Cell Antibody-dependent Cell-mediated Cytotoxicity Research Articles

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

Xianling Lianxia formula improves the efficacy of trastuzumab by enhancing NK cell-mediated ADCC in HER2-positive BC

Trastuzumab has improved survival rates in human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC), but drug resistance leads to treatment failure. Natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC) represents an essential antitumor immune mechanism of trastuzumab. Traditional Chinese medicine (TCM) has been used for centuries to treat diseases because of its capacity to improve immune responses. Xianling Lianxia formula (XLLXF), based on the principle of “strengthening body and eliminating toxin”, exhibits a synergistic effect in the trastuzumab treatment of patients with HER2-positive BC. Notably, this synergistic effect of XLLXF was executed by enhancing NK cells and ADCC, as demonstrated through in vitro co-culture of NK cells and BC cells and in vivo intervention experiments. Mechanistically, the augmented impact of XLLXF on NK cells is linked to a decrease in cytokine inducible SH2 containing protein (CISH) expression, which in turn activates the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 5 (STAT5) pathway. Collectively, these findings suggested that XLLXF holds promise for enhancing NK cell function and sensitizing patients with HER2-positive BC to trastuzumab.

Inhibiting N-glycan processing increases the antibody binding affinity and effector function of human natural killer cells.

Novel approaches are required to improve the efficacy of immunotherapies and increase the proportion of patients who experience a benefit. Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to the efficacy of many monoclonal antibodies therapies. Natural killer (NK) cells mediate ADCC, though responses are highly variable and depend on prior treatment as well as other factors. Thus, strategies to increase NK cell activity are expected to improve multiple therapies. Both cytokine treatment and NK cell receptor engineering are being explored to increase ADCC. Post-translational modifications, including glycosylation, are widely recognized as mediators of cellular processes but minimally explored as an alternative strategy to increase ADCC. We evaluated the impact of treatment with kifunensine, an inhibitor of asparagine-linked (N-)glycan processing, on ADCC using primary and cultured human NK cells. We also probed affinity using binding assays and CD16a structure with nuclear magnetic resonance spectroscopy. Treating primary human NK cells and cultured YTS-CD16a cells with kifunensine doubled ADCC in a CD16a-dependent manner. Kifunensine treatment also increased the antibody-binding affinity of CD16a on the NK cell surface. Structural interrogation identified a single CD16a region, proximal to the N162 glycan and the antibody-binding interface, perturbed by the N-glycan composition. The observed increase in NK cell activity following kifunensine treatment synergized with afucosylated antibodies, further increasing ADCC by an additional 33%. These results demonstrate native N-glycan processing is an important factor that limits NK cell ADCC. Furthermore, optimal antibody and CD16a glycoforms are defined that provide the greatest ADCC activity.

163P Anti-tumor activity of cetuximab plus avelumab in non-small cell lung cancer patients involves innate immunity activation: Findings from the CAVE-lung trial

We recently conducted Cetuximab-AVElumab-Lung (CAVE-Lung), a proof-of-concept, translational and clinical trial, to evaluate the combination of two IgG1 monoclonal antibodies (mAb): avelumab, an anti-PD-L1 drug, and cetuximab, an anti-epidermal growth factor receptor (EGFR) drug, as second- or third-line treatment in non-small cell lung cancer (NSCLC) patients. We have reported clinically relevant anti-tumor activity in 6/16 patients. Clinical benefit was accompanied by Natural Killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC).

