persistence.

[0316] In some embodiments, flow cytometry and PCR are used to measure in vivo persistence and tumor cell toxicity. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time. In some embodiments, persistence is quantified by calculating the relative change of each gRNA compared to the gRNA polynucleotides from negative control CAR-T cells over time. In some embodiments, persistence is quantified by calculating the relative change of each gRNA over time at at least 3 time points and calculating the slope of the relative change. In some embodiments, clinical imaging and / or bone marrow evaluation are used to quantify tumor cell toxicity.

[0317] In some embodiments, mutant CAR-T cell persistence is determined based on relative abundance, and mutant CAR-T cells with high relative abundance are then tested for efficacy in killing cancer (e.g., in vitro or in vivo).

[0318] In some embodiments, the efficacy is measured for all mutant CAR-T cell libraries administered to the patient. For example, if the patient shows improved signs (e.g., tumor or cancer burden reduction, improved life expectancy, etc.) after administering the mutant CAR-T cell library, then the library as a whole may be effective.

[0319] Generation of mutant NK cell libraries

[0320] In some embodiments, the method for producing a mutant CAR-NK cell library described herein may be applicable to producing a mutant natural killer cell library. In some embodiments, the method includes transfecting a gRNA vector library into a plurality of NK cells (e.g., CAR-NK cells) extracted from a subject (e.g., using the method described herein). In some embodiments, NK cells are also transfected with a CAR vector described herein.

[0321] In some aspects, the disclosure provides a method for producing a mutant CAR-NK cell library. In some embodiments, the method includes: (a) obtaining CAR-NK cells (e.g., previously prepared CAR-NK cells), and (b) transfecting CAR-NK cells with a gRNA vector library (e.g., as described herein). In some embodiments, the obtained CAR-NK cells have been previously activated. In some embodiments, the method includes activating CAR-NK cells. In some embodiments, CAR-NK cells express CRISPR proteins. In some embodiments, the method also includes introducing mRNA or CRISPR proteins encoding CRISPR proteins into CAR-NK cells (e.g., by electroporation).

[0322] In some aspects, the present application discloses a method for generating a mutant CAR-NK cell library, the method comprising: (a) activating NK cells (e.g., as described above (e.g., using anti-CD3 and anti-CD28 antibodies)), (b) transfecting NK cells with a gRNA vector library as described above, (c) transfecting NK cells with a CAR vector as described in any one of the claims above, and (d) introducing mRNA encoding a CRISPR protein or a CRISPR protein into the NK cells (e.g., by electroporation).

[0323] 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, the introduction of mRNA or CRISPR protein encoding CRISPR protein into NK cells is performed 1 to 10 days after gRNA vector and / or CAR vector transfection. In some embodiments, the introduction of mRNA or CRISPR protein encoding CRISPR protein into NK cells is performed 3 to 7 days after gRNA vector and / or CAR vector transfection. In some embodiments, the introduced CRISPR protein is CRISPR Cas9 protein. In some embodiments, the introduced CRISPR protein is introduced as a ribonucleoprotein. In some embodiments, CRISPR protein mRNA is expressed by a gRNA vector or a CAR vector.

[0324] In some embodiments, the method further comprises collecting NK cells from a subject (e.g., a subject as described above), for example, which NK cells can then be modified using, for example, one or more (e.g., all) of steps (a) to (d) above.

[0325] In some embodiments, NK cells are collected using any suitable method (eg, including any suitable method described above).

[0326] In some embodiments, the method further comprises purifying NK cells comprising the CAR vector and comprising a CRISPR-induced genetic mutation (e.g., comprising any suitable purification method described above).

[0327] In some embodiments, the gRNA vector library is transduced into CAR-NK cells at the above MOI. In some embodiments, the CAR vector is transduced at the above MOI.

[0328] In some embodiments, the present application discloses a library of mutant CAR-NK cells generated using any of the methods described herein.

[0329] Identification of the effect of CRISPR on CAR-NK cell function.

[0330] In some embodiments, the present disclosure provides methods for identifying mutated (e.g., by CRISPR) CAR-NK cells with improved efficacy.

[0331] In some aspects, the present application discloses a method for identifying gRNA polynucleotides associated with CAR-NK cell efficacy in vivo, the method comprising: (a) administering a mutant CAR-NK cell library (e.g., a mutant CAR-NK cell library disclosed herein or a mutant CAR-NK cell library produced by the method described herein) to a subject, (b) collecting one or more samples containing multiple mutant CAR-NK cells from a subject (e.g., by leukocyte apheresis), (c) sequencing the gRNA polynucleotides from the mutant CAR-NK cells collected in (b) (e.g., using any reasonable method, such as Illumina sequencing), and (d) evaluating the changes in the relative abundance of each gRNA polynucleotide based on the sequencing in (c). For example, the gRNA associated with CAR-NK cell efficacy is a gRNA that induces mutations that increase CAR-NK cell proliferation and / or tumor toxicity. The technician will understand that after administering a mutant CAR-NK cell library to a subject, the increased relative abundance of a given gRNA indicates that a given gRNA causes a mutation that increases CAR-NK cell efficacy (e.g., increases persistence).

[0332] The skilled person will appreciate that mutant CAR-NK cells (e.g., mutant CAR-NK cells in a mutant CAR-NK cell library) can be replicated in a subject. Therefore, the multiple mutant CAR-NK cells collected in step b above may include mutant CAR-NK cells administered to a subject and / or offspring of mutant CAR-NK cells administered to a subject.

[0333] The CAR-NK cells described herein include immune cells comprising any CAR polypeptide described herein (and optionally an antibody agent or cytokine), or a nucleic acid encoding any CAR polypeptide described herein (and optionally an antibody agent or cytokine).

[0334] In some embodiments, the methods described herein include administering to a subject an effective amount of activated CAR-NK cells described herein (e.g., mutant CAR-NK cell libraries) to identify mutant CAR-NK cells with improved efficacy. In some embodiments, the methods described herein include administering to a subject an effective amount of activated CAR-NK cells described herein (e.g., mutant CAR-NK cell libraries) to alleviate the symptoms of the disease.

[0335] In some embodiments, the composition of NK cells can be administered to a subject as described above (e.g., in the same or similar manner as performed using mutant CAR-T cells).

[0336] In some embodiments, the mutant CAR-NK cell library administered to a subject comprises a whole genome CAR-NK cell mutant library (e.g., a whole genome generated using a whole genome gRNA vector library as described above for mutant CAR-T cells).

[0337] In some embodiments, the CAR of the CAR-NK cell comprises an antigen binding domain (e.g., an antigen binding domain as described herein) that binds to an antigen expressed by cancer. In some embodiments, CAR comprises an antigen binding domain that binds to any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and claudin 6, an antigen binding domain that binds to any one of CD19 / CD79b, BCMA / TACI, or a TriPRIL antigen binding domain. In some embodiments, the antigen binding domain binds to mesothelin.

[0338] In some embodiments, one or more samples collected from a subject are collected from any reasonable tissue or fluid using any reasonable means as described above.

[0339] In some embodiments, methods of collecting mutated CAR-NK cells from a subject, extracting genomic DNA and RNA from CAR-NK cells, amplifying gRNA polynucleotides and / or sequencing gRNA polynucleotides, determining relative abundance, and determining CAR-NK cell potency are described above with respect to CAR-T cells, and can be similarly applied to CAR-NK cells.

[0340] All patents and other publications cited throughout this application; including references, issued patents, published patent applications, and co-pending patent applications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methods described in such publications that can be used in conjunction with the technology described herein. These publications are provided solely for their disclosures prior to the filing date of this application. Any disclosure in this regard should not be construed as an admission that the inventor is not entitled to antedate such disclosures by reason of prior art or for any other reason. All statements as to dates or representations as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0341] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. As will be appreciated by those skilled in the relevant art, although the specific embodiments and embodiments of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure. For example, although the method steps or functions are presented in a given order, alternative embodiments may perform functions in different orders, or may perform functions substantially simultaneously. The teachings of the disclosure provided herein may be appropriately applied to other processes or methods. The various embodiments described herein may be combined to provide additional embodiments. If necessary, some aspects of the present disclosure may be modified to provide yet other embodiments of the present disclosure using the composition, function and concept in the above references and applications. In addition, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the biological or chemical effects in terms of species or amount. According to the detailed description, these and other changes may be made to the present disclosure. All of these modifications are intended to be included within the scope of the appended claims.

[0342] The specific elements of any of the foregoing embodiments may be combined or replaced with the elements in other embodiments. In addition, although the advantages associated with certain 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 need to exhibit such advantages to fall within the scope of the present disclosure.

[0343] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting.

[0344] Example

[0345] Example 1: Vector design of gRNA vector and CAR vector

[0346] Two different vectors were designed that together provide positive selection using a clinically feasible magnetic bead system as well as CAR expression, and expression of two guides (a suicide gene and a selectable marker) that enable specific knockout of two genes by CRISPR / Cas9, one of which is used for negative selection in the clinically feasible magnetic bead system.

