Allogeneic Setting Research Articles

Towards the standardization of methods of tissue processing for the isolation of mesenchymal stromal cells for clinical use

Multipotent mesenchymal stromal cells (MSCs) are currently the most extensively studied type of adult stem cells in advanced stages of development in the field of regenerative medicine. The biological properties of MSCs have generated great hope for their therapeutic use in degenerative and autoimmune conditions that, at present, lack effective treatment options. Over the last decades, MSCs have been typically obtained from adult bone marrow, but the extraction process is highly invasive and the quality and numbers of isolated cells is drastically influenced by patient age, medication and associated comorbidities. Therefore, there is currently an open discussion on the convenience of allogeneic over autologous treatments, despite potential disadvantages such as rejection by the host. This shift to the allogeneic setting entails the need for high production of MSCs to ensure availability of sufficient cell numbers for transplantation, and therefore making the search for alternative tissue sources of highly proliferative MSC cultures with low levels of senescence occurrence, which is one of the greatest current challenges in the scale up of therapeutic cell bioprocessing. Herein we (i) present the main isolation protocols of MSCs from bone marrow, adipose tissue and Wharton’s jelly of the umbilical cord; and (ii) compare their qualities from a bioprocess standpoint, addressing both quality and regulatory aspects, in view of their anticipated clinical use.

Role of ex vivo Expanded Mesenchymal Stromal Cells in Determining Hematopoietic Stem Cell Transplantation Outcome

Overall, the human organism requires the production of ∼1 trillion new blood cells per day. Such goal is achieved via hematopoiesis occurring within the bone marrow (BM) under the tight regulation of hematopoietic stem and progenitor cell (HSPC) homeostasis made by the BM microenvironment. The BM niche is defined by the close interactions of HSPCs and non-hematopoietic cells of different origin, which control the maintenance of HSPCs and orchestrate hematopoiesis in response to the body’s requirements. The activity of the BM niche is regulated by specific signaling pathways in physiological conditions and in case of stress, including the one induced by the HSPC transplantation (HSCT) procedures. HSCT is the curative option for several hematological and non-hematological diseases, despite being associated with early and late complications, mainly due to a low level of HSPC engraftment, impaired hematopoietic recovery, immune-mediated graft rejection, and graft-versus-host disease (GvHD) in case of allogenic transplant. Mesenchymal stromal cells (MSCs) are key elements of the BM niche, regulating HSPC homeostasis by direct contact and secreting several paracrine factors. In this review, we will explore the several mechanisms through which MSCs impact on the supportive activity of the BM niche and regulate HSPC homeostasis. We will further discuss how the growing understanding of such mechanisms have impacted, under a clinical point of view, on the transplantation field. In more recent years, these results have instructed the design of clinical trials to ameliorate the outcome of HSCT, especially in the allogenic setting, and when low doses of HSPCs were available for transplantation.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Rapid generation of monocyte-derived antigen-presenting cells with dendritic cell-like properties.

One of the major challenges in cellular therapy is the establishment and validation of simple and fast production protocols meeting good manufacturing practice (GMP) requirements. Dendritic cells (DCs) are of particular therapeutic interest, due to their critical role in T cell response initiation and regulation. Conventional wisdom states that DC generation from monocytes is a time-consuming protocol, taking up to 7-9 days. This study systematically screened and validated numerous culture components and conditions to identify the minimal requirements, which can give rise to functional monocyte-derived antigen-presenting cells (MoAPCs) in less than 48 h (36 h MoAPC). A total of 36 h MoAPCs were evaluated in terms of surface marker expression, endocytic capability, and induction of antigen-specific T cell expansion via flow cytometry. Screening of media compositions, glucose concentrations, and surface marker kinetics, particularly DC-SIGN as a DC-specific marker, allowed the generation of DC-like APCs in 36 h (36 h MoAPCs). A total of 36 h MoAPCs displayed a similar phenotype to 48 h MoAPC and standard 7 d MoDCs in terms of HLA-DP,DQ,DR, CD83, and DC-SIGN expression, while CD1a was preferentially expressed in standard MoDCs. Functional evaluation revealed that 36 h MoAPCs displayed reduced endocytosis capabilities and IL-12p70 production. However, 36 h MoAPCs were able to induce T cell expansion both in an allogenic and antigen-specific setting. Our results indicate that mature 36 h MoAPCs possess DC-like capabilities by inducing antigen-specific T cell responses. This study has important implications for the generation of DC-based cellular therapies, allowing a more cost and time-efficient generation of APCs.