Anti-tumor activity of cetuximab plus avelumab in non-small cell lung cancer patients involves innate immunity activation: findings from the CAVE-Lung trial

BackgroundWe recently conducted Cetuximab-AVElumab-Lung (CAVE-Lung), a proof-of-concept, translational and clinical trial, to evaluate the combination of two IgG1 monoclonal antibodies (mAb): avelumab, an anti-PD-L1 drug, and cetuximab, an anti-epidermal growth factor receptor (EGFR) drug, as second- or third-line treatment in non-small cell lung cancer (NSCLC) patients. We have reported clinically relevant anti-tumor activity in 6/16 patients. Clinical benefit was accompanied by Natural Killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC). Among the 6 responding patients, 3 had progressed after initial response to a previous treatment with single agent anti-PD-1, nivolumab or pembrolizumab.MethodsWe report long-term clinical follow-up and additional findings on the anti-tumor activity and on the immune effects of cetuximab plus avelumab treatment for these 3 patients.ResultsAs of November 30, 2021, 2/3 patients were alive. One patient was still on treatment from 34 months, while the other two patients had progression free survival (PFS) of 15 and 19 months, respectively. Analysis of serially collected peripheral blood mononuclear cells (PBMC) revealed long-term activation of NK cell-mediated ADCC. Comprehensive genomic profile analysis found somatic mutations and germline rare variants in DNA damage response (DDR) genes. Furthermore, by transcriptomic analysis of The Cancer Genome Atlas (TCGA) dataset we found that DDR mutant NSCLC displayed high STING pathway gene expression. In NSCLC patient-derived three-dimensional in vitro spheroid cultures, cetuximab plus avelumab treatment induced additive cancer cell growth inhibition as compared to single agent treatment. This effect was partially blocked by treatment with an anti-CD16 mAb, suggesting a direct involvement of NK cell activation. Furthermore, cetuximab plus avelumab treatment induced 10-, 20-, and 20-fold increase, respectively, in the gene expression of CCL5 and CXCL10, two STING downstream effector cytokines, and of interferon β, as compared to untreated control samples.ConclusionsDDR mutations may contribute to DDR-induced STING pathway with sustained innate immunity activation following cetuximab plus avelumab combination in previously treated, PD-1 inhibitor responsive NSCLC patients.

216 Multiplex base editing of NK cell to enhance cancer immunotherapy

BackgroundNatural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody-dependent cell-mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. NK cells are also known for their technical and biological challenges which result in low editing efficiencies, compared to T cells and other immune cells.MethodsBase editing (BE) is a CRISPR-Cas9 based genome editing technology that allows precise single base transitions. Previously, we reported a high efficiency method for multiplex engineering of T cells using BE and thus reasoned that applying similar concepts in NK cells may offer an opportunity to alter many genes simultaneously at higher efficiency through multiplex base editing. We thus selected a panel of genes bearing critical roles in NK cell function for immunotherapy, including inhibitory intracellular regulator AHR and CISH, inhibitory checkpoint receptor KLRG1, TIGIT, KLRC1, and PDCD1, and Fc receptor CD16A. CD16A is responsible for NK cell ADCC and is regulated via cleavage upon NK activation. Non-cleavable CD16A improves ADCC killing and can be achieved through single-base substitution with BE.ResultsUsing the adenosine BE (ABE8e), we achieved multiplex editing (6 genes) rates up to 99% and 95% editing/knockout at DNA and protein levels, respectively. Notably, we assessed for reduction in editing efficiency when additional genes were targeted and found no significant reduction in editing efficiencies when targeting up to 6 genes simultaneously. Moreover, functional evaluation of non-cleavable CD16A NK cells revealed up to 35% increase of cytotoxicity against Raji cells.ConclusionsWe were able to achieve high multiplex editing efficiency in primary human NK cells using ABE8eand there was no significant decrease of editing efficiency as the number of gene of interest increases, up to 6 genes in total. Functional assay confirmed increased NK cell cytotoxicity against tumor cells. Our end goal is to achieve high efficiency multiplex editing in CAR-expressing NK cells to further improve NK cell activity and toxicity for cancer immunotherapy.ReferenceWebber B, Lonetree C, Kluesner M, et al. Highly efficient multiplex human T cell engineering without double-strand breaks usingCas9 base editors. Nat Commun 2019;10:5222.

Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2+ pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice

BackgroundChildren with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an...