[0347] The CAR vector is composed of the backbone of the third-generation self-inactivating (SIN) lentiviral vector ( Figure 1 A). A promoter (e.g., EF1a) is used to drive the expression of CAR. The CAR sequence is followed by a 2A ribosomal skipping element, and the 2A ribosomal skipping element is followed by a reporter gene (truncated CD34 lacking the intracellular domain) that can be detected and selected by surface expression staining.

[0348] A dual-guide vector backbone (e.g., a gRNA vector) enables the use of a library of guides. The vector backbone encodes 2 guides; one is static (TRAC) and is capable of knocking out the TCR / CD3 complex on gene-edited T cells; the lack of CD3 enables clinically feasible negative selection, thereby selecting for effector populations in which successful transduction and gene editing with CRISPR / Cas9 occurs. In this exemplary embodiment, the guide RNA on the vector is inverted; in another embodiment, the two guides are constructed in tandem ( Figure 1 B).

[0349] EF1a and EF1a-short (EFS) were used for CAR and reporter expression. Expression of the reporter gene LNGFR and the suicide gene was driven by a weaker promoter (PGK) to avoid overexpression of the suicide gene, which could leak and trigger apoptosis of the cell product.

[0350] The CAR vector and the dual-guide vector are co-transduced into the same T cell population. The final "active" research target product is CAR+CD34+LNGFR+CD3-T cells. To enhance safety, a suicide gene (e.g., icaspase9) can be included in the dual-guide vector.

[0351] Example 2: gRNA library design and generation

[0352] Gene selection

[0353] Using genomic, transcriptional, and functional data from human and murine T cells, an initial list of 135 genes suitable for CRISPR / Cas9 knockout and hypothesized to improve CAR-T cell function was selected (Table 2). These genes include checkpoint proteins (e.g., PD-1, encoded by PDCD1), as well as genes in which mutations are observed in subjects with lymphoproliferative diseases or subjects with abnormal responses to CAR-T cells (e.g., TET2 and cbl-b).

[0354] Determine sgRNA library size

[0355] The library size is calculated based on the feasibility of preparation and the dose of cells to be administered (usually 100×106) and the number of cells that can be reasonably recovered from the patient after infusion (usually 50×106 to 100×106). Targeting the entire genome will result in suboptimal depth and low signal-to-noise ratio. On the other hand, targeting <10 genes provides less information about which candidate gene knockouts are optimal. Targeting 100 to 500 genes (with guide redundancy to improve the signal-to-noise ratio) provides coverage depth, reduces the randomness of the number of T cells recovered after infusion, and provides a competitive system within and between different patients to strictly evaluate the role of each candidate gene. The library also contains controls targeting genes that are expected to have any effect on T cell biology.

[0356] sgRNA library design

[0357] For the selected 135 genes, the primary transcription ID of each gene in human T cells (e.g., encoded by SEQ ID NO: 1 to 135) was used to design a library of 8 sgRNAs per gene. All possible guides were designed using CRISPick using these transcripts. A total of 34,318 guides were designed. The on-target activity of these guides was scored using Rule Set 3 (RS3) and sequence and target information and Chen2013tracr (PMID: 24360272). The off-target activity of the guide was also scored using the Tier-agnostic 1 mismatch aggregated CFD score, which is independent of the hierarchy. 2009 guides were filtered out based on clonal incompatibility (e.g., BsmBI site) and polyT. Five guides had >10,000 off-target sites, including 1 mismatch. 277 guides with off-targets in 58 tumor suppressor genes were filtered out, as defined in Lenoir et al. Nature communications 12.1 (2021): 1-15. 118 guides that were marked as targeting genomic regions with high variation in the human population were also filtered out. 5561 guides that targeted outside the 2% to 85% protein window were also filtered out. To avoid mixed guides, 4771 guides with a Cutting Frequency Determination (CFD) score < 0.27 were filtered out.

[0358] After applying all filters, the top 8 guides for each gene were selected by RS3 scoring. The library has a total of 1080 gene-targeting guides (Table 4). 100 effective intergenic controls were also included, bringing the total number of guides to 1180.

[0359] sgRNA library generation

[0360] In order to produce the sgRNA library, the oligonucleotides of each sgRNA are synthesized on the chip and then amplified using PCR primers containing BsmBI compatible cloning sequences. The amplified oligonucleotide pool is connected to the above-mentioned sgRNA vector, and the connection product is transformed into electrocompetent E. coli. Then the transformed E. coli is plated on solid agar with a certain density to achieve a density of 1000 times representation of each sgRNA in the library, and grown overnight. The colony is scraped off the plate, and the merged sgRNA plasmid is extracted. The plasmid library is sequenced to confirm the sgRNA representativeness, and then transformed into electrocompetent E. coli and frozen in glycerol medium. Then the glycerol-preserved E. coli aliquots are grown overnight in liquid culture, and plasmids are extracted from the amplified E. coli. The merged sgRNA library lentivirus is prepared using standard 3rd generation lentiviral vector production.

[0361] Example 3: Generation and administration of mutant CAR-T cell libraries

[0362] T cells are collected from human subjects by standard leukapheresis procedures. Peripheral blood mononuclear cells are isolated and T cells are purified. T cells are then activated using anti-CD3 / anti-CD28 antibodies (optionally antibody-coated beads) and subsequently transduced with two vectors; the MOI of the dual-guide vector is capped at 1, and is optionally 0.5 or close to 0.5. The MOI of the CAR vector is as high as 20.

[0363] Cas9 is introduced into CAR-T cells in the form of mRNA encoding Cas9 or in the form of ribonucleoprotein (RNP), in which soluble Cas9 protein is complexed with non-targeting guide RNA that has no predicted target site in the genome. Whether by mRNA or RNP, Cas9 is electroporated into T cells 3 to 7 days after lentiviral transduction.

[0364] T cells are further expanded and subsequently selected through a series of magnetic bead processes. First, the product is negatively selected for CD3 expression using a system such as CliniMACS to eliminate T cells. The CD3 negative fraction is then positively selected for CD34 expression using a system such as CliniMACS. CD34+CD3- cells are considered the research product for infusion.

[0365] Flow cytometric analysis was used to determine the number of CAR+ cells based on CD34+ cells, as well as the number of successfully transduced and gene-edited cells based on CD3-negative and LNGFR+.

[0366] CAR T cells are administered based on the dose of CAR+ cells, ranging from 10 × 10 6 (likely suboptimal dose levels) to 100×10 6 (usual dose level) to 300×10 6 (The usual maximum tolerated dose for CAR T cells.) Library representativeness is measured as a baseline to assess bias in the preparation, rather than as part of dose selection itself.

[0367] Example 4: Determining the efficacy of mutant CAR-T cells

[0368] According to the event schedule in the clinical scheme, samples of mutant T cells are collected from subjects by venipuncture, apheresis or tumor resection (usually the 3rd day, the 7th day, the 10th day, the 14th day, the 21st day, the 28th day, the 2nd month, the 3rd month and the 6th month). T cells are separated by standard operations. CAR+T cells are purified based on the expression of CD34, and the expression of CD3 and other T cell markers such as CD4 and CD8 of CAR+T cells are analyzed. Genomic DNA is separated from purified CAR+T cells for downstream analysis. The sgRNA region from CAR+T cells is amplified using PCR primers, which are specific to the sequences flanking the sgRNA box of the library. Nextgen sequencing is then used to sequence the PCR amplicon to quantify the abundance of each individual sgRNA in the library, and the abundance of each guide in the post-infusion CAR+T cells is compared with the abundance value in the CAR+T cells before infusion.

[0369] This approach also allows single-cell transcriptome profiling of CAR-T cells isolated from patients and capture of sgRNAs to allow linking gene deletion to transcriptional phenotype analysis. A standard droplet-based single-cell whole transcriptome analysis platform, such as 10x Genomics, is used in combination with direct capture of sgRNA sequences from cellular RNA using primer sets specific for the sgRNA region.

[0370] Example 5: Knockout of multiple genes in primary T cells using dual guide cassette vectors

[0371] 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 3AOn day 1, activated T cells were transduced with guide RNA vectors containing sgRNAs targeting CD5 and TRAC, important components of the T cell receptor complex (TCR) ( Figure 3B ). The next day, the transduced cells were debeaded (anti-CD3 / CD28 beads removed), electroporated with Cas9, and given fresh medium containing IL-2 every two days. On day 10, the transduced T cells were analyzed for LNGFR expression and TCR and CD5 knockout. ( Figures 3C to 3E After staining for cell surface expression of TCR, CD5, and LNGFR, naive and transduced T cells were analyzed by flow cytometry. The results showed that non-transduced naive T cells maintained TCR expression ( Figure 1 C). However, cells transduced with the guide RNA vector showed knockout of the T cell receptor complex, which was caused by expression of sgRNA targeting TRAC 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 (CD5sgRNA#1 or CD5 sgRNA#2). The transduced T cells were stained for CD5 using a fluorescent antibody that detects CD5 and analyzed by flow cytometry. An isotype control (a similar fluorescent antibody that lacks specificity for CD5) was used as a negative control. The results showed loss of CD5 surface expression in T cells transduced with CD5sgRNA#2, but maintained CD5 expression in T cells transduced with CD5 sgRNA#1. In summary, the strong CD5 knockout observed with CD5 sgRNA#2 indicates proper function of the guide RNA vector. The maintained CD5 expression observed with CD5 sgRNA#1 indicates poor sgRNA function and does not reflect the guide RNA vector itself. Untransduced naive T cells and T cells transduced with gRNA vectors were stained for expression of the LNGFR cell surface marker. The results show that LNGFR is only expressed in T cells transduced with gRNA vectors ( Figure 3E ).

