scholarly journals Cryopreserved CAR-like NK Cells Pre-Complexed with the CD30/CD16A Bispecific Innate Cell Engager (ICE®) AFM13 for the Treatment of CD30 + Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3992-3992
Author(s):  
Uwe Reusch ◽  
Kristina Ellwanger ◽  
Ivica Fucek ◽  
Thomas Müller ◽  
Ute Schniegler-Mattox ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Tumor Cells ◽  
Stock Options ◽  
Nk Cell ◽  
Safety And Efficacy ◽  
Cell Product ◽  
High Affinity ◽  
Thaw Cycle ◽  
Privately Held

Abstract AFM13 is a bispecific tetravalent innate cell engager (ICE®) with two binding sites for CD16A and CD30 on immune cells and tumor cells, respectively. AFM13 is designed to potently activate anti-tumoral responses of natural killer (NK) cells, through antibody-dependent cellular cytotoxicity (ADCC) and macrophages via antibody-dependent cellular phagocytosis (ADCP) towards CD30 + tumor cells. It is widely known that the number of NK cells and their anti-tumor activity are often compromised in patients with cancer, including CD30 + malignancies such as Hodgkin lymphoma (HL) (Reiners, K.S. et al., Mol Ther 2013;21:895-903). Accumulating evidence suggests that the selective, high affinity binding of ICE® to CD16A enables stable pre-complexing with NK cells to enhance ADCC. Early clinical studies with AFM13 have demonstrated safety and efficacy, both as mono- and combination therapy with an anti-PD-1 checkpoint inhibitor (pembrolizumab) (Bartlett, N.L. et al., Blood 2020;136(21):2401-2409), in patients with relapsed or refractory (R/R) HL or peripheral T cell lymphoma (PTCL). Currently, AFM13 monotherapy is being evaluated in a registration-directed Phase 2 study to treat patients with PTCL (NCT04101331). Additionally, a Phase 1b/2a study in patients with PTCL (NCT03192202) previously demonstrated that NK cell numbers pre- and post-dosing of AFM13 may positively correlate with anti-tumoral response (Sawas, A. et al., Blood 2020;136(1):25-26). Therefore, the safety and efficacy of freshly prepared cord blood-derived allogeneic NK cells pre-complexed with AFM13, followed by three weekly infusions of AFM13 monotherapy, is currently being evaluated in patients with R/R CD30 + lymphomas in a Phase 1 dose escalation study (NCT04074746). The first four enrolled patients, all with HL, have shown a response rate of 100% without any safety concerns (Rezvani, K. et al., presentation AACR 2021). To further develop this CAR-like NK cell immunotherapeutic towards an off-the-shelf product, we evaluated the feasibility of cryopreservation of pre-complexed NK cells and assessed the biological activity of such cryopreserved and pre-complexed NK cells after thawing. In this study, the cell surface retention, suitability for NK cell preloading, and cytotoxic activity of AFM13 and other CD30-specific antibody formats was assessed. Furthermore, the compatibility of these formats with cryopreservation of the resulting CAR-like NK cell product was investigated. Cell surface retention assays on primary NK cells confirm substantially slower dissociation kinetics of the tetravalent bispecific ICE® compared to corresponding IgG1 or Fc-enhanced IgG1, enabling efficient and durable pre-complexing of NK cells with ICE®. Surface plasmon resonance analyses confirm long retention of ICE® independent of the CD16A polymorphism. More importantly, these assays demonstrate that the high ADCC potency and efficacy of NK cells, pre-complexed with CD16A-specific tetravalent ICE®, is maintained after one freeze-thaw cycle. These data suggest that high-affinity pre-complexing of adoptive NK cells with bispecific, CD16A-selective ICE® could be a novel cryopreserved off-the-shelf NK cell product for the effective depletion of tumor cells without the limitations and potential risks associated with the CAR-NK cell technology. Disclosures Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ellwanger: Affimed: Current Employment, Current holder of stock options in a privately-held company. Fucek: Affimed: Current Employment, Current holder of stock options in a privately-held company. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schniegler-Mattox: Affimed: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed: Current Employment, Current holder of stock options in a privately-held company. Tesar: Affimed: Current Employment, Current holder of stock options in a privately-held company. Koch: Affimed: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals 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

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3344-3344
Author(s):  
Jana-Julia Götz ◽  
Jens Pahl ◽  
Nanni Schmitt ◽  
Thomas Müller ◽  
Torsten Haneke ◽  
...  
Keyword(s):  
Nk Cells ◽  
Stock Options ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Activation ◽  
Nk Cell ◽  
Cytokine Release ◽  
High Affinity ◽  
Privately Held

Abstract 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. Disclosures Götz: 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.