Human NK Cells in Autologous Hematopoietic Stem Cell Transplantation for Cancer Treatment

Simple SummaryNatural killer (NK) cells are key elements of the innate immune system that have the ability to kill transformed (tumor and virus-infected) cells without prior sensitization. Hematopoietic stem cell transplantation (HSCT) is a medical procedure used in the treatment of a variety of cancers. The early reconstitution of NK cells after HSCT and their functions support the therapeutic potential of these cells in allogenic HSCT. However, the role of NK cells in autologous HSCT is less clear. In this review, we have summarized general aspects of NK cell biology. In addition, we have also reviewed factors that affect autologous HSCT outcome, with particular attention to the role played by NK cells.Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes with the ability to recognize and kill malignant cells without prior sensitization, and therefore, they have a relevant role in tumor immunosurveillance. NK cells constitute the main lymphocyte subset in peripheral blood in the first week after hematopoietic stem cell transplantation (HSCT). Although the role that NK cells play in allogenic HSCT settings has been documented for years, their significance and beneficial effects associated with the outcome after autologous HSCT are less recognized. In this review, we have summarized fundamental aspects of NK cell biology, such as, NK cell subset diversity, their effector functions, and differentiation. Moreover, we have reviewed the factors that affect autologous HSCT outcome, with particular attention to the role played by NK cells and their receptor repertoire in this regard.

Is Adoptive Cellular Therapy With Non-T-Cell Immune Effectors the Future?

Success from checkpoint blockade and adoptive cell therapy has brought a new hope in cancer immunotherapy. Adoptive cell therapy involves the isolation of immune cells, ex vivo activation and/or expansion, and reinfusion into the patients, and their effect can be dramatically increased by the incorporation of chimeric antigen receptors specific to molecules expressed on tumor cells. Chimeric antigen receptor T cells have shown exciting results in the treatment of liquid malignancies; nevertheless, they suffer from limitations including severe adverse effects such as cytokine release syndrome and neurotoxicity seen in patients as well as a potential for causing graft-versus-host disease in an allogeneic setting. It is thus imperial to explore innate immune cells including natural killer cells, macrophages, natural killer T cells, and γδ T cells. Here, we provide a broad overview of the major innate immune cells and their potential for adoptive cell therapy and chimeric antigen receptor engineering.

Epcoritamab induces potent anti-tumor activity against malignant B-cells from patients with DLBCL, FL and MCL, irrespective of prior CD20 monoclonal antibody treatment

Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.

203 - ATA3219: A Potent Next-Generation Allogeneic Off-the-Shelf CD19 CAR-T Therapy without the Need for Gene Editing

INTRODUCTION The development of allogeneic CD19 chimeric antigen receptor (CAR) T cells from healthy donors is a significant focus in cell therapy and is anticipated to overcome the technical and logistical challenges associated with autologous CAR-T cells. Unlike gene-edited approaches, which require inactivation of the endogenous αβ T cell receptor to reduce the risk of Graft-versus-Host Disease (GvHD), allogeneic Epstein-Barr Virus (EBV)-targeted T cells represent a clinically-advanced treatment modality that, to-date, has demonstrated a favorable safety profile with limited risks of GvHD or cytokine release syndrome [Prockop et al. JCI, 2020; Prockop et al. Blood, 2019] . As an allogeneic CAR T cell platform, EBV T cells represent a unique composition that retains critical transducibility and function, and minimizes risks for GvHD and other host interactions, without requiring complex gene editing or other cell engineering approaches to facilitate use in the allogeneic setting. Recent clinical experience with allogeneic CD19 CAR-modified EBV T cells have further supported safe and effective clinical experience in the context of B cell malignancies [Curran KJ et al. TCT 2020]. Recent advances in next-generation stimulatory domains also represent potential for improvement on current CAR-T therapies. Specifically, a modified CD3ζ domain retaining signaling capacity in 1 of 3 immune-receptor-tyrosine-based-activation-motif (ITAM) regions (referred to as 1XX) is designed to extend functional persistence without compromising potency via calibration of antigen induced CAR signaling intensity to more physiologic levels [Feucht et al. Science Trans Med 2018]. Here, we describe the first preclinical evaluation of ATA3219, a next-generation allogeneic CD19 CAR T cell therapy, combining a non-edited allogeneic EBV T cell approach with a CAR signaling domain designed to improve upon the currently clinically validated CD19 targeted CAR therapies. METHODS and RESULTS We generated EBV T cells engineered with a CD19-targeted CAR containing a modified CD3ζ signaling domain, 1XX (CD19-1XX CAR+ EBV T cells). CD19-1XX CAR+ EBV T cells demonstrate high CAR expression, polyfunctionality, expansion and in vitro potency through HLA-independent killing of CD19+ targets. Furthermore, CD19-1XX CAR+ EBV T cells demonstrate highly potent antitumor activity in an established disseminated tumor model of acute lymphoblastic leukemia and is associated with long-term persistence of the product. No treatment-related toxicities were observed in this animal model. CONCLUSIONS This preclinical dataset for CD19-1XX CAR+ EBV T cells demonstrate, persistence, polyfunctional phenotype and efficient targeting of CD19-expressing tumor cells, both in vitro and in vivo, with limited allocytoxicity against antigen-negative, HLA-mismatched targets. These findings support advancing ATA3219 to clinical evaluation. Disclosures Pham: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Spindler: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Hwang: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Brito: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Bulliard: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Aftab: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

