Gene-regulating compositions and methods for improved immunotherapy
Inventors
Benson, Micah • Merkin, Jason J. • Kryukov, Gregory V. • Shenker, Solomon Martin • Schlabach, Michael R. • Tubo, Noah Jacob
Assignees
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Publication Number
US-11421228-B2
Publication Date
2022-08-23
Expiration Date
Abstract
The present disclosure provides methods and compositions related to the modification of immune effector cells to increase therapeutic efficacy. In some embodiments, immune effector cells modified to reduce expression of one or more endogenous target genes, or to reduce one or more functions of an endogenous protein to enhance effector functions of the immune cells are provided. In some embodiments, immune effector cells further modified by introduction of transgenes conferring antigen specificity, such as exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) are provided. Methods of treating a cell proliferative disorder, such as a cancer, using the modified immune effector cells described herein are also provided.
Core Innovation
The invention relates to engineered human tumor infiltrating lymphocytes (TILs) and engineered CRISPR/Cas and guide RNA (gRNA) components for a gene-regulating system in immune effector cells. The engineered human TIL comprises a modified endogenous ZC3H12A gene and a Cas endonuclease and/or a gRNA, wherein ZC3H12A protein expression and/or function is reduced relative to an unmodified human TIL expressing an endogenous ZC3H12A gene, and anti-tumor efficacy is increased relative to the unmodified human TIL.
The disclosure includes Cas endonucleases, including Cas mutants, and gRNAs configured to reduce off-target activity and to regulate gene expression and/or function of endogenous targets. It also describes Cas mutants with altered PAM/PFS specificity, directed evolution, PAM length options, and sequence complementarity/identity rules, together with crRNA and tracr RNA features, sgRNA versus dual-gRNA formats, and spacer-targeting elements defined for endogenous targets.
The document further describes approaches to minimize off-target effects and to improve gRNA stability and reduce innate immune activation. It includes chemical and sequence modifications to nucleosides/nucleotides, end modifications for RNA, and general compositions such as polynucleotides, vectors, kits, and pharmaceutical compositions for delivery into immune effector cells such as T cells and tumor-infiltrating lymphocytes (TILs).
Claims Coverage
The independent claim set centers on an engineered human TIL with reduced ZC3H12A expression and/or function and increased anti-tumor efficacy using a Cas endonuclease and/or a gRNA. The claim set includes six inventive features, with dependent refinements specifying alternative Cas endonuclease forms, constraining target sequence options, optionally adding additional modified endogenous target genes, optionally adding an exogenous transgene expressing an immune-activating molecule, and optionally providing a pharmaceutical composition containing the engineered TIL.
Engineered human TIL with reduced endogenous ZC3H12A and increased anti-tumor efficacy
A human tumor infiltrating lymphocyte (TIL) comprising a modified endogenous ZC3H12A gene and a Cas endonuclease and/or a guide RNA (gRNA), wherein ZC3H12A protein expression and/or function is reduced relative to an unmodified human TIL expressing an endogenous ZC3H12A gene, and anti-tumor efficacy is increased relative to the unmodified TIL.
Selected Cas endonuclease forms
The Cas endonuclease is selected from a wild-type Cas protein with two active domains, a Cas nickase mutant with one active domain, a deactivated Cas protein (dCas) associated with a heterologous protein, or a Cas ortholog.
ZC3H12A target sequence constrained to SEQ ID NOs 1065-1264
The target sequence is chosen from SEQ ID NOs 1065-1264.
Exogenous transgene encoding an immune-activating molecule
The human TIL further comprises an exogenous transgene expressing an immune-activating molecule chosen from cytokine, chemokine, co-stimulatory molecule, activating peptide, antibody, or antigen-binding fragment.
Additional modified endogenous target gene selected from an enumerated set
The human TIL further comprises a modified endogenous target gene selected from a specified group of genes including IKZF1 and others listed such as IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, BCOR, BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, MAP4K1, and NR4A3.
Pharmaceutical composition containing the engineered TIL
A pharmaceutical composition that contains the human TIL described as comprising a modified endogenous ZC3H12A gene and a Cas endonuclease and/or a gRNA with reduced ZC3H12A expression and/or function and increased anti-tumor efficacy.
Overall, the claim set covers an engineered human TIL with reduced ZC3H12A expression and/or function and increased anti-tumor efficacy using a Cas endonuclease and/or a gRNA, with dependent refinements specifying alternative Cas endonuclease forms, constraining ZC3H12A target sequence options to SEQ ID NOs 1065-1264, optionally adding additional modified endogenous target genes, optionally adding an exogenous transgene expressing an immune-activating molecule, and optionally providing pharmaceutical compositions containing the engineered TIL.
Stated Advantages
Increased anti-tumor efficacy relative to an unmodified human TIL expressing an endogenous ZC3H12A gene.
Resistance to T cell exhaustion, with increased cytokine production, increased proliferation, increased target cell lysis, and increased expression of pro-inflammatory/cytolytic factors such as granzyme B, perforin, and granulysin.
Off-target activity is reduced.
gRNA stability is improved and innate immune activation is reduced.
Anti-tumor memory and epitope spreading are associated with ZC3H12A inhibition/deletion.
Cytokine production is improved, including IL-2 and IFN-γ, upon multi-gene editing.
MAP4K1 editing enhances cytokine production in naive human T cells.
Documented Applications
Measuring exhaustion during in vitro/ex vivo TIL manufacturing, including after harvest and between expansion rounds.
Measuring exhaustion in vivo after transfer into subjects.
Engineered human tumor infiltrating lymphocytes (TILs) for improving anti-tumor efficacy compared to unmodified human TILs expressing an endogenous ZC3H12A gene.
Therapeutic applications of the engineered immune effector cells including T cells and tumor-infiltrating lymphocytes (TILs), including kits and pharmaceutical compositions.
In vivo and in vitro validation of CRISPR-edited immune effector cells, including adoptively transferred CD19 CAR-T cells, with NGS-measured edit efficiencies for MAP4K1 and NR4A3 linked to tumor growth inhibition.
In vivo screening of dual sgRNA retroviral libraries to identify gene pair combinations affecting tumor infiltration and fitness and anti-tumor efficacy.
Anti-tumor efficacy evaluation in multiple mouse xenograft and syngeneic models.
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