Allogeneic Products Research Articles

Role of Cytokines and Growth Factors in the Manufacturing of iPSC-Derived Allogeneic Cell Therapy Products.

Cytokines and other growth factors are essential for cell expansion, health, function, and immune stimulation. Stem cells have the additional reliance on these factors to direct differentiation to the appropriate terminal cell type. Successful manufacturing of allogeneic cell therapies from induced pluripotent stem cells (iPSCs) requires close attention to the selection and control of cytokines and factors used throughout the manufacturing process, as well as after administration to the patient. This paper employs iPSC-derived natural killer cell/T cell therapeutics to illustrate the use of cytokines, growth factors, and transcription factors at different stages of the manufacturing process, ranging from the generation of iPSCs to controlling of iPSC differentiation into immune-effector cells through the support of cell therapy after patient administration.

Clinical manifestations of allergic reactions and immunoglobulin E levels on allogeneic freeze-dried platelet-rich plasma (PRP) application

Link of Video Abstract: https://youtu.be/yS431sCL3VQ Background: Several studies have reported that modern wound dressings offer better results. Allogeneic PRP products in the form of injections and gels are available in the market, which helps accelerate wound healing without triggering an immunological reaction. Meanwhile, this study was carried out to investigate the Allogeneic Freeze-Dried PRP form, which is more practical, efficient, and safer. This study aims to determine the effect of Allogeneic Freeze-Dried PRP on human split-thickness skin graft donor wounds on clinical manifestations of allergic reactions and levels of Immunoglobulin E (IgE). Methods: A randomized Pre- and Post-test Control-Trial Group Design was conducted on patients with split-thickness skin graft donor wounds using Allogeneic Freeze-Dried PRP dressings at Dr. Soetomo General Hospital, Surabaya. Allergic manifestations were reviewed during the observation period of up to 4 days, and IgE levels were measured pre-test and 60 minutes post-application. A statistical comparison of results was carried out between the trial and control groups that used paraffin dressings. Results: A total of 36 patients were used as subjects, with 19 (52.78%) in the trial group and 17 (47.22%) in the control group, and their parameters were generally homogeneous. There were no clinical manifestations of allergic, changes in vital signs, and significant differences in IgE levels before and after application statistically in all study subjects (p>0.05). Conclusion: Application of Allogeneic Freeze-Dried PRP to human split-thickness skin graft donor wounds did not generate clinical manifestations of allergic reactions and an increase in IgE.

Cryopreservation of Allogeneic Hematopoietic Cell Products During COVID-19 Pandemic: Graft Characterization and Engraftment Outcomes

