Mediated Antibody Dependent Cellular Cytotoxicity Research Articles

Abstract B025: MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment

Abstract Background Dendritic cells (DC) are the primary antigen presenting cells in the body responsible for processing and presenting MHC Class I and II antigens to initiate adaptive T cell response against cancer. In addition to MHC, DC also express non-classical receptors such as CD1d to activate invariant TCR α chain natural killer T (iNKT) cells. The conventional type-I dendritic cells (cDC1) presenting MHC class-II tumor associated antigens within tumor microenvironment (TME) have shown robust efficacy in mouse models of breast cancer (BC). Here, we have extended prior knowledge to deliver DC-based therapeutics, co-presenting class II antigens and CD1d directly to TME to show the potential of co-stimulation of multiple effector T cells in combination of tumor specific antibody (IgG1) mediating antibody dependent cellular cytotoxicity (ADCC). We hypothesize that merger of adaptive and innate responses will attain greater pathologic response in pre-clinical and clinical settings to design futuristic therapeutics against BC. Methods Present study uses variety of pre-clinical mouse models of BC and other types of cancer to test the therapeutic efficacy of intratumoral delivery of mouse bone marrow-derived conventional type I (CD103+ CD1d+) polarized DC pulsed with MHC class II HER2-derived peptides (HER2-DC1) in combination with systemic delivery of tumor specific IgG1 (aSEMA4D) to evoke ADCC. Cellular mechanism of response was evaluated using downstream methods including multiplex immunofluorescence, flow cytometry, and transcriptomics assays. Results We exhibit that specifically intratumoral combination therapy of HER2-DC1 expressing CD1d and aSEMA4D primes both innate and adaptive immune responses to completely regress tumor in 75-80% of mice with significant survival advantage in comparison to mice that received no therapy or monotherapies. The early CD4 Th1 response through interferon gamma combined with ADCC played a critical role in the decrease of pro-tumorigenic myeloid cells and increase in anti-tumor effector cells with a major contribution from classical CD4+ NKT cells within the TME. Interestingly, intratumoral combination therapy intensified DC1 migration from primary tumor to secondary tumor sites along with dynamic changes in subpopulations of DC with an associated increase in NKT cells at untreated tumor sites to display abscopal effect. Likewise, early data from phase I/II clinical trial illustrated great potential of cDC1 and IgG1 combination therapy to recruit anti-tumor effector cells such CD8 and NKT cells to mediate cancer eradication in HER2+ BC setting. Conclusions Specifically, intratumoral delivery of HER2-DC1 in combination with anti-tumor antibody mediating ADCC shows great promise for future BC clinical trials and improved patient outcomes. Furthermore, present study highlighted the role of NKT cells in favorable anti-tumor response. Our future studies are focused on understanding the crosstalk between DC and NKT to modulate the function of various cell types in TME to design improved cancer immunotherapies. Citation Format: Saurabh K Garg, Colin Snyder, Ganesan Ramamoorthi, Elizabeth E Evans, Amy L Aldrich, Namrata Gautam, Doris Wiener, Marie C Lee, Hyo S Han, Koski K Gary, Brian J Czerniecki. MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B025.

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Meta-Analysis of Randomized Controlled Trials to Evaluate the Incidence of Pneumonia, Upper Respiratory Tract Infections and Febrile Neutropenia in Patients with Multiple Myeloma Treated with Isatuximab

