Optimizing NK-92 Serial Killers: Gamma Irradiation, CD95/FasLigation, And NK Or LAK Attack Limit Cytotoxic Efficacy

Background: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. Methods: To evaluate serial killing, we used 51 Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-hours. Results: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in eight hours. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost >50% activity one day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, one day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. Conclusions: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Combination of Three Unique Anti-Tumor Modalities Engineered into iPSC-Derived T Cells Demonstrate a Synergistic Effect in Overcoming Tumor Heterogeneity and Cancer Escape

Chimeric antigen receptor (CAR) is known to trigger an effective immune response through surface antigen recognition enhanced by T-cell activation signal one (ex. CD3) and signal two (ex. CD28); however, targeting neoantigens and intracellular antigens remains a challenge. On the other hand, the T-cell receptor (TCR) can target neo/intracellular antigens presented by MHC molecules, but often the response is not as potent. The CD16 Fc receptor, which is naturally expressed on NK cells, mediates antibody-dependent cellular cytotoxicity (ADCC), but its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16 (tri-modal), to determine whether challenges associated with the treatment of heterogeneous tumors may be overcome. To evaluate the benefit of the three distinct targeting modalities, we tested the functional activity of individual and various combinations of i) anti-CD19 CAR, anti-MICA/B CAR, and anti-BCMA CAR, ii) high-affinity, non-cleavable CD16 (hnCD16), and iii) MR1 and NYESO1 TCR modalities in iT cells. All tested combinations successfully expressed the designated edits and differentiated into iT cells (T-lymphocytes > 95%). Initially, we tested CD19 CAR and MR1 TCR in a 9-day serial killing assay of Nalm6 leukemia cells (CD19 high, MR1 +), where we observed CD19 CAR-iT cells induce prompt CAR-mediated tumor growth inhibition (TGI), saw similar effective killing by MR1 TCR-iT cells but with a 24-hr delay, and observed the most effective response of tumor cell elimination when both where combined in the same iT cell population (relative tumor counts; Day 1, no stim: 2.6, CAR: 0.28, TCR: 1.02, CAR+TCR: 0.02; Day 2, no stim: 5.36, CAR: 0.25, TCR: 0.05, CAR+TCR: 0.02). All conditions (CAR, TCR, and CAR+TCR) reached and maintained complete TGI by Day 9 of the assay (relative tumor count, Day 9, no stim: 50.97, all other stim conditions: <0.01). In line with killing kinetics, the time for the activation marker CD25 upregulation differed between CAR and TCR (peak time and percentage of CD25 +, CAR: Day 1, 49.9%; TCR: Day 5, 74.6%). Co-triggering of CAR and TCR in combination revealed quickest, highest, and sustained CD25 upregulation levels (CD25 +, CAR+TCR Day 1: 62.5%, Days 3 to 9: >90%), indicating a synergistic effect and compatibility between CAR and TCR. Assessing anti-MICA/B CAR and hnCD16, we confirmed the hnCD16-mediated response in iT cells in the presence of anti-MICA/B CAR when crosslinking hnCD16 via biotinylated anti-CD16 antibody with streptavidin (phosphorylated CD3zeta peaked at 10 min upon ADCC triggering), indicating the compatibility between a CAR-iT cell and the hnCD16 motif. Lastly, combining iT cells expressing anti-BCMA CAR + MR1 TCR + hnCD16 with daratumumab (anti-CD38 mAb) in a 9-day serial killing assay demonstrated the best TGI among the groups with a near-elimination of transgenic Nalm6 cells (area under curve, no stim: 30.29, TCR: 1.564, CAR: 0.7087, hnCD16+mAb: 1.452, trimodal+mAb: 0.5824). To assess the function of the tri-modal iT cells in vivo, we used a disseminated xenograft model of B-cell leukemia where a heterogenous mixture of transgenic Nalm6 leukemia cells was used to mimic tumor heterogeneity. Assessment of the bone marrow revealed the unique capacity of each target modality to eliminate its target designated Nalm6 leukemia group, with tri-modal iT cells effectively clearing all populations (Figure 1). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Figure 1 Figure 1. Disclosures Yang: Fate Therapeutics, Inc.: Current Employment. Lin: Fate Therapeutics, Inc.: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Pribadi: Fate Therapeutics, Inc.: Current Employment. Chu: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Mehta: Fate Therapeutics, Inc.: Current Employment. Avramis: Fate Therapeutics, Inc.: Current Employment. Whitlock: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Witty: Fate Therapeutics, Inc.: Current Employment. Peralta: Fate Therapeutics, Inc.: Current Employment. Hosking: Fate Therapeutics: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

Introduction As the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells. Methods NK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use. Results Overall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016). Conclusions Our data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. Figure 1 Figure 1. Disclosures Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

894 The bispecific innate cell engagers AFM13 (CD30/CD16A) and AFM24 (EGFR/CD16A) increase the fraction of tumor target-responsive NK cells and boost serial killing