The COVID-19 pandemic triggered the deployment of unfamiliar measures to safeguard successful allogeneic hematopoietic cell transplantation (allo-HCT). Among these measures, cryopreservation offered logistical benefits that could outlast the pandemic, including graft availability and timely clinical service. The purpose of this study was to evaluate graft quality and hematopoietic reconstitution in patients transplanted with cryopreserved allogeneic stem cell products during the COVID-19 pandemic. We evaluated 44 patients who underwent allo-HCT using cryopreserved grafts consisting of hematopoietic progenitor cells (HPC) apheresis (A) and bone marrow (BM) products at Mount Sinai Hospital. Comparative analyses of 37 grafts infused fresh during the one-year period preceding the pandemic were performed. Assessment of cellular therapy products included total nucleated cell and CD34+ cell enumeration, viability, and post-thaw recovery. The primary clinical endpoint was the evaluation of engraftment (absolute neutrophil count [ANC] and platelet count) and donor chimerism (presence of CD33+ and CD3+ donor cells) at day +30 and +100 post-transplant. Adverse events related to cell infusion were also analyzed. Patient characteristics were comparable between the fresh and cryopreserved groups with 2 exceptions in the HPC-A cohort: the number of patients in the cryopreserved group that received haploidentical grafts was 6 times that in the fresh group, and the number of patients in the fresh group with a Karnofsky performance score >90 was double that in the cryopreserved group. The quality of HPC-A and HPC-BM products was not affected by cryopreservation, and all grafts met the release criteria for infusion. The pandemic did not affect the time between collection and cryopreservation (median, 24 hours) and time in storage (median, 15 days). Median time to ANC recovery was significantly delayed in recipients of cryopreserved HPC-A (15 vs 11 days, P=.0121), and there was a trend toward delayed platelet engraftment (24 vs 19 days, P=.0712). The delay in ANC and platelet recovery was not observed when only matched graft recipients were compared. Cryopreservation did not affect the ability of HPC-BM grafts to engraft and reconstitute hematopoiesis, and there was no difference in the rates of ANC and platelet recovery. Achievement of donor CD3/CD33 chimerism was not affected by cryopreservation of either HPC-A or HPC-BM products. Graft failure was observed in only 1 case, a recipient of cryopreserved HPC-BM. Three recipients of cryopreserved HPC-A grafts died before ANC engraftment from infectious complications. Remarkably, 22% of our studied population had myelofibrosis, and almost half received cryopreserved HPC-A grafts with no graft failure observed. Finally, patients receiving cryopreserved grafts were at a higher risk of infusion-related adverse events than those receiving fresh grafts. Cryopreservation of allogeneic grafts results in adequate product quality with minimal impact on short-term clinical outcomes, except for an increased risk of infusion-related adverse events. Cryopreservation is a safe option in terms of graft quality and hematopoietic reconstitution with logistical benefits, but additional data are needed to determine long-term outcomes and assess whether this is a suitable strategy for at-risk patients.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Abstract 2904: CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice

Abstract While chimeric antigen receptor (CAR)-engineered immune cell therapies have been at the forefront of cancer immunotherapy for hematological malignancies, patients with solid tumors have yet to benefit from such therapies. Engineered, off-the-shelf, allogeneic natural killer (NK) cells are particularly attractive as cell therapies for solid tumors given their clinical safety, efficacy, and multimodal recognition of tumor cells. We describe here the pre-clinical pharmacokinetics, efficacy, biodistribution and safety of CAT-179, a novel allogeneic, cryopreserved CAR-NK cell therapy, in naïve animals as well as multiple xenograft models of HER2-amplified ovarian and gastric cancer. CAT-179 cells are engineered to express an optimized HER2-directed CAR to effectively target tumor cells, a transforming growth factor b (TGFβ) dominant negative receptor (DNR) to protect against TGFβ-mediated immunosuppression, and interleukin-15 (IL-15) to enhance NK cell persistence. A single intravenous (IV) dose of CAT-179 resulted in IL-15-dependent (p<0.0001) expansion and persistence of CAT-179 cells for at least 140 days in NOD-scid IL2Rgnull (NSG) mice, peaking at approximately 15,000 cells/µL of blood (by day 60). Persisting CAT-179 NK cell levels at day 60 were functionally active and significantly reduced tumor burden when challenged intraperitoneally (IP) with HER2+ SKOV3-luc tumor cells. No significant changes in body weight or condition were observed during this study. The therapeutic anti-tumor activity of CAT-179 against established tumors was assayed using two different xenograft models. In the first model, NSG mice were given an IP dose of 1 million SKOV3-luc tumor cells (derived from ovarian cancer) followed by either 4 million CAT-179 or control NK cells on days 4, 11 and 18. CAT-179 dosed animals showed a rapid and sustained 95% decline in tumor burden (p<0.0001) and a significant extension in survival relative to animals dosed with control NK cells (p<0.0001). In the second model, 1 million HER2+ N87 cancer cells (derived from gastric cancer) were implanted subcutaneously into the right flank of NSG mice. When tumors reached 70mm3, a single IV dose of 2M CAT-179 or control NK cells was administered. CAT-179 dosed animals showed a 96% durable tumor regression and significant survival benefit relative to animals dosed with control NK cells (p<0.0001). Efficacy strongly correlated with the circulating levels of CAT-179, which significantly infiltrated the tumor xenograft. Our pre-clinical results demonstrate the potential of CAT-179 as a novel, durable, and off-the-shelf cell therapy to overcome the challenges associated with solid tumors and provide quantitative insights into pharmacokinetics, pharmacodynamics and anti-tumor activity of engineered NK cells expressing CAR, TGFb DNR and IL-15. Citation Format: Bashar Hamza, Angela Nunez, Marilyn Marques, Alexia Barandiaran, Henry Moreno, Finola Moore, Meghan Walsh, Eugene Choi, Kisha Pradhan, Krista Daniel, Jennifer Johnson, Charlotte Franco, Andres Alvarez, Karl Malakian, Keith H. Wong, Joseph Gold, Vipin Suri, Dominic Picarella. CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2904.