Abstract 952: The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP

Abstract MCLA-129 is an ADCC-enhanced common light chain bispecific human IgG1 Biclonics® antibody specifically targeting the receptor tyrosine kinases EGFR and c-MET. It inhibits activation and downstream signaling of EGFR and c-MET induced by their respective ligands EGF and HGF and promotes elimination of tumor cells via Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Antibody-Dependent Cell-Mediated Phagocytosis (ADCP). MCLA-129 was developed to overcome c-MET signaling-dependent EGFR TKI-resistance mechanisms and was identified during an unbiased screening campaign with a focus on inhibition of ligand-induced proliferation and migration of cancer cells. Selectivity of MCLA-129 for tumor cells is achieved by simultaneously targeting both EGFR and c-MET. MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and consequently inhibits their ligand-induced phosphorylation. MCLA-129 was shown to bind critical residues on EGFR and c-MET for EGF and HGF ligand binding. GlymaxX® low fucose glycoengineering technology was used to enhance the mononuclear cell ADCC activity of the bispecific antibody. MCLA-129 demonstrated potent dose-dependent ADCC and ADCP against all NSCLC cell lines tested, which positively correlated with the EGFR and c-MET expression levels on the target cells. Significant inhibition of tumor growth of NCI-H1975EGFR L858R, T790M and HCC827/ER1EGFR del (E746, A750), c-MET amplified NSCLC cell line derived tumors was observed following MCLA-129 treatment. MCLA-129 can overcome HGF-mediated EGFR-TKI resistance, as demonstrated by testing a non-ADCC enhanced version of this antibody in a panel of NSCLC cell lines in vitro and orthotopic tumors in vivo showing that it inhibits EGFR and c-MET activity. Significant tumor regression was observed following treatment of immunodeficient xenograft NSG-hHGFki mice bearing orthotopic tumors established from HCC827EGFR del (E746, A750) cells. In addition, in an acquired erlotinib EGFR TKI resistance model, treatment of mice harboring tumors as big as &amp;gt;500 mm3 led to significant reduction of tumor size which persisted after the treatment period. Taken together these data demonstrate that MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action, including inhibition of c-MET and EGFR signaling, ADCC and ADCP. MCLA-129 holds promise as a potential treatment for patients with NSCLC and other cancers, and warrants clinical evaluation. Citation Format: David J. de Gorter, Alexandre Deshiere, Martijn van Rosmalen, Christian Wohn, Berina Eppink, Tristan Gallenne, Rinse Klooster, Li Mao, Wenxin Xu, Liang Deng, Qingyu Shu, Wei Liu, John de Kruif, Mario Di Matteo, Massimiliano Mazzone, Mark Throsby, Cecile A. Geuijen. The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP [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 952.

ADCC against MICA/B Is Mediated against Differentiated Oral and Pancreatic and Not Stem-Like/Poorly Differentiated Tumors by the NK Cells; Loss in Cancer Patients due to Down-Modulation of CD16 Receptor.

Simple SummaryNatural Killer cells are known to eliminate tumors directly or via antibody dependent cellular cytotoxicity. The complete modes and mechanisms of such killings are yet to be delineated. It is also unclear at what stages of tumor differentiation NK cells are capable of mediating the two modes of tumor killing. In this report we provide evidence that NK cells mediate killing of both stem-like/poorly differentiated tumors and well-differentiated tumors via direct cytotoxicity and antibody dependent cellular cytotoxicity, respectively. By using antibodies to MICA/B, EGFR and PDL1 surface receptors expressed on well-differentiated but not on stem-like/poorly differentiated tumors we demonstrate significant NK cell mediated antibody dependent cellular cytotoxicity in the absence of direct killing. In addition, our results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and antibody dependent cellular cytotoxicity, resulting in the competitive use of these receptors in either direct killing or antibody dependent cellular cytotoxicity.Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC).

