Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells

Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.

PI3K δ /γ Inhibition Enhances the Expansion and Anti-Tumor Cytotoxicity of CART Cells for CLL Patients

Background: While CD19-targeted chimeric antigen receptor (CAR) based T cell therapy has shown promise in the treatment of chronic lymphocytic leukemia (CLL), overall efficacy is limited due to impaired T-cell fitness. We have previously shown that dual inhibition of PI3Kδ and PI3Kγ enhanced mitochondrial mass and ex vivo expansion of central and stem cell memory T cells from CLL patients(Funk, 2019,Journal for Immunotherapy of Cancer). In this study, we hypothesized that pharmacological inhibition of these pathways during ex vivo culture would increase the expansion and in vivo anti-tumor cytotoxicity. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of CLL patients by ficol hypaque centrifugation. T cells were negatively selected using MACS beads and transduced with CD19 CAR lentivirus (encoding CD28 or 41BB co-stimulatory domains) and stimulated with anti-CD3/CD28 beads in media containing 30 U/mL interleukin-2 with or without the PI3Kδ/γ inhibitor duvelisib (Duv) for 15 days. NOG mice were engrafted with the OSU-CLL cell line for 14 to 18 days with tumor burden measured by flow cytometry of blood samples from the mice, comprising a mean 0.15% of peripheral nucleated cell content. Control-CART or Duv-CART were injected by tail vein injection. Frequencies of CART, OSU-CLL cells and immune checkpoint molecule expression of CART or T cells in blood were measured by serial flow cytometry. Kaplan-Meier survival plots were represented as recipient survival on the indicated days Results: Treatment with either CD28 or 4-1BB Duv-CART cells led to significantly prolonged survival relative to control-CART (Figure 1, P<0.05). Recipients of Duv-CART cleared circulating OSU-CLL faster than control CART (Figure 2. P<0.05 at day26 for CD28 CART) and exhibited greater peak expansion and persistence of total CART and CD8+ CART. Recipients of Duv-CART cells had significantly greater in vivo persistence and expansion of total CART and CD8+ CART (Figure 3, P<0.001, day14 after CART were infused). Consistent with improved survival, both CD28 Duv-CART and 4-BB Duv-CART show reduced expression of LAG3, TIM3 and PD1 in the CD4 (Figure 4) and CD8+ subsets at earlier time point in vivo. (* p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). Conclusions: Inhibition of PI3Kd/g during CART cell culture decreased the expression of immune checkpoint molecules and enhanced in vivo expansion leading to greater efficacy in eliminating CLL. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

789 Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule to enhance targeted tumor cytotoxicity

BackgroundNatural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Tumor specific activation of NK cells is thus an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies), each of which has inherent disadvantages in this application. Thus, alternative approaches are warranted. Bicycle® are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to the key activating receptors, NKp46 and CD16a. When chemically coupled to tumor antigen binding Bicycles this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules Bicycle® natural killer- tumor-targeted immune cell agonists (NK-TICAs™) and we will describe their discovery and evaluation in this presentation.MethodsUsing our unique phage display screening platform, we have identified high affinity, selective binders to NKp46 and CD16a. By conjugating the Bicycle® NK cell-engaging binders to a model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle NK-TICAs™ to induce NK cell activation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays.ResultsWe have developed a novel modular compound with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compound, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced EphA2-expressing tumor cytotoxicity, in a dose dependent manner.ConclusionsBicycle NK-TICAs™ are novel therapeutic agents capable of enhancing the landscape of immune oncology. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote modulation of NK cells, and infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for application of the Bicycle technology to drive NK cell-mediated tumor immunity.

Novel ROS-Responsive Marine Biomaterial Fucoidan Nanocarriers with AIE Effect and Chemodynamic Therapy