[0372] Example 6: Determination of guide RNA polynucleotide abundance in the pooled library by sequencing

[0373] 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 selectable marker ( Figures 4A to 4B)). On day 5, the transduced cells were de-beaded (anti-CD3 / CD28 beads removed), electroporated with Cas9, and supplemented with fresh medium containing IL-2. On day 8, T cells transduced with pMGH354-CP1780 were not treated (0nM Rimducid), treated with vehicle only (DMSO control), or treated with different concentrations of Rimducid that activated the iCasp9 safety switch. Each experimental condition was performed in triplicate.

[0374] Response to Rimducid was assessed by measuring the change in the proportion of cells expressing the guide RNA vector (CAR+ve) after treatment ( Figure 4C ). At 24 and 48 hours after Rimducid treatment, CountBright TM Absolute counting beads and live / dead cell stain were mixed with cell samples and analyzed by flow cytometry. The absolute number and proportion of CAR+ve live cells in the sample were calculated by comparing the ratio of bead events to live cell events. The results showed the proportion of CAR+ve cells maintained in the untreated and vehicle-treated control groups. However, the Rimducid-treated group showed a loss of CAR+ve cells over time. This indicates the proper expression and function of the iCasp9 safety switch in T cells transduced with pMGH354-CP1780.

[0375] Example 7: CAR-T cell CRISPR library and use in humans with solid tumors

[0376] Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for patients with hematologic malignancies, achieving durable complete remissions in approximately 40% of patients with refractory lymphomas. However, some patients ultimately relapse, and CAR T cells have so far been relatively unsuccessful in patients with advanced solid tumors. While some of this acquired resistance is explained by loss of the CAR target antigen, the major driver of treatment failure in both liquid and solid tumors is the inability of CAR T cells to persist and maintain durable effector capacity 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 critical barrier to CAR T cell efficacy. In solid tumors, this program is amplified by the suppressive tumor microenvironment (1,2). Therefore, it is critical to identify genes and pathways that can enhance CAR T cell efficacy in both solid and liquid tumors in vivo, where factors driving T cell dysfunction are at work.

[0377] In theory, a variety of genes and pathways could be modulated to improve CAR-T cell responses. However, there are so many candidate genes that it is essentially impossible for the field to individually validate each potential candidate in separate Investigational New Drug (IND) and Phase 1 clinical trials of CAR T cells. Although data from in vitro and in vivo studies provide valuable insights into factors that determine CAR T cell function, parallel in vitro and in vivo genetic screens in solid and liquid cancer models have shown that targets identified by in vitro screens do not always correlate with in vivo screens (3,4). Some target genes identified as regulators of T cell function in in vitro genetic screens, such as RASA2, were not identified as top targets in the in vivo screens disclosed herein (e.g., Fig. 10B )(5). In addition, preclinical in vivo models of CAR T cell efficacy do not always predict clinical performance. Therefore, genetic screening of genes that regulate CAR T cell function in human patients may be the only way to identify genes that will improve clinical performance. In order to meet this demand, this embodiment screens selected target genes in CAR T cells administered to patients, and performs IND-enabling studies to initiate the process. The inventors believe that the use of large-scale, systematic genetic screening in cancer patients can identify regulators of CART cell function in solid tumors. The limited loss-of-function (LOF) CRISPR screening group described herein is used to identify key regulators of T cell performance in human patients.

[0378] The therapeutic drug product has autologous T lymphocytes transduced with two lentiviral vectors - one encoding a CRISPR guide library (Mario library) with sgRNAs targeting 135 genes related to T cell function, and the other encoding a chimeric antigen receptor (CAR) targeting mesothelin (using an SS1 scFv conjugate) (Meso-Mario-CART cells). Such product preparation is designed so that each mesothelin-targeting CAR T cell expresses a single variable sgRNA for a specific gene, as well as a single fixed sgRNA for the TCR alpha chain gene (TRAC). The entire limited set of knockout Meso-Mario-CAR T cells is delivered to each patient ( Figure 5 ). The diseases being treated are very advanced mesothelin-expressing cancers, including ovarian and pancreatic cancer. Specific mesothelin CAR constructs have previously been tested in a Phase 1 clinical trial in these patient populations (SS1-BBz) in both RNA-electroporated T cells and lentiviral-transduced T cells (6-12).

[0379] Mesothelin is highly expressed in ovarian cancer cells compared to normal cells, making it an attractive antigen for CAR T cell targeting. Ovarian cancer is the fifth leading cause of cancer death in American women (American Cancer Society, 2022). Ninety percent of ovarian cancers are epithelial, the most common being serous epithelial carcinoma. Most serous epithelial carcinomas are diagnosed at stage III (51%) or stage IV (29%), with 5-year cause-specific survival rates of 42% and 26%, respectively (13). Current treatment options combine debulking surgery, drug therapy, and radiation therapy. However, 70% of ovarian cancer patients relapse after primary cytoreductive surgery and standard first-line chemotherapy (14). The median survival of patients with platinum-resistant or refractory high-grade epithelial ovarian cancer is 9 to 12 months (15). Such a poor prognosis makes this indication an unmet medical need. Mesothelin is expressed in 75% of high-grade serous ovarian cancers, making it a suitable target for immunotherapy (16).

[0380] Another malignancy that highly expresses mesothelin is pancreatic adenocarcinoma (16). Pancreatic cancer is one of the most lethal solid tumor malignancies. The five-year survival rate is 11%, and approximately 50,000 deaths are expected in 2022 (17). Only 10% to 15% of patients have resectable disease at the time of diagnosis (18). For patients with locally advanced and unresectable disease, treatment options typically consist of multiple doses of chemotherapy, of which 80% of patients do not elicit a response sufficient to qualify for surgery (18). Finally, treatment of metastatic pancreatic cancer consists of cytotoxic regimens with a median overall response of 6 to 11 months.

[0381] Previous trials of mesothelin-directed CAR T cells using SS1-derived single-chain variable fragments as CAR binding domains have demonstrated safety, but overall lack of clinically meaningful efficacy in patients with advanced ovarian or pancreatic cancer (6-12). In part, this is thought to be due to poor persistence of CAR T cells and the occurrence of T cell dysfunction in the tumor microenvironment. There are hundreds of candidate genes and pathways that can alter T cell biology; it is unclear whether it is possible to conduct a single first-in-human study or randomized study in a separate clinical trial or drug development program to test modifications of each of these candidate pathways. When the therapeutic product is a genetically modified cell, the gene marker itself can be used to test a product library with different genetic modifications and subsequently identify the highest implanted cell product after infusion. In this embodiment, each patient provides information about which genetically modified CAR T cell confers the highest benefit to the individual. It may also be possible to identify the highest implantation and / or most persistent CAR T product in patients and in two diseases expressing the same target antigen (mesothelin in this case).

[0382] CRISPR screening is based on the use of a lentiviral library of guide RNAs combined with transient expression of the Cas9 nuclease to generate a pool of cells, each cell having a single gene knocked out from the library. Therefore, CRISPR screening in human patients can be used to quickly identify which gene knockouts from the selected and displayed gene library are involved in altering T cell biology, resulting in improved implantation, persistence and response in patients with advanced solid tumors expressing mesothelin. The inventors believe that Meso-Mario CAR T cells provide an opportunity for expected benefits for individual patients because the therapeutic product "hit the target multiple times", thereby producing CAR T cells (based on the SS1-BBzCAR construct) that may have improved effector function and still have a defined specificity, which has previously been shown to be safe (6,7). In addition to the expected benefits of each individual human subject in this study, using this method, a single gene knockout can be quickly identified, thereby conferring greater implantation, persistence and response in patients or in both diseases; if this is the case, a more typical drug development approach incorporating specific single gene knockouts in the Meso-CAR-T context can be performed separately to maximize the benefits to the largest number of patients.

[0383] Product Description and Design

[0384] The therapeutic product is autologous T lymphocytes transduced with two lentiviral vectors—one lentiviral vector encoding a CRISPR sgRNA library targeting 135 genes related to T cell function (Table 1) (Mario library) and TCR α chain with endogenous TCR removed (TRAC), and the other lentiviral vector encoding a chimeric antigen receptor (CAR) targeting mesothelin (Meso-Mario-CART cells).

[0385]

[0386] Example 6 Table 1: Genes contained in the sgRNA library (Mario library) of Meso-Mario-CART cell products.

