10-Year Stability Assessment of Cryopreserved, Engineered iPSC Banks: Genetic and Phenotypic Characterization
Induced pluripotent stem cells (iPSCs) represent a promising renewable source of starting material for the mass production of uniform and consistent multiplexed-engineered cellular therapies for off-the-shelf therapeutic use, including for cancer, autoimmune disease and regenerative medicine. Over the past decade, we have developed a robust proprietary iPSC platform, where cellular reprogramming, maintenance of pluripotency in the naïve state, and single-cell culture in a feeder-free environment is enabled by stage-specific, small molecule combinations to block differentiation, enhance survival, and support self-renewal of iPSCs (Valamehr et al., Stem Cell Reports 2014). We have applied our iPSC product platform to generate clonal master iPSC banks, and have used these banks for cGMP manufacture of multiplexed-engineered, off-the-shelf natural killer and T-cell product candidates now in clinical studies. Establishing a cell banking paradigm, including a bank stability program, to ensure the long-term availability and viability of clonal master iPSC banks for drug product manufacturing is critical to ensure safety, efficacy, regulatory compliance, and manufacturing demands throughout a product’s life cycle. To this end, we have performed long-term stability studies of cryopreserved iPSC banks, including at 2, 5 and 10 years from formation. Our data show that different somatic cells can be efficiently reprogrammed to the pluripotent state, frozen in a controlled environment, and maintained for at least 10 years. Cryopreserved iPSC banks were thawed and tested for critical quality attributes including viability, recovery, purity, and potency. In addition, the stability of cryopreserved, multiplexed-engineered iPSC lines was examined following thaw and stress-inducing manipulations, including further rounds of genetic engineering, single-cell subcloning and expansion in feeder-free culture, and cryopreservation to establish secondary iPSC banks. The data to be presented will collectively show that cryopreserved iPSC banks maintain viability (>90%), purity (>90%), potency (tri-lineage differentiation), phenotypic and genetic stability (genome integrity and transgene expression stability) over a long period of time without significant deterioration. Therefore, clonal master iPSC banks created using our proprietary iPSC platform and maintained under our proprietary banking paradigm can serve as a renewable source of starting material over the long term for the mass production of off-the-shelf cell therapies.
Robust T-Cell Cellular Reprogramming and Single-Cell Engineering Platform Overcomes Inconsistencies and Heterogeneity Associated with Engineering Primary T Cells
Adoptive transfer of primary T cells expressing chimeric antigen receptors (CARs) has shown great promise in treating hematologic malignancies. However, application of CAR T-cell therapy is limited by several challenges, including cell product inconsistencies and heterogeneity resulting from engineering primary T-cell populations as part of drug product manufacture. The use of clonally-derived, master induced pluripotent stem cell (iPSC) lines is an attractive source for the renewable manufacture of precisely-engineered, homogenous CAR T-cell products that can be fully characterized, stored, and administered on-demand for broad patient access. Furthermore, generation of such master iPSC lines from donor T cells (TiPSCs) allows for the unique opportunity to use the pre-rearranged TCR locus as an ideal approach to facilitate T-cell receptor alpha constant (TRAC) locus-targeted genetic editing to enhance CAR-mediated activity. Reprogramming of donor T cells has historically been inefficient though and iPSC-derived T cells often exhibit poor functional attributes. Here we evaluate our non-integrating proprietary T-cell cellular reprogramming platform for the efficient generation of high-quality naïve TiPSCs using current good manufacturing practice (cGMP)-grade processes, procedures, and materials. Donor T cells were sourced from leukapheresis blood collections following consent and screening for infectious disease markers. The aß T-cell population was isolated and banked in animal component-free cryopreservation medium. These parental banks were characterized for purity, safety, identity, viability, and recovery. cGMP parental cell banks from nine independent donors were assessed for reprogramming efficiency and naïve iPSC generation using our proprietary reprogramming platform (Valamehr et al., 2014). TiPSCs were efficiently generated from all nine cGMP parental cell banks regardless of donor background or attributes. The fraction of TiPSCs reached >80% by day 28 for most donors (n=7), whereas two donors exhibited lower TiPSC fraction (24 and 58%). In each case, this output of TiPSCs represented a significant increase over other donor T-cell reprogramming methods. To test the potential to precisely engineer and single-cell select TiPSCs for the generation of clonally-derived, master iPSC lines, TiPSCs from four cGMP parental cell banks were engineered by CRISPR-mediated CAR insertion into the TCR alpha constant locus. TiPSCs from all four cGMP banks showed robust knock-in efficiency (20-60%) and maintained uniform pluripotent phenotypes and genomic stability post genetic engineering. We next isolated and sorted single-cell TiPSCs into 96-well plates, and extensively characterized each clonal TiPSC population following expansion. Hundreds of TiPSC clones were successfully generated and cryopreserved for characterization and assessment. Cryopreserved TiPSC clones showed robust recovery upon thaw (viability >75%) and maintained normal karyotype (100% of clones tested). In contrast to engineering cell populations as part of each manufacturing campaign for primary CAR T-cell production, this study highlights the potential to use clonally-derived, master engineered TiPSC lines as a renewable source for the consistent and uniform manufacture of off-the-shelf CAR T cells for therapeutic applications.