Abstract 4059: Developing an allogeneic iPSC derived macrophage cell therapy for oncology

Abstract The full potential of cell therapy has yet to be realized in solid tumor indications and new approaches are urgently required. Macrophages are innate immune cells that can kill tumor cells and orchestrate the anti-tumor immune response. Macrophage cell therapy is an exciting new approach to treat cancer with the aim to harness the powerful activity of macrophages to reignite the immune system. Patient derived macrophages, however, are difficult to genetically engineer and do not proliferate, which makes generating high-quality cells at clinically relevant scales challenging. To address this, we have developed an induced pluripotent stem cell (iPSC) approach to macrophage cell therapy. These cells can be genetically engineered at the iPSC stage and then differentiated into billions of highly functional iPSC derived macrophages (iMACs). This allogenic cell therapy product can then be cryopreserved and stored for immediate use in the clinic when the patient is ready. Here we show that iMACs function like normal macrophages. They migrate towards tumor cells, respond to challenges through innate immune receptors, and produce immune recruiting and activating cytokines and chemokines. iMACs also express high levels of antibody receptors and we show that these cells can be directed to kill tumors in vitro and in vivo via antibody dependent cellular phagocytosis. Furthermore, we have conducted a robust screen to develop a novel CAR that is optimized for use in iMACs. These receptors can be engineered into iMACs at the iPSC stage and trigger robust tumor cell killing. In summary, these data highlight the therapeutic potential of iMACs in oncology and support the further development of this technology for clinical use. Citation Format: Huafeng Wang, May Sumi, Christine Huh, Fereshteh Parviz, Jessica Mastroianni, Jane Healy, Nicole Stevens, Leah Mitchell, Susanne Lang, Dan Kaufman, Robert Hollingsworth, David T. Rodgers. Developing an allogeneic iPSC derived macrophage cell therapy for oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4059.

Abstract 4096: Assessing individual variability in efficacy and toxicity of autologous and allogeneic chimeric antigen receptor T-cell immunotherapy using a PBMC-humanized mouse model