[0387] Design of CRISPR sgRNA libraries

[0388] The CRISPR library was designed to maximize safety, benefit prospects, and understanding of the field by optimally deconvoluting the data obtained from each treated subject. 135 genes were selected based on literature searches and tested genes (i.e., IFNG, ITK, PDCD1, HAVCR2) Figure 6). The major transcript of each gene was then selected, and 34,318 guides were designed using CRISPick (30). To minimize the potential off-target effects of sgRNA guides, guides with predicted off-target cleavage in tumor suppressor genes were subsequently removed (31). Guide RNAs targeting regions of high variability in the human population were removed. Cutting frequency determination (CFD) scoring was used to mitigate the risk of off-target activity for any given guide (30). Guides with an aggregate CFD off-target score ≤ 0.27 were removed. For each gene, Rule Set 3 scoring was used, which integrates multiple sequence features that contribute to the expression of sgRNAs and the generation of on-target gene editing, to select 8 sgRNAs per gene (32). Finally, 100 effective intergenic control guides were included in the library for a total of 1180 guides. When combined with Cas9 protein (which will be delivered as mRNA by electroporation), each cell transduced with the Mario library is expected to have two genes knocked out: the TCRα chain and a single gene from the library.

[0389] Lentiviral vector design

[0390] Two lentiviral vector constructs were designed for the safe delivery of a CRISPR sgRNA library (pMario) and a chimeric antigen receptor targeting mesothelin (pCAR, Fig. 7A ). For constructs containing CRISPR libraries (pMario), another layer of safety is added by incorporating a safety switch based on inducible caspase 9 (iCasp9), which can eliminate CAR T cells in the event of severe adverse events or CAR T cell clonal expansion (outgrowth). In order to optimize the expression of two sgRNAs, a dual sgRNA box was designed, in which the sgRNA library was cloned into position 1, and position 2 contained sgRNAs of the T cell receptor α constant gene (TRAC). Because the assembly of the natural T cell receptor / CD3 complex requires a complete TCRα chain, knocking out TRAC leads to the loss of expression of the entire natural TCR / CD3 complex. Therefore, the guide targeting TRAC allows negative selection of edited cells during preparation (using CliniMACs to perform CD3-based magnetic selection on research products). Clinically, this selection of CD3-negative T cells also minimizes the risk of enhancing autoreactive T cells (based on retained TCRs with undefined specificity). Finally, a truncated low-affinity nerve growth factor receptor (tLNGFR) reporter gene was included to allow flow cytometry-based counting, recovery, and cell enrichment after infusion and to facilitate further analysis of enriched and depleted sgRNAs. Fig. 7A ) is designed to contain a CAR construct consisting of SS1scFv and 4-1BB intracellular co-stimulatory domains that bind to mesothelin (6-11). The construct also contains a CD34t reporter to allow for administration and post-infusion monitoring of CAR+T cells.

[0391] Validation of sgRNA dual cassette and reporter system

[0392] To determine reporter expression and editing efficiency of the dual sgRNA cassette, bead-activated T cells (CD3 / CD28 beads at a 3:1 bead:T cell ratio) were transduced with dual guide vectors encoding sgRNAs targeting CD5 and TRAC. After bead removal, cells were electroporated with Cas9 mRNA. Naive and transduced T cells were then analyzed by flow cytometry for cell surface expression of tLNGFR, CD3, and CD5 ( Figures 7B to 7D ). An isotype control, which is a similar fluorescent antibody lacking specificity for the target being analyzed, was used as a negative control. The results showed that LNGFR was expressed in T cells transduced with the dual-guide vector ( Figure 7B Uninduced, naive T cells maintained expression of the TCR (CD3); however, cells transduced with the dual-guide vector lacked CD3 expression due to successful targeting of TRAC with sgRNA following Cas9 electroporation ( Figure 7C ). LNFGR+ cells also lacked CD5 expression, indicating proper function of the dual guide cassette vector.

[0393] Generation of Meso-Mario-CART cells for in vivo testing

[0394] The production scheme of 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. The cells were then transfected with Cas9 mRNA by electroporation on day 5. The cells were expanded to day 10, and CD3-negative cells were enriched by depleting CD3-positive cells using an APC-CD3 selection kit. After two rounds of selection, the protocol was able to produce a final product with up to 88% double-transduced cells (mCherry+CD3-) ( Figures 8A to 8C ).

[0395] Initial in vivo studies of CRISPR screening

[0396] In preparation for the human CRISPR screen, a preliminary CRISPR screen was performed in NSG mice ( Fig. 9). The Mario sgRNA library targeting 135 selected related T cell regulators was generated, with 8 sgRNAs per gene. T cells were transduced with the library and mesothelin CAR, and electroporated with Cas9 mRNA as described. T cells transduced with the library were transferred into mesothelin + ASPC1 tumor-bearing mice. After 14 days, CAR T cells were isolated from the tumor after digestion and positive selection for human CD45. Data was generated by sgRNA PCR amplification from gDNA in isolated T cells, followed by next-generation sequencing (NGS).

[0397] CAR T cells recovered from tumors and spleens of ASPC1 tumor-bearing mice demonstrated the ability to engraft large numbers of tumor-infiltrating mesothelin CAR T cells and to efficiently capture T cells from tumors and spleens, allowing for technically robust in vivo screening experiments, even in small numbers of animals ( Fig. 10A ). The distribution of sgRNAs in recovered CAR T cells demonstrated that the complete CRISPR library could be captured and the guides were well represented in the recovered cell pools.

[0398] These results suggest that the top hits from the in vivo screen are actually the opposite of the top predicted hits from published genome-wide CRISPR screens performed in vitro (5). In these results, in vivo knockout of IL2RA was enriched, whereas knockout of RASA2 was depleted ( Fig. 10B ). In previous in vitro screening, RASA2 was the most enriched gene knockout in T cells. In addition, these data illustrate the use of in vitro models to predict the best gene knockout targets for enhancing CAR T cell function, which may not actually predict in vivo function or activity. Similar results were observed using BCMACAR, indicating that the effect of gene knockout is not CAR-specific (see Example 8). Generation of large-scale sgRNA plasmid libraries

[0399] Based on the preclinical data results, the feasibility of generating a plasmid library (Mario library) of sufficient quality to achieve current good manufacturing practice (cGMP)-grade lentiviral vector preparations on a large scale and using an investigational new drug (IND)-ready approach was evaluated. To evaluate the quality of the large-scale guide library, the guide distribution of the original plasmid library stock was compared to the large-scale IND-ready plasmid preparation. In the large-scale preparation, the guide abundance in the plasmid was first analyzed using NGS of PCR products flanking the guide region. Primary human T cells were then transduced with lentivirus prepared in parallel using either the original plasmid initial library stock or the large-scale plasmid preparation.

[0400] The results showed that the large-scale plasmid preparation process did not significantly change the distribution of plasmids in the library ( Fig.11A In addition, the distribution of guides in transduced T cells did not change significantly ( Fig. 11B ), indicating that plasmid preparations can be used to prepare lentiviral and cell products suitable for clinical-grade screening studies. Fig.12 An overview of the cGMP-compliant manufacturing process for Meso-Mario-CAR T cell products is provided. Fig.12 ).

[0401] Treatment options

[0402] Human patients can be given 3 days of lymphodepleting chemotherapy starting about 5 days before the infusion of Meso-Mario-CART cells on day 0 ( Fig.13 Lymphodepletion can be administered as an outpatient or inpatient at the discretion of the healthcare provider. Following CAR T-cell infusion, patients were monitored for adverse events, clinical status, and laboratory parameters. Fig.13 and Fig.14 ) for up to 24 months. The study samples were analyzed using the methods described herein (e.g., the methods in Examples 4 to 6).

[0403] References

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[0405] 2. Bailey SR, Vatsa S, Larson RC, Bouffard AA, Scarfò I, Kann MC, et al. Blockade or Deletion of IFNγ Reduces Macrophage Activation without Compromising CAR T cell Function in Hcmatologic Malignancics. Blood Canccr Discov[Internet]. 2022 Mar 1[Cited 2023 Mar 2]; 3(2): 136-53. Available from: https: / / aacrjournals.org / bloodcancerdiscov / article / 3 / 2 / 136 / 681793 / Blockade-or-Deletion-of-IFN-Rcduccs-Macrophagc

[0406] 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]. 2022 Feb 1[Cited 2023 Mar 2]; 12(2): 432-49. Available from: https: / / aacrjournals.org / cancerdiscovery / article / 12 / 2 / 432 / 678482 / An-In-Vivo-CRISPR-Screening-Platform-for

[0407] 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]. 2020 May [cited 2023 Mar 2]; 26(5): 782-792.c7. Available from: https: / / linkinghub.clscvicr.com / retricvc / pii / S1934590920301429

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Claims

1. A composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by a sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

2. The composition of claim 1, comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 10 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

3. The composition of claim 1 or claim 2, comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 20 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by a sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

4. The composition of any one of claims 1 to 3, comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 50 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

5. The composition of any one of claims 1 to 4, comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 100 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

6. The composition of any one of claims 1 to 5, comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least 135 gRNA polynucleotides each comprise a homology region complementary to the sense strand or antisense strand of a different gene encoded by the sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

7. A composition comprising a plurality of guide RNA (gRNA) polynucleotides, wherein at least two gRNA polynucleotides each comprise a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

8. The composition of claim 7, wherein each of the at least 10 gRNA polynucleotides comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

9. The composition of claim 7 or claim 8, wherein each of the at least 20 gRNA polynucleotides comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

10. The composition of any one of claims 7 to 9, wherein each of the at least 50 gRNA polynucleotides comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

11. The composition of any one of claims 7 to 10, wherein each of at least 100 gRNA polynucleotides comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

12. The composition of any one of claims 7 to 11, wherein each of at least 135 gRNA polynucleotides comprises a homology region complementary to a different gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof.