Mimicking An Induced Self Phenotype by Coating Lymphomas with the Nkp30-Ligand B7-H6 Promotes Antitumoral Natural Killer Cell Cytotoxicity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 103-103
Author(s):  
Christian Kellner ◽  
Tina Maurer ◽  
Daniela Hallack ◽  
Roland Repp ◽  
Jan G.J. van de Winkel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Surface Density ◽  
Nk Cell ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

Abstract Abstract 103 Induced self-expression of ligands for stimulatory receptors facilitates natural killer (NK) cell-mediated elimination of stressed cells. Stimulatory receptors include Natural killer group 2 member D (NKG2D) and Nkp30, which control cytotoxic activities of NK cells and are important in immune surveillance against tumors. Specific modulation of NK cell cytotoxicity by selectively increasing the surface density of activating ligands on tumor cells may therefore represent an innovative approach to develop novel treatment strategies. A novel fusion protein was designed to enhance NK cell-based immune responses against B-lineage lymphomas by increasing the cell surface density of the recently identified Nkp30 ligand B7-H6 on tumor cells. The recombinant protein consisted of the ectodomain of B7-H6 and a CD20-directed human single chain fragment variable (scFv) as targeting device. The resulting fully-human protein designated B7-H6:CD20-scFv was eukaryotically expressed and purified by affinity chromatography. B7-H6:CD20-scFv indeed had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 antigen and to the Nkp30 receptor. CD20-positive lymphoma cells opsonized with B7-H6:CD20-scFv alerted human NK cells as indicated by upregulated surface expression levels of the early inducible activation marker CD69. Activation was accompanied by induced CD107a cell surface exposure indicating enhanced NK cell degranulation. In cytotoxicity assays using human NK cells from healthy donors as effector cells, B7-H6:CD20-scFv triggered killing of lymphoma-derived B-cell lines. B7-H6:CD20-scFv was active in a strictly antigen-specific manner as demonstrated by blocking experiments and was not able to mediate killing of cell lines not expressing the CD20 target antigen. B7-H6:CD20-scFv mediated killing of lymphoma cells in a dose-dependent manner starting at nanomolar concentrations. Target cell death induced by B7-H6:CD20-scFv occurred by apoptosis and involved caspase cleavage. Moreover, B7-H6:CD20-scFv induced NK cell-mediated lysis of fresh tumor cells from 8/8 CLL and 5/5 MCL patients with variable CD20 expression levels. In comparison to ULBP2:CD20-scFv, a similarly constructed fusion protein of the NKG2D ligand ULBP2 and a CD20-directed scFv, the B7-H6:CD20-scFv had a lower potency (EC50 values for B7-H6:CD20-scFv and ULBP2:CD20-scFv were 100 and 4 nM, respectively) but nevertheless achieved similar maximum extents of lysis. Interestingly, when B7-H6:CD20-scFv was added together with ULBP2:CD20-scFv to a mixture of NK cells and target cells, synergistic cytotoxic effects were induced. The combined treatment resulted in a higher percentage of NK cells that responded and exposed the degranulation marker CD107a on the cell surface in comparison to samples containing only one of the two agents. As a consequence a significantly higher extent of lysis was achieved. These results strongly indicate a co-operation between Nkp30 and NKG2D signalling which use different downstream signalling pathways. Thus, mimicking an induced self phenotype of tumors by coating lymphomas with B7-H6:CD20-scFv either alone or in combination with molecules triggering NKG2D may provide an innovative strategy to enhance specific anti-tumoral NK cell cytotoxicity. Disclosures: van de Winkel: Genmab: Employment. Parren:Genmab BV: Employment. Peipp:Genmab: Consultancy.