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a revolutionary therapeutic to fight cancer. However, side effects such as cytokine-release syndrome and neurotoxicity often accompany and remain unpredictable. While autologous CAR-Ts are FDA-approved, allogeneic CAR-Ts are in active development in pursuit of “off-the-shelf” availability and reduced manufacturing costs. However, the same concern for adverse events applies. Importantly, how the immune characteristics of the patient may predispose towards toxicity is not well modeled. Further, it is unclear how autologous versus allogeneic products interact with the patient immune system. Toxicity does not correlate with in vitro characterization of the CAR-Ts, adding unpredictability to the toxicity and efficacy. These highlight the need for in vivo mouse models to explore biological variation, predictive biomarkers, and therapeutic intervention. Therefore, we developed a humanized mouse model using NSG™-MHC Class I/II double knock-out (DKO) mice engrafted with human PBMCs to access individual variability in CAR-T-induced toxicity and efficacy. DKO mice are optimal for assessing human cytokine induction from CAR-Ts as they show delayed onset of GvHD and low baseline cytokine levels. To test donor variation in autologous CAR-T toxicity, we humanized mice and treated with autologous CD19 CAR-Ts containing the CD28 costimulatory domain and CD3z chain from two human PBMC donors (Donor A and B). CAR-T successfully eliminated human B cells from both PBMC donors in vivo. We observed drastic body weight loss and 83% of Donor A mice (engrafted with 17M PBMCs) reached the humane endpoint. In contrast, Donor B mice did not experience body weight loss. CAR-T induced human cytokines such as IFN-ꝩ, IL-2, IL-4, IL-6, IL-10, TNFα, MIG, MIP-1a, and IP-10, recapturing clinical data. To test donor variation in allogeneic CAR-T toxicity, we engrafted mice with 10M PBMCs from 6 healthy donors and treated with allogeneic CAR-Ts from Donor A or B. Allogenic efficacy was greater in Donor B CAR- than Donor A CAR-Ts. Donor-specific variability was observed; mice humanized with one of the six donors, experienced significant body weight loss only from the Donor B CAR-Ts but not from Donor A CAR-Ts. Principal component analysis of cytokine data revealed that Donor B CAR-Ts induced more distinguished cytokine responses than Donor A CAR-Ts, driven by IFN-ꝩ and IL-10. Lastly, the data indicates that the CAR-T cells show poor expansion in allogeneic treatment, corresponding to the current literature. PBMC-humanized mice provide an in vivo platform to assess the toxicity and efficacy of autologous and allogeneic CAR-T treatment. This platform can be used to preclinically assess cytokine induction from the interaction between the patient immune system and allogeneic CAR-Ts from healthy donors. Citation Format: Won Lee, Destanie Rose, Katelyn Burleigh, Blair Armstrong, Heather Gustafson, Rebecca Gardner, James G. Keck, Jiwon Yang. Assessing individual variability in efficacy and toxicity of autologous and allogeneic chimeric antigen receptor T-cell immunotherapy using a PBMC-humanized mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4096.

Abstract 1773: Preclinical characterization of allogeneic Vγ9Vδ2 HER2-TAC T cells for the treatment of HER2-positive solid tumors

Abstract Background: The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the redirection of T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the goal to elicit safe and durable anti-tumor responses. TAC01-HER2, a first-in-class, autologous TAC T cell product targeting HER2 (ERBB2), has entered a phase I/II clinical trial in patients with HER2-positive solid tumors. Here, we describe the development of an allogeneic HER2-TAC T cell product based on Vγ9Vδ2 (γδ) T cells which belong to a subset of T cells that recognize target cells in a human leukocyte antigen (HLA) independent manner. Thus, γδ T cells do not cause GvHD and have the potential for allogeneic cell therapy applications. Materials and Methods: A variety of in vitro and in vivo assays were used to evaluate the potency and safety of HER2-TAC γδ T cells generated from multiple donors. In vitro assays included flow cytometric analysis determining the γδ T cell phenotype, intracellular cytokines, CD69 upregulation, and T cell proliferation. Anti-tumor cytotoxicity was assessed via real-time microscopy-based co-culture assays. Mixed lymphocyte reactions (MLR) were performed to measure cytokine production and proliferation of HER2-TAC γδ T cells in response to HLA mismatches between unrelated donors. In vivo studies examined the anti-tumor effect of HER2-TAC γδ T cells against established solid HER2-expressing tumors. Results: HER2-TAC γδ T cells selectively reacted to HER2-expressing tumor cells in co-culture, as demonstrated by CD69 upregulation, intracellular cytokine production, increase in proliferation, and cytotoxicity. In contrast, HER2-TAC γδ T cells failed to show activity in MLR assays, potentially indicating that HER2-TAC γδ T cells are free of GvH reactivity. These MLR assays comprised dendritic cells that represent the major HLA subtypes found in North America. In addition, HER2-TAC γδ T cells showed strong anti-tumor efficacy in HER2-positive tumor xenograft models without signs of toxicity. Conclusions: The in vitro and in vivo data confirm strong and specific activity of HER2-targeted TAC γδ T cells against HER2-expressing tumor models and highlights the potential of the TAC platform in the development of an allogeneic product for therapeutic applications in solid tumors. Citation Format: Suzanna L. Prosser, Stacey X. Xu, Ling Wang, Ritu R. Randhawa, Sailaja Pirati, Laura Ravensbergen, Seungmi Yoo, Miyoung Jung, Laurentia Gheorghiu, Angel Gomez, Gurleen Sandhu, Chris Ayers, Donna Rill, Christopher W. Helsen, Andreas G. Bader. Preclinical characterization of allogeneic Vγ9Vδ2 HER2-TAC T cells for the treatment of HER2-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1773.