Engineering a Potent Cancer Vaccine Using Immunoproteasome-Expressing Mesenchymal Stromal Cells

Dendritic cells (DCs) excel at cross-presenting antigens, but their effectiveness as cancer vaccine is limited. Here, we describe an innovative vaccine approach using mesenchymal stromal cells (MSCs) engineered to express the immunoproteasome (IPr) complex (MSC-IPr). Such modification instilled efficient antigen cross-presenting abilities associated with enhanced major histocompatibility complex (MHC)I and CD80 expression, de novo expression of interleukin-12 along with higher chemokine secretion. In addition, the cross-presentation capacity of MSC-IPr is highly dependent on AMPK signaling, oxidative phosphorylation and production of reactive oxygen species. Compared to DCs, MSC-IPr hold the ability to cross-present a vastly different epitope repertoire, which translates into potent re-activation of T-cell immunity against EL4 lymphoma and B16 melanoma tumors. Moreover, therapeutic vaccination of animals with pre-established tumors efficiently controls cancer growth, an effect further enhanced when combined with antibodies targeting PD1, CTLA4, LAG3 or 4-1BB under both autologous and allogeneic settings. MSC-IPr constitute therefore a novel subset of non-hematopoietic antigen-presenting cells suitable for the future design of universal cell-based cancer vaccines.

Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

LIR-1 educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

ObjectiveKIR and NKG2A receptors educate human NK cells to stay responsive to cells with diminished HLA class I. Here, we addressed whether the HLA class I‐binding receptor LIR‐1 (LILRB1/ILT2/CD85j), which is widely expressed on human NK cells, can mediate education and contribute to antitumor functions of NK cells.MethodsHealthy donor NK cells either unstimulated, overnight cytokine‐activated or ex vivo‐expanded were used to target human cell lines. Phenotype and function were analysed using flow cytometry and 51Cr‐release assays.ResultsWe found that the inhibitory receptor LIR‐1 can mediate NK cell education under specific conditions. This novel finding was exclusive to expanded NK cells and further characterisation of the cells revealed high expression of granzyme B and DNAM‐1, which both previously have been linked to NK cell education. Corroborating the rheostat education model, LIR‐1 co‐expression with an educating KIR further increased the responsiveness of expanded NK cells. Inversely, antibody masking of LIR‐1 decreased the responsiveness. LIR‐1+ expanded NK cells displayed high intrinsic ADCC that, in contrast to KIR and NKG2A, was not inhibited by HLA class I.ConclusionThese findings identify a unique NK cell subset attractive for adoptive cell therapy to treat cancer. Given that LIR‐1 binds most HLA class I molecules, this subset may be explored in both autologous and allogeneic settings to innately reject HLA class I‐ tumor cells as well as HLA class I+ target cells when combined with antitumor antibodies. Further studies are warranted to address the potential of this subset in vivo.

Use of Sysmex Hematology Analyzer based Hematopoietic Progenitor Cell (XN-HPC) Count in Allogeneic Peripheral Blood Stem Cell Transplant Setting; Is It Substantially Reliable?