Introduction Multiple myeloma is a cancer of plasma cell and accounts for approximately 13% of all haematologic malignancies. Isatuximab is an anti CD38 IgG1 monoclonal antibody which binds to and kills myeloma cells through antibody-dependent cellular-mediated cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and direct induction of apoptosis. The incorporation of isatuximab to standard treatment in patients with relapsed refractory multiple myeloma (RRMM) has shown to significantly improve efficacy, with notable toxicities. We performed a combined analysis of randomized controlled trials (RCT) to determine the risk of pneumonia and upper respiratory tract infections (URTIs) and febrile neutropenia in patients with RRMM treated with isatuximab. Methods We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing isatuximab in patients with relapsed or refractory multiple myeloma that mention pneumonia, URTIs and febrile neutropenia as adverse effects were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Random effects model was applied. Results A total of 600 patients with RRMM from 2 phase III RCTs (ICARIA-MM and IKEMA) were eligible. ICARIA-MM and IKEMA compared isatuximab (I) + pomalidomide (P) + dexamethasone (d) vs Pd, and isatuximab (I) + Carfilzomib (K) + dexamethasone (d) vs Kd respectively. The randomization ratios were 1:1 in ICARIA-MM, and 3:2 in IKEMA studies. The I 2 statistic for heterogeneity was 0, suggesting homogeneity among RCT. The incidence of any-grade pneumonia was 30.1% in study group vs 25.8% in control group (RR, 1.19; 95% CI: 0.91 - 1.54; P=0.20). High-grade pneumonia rate was nearly 4% higher in isatuximab group compared to control arm but RR was not statistically significant at 1.26 (95% CI: 0.90 - 1.75; P = 0.18). More events of any-grade URTIs were reported in study arm (36.8%) versus 23.2% in control arm with RR of 1.55 (95% CI: 1.20- 2.01; P = 0.0008). The incidence of high-grade URTIs was low in both groups: 3.3% in study group versus 1.47% in control arm (RR, 2.25; 95% CI: 0.72 - 6.98; P = 0.16). Febrile neutropenia was noted in 20 (6%) of patients in study group compared to 5 (1.8%) in control group with RR of 3.52 (95% CI: 1.40- 8.83; P = 0.007). Conclusions Our meta-analysis showed that the addition of isatuximab to standard anti-myeloma regimen contributed to higher incidence of any-grade URTIs and febrile neutropenia in patients with RRMM, with RR of 1.55 for any-grade URTIs and RR of 3.5 for febrile neutropenia. However, risks of high-grade URTIs, any-grade and high-grade pneumonia were found to be statistically not significant between the two groups. Early recognition and proper supportive care are required to improve any-grade URTIs and febrile neutropenia.

Long-term outcomes and circulating tumor DNA analysis from a phase I/II study of lenalidomide and obinutuzumab with CHOP for newly diagnosed diffuse large B-cell lymphoma.

7553 Background: Diffuse large B-cell lymphoma (DLBCL) can be cured with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP), but a third of patients (pts) experience treatment failure. Randomized trials comparing RCHOP with modified regimens, including replacing R with obinutuzumab (O, CD20 antibody, Vitolo JCO 2017) and adding lenalidomide (L) to RCHOP (Nowakowski JCO 2021), have not significantly improved outcomes. The combination of L and O may have synergy via an enhanced NK-cell mediated antibody dependent cellular cytotoxicity. We hypothesized combining L and O with CHOP (LOCHOP) would be an effective and well tolerated treatment of DLBCL. Methods: In this single center phase Ib/II study (NCT02529852) pts with untreated CD20+ DLBCL received 6 cycles of CHOP with O 1000mg IV on days (D) 1, 8, and 15 during cycle 1 and D1 during cycles 2-6, and L 15 mg orally on D1-14 of each cycle. Growth factor and thromboembolism prophylaxis were mandated. The primary objectives were to determine the maximum tolerated dose (MTD) of L and efficacy of LOCHOP. Plasma samples were collected for circulating tumor DNA (ctDNA) analysis. Results: Fifty-three pts were enrolled, 6 in phase Ib and 47 in phase II. No dose limiting toxicities were experienced in phase Ib at an MTD of 15 mg of L. Median age was 62 years, 35 (66%) pts had stage III/IV disease, 12 (23%) an IPI > 2, and 26 (49%) and 22 (42%) were germinal center (GC) and non-GC subtype (5 not classified) by Hans algorithm. At end of therapy, 50 pts were evaluable, overall response: 49 (98%), complete response: 45 (90%). Median follow up was 4.5 years and 4-year progression free and overall survival rates were 87.4% and 91.3%. No characteristics were associated with differences in outcomes. Any grade and grade 3-4 adverse events (AEs) were experienced by 100% and 70% of pts. Grade 3-4 AEs included neutropenia (38%), thrombocytopenia (17%), fatigue (13%), neutropenic fever (13%), and infection (9%). Four (8%) pts developed venous thromboembolism. At study entry, 31/33 (94%) profiled pts had detectable ctDNA with CAPP-Seq, 24% had high ctDNA ( > log 2.5 hGe/ml) and 24/31 (77%) were evaluable by LymphGen classifier: molecular subtypes included EZB (5, 21%), ST2 (5, 21%), MCD (4, 17%), and other (9, 38%). All 5 EZB and 4/5 ST2 pts were GC subtype while 2/4 MCD pts were non-GC (1 GC, 1 not classified). With LOCHOP, 13/18 (72%) and 11/15 (73%) pts achieved early and major molecular response (Kurtz JCO 2018). By PhasED-Seq (Kurtz Nat Biotechnol 2021), 16/18 (89%) had no detectable ctDNA after ≥5 cycles. Conclusions: LOCHOP demonstrates high efficacy and tolerability in newly diagnosed DLBCL, leading to a high rate of undetectable minimal residual disease by ctDNA. Noninvasive molecular subtyping is feasible as a supplement to tissue diagnosis and should be incorporated in future studies aiming to improve on RCHOP. Clinical trial information: NCT02529852.