Genetic engineering strategies to enhance antitumor reactivity and reduce alloreactivity for allogeneic cell-based cancer therapy.

The realm of cell-based immunotherapy holds untapped potential for the development of next-generation cancer treatment through genetic engineering of chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies for targeted eradication of cancerous malignancies. Such allogeneic "off-the-shelf" cell products can be advantageously manufactured in large quantities, stored for extended periods, and easily distributed to treat an exponential number of cancer patients. At current, patient risk of graft-versus-host disease (GvHD) and host-versus-graft (HvG) allorejection severely restrict the development of allogeneic CAR-T cell products. To address these limitations, a variety of genetic engineering strategies have been implemented to enhance antitumor efficacy, reduce GvHD and HvG onset, and improve the overall safety profile of T-cell based immunotherapies. In this review, we summarize these genetic engineering strategies and discuss the challenges and prospects these approaches provide to expedite progression of translational and clinical studies for adoption of a universal cell-based cancer immunotherapy.

Immune Responses to Sequential Binocular Transplantation of Allogeneic Retinal Progenitor Cells to the Vitreous Cavity in Mice.

Intravitreal transplantation of allogeneic human retinal progenitor cells (hRPCs) holds promise as a treatment for blinding retinal degenerations. Prior work has shown that neural progenitors are well-tolerated as allografts following single injections; however, sequential delivery of allogeneic cells raises the potential risk of host sensitization with subsequent immune rejection of grafts. The current study was designed to assess whether an immune response would be induced by repeated intravitreal transplants of allogeneic RPCs utilizing the mouse animal model. We injected murine retinal progenitor cells (gmRPCs), originally derived from donors with a C57BL/6 genetic background, into BALB/c recipient mice in order to provide safety data as to what might be expected following repeated treatment of patients with allogeneic human cell product. Immune responses to gmRPCs were mild, consisting of T cells, B cells, neutrophils, and natural killer cells, with macrophages clearly the predominating. Animals treated with repeat doses of gmRPCs did not show evidence of sensitization, nor was there immune-mediated destruction of the grafts. Despite the absence of immunosuppressive treatments, allogeneic gmRPC grafts survived following repeat dosing, thus providing support for the preliminary observation that repeated injection of allogeneic RPCs to the vitreous cavity is tolerated in patients with retinitis pigmentosa.

Assessing deviations for HPCs obtained during COVID-19 (ADHOC): Evaluating impact of the COVID-19 pandemic on cellular therapy products and processes, the BEST collaborative study.