Allogeneic stem cell transplantation remains the only curative option for various benign and malignant hematological disorders. The procedure of allogeneic stem cell transplantation entails the administration of adequate doses of hematopoietic progenitor cells, aiming for complete and sustained hematopoietic reconstitution. Hematopoietic progenitors are obtained either through a bone marrow harvest or from GCSF-mobilized peripheral blood of the identified donor. Over the years various modalities have been utilized to establish the adequacy of harvested stem cell product e.g. Colony-forming unit– granulocyte monocyte (CFU-GM) assay, total mononuclear cell count (MNC), and CD34+ cell count

Pre-Transplant Marital Status and Hematopoietic Cell Transplantation Outcomes

Evidence about the impact of marital status before hematopoietic cell transplantation (hct) on outcomes after hct is conflicting. We identified patients 40 years of age and older within the Center for International Blood and Marrow Transplant Research registry who underwent hct between January 2008 and December 2015. Marital status before hct was declared as one of: married or living with a partner, single (never married), separated or divorced, and widowed. We performed a multivariable analysis to determine the association of marital status with outcomes after hct. We identified 10,226 allogeneic and 5714 autologous hct cases with, respectively, a median follow-up of 37 months (range: 1-102 months) and 40 months (range: 1-106 months). No association between marital status and overall survival was observed in either the allogeneic (p = 0.58) or autologous (p = 0.17) setting. However, marital status was associated with grades 2-4 acute graft-versus-host disease (gvhd), p < 0.001, and chronic gvhd, p = 0.04. The risk of grades 2-4 acute gvhd was increased in separated compared with married patients [hazard ratio (hr): 1.13; 95% confidence interval (ci): 1.03 to 1.24], and single patients had a reduced risk of grades 2-4 acute gvhd (hr: 0.87; 95% ci: 0.77 to 0.98). The risk of chronic gvhd was lower in widowed compared with married patients (hr: 0.82; 95% ci: 0.67 to 0.99). Overall survival after hct is not influenced by marital status, but associations were evident between marital status and grades 2-4 acute and chronic gvhd. To better appreciate the effects of marital status and social support, future research should consider using validated scales to measure social support and patient and caregiver reports of caregiver commitment, and to assess health-related quality of life together with health care utilization.

Generation of Multiplexed Engineered, Off-the-Shelf CAR T Cells Uniformly Carrying Multiple Anti-Tumor Modalities to Prevent Tumor Relapse

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (&amp;gt;100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P&amp;lt;0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P&amp;lt;0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

5-Azacytidine Depletes Hematopoietic Stem Cells and Synergizes with an Anti-CD117 Antibody to Augment Engraftment of Donor Stem Cells in Immunocompetent Mice

