NK Cell Engager Research Articles

Leveraging Natural Killer Cell Innate Immunity against Hematologic Malignancies: From Stem Cell Transplant to Adoptive Transfer and Beyond

Numerous recent advancements in T-cell based immunotherapies have revolutionized the treatment of hematologic malignancies. In the race towards the first approved allogeneic cellular therapy product, there is growing interest in utilizing natural killer (NK) cells as a platform for off-the-shelf cellular therapies due to their scalable manufacturing potential, potent anti-tumor efficacy, and superior safety profile. Allogeneic NK cell therapies are now being actively explored in the setting of hematopoietic stem cell transplantation and adoptive transfer. Increasingly sophisticated gene editing techniques have permitted the engineering of chimeric antigen receptors, ectopic cytokine expression, and tumor recognition signals to improve the overall cytotoxicity of NK cell therapies. Furthermore, the enhancement of antibody-dependent cellular cytotoxicity has been achieved through the use of NK cell engagers and combination regimens with monoclonal antibodies that act synergistically with CD16-expressing NK cells. Finally, a greater understanding of NK cell biology and the mechanisms of resistance have allowed the preclinical development of NK checkpoint blockade and methods to modulate the tumor microenvironment, which have been evaluated in early phase trials. This review will discuss the recent clinical advancements in NK cell therapies in hematologic malignancies as well as promising avenues of future research.

The Novel Trifunctional Anti-BCMA NK Cell Engager SAR'514 Has Potent in-Vitro and in-Vivo Anti-Myeloma Effect through Dual NK Cell Engagement

Multiple Myeloma (MM) is the second most prevalent hematopoietic malignancy, representing 10% of total blood cancers. Despite the emergence of new therapies in the recent years, it is still an incurable disease, with a 5-year overall survival of 56% in patients (https://cancerstatisticscenter.cancer.org/). Thus, innovative approaches that would be safe, potent and with long lasting beneficial effects are required. BCMA (B Cell Maturation Antigen) is a cell surface receptor highly and selectively expressed on normal and malignant plasma cells and which function is to promote cell proliferation and survival upon binding of its ligands APRIL (A PRoliferation Inducing Ligand) and BAFF (B cell Activating Factor). BCMA has a very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care such as anti-CD38 therapies, or even BCMA-targeting agents (Cohen AD et al. J Clin Invest 2019). The ability of NK cells to intrinsically kill tumor cells, leaving healthy cells unharmed, with minimal pro-inflammatory cytokine release induction as compared to T cell-based therapies makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. We developed a NK Cell Engager (NKCE) SAR'514 that activates NK cells through a dual engagement of NKp46 and CD16a, two major NK cell activating receptors highly expressed on NK cells in MM patients, and which redirects the activated NK cells to engage and kill BCMA+ tumor cells. The trifunctional NK cell engager molecule SAR'514 was designed to specifically and efficiently activate NK cells to induce potent cytotoxic activity against BCMA+ MM cells. SAR'514 exhibits high affinity for the 3 targets BCMA, CD16 and NKp46. The ability of SAR'514 to induce NK cell-mediated cytotoxicity in vitro was tested against a panel of MM cell lines with low, medium and high BCMA expression levels and compared to an afucosylated reference compound. SAR'514 exhibited a strong and similar potency against all tested cell lines and a better potency compared to this comparator (EC50 0.4 pM vs 7.8 pM) demonstrating the superiority of a dual targeting NK cell engager molecule as compared to an antibody approach. An optimized NK cell activation was obtained with a dual engagement of both NKp46 and CD16a compared to single CD16a or NKp46 engagement or the combination of two molecules engaging CD16a or NKp46. Binding of SAR'514 on NK cells promoted a strong NK cell activation and induced cytotoxic activity only in the presence of MM target cells with no fratricide effect on NK cells. This antitumor activity is associated with very low IL-1b, IL-6, TNFa and IFNg cytokine release (in the low pg/mL range) in human PBMC and whole blood settings in the presence of BCMA+ tumor target cells, and a good safety profile. In addition, the in vivo anti-tumor activity of an anti-murine NKp46 surrogate NK cell engager molecule at 0.05, 0.5 and 5 mg/kg was investigated versus a control isotype in huFcgR transgenic mice engrafted with the EL4-huBCMA murine thymoma model. As compared to the control group in which only 20% of mice survived with a median survival day of 44.5 days, SAR'514 induced a significant mouse survival from 0.5 mg/kg to 5 mg/kg with an overall survival of 90% and a median survival day over 90 days. In summary, these results demonstrate the efficacy of SAR'514 for controlling MM tumors in vivo and provide consistent support for its clinical development.