Development of Next-Generation, Off-the-Shelf CAR T-Cell Immunotherapies for Solid Tumors
Despite inducing promising clinical outcomes in patients with relapsed / refractory hematologic malignancies, the adoptive transfer of engineered T cells armed with a chimeric antigen receptor (CAR) has been less effective against advanced solid tumors. Specific challenges that have emerged include lack of tumor-exclusive antigen targets, antigen heterogeneity, and functional suppression resulting from the tumor microenvironment. In addition, inherent limitations associated with patient- and donor-derived T-cell therapies limit the breadth of their therapeutic application, including the potential to reach patients earlier in care. Induced pluripotent stem cells (iPSCs) can serve as renewable starting material for the mass production of off-the-shelf, cell-based cancer immunotherapies. We have previously developed FT819, a first-of-kind off-the-shelf CAR T-cell therapy derived from a clonal master iPSC line engineered to uniformly express a novel CD19 1XX-CAR driven by the endogenous T-cell receptor (TCR) a promoter at the T-cell receptor a constant (TRAC) locus. FT819 was designed to promote antigen specificity, improved safety by knocking out the TCR to eliminate the possibility of graft versus host disease (GVHD), and enable off-shelf availability. Here, we describe a novel multiplexed-engineered, off-the-shelf CAR T-cell therapy derived from a clonal master iPSC line for the treatment of solid tumors. The master iPSC line incorporates seven functional modalities: 1) a CAR for direct targeting of tumor antigen; 2) a cytokine receptor fusion protein for enhanced T-cell activity; 3) a high-affinity, non-cleavable CD16 (hnCD16) for enhanced antibody-dependent cellular cytotoxicity (ADCC) in combination with tumor-targeting monoclonal antibodies; 4) an engineered modality for enhanced trafficking; 5) a novel chimeric protein for enhanced functionality in response to tumor microenvironment resistance signaling; 6) CD38 deletion for enhanced metabolic fitness; and 7) TRAC-targeted TCR deletion for eliminating the risk of GVHD. Utilizing our proprietary iPSC product platform, a clonal master iPSC line was derived by CRISPR-mediated knock-in of functional modalities into the TRAC and CD38 loci. Single iPSCs were assessed and selected for specific targeted integration at the desired loci without random donor template integration; transgene copy numbers were confirmed by droplet digital PCR as well as genome stability by karyotyping and microarray analysis. We next demonstrated that T cells derived from the clonal master iPSC line exhibited improved expansion during differentiation, including without the need for cytokine support. In vitro functional studies including antigen-specific cytokine release assays, cytotoxic T lymphocytes assays, mixed lymphocyte reaction, and serial restimulation assays demonstrated enhanced efficacy. Our data validate the potential of multi-loci engineering of iPSCs to create master multiplexed-engineered iPSC lines and produce next-generation, iPSC-derived CAR-T therapies against solid tumor.