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

Antibody-dependent cell-mediated cytotoxicity (ADCC) in familial myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

ABSTRACT Background Chronic fatigue syndrome (CFS) is an illness of unknown origin that may have familial risks. Low natural killer (NK) lymphocyte activity was proposed as a risk for familial CFS in 1998. Since then, there have been many studies of NK lymphocytes in CFS in general populations but few in familial CFS. Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK lymphocytes helps control viral infections. ADCC is affected by variant CD16A receptors for antibody that are genetically encoded by FCGR3A. Methods This report characterizes ADCC effector NK cell numbers, ADCC activities, and FCGR3A variants of five families each with 2–5 CFS patients, their family members without CFS and unrelated controls. The patients met the Fukuda diagnostic criteria. We determined: CD16Apositive blood NK cell counts; EC50s for NK cell recognition of antibody; ADCC lytic capacity; FCGR3A alleles encoding CD16A variants, ROC tests for biomarkers, and synergistic risks. Results CFS patients and their family members had fewer CD16Apositive NK cells, required more antibody, and had ADCC that was lower than the unrelated controls. CFS family members were predominantly genetically CD16A F/F s for the variant with low affinity for antibodies. ROC tests indicated unsuitability of ADCC as a biomarker for CFS because of the low ADCC of family members without CFS. Familial synergistic risk vs. controls was evident for the combination of CD16Apositive NK cell counts with ADCC capacity. Conclusions Low ADCC may be a risk factor for familial CFS. Furthermore, characterization of familial CFS represents an opportunity to identify pathogenic mechanisms of CFS.

922P EACH: A phase II study evaluating the safety and anti-tumour activity of avelumab and cetuximab in recurrent/metastatic squamous cell carcinomas

Patients with R/M SCC have low response rates to second line therapies, including PD-1 inhibitors nivolumab and pembrolizumab, representing an area of unmet clinical need. Cetuximab has modest activity as a single agent but potentiates the activity of radiotherapy in locally advanced head & neck SCC (HNSCC) and chemotherapy in R/M HNSCC. Cetuximab initiates Natural Killer cell antibody-dependent cell-mediated cytotoxicity, resulting in an anti-tumour immune response and the potential to augment the activity of PD-1/PD-L1 inhibition.

Abstract 3251: Assessment of NK cell-mediated killing and phenotypic analysis using advanced flow cytometry and an optimized multiplexed assay

Abstract In this study, a novel multiplexed assay was developed that simultaneously measures NK cell phenotype and function in relation to tumor target killing. Natural Killer (NK) cells are an essential part of the innate immune system, interface with the adaptive immune system, and play a critical role in tumor immune surveillance and anti-tumor responses. Although chimeric antigen receptor (CAR)-modified T cells have proven to be highly effective as an anti-cancer therapeutic, there are potential problems associated with their use, particularly the risk of inducing graft-versus-host disease (GVHD) resulting in aberrant cytokine release. In contrast to T cells, NK cells are potent cytotoxic effector cells that do not require MHC restriction and have a low risk of inducing GVHD. Recent advances in the development of CAR-modified NK cells have shown great promise for clinical utilization in cancer immunotherapy with capability for both direct tumor killing as well as for use in combination therapy with monoclonal antibodies to boost NK cell antibody-dependent cell-mediated cytotoxicity (ADCC). A critical process in the development of new NK cell-mediated therapeutics is the ability to expediently assay, screen, and analyze data for NK cell activation and tumor killing. To address this need, a novel multiplexed assay was developed that measures tumor target cell killing, target-dependent expression of activation markers (CD69 and CD25), and the quantitation of secreted granzyme B, IFNγ, and TNFα in a single well of a microtiter plate. To validate the assay, a direct NK cell-tumor cell killing model was used. Purified human NK cells were mixed with target cells at different ratios. Tumor cell killing, NK cell activation status, and quantification of secreted proteins were measured at 4 and 24 hours after co-culture. Assay data acquisition was performed on Intellicyt iQue3® platform for advanced flow cytometry. Data analysis and visualization was performed using the integrated ForeCyt® software package. Tumor cell killing along with granzyme B release, was evident by 4 hours following co-culture, and both were increased with the increase in effector to target ratio (E:T). Killing was further enhanced when NK cells were stimulated with IL-2 and IL-15. As expected, an increase in CD69 and CD25 expression was also observed following cytokine stimulation with further increase upon co-culture of NK cells with tumor cell targets, along with increased levels of secreted IFNγ and TNFα. Further validation of the assay using an ADCC model was also performed. These data show that a miniaturized, multiplexed NK cell killing assay, combined with high throughput flow cytometry, is a powerful tool to rapidly screen CAR-NK cell effector functions and other NK cell based therapeutics. Citation Format: Julie Lovchik, Sarita Chauhan, John O'Rourke. Assessment of NK cell-mediated killing and phenotypic analysis using advanced flow cytometry and an optimized multiplexed assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3251.