IPH6501 Is a Novel NKp46-Targeting Tetraspecific Antibody-Based Natural Killer Cell Engager Therapeutic (ANKET) Armed with a Non-Alpha IL-2 Variant and Developed for the Treatment of CD20-Positive Malignancies

CD20 is expressed by >90% of B-cell non-Hodgkin's lymphomas (NHL). Several generations of CD20-targeting monoclonal antibodies including rituximab, ofatumumab, and obinutuzumab have been widely used for B-cell malignancy therapies. Despite the recent approvals of novel CD20-targeting agents, new alternatives and strategies are still required for patients, which are relapsing or refractory after several lines of treatment. High circulating NK cell numbers have been associated with better clinical responses to anti-CD20- targeting monoclonal antibodies, supporting the role of NK cells in efficacy of these treatments. IPH6501, a novel antibody-based NK cell engager therapeutics (ANKET), is a single tetraspecific molecule engaging two NK cell activating receptors NKp46 and CD16a (FcγRIIIa), the β chain (CD122) of the interleukin-2 receptor (IL-2R), and the CD20 antigen expressed on malignant B cells. The IL-2R binding element incorporated in IPH6501 is an IL-2 variant (IL-2v) designed with point mutations that abolish binding to the IL-2R-α chain (CD25), with the goal of limiting toxicity and interaction with Tregs. IL-2v incorporated into IPH6501 is directed towards NK cells through the binding with high affinity to NKp46 and CD16a, providing its ability to interact with IL-2R preferentially on NK cells and to promote their activation and proliferation at pM doses. IPH6501 was designed to induce NK cell mediated-cytotoxicity and cytokine secretion by co-engaging CD16a and NKp46. Only the binding of IPH6501 to CD20, bridging the NK cells to the target cells, was able to trigger the cytotoxic activity of NK cells. Non-saturating doses of IPH6501 on NK cells and on CD20+ cells were sufficient to promote maximal killing activity. Furthermore, IPH6501 promoted NK cell cytotoxicity against tumor cells expressing very low levels of CD20. IPH6501 also demonstrated superiority to control tumor cell growth in vitro, as compared to the CD20-targeting clinical benchmark antibodies rituximab and the Fc-optimized obinutuzumab. In vivo treatment with a mouse surrogate of IPH6501 induced peripheral NK cell proliferation and activation, and demonstrated potent antitumor efficacy in a xenograft mouse model using the aggressive human B-lymphoma CD20+ RAJI cell line engrafted subcutaneously. In non-human primate, a well-tolerated dose of IPH6501 led to peripheral NK cell expansion and to the depletion of CD20+ B cells in the circulation and within lymphoid tissues, with minimal systemic cytokine release. In conclusion, IPH6501 is a first-in-class CD20-targeting tetraspecific NK cell engager designed to promote NK cell proliferation and specific cytotoxicity against CD20+ cells. IPH6501 is thus a promising biologic designed to harness the anti-tumor functions of NK cells in CD20+ B cell malignancies. IND-enabling studies are ongoing, and IPH6501 is expected to enter FIH clinical study in 2023.

Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma

The re-direction of natural killer (NK) cells is an emerging strategy for cancer immunotherapy. We describe the development and characterization of the so far smallest NK cell re-directing hBCMA x CD16 dual engagers for potential multiple myeloma treatment based on combinations of 7 kDa non-immunoglobulin affibody affinity proteins (Fig. 1). Affibodies to human CD16a protein (low affinity Fc-gamma receptor IIIa) were selected by phage display which resulted in the identification of binders with individual affinities (KD) for CD16a in the range of 100 nM to 3 µM, depending on the CD16 allotype (158V/F) and clone used in the analysis. Pairwise epitope binding experiments showed that binders with non-overlapping epitopes could be identified, allowing for the construction of bi-paratopic heterodimers with subnanomolar affinity for CD16a. In addition, a bivalent construct based on the variant with the highest monovalent affinity, denoted A10, showed subnanomolar apparent affinity (avidity) for both CD16a allotypes. The binding epitope of A10 was shown not to overlap with the Fc (hinge) binding activity of CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding affibody constructs containing one or two CD16a binding affibodies were genetically fused to a high-affinity (KD of ca. 7 nM) hBCMA-specific affibody, or a null-variant thereof. These 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently induce synapse formation, activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines (Fig. 2), without evidence of NK cell fratricide. Hence, we report a novel class of versatile and uniquely small NK cell engagers with potent and specific binding properties and functional profiles. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multifunctional NK Cell-Engaging Antibodies Targeting EGFR and NKp30 Elicit Efficient Tumor Cell Killing and Proinflammatory Cytokine Release.