FT536: A First-of-Kind, Off-the-Shelf CAR-iNK Cell Product Candidate for Solid Tumors Designed to Specifically Target MICA/B Stress Proteins and Overcome Mechanisms of Tumor Evasion
The advent of chimeric antigen receptor (CAR)-T cell therapies has revolutionized the treatment of hematological malignancies; however, broader therapeutic success has been challenged by observed toxicities, including on-target, off-tumor engagement of non-cancerous cells, limited tumor antigen expression and availability, and the ineffectiveness of single-antigen targeted CAR T cells to eradicate heterogeneous tumors. In addition, the inherent variability that arises from the use of patient- and donor-sourced T cells and the engineering of these T-cell populations as part of each manufacturing campaign results in significant drug product inconsistencies, which can impact safety, efficacy, and therapeutic reach. We have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell product candidate that expresses a novel CAR and ubiquitously targets cancer cells through canonical stress ligand recognition (Figure 1A). FT536 recognizes the conserved a3 domain of the pan-tumor associated MICA and MICB (MICA/B) stress proteins, a novel targeting strategy that mitigates a key tumor immune evasion mechanism. In addition to the CAR, FT536 is derived from a clonal master iPSC line that incorporates multiple genetic edits to enhance NK cell effector function, persistence, and multi-antigen targeting. Starting with a clonal master iPSC line, the cGMP manufacturing process of FT536 is analogous to pharmaceutical drug product development, and consistently and uniformly delivers greater than 4x107 cellular fold expansion per manufacturing campaign. FT536 drug product can be cryopreserved, stored, and delivered to clinical sites for thaw-and-infusion to patients in the out-patient setting. Preclinical assessment of the product candidate’s unique CAR modality demonstrated potent antigen-specific cytolytic activity against an array of solid and hematological tumor cell lines. FT536 is also armed with a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor, which provides the potential to target additional tumor antigens in combination with tumor-targeting antibodies. In addition, FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models (Figure 1B). An Investigational New Drug (IND) application for FT536 was allowed by the U.S. Food and Drug Administration (FDA) in December 2021, and a first-in-human clinical study of FT536 as monotherapy and in combination with tumor-targeting monoclonal antibody therapy for the treatment of multiple solid tumor indications is expected to commence in mid-2022.
(A) MICA/B CAR containing primary T cells demonstrated pan-tumor reactivity and superior IFN? cytokine responses compared to control and NKG2D CAR containing primary T cells. (B) FT536 significantly reduced the number of lung and liver (not shown) metastases compared to CAR negative iNK control cells in a murine metastatic melanoma model using B16/F10 cells engineered to overexpress human MICA.
Induced pluripotent stem cells (iPSCs) represent a promising renewable source of starting material for the mass production of uniform and consistent multiplexed-engineered cellular therapies for off-the-shelf therapeutic use, including for cancer, autoimmune disease and regenerative medicine. Over the past decade, we have developed a robust proprietary iPSC platform, where cellular reprogramming, maintenance of pluripotency in the naïve state, and single-cell culture in a feeder-free environment is enabled by stage-specific, small molecule combinations to block differentiation, enhance survival, and support self-renewal of iPSCs (Valamehr et al., Stem Cell Reports 2014). We have applied our iPSC product platform to generate clonal master iPSC banks, and have used these banks for cGMP manufacture of multiplexed-engineered, off-the-shelf natural killer and T-cell product candidates now in clinical studies. Establishing a cell banking paradigm, including a bank stability program, to ensure the long-term availability and viability of clonal master iPSC banks for drug product manufacturing is critical to ensure safety, efficacy, regulatory compliance, and manufacturing demands throughout a product’s life cycle. To this end, we have performed long-term stability studies of cryopreserved iPSC banks, including at 2, 5 and 10 years from formation. Our data show that different somatic cells can be efficiently reprogrammed to the pluripotent state, frozen in a controlled environment, and maintained for at least 10 years. Cryopreserved iPSC banks were thawed and tested for critical quality attributes including viability, recovery, purity, and potency. In addition, the stability of cryopreserved, multiplexed-engineered iPSC lines was examined following thaw and stress-inducing manipulations, including further rounds of genetic engineering, single-cell subcloning and expansion in feeder-free culture, and cryopreservation to establish secondary iPSC banks. The data to be presented will collectively show that cryopreserved iPSC banks maintain viability (>90%), purity (>90%), potency (tri-lineage differentiation), phenotypic and genetic stability (genome integrity and transgene expression stability) over a long period of time without significant deterioration. Therefore, clonal master iPSC banks created using our proprietary iPSC platform and maintained under our proprietary banking paradigm can serve as a renewable source of starting material over the long term for the mass production of off-the-shelf cell therapies.