Combination of monalizumab and cetuximab in recurrent or metastatic head and neck cancer patients previously treated with platinum-based chemotherapy and PD-(L)1 inhibitors.

6516 Background: Monalizumab is a first-in-class immune checkpoint inhibitor targeting Natural Killer Group 2A (NKG2A), which is expressed on subsets of Natural Killer (NK), gd T and tumor-infiltrating CD8+T cells. NKG2A blockade promotes innate anti-tumor immunity mediated by NK and CD8+T cells and enhances NK cell antibody-dependent cell-mediated cytotoxicity induced by cetuximab. In a Phase I study, the combination of monalizumab and cetuximab was well tolerated. In an initial expansion cohort 1 of 40 patients (pts) who had progressed after platinum-based therapy, we reported an overall response rate (ORR) of 27.5%, a 4.5 month median PFS and an 8.5 month median OS. In a subset of patients (n=18) previously treated with PD-(L)1 inhibitors (IO), corresponding results were 17%, 5.1, and 14.1 months, respectively (ESMO 2019). Here we present data from a second expansion cohort 2 (n=40) conducted specifically in the post-IO setting to independently confirm the cohort 1 results. Methods: Eligible patients had R/M SCCHN previously treated with platinum and a PD-(L)1 inhibitor. Pts received monalizumab 750 mg q2weeks and cetuximab according to the label until progression or toxicity. Cohort 2 was designed as a confirmatory multicenter single arm phase II study, with a pre-planned total of 40 patients. The primary endpoint was ORR assessed per RECIST 1.1. Results: As of January 31, 2020, 40 pts have been treated in cohort 2. Median follow-up is 7.3 months (range, 1.9-13.6+). Eight (8) pts have a confirmed partial response (PR); ORR is 20% [95% confidence interval: 11-35]. Median time to response is 1.6 months [1.6-5.3]. At the time of data analysis, 3 pts were still in PR and 3 pts had stable disease continue on treatment. PFS and OS are still immature. Conclusions: In pts previously treated with platinum and PD-(L)1 inhibitors, the combination of monalizumab and cetuximab demonstrated promising activity. The second extension cohort confirmed prospectively the ORR reported in cohort 1. A randomized phase III trial of monalizumab and cetuximab is planned in this platinum and IO-pretreated SCCHN population. Clinical trial information: NCT02643550 .

Mechanisms of Resistance to NK Cell Immunotherapy.

Immunotherapy has recently been a major breakthrough in cancer treatment. Natural killer (NK) cells are suitable targets for immunotherapy owing to their potent cytotoxic activity that may target cancer cells in a major histocompatibility complex (MHC) and antigen-unrestricted manner. Current therapies targeting NK cells include monoclonal antibodies that promote NK cell antibody-dependent cell-mediated cytotoxicity (ADCC), hematopoietic stem cell transplantation (HSCT), the adoptive transfer of NK cells, the redirection of NK cells using chimeric antigen receptor (CAR)-NK cells and the use of cytokines and immunostimulatory drugs to boost the anti-tumor activity of NK cells. Despite some encouraging clinical results, patients receiving these therapies frequently develop resistance, and a myriad of mechanisms of resistance affecting both the immune system and cancer cells have been reported. A first contributing factor that modulates the efficacy of the NK cell therapy is the genetic profile of the individual, which regulates all aspects of NK cell biology. Additionally, the resistance of cancer cells to apoptosis and the immunoediting of cancer cells, a process that decreases their immunogenicity and promotes immunosuppression, are major determinants of the resistance to NK cell therapy. Consequently, the efficacy of NK cell anti-tumor therapy is specific to each patient and disease. The elucidation of such immunosubversive mechanisms is crucial to developing new procedures and therapeutic strategies to fully harness the anti-tumor potential of NK cells.