The success of allogeneic hematopoietic stem cell transplantation is dependent on a world-wide network of collection centers providing donations that predominantly have been infused as fresh cells. The logistics chain that supports the just-in-time delivery model for stem cell and immunotherapy products was severely stressed by the COVID pandemic, and in early 2020 a number of national and international bodies recommended that cells should be cryopreserved at the collection or transplant center to avoid interruptions in their acquisition or delivery to patients who had started conditioning. To assess the potential consequences of such pandemic-related deviations to normal practice, we surveyed nine international laboratories to determine if the characteristics or transplant outcomes of allogeneic stem cell donations differed in the immediate periods before and after the switch to routine cryopreservation. Nine centers on two continents provided data for 72 HSC donations just before, and 71 just after, switching to cryopreservation for allogeneic HSC products. No statistically significant differences between the period before and after cryopreservation were noted for time from product collection to receipt, product temperature at receipt, or CD34+ cell viability at receipt. There was an indication of slower absolute neutrophil count recovery after cryopreservation was required (mean time of 15 vs. 17.6 days). While there were no apparent changes to most parameters studied, there was an indication of slower neutrophil engraftment that will need to be examined in larger, longer term studies.

Sterility Testing for Hematopoietic Stem Cells.

Over the last two decades, rapid technological advances have led to the wide adoption of cell and gene therapy products for the treatment of a variety of disease states. In this study, we reviewed the literature between 2003 and 2021 to provide a summary of overarching trends associated with microbial contamination in hematopoietic stem cells (HSCs) derived from peripheral blood, bone marrow, and cord blood. We provide a brief background on the regulatory context for human cells, tissues, and cellular and tissue-based products (HCT/Ps) as regulated by the US Food and Drug Administration (FDA), sterility testing expectations for autologous (Section 361) and allogeneic (Section 351) HSC products, and discuss clinical risks associated with the infusion of a contaminated HSC product. Finally, we discuss the expectations for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) for the manufacturing and testing of HSC based on Section 361 and Section 351 categorization, respectively. We provide commentary on what is practiced in the field and discuss the critical need for updates to professional standards that keep pace with advancing technologies with an aim to clarify expectations for manufacturing and testing facilities to improve standardization across institutions.

Safety and efficacy of first-in-man intrathecal injection of human astrocytes (AstroRx®) in ALS patients: phase I/IIa clinical trial results

BackgroundMalfunction of astrocytes is implicated as one of the pathological factors of ALS. Thus, intrathecal injection of healthy astrocytes in ALS can potentially compensate for the diseased astrocytes. AstroRx® is an allogeneic cell-based product, composed of healthy and functional human astrocytes derived from embryonic stem cells. AstroRx® was shown to clear excessive glutamate, reduce oxidative stress, secrete various neuroprotective factors, and act as an immunomodulator. Intrathecal injection of AstroRx® to animal models of ALS slowed disease progression and extended survival. Here we report the result of a first-in-human clinical study evaluating intrathecal injection of AstroRx® in ALS patients.MethodsWe conducted a phase I/IIa, open-label, dose-escalating clinical trial to evaluate the safety, tolerability, and therapeutic effects of intrathecal injection of AstroRx® in patients with ALS. Five patients were injected intrathecally with a single dose of 100 × 106 AstroRx® cells and 5 patients with 250 × 106 cells (low and high dose, respectively). Safety and efficacy assessments were recorded for 3 months pre-treatment (run-in period) and 12 months post-treatment (follow-up period).ResultsA single administration of AstroRx® at either low or high doses was safe and well tolerated. No adverse events (AEs) related to AstroRx® itself were reported. Transient AEs related to the Intrathecal (IT) procedure were all mild to moderate. The study demonstrated a clinically meaningful effect that was maintained over the first 3 months after treatment, as measured by the pre-post slope change in ALSFRS-R. In the 100 × 106 AstroRx® arm, the ALSFRS-R rate of deterioration was attenuated from − 0.88/month pre-treatment to − 0.30/month in the first 3 months post-treatment (p = 0.039). In the 250 × 106 AstroRx® arm, the ALSFRS-R slope decreased from − 1.43/month to − 0.78/month (p = 0.0023). The effect was even more profound in a rapid progressor subgroup of 5 patients. No statistically significant change was measured in muscle strength using hand-held dynamometry and slow vital capacity continued to deteriorate during the study.ConclusionsOverall, these findings suggest that a single IT administration of AstroRx® to ALS patients at a dose of 100 × 106 or 250 × 106 cells is safe. A signal of beneficial clinical effect was observed for the first 3 months following cell injection. These results support further investigation of repeated intrathecal administrations of AstroRx®, e.g., every 3 months.Trial Registration: NCT03482050.