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.

Abstract PO001: Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC)

Abstract BACKGROUND: GPC3 is highly expressed in HCC while it is hardly expressed in adult normal tissues except placenta. Given the presence of dysfunctional NK cells in the HCC tumor microenvironment that contribute to poor anti-tumor immune responses, we sought to develop an iNK cell therapy in combination with GPC3 targeted NK cell engagers (NKE). FLEX-NK™ is a proprietary tetravalent IgG1-like multifunctional antibody NKE platform with a novel FLEX-linker to allow for simultaneous binding to different antigens on different cells. CYT-303 was constructed to engage NK cells via the activation receptor NKp46 and to target cancer cells via GPC3. We hypothesized that CYT-303 may be active in the tumor microenvironment where there are sufficient number and function of endogenous NK cells, but that this activity may be augmented by fresh functional NK cells. iNK cells derived from iPSC, a uniform starting material with unlimited self-renewal capabilities, can be expanded to produce a universal off-the-shelf allogeneic therapy. We investigated the combination of CYT-303 to enhance tumor specificity and potency of iNKs. METHODS: CYT-303 was expressed in CHOZEN cells and purified by step column chromatography. iNK’s were differentiated from iPSC’s and expanded using irradiated feeder cells. PB-NK cells were purified from healthy donor blood. iNK and PB-NK cytotoxicity against Hep3B tumors was evaluated in the presence of CYT-303 or isotype control antibodies at a fixed E/T ratio. CYT-303 pharmacokinetics in mice was evaluated following a single intravenous injection. Anti-tumor efficacy of the iNK combination with CYT-303 was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors following intra-tumoral injection of iNKs and intravenous dosing with CYT-303. Blood alpha-fetoprotein (AFP) levels were assessed by immunoassay. Cytokine release assessments for CYT-303 was evaluated in an in-vitro PBMC activation assay. RESULTS: iNK cells express high levels of multiple activation receptors including NKp46, but low levels of CD16. Despite low expression of CD16, natural cytotoxicity of iNK’s against Hep3B tumors was higher than with fresh PB-NK and CYT-303 redirected killing of Hep3B tumors was comparable to PB-NK’s. The CYT-303 pharmacokinetics study in mice showed a terminal half-life of 17 hrs. In a Hep3B tumor model in NSG-hIL-15 mice, the combination of CYT-303 and iNK’s showed significantly greater tumor growth inhibition compared to iNK’s alone plus an IgG1 isotype control. Blood AFP levels decreased in the CYT-303 plus iNK combination compared to iNK’s alone. Cytokine release assessment of CYT-303 in the human PBMC assay showed no evidence of cytokine release while high levels of cytokine release was observed with anti-CD28 (TGN1412) and CD3 antibody controls. CONCLUSIONS: The combination of NK cell engager CYT-303 and iNK cells is active in preclinical models of HCC with low levels of cytokine release. These data support further development of CYT-303 and iNKs to engage and empower NK cells as an off the shelf therapeutic approach in HCC. Citation Format: Antonio Arulanandam, Liang Lin, Hao-Ming Chang, Harish Potu, Vishal Khairnar, David Zou, Melissa Triggiano, Nejmi Dilmac, Yinan Yang, Shira Kahlon, Stanley Frankel, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Yoav Hershkovitz, Yaron Ilan, Wei Li. Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO001.