Robust T-Cell Cellular Reprogramming and Single-Cell Engineering Platform Overcomes Inconsistencies and Heterogeneity Associated with Engineering Primary T Cells
Adoptive transfer of primary T cells expressing chimeric antigen receptors (CARs) has shown great promise in treating hematologic malignancies. However, application of CAR T-cell therapy is limited by several challenges, including cell product inconsistencies and heterogeneity resulting from engineering primary T-cell populations as part of drug product manufacture. The use of clonally-derived, master induced pluripotent stem cell (iPSC) lines is an attractive source for the renewable manufacture of precisely-engineered, homogenous CAR T-cell products that can be fully characterized, stored, and administered on-demand for broad patient access. Furthermore, generation of such master iPSC lines from donor T cells (TiPSCs) allows for the unique opportunity to use the pre-rearranged TCR locus as an ideal approach to facilitate T-cell receptor alpha constant (TRAC) locus-targeted genetic editing to enhance CAR-mediated activity. Reprogramming of donor T cells has historically been inefficient though and iPSC-derived T cells often exhibit poor functional attributes. Here we evaluate our non-integrating proprietary T-cell cellular reprogramming platform for the efficient generation of high-quality naïve TiPSCs using current good manufacturing practice (cGMP)-grade processes, procedures, and materials. Donor T cells were sourced from leukapheresis blood collections following consent and screening for infectious disease markers. The aß T-cell population was isolated and banked in animal component-free cryopreservation medium. These parental banks were characterized for purity, safety, identity, viability, and recovery. cGMP parental cell banks from nine independent donors were assessed for reprogramming efficiency and naïve iPSC generation using our proprietary reprogramming platform (Valamehr et al., 2014). TiPSCs were efficiently generated from all nine cGMP parental cell banks regardless of donor background or attributes. The fraction of TiPSCs reached >80% by day 28 for most donors (n=7), whereas two donors exhibited lower TiPSC fraction (24 and 58%). In each case, this output of TiPSCs represented a significant increase over other donor T-cell reprogramming methods. To test the potential to precisely engineer and single-cell select TiPSCs for the generation of clonally-derived, master iPSC lines, TiPSCs from four cGMP parental cell banks were engineered by CRISPR-mediated CAR insertion into the TCR alpha constant locus. TiPSCs from all four cGMP banks showed robust knock-in efficiency (20-60%) and maintained uniform pluripotent phenotypes and genomic stability post genetic engineering. We next isolated and sorted single-cell TiPSCs into 96-well plates, and extensively characterized each clonal TiPSC population following expansion. Hundreds of TiPSC clones were successfully generated and cryopreserved for characterization and assessment. Cryopreserved TiPSC clones showed robust recovery upon thaw (viability >75%) and maintained normal karyotype (100% of clones tested). In contrast to engineering cell populations as part of each manufacturing campaign for primary CAR T-cell production, this study highlights the potential to use clonally-derived, master engineered TiPSC lines as a renewable source for the consistent and uniform manufacture of off-the-shelf CAR T cells for therapeutic applications.
