Allogeneic Cell Therapy Research Articles

Macroscopic membranes self‐assembled by alginate and a cationic and amphiphilic peptide for cell culture

AbstractPeptides self‐assembly systems have been explored over the past years in the context of various biological applications, such as tissue engineering and drug delivery. Here we present the development of hierarchically ordered planar and spherical macroscopic membranes constructed by an amphiphilic and cationic β‐sheet peptide that spontaneously assembles with the negatively charged alginate biopolymer. These two components generate a condensed film that developed to exhibit perpendicularly oriented fibers under specific conditioning over days. Here we examined the suitability of these spherical and planar membranes to serve as substrates for cell culture applications. Planar and spherical membranes were stable in the cell culture Dulbecco's modified eagle medium, at 37°C, over days. Breast‐cancer cells seeded on the peptide and alginate sides of fully developed and non‐fully developed planar membrane were viable for more than 2 weeks. In addition, cells were injected into spherical membranes and detected viable close to the rim even at 3 weeks. These results demonstrate the suitability of planar and spherical membranes to serve as a platform, for example, in studying cancer cells in unique niches and alternatively in potential allogeneic cell therapies.

Alloreactivity of Allogeneic Mesenchymal Stem/Stromal Cells and Other Cellular Therapies: A Concise Review.

Cellular therapies, deemed live medicine, have brought a wave of new generation biological therapies to treat previously untreatable diseases such as cancers and degenerative diseases like osteoarthritis. These cellular therapies have gained significant recognition in clinical research. The area has been further strengthened with the approval of Chimeric Antigen Receptor added on T cells (CAR-T) therapies by the regulatory authorities USA's Food and Drugs Administration (FDA), European Medical Agency (EMA), the Australian Therapeutic Goods Administration (TGA), and in many countries in 2017 to treat hematological cancers. Another milestone was achieved when allogeneic Mesenchymal Stem Cell- (MSC-) based therapy was approved by the EMA to treat Chrohn's disease in 2018. Allogeneic donor-derived MSC therapies in particular hold great promise and real hope because of their ‘off-the shelf' availability and accessibility for patients in need of urgent treatment. So far, thousands of clinical trials have explored the safety and efficacy of both autologous and allogeneic cell therapies, deeming them safe, however with varying degrees of efficacy. In the current pandemic, clinical trials have begun in many parts of the world to treat severe cases of COVID with MSCs. However, the risk of tissue rejection and the development of undesirable effects due to alloreactivity of allogeneic cells are currently not adequately addressed. Therefore, this warrants careful investigation and detailed reporting of such events by clinical researchers. This review aims at discussing the current landscape of approved allogeneic MSCs along with a few other cellular therapies. We explore any possible reactivity reported to inform the readers of any safety concern and on the efficacy of such therapies.

The metabolism of cells regulates their sensitivity to NK cells depending on p53 status

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies.

Cell Culture Process Scale-Up Challenges for Commercial-Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.

Allogeneic Cell Combination Therapy Ameliorates Chronic Kidney Disease-Induced Heart Failure with Preserved Ejection Fraction.

BackgroundLeft ventricular hypertrophy and heart failure with preserved ejection fraction (HFpEF) are primary manifestations of the cardiorenal syndrome in patients with chronic kidney disease (CKD). Therapies that improve morbidity and mortality in HFpEF are lacking. Cell-based therapies promote cardiac repair in ischemic and non-ischemic cardiomyopathies. We hypothesized that cell-based therapy ameliorates CKD-induced HFpEF.Methods and ResultsYorkshire pigs (n = 26) underwent 5/6 embolization-mediated nephrectomy. CKD was confirmed by increased creatinine and decreased glomerular filtration rate (GFR). Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks. HFpEF was evident at 4 weeks by increased LV mass, relative wall thickening, end-diastolic pressure, and end-diastolic pressure-volume relationship, with no change in ejection fraction (EF). Four weeks post-embolization, allogeneic (allo) bone marrow-derived mesenchymal stem cells (MSC; 1 × 107 cells), allo-kidney-derived stem cells (KSC; 1 × 107 cells), allo-cell combination therapy (ACCT; MSC + KSC; 1:1 ratio; total = 1 × 107 cells), or placebo (Plasma-Lyte) was delivered via intra-renal artery. Eight weeks post-treatment, there was a significant increase in MAP in the placebo group (21.89 ± 6.05 mmHg) compared to the ACCT group. GFR significantly improved in the ACCT group. EF, relative wall thickness, and LV mass did not differ between groups at 12 weeks. EDPVR improved in the ACCT group, indicating decreased ventricular stiffness.ConclusionsIntra-renal artery allogeneic cell therapy was safe in a CKD swine model manifesting the characteristics of HFpEF. The beneficial effect on renal function and ventricular compliance in the ACCT group supports further research of cell therapy for cardiorenal syndrome.