The power of combining iPSC-derived NK cell therapy & NK-cell engager antibodies

Manufacturing of individualized neoantigen-specific therapies for patients takes place one batch at a time. The existing regulatory mechanisms for post-approval changes for traditional products can be applied for limited types of changes to either controls or manufacturing processes for some individualized Advanced Therapy Medicinal Products. However, these existing mechanisms become untenable given the rapid pace of evolution among these therapies and constant innovations in manufacturing technologies (such as next-generation sequencing and bioinformatics workflows). There is a need for a novel regulatory framework for these therapies and mechanisms of pre- and post-approval lifecycle management (such as a predetermined change control plan) that allow for streamlined process updates, to ensure that patients receive products manufactured using the best technologies and most accurate data based on a continuous improvement mindset. The application of relevant medical device regulatory mechanisms to individualized neoantigen-specific therapies is proposed here, as devices have mechanisms for iterative improvements managed in a risk-based approach. While there are ongoing attempts at harmonization of regulatory expectations for lifecycle management, new regulatory mechanisms (and leveraging existing frameworks where available) are needed to avoid some existing pitfalls. A novel regulatory framework is proposed to consider the genomics and bioinformatics workflows as integral parts of the design and end-to-end manufacture of individualized neoantigen-specific immunotherapies.

Abstract 2749: iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples

Abstract Cancer immunotherapy has transformed the oncology landscape and shown promising therapeutic results in the clinic but it is also clearly facing multiple limitations. So far, the focus of immunotherapies has mainly been on the modulation of T-cells. Natural killer (NK) cells offer promising perspectives for immunotherapies as they directly kill tumor cells and produce pro-inflammatory cytokines to activate and recruit T-cells and other immune cells. Thus, NK cells are considered key players in the anti-tumor immune response that can make a “cold” tumor “hot”. However, in the tumor microenvironment (TME) of cancer patients, the low numbers of NK cells present and their exhaustion due to local immunosuppression often limit tumor cell killing. The development of NK based cell therapy products can overcome these limitations, either as single therapy or in combination with biologics such as e.g. NK cell engagers or monoclonal antibodies for boosting ADCC (antibody dependent cell cytotoxicity). To overcome the main hurdle of current autologous cell therapies, the EVOcells Oncology platform aims at developing a streamlined manufacturing process for iPSC-derived immune effector cells. The goal is to develop “off-the-shelf” cell therapy products for clinical use in large patient populations. We are currently establishing a multi-asset portfolio combining multiple immune effector cell types with different genetic editing strategies that serve to optimize this approach further. iPSC-derived NK cells (iNK cells) offer an attractive option for improving both therapeutic efficacy and safety of CAR cell therapies. Our robust, feeder-free production protocol produces iNK cells that can be reliably frozen with good recovery rates. We have generated multiple genetically modified iNK cells and have validated that the genetic modifications boost iNK function. At the end of the production process, iNK cells show phenotypic properties and single cell RNA sequencing profiles comparable to blood-derived NK cells. Importantly, iNK cells are fully functional with the ability to produce key pro-inflammatory cytokines and to form lytic immunological synapses leading to efficient killing of cancer cell lines. In addition to the killing of established tumor cell lines, we also evaluated the ability of iNK cells to kill primary leukemia cells ex vivo by using blood samples from B-cell chronic lymphocytic leukemia patients. In combination with a standard of care anti-CD20 antibody, iNK cells demonstrated the ability to kill primary leukemia cells via ADCC. Taken together, these promising results obtained with iNK cells show the potential of the EVOcells Oncology platform to deliver the next generation of cell therapies in oncology paving the way to develop a portfolio of iPSC-derived immune effector cells. Citation Format: Michael Esquerré, Audrey Holtzinger, Mélanie Pichery, Stefanie Pfaender, Saniye Yumlu, Mandy Richter, Delphine Betous, Oriane Bombarde, Mylène Gador, Nadja Sailer, Michael Paillasse, Loïc Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2749.