Monoclonal antibody (mAb)-targeting of CD117 (c-Kit) present on hematopoietic stem cells (HSC) is an emerging conditioning strategy for hematopoietic cell transplantation (HCT) to replace standard-of-care alkylator and radiation therapy used to deplete recipient HSC and permit donor HSC engraftment. In preclinical and clinical studies anti-CD117 mAbs, which inhibit stem cell factor (SCF) from binding to CD117, have shown single agent efficacy in permitting donor HSC engraftment in immunocompromised recipients. However, preclinical data suggest that anti-CD117 mAb, ACK2, used as a single agent is insufficient to enable HSC engraftment in immunocompetent mice. Hence, there is a desire to potentiate the effect of naked anti-CD117 mAbs to permit targeted elimination of host HSC for a range of immune competent bone marrow (BM) states, from monogenic disorders, e.g., hemoglobinopathies, to BM clonal malignant diseases. Here, we show that ACK2 synergizes with the widely used hypomethylating agent, 5-Azacytidine (AZA), to deplete HSC and enable robust long-term HSC engraftment in immunocompetent mice. The cell depleting effect of AZA or ACK2+AZA was first tested on mature and primitive hematopoietic cells of C57BL/6 (B6) mice. Mice received AZA at 5mg/kg i.p. for 5 days either alone or combined with ACK2 at 500 µg i.v. 5 days prior to AZA. BM and spleen were harvested days 6, 10 and 20 after the first AZA dose and analyzed by extended immunophenotype. For HCT studies both congenic [B6 (H-2b, Thy1.1, CD45.1) into B6 recipients (H-2b, Thy1.1, CD45.1/CD45.2)] and allogeneic [B6 (H-2b, Thy1.1, CD45.1) into BALB.B recipients (H-2b, Thy1.2, CD45.2)] transplantation mouse models were tested. Single agent AZA induced rapid depletion of CD117+ cells in the BM, including LT-HSC (Lin-Sca+Kit+[LSK]CD150+CD48-) and ST-HSC (LSKCD150-CD48-). In vivo HSC depletion by AZA has not been previously reported, and was surprising given that most HSC are quiescent, and non-proliferating cells are known to be insensitive to the toxicities of AZA. Examination of HSC populations in the spleen did not suggest that mobilization of LT- or ST-HSC to secondary organs caused the decrease in BM. Rather, AZA-induced myelosuppression was followed by rapid expansion of myeloid-biased MPP and increased HSC proliferation as assessed by Ki67 intracellular protein expression. We hypothesized, that the in vivo HSC-depletive effects of AZA might result from increased sensitivity of proliferating HSC to AZA. Hence, the in vitro effects of different AZA concentrations were tested on proliferating HSC. In both mouse and human HSC cultures, supplemented with SCF and TPO, we observed that AZA reduced the number of cells in a dose dependent manner and decreased cell viability at concentrations of ≥ 0.5 µg/ml. In vivo, combined ACK2+AZA led to more robust HSC depletion and prolonged time to HSC recovery compared to AZA alone. We then evaluated if blockade of CD117 with its ligand SCF by ACK2 was the cause of this prolonged HSC-depletion. 2B8 mAb, which also binds mouse CD117, but only partially inhibits CD117/SCF signaling, did not result in augmented in vivo HSC depletion (Figure 1A). We further tested if immunocompetent mice treated with ACK2+AZA could be "rescued" with exogenous recombinant SCF. SCF-treatment resulted in faster recovery of LT-HSC. Together these studies suggest that the synergy of ACK2+AZA on the depth and duration of HSC-depletion depends of the disruption of CD117/SCF binding. Finally, we tested ACK2+AZA conditioning on donor stem cell engraftment in immunocompetent mice. Transplantation of 15-20x106 WBM cells following ACK2+AZA conditioning led to significantly increased stable mean multilineage donor chimerism of &amp;gt;60% in the congenic (Figure 1B) and &amp;gt;20% in the allogeneic setting. Single agent AZA enabled sustained donor engraftment with levels of myeloid donor chimerism of 10-15%, serving as proof of principle that AZA treatment can indeed clear HSC niches and permit engraftment of true HSC. Similar to WBM grafts, 5x104 purified HSC (LSK) engrafted successfully in the ACK2+AZA group, reaching long-term (26 weeks) mean donor myeloid chimerism &amp;gt;25% in both congenic and allogeneic settings. Our studies reveal a previously unknown effect of AZA on BM HSC and provide a platform for the clinical use of a novel less toxic non-myeloablative conditioning strategy for HCT, which is aimed at the rapid translation into clinical practice. Disclosures Pang: Jasper Therapeutics, Inc: Current Employment, Current equity holder in private company. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

ATA3219: A Potent Next-Generation Allogeneic Off-the-Shelf CD19-CAR T Therapy without the Need for Gene-Editing

