Modified cells and methods of therapy
Inventors
Moriarity, Branden • Webber, Beau • Choudhry, Modassir • Rosenberg, Steven A. • Palmer, Douglas C. • Restifo, Nicholas P.
Assignees
Intima Bioscience Inc • University of Minnesota System • US Department of Health and Human Services
Publication Number
US-10406177-B2
Publication Date
2019-09-10
Expiration Date
2036-07-29
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Abstract
Genetically modified compositions, such as non-viral vectors and T cells, for treating cancer are disclosed. Also disclosed are the methods of making and using the genetically modified compositions in treating cancer.
Core Innovation
The invention discloses genetically modified compositions, such as non-viral vectors and T cells, for treating cancer. These engineered cells comprise at least one gene disruption and at least one non-virally integrated T cell receptor (TCR) sequence, where the gene disrupted can be an immune checkpoint gene such as PD-1. The TCR sequence can be single or multiple chains, capable of recognizing antigen in the context of major histocompatibility complex (MHC) class I or II and binding to cancer cells, including those presenting mutations identified by whole-exomic sequencing.
The gene disruption is achieved by non-viral methods including CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL, facilitating insertion of the exogenous TCR sequence near double strand break regions in checkpoint genes. The insertion can disrupt one or more genes and can be assisted by homologous recombination enhancers that inhibit non-homologous end joining, thereby improving targeted integration efficiency.
The engineered cells can be primary, immune, stem, or progenitor cells, including hematopoietic progenitor cells. They can be autologous or non-autologous and are compatible with good manufacturing practices (GMP). The compositions and methods also include reducing cellular toxicity during genetic modification by altering cellular responses to exogenous polynucleic acids, for example using caspase inhibitors and inhibitors of cytosolic DNA sensing pathways.
Claims Coverage
The independent claims cover methods of treating cancer by administering engineered primary lymphocytes with targeted gene disruptions and TCR sequence insertions via CRISPR systems.
Targeted gene disruption using CRISPR in cytokine inducible SH2-containing protein (CISH) gene
A method of treating cancer comprising administering engineered primary lymphocytes with a gene disruption at exon 2 or 3 of the human CISH gene by CRISPR-induced targeted double strand break, with a guide RNA hybridizing to CISH sequences including those with at least 80% identity to SEQ ID NOS: 75-86, and achieving at least 60% integration efficiency.
Use of autologous tumor infiltrating lymphocytes
The engineered primary lymphocytes are autologous tumor infiltrating lymphocytes expanded to at least approximately 5×107 cells before genomic disruption.
Efficient and specific guide RNA targeting
The guide RNA hybridizes within 10 base pairs of a PAM site in CISH gene, sequences contain GA or GG motifs, and specific sequence identities to SEQ ID NO: 82.
High viability and cell number for therapy
The therapeutic composition comprises engineered lymphocytes with at least 70% viability and at least about 1×109 cells for administration.
Combination therapy and cancer applicability
Treatment may include administration of cyclophosphamide and/or fludaribine, rapid expansion protocol of lymphocytes, and applicability to various cancers including gastrointestinal cancers and solid tumors in humans.
The independent claims focus on cancer treatment methods using ex vivo engineered primary lymphocytes, particularly tumor infiltrating lymphocytes, with CRISPR-mediated targeted gene disruption of CISH and TCR insertion, using guide RNAs targeting specific CISH sequences with high integration efficiency, combined with expansion protocols and optionally other therapies. The features cover guide RNA design, cell viability, cell numbers, and applicability to solid tumor cancer treatment.
Stated Advantages
The disclosed methods and compositions provide high efficiency gene transfer and expression with increased cell survival rates and low toxicity.
The method favors homology directed repair over non-homologous end joining, facilitating efficient homologous recombinants recovery and expansion.
Use of modifying compounds reduces cytotoxicity induced by exogenous polynucleic acids, improving viability of engineered cells during genome engineering.
Non-viral gene editing methods with homologous recombination enhancers increase insertion efficiency of exogenous TCR into endogenous checkpoint genes.
The approach expands the therapeutic potential of adoptive cell transfer for a broad range of cancer types, including solid tumors.
Documented Applications
Identification of cancer-specific T cell receptors that recognize unique immunogenic mutations in a patient's cancer for personalized cancer therapy.
Engineering primary T cells, tumor infiltrating lymphocytes, or progenitor cells by non-viral insertion of TCRs targeting cancer neoantigens.
Treatment of various cancers including melanoma, gastrointestinal cancer, breast cancer, prostate cancer, myeloma, lymphoma, sarcoma, pancreatic, liver, lung, thyroid, brain, hematopoietic, esophageal, bone, bladder, cervical, and ovarian cancers.
Use in autologous or allogenic transplantation of engineered immune cells to treat cancer.
Reduction of cytotoxicity in engineered cells by modulating innate immune sensing pathways to improve genetic engineering outcomes.
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