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.

CD38 Down-Regulation on Ex Vivo Activated and Expanded NK Cells for Cell Therapy Persists after Infusion

Introduction: Immunotherapies are gaining more and more importance in the treatment of multiple myeloma (MM). Antibodies directed against MM antigens like CD38, SLAMF7 or BCMA are used either in their natural form, conjugated to drugs, or in the form of bispecific T-cell engagers. Cellular therapies make use of cytotoxic lymphocytes, i.e. T cells or NK cells that can also be modified to express chimeric antigen receptors to target MM cells. Combinations of antibody and cellular therapies could further improve the outcome as, for example, NK cells can mediate antibody dependent cellular cytotoxicity (ADCC). However, NK cells also express CD38 and SLAMF7 and would be targeted by the therapeutic antibodies against these antigens. We have recently reported our clinical study infusing multiple doses of ex vivo activated and expanded autologous NK cells in six patients with MM post autologous stem-cell transplantation (EudraCT 2010-022330-83). Here, we report results of a phenotypic analysis of the ex vivo expanded NK cells and peripheral blood NK cells before and after infusion with implications for possible combination therapies.Methods: Ex vivo activated and expanded NK cells and NK cells in peripheral blood of the patients were analyzed by multiparameter flow cytometry. Peripheral blood cells were taken from the non-NK cell infusion arm before and at three different timepoints after infusion. NK-cell sub-populations within these samples were analyzed using t-SNE clustering.Results: Upon ex vivo activation and expansion, we observed that the NK cells gained a unique activated phenotype including populations of CD56 brightCD16 +Ki67 +HLA-DR + NK cells. Interestingly, these NK cells showed a reduced expression of CD38 compared to peripheral blood NK cells. Clustering analyses of data from peripheral blood samples revealed the gradual appearance of a new NK cell population with a similar phenotype in a dose-dependent fashion over four hours following infusion of the NK cell product. Infused NK cells could be detected in circulation up to four weeks after the last infusion. Like the NK cell infusion product, these cells expressed little to none CD38, high levels of NKG2D, 2B4, TIM-3, and TIGIT and similar levels of SLAMF7 compared to peripheral blood NK cells.Conclusions: The persistent high expression of CD16 and the low expression of CD38 in infused NK cells offers the choice to combine ex vivo activated and expanded NK cells with anti-CD38 antibody therapy without concern for antibody-mediated NK-cell death. Based on these findings, we have started a clinical trial testing this combined therapy (NCT04558931). DisclosuresNahi: XNK Therapeutics AB: Consultancy. Chrobook: XNK Therapeutics AB: Consultancy. Meinke: XNK Therapeutics AB: Consultancy, Current holder of stock options in a privately-held company. Gilljam: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Stellan: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Walther-Jallow: XNK Therapeutics: Other: Shareholder in the company. Liwing: XNK Therapeutics AB: Current Employment. Gahrton: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company; Fujimoto Pharmaceutical Corporation Japan: Membership on an entity's Board of Directors or advisory committees. Ljungman: Takeda: Consultancy, Other: Endpoint committee, speaker; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy; Janssen: Other: Investigator. Ljunggren: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Alici: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company.

Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E

Introduction: Natural killer (NK) cells are powerful immune effectors which induce direct cytotoxicity, promote adaptive immune responses and mediate antibody dependent cellular cytotoxicity (ADCC). Enhancement of NK cell activity against cancer is currently the focus of intense research efforts and strategies include CAR-NK, stimulatory antibodies, cytokines and checkpoint inhibitors.Upregulation of exportin-1 (XPO1) is common in human cancers and high expression is negatively associated with survival in various cancers including diffuse large B cell lymphoma (DLBCL). Targeted inhibition of XPO1 by the selective inhibitor selinexor leads to cancer cell death via accumulation of tumour suppressor proteins in the nucleus, dysregulation of growth regulatory proteins and blockade of oncogene protein translation. The therapeutic efficacy of XPO1 inhibition has led to FDA approval of the oral XPO1 inhibitor selinexor for the treatment of multiple myeloma and DLBCL. The effect of selinexor on NK cell activity has not previously been investigated and was therefore addressed in this study.Methods: The B lymphoma cell lines JeKo-1, SU-DHL-4 and Ramos were incubated with selinexor (50-2000nM) for 18 hours before analysis. Flow cytometry was used to assess cell surface expression of activating and inhibitory ligands for NK cells. For NK based assays, peripheral blood derived NK cells were isolated from healthy donors and incubated with IL-15 (1ng/ml) overnight prior to co-culture with target lymphoma cells for a further 4 hours. Cytotoxicity was assessed using propidium iodide staining of target cells and degranulation of NK cells was assessed by measurement of CD107a. Whole blood samples from colorectal cancer patients (n=11) at pre-treatment and 3 weeks post selinexor monotherapy were assessed by flow cytometry for CD45+CD3-CD19-CD56+ NK cells.Results: Selinexor pre-treatment of target lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4 (2.2 Fold increase, p<0.01), JeKo-1 (2 Fold increase, p<0.01) and Ramos (1.7 Fold increase, p<0.01) cells. In accordance with this, selinexor pre-treatment of target cells also increased the activation of NK cells against SU-DHL-4, JeKo-1 and Ramos cells as measured by CD107a expression in both CD56 bright and CD56 dim NK cell sub-groups.To identify the mechanism behind this, we measured expression of activating and inhibitory ligands for NK cells on SU-DHL-4 cells after incubation with selinexor. No significant changes in expression of activating ligands (MICA/B, ULBP-2/5/6, ULBP-1, Vimentin, B7H6, CD54) were evident. In contrast, selinexor significantly (p<0.001) reduced the surface expression of HLA-E on SU-DHL-4 cells by 50%. Selinexor mediated downregulation of HLA-E was also evident in Ramos (60% reduction, p<0.001) and JeKo-1 cells (20% reduction, p<0.01). HLA-E binds the ITIM containing receptor NKG2A, a key inhibitory receptor for NK cells and subsets of T cells. In accordance with this, selinexor pre-treatment of SU-DHL-4 cells selectively increased NKG2A+ NK cell activation (p<0.01) following co-culture.To examine the effect of selinexor on NK cells in patients, we assessed the proportion of NK cells in the peripheral blood of 11 colorectal cancer patients at pre-treatment and three weeks post selinexor monotherapy. % NK cells of CD45+ peripheral blood lymphocytes following treatment with selinexor was increased 2-fold (Median 5% pre-treatment vs 10% post selinexor). In addition, increased abundance of the less mature and less cytotoxic CD56 bright subset of NK cells was associated with poor response to therapy (Median 4% responders (n=3) vs 20% non-responders (n=8)). Larger patient datasets are required to confirm these effects and this analysis is currently ongoing. The effect of selinexor on NK cells in patients with lymphoma is also currently under investigation.Conclusions:The NKG2A:HLA-E axis is a novel immune checkpoint target and our data identifies that selinexor sensitises lymphoma cells to NK cell mediated killing via disruption of this interaction. In addition, we provide initial evidence that NK cells may be associated with clinical response to selinexor. This data indicates that NK cells may contribute to the therapeutic efficacy of selinexor and that selinexor may synergise with NK cell targeted therapies for the treatment of lymphoma. DisclosuresWalker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Johnson: Morphosys: Honoraria; Kymera: Honoraria; Kite Pharma: Honoraria; Incyte: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Bristol-Myers: Honoraria; Epizyme: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Honoraria; Takeda: Honoraria; Oncimmune: Consultancy; Janssen: Consultancy. Cragg: BioInvent International: Consultancy, Research Funding; GSK: Research Funding; UCB: Research Funding; iTeos: Research Funding; Roche: Research Funding. Forconi: Novartis: Honoraria; Roche: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Gilead: Research Funding. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Blunt: Karyopharm Therapeutics: Research Funding.