Non-Conventional Allogeneic Anti-BCMA Chimeric Antigen Receptor-Based Immune Cell Therapies for Multiple Myeloma Treatment.

MM, characterized by the progressive accumulation of clonal plasma cells in bone marrow, remains a severe medical problem globally. Currently, almost all MM patients who have received standard treatments will eventually relapse. Autologous anti-BCMA CAR-T cells are one of the FDA-approved immunotherapy cell-based products for treating adults with relapsed or refractory (r/r) multiple myeloma. However, this type of CAR-T cell product has several limitations, including high costs, long manufacturing times, and possible manufacturing failure, which significantly hinder its wider application for more patients. In this review, we summarized the current development stage of applying other types of immune cells to bring the anti-BCMA CAR-T therapy from autologous to allogeneic. In general, anti-BCMA CAR gene-edited αβ T cells and CAR-Natural Killer (NK) cells are at the forefront, with multiple clinical trials ongoing, while CAR-γδ T cells and CAR-invariant Natural Killer T (iNKT) cells are still in pre-clinical studies. Other immune cells such as macrophages, B cells, and dendritic cells have been mainly developed to target other antigens and have the potential to be used to target BCMA. Nevertheless, additional regulatory requirements might need to be taken into account in developing these non-conventional allogenic anti-BCMA CAR-based cell products.

Safety and Clinical Effects of a Muse Cell-Based Product in Patients With Amyotrophic Lateral Sclerosis: Results of a Phase 2 Clinical Trial

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of motor neurons. Multilineage-differentiating stress-enduring (Muse) cells are unique endogenous stem cells that show therapeutic effects on motor function in ALS mouse models. We conducted a single-center open phase II clinical trial to evaluate the safety and clinical effects of repeated intravenous injections of an allogenic Muse cell-based product, CL2020, in patients with ALS. Five patients with ALS received CL2020 intravenously once a month for a total of six doses. The primary endpoints were safety and tolerability, and the secondary endpoint was the rate of change in the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score. In addition, serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), sphingosine-1-phosphate (S1P), cerebrospinal fluid chitotriosidase-1 (CHIT-1), and neurofilament light chain (NfL) levels were evaluated. The CL2020 treatment was highly tolerated without serious side effects. The ALSFRS-R score change trended upward at 12 months post-CL2020 treatment compared with that at 3 months pre-administration, but the difference was not statistically significant. Among five patients diagnosed with ALS, three exhibited a decrease in the rate of ALSFRS-R score change, one demonstrated an increase, and another showed no change. In addition, the patients’ serum IL-6 and TNF-α levels and cerebrospinal fluid CHIT-1 and NfL levels increased for up to 6 months post-treatment; however, their serum S1P levels continuously decreased over 12 months. These findings indicate a favorable safety profile of CL2020 therapy. In the near future, a double-blind study of a larger number of ALS patients should be conducted to confirm the efficacy of ALS treatment with CL2020.