INTRODUCTION The development of allogeneic CD19 chimeric antigen receptor (CAR) T cells from healthy donors is a significant focus in cell therapy and is anticipated to overcome the technical and logistical challenges associated with autologous CAR-T cells. Unlike gene-edited approaches, which require inactivation of the endogenous αβ T cell receptor to reduce the risk of Graft-versus-Host Disease (GvHD), allogeneic Epstein-Barr Virus (EBV)-targeted T cells represent a clinically-advanced treatment modality that, to-date, has demonstrated a favorable safety profile with limited risks of GvHD or cytokine release syndrome [Prockop et al. JCI, 2020; Prockop et al. Blood, 2019] . As an allogeneic CAR T cell platform, EBV T cells represent a unique composition that retains critical transducibility and function, and minimizes risks for GvHD and other host interactions, without requiring complex gene editing or other cell engineering approaches to facilitate use in the allogeneic setting. Recent clinical experience with allogeneic CD19 CAR-modified EBV T cells have further supported safe and effective clinical experience in the context of B cell malignancies [Curran KJ et al. TCT 2020]. Recent advances in next-generation stimulatory domains also represent potential for improvement on current CAR-T therapies. Specifically, a modified CD3ζ domain retaining signaling capacity in 1 of 3 immune-receptor-tyrosine-based-activation-motif (ITAM) regions (referred to as 1XX) is designed to extend functional persistence without compromising potency via calibration of antigen induced CAR signaling intensity to more physiologic levels [Feucht et al. Science Trans Med 2018]. Here, we describe the first preclinical evaluation of ATA3219, a next-generation allogeneic CD19 CAR T cell therapy, combining a non-edited allogeneic EBV T cell approach with a CAR signaling domain designed to improve upon the currently clinically validated CD19 targeted CAR therapies. METHODS and RESULTS We generated EBV T cells engineered with a CD19-targeted CAR containing a modified CD3ζ signaling domain, 1XX (CD19-1XX CAR+ EBV T cells). CD19-1XX CAR+ EBV T cells demonstrate high CAR expression, polyfunctionality, expansion and in vitro potency through HLA-independent killing of CD19+ targets. Furthermore, CD19-1XX CAR+ EBV T cells demonstrate highly potent antitumor activity in an established disseminated tumor model of acute lymphoblastic leukemia and is associated with long-term persistence of the product. No treatment-related toxicities were observed in this animal model. CONCLUSIONS This preclinical dataset for CD19-1XX CAR+ EBV T cells demonstrate, persistence, polyfunctional phenotype and efficient targeting of CD19-expressing tumor cells, both in vitro and in vivo, with limited allocytoxicity against antigen-negative, HLA-mismatched targets. These findings support advancing ATA3219 to clinical evaluation. Disclosures Pham: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Spindler:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Hwang:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Brito:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Bulliard:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Aftab:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

Universal: An Allogeneic First-in-Human Study of the Anti-Bcma ALLO-715 and the Anti-CD52 ALLO-647 in Relapsed/Refractory Multiple Myeloma

Universal: An Allogeneic First-in-Human Study of the Anti-Bcma ALLO-715 and the Anti-CD52 ALLO-647 in Relapsed/Refractory Multiple Myeloma

IN-DEPTH ASSESSMENT OF ISCHEMIA/REPERFUSION INJURY IN A MURINE MODEL OF HINDLIMB TRANSPLANTATION

Background: Static cold storage (SCS) still represents the gold standard for organ and tissue preservation in transplantation. Ischemia/reperfusion injury that occurs due to the lack of oxygen and nutrient supply and subsequent generation of oxygen radicals contributes crucially to the damage occurring during this time period. In this study, we investigated tissue damage at different durations of SCS in a sophisticated murine VCA model. Methods: 6 to 10-week-old mice were used for syngeneic (C57BL/6àC57BL/6) and allogeneic (Balb/càC57BL/6) orthotopic hindlimb transplants. Hindlimb grafts were subjected to 6, 12, or 24h of SCS. In the allogeneic setting, animals received rapamycin 1mg/kg (i.p.) after recovery from surgery. Biopsies of the different components of the VCA graft were taken 24h after reperfusion and further analyzed by histology, qRT-PCR and ELISA. Results: All animals tolerated up to 24h of SCS without increased mortality. Histologic analysis revealed that the amount of cellular infiltration did correlate with ischemic time as well as the number of apoptotic cells. Compared to skin, muscle tissue was more susceptible to ischemic injury. In syngeneic animals, expression of IL-1a, IL-1b, MCP-1, CD-28, and FoxP3 in the skin increased with duration of SCS, while a trend toward decrease was seen in allogeneic animals. In muscle tissue, an increase in expression of CXCL-1, CXCL-2, MCP-1, IL-1b, IL-6, and IL-10 was seen for both, the syn- and allogeneic group, that correlated with the SCS duration. Compared to baseline, serum levels of ALT, AST, CK and creatinine were elevated, however, no clear trend toward an increase after prolonged storage was seen. Conclusion: Ischemia/reperfusion injury still poses a major obstacle in transplantation substantially limiting the time between tissue procurement and implantation. The current study demonstrates the susceptibility of muscle tissue and displays different dynamics of RNA expression depending SCS duration and MHC barrier. This study was supported by the Department of Defense (Award Number W81XW1810794).

CAR-NK cells: A promising cellular immunotherapy for cancer.