BackgroundThe use of bispecific natural killer (NK) cell engagers has emerged as a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC). Among these, tetravalent, bispecific innate cell engagers (ICE®) with specificity for the activating receptor CD16A selectively triggering innate responses from NK cells or macrophages represent the most clinically advanced concept. The CD30/CD16A specific ICE® AFM13, has shown efficacy in patients with CD30+ lymphomas as monotherapy1 and combination therapy with check-point inhibitors2 and most recently in combination with adoptive NK cell therapy.3 The EGFR/CD16A specific ICE® AFM24, targeting a variety of solid tumors like colorectal, or lung cancer with a unique mode of action independent of EGFR signaling inhibition, is currently evaluated in an ongoing Ph1/2a clinical study.MethodsWe used a microchip-based screening with single cell resolution4 to elucidate the dynamic responses of individual NK cells towards tumor target cells upon treatment with AFM13 or AFM24.ResultsWe found that AFM13 and AFM24 mediated potent activation of NK cells, leading to increased responsive cytotoxic NK cells and significantly increased the number of NK cells that exerted engagement with multiple target cells rendering these NK cells serial killers. Strikingly, bispecific ICE® molecules triggered stronger cytotoxic responses compared to monoclonal antibodies. One suggested strategy to boost killing by NK cells is to use molecular inhibitors or protein constructs that prevent shedding of CD16.5 However, previous results have shown that this can lead to impaired detachment from target cells, reducing the capacity for an individual NK cell to form serial contacts to target cells.6 We observed that the elevated NK cell killing induced by ICE® molecules was largely conserved when cells were treated with the shedding inhibitor Batimastat. Analysis of the functional dynamics of NK cells revealed that inhibition of CD16 shedding prevented NK cell detachment from target cells, resulting in cell cluster formation. This might strongly impact targeting of distant tumor cells by an individual NK cell thus limiting its anti-tumoral activity.ConclusionsIn conclusion, we show that both AFM13 and AFM24 increase the fraction of tumor-target responsive NK cells and boost serial killing of target cells by individual NK cells. Based on these data, ICE® molecules can be characterized as potent anti-tumoral agents leveraging the enormous potential of NK cells while maintaining crucial features of NK cell biology.AcknowledgementsWe thank members of the Önfelt lab for their valuable help and feedback.ReferencesSawas A, Elgedawe H, Vlad G, Lipschitz M, Chen P-H, Rodig SJ, et al. Clinical and biological evaluation of the novel CD30/CD16A tetravalent bispecific antibody (AFM13) in relapsed or refractory CD30-positive lymphoma with cutaneous presentation: a biomarker phase Ib/IIa study (NCT03192202). Blood 2018;132(Supplement 1):2908–2908.Bartlett NL, Herrera AF, Domingo-Domenech E, Mehta A, Forero-Torres A, Garcia-Sanz R, et al. A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma. Blood 2020. Blood 2020;136(21):2401–2409.Kerbauy LN, Marin ND, Kaplan M, Banerjee PP, Berrien-Elliott MM, Becker-Hapak M, et al. Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated blood and cord blood–derived NK cells facilitates CAR-like responses against CD30 + malignancies. Clin Cancer Res Epub 2021.Guldevall K, Brandt L, Forslund E, Olofsson K, Frisk TW, Olofsson PE, et al. Microchip screening platform for single cell assessment of NK cell cytotoxicity. Front Immunol 2016;7:119.Romee R, Foley B, Lenvik T, Wang Y, Zhang B, Ankarlo D, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 2013;121(18):3599–608.Srpan K, Ambrose A, Karampatzakis A, Saeed M, Cartwright ANR, Guldevall K, et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J Cell Biol 2018;217(9):3267–83.Ethics ApprovalThis work was performed with NK cells from healthy anonymous blood donors, which requires no ethical permit according to local regulations.

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

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.

On serial killers: the collective's search for understanding

I will begin by examining the term serial itself and its importance in the notion of the serial killer. What does the term serial say? It draws on series - a term often used in reference to novels, films and, quite literally, the television series. Series carries notions of multiple segments that are all linked in some way. There is an ongoing nature to the series - when one segment ends, another begins. The series creates anticipation, anxiety of what is to come and a hope for closure. As "serial killer" is a relatively new term it becomes possible to trace its inception and examine what is being revealed in this naming process. I then go to illustrate how this term serial is what sets serial killing apart from other forms of multiple murder (such as mass murder, spree killing, terrorism and assassination). I will explore the features of the serial killer that appear to make it unique to the collective by developing the language of the accounts of understanding the serial killer: the profiling account, the study of numbers, facts and statistics as a way to apprehend and comprehend the serial killer; the logical extension of society account, where the serial killer is a reflection of society from concerns with the individual to celebrity and consumerism; and the serial killer account, where the serial killer explains his own motivations. In looking at these accounts it becomes possible to see the cliches that are used to discuss the serial killer and how these reveal thoughts and fears of the collective, rather than providing the insight on the serial killer that the accounts are seeking.

On serial killers: the collective's search for understanding

I will begin by examining the term serial itself and its importance in the notion of the serial killer. What does the term serial say? It draws on series - a term often used in reference to novels, films and, quite literally, the television series. Series carries notions of multiple segments that are all linked in some way. There is an ongoing nature to the series - when one segment ends, another begins. The series creates anticipation, anxiety of what is to come and a hope for closure. As "serial killer" is a relatively new term it becomes possible to trace its inception and examine what is being revealed in this naming process. I then go to illustrate how this term serial is what sets serial killing apart from other forms of multiple murder (such as mass murder, spree killing, terrorism and assassination). I will explore the features of the serial killer that appear to make it unique to the collective by developing the language of the accounts of understanding the serial killer: the profiling account, the study of numbers, facts and statistics as a way to apprehend and comprehend the serial killer; the logical extension of society account, where the serial killer is a reflection of society from concerns with the individual to celebrity and consumerism; and the serial killer account, where the serial killer explains his own motivations. In looking at these accounts it becomes possible to see the cliches that are used to discuss the serial killer and how these reveal thoughts and fears of the collective, rather than providing the insight on the serial killer that the accounts are seeking.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.