Development of Next-Generation, Off-the-Shelf CAR T-Cell Immunotherapies for Solid Tumors
Despite inducing promising clinical outcomes in patients with relapsed / refractory hematologic malignancies, the adoptive transfer of engineered T cells armed with a chimeric antigen receptor (CAR) has been less effective against advanced solid tumors. Specific challenges that have emerged include lack of tumor-exclusive antigen targets, antigen heterogeneity, and functional suppression resulting from the tumor microenvironment. In addition, inherent limitations associated with patient- and donor-derived T-cell therapies limit the breadth of their therapeutic application, including the potential to reach patients earlier in care. Induced pluripotent stem cells (iPSCs) can serve as renewable starting material for the mass production of off-the-shelf, cell-based cancer immunotherapies. We have previously developed FT819, a first-of-kind off-the-shelf CAR T-cell therapy derived from a clonal master iPSC line engineered to uniformly express a novel CD19 1XX-CAR driven by the endogenous T-cell receptor (TCR) a promoter at the T-cell receptor a constant (TRAC) locus. FT819 was designed to promote antigen specificity, improved safety by knocking out the TCR to eliminate the possibility of graft versus host disease (GVHD), and enable off-shelf availability. Here, we describe a novel multiplexed-engineered, off-the-shelf CAR T-cell therapy derived from a clonal master iPSC line for the treatment of solid tumors. The master iPSC line incorporates seven functional modalities: 1) a CAR for direct targeting of tumor antigen; 2) a cytokine receptor fusion protein for enhanced T-cell activity; 3) a high-affinity, non-cleavable CD16 (hnCD16) for enhanced antibody-dependent cellular cytotoxicity (ADCC) in combination with tumor-targeting monoclonal antibodies; 4) an engineered modality for enhanced trafficking; 5) a novel chimeric protein for enhanced functionality in response to tumor microenvironment resistance signaling; 6) CD38 deletion for enhanced metabolic fitness; and 7) TRAC-targeted TCR deletion for eliminating the risk of GVHD. Utilizing our proprietary iPSC product platform, a clonal master iPSC line was derived by CRISPR-mediated knock-in of functional modalities into the TRAC and CD38 loci. Single iPSCs were assessed and selected for specific targeted integration at the desired loci without random donor template integration; transgene copy numbers were confirmed by droplet digital PCR as well as genome stability by karyotyping and microarray analysis. We next demonstrated that T cells derived from the clonal master iPSC line exhibited improved expansion during differentiation, including without the need for cytokine support. In vitro functional studies including antigen-specific cytokine release assays, cytotoxic T lymphocytes assays, mixed lymphocyte reaction, and serial restimulation assays demonstrated enhanced efficacy. Our data validate the potential of multi-loci engineering of iPSCs to create master multiplexed-engineered iPSC lines and produce next-generation, iPSC-derived CAR-T therapies against solid tumor.
FT536: A First-of-Kind, Off-the-Shelf CAR-iNK Cell Product Candidate for Solid Tumors Designed to Specifically Target MICA/B Stress Proteins and Overcome Mechanisms of Tumor Evasion
The advent of chimeric antigen receptor (CAR)-T cell therapies has revolutionized the treatment of hematological malignancies; however, broader therapeutic success has been challenged by observed toxicities, including on-target, off-tumor engagement of non-cancerous cells, limited tumor antigen expression and availability, and the ineffectiveness of single-antigen targeted CAR T cells to eradicate heterogeneous tumors. In addition, the inherent variability that arises from the use of patient- and donor-sourced T cells and the engineering of these T-cell populations as part of each manufacturing campaign results in significant drug product inconsistencies, which can impact safety, efficacy, and therapeutic reach. We have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell product candidate that expresses a novel CAR and ubiquitously targets cancer cells through canonical stress ligand recognition (Figure 1A). FT536 recognizes the conserved a3 domain of the pan-tumor associated MICA and MICB (MICA/B) stress proteins, a novel targeting strategy that mitigates a key tumor immune evasion mechanism. In addition to the CAR, FT536 is derived from a clonal master iPSC line that incorporates multiple genetic edits to enhance NK cell effector function, persistence, and multi-antigen targeting. Starting with a clonal master iPSC line, the cGMP manufacturing process of FT536 is analogous to pharmaceutical drug product development, and consistently and uniformly delivers greater than 4x107 cellular fold expansion per manufacturing campaign. FT536 drug product can be cryopreserved, stored, and delivered to clinical sites for thaw-and-infusion to patients in the out-patient setting. Preclinical assessment of the product candidate’s unique CAR modality demonstrated potent antigen-specific cytolytic activity against an array of solid and hematological tumor cell lines. FT536 is also armed with a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor, which provides the potential to target additional tumor antigens in combination with tumor-targeting antibodies. In addition, FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models (Figure 1B). An Investigational New Drug (IND) application for FT536 was allowed by the U.S. Food and Drug Administration (FDA) in December 2021, and a first-in-human clinical study of FT536 as monotherapy and in combination with tumor-targeting monoclonal antibody therapy for the treatment of multiple solid tumor indications is expected to commence in mid-2022.
(A) MICA/B CAR containing primary T cells demonstrated pan-tumor reactivity and superior IFN? cytokine responses compared to control and NKG2D CAR containing primary T cells. (B) FT536 significantly reduced the number of lung and liver (not shown) metastases compared to CAR negative iNK control cells in a murine metastatic melanoma model using B16/F10 cells engineered to overexpress human MICA.
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