Down-Regulation of Glucocorticoid-Induced TNF Receptor (GITR) Ligand on Human Tumors by Proteolytic Shedding Increases Anti-Tumor Reactivity of NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 926-926
Author(s):  
Katrin M. Baltz ◽  
Matthias Krusch ◽  
Tina Baessler ◽  
Mercedes Kloss ◽  
Ingrid Kumbier ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cellular Proliferation ◽  
Nk Cell ◽  
Soluble Form ◽  
Tnf Receptor ◽  
Ifn Gamma ◽  
Effector Mechanisms

Abstract Members of the TNF superfamily mediate multiple cellular functions, including cellular proliferation, differentiation and cell death. Many members of this protein family are shed from the cell surface as soluble forms, which affects cell-cell interactions by reduction of ligand densities and distally modulates effector cells bearing the respective receptor. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and NFkappaB and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while tumor cells express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell-mediated anti-tumor effector mechanisms like cytotoxicity and IFN-gamma production. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL) detectable in culture supernatants by ELISA. Furthermore, we found elevated levels of sGITRL in sera of patients with various malignancies compared to healthy controls. We demonstrate that the release of GITRL from tumor cells can be blocked by inhibition of metalloproteinases, concomitantly causing accumulation of GITRL on the tumor cell surface as determined by FACS analysis. Upregulated GITRL surface expression further increased inhibition of NK cell anti-tumor effector mechanisms, while, in contrast, presence of sGITRL in cocultures of GITRL-expressing tumor cells and GITR-positive NK cells enhanced NK cell cytotoxicity and IFN-gamma production. Thus, in line with the results obtained with other TNF family members, conversion of membrane bound GITRL to its soluble form is associated with downregulation of its function, potentially due to blocking its cognate receptor. Thus, release of sGITRL substantially influences the interaction of tumor cells with NK cells. In addition, determination of sGITRL levels may be implemented as a diagnostic marker in patients with malignancies. Further prospective studies are currently being conducted addressing the value of GITRL as a tumor marker in different tumor entities.

Natural Killer Cell Death Mediated By NKG2D-Trogocytosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 18-18
Author(s):  
Kyohei Nakamura ◽  
Masafumi Nakayama ◽  
Mitsuko Kawano ◽  
Tomonori Ishii ◽  
Hideo Harigae ◽  
...  
Keyword(s):  
Cell Death ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dependent Manner

Abstract Introduction The activating receptor, NKG2D, plays crucial role in natural killer (NK) cell-mediated effector function. NKG2D is involved not only in host defense against tumor and viral infection, but also in autoimmune diseases by recognizing stress-induced self-ligands (NKG2DLs). However, the negative feedback regulation of NKG2D has not been fully understood. It has been reported that NK cells undergo rapid apoptosis upon interaction with NK-sensitive tumor cells, suggesting that activation-induced NK cell death can be triggered in certain situations. In this study, we aimed to elucidate underlying mechanism of NK cell death, especially focused on NKG2D-NKG2DLs interaction. Methods NK cells were purified from splenocytes of C57BL/6, perforin-/-, and DAP10-/-/12-/- mice, and cultured with rhIL-2 (1000 U/ml) for 5 days. We used these IL-2-activated NK cells as effector cells and three target cell lines: mouse T cell lymphoma RMA cells (RMA), RMA lacking MHCI expression (RMA-S), and RMA stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). CFSE-labeled NK cells were co-cultured with target cells for 2 hours, and stained with anti-NK1.1 mAb propidium iodide (PI). The percentage of PI-positive cells within CFSE+ NK1.1+ population was measured by flowcytometry. The cell surface expression of Rae-1 on NK cells after co-culture with target cells was evaluated by flowcytometry and confocal microscopy. Results NK cells from WT mice rapidly underwent cell death when co-cultured with Rae-1δ, but not with RMA or RMA-S, suggesting that NKG2D-Rae-1 interaction is involved in NK cell death. NK cells from perforin-/-, and DAP10-/-/12-/- mice did not undergo cell death, highlighting the importance of the NKG2D pathway for NK cell death. However, cross-linking of NKG2D receptor failed to induce NK cell death. To understand underlying the mechanism of NK cell death, we evaluated the cell surface expression of NKG2DLs on NK cells after co-culture with tumor cells. We found that cell surface expression of Rae-1 on NK cells was remarkably induced after co-culture with RMA/Rae-1δ, but no with RMA or RMA-S, implying that these Rae-1-positive NK cells may be lysed by NK cells through NKG2D-induced perforin pathway. The cell surface induction of Rae-1 on NK cells was very rapid (within 5min), and it occurred cell-cell contact dependent manner. Interestingly, NK cells from C57/BL6 mice rapidly became BALB/c allele Rae-1γ-positive after co-culture with RMA/Rae-1γ, indicating that NK cells acquire tumor-derived Rae-1. Consistently, acquisition of Rae-1 by NK cells was confirmed by confocal microscopy. Therefore, NK cells rapidly dress tumor-derived Rae-1 after interaction with tumor cells through intercellular membrane transfer, namely trogocytosis. Trogocytosis of Rae-1 was significantly inhibited in NK cells from DAP10-/-/12-/- mice and by chemical inhibitors of PI3K and Syk, indicating that it requires NKG2D-signaling. To confirm whether Rae-1-dressed NK cells can be recognized and lysed by other NK cells, we used sort-purified Rae-1-dressed NK cells as target cells. Rae-1-dressed NK cells were lysed by WT NK cell in an E/T-ratio dependent manner through NKG2D-induced perforin pathway. Furthermore, adoptively transferred Rae-1-dressed NK cells were significantly eliminated in Rag-1-deficient mice, indicating that Rae-1-dressed NK cells are also recognized and lysed in vivo. Conclusion Upon interaction with NKG2DLs-expressing tumor cells NK cells rapidly dress tumor-derived NKG2DLs, and subsequently undergo fratricide. Our results provide novel insights into activation-induced NK cell death via dynamic intercellular communications. Disclosures: No relevant conflicts of interest to declare.

