Back to the Future: Spatiotemporal Determinants of NK Cell Antitumor Function

NK cells play a crucial role in host protection during tumorigenesis. Throughout tumor development, however, NK cells become progressively dysfunctional through a combination of dynamic tissue-specific and systemic factors. While a number of immunosuppressive mechanisms present within the tumor microenvironment have been characterized, few studies have contextualized the spatiotemporal dynamics of these mechanisms during disease progression and across anatomical sites. Understanding how NK cell immunosuppression evolves in these contexts will be necessary to optimize NK cell therapy for solid and metastatic cancers. Here, we outline the spatiotemporal determinants of antitumor NK cell regulation, including heterogeneous tumor architecture, temporal disease states, diverse cellular communities, as well as the complex changes in NK cell states produced by the sum of these higher-order elements. Understanding of the signals encountered by NK cells across time and space may reveal new therapeutic targets to harness the full potential of NK cell therapy for cancer.

Engineering multi-functional protein biologics through PAL-mediated hydrazide ligation

Attributed to the -effects, hydrazides are nucleophilic despite being non-basic. Herein, we report that the hydrazide nucleophiles are effective acyl acceptors in the ligation reactions catalyzed by peptidyl asparaginyl ligases (PALs). Because hydrazides are easily functionalizable, hydrazide ligation is highly versatile. Interestingly, the linkages formed with the hydrazide substrates have varying degrees of liability toward the PAL enzyme, with some being remarkably resistant and thus ensuring nearly irreversible ligation. Using the hydrazide ligation method, we labelled an EGFR-targeting affibody-Fc fusion protein with various functional moieties to generate selective and potent cancer-imaging and therapeutic agents. Irreversible hydrazide ligation also allowed a sequential ligation scheme to be conducted on a protein. Using this scheme, quadruple FITC labels were introduced onto the N- and C-termini of the affibody-Fc protein to yield a bi-specific engager for Car-NK cell therapy. Our work expands the substrate scope of PAL enzymes and further point to their promise as a precision manufacturing tool for multi-functional protein biologics.

Immune Combination Therapy With NK Cell and Pembrolizumab Showed Therapeutic Efficacy in Treating Advanced Solid Tumors

Background：Natural killer cells are innate cytotoxic lymphocytes that play an important role in the anti-tumor immune response. However, in the microenvironment of solid tumors, the effector functions of NK cells are often impaired by the induction of immune checkpoint inhibitors, including PD-1. Methods: In this study, we conducted a two-phase study treating advanced solid patients with NK cell therapy (phase 1) or NK and anti-PD-1 inhibitor, pembrolizumab (phase 2).Results: After treatment, only 3 of 9 patients achieved stable disease after accepting NK cell therapy in the phase 1 study. While in the phase 2 study, 4 patients achieved stable diseases and 1 patient achieved partial response. Remarkably, no severe adverse event was observed in patients treated by NK cell and pembrolizumab combination therapy.Conclusion: The results in our study indicated that immune combination therapy with NK cell and pembrolizumab might be a promising and safe approaches to treating advanced solid tumors.

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients. Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs). Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape. Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis. Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. Disclosures No relevant conflicts of interest to declare.

799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

Adoptive NK Cell Therapy: A Promising Treatment Prospect for Metastatic Melanoma

Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, “activated” autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future.

Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6–8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

NK Cell Therapy: A Rising Star in Cancer Treatment

Immunotherapy has become a robust and routine treatment strategy for patients with cancer; however, there are efficacy and safety issues that should be resolved. Natural killer (NK) cells are important innate immune cells that have attracted increasing attention owing to their major histocompatibility complex-independent immunosurveillance ability. These cells provide the first-line defense against carcinogenesis and are closely related to cancer development. However, NK cells are functionally suppressed owing to multiple immunosuppressive factors in the tumor microenvironment; thus, releasing the suppressed state of NK cells is an emergent project and a promising solution for immunotherapy. As a result, many clinical trials of NK cell therapy alone or in combination with other agents are currently underway. This review describes the current status of NK cell therapy for cancer treatment based on the effector function and releasing the inhibited state of NK cells in the cancer microenvironment.