Safety study of allogeneic mesenchymal stem cell therapy in animal model.

Intravenous (IV) infusion of mesenchymal stem cells (MSCs) from nascent tissues like Wharton's Jelly of the umbilical cord is reported to offer therapeutic effects against chronic diseases. However, toxicological data essential for the clinical application of these cells are limited. Thus, this study aimed to determine the safety of IV infusion of Wharton's Jelly derived MSCs (WJ-MSCs) in rats. Fifteen male Sprague–Dawley rats were randomised into the control or treatment group. Each group received an equal volume of saline or WJ-MSC (10 × 106 cell/kg) respectively. The animals were evaluated for physical, biochemical and haematological changes at Week 0, 2, 4, 8 and 12 during the 12-week study. Acute toxicity was performed during Week 2 and sub-chronic toxicity during Week 12. At the end of the study, the relative weight of organs was calculated and histology was performed for lung, liver, spleen and kidney. The findings from physical, serum biochemistry and complete blood count demonstrated no statistically significant differences between groups. However, pathological evaluation reported minor inflammation in the lungs for all groups, but visible healing and resolution of inflammation were observed in the treatment group only. Additionally, the histological images of the treatment group had significantly improved pulmonary structures compared to the control group. In summary, the IV administration of WJ-MSC was safe in the rats. Further studies are needed to determine the long-term safety of the WJ-MSC in both healthy and diseased animal models.

Immunosuppressive Properties of Epidermal Keratinocytes Differ According to Their Immaturity Status.

Preservation of a functional keratinocyte stem cell pool is essential to ensure the long-term maintenance of epidermis integrity, through continuous physiological renewal and regeneration in case of injury. Protecting stem cells from inflammation and immune reactions is thus a critical issue that needs to be explored. Here, we show that the immature CD49fhigh precursor cell fraction from interfollicular epidermis keratinocytes, comprising stem cells and progenitors, is able to inhibit CD4 + T-cell proliferation. Of note, both the stem cell-enriched CD49fhigh/EGFRlow subpopulation and the less immature CD49fhigh/EGFRhigh progenitors ensure this effect. Moreover, we show that HLA-G and PD-L1 immune checkpoints are overexpressed in CD49fhigh precursors, as compared to CD49flow differentiated keratinocytes. This potency may limit immune reactions against immature precursors including stem cells, and protect them from exacerbated inflammation. Further exploring this correlation between immuno-modulation and immaturity may open perspectives in allogenic cell therapies.

Genome Editing of Pluripotent Stem Cells for Adoptive and Regenerative Cell Therapies

The explosion of interest in CRISPR-Cas systems over recent years has delivered rapid advances in precision genome engineering technologies that hold enormous potential for application across the biotechnology sector. Within the cell therapy space, adoptive immunotherapies continue to lead the translation of increasingly sophisticated genetic engineering strategies toward clinical application. Although early focus was placed on engineering primary immune cells collected from patient blood, the democratization of these therapeutics will require the development of off-the-shelf allogeneic cell therapy products that can be manufactured at scale. Pluripotent stem cells (PSCs) represent an attractive substrate for such products, and many of the challenges that must be overcome to realize the application of PSCs in adoptive cell therapies are common with those holding back regenerative cell-replacement therapies. This article discusses how genome editing can overcome these challenges and highlights the potential of rapidly advancing second-generation technologies in the development of novel cell-based therapeutics.