Improving Immunotherapy Against B-Cell Malignancies Using γδ T-Cell-specific Stimulation and Therapeutic Monoclonal Antibodies.

Tumor antigen-targeting monoclonal antibodies (mAbs) are an important element of current cancer therapies. Some of these therapeutic mAbs enable antibody-dependent cell mediated cytotoxicity (ADCC) against tumor cells. However, cancer-related functional impairment of immune effector cells may limit the clinical efficacy of antibody treatments. We reckoned that combining mAbs with cell-based immunotherapies would provide a clinically relevant synergism and benefit for cancer patients. Here, we focus on γδ T cells, as earlier studies demonstrated that γδ T-cell-based therapies are safe and promising for several types of malignancies. Similar to natural killer cells, their antitumor effects can be enhanced using antibodies, and they could, therefore, become a versatile effector cell platform for use with a variety of licensed therapeutic mAbs against cancer. In this study, we explore the potential of a combination therapy of activated γδ T cells with rituximab and the more recently developed mAbs (obinutuzumab and daratumumab) in different B-cell malignancies in vitro. Obinutuzumab outperformed the other mAbs with regard to direct target cell lysis and ADCC by γδ T cells in several CD20 cell lines and primary lymphoma specimens. We demonstrate that comparatively few CD16 γδ T cells are sufficient to mediate a strong ADCC. Using Fc-receptor-positive B-cell lymphomas as target cells, ADCC cannot be blocked by high concentrations of immunoglobulins or anti-CD16 antibodies, but both substances can promote cell mediated target cell lysis. This study expands on earlier reports on the therapeutic potential of distinctive tumor antigen-targeting mAbs and facilitates the understanding of the mechanism and potential of ADCC by γδ T-cell subsets.

Imlifidase Inhibits HLA Antibody-mediated NK Cell Activation and Antibody-dependent Cell-mediated Cytotoxicity (ADCC) In Vitro.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important pathway responsible for antibody-mediated rejection (AMR). Imlifidase (IdeS) cleaves human IgG into F(ab')2 and Fc fragments, potentially inhibiting ADCC. Here we examined the effect of IdeS on allo-antibody-mediated NK cell activation (Allo-CFC) and ADCC in vitro. For Allo-CFC, normal whole blood was incubated with third-party peripheral blood mononuclear cells (PBMCs) pretreated with anti-HLA antibody positive (HS) or negative (NC) sera to measure IFNγ+ NK cell%. For ADCC, normal PBMCs were incubated with Farage B (FB) cells with HS or NC sera to measure 7-AAD+ lysed FB cell%. To assess the effect of IdeS on these assays, serum-treated PBMCs (Allo-CFC-1) and serum used for PBMC pretreatment (Allo-CFC-2) in Allo-CFC, and serum used for ADCC were preincubated with IdeS. Sera from IdeS-treated patients were also tested for Allo-CFC (Allo-CFC-3). IFNγ+ NK cell% were significantly elevated in HS versus NC sera in Allo-CFC-1 (10 ± 3% versus 2 ± 1%, P = 0.001), Allo-CFC-2 (20 ± 10% versus 4 ± 2%, P = 0.01) and 7AAD+ FB cell% (11 ± 3% versus 4 ± 2%, P = 0.02) in ADCC. These were significantly reduced by IdeS treatment. Patient sera with significantly reduced anti-HLA antibody levels at 1 day postimlifidase lost the capacity to activate NK cells in Allo-CFC-3, but those at 1-3 months postimlifidase regained the capacity. IdeS inhibited NK cell activation and ADCC in vitro and in treated patients. These results and reported inhibition of complement activating anti-HLA antibodies by IdeS suggest its possible role in treatment of AMR.