13. The composition of any one of claims 1 to 12, wherein the composition comprises at least 2 gRNA polynucleotides for each gene sequence, wherein the at least 2 gRNA polynucleotides for each gene sequence comprise different sequences.

14. The composition of any one of claims 1 to 12, comprising 8 gRNA polynucleotides for each gene sequence, wherein the 8 gRNA polynucleotides for each gene sequence comprise different sequences.

15. The composition of any one of claims 1 to 14, wherein at least 2 or at least 8 gRNA polynucleotides for each gene sequence are complementary to non-overlapping regions of the same gene sequence.

16. The composition of any one of claims 1 to 15, comprising one or more negative control gRNA polynucleotides.

17. The composition of claim 16, wherein the negative control gRNA polynucleotide comprises a randomly generated homology region.

18. The composition of claim 17, wherein the negative control gRNA polynucleotide comprises a homology region complementary to a gene not known to be involved in T cell function.

19. The composition of claim 17, wherein the negative control gRNA polynucleotide comprises a homologous region, which is a complementary intergenic region.

20. The composition of any one of claims 1 to 19, comprising at least one gRNA polynucleotide comprising the sequence of any one of SEQ ID NOs: 136 to 1315.

21. The composition of any one of claims 1 to 20, comprising at least 10 gRNA polynucleotides each containing a different sequence of any one of SEQ ID NOs: 136 to 1315.

22. The composition of any one of claims 1 to 21, comprising at least 100 gRNA polynucleotides each containing a different sequence of any one of SEQ ID NOs: 136 to 1315.

23. The composition of any one of claims 1 to 22, comprising at least 1000 gRNA polynucleotides each containing a different sequence of any one of SEQ ID NOs: 136 to 1315.

24. A composition comprising multiple gRNA polynucleotides containing sequences of SEQ ID NO: 136 to 1315.

25. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 200,000 gRNA polynucleotides having different sequences.

26. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 100,000 gRNA polynucleotides having different sequences.

27. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 50,000 gRNA polynucleotides having different sequences.

28. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 25,000 gRNA polynucleotides having different sequences.

29. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 15,000 gRNA polynucleotides having different sequences.

30. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 10,000 gRNA polynucleotides having different sequences.

31. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 5,000 gRNA polynucleotides having different sequences.

32. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 2,000 gRNA polynucleotides having different sequences.

33. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 1,080 gRNA polynucleotides having different sequences.

34. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 500 gRNA polynucleotides having different sequences.

35. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 250 gRNA polynucleotides having different sequences.

36. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 100 gRNA polynucleotides having different sequences.

37. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 50 gRNA polynucleotides having different sequences.

38. The composition of any one of claims 1 to 24, wherein the plurality of guide RNA polynucleotides consists of up to 10 gRNA polynucleotides having different sequences.

39. The composition of any one of claims 1 to 38, wherein at least some of the plurality of gRNA polynucleotides do not cross-react with more than one gene.

40. The composition of any one of claims 1 to 39, wherein the composition does not comprise a gRNA homology region complementary to the sense strand or antisense strand of a gene encoding p53, Rb, PTEN, BRCA1 and / or BRCA2, or a variant thereof.

41. The composition of any one of claims 1 to 39, wherein the composition does not comprise a gRNA complementary to the sense strand 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.

42. The composition of any one of claims 1 to 41, wherein the multiple gRNA polynucleotides are Cas9 protein gRNA polynucleotides or Cas12 protein gRNA polynucleotides.

43. The composition of any one of claims 1 to 42, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of the gene encoded by IL2RA (SEQ ID NO: 65) or a variant thereof.

44. The composition of any one of claims 1 to 43, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by GATA3 (SEQ ID NO: 46) or a variant thereof.

45. The composition of any one of claims 1 to 44, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by AGPS (SEQ ID NO: 3) or a variant thereof.

46. ​​The composition of any one of claims 1 to 45, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by PTPN2 (SEQ ID NO: 98) or a variant thereof.

47. The composition of any one of claims 1 to 46, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by LAG3 (SEQ ID NO: 76) or a variant thereof.

48. The composition of any one of claims 1 to 47, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by PDCD1 (SEQ ID NO: 91) or a variant thereof.

49. The composition of any one of claims 1 to 48, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by TGFBR2 (SEQ ID NO: 121) or a variant thereof.

50. The composition of any one of claims 1 to 49, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by RARA (SEQ ID NO: 99) or a variant thereof.

51. The composition of any one of claims 1 to 50, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by SmarcB1 (SEQ ID NO: 113) or a variant thereof.

52. The composition of any one of claims 1 to 51, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof.

53. The composition of any one of claims 1 to 52, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by RunX (SEQ ID NO: 107) or a variant thereof.

54. The composition of any one of claims 1 to 53, wherein at least one gRNA polynucleotide comprises a homology region complementary to the sense strand or antisense strand of a gene encoded by TCF7 (SEQ ID NO: 118) or a variant thereof.

55. The composition of any one of claims 1 to 54, wherein at least two gRNA polynucleotides each comprise a homology region complementary to the sense strand 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.

56. The composition of any one of claims 1 to 54, wherein at least 5 gRNA polynucleotides each comprise a homology region complementary to the sense strand 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.

57. The composition of any one of claims 1 to 54, wherein each of the at least 7 gRNA polynucleotides comprises a homology region complementary to the sense strand 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.

58. The composition of any one of claims 1 to 54, wherein each of the 10 gRNA polynucleotides comprises a homology region complementary to the sense strand 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.

59. The composition of any one of claims 1 to 54, wherein each of the 14 gRNA polynucleotides comprises a homology region complementary to the sense strand 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.

60. The composition of any one of claims 1 to 54, comprising: a first gRNA polynucleotide comprising a homology 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 comprising a homology region complementary to the sense strand 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 strand 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 strand 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 strand 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 strand 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 strand 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 strand 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 strand 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 strand or antisense strand of a gene encoded by CDKN1B (SEQ ID NO: 18) or a variant thereof or a variant thereof; an eleventh gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of a gene encoded by RunX (SEQ ID NO: 107) or a variant thereof; and / or A twelfth gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of a gene encoded by TCF7 (SEQ ID NO: 118) or a variant thereof.

61. The composition of any one of claims 1 to 60, wherein the composition does not comprise a gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of the gene encoded by RASA2 (SEQ ID NO: 102).

62. The composition of any one of claims 1 to 61, wherein the composition does not comprise a gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of such a gene, wherein the gene is 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.

63. The composition of any one of claims 1 to 61, wherein the composition does not comprise a gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of such genes, wherein the genes are 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.

64. The composition of any one of claims 1 to 61, wherein the composition does not comprise a gRNA polynucleotide comprising a homology region complementary to the sense strand or antisense strand of such a gene, wherein the gene is encoded by at least five 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.

65. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 15,000 different gene sequences.

66. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 10,000 different gene sequences.

67. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 5,000 different gene sequences.

68. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 2,500 different gene sequences.

69. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 1,000 different gene sequences.

70. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 500 different gene sequences.

71. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 200 different gene sequences.

72. The composition of any one of claims 1 to 64, wherein the plurality of gRNA polynucleotides comprises sequences complementary to up to 135 different gene sequences.

73. The composition of any one of claims 1 to 72, which is used for human CAR-T cell screening.

74. A guide RNA polynucleotide comprising the sequence of any one of SEQ ID NOs: 136 to 1315.

75. A plasmid comprising any one of the gRNA polynucleotides of the composition of any one of claims 1 to 72 or the guide RNA polynucleotide of claim 74.

76. A plasmid library comprising at least 2 plasmids according to claim 75.

77. The plasmid library of claim 76, wherein at least some of the at least 2 plasmids comprise the gRNA polynucleotide of the composition of any one of claims 1 to 72 or the guide RNA polynucleotide of claim 74.

78. The plasmid library of claim 76, wherein each plasmid comprises a gRNA polynucleotide of the composition of any one of claims 1 to 72 or a guide RNA polynucleotide of claim 74.

79. The plasmid library of any one of claims 75 to 78, comprising each of SEQ ID NOs: 136 to 1315 encoded on different plasmids.

80. gRNA vector, the gRNA vector comprising: (i) a first gRNA polynucleotide comprising a homology region complementary to a sense strand or an antisense strand encoded by a TCR / CD3 complex of a T cell (any one of SEQ ID NOs: 1383 to 1389); and (ii) a second gRNA polynucleotide.

81. The gRNA vector of claim 80, wherein the second gRNA polynucleotide comprises any one of the gRNA polynucleotides of the composition of any one of claims 1 to 72 or the guide RNA of claim 74.

82. The gRNA vector of claim 80 or claim 81, wherein the first gRNA polynucleotide and the second gRNA polynucleotide are in opposite orientations.