Advances in Allogeneic Cancer Cell Therapy and Future Perspectives on "Off-the-Shelf" T Cell Therapy Using iPSC Technology and Gene Editing.

The concept of allogeneic cell therapy was first presented over 60 years ago with hematopoietic stem cell transplantation. However, complications such as graft versus host disease (GVHD) and regimen-related toxicities remained as major obstacles. To maximize the effect of graft versus leukemia, while minimizing the effect of GVHD, donor lymphocyte infusion was utilized. This idea, which was used against viral infections, postulated that adoptive transfer of virus-specific cytotoxic T lymphocytes could reconstitute specific immunity and eliminate virus infected cells and led to the idea of banking third party cytotoxic T cells (CTLs). T cell exhaustion sometimes became a problem and difficulty arose in creating robust CTLs. However, the introduction of induced pluripotent stem cells (iPSCs) lessens such problems, and by using iPSC technology, unlimited numbers of allogeneic rejuvenated CTLs with robust and proliferative cytotoxic activity can be created. Despite this revolutionary concept, several concerns still exist, such as immunorejection by recipient cells and safety issues of gene editing. In this review, we describe approaches to a feasible “off-the-shelf” therapy that can be distributed rapidly worldwide. We also offer perspectives on the future of allogeneic cell cancer immunotherapy.

Development of Stem Cell-Derived Immune Cells for Off-the-Shelf Cancer Immunotherapies.

Cell-based cancer immunotherapy has revolutionized the treatment of hematological malignancies. Specifically, autologous chimeric antigen receptor-engineered T (CAR-T) cell therapies have received approvals for treating leukemias, lymphomas, and multiple myeloma following unprecedented clinical response rates. A critical barrier to the widespread usage of current CAR-T cell products is their autologous nature, which renders these cellular products patient-selective, costly, and challenging to manufacture. Allogeneic cell products can be scalable and readily administrable but face critical concerns of graft-versus-host disease (GvHD), a life-threatening adverse event in which therapeutic cells attack host tissues, and allorejection, in which host immune cells eliminate therapeutic cells, thereby limiting their antitumor efficacy. In this review, we discuss recent advances in developing stem cell-engineered allogeneic cell therapies that aim to overcome the limitations of current autologous and allogeneic cell therapies, with a special focus on stem cell-engineered conventional αβ T cells, unconventional T (iNKT, MAIT, and γδ T) cells, and natural killer (NK) cells.

Current Perspectives on "Off-The-Shelf" Allogeneic NK and CAR-NK Cell Therapies.

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Clofarabine-fludarabine-busulfan in HCT for pediatric leukemia: an effective, low toxicity, TBI-free conditioning regimen

AbstractWe prospectively studied clofarabine-fludarabine-busulfan (CloFluBu)-conditioning in allogeneic hematopoietic cell therapy (HCT) for lymphoid and myeloid malignancies and hypothesized that CloFluBu provides a less toxic alternative to conventional conditioning regimens, with adequate antileukemic activity. All patients receiving their first HCT, from 2011-2019, were included and received CloFluBu. The primary endpoint was event-free survival (EFS). Secondary endpoints were overall survival (OS), graft-versus-host disease (GvHD)-relapse-free survival (GRFS), treatment-related mortality (TRM), cumulative incidence of relapse (CIR), acute and chronic GvHD (aGvHD and cGvHD), and veno-occlusive disease (VOD). Cox proportional hazard and Fine and Gray competing-risk models were used for data analysis. One hundred fifty-five children were included: 60 acute lymphoid leukemia (ALL), 69 acute myeloid leukemia (AML), and 26 other malignancies (mostly MDS-EB). The median age was 9.7 (0.5 to 18.6) years. Estimated 2-year EFS was 72.0% ± 6.0 in ALL patients, and 62.4% ± 6.0 in AML patients. TRM in the whole cohort was 11.0% ± 2.6, incidence of aGvHD 3 to 4 at 6 months was 12.3% ± 2.7, extensive cGvHD at 2 years was 6.4% ± 2.1. Minimal residual disease-positivity prior to HCT was associated with higher CIR, both in ALL and AML. CloFluBu showed limited toxicity and encouraging EFS. CloFluBu is a potentially less toxic alternative to conventional conditioning regimens. Randomized prospective studies are needed.