1134P - Monalizumab in combination with cetuximab in patients (pts) with recurrent or metastatic (R/M) head and neck cancer (SCCHN) previously treated or not with PD-(L)1 inhibitors (IO): 1-year survival data

BackgroundMonalizumab is a first in class immune checkpoint inhibitor targeting Natural Killer Group 2A (NKG2A), which is expressed as a heterodimer with CD94 on subsets of Natural Killer (NK), γδ T and tumor-infiltrating CD8+ T cells (André et al. Cell 2018). The NKG2A ligand, HLA-E, is upregulated in cancer, including SCCHN. NKG2A blockade promotes innate anti-tumor immunity mediated by NK and CD8+ T cells and enhances human NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) induced by cetuximab. This dual targeting could provide greater antitumor activity than cetuximab alone. MethodsThe multicenter phase II trial tested the combination of monalizumab and cetuximab in pts with R/M SCCHN. Pts received monalizumab 10mg/kg q2weeks and cetuximab according to the label until disease progression or unacceptable toxicity. Pts were required to be progressing after platinum-based chemotherapy and to have received ≤2 prior lines of therapy in the R/M setting. Previous treatment with IO was allowed. The primary endpoint was Overall Response Rate (ORR) per RECIST. ResultsAll 40 pts received prior platinum-based chemotherapy, 18 (45%) prior IO and 5 (13%) prior cetuximab. ORR was 27.5% [95% confidence interval: 16-43] with 11 confirmed responses (1 complete + 10 partial) observed in both IO naive 36% [20-57] and IO pretreated pts 17% [6-39]. As of April 17, 2019, with a median follow-up of 17 months (mo), in all pts, IO naïve and IO pretreated pts respectively, median duration of response was 5.6, 5.3 and 5.6mo, PFS was 4.5, 3.9 and 5.1mo and OS was 8.3, 7.8 and 12.8mo. The 12mo OS estimate is 44% [31-63]. No new safety signal emerged. Pre and post treatment tumor biopsies were collected and RNA seq analyses are ongoing. ConclusionsIn a cohort of 40 patients of heavily pretreated SCCHN patients, monalizumab and cetuximab combination demonstrated high response rate, good duration of response, and promising PFS and OS as well as good safety profile. An additional R/M SCCHN cohort of 40 patients who received both platinum-based chemotherapy and IO is being enrolled in this study. Clinical trial identificationNCT02643550. Legal entity responsible for the studyInnate Pharma. FundingInnate Pharma. DisclosureR.B. Cohen: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Innate Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Genocea; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Merck. M.R. Posner: Advisory / Consultancy: Merck; Advisory / Consultancy: Cel-Sci; Shareholder / Stockholder / Stock options: Promedior; Licensing / Royalties: Bet Isreal-Deaconess Médical Centre, Arizona Stat. J.R. Bauman: Advisory / Consultancy: Pfizer; Advisory / Consultancy: Bayer. C. Even: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Innate Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Genocea; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Heat; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Alkermes; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Arrys; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Takeda; Honoraria (self), Advisory / Consultancy: Cantargia; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Xencor. E. Saada-Bouzid: Advisory / Consultancy: AstraZeneca. T. Seiwert: Honoraria (self): Aduro biotech; Honoraria (self): AstraZeneca; Honoraria (self): Bayer; Honoraria (self), Research grant / Funding (institution): BMS; Honoraria (self): Celgene; Honoraria (self): Innate; Honoraria (self): Loxo Oncology; Honoraria (self), Research grant / Funding (institution): Merck; Honoraria (self): Nanobiotix; Research grant / Funding (institution): Jounce. F. Calmels: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. R. Zerbib: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. P. André: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. A. Boyer-Chammard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Innate Pharma. J. Fayette: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy: Merck Serono; Honoraria (self), Advisory / Consultancy: Innate Pharma; Honoraria (self), Advisory / Consultancy: Biogen. All other authors have declared no conflicts of interest.