Abstract 3436: Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells

Abstract Given that CD38 is a clinically validated target for NK cell mediated cytotoxicity in multiple myeloma, we sought to leverage our FLEX NKTrademark platform to create a NK engager antibody targeting CD38. FLEX NKTrademark is a proprietary platform for production of tetravalent multifunctional NK engager antibodies with a novel FLEX linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. NK engagement and activation is mediated through a binder directed against the natural cytotoxicity receptor NKp46. With the FLEX NKTrademark scaffold, we created two novel tetravalent NK cell engagers targeting CD38, one with a wild type (WT) Fc and one with a mutant null Fc. Both CD38 NKp46 engagers showed dose dependent binding to CD38 expressing multiple myeloma cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. Both engagers also bound multiple myeloma cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb)or daratumumab alone. Epitope mapping studies for our anti-CD38 mAb using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for our anti-CD38 antibody binding to CD38, suggesting a distinct epitope detected by our antibody. Interestingly, the NKp46 monoclonal antibody alone without a functional Fc induced peripheral blood NK cell cytolysis of the multiple myeloma cells, consistent with a prior report that NKp46 plays a key role in NK-cell mediated killing of myeloma cells. Both CD38 NKp46 engagers showed further enhanced dose dependent NK cell redirected cytolysis and degranulation against multiple myeloma cells compared to anti-NKp46 monoclonal antibody. The CD38 NKp46 engagers demonstrated higher cytotoxicity than the anti-CD38 binder mAb or daratumumab alone. The CD38 NKp46 engager with the WT Fc is more potent in induction of TNF-α and IFN-ɣ production compared to daratumumab and the engager with the mutant Fc. No IL-1β or IL-6 was induced by the engagers or daratumumab. Daratumumab treatment resulted in NK cell fratricide, while no fratricide was observed with the CD38-NKp46 NK engager with the mutant Fc. In peripheral blood mononuclear cell hemato-toxicity studies depletion of monocytes and NK cells were observed with daratumumab but no depletion was observed with the CD38 NKp46 engager with the mutant Fc. These results suggest that the CD38 NKp46 engagers have a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma that’s distinct from daratumumab. Citation Format: Liang Lin, Hao-Ming Chang, Cecilia Nakid, Stanley Frankel, Dennis Wu, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Jean-Christophe Bories, Antonio Arulanandam, Wei Li. Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3436.

Abstract 2893: Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format

Abstract Bispecific antibodies are being explored as a means to modulate responses of the immune system to tumor infiltration. They are emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. Besides tumor-targeted and dual immunomodulators, the most important class of therapeutically relevant bispecific antibodies are engagers of cytotoxic immune cells to kill tumor cells. These are mainly engager of T-cells (BiTE´s), but also NK-cell engager several of which have entered clinics with Blinatumomab (anti-CD3/anti-CD19) being FDA-approved for the treatment of Acute Lymphoblastic Leukemia. Several studies are under way to address potential combinatorial effects with other compounds in clinics. In order to facilitate analysis of the cytotoxic impact of BiTE´s in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK-cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. Our data support the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents. Citation Format: Carla N. Castro, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert. Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2893.

Abstract 4233: Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity

Abstract The tumor specific activation of natural killer (NK) cells is 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). NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles® are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to identify Bicycles® that bind specifically to the key activating receptor, NKp46. 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 NK-TICAs and we will describe herein their discovery and evaluation.We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner.In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for the application of our Bicycle technology to drive NK cell-mediated tumor immunity. Citation Format: Fay J. Dufort, Christopher J. Leitheiser, Gemma Mudd, Julia Kristensson, Alexandra Rezvaya, Katie Gaynor, Sandra Uhlenbroich, Liudvikas Urbonas, Heather Scott, Liuhong Chen, Helen Harrison, Michael Skynner, Kevin McDonnell, Philip E. Brandish, Nicholas Keen. Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4233.

Abstract 4247: Development of NK humanized mice models for the in vivo evaluation of NK cell engagers

Abstract Novel immunotherapeutic strategies targeting Natural Killer (NK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and there is a pressing need to have suitable mouse models for the evaluation of these therapeutics. To date, there are no validated in vivo mouse models that support the development of fully functional human NK cells. In recent years, the adoptive transfer of human peripheral mononuclear blood cells or CD34+ stem cells into highly immunodeficient mouse strains mostly allowed the evaluation of T cell-based therapies. However, these humanized mouse models have limitations and do not show an optimal development of innate immune cells such as NK, dendritic or myeloid cells. This is mostly due to the poor interspecies cross-reactivity of key factors necessary for cell development and maturation, like cytokines and growth factors.Here, we report the development and characterization of robust in vivo models that allow the engraftment and maintenance of fully functional human NK (huNK) cells, using several strains of immunodeficient transgenic mice for human cytokines (IL-15). The optimal parameters (source of immune cells, pre-conditioning regimen, route of administration) were identified. These models were characterized and validated by flow cytometry, and in vivo efficacy studies using an anti-CD20 Ab.In summary, we developed several murine models that exhibit significant numbers of functional huNK cells maintained over time in vivo. Different sources of human immune cell (CD34+ HSC, huNK cells) were used to implant immunodeficient huIL15 transgenic mice. HuNK cells were monitored by flow cytometry in all tested models. We showed huNK engraftment in both irradiated and non-irradiated models and confirmed the maintenance of expression of important functional NK markers on these cells. Efficacy studies performed in disseminated models showed promising results as efficacy of an anti-CD20 Ab, rituximab, was improved in presence of huNK cells, thus confirming the engagement of huNK cells in vivo in these models. Overall, we have developed several models that can sustain fully functional huNK cells over a period of several weeks that can be proficiently recruited in vivo by various NK-based therapeutics. Citation Format: Pauline Rettman, Dorothée Bourges, Laure-Marie Meyer, Ravi Rangara, Anna Ponchet-Lac, Nicolas Moindrot, Sukhvinder Sidhu, Céline Nicolazzi. Development of NK humanized mice models for the in vivo evaluation of NK cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4247.