Natural Killer (NK) cells and CD8+ cytotoxic T cells are two types of immune cells that can kill target cells through similar cytotoxic mechanisms. With the remarkable success of chimeric antigen receptor (CAR)-engineered T (CAR-T) cells for treating haematological malignancies, there is a rapid growing interest in developing CAR-engineered NK (CAR-NK) cells for cancer therapy. Compared to CAR-T cells, CAR-NK cells could offer some significant advantages, including: (1) better safety, such as a lack or minimal cytokine release syndrome and neurotoxicity in autologous setting and graft-versus-host disease in allogenic setting, (2) multiple mechanisms for activating cytotoxic activity, and (3) high feasibility for ‘off-the-shelf’ manufacturing. CAR-NK cells could be engineered to target diverse antigens, enhance proliferation and persistence in vivo, increase infiltration into solid tumours, overcome resistant tumour microenvironment, and ultimately achieve an effective anti-tumour response. In this review, we focus on recent progress in genetic engineering and clinical application of CAR-NK cells, and discuss current challenges and future promise of CAR-NK cells as a novel cellular immunotherapy in cancer.

Abstract 2216: Combinational strategy targeting B cell malignancy using iPSC engineered CAR-NK (FT596) and CAR-T cell (FT819) platforms with therapeutic antibody to achieve an effective deep and durable response

Abstract The use of induced pluripotent stem cells (iPSCs) to derive immune effector cells offers distinct advantages for immune therapy over existing patient- or donor- derived platforms, not only in terms of scalable manufacturing and precision genetic engineering at the clonal level, but also in allowing the generation and combinational use of multiple effector cell types each with distinct characteristics. Taking cues from the natural propagation of innate to adaptive effector responses, here we describe the combined use of multi-engineered iPSC derived Natural Killer (iNK) and T (iT) cell platforms in order to exploit the unique properties of each cell type to achieve both depth and durability of response for hematological malignancies. As innate cells, NK cells are characterized by the capacity for spontaneous reactivity, either in response to cell surface antigen or downregulation of class I MHC, and the rapid kinetic under which NK cells operate make them an ideal candidate to achieve a depth of response that outpaces the limiting effects of cytokine support. FT596 was developed as a dual-targeted iNK cell platform engineered to express both a CD19-directed, NK cell-optimized (NKG2D-2B4-CD3ζ) chimeric antigen receptor (CAR) and a high-affinity, non-cleavable Fc receptor (hnCD16), enabling multi-targeting through combination with therapeutic antibodies. The activity of each receptor is further enhanced by the expression of an IL15-IL15Ra fusion receptor, which also allows the cells to expand in the absence of exogenous cytokine support and prolongs cell survival in vitro and in vivo. T cells are exquisitely specific and undergo rapid clonal expansion and differentiation in response to target antigen, and antigen driven persistence has been demonstrated as a key determinant in efficacy in primary CAR-T cell immune therapy. FT819 is an iT cell platform engineered to express a functionally optimized CD19-CAR (1XX) that has been genome edited into the T cell receptor (TCR) alpha constant (TRAC) locus to provide ideal CAR activity and to prevent TCR expression, thereby avoiding the complications of GVH reactivity in an allogeneic setting. In vivo, both FT596 and FT819 showed stable levels of tumor cell clearance against the CD19+ acute lymphoblastic leukemia cell line NALM6, comparable to that of primary CAR19 T cells (p*&amp;lt;0.0001 for FT596, FT819 or Primary CART vs NALM6 alone). FT596 also shows enhanced clearance of CD19+CD20+ Burkitts lymphoma cell line RAJI when used in combination with rituximab (p=0.0002 vs rituximab alone). Collectively, these studies suggest a compounded anti-tumor effect can be achieved utilizing the inherent properties of engineered CAR-iNK cells together with therapeutic antibody combined with engineered CAR-iT cells which will be highlighted in this presentation. Citation Format: Jode P. Goodridge, John W. Reiser, Ryan Bjordahl, Milli Mandal, Chia-wei Chang, Raedun Clarck, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Frank Cichocki, Hui-ting Hsu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Mamlberg, Bruce Walcheck, Jeffrey S. Miller, Dan Kaufman, Bahram Valamehr. Combinational strategy targeting B cell malignancy using iPSC engineered CAR-NK (FT596) and CAR-T cell (FT819) platforms with therapeutic antibody to achieve an effective deep and durable response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2216.