Platelets Impair NK Cell Immunosurveillance Of Metastasizing Tumor Cells By Altering Surface Expression and Shedding Of Ligands For The Activating Immunoreceptor NKG2D

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3488-3488
Author(s):  
Stefanie Raab ◽  
Korbinian Nepomuk Kropp ◽  
Alexander Steinle ◽  
Lothar Kanz ◽  
Hans-Georg Kopp ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Nk Cell ◽  
Surface Expression ◽  
Class I ◽  
Target Cells ◽  
Missing Self

Abstract NK cells play an important role in the immunosurveillance of tumor cells. The mechanisms leading to NK cell activation are described by the ‘missing-self’ and “induced-self’ hypotheses, implying that cells with low or absent expression of MHC class I and stress-induced expression of ligands for activating receptors like e.g. NKG2D (NKG2DL) are preferentially recognized and eliminated by NK cells. Besides the direct interaction with their target cells, NK activity is further influenced by various other hematopoietic cells. In mouse models, thrombocytopenia impairs metastasis, and this is reversed by additional depletion of NK cells. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is still fragmentary. We recently reported that release of TGF-β by platelets upon their interaction with (metastasizing) tumor cells downmodulates NKG2D on NK cells (Kopp et al., Cancer Res. 2009; Placke et al., J Innate Immun. 2011). Moreover, platelets transfer “healthy” MHC class I to the tumor cell surface. Thus, platelets may facilitate metastasis by interfering with both, “induced and missing self’ NK cell recognition. Here we provide evidence for a yet unknown mechanism by which platelets further impair NKG2D-mediated immunosurveillance. Tumor cells were incubated with platelets from healthy donors resulting in coating of tumor cells and activation of the platelets, or treated with platelet-derived soluble factors (releasate) obtained either by tumor cell-induced platelet activation (TCIPA) or the platelet agonist thrombin. Presence of platelet derived factors derived either from coating of tumor cells or contained in platelet releasate substantially reduced NKG2DL surface expression on tumor cells. This was paralleled by enhanced levels of soluble NKG2DL in culture supernatants, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Diminished NKG2DL surface expression resulted in decreased NKG2D-dependent cytotoxicity of NK cells as revealed by blocking experiments using NKG2D antibody and NKG2DL-specific F(ab)2 fragments targeting the specific modulated NKG2DL. Our data thus identify induction of NKG2DL shedding as novel mechanism by which interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals iPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1692-1692
Author(s):  
Benjamin Goldenson ◽  
Manuel Fierro ◽  
Somayeh Pouyanfard ◽  
Dan S Kaufman
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Checkpoint ◽  
Stock Options ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Anti Tumor Activity ◽  
Acute Myeloid ◽  
Privately Held

Abstract Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML. AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01). To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC. To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, "off-the-shelf" cell therapy approach that can be translated into novel clinical therapies. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

scholarly journals Engineering CD70-Directed CAR-NK Cells for the Treatment of Hematological and Solid Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1691-1691
Author(s):  
Eugene Choi ◽  
Jae-Woong Chang ◽  
Joshua Krueger ◽  
Walker S Lahr ◽  
Emily Pomeroy ◽  
...  
Keyword(s):  
Nk Cells ◽  
Stock Options ◽  
Cell Activation ◽  
Nk Cell ◽  
Expression Cassette ◽  
Publicly Traded ◽  
Cell Therapies ◽  
The Past ◽  
Car T ◽  
Privately Held