CAR-T cell therapy in India requires a paradigm shift in training, education and health care processes

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of some kinds of cancers. Hundreds of companies and academic institutions are collaborating to develop gene-modified cell therapies using novel targets, different cell types, and manufacturing processes of autologous and allogenic cell therapies. The individualized, custom-made autologous CAR-T cell production platform remains a significant limiting factor for its large-scale clinical application. In this respect, the advances in standardization and automation of the process can have considerable impact on cost reduction. Development of off-the-shelf, ready-to-use universal killer cells can enable scaling up. Despite the wide use of this cell therapy in the United States, Europe and China, its development is limited in developing countries in Southeast Asia, Africa and Latin America. In this review, we focus on good manufacturing practices–compliant manufacturing requirements, operational logistics, and regulatory processes that need to be considered for high-quality gene-modified cell therapies from an Indian perspective. We also list the potential strategies to overcome challenges associated with translation to affordability and scalability.

Outcomes with “Off the Shelf” Allogeneic CD19 Chimeric Antigen Receptor T Cell Therapy for Hematological Malignancies: A Systematic Review and Meta-Analysis

Background:Chimeric antigen receptor T cell (CAR-T) therapy is an adoptive T cell immunotherapy that employs the genetically modified T cell to attack the cancer cell. It is widely studied across various hematological and solid organ malignancies. Several autologous CD19 CAR-T cell therapy constructs are now approved for various B cell lymphomas, including aggressive B cell lymphomas, indolent follicular lymphoma and mantle cell lymphoma, and acute lymphoblastic leukemia (ALL). Autologous CD19 CAR-T cell therapy has unprecedented success in relapsed and refractory disease. Long time to manufacture (2-5 weeks) and manufacture failure are challenges associated with risk of interim death and deterioration of CAR-T candidates with rapidly progressive disease. T cell fitness of the autologous product in heavily pretreated patients is also potentially compromised. To overcome these shortcomings, universal “off the shelf” allogeneic CAR-T cell therapy constructs are being developed and studied. Donor sources include healthy donors and cord or induced pluripotent stem cells (iPSCs). These CAR-T constructs have additional gene modifications to mitigate the risk of rejection and graft versus host disease (GVHD). We performed a systematic review and meta-analysis to assess the safety and efficacy of allogeneic CD19 CAR-T cell therapy.Methods:Four databases (Web of Science/MEDLINE/PubMed, Embase, and Cochrane Registry of Controlled Trials) were searched for this systematic review and meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines using MeSH terms and keywords for “Receptors, Chimeric antigen” OR “Artificial-T-cell receptor” OR “immunotherapy, adoptive” OR “CD-19”. Our search produced 3506 articles and after removing duplicates, 2243 records were screened. After excluding reviews and irrelevant articles, we included 8 prospective trials of allogeneic CD-19 CAR-T cell therapy enrolling two or more than two patients from Jan 2013 to Nov 2020. We also searched ASH 2020 abstracts to include any additional trials. The methodological quality of the included studies was evaluated using NIH quality assessment tool. Inter-study variance was calculated using the Der Simonian-Laird Estimator. Proportions along with 95% confidence Interval (CI) were extracted to compute pooled analysis using the ‘meta’ package by Schwarzer et al. in the R programming language (version 4.16-2).Results:A total of 68 patients from 8 studies were evaluated. Median age was 22.5 (4.8-64) years. (Table 1) The median follow-up time was 10 (2-18) months with median number of prior therapies of 3.2 (2-11) as reported by 5 studies. Underlying diagnosis was acute lymphocytic lymphoma (n=49, 72%), chronic lymphocytic leukemia (n=6, 9%), and non-Hodgkin lymphoma (n=13, 19%). The pooled overall response rate (ORR) was 77% (95% CI 0.63-0.89, I 2 =22%, n=68) with a complete response (CR) of 75% (95% CI 0.57-0.90, I 2 =48%, n=65). The pooled incidence of cytokine release syndrome grade I/II and grade III/IV was 53% (95% CI 0.16-0.89, I 2 =89%, n=65) and 10% (95% CI 0.01-0.25, I 2 =50%, n=65) respectively. Neurotoxicity grade I/II was 12% (95%CI 0.01-0.30, I 2 =47%, p=0.09, n=47) and GVHD grade I/II was 8% (95%CI 0.01-0.19, I 2 =0%, p=0.57 n=53). None of the clinical trials reported the duration of response.Conclusion:“Off the shelf” universal CAR-T therapy is early in development. Our available data suggest that allogeneic CD19 CAR-T constructs offer high ORR and CR rates with acceptable safety profiles. GVHD was mainly low grade (grade I-II). Given these findings, allogeneic CAR-T cell therapy is an attractive option to improve timely access compared to available autologous therapy. Extensive preclinical research to develop novel constructs and several phase I/II clinical trials are ongoing to shape the future of “off the shelf” CAR-T cell therapy. [Display omitted] DisclosuresHoffmann: Pharmcyclics: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria. Abhyankar: Incyte/Therakos: Consultancy, Research Funding, Speakers Bureau. McGuirk: Fresenius Biotech: Research Funding; Gamida Cell: Research Funding; Pluristem Therapeutics: Research Funding; EcoR1 Capital: Consultancy; Novartis: Research Funding; Astelllas Pharma: Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Bellicum Pharmaceuticals: Research Funding; Allovir: Consultancy, Honoraria, Research Funding.