Chemodynamic therapy (CDT) has been widely used in the treatment of many kinds of tumors, which can effectively induce tumor cell apoptosis by using produced reactive oxygen species (ROS). In this paper, ROS-sensitive multifunctional marine biomaterial natural polysaccharide nanoparticles (CT/PTX) were designed. Aggregation-induced emission (AIE) molecules tetraphenylethylene (TPE) labeled and caffeic acid (CA) modified fucoidan (FUC) amphiphilic carrier material (CA-FUC-TK-TPE, CFTT) was fabricated, in which the thioketal bond was used as the linkage arm between TPE and fucoidan chain, giving the CFTT material ROS sensitivity. In addition, amphiphilic carrier material (FUC-TK-VE, FTVE) composed of thioketal-linked vitamin E and fucoidan was synthesized. The mixed carrier material CFTT and FTVE self-assembled in water to form nanoparticles (CT/PTX ) loaded with PTX and Fe3+. CT/PTX nanoparticles could induce ROS oxidative stress in tumor sites through the CDT effect induced by Fe3+. The CDT effect was combined with the chemotherapeutic drug PTX to achieve tumor inhibition. In vitro cell studies have proved that CT/PTX nanoparticles have excellent cell permeability and tumor cytotoxicity. In vivo antitumor experiments confirmed effective antitumor activity and reduced side effects.

Preclinical Development of Inducible MyD88/CD40 (iMC)-Enhanced Chimeric Antigen Receptor Natural Killer (GoCAR-NK) Cells to Target BCMA+ Tumors

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a >70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

101 Engineering gamma/delta T cells with the T-Cell antigen coupler receptor effectively induces antigen-specific tumor cytotoxicity in vitro and in vivo

BackgroundEngineered T cell therapies have revolutionized treatment of relapsed refractory haematological malignancies, however the cost of treatment for autologous products remains a significant challenge to their widespread use. The high cost is driven largely by the need for personalized manufacturing of autologous cell products. A non-conventional class of T cells, the gamma/delta T cell, can be safely transplanted into an unrelated recipient without inducing graft-versus host disease,1 making them an ideal candidate for mass-manufactured off-the-shelf T cell therapies. We have previously described a novel method of directing conventional alpha/beta T cells towards tumour targets by co-opting the T cell receptor using the T cell Antigen Coupler (TAC) receptor.2 Here, we describe the use of TAC receptors to engineer antigen-specific reactivity into gamma/delta T cells, resulting in highly potent anti-tumor cytotoxicity.MethodsEngineered gamma/delta T cells were manufactured by activating PBMCs with Zoledronate and IL-2. The TAC transgene was introduced into T cells using either VSV-G pseudotype lentivirus or GALV-psuedotyped gamma-retrovirus vectors.Through optimization studies, we determined transduction was highest 24 hours post-activation for lentivirus and 72 hours post-activation for gamma-retrovirus. Cultures were fed with IL-2 supplemented media every 2 – 3 days and enriched on Day 14 to >99% gamma/delta T cell purity using CD4/CD8 magnetic-activated cell sorting depletion (Miltenyi Biotec).ResultsBoth methods of gene transfer tested for our pilot study yielded excellent gene transduction (40% - 70%). Using lentivirus-engineered gamma/delta T cells, we demonstrated that the TAC receptor re-directs gamma/delta T cells to attack tumors in an antigen-specific manner. The presence of the TAC receptor did not interfere with lysis of tumor cells via the natural tumor-reactive gamma/delta T cell receptors. Importantly, TAC-engineered gamma/delta T cells displayed robust cytotoxicity at very low effector:target ratios (<1) and caused regression of human tumor xenografts that were otherwise resistant to non-engineered gamma/delta T cells. Curiously, gamma/delta T cell manufacturing was sensitive to the quality of the lentivirus product, where products with low titers were associated with outgrowth of conventional alpha/beta T cells. Outgrowth of alpha/beta T cells was not observed with gamma-retroviruses. We are presently evaluating the anti-tumor activity of gamma-retrovirus-engineered gamma/delta T cells.ConclusionsOff-the-shelf engineered gamma/delta T cells represent a strategy to reduce manufacturing cost and may represent the next generation of engineered T cell therapies.TAC receptors provide a robust tool for directing gamma/delta T cells to attack tumors that are otherwise resistant to gamma/delta T cells and should be evaluated further.AcknowledgementsThis work was supported by the Samuel Family Foundation, the Ontario Centres of Excellence and Triumvira Immunologics.Ethics ApprovalThe study was approved by McMaster’s Animal Research Ethics Board, AUP#19-02-10.ReferencesArruda LCM, Gaballa A, Uhlin M. Impact of γδ T cells on clinical outcome of hematopoietic stem cell transplantation: systematic review and meta-analysis. Blood Adv 2019;3(21):3436–3448.Helsen CW, Hammill JA, Lau VWC, et al. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 2018;9(1):3049.