83. The gRNA vector of claim 82, wherein the first gRNA polynucleotide and the second gRNA polynucleotide are in a tandem orientation.

84. The gRNA vector of any one of claims 80 to 83, wherein each gRNA coding sequence is operably linked to a promoter.

85. The gRNA vector of claims 80 to 84, wherein the promoter is a constitutively active promoter or an inducible promoter.

86. The gRNA vector of claim 84 or 85, wherein the promoter is selected from U6 and EF1a promoters.

87. The gRNA vector of claim 84 or claim 85, wherein the promoter is a U6 promoter or an H1 promoter, optionally wherein the first gRNA polynucleotide is operably linked to the U6 promoter and the second gRNA polynucleotide is operably linked to the H1 promoter.

88. The gRNA vector of any one of claims 80 to 87, further comprising a selectable marker, optionally wherein the selectable marker is compatible with human use.

89. The gRNA vector of claim 88, wherein the selectable marker is selected from LGNFR, EGFR, CD19, CD20, CD34, and truncated forms thereof.

90. The gRNA vector of claim 88 or claim 89, wherein the selectable marker is operably linked to a promoter, which is optionally a weak promoter.

91. The gRNA vector of claim 90, wherein the promoter is selected from PGK, CMV, EF1a and a tissue-specific promoter.

92. The gRNA vector of any one of claims 82 to 91, wherein the vector is an adeno-associated vector, a retroviral vector, or a lentiviral vector.

93. The gRNA vector of claim 92, wherein the lentiviral vector is a third generation self-inactivating (SIN) lentiviral vector.

94. The gRNA vector of any one of claims 80 to 93, wherein the vector comprises a suicide gene.

95. The gRNA vector of claim 94, wherein the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20 and tHer2.

96. The gRNA vector of any one of claims 80 to 95, comprising the nucleic acid sequence of any one of SEQ ID NO: 1318.

97. The gRNA vector of any one of claims 80 to 96, further comprising a polynucleotide encoding a CRISPR protein, wherein the CRISPR protein is optionally Cas9 or Cas12.

98. A gRNA vector library comprising at least 2 gRNA vectors according to any one of claims 80 to 97.

99. The gRNA vector library described in claim B3, wherein each vector comprises the gRNA polynucleotide of the composition described in any one of claims 1 to 72 or the guide RNA described in claim 74.

100. The gRNA vector library of claim 98 or claim 99, wherein at least 10 vectors of the library each comprise a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology regions are 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.

101. The gRNA vector library of claim 98 or claim 99, wherein each of the at least 20 vectors of the library comprises a different gRNA polynucleotide homology region, and the different gRNA polynucleotide homology regions are 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.

102. The gRNA vector library of claim 98 or claim 99, wherein at least 50 vectors of the library each comprise a different gRNA polynucleotide homology region, wherein the different gRNA polynucleotide homology regions are 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.

103. The gRNA vector library of claim 98 or claim 99, wherein each of the at least one hundred vectors of the library comprises a different gRNA polynucleotide homology region, and the different gRNA polynucleotide homology regions are complementary to the sense strand or antisense strand of different genes encoded by any one of SEQ ID NOs: 1 to 135 or variants thereof.

104. The gRNA vector library of claim 98, wherein each of the at least one hundred and thirty-five vectors of the library comprises a different gRNA polynucleotide homology 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.

105. The gRNA vector library of any one of claims 98 to 104, wherein the gRNA vector library is a current good manufacturing practice (cGMP) grade gRNA vector library.

106. The gRNA vector library of claim 105, wherein the cGMP-grade gRNA vector library is a cGMP-grade gRNA lentiviral gRNA vector library.

107. A CAR vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

108. The CAR vector of claim 107, wherein the vector is a self-inactivating vector.

109. The CAR vector of claim 107 or claim 108, wherein the vector is an adeno-associated vector, a retroviral vector, or a lentiviral vector.

110. The CAR vector of claim 109, wherein the vector is a third generation self-inactivating (SIN) lentiviral vector.

111. The CAR vector of any one of claims 107 to 110, wherein the CAR is operably linked to a promoter, which is optionally an EF1a promoter or an EF1a-short promoter.

112. The CAR vector of any one of claims 107 to 111, wherein the vector comprises a suicide gene.

113. The CAR vector of claims 107 to 112, wherein the suicide gene is selected from icaspase9, tEGFR, tCD29, CD20 and tHer2.

114. The CAR vector of any one of claims 107 to 113, wherein the vector comprises a reporter.

115. The CAR vector of claim 114, wherein the reporter is selected from truncated CD34, tEGFR, tCD19, tCD20, tCD34 and tHer2.

116. The CAR vector of any one of claims 107 to 115, wherein 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, and claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is a TriPRIL antigen binding domain.

117. The CAR vector of any one of claims 107 to 116, wherein the CAR comprises a polynucleotide encoding an antigen binding domain that binds to mesothelin.

118. The CAR vector of any one of claims 107 to 117, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.

119. The CAR vector of any one of claims 107 to 118, wherein the transmembrane domain is selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDlla , CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2Rβ, IL2Rγ, I L7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, I TGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD15 0, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

120. The CAR vector of any one of claims 107 to 119, wherein the intracellular signaling domain is selected from CD28, 4-1BB, CD27, TCR-ζ, FcR-γ, FcR-β, CD3-γ, CD3-θ, CD3-σ, CD3-η, CD3-ε, CD3-ζ, CD22, CD79a, CD79b, and CD66d.

121. The CAR vector of any one of claims 107 to 120, wherein a 2A ribosomal skipping element or an internal ribosome entry site (IRES) is downstream of the CAR.

122. The CAR vector of any one of claims 107 to 121, wherein the CAR vector comprises the sequence of SEQ ID NO: 1319.

123. A composition comprising the gRNA vector of any one of claims 75 to 106 and the CAR vector of any one of claims 107 to 122.

124. CAR-T cells comprising the composition of claim 123.

125. CAR-T cells, comprising one or more of the following: a mutation in a gene corresponding to a gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof, a gRNA polynucleotide of a composition of any one of claims 1 to 72 or 74, a plasmid of claims 75 to 79, and a gRNA vector of any one of claims 80 to 97.

126. The CAR-T cell of claim D1.0, wherein the CAR-T cell comprises the CAR vector of any one of claims 107 to 122.

127. The CAR-T cell of any one of claims 124 to 126, wherein the mutation is a loss-of-function mutation.

128. The CAR-T cell of any one of claims 125 to 126, wherein the mutation is an insertion or a deletion.

129. The CAR-T cell of any one of claims 125 to 126, wherein the insertion or deletion mutation results in a frameshift.

130. The CAR-T cell of any one of claims 125 to 129, further comprising an mRNA encoding a CRISPR protein.

131. The CAR-T cell of any one of claims 125 to 130, wherein the CAR-T cell is CD34 positive and CD3 negative.

132. The CAR-T cell of claim 131, wherein the T cells used to prepare the CAR-T cells are from a mammalian subject.

133. The CAR-T cell of claim 132, wherein the T cells used to prepare the CAR-T cells are from a human subject.

134. The CAR-T cell of claim 133, wherein the T cells used to generate the CAR-T cells are collected using leukapheresis.

135. The CAR-T cell of any one of claims 124 to 134, wherein the CAR-T cell is allogeneic.

136. The CAR-T cell of any one of claims 124 to 134, wherein the CAR-T cell is autologous.

137. The CAR-T cell of any one of claims 131 to 134, wherein the CAR-T cell is activated.

138. The CAR-T cell of claim 137, wherein the CAR-T cell is activated using anti-CD3 antibody and anti-CD28 antibody.

139. The CAR-T cell of claim 137, wherein the CAR-T cell is activated using an anti-CD3 antibody.

140. The CAR-T cell of claim 137, wherein the CAR-T cell is activated using anti-CD3 antibody and IL-2.

141. The CAR-T cell of claim 137, wherein the CAR-T cell is activated using anti-CD3 antibody, IL-2, and PHA.

142. The CAR-T cell of any one of claims 137 to 141, wherein the anti-CD3 antibody, anti-CD28 antibody, IL2, and PHA are soluble or plate-bound.

143. The CAR-T cell of any one of claims 137 to 142, wherein the CRISPR protein is Cas9 or Cas12.

144. The CAR-T cell of any one of claims 124 to 143, wherein the cell does not comprise a DNA polynucleotide encoding a CRISPR protein.

145. The CAR-T cell of any one of claims 124 to 144, wherein the CAR-T cell is CD34 positive, CD3 negative, and LNGFR positive.

146. A mutant CAR-T cell library comprising a plurality of CAR-T cells, wherein the plurality of CAR-T cells are selected from the CAR-T cells described in any one of claims 124 to 145.

147. The mutant CAR-T cell library of claim 146, wherein at least 2 CAR-T cells of the library each comprise a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region 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.

148. The mutant CAR-T cell library of claim 146, wherein at least 10 CAR-T cells of the library each comprise a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region 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.