AFM28, a Novel Bispecific Innate Cell Engager (ICE ®), Designed to Selectively Re-Direct NK Cell Lysis to CD123+ Leukemic Cells in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are common forms of adult hematologic malignancies with incidences of approximately 1-6/100.000/year. Progress in the management of AML and high-risk MDS has lagged behind other hematological malignancies, and, in particular, treatment for relapsed or refractory (R/R) disease and minimal residual disease (MRD) are needed to induce long-term remission.Here we describe the preclinical development of AFM28, a novel bispecific Innate Cell Engager (ICE ®) developed using the Redirected Optimized Cell Killing (ROCK ®) platform, designed to specifically activate NK cells and induce depletion of leukemic blasts and LSCs. Natural killer (NK) cell-based innate immunotherapy is emerging as a promising treatment option in AML and MDS based on the demonstrated susceptibility of leukemic blasts for NK cell killing and clinical activity of allogeneic NK cell therapy in R/R disease. Depletion of leukemic stem cells (LSCs) alongside leukemic blasts is considered a critical goal of treatment in order to eradicate MRD and prevent relapse. Accordingly, treatments that effectively target both cell types hold the potential to induce long-term remission in patients with AML or MDS.AFM28 binds the surface antigen CD123, which is almost universally expressed on blasts and LSCs in AML and MDS, and selectively targets CD16A expressed on NK cells, with no detectable binding to CD16B expressed on neutrophils. Through high affinity, bivalent binding to CD16A in a region distinct from the Fc binding site of IgGs, AFM28 remains stably bound to NK cells for extended periods and potently induces antibody-dependent cell-mediated cytotoxicity (ADCC) towards CD123+ AML and MDS cells. When compared with conventional anti-CD123 antibodies, including Fc-enhanced IgG1, AFM28 mediates more potent lysis of tumor cells even with low levels of CD123 expression. Furthermore, in contrast to conventional monoclonal CD123 antibodies, AFM28-induced lysis is not affected by the addition of competing IgG, which otherwise could impact efficacy. Similarly, ex vivo analysis of NK cell activation and inflammatory cytokine release in the ID.Flow human whole-blood loop system demonstrates that AFM28, compared with Fc-enhanced IgG1, produces 100-fold more potent NK cell activation associated with dose-dependent release of pro-inflammatory IL-6, TNFα, and IFNγ, concomitant with depletion of CD123+ plasmacytoid dendritic cells (pDCs) and basophils. In contrast, cytokine release was substantially lower than observed with a CD123-directed T cell-engaging bispecific antibody suggesting a low risk of cytokine release syndrome with AFM28. In vivo, AFM28 demonstrates a monoclonal antibody-like pharmacokinetic profile in mice and cynomolgus monkeys. A pre-clinical toxicology model in cynomolgus monkey suggested that AFM28 is safe and well tolerated and demonstrated pharmacodynamic activity.In summary, these data demonstrate AFM28 could be a novel treatment modality for AML and MDS that has good tolerability and the potential to effectively target MRD by depleting leukemic blasts and LSCs. AFM28 is currently being prepared for first-in-human clinical investigation. High affinity binding, potent induction of NK cell activation, and extended cell surface retention suggest AFM28 endows NK cells with CAR-like properties and may hold particular promise when combined with allogeneic NK cell therapy, for example within a pre-complexed NK cell product. DisclosuresGötz: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schmitt: Affimed GmbH: Research Funding. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Haneke: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Kozlowska: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Sarlang: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Knackmuss: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Peters: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ross: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Nowak: AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; Takeda: Honoraria. Hofmann: Affimed GmbH: Research Funding. Merz: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company.