Abstract Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen. CD70 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in >70% CAR integration/expression and >80% knockout of CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing. 1 Perna et al. 2017, Cancer Cell. 32:506-519. 2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772. Disclosures Choi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.

scholarly journals 191 GAPDH knock-in of high affinity CD16 in iPSC derived NK cells drives high-level expression and increased anti-tumor function

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A203-A203
Author(s):  
Rithu Pattali ◽  
Kaitlyn Izzo ◽  
Edward Goncz ◽  
Steven Sexton ◽  
Kevin Wasko ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Tumor Spheroid ◽  
Killing Assay ◽  
High Affinity ◽  
Serial Killing ◽  
High Level ◽  
Tumor Cell Killing

BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.

scholarly journals Characterization of Aleta-001, a CAR T Engager Designed to Optimize Responses and Prevent Relapses from CAR-19 Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1713-1713
Author(s):  
Paul Rennert ◽  
Lihe Su ◽  
Lan Wu ◽  
Roy Lobb ◽  
Christine Ambrose
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Tumor Cells ◽  
Stock Options ◽  
Phase 1 ◽  
Complete Response ◽  
Car T ◽  
Cd19 Expression ◽  
Privately Held

Abstract Adoptive T cell therapies directed to the B cell malignancy antigen CD19 (CAR19 T cells) have transformed the care of otherwise refractory, last-line leukemia and lymphoma patients. The overall response rates achieved are very high, routinely above 60%, and a substantial number of responding patients have durable responses that can last many years. However, 50% of responders relapse within 6 months, and outcomes for these relapsed patients are poor. Relapses occur most often in patients whose initial CAR19 T cell expansion is suboptimal, and whose tumor cells reduce or lose expression of the target antigen, CD19. We have created a CAR T Engager protein that is designed to improve responses to anti-CD19 CAR T treatment and is further designed to prevent relapses. This modular protein contains an anti-CD20 llama VHH linked to an optimized CD19 protein and further linked to an anti-albumin llama VHH. Thus, this CAR T Engager, called Aleta-001, binds to CD20 on B cell tumor cells, displays CD19 on the tumor cell surface thereby activating anti-CD19 CAR T cells, and binds to albumin, providing for a long half-life upon injection. Aleta-001 increases CD19 antigen density and/or replaces lost CD19 expression by coating cell surface CD20 with CD19. Here we present studies that establish the efficacious dose to support the upcoming Phase 1/2 clinical trial to be run in collaboration with Cancer Research UK. Extensive in vitro modeling established that the Aleta-001 CAR T Engager specifically bound to CD20-positive/CD19 negative lymphoma cells, JeKo-1-CD19KO, that represent the phenotype of a lymphoma relapsing after losing CD19 expression. In the presence of CAR19 T cells, the Engager protein mediated cytotoxicity against the JeKo-1-CD19KO cells at sub-nM concentrations. The JeKo-1-CD19KO cells induced a rapid and lethal lymphoma when implanted into NSG mice. Administration of the Aleta-001 CAR T Engager and CAR19 T cells eliminated the JeKo-1-CD19KO lymphoma at administered protein concentrations as low as 0.5mg/kg. These results support the clinical development of the Aleta-001 Engager protein. The protein is designed to be administered to patients who have received CAR19 T cell therapy and who fail to achieve a complete response at the time of their first clinical evaluation, or who relapse from a complete response thereafter. Patients who are enrolled will be dosed with the Aleta-001 Engager protein every two weeks. Aleta-001 has entered GMP production and IND enabling studies and will then enter Phase 1 dose escalation clinical studies in the UK. Figure 1 Figure 1. Disclosures Rennert: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Su: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Wu: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company. Lobb: Aleta Biotherapeutics Inc.: Consultancy, Current holder of stock options in a privately-held company. Ambrose: Aleta Biotherapeutics Inc.: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Cytokine-Induced Memory-Like NK Cells with High Reactivity against Acute Leukemia Blasts and Solid Tumor Cells Suitable for Adoptive Immunotherapy Approaches

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1577
Author(s):  
Matteo Tanzi ◽  
Michela Consonni ◽  
Michela Falco ◽  
Federica Ferulli ◽  
Enrica Montini ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Lymphoid Leukemia ◽  
Hematopoietic Stem ◽  
Solid Malignancies

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients’ myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients’ non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.

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