149. The mutant CAR-T cell library of claim 146, wherein each of the at least 20 CAR-T cells in the library comprises a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region complementary to a different sense strand or antisense strand encoded by any one of SEQ ID NOs: 1 to 135 or a variant thereof.

150. The mutant CAR-T cell library of claim 146, wherein each of the at least 50 CAR-T cells of the library comprises a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region 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.

151. The mutant CAR-T cell library of claim 146, wherein each of the at least 100 CAR-T cells in the library comprises a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region 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.

152. The mutant CAR-T cell library of claim 146, wherein each of the 135 CAR-T cells of the library comprises a different gRNA polynucleotide, wherein the different gRNA polynucleotides comprise a homology region 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.

153. The mutant CAR-T cell library of claim 146, wherein at least some of the mutant CAR-T cells comprise a gRNA containing a homology region to any one of SEQ ID NOs: 136 to 1315, and each of SEQ ID NOs: 136 to 1315 is present in at least one mutant CAR-T cell of the library.

154. The mutant CAR-T cell library of claim 146, wherein each of SEQ ID NOs: 136 to 1315 is present in at least one CAR-T cell.

155. A method for generating a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting the T cell with the gRNA vector library described in any one of claims 98 to 104; (c) transfecting the T cell with the CAR vector of any one of claims 107 to 122; and (d) introducing mRNA encoding the CRISPR protein or the CRISPR protein into the T cell.

156. A method for generating a mutant CAR-T cell library, comprising: (a) activating T cells; (b) transfecting the T cell with the gRNA vector library described in any one of claims 98 to 104; and (c) transfecting the T cell with the CAR vector of any one of claims 107 to 122; Wherein the gRNA vector or the CAR vector comprises a polynucleotide encoding a CRISPR protein.

157. The method of claim 155 or 156, further comprising collecting T cells from the subject.

158. The method of any one of claims 155 to 157, further comprising purifying T cells comprising the CAR vector and comprising a CRISPR-induced genetic mutation.

159. The method of any one of claims 155-158, wherein the T cells are collected using leukapheresis.

160. The method of any one of claims 155-159, wherein the T cells are collected from a human or mouse subject.

161. The method of any one of claims 155 to 160, wherein the T cells are activated using anti-CD3 antibodies and anti-CD28 antibodies.

162. The method of any one of claims 155 to 160, wherein the gRNA vector library is transduced at a multiplicity of infection (MOI) of 0.1 to 1.

163. The method of claim 162, wherein the MOI is between 0.3 and 0.

7.

164. The method of claim 162, wherein the MOI is between 0.4 and 0.

6.

165. The method of claim 162, wherein the MOI is 0.

5.

166. The method of any one of claims 155 to 165, wherein the CAR vector is transduced with an MOI of up to 20.

167. The method of any one of claims 155 to 166, wherein the CRISPR protein is Cas9 or Cas12.

168. The method of any one of claims 155 to 167, wherein CRISPR protein mRNA or CRISPR protein is introduced using electroporation.

169. The method of any one of claims 155 to 167, wherein the CRISPR mRNA is expressed by the gRNA vector or the CAR vector.

170. The method of claim 168, wherein electroporation is performed 3 to 7 days after transduction with the gRNA vector and the CAR vector.

171. The method of any one of claims 155 to 170, further comprising selecting T cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

172. The method of claim 171, wherein selection is performed using magnetic beads and / or flow cytometric sorting.

173. A mutant CAR-T cell library produced using the method of any one of claims 155 to 171.

174. A method for identifying a gRNA polynucleotide associated with in vivo CAR-T cell efficacy, comprising: (a) administering to a subject the mutant CAR-T cell library of any one of claims 146 to 154 or 173; (b) collecting one or more samples comprising a plurality of mutant CAR-T cells of the mutant CAR-T cell library from the subject; (c) sequencing the gRNA polynucleotides from the mutant CAR-T cells collected in (b); and (d) Evaluation of changes in the relative abundance of each gRNA polynucleotide based on sequencing in (c).

175. The method of claim 174, further comprising: (e) identifying genes associated with gRNA polynucleotides with 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 cells to the subject.

176. The method of claim 174 or claim 175, wherein the mutant CAR-T cell library comprises a whole genome CAR-T cell mutant library.

177. The method of any one of claims 174 to 176, wherein the library comprises negative control CAR-T cells.

178. The method of claim 177, wherein the negative control CAR-T cells comprise the negative control gRNA polynucleotide of any one of claims 17 to 19.

179. The method of any one of claims 175 to 178, wherein the subject is a human.

180. The method of any one of claims 175-178, wherein the subject has cancer.

181. The method of claim 180, wherein the cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, and B-cell lymphoma.

182. The method of any one of claims 180 to 181, wherein the CAR comprises an antigen binding domain that binds to an antigen expressed by the cancer.

183. The method of claim 182, wherein the CAR comprises an antigen binding domain selected from CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is TriPRIL.

184. The method of any one of claims 174-183, wherein the sample is collected from blood of the subject.

185. The method of any one of claims 174-183, wherein at least 3 samples are collected from the subject.

186. The method of any one of claims 174-183, wherein at least 5 samples are collected by venipuncture, ablation, or tumor resection.

187. The method of any one of claims 174 to 186, wherein after step (a), a sample is collected from the subject at one or more of day 3, day 7, day 10, day 14, day 21, day 28, month 2, month 3, and month 6 after administration of the mutant CAR-T cell library.

188. The method of claim 187, wherein after step (a), a sample is collected from the subject on day 21 after administration of the mutant CAR-T cell library.

189. The method of any one of claims 174 to 187, further comprising extracting mutant CAR T cells from the sample prior to sequencing.

190. The method of claim 189, wherein the extraction is performed by isolating T cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

191. The method of any one of claims 174 to 190, further comprising isolating genomic DNA from the mutant CAR-T cells.

192. The method of any one of claims 174 to 191, further comprising isolating the gRNA polynucleotide from the mutant CAR-T cells.

193. The method of any one of claims 174 to 192, further comprising isolating RNA from the mutant CAR-T cells, and optionally sequencing the RNA.

194. The method of any one of claims 174 to 193, further comprising amplifying the gRNA polynucleotide of the mutant CAR-T cell to produce an amplicon of any one thereof.

195. The method of claim 194, wherein the amplified gRNA polynucleotide is genomic DNA, vector DNA or RNA.

196. The method of claim 194 or claim 195, wherein amplification is performed using primers of SEQ ID NOs: 1324 to 1325.

197. The method of any one of claims 174 to 196, wherein the gRNA polynucleotide or an amplicon thereof is sequenced.

198. The method of claim 197, wherein the gRNA polynucleotide or an amplicon thereof is sequenced using next generation sequencing.

199. The method of any one of claims 174 to 198, wherein the potency of each mutant CAR-T cell is evaluated based on in vivo persistence and tumor cytotoxicity.

200. The method of claim 199, wherein in vivo persistence and tumor cytotoxicity are measured using flow cytometry and PCR.

201. The method of any one of claims 174 to 198, wherein persistence is quantified by calculating the relative change of each gRNA over time.

202. The method of claim 201, wherein persistence is quantified by calculating the relative change over time for each gRNA compared to the gRNA polynucleotide from a negative control CAR-T cell.

203. The method of any one of claims 201, wherein persistence is quantified by calculating the relative change over time for each gRNA over at least 3 time points and calculating the slope of the relative change.

204. The method of claim 203, wherein tumor cytotoxicity is quantified using clinical imaging and / or bone marrow assessment. 205.CAR natural killer (NK) cells comprising the composition of claim 123.

206. CAR-NK cells, comprising one or more of the following: a mutation in a gene corresponding to a gene sequence of any one of SEQ ID NOs: 1 to 135 or a variant thereof, a gRNA polynucleotide of a composition of any one of claims 1 to 72 or 74, a plasmid of any one of claims 75 to 79, or a gRNA vector of any one of claims 80 to 106; and optionally a CAR vector of any one of claims 107 to 123.

207. The CAR-NK cell of claim 206, wherein the mutation is a loss-of-function mutation.

208. The CAR-NK cell of any one of claims 205 to 207, wherein the mutation is an insertion or a deletion.

209. The CAR-NK cell of any one of claims 205 to 208, wherein the insertion or deletion mutation results in a frameshift.

210. The CAR-NK cell of any one of claims 205 to 209, further comprising mRNA encoding a CRISPR protein and / or a CRISPR protein.

211. The CAR-NK cell of any one of claims 205 to 210, wherein the NK cell is CD34 positive and CD3 negative.

212. The CAR-NK cell of claim 211, wherein the NK cells used to prepare the NK cells are from a mammalian subject.

213. The CAR-NK cell of claim 212, wherein the NK cells used to prepare the NK cells are from a human subject.

214. The CAR-NK cells of claim 213, wherein the NK cells used to produce the NK cells are collected from blood, optionally umbilical cord blood or peripheral blood.

215. The CAR-NK cell of any one of claims 211 to 214, wherein the NK cell is activated.

216. The CAR-NK cell of claim 215, wherein the NK cells are activated using irradiated feeder cells, IL-2, and IL-15.

217. The CAR-NK cells of claim 216, wherein the NK cells are activated using irradiated K562 cells.

218. The CAR-NK cell of claim 216 or 217, wherein the NK cell is activated using IL-21.

219. The CAR-NK cells of claim 216, wherein the NK cells are activated using 4-1BBL, IL-2, and irradiated K562 cells expressing membrane-bound IL-15.