A Novel Stealth Strategy That Activates Adoptively Transferred Allogeneic Immune Cells and Avoids Rejection for Off-the-Shelf Cell-Based Cancer Therapy

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition.To this end, allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, since HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells, and since the ligand for CD47, SIRPα, is known to be expressed on macrophages and dendritic cells and not on human NK cells, the observed effects of these immune-modulating strategies may not translate into the patient-treatment setting. In assessment of their protective effects in a defined human system, we found that HLA-E or CD47 overexpression on class I HLA null human cells offer only partial protection in evading various human NK cell compartments. We found that class I HLA-null K562 cells engineered to over-express CD47 were ineffective in inhibiting NK cells (0 to 7% inhibition). Separately, K562 cells engineered to over-express HLA-E, while effective in inhibiting NKG2A+ NK cells (90.2% +/- 3.7% inhibition), were unable to completely inhibit CD56 dim NK cells (33.2% +/- 29.6% inhibition) and not only failed, but instead activated, NKG2C+ NK cells (167% +/- 69% activation). Our data highlight the limitations of engineered CD47 and HLA-E modalities in suppressing broad populations of NK cells in clinically relevant settings.We therefore evaluated expression of the alloimmune defense receptor (ADR) that uniquely targets alloreactive immune cells (Mo et al. Nat Biotechnol 2021). We have shown that the expression of ADR has the potential to evade host immune cells without the need for further genetic editing such as class I HLA deletion. To determine its applicability for off-the-shelf cell therapy, ADR expression was engineered into induced pluripotent stem cells (iPSCs) and iPSC-derived CAR-NK (CAR-iNK) cells were generated. CAR-iNK cells carrying the ADR modality (ADR+ CAR-iNK cells) showed normal patterns of differentiation (>99% CD56+ with co-expression of NK cell receptors such as NKG2D, NKp30 and NKp46), suggesting that ADR expression did not disrupt hematopoiesis or the expansion of iNK cells. Additionally, ADR+ and ADR-negative CAR-iNK cells produced similar cytotoxicity against tumor cells. We next tested the ability of ADR to provide resistance to alloimmune rejection by coculturing ADR+ CAR-iNK cells with allogeneic pBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, ADR+ CAR-iNK cells maintained durable persistence throughout the entire duration of the MLR assay and suppressed the expansion of alloreactive T- and NK-cells in comparison to the control arm (Figure 1).Collectively, initial preclinical studies suggest that ADR-modified CAR-iNK cells resist host immune cell rejection, while eliciting a durable anti-tumor response. Our preliminary data show evidence toward a promising off-the-shelf solution for elimination of broad pools of alloreactive T- and NK- effector cells in the clinical setting without the need for lympho-depleting conditioning or genetic editing strategies. [Display omitted] DisclosuresWilliams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa.Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy.Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane.The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%).The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. DisclosuresPatel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

Multicenter, Open-Label, Phase 3 Study of Tabelecleucel for Solid Organ or Allogeneic Hematopoietic Cell Transplant Recipients with Epstein-Barr Virus-Driven Post Transplant Lymphoproliferative Disease after Failure of Rituximab or Rituximab and Chemotherapy (ALLELE)