A New Functional Screening Platform Identifies Colistin Sulfate as an Enhancer of Natural Killer Cell Cytotoxicity.

Simple SummaryThe use of small compounds in cancer immunotherapy has been limited so far. Her we screen for drugs that enhanced the ability of immune cells to kill tumor cells and identified the molecule Colistin Sulfate as a booster of immune activity.Due to their crucial role in tumor immunity, NK cells have quickly became a prime target for immunotherapies, with the adoptive transfer of NK cells and the use of NK cell engagers quickly moving to the clinical stage. On the other hand, only a few studies have focused on small molecule drugs capable of unleashing NK cells against cancer. In this context, repurposing small molecules is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we developed a new platform to screen small molecule compounds based on a high-throughput luciferase-release cytotoxicity assay. We tested 1200 FDA approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cells’ cytotoxic potential. We found that the antibiotic colistin sulfate increased the cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.

Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia.

Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αβT/B-depleted haplo-HSCT. The NK cell-resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.

126P Novel B7-H3 targeting dual nanobody NK cell engagers display robust activity against a broad spectrum of solid and hematologic malignancies

B7-H3 is a checkpoint molecule under intense investigation as an immune therapy target. We previously showed that dual camelid nanobody tri-specific killer engager (TriKE) (GTB-5550) specifically bound B7-H3 on PC3/C4-2 prostate cancer (PCa) cells and activated peripheral blood (PB) NK cells. We have since developed a dual camelid bispecific killer engager (BiKE) targeting B7-H3 and show that both GTB-5550, which harbors wild-type IL-15, and BiKE display broad activity against B7-H3-expressing tumors.

Bi-specific and Tri-specific NK Cell Engagers: The New Avenue of Targeted NK Cell Immunotherapy.

Natural killer (NK) cell-mediated cancer immunotherapy has grown significantly over the past two decades. More recently, multi-specific engagers have been developed as cancer therapeutics to effectively arm endogenous NK cells to more potently induce specific cytolytic responses against tumor targets. This review explores the bi- and tri-specific NK/tumor engagers that are emerging as a new generation of immunotherapeutics. These molecules vary in configuration, but they typically have small molecular weights and domains that engage specific tumor antigens and NK cell-activating receptors such as CD16, NKp30, NKp46, and NKG2D. They have demonstrated compelling potential in boosting NK cell cytotoxicity against specific tumor targets. This highly adaptable off-the-shelf platform, which in some formats also integrates cytokines, is poised to revolutionize targeted NK cell immunotherapy, either as a monotherapy or in combination with other effective anti-cancer therapies.

Abstract 1872: Generation of bispecific and trispecific T cell engagers (TCEs) and NK cell engagers (NKE)s without prior IgG reengineering