Leveraging Natural Killer Cell Innate Immunity against Hematologic Malignancies: From Stem Cell Transplant to Adoptive Transfer and Beyond

Numerous recent advancements in T-cell based immunotherapies have revolutionized the treatment of hematologic malignancies. In the race towards the first approved allogeneic cellular therapy product, there is growing interest in utilizing natural killer (NK) cells as a platform for off-the-shelf cellular therapies due to their scalable manufacturing potential, potent anti-tumor efficacy, and superior safety profile. Allogeneic NK cell therapies are now being actively explored in the setting of hematopoietic stem cell transplantation and adoptive transfer. Increasingly sophisticated gene editing techniques have permitted the engineering of chimeric antigen receptors, ectopic cytokine expression, and tumor recognition signals to improve the overall cytotoxicity of NK cell therapies. Furthermore, the enhancement of antibody-dependent cellular cytotoxicity has been achieved through the use of NK cell engagers and combination regimens with monoclonal antibodies that act synergistically with CD16-expressing NK cells. Finally, a greater understanding of NK cell biology and the mechanisms of resistance have allowed the preclinical development of NK checkpoint blockade and methods to modulate the tumor microenvironment, which have been evaluated in early phase trials. This review will discuss the recent clinical advancements in NK cell therapies in hematologic malignancies as well as promising avenues of future research.

Key considerations & challenges in developing flow cytometry assays for cell & gene therapy products

Flow cytometric assays are a vital component of the cell and gene therapy analytics toolbox. In both autologous and allogeneic cell therapy products, key drug product parameters are now evaluated using flow cytometric assays. The complexity of this technique and lack of true reference standards has led to unique challenges during the adoption of flow cytometry assays in quality control as well as during their validation. This article discusses key considerations during assay development and the main challenges in adoption of flow cytometry assays in a quality control laboratory. Further, this article discusses key areas of innovation that can drive the improvement of flow cytometry assays in the future. 

Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

Clinical Pharmacology and Determinants of Response to UCART19, an Allogeneic Anti-CD19 CAR-T Cell Product, in Adult B-cell Acute Lymphoblastic Leukemia.

UCART19 is an "off-the-shelf" genome-edited anti-CD19 chimeric antigen receptor (CAR)-T cell product, manufactured from unrelated healthy donor cells. UCART19 was administered to 25 adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) in the CALM trial. All patients underwent lymphodepletion with fludarabine and cyclophosphamide ± alemtuzumab and received one of three ascending doses of UCART19. Given the allogeneic nature of UCART19, we analyzed the impact of lymphodepletion, HLA disparities, and host immune system reconstitution on its kinetics, along with other factors known to affect autologous CAR-T cell clinical pharmacology. Responder patients (12/25) had higher UCART19 expansion (C max) and exposure (AUCTlast) than nonresponders (13/25), as measured by transgene levels in peripheral blood. The persistence of CAR+ T cells did not exceed 28 days in 10/25 patients and lasted beyond 42 days in 4/25. No significant correlation was found between UCART19 kinetics and administered cell dose, patient and product characteristics or HLA disparities. However, the number of prior lines of therapy and absence of alemtuzumab negatively impacted UCART19 expansion and persistence. Alemtuzumab exposure positively affected IL7 and UCART19 kinetics, while negatively correlating with host T lymphocyte AUC0-28. UCART19 expansion is a driver of response in adult patients with R/R B-ALL. These results shed light on the factors associated with UCART19 kinetics, which remain highly affected by the impact of alemtuzumab on IL7 and host-versus-graft rejection. First description of the clinical pharmacology of a genome-edited allogeneic anti-CD19 CAR-T cell product showing the crucial role of an alemtuzumab-based regimen in sustaining UCART19 expansion and persistence through increased IL7 availability and decreased host T lymphocyte population.

Mechanistic Modeling of the Interplay Between Host Immune System, IL-7 and UCART19 Allogeneic CAR-T Cells in Adult B-cell Acute Lymphoblastic Leukemia.

A mathematical mechanistic pharmacokinetic/pharmacodynamic model supports and captures quantitatively the beneficial impact of lymphodepleting patients before the infusion of an allogeneic CAR-T cell product. Mediation through IL-7 increase and host T lymphocytes decrease is underlined, and the model can be further used to optimize CAR-T cell therapies lymphodepletion regimen.