Multicenter, Open-Label, Phase 3 Study of Tabelecleucel for Solid Organ or Allogeneic Hematopoietic Cell Transplant Recipients with Epstein-Barr Virus-Driven Post Transplant Lymphoproliferative Disease after Failure of Rituximab or Rituximab and Chemotherapy (ALLELE)

Characterization of WU-CART-007, an Allogeneic CD7-Targeted CAR-T Cell Therapy for T-Cell Malignancies

BackgroundT-cell Acute Lymphoblastic Leukemia (T-ALL) / Lymphoblastic Lymphoma (LBL) represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Development of CAR-T cell therapies for T-cell cancers has been complicated by induction of fratricide and the high risk of malignant cell contamination of the drug product in the autologous setting. Previously, Cooper et. al., demonstrated that CRISPR/Cas9 gene-editing to delete CD7 prevented self-killing, and deletion of the T-cell receptor alpha constant (TRAC) enabled the use of healthy donor-derived T-cells to manufacture CD7-targeted CAR-T cells without risk of malignancy and mitigating the risk of GvHD. Here we present preclinical data supporting the safety and efficacy of WU-CART-007, an IND ready, off-the-shelf, and fratricide resistant CD7-targeted CAR-T cell for the treatment of CD7+ T-cell malignancies.MethodsWU-CART-007 was manufactured using T cells isolated from healthy donors by deletion of CD7 and TRAC, followed by CAR transduction, cell expansion, depletion of residual TCRa/b+ cells and cryopreservation. Donors were confirmed negative for a panel of adventitious viruses. CD7/TRAC deletion and CAR transduction were confirmed by flow cytometry. Off-target editing profile was assessed by GUIDE-Seq. The binding kinetics to human CD7 were conducted by bio-layer interferometry and CD7 selectivity was confirmed by cell microarray with a library of HEK-293 cells expressing approximately 6000 human proteins. The in vitro activity of WU-CART-007 was interrogated by co-culture with human CD7+ CCRF-CEM T-ALL cells and the potential on-target, off-tumor activity was assessed by co-culture with a panel of immune and non-immune primary human cells. In vivo anti-tumor functionality was confirmed in immunocompromised NSG mice after the establishment of low or high tumor burden CCRF-CEM xenografts engineered to express green fluorescent protein (GFP) and click beetle red (CBR) luciferase. The impact of WU-CART-007 on normal hematopoiesis was assessed using CD34+ humanized NCG mice.ResultsSeveral successful full-scale manufacturing runs were completed with consistently high dual CD7/TRAC deletion, transduction efficiency, and cell viability. Drug product was primarily composed of a T cell memory phenotype. Off- target nuclease analysis by GUIDE-seq and targeted NGS confirmed no evidence of off-target editing events. The WU-CART-007 scFv exhibited high affinity and exquisite specificity for human CD7. In vitro co-incubation experiments confirmed strong cytotoxicity against CD7-expressing cells including CCRF-CEM T-ALL cells, primary T and NK cells, but not CD7- cells such as myeloid cells, B cells, hepatocytes, astrocytes, cardiomyocytes, epithelial cells, and endothelial cells. Importantly, no cytotoxicity was observed against hematopoietic progenitor cells in human bone marrow or cord blood following co-incubation with WU-CART-007. Similarly, WU-CART-007 treatment of a non-tumor bearing humanized mouse model resulted in transient reductions in CD7+ cells (T-cells and NK cells) but not CD7- cells (myeloid and B cells), and the impacted cells recovered after circulating WU-CART-007 cells were no longer detectable. Assessment of in vivo anti-tumor efficacy revealed that WU-CART-007 effectively inhibited tumor progression (>99% TGI) in both low and high burden CCRF-CEM tumor models and improved survival in a dose-dependent manner, while CAR- cells were inactive, confirming CD7-dependent activity.ConclusionsThese preclinical studies support the use of WU-CART-007 in clinical trials and highlight the potential of WU-CART-007 to be a well-tolerated and active therapy for patients with CD7+ T-cell malignancies. A first in human Phase 1/2 trial in patients with R/R T-ALL/LBL is currently open for enrollment (NCT# 04984356). DisclosuresNo relevant conflicts of interest to declare.