Abstract T cell and NK cell-redirecting antibodies are emerging as promising modalities in targeted immunotherapy. New technologies are overcoming the limitations of 1st generation immune cell-redirecting bispecifics, especially extending half-life, reducing immunogenicity and improving safety. For example, in the field of T cell engagers (TCEs), a plethora of molecular architectures has entered the clinic, the vast majority generated by genetic engineering of an IgG to bind both a tumor-associated antigen (TAA) and CD3 or 4-1BB. Similarly, recombinant DNA technology enables the C-terminal fusion of a cytokine (e.g. IL-2 or IL-15) to an IgG to obtain NK cell engagers (NKEs). We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for attachment of cytotoxic payloads,1 providing antibody-drug conjugates (ADCs) with significantly enhanced therapeutic index. This technology, known as GlycoConnect™, has been adapted by various drug developers (e.g. ADC Therapeutics, Mersana Therapeutics and others) and is now applied in two (2) clinical programs as well as multiple late-stage preclinical programs. Here we demonstrate how GlycoConnect™ technology can be applied to generate TCEs or NKEs by selective attachment of scFv (anti-CD3, anti-CD28) or cytokines (IL-15), with concomitant Fc-silencing. Moreover, an existing bispecific TCE (TAA/CD3) can be further boosted by attachment of anti-CD28 scFv for further T cell activation. Molecular architectures can be readily tailored to 2:2 or 2:1 ratio (TAA:scFv or cytokine) to mitigate adverse events like cytokine release syndrome, and even 2:1:1 formats. Structural and functional studies will be presented to corroborate the architecture and biological activity of these GlycoConnect™ bispecifics and trispecifics, in terms of binding, immune cell engagement and cell-killing potential. 1van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. Citation Format: Remon van Geel, Floris van Delft. Generation of bispecific and trispecific T cell engagers (TCEs) and NK cell engagers (NKE)s without prior IgG reengineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1872.

Natural killer cell engagers in cancer immunotherapy: Next generation of immuno‐oncology treatments

Immuno-oncology is revolutionizing the treatment of cancers, by inducing the recognition and elimination of tumor cells by the immune system. Recent advances have focused on generating or unleashing tumor antigen-specific T-cell responses, leading to alternative treatment paradigms for many cancers. Despite these successes, the clinical benefit has been limited to a subset of patients and certain tumor types, highlighting the need for alternative strategies. One innovative approach is to broaden and amplify antitumoral immune responses by targeting innate immunity. Particularly, the aim has been to develop new antibody formats capable of stimulating the antitumor activity of innate immune cells, boosting not only their direct role in tumor elimination, but also their function in eliciting multicellular immune responses ultimately resulting in long-lasting tumor control by adaptive immunity. This review covers the development of a new class of synthetic molecules, natural killer cell engagers (NKCEs), which are built from fragments of monoclonal antibodies (mAbs) and are designed to harness the immune functions of NK cells in cancer. As currently shown in preclinical studies and clinical trials, NKCEs are promising candidates for the next generation of tumor immunotherapies.

Harnessing CD16-Mediated NK Cell Functions to Enhance Therapeutic Efficacy of Tumor-Targeting mAbs.

Simple SummaryNatural Killer (NK) cells play a major role in cancer immunotherapy based on tumor-targeting mAbs. NK cell-mediated tumor cell killing and cytokine secretion are powerfully stimulated upon interaction with IgG-opsonized tumor cells, through the aggregation of FcγRIIIA/CD16 IgG receptor. Advances in basic and translational NK cell biology have led to the development of strategies that, by improving mAb-dependent antitumor responses, may overcome the current limitations of antibody therapy attributable to tolerance, immunosuppressive microenvironment, and genotypic factors. This review provides an overview of the immunotherapeutic strategies being pursued to improve the efficacy of mAb-induced NK antitumor activity. The exploitation of antibody combinations, antibody-based molecules, used alone or combined with adoptive NK cell therapy, will be uncovered. Within the landscape of NK cell heterogeneity, we stress the role of memory NK cells as promising effectors in the next generation of immunotherapy with the aim to obtain long-lasting tumor control.Natural killer (NK) cells hold a pivotal role in tumor-targeting monoclonal antibody (mAb)-based activity due to the expression of CD16, the low-affinity receptor for IgG. Indeed, beyond exerting cytotoxic function, activated NK cells also produce an array of cytokines and chemokines, through which they interface with and potentiate adaptive immune responses. Thus, CD16-activated NK cells can concur to mAb-dependent “vaccinal effect”, i.e., the development of antigen-specific responses, which may be highly relevant in maintaining long-term protection of treated patients. On this basis, the review will focus on strategies aimed at potentiating NK cell-mediated antitumor functions in tumor-targeting mAb-based regimens, represented by (a) mAb manipulation strategies, aimed at augmenting recruitment and efficacy of NK cells, such as Fc-engineering, and the design of bi- or trispecific NK cell engagers and (b) the possible exploitation of memory NK cells, whose distinctive characteristics (enhanced responsiveness to CD16 engagement, longevity, and intrinsic resistance to the immunosuppressive microenvironment) may maximize therapeutic mAb antitumor efficacy.