Antibody Therapies for Acute Myeloid Leukemia: Unconjugated, Toxin-Conjugated, Radio-Conjugated and Multivalent Formats.

In recent decades, therapy for acute myeloid leukemia (AML) has remained relatively unchanged, with chemotherapy regimens primarily consisting of an induction regimen based on a daunorubicin and cytarabine backbone, followed by consolidation chemotherapy. Patients who are relapsed or refractory can be treated with allogeneic hematopoietic stem-cell transplantation with modest benefits to event-free and overall survival. Other modalities of immunotherapy include antibody therapies, which hold considerable promise and can be categorized into unconjugated classical antibodies, multivalent recombinant antibodies (bi-, tri- and quad-specific), toxin-conjugated antibodies and radio-conjugated antibodies. While unconjugated antibodies can facilitate Natural Killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), bi- and tri-specific antibodies can engage either NK cells or T-cells to redirect cytotoxicity against AML targets in a highly efficient manner, similarly to classic ADCC. Finally, toxin-conjugated and radio-conjugated antibodies can increase the potency of antibody therapies. Several AML tumour-associated antigens are at the forefront of targeted therapy development, which include CD33, CD123, CD13, CLL-1 and CD38 and which may be present on both AML blasts and leukemic stem cells. This review focused on antibody therapies for AML, including pre-clinical studies of these agents and those that are either entering or have been tested in early phase clinical trials. Antibodies for checkpoint inhibition and microenvironment targeting in AML were excluded from this review.

EACH: A randomised phase II study evaluating the safety and anti-tumour activity of the combination of avelumab and cetuximab relative to avelumab monotherapy in recurrent/metastatic head and neck squamous cell cancer.

TPS6091 Background: Patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) have low response rates to licensed second line therapies, including PD-1 inhibitors nivolumab and pembrolizumab, and represent an area of unmet clinical need. The chimeric IgG1 epithelial growth factor receptor (EGFR) monoclonal antibody cetuximab potentiates the activity of radiotherapy in locally advanced HNSCC and chemotherapy in R/M HNSCC and is also licensed with modest activity as a single agent. Cetuximab initiates Natural Killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in an anti-tumour immune response and the potential to augment the activity of PD-1/PD-L1 inhibition. EACH aims to examine the safety and efficacy of the potentially synergistic interaction between cetuximab and avelumab, a fully human IgG1 anti-PD-L1 monoclonal antibody in R/M HNSCC. Methods: EACH is a randomised phase II trial preceded by a safety run-in phase. Eligible patients have histologically or cytologically confirmed measurable recurrent or metastatic squamous cell carcinoma of any site in the safety run-in phase, and HNSCC in phase II, that is considered incurable by local therapies. The safety run-in has a single arm de-escalating design, aiming to establish the safety of cetuximab with avelumab and determine the optimal dose of cetuximab within this combination. The safety run-in has a dosing schedule of avelumab (10 mg/kg) + cetuximab (500 mg/m2) intravenously every 2 weeks, with de-escalation of cetuximab to 400 mg/m2 and 300 mg/m2 if necessary. The safety run-in phase commenced recruitment in July 2018 and is ongoing. The phase II component will randomize 114 HNSCC patients between either avelumab + cetuximab at the dose determined by the safety run-in phase or avelumab (10 mg/kg) alone. Treatment will be in 4-week cycles for up to one year. The primary endpoint in the safety run-in phase is the occurrence of dose limiting toxicities, and in phase II is Disease Control Rate at 24 weeks, using iRECIST. Blood and fresh tissue will be collected for exploratory translational studies, which will focus on the identification of potential novel predictive biomarkers for response. Clinical trial information: NCT03494322.