Modified cells and methods of therapy
Inventors
Moriarity, Branden • Webber, Beau • Choudhry, Modassir • McIvor, R. Scott • Largaespada, David • 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-11147837-B2
Publication Date
2021-10-19
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, including non-viral vectors and T cells, for treating cancer, and methods of making and using these genetically modified compositions in cancer treatment. It emphasizes engineered cells that comprise at least one gene disruption and at least one non-virally integrated T cell receptor (TCR) sequence, where the gene can be disrupted by the non-viral TCR sequence. The genes disrupted can be checkpoint genes such as PD-1, CTLA4, or others involved in immune checkpoints.
The disclosed engineered cells can be primary or immune cells including T cells, stem cells, and progenitor cells, and can be autologous or non-autologous to the subject. The TCR sequences can be engineered, comprising two or more chains like alpha and beta, recognizing antigens in the context of MHC molecules and specifically cancer mutations identified for example by whole-exomic sequencing. Methods of making these engineered cells involve non-viral introduction of exogenous TCR sequences flanked by recombination arms complementary to genomic sequences, facilitating double-stranded break repair and insertion by homologous recombination possibly assisted by HR enhancers such as viral proteins or chemical inhibitors.
The problem being solved arises from limitations of current cancer therapies, where many tumor types are resistant to chemotherapy, radiotherapy or biotherapy especially in advanced stages. While genetic engineering of lymphocytes to target tumors has shown remission in hematologic tumors, broad application to solid tumors is challenged by the lack of identified tumor-specific molecules useful for targeting and destroying tumors. The present invention provides innovative non-viral approaches such as CRISPR, TALEN, or transposon-based systems to insert transgenes encoding cancer-specific TCRs into T cells for treating any type of cancer by targeting unique immunogenic mutations.
Claims Coverage
The independent claims focus on methods and compositions for producing ex vivo populations of lymphocytes with PD-1 gene disruption using modified guide RNAs and Cas9 nucleases delivered by nucleofection or electroporation, and the expanded lymphocytes for cancer treatment.
Use of a modified guide RNA with 2′-O-methyl, 3′ phosphorothioate modifications
The modified gRNA has increased stability compared to unmodified gRNA and is engineered to specifically bind a PD-1 gene PAM adjacent sequence. The modified gRNA is delivered with a polynucleotide encoding Cas9 by nucleofection or electroporation into primary lymphocytes.
Efficient PD-1 gene cleavage and knockdown in primary lymphocytes
Cas9-mediated cleavage of the PD-1 gene occurs in at least 65% of primary lymphocytes as quantified by TIDE analysis absent selection, reducing or suppressing PD-1 protein production, thus generating a population of PD-1 knockdown lymphocytes.
Expansion of PD-1 knockdown lymphocytes ex vivo
The method includes expanding the population of PD-1 knockdown lymphocytes, producing an ex vivo population of lymphocytes with high proportions of PD-1 knockdown cells.
Production of therapeutic ex vivo lymphocyte populations
The method and compositions result in ex vivo lymphocyte populations comprising at least about 5×10^6 to 5×10^9 viable lymphocytes with high PD-1 knockdown percentage, suitable for immunotherapy.
Combination of PD-1 disruption with additional genetic alterations
The ex vivo lymphocyte population can include additional genomic alterations, for example in T-cell receptor components such as alpha or beta chains for further engineering of cellular function.
Methods of treating cancer by administering engineered PD-1 knockdown lymphocytes
The claims cover methods of treating various cancers including leukemias, myelomas, sarcomas, melanomas, gastrointestinal, lung and breast cancers by administering the engineered lymphocyte populations.
Preparation methods for modified primary T cells or tumor infiltrating lymphocytes (TILs) with reduced PD-1 expression
Methods include ex vivo modification of primary circulating T cells or TILs by delivering modified gRNAs and Cas9 polynucleotides via nucleofection or electroporation, achieving at least 65% PD-1 knockdown with minimal cell death, followed by expansion of the modified cells.
The independent claims cover the use of chemically modified guide RNAs and Cas9 endonuclease delivered by electroporation or nucleofection for efficient ex vivo PD-1 gene disruption in primary lymphocytes, methods for expanding the modified lymphocytes, and their use in cancer immunotherapy, including methods to prepare primary circulating T cells or TILs with high PD-1 knockdown and minimal toxicity.
Stated Advantages
High efficiency gene transfer and expression of TCR transgenes with increased cell survival rates.
Favors homology directed repair (HDR) over non-homologous end joining (NHEJ) to improve precise gene editing.
Reduced cellular toxicity during genome engineering leads to improved viability and expansion of engineered cells.
Non-viral integration methods provide safer and scalable production of engineered cells compatible with good manufacturing practices (GMP).
Documented Applications
Treatment of cancer including hematologic tumors and solid tumors by administration of engineered T cells expressing cancer-specific TCRs that recognize unique immunogenic mutations.
Use of engineered cells in autologous and allogenic transplantation for cancer therapy.
Treatment of infections, autoimmune disorders, or graft-versus-host disease (GVHD) using engineered immune cells.
Methods of gene editing primary T cells for adoptive cell transfer-based immunotherapy.
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