220. The CAR-NK cell of any one of claims 205 to 219, wherein the CRISPR protein is Cas9 or Cas12.

221. The CAR-NK cell of any one of claims 205 to 220, wherein the NK cell does not comprise a DNA polynucleotide encoding a CRISPR protein.

222. The CAR-NK cell of any one of claims 205 to 221, wherein the NK cell is CD34 positive, CD3 negative, and LNGFR positive.

223. A mutant CAR-NK cell library comprising a plurality of NK cells, wherein the NK cells of the library are selected from the NK cells described in any one of claims 205 to 222.

224. The mutant CAR-NK cell library of claim 223, wherein at least 2 CAR-NK cells of the library each contain a different gRNA polynucleotide, and the different gRNA polynucleotides contain homologous regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants.

225. The mutant CAR-NK cell library of claim 223, wherein at least 10 CAR-NK cells of the library each contain a different gRNA polynucleotide, and the different gRNA polynucleotides contain homologous regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants.

226. The mutant CAR-NK cell library of claim 223, wherein each of the at least 20 CAR-NK cells in the library comprises a different gRNA polynucleotide, and the different gRNA polynucleotides comprise a homologous region complementary to a different gene sequence of any one of SEQ ID NO: 1 to 135 or a variant thereof.

227. The mutant CAR-NK cell library of claim 223, wherein at least 50 CAR-NK cells of the library each contain a different gRNA polynucleotide, and the different gRNA polynucleotides contain homologous regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants.

228. The mutant CAR-NK cell library of claim 223, wherein at least 100 CAR-NK cells of the library each contain a different gRNA polynucleotide, and the different gRNA polynucleotides contain homologous regions complementary to different gene sequences of any one of SEQ ID NO: 1 to 135 or its variants.

229. The mutant CAR-NK cell library of claim 223, wherein each of the 135 CAR-NK cells in the library comprises a different gRNA polynucleotide, and the different gRNA polynucleotides comprise a homologous region complementary to a different gene sequence of any one of SEQ ID NO: 1 to 135 or a variant thereof.

230. The mutant CAR-NK cell library of claim 223, wherein SEQ ID NOs: 136 to 1315 are each present in at least one NK cell.

231. A method for producing a mutant CAR-NK cell library, comprising: (a) activating CAR-NK cells; (b) transducing the CAR-NK cells with the gRNA vector library described in any one of claims 98 to 104; (c) transducing the CAR-NK cell with the CAR vector of any one of claims 107 to 122; and (d) introducing mRNA encoding CRISPR protein or CRISPR protein into the NK cells.

232. A method for producing a mutant CAR-NK cell library, comprising: (a) activating CAR-NK cells; (b) transducing the CAR-NK cells with the gRNA vector library described in any one of 98 to 104; as well as (c) transducing the CAR-NK cell with the CAR vector of any one of claims 107 to 122; Wherein the gRNA vector or the CAR vector comprises a polynucleotide encoding a CRISPR protein.

233. The method of claim 231 or 232, further comprising collecting CAR-NK cells from the subject.

234. The method of any one of claims 231 to 233, further comprising purifying CAR-NK cells comprising the CAR vector and comprising a CRISPR-induced genetic mutation.

235. The method of any one of claims 231 to 234, wherein the CAR-NK cells are collected using leukapheresis.

236. The method of any one of claims 231 to 235, wherein the CAR-NK cells are collected from a human or mouse subject.

237. The method of any one of claims 231 to 236, wherein the CAR-NK cells are activated using anti-CD3 antibodies and anti-CD28 antibodies.

238. The method of any one of claims 231 to 236, wherein the gRNA vector library is transduced at a multiplicity of infection (MOI) of 0.1 to 1.

239. The method of claim 238, wherein the MOI is between 0.3 and 0.

7.

240. The method of claim 238, wherein the MOI is between 0.4 and 0.

6.

241. The method of claim 238, wherein the MOI is 0.

5.

242. The method of any one of claims 231 to 241, wherein the CAR vector is transduced with an MOI of up to 20.

243. The method of any one of claims 231 to 242, wherein the CRISPR protein is Cas9 or Cas12.

244. The method of any one of claims 231 to 243, wherein the CRISPR protein mRNA or the CRISPR protein is introduced using electroporation.

245. The method of any one of claims 231 to 244, wherein the CRISPR mRNA is expressed by the gRNA vector or the CAR vector.

246. The method of claim 245, wherein electroporation is performed 3 to 7 days after transduction with the gRNA vector and the CAR vector.

247. The method of any one of claims 231 to 246, further comprising selecting NK cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

248. The method of claim 247, wherein selection is performed using magnetic beads and / or flow cytometric sorting.

249. A mutant CAR-NK cell library produced using the method described in any one of claims 231 to 247.

250. A method for identifying a mutation that improves CAR-NK cell efficacy in vivo, comprising: (a) administering to a subject the mutant CAR-NK cell library of any one of claims 223 to 230 or 249; (b) collecting one or more samples of a plurality of mutant CAR-NK cells comprising a mutant NK cell library from the subject; (c) sequencing the gRNA polynucleotides from the mutant CAR-NK cells collected in (b); and (d) Evaluation of changes in the relative abundance of each gRNA polynucleotide based on sequencing in (c).

251. The method of claim 250, further comprising: (e) identifying genes associated with gRNA polynucleotides with increased relative abundance; (f) modifying the NK cell to contain the mutation in the gene identified in (e), thereby producing a modified NK cell; and (g) administering the modified NK cells to the subject.

252. The method of claim 250, wherein the mutant CAR-NK cell library comprises a whole genome NK cell mutant library.

253. The method of any one of claims 250 to 252, wherein the library comprises negative control CAR-NK cells.

254. The method described in claim K3, wherein the negative control CAR-NK cells comprise the negative control gRNA polynucleotide described in any one of claims 17 to 19.

255. The method of any one of claims 250 to 254, wherein the subject is a human.

256. The method of any one of claims 250-254, wherein the subject has cancer.

257. The method of claim 256, wherein the cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, breast cancer, AML, multiple myeloma, and B-cell lymphoma.

258. The method of any one of claims 256 to 257, wherein the CAR comprises an antigen binding domain that binds to an antigen expressed by the cancer.

259. The method of claim 258, wherein the CAR comprises an antigen binding domain selected from CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, and claudin 6, binds to any pair of CD19 / CD79b, BCMA / TACI, or is TriPRIL.

260. The method of any one of claims 250-259, wherein the sample is collected from blood of the subject.

261. The method of any one of claims 25-259, wherein at least 3 samples are collected from the subject.

262. The method of any one of claims 250-259, wherein at least 5 samples are collected by venipuncture, ablation, or tumor resection.

263. The method of any one of claims 250 to 262, wherein after step (a), a sample is collected from the subject at one or more of day 3, day 7, day 10, day 14, day 21, day 28, month 2, month 3, and month 6.

264. The method of any one of claims 250 to 263, further comprising extracting the mutant NK cells from the sample prior to sequencing.

265. The method of claim 264, wherein the extraction is performed by isolating NK cells that are CD3 negative, CD34 positive, and optionally LNGFR positive.

266. The method of any one of claims 250 to 265, further comprising isolating genomic DNA from the mutant NK cells.

267. The method of any one of claims 250 to 266, further comprising isolating gRNA polynucleotides from the mutant NK cells.

268. The method of any one of claims 250 to 267, further comprising isolating RNA from the mutant NK cells and optionally sequencing the RNA.

269. The method of any one of claims 250 to 268, further comprising amplifying the gRNA polynucleotide of the mutant NK cell to produce an amplicon of any one thereof.

270. The method of claim 269, wherein the amplified gRNA polynucleotide is genomic DNA, vector DNA or RNA.

271. The method of claim 269 or claim 270, wherein amplification is performed using primers of SEQ ID NOs: 1324 to 1325.

272. The method of any one of claims 250 to 271, wherein the gRNA polynucleotide or an amplicon thereof is sequenced.

273. The method of claim 272, wherein the gRNA polynucleotide or its amplicon is sequenced using next generation sequencing.

274. The method of any one of claims 250 to 273, wherein the potency of each mutant NK cell is evaluated based on in vivo persistence and tumor cytotoxicity.

275. The method of claim 274, wherein in vivo persistence and tumor cytotoxicity are measured using flow cytometry and PCR.

276. The method of any one of claims 250 to 273, wherein persistence is quantified by calculating the relative change of each gRNA over time.

277. The method of claim 276, wherein persistence is quantified by calculating the relative change over time for each gRNA compared to the gRNA polynucleotide from a negative control NK cell.

278. The method of any of claims 276, wherein persistence is quantified by calculating the relative change over time for each gRNA over at least 3 time points and calculating the slope of the relative change.

279. The method of claim 276, wherein tumor cytotoxicity is quantified using clinical imaging and / or bone marrow assessment.

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