117 FT536 Path to IND: Ubiquitous targeting of solid tumors with an off-the-shelf, first-of-kind MICA/B-specific CAR-iNK cellular immunotherapy

BackgroundChimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment, but it is associated with significant dose-limiting toxicities, restricted tumor targeting (limited by specific antigen expression), and, notably, a lack of multi-antigen targeting capability to mitigate tumor associated immune evasion and heterogeneity. Furthermore, dysfunctional starting material, product inconsistency, and small manufacturing lot size limits the application and on-demand availability of CAR-T cell therapy.MethodsTo overcome these considerable limitations, we have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell with a novel CAR that ubiquitously targets cancer cells through canonical stress ligand recognition. We have previously reported FT536 recognizes the conserved α3 domain of the pan-tumor associated antigens MICA and MICB (MICA/B), and is derived from a renewable master iPSC line that contains multiplexed genetic edits to enhance effector cell functionality, persistence, and multi-antigen targeting capabilities via high affinity non cleavable CD16 (hnCD16) mediated antibody dependent cellular cytotoxicity (ADCC). Here we preview the nonclinical study for the investigational new drug (IND) application for FT536.ResultsUtilizing a manufacturing process analogous to pharmaceutical drug product development, we demonstrate FT536 can be consistently and uniformly produced with a greater than 4x10E7 fold cellular expansion per manufacturing campaign. Furthermore, FT536 can be cryopreserved at clinical scale to support off-the-shelf clinical application, with rapid product thaw and immediate patient infusion in an out-patient setting. Functional evaluation demonstrated that FT536 uniquely possesses potent and persistent antigen specific cytolytic activity against an array of solid and hematological tumor lines. Through its hnCD16 modality, FT536 can be utilized in combination with monoclonal antibodies to provide multi-antigen targeting capabilities and in conjunction with chemotherapeutics and/or radiation that augment surface MICA/B expression. In addition, directly thawed and infused FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models, in which tumor control was further enhanced in combination with a therapeutic antibody (figure 1). Finally, ongoing studies utilizing a lung adenocarcinoma model have highlighted the sustained persistence of FT536 in lung tissue up to 33 days following a single dose infusion without the need for exogenous cytokine support.Abstract 117 Figure 1FT536 provides statistically significant in vivo anti-tumor activity which is enhanced in combination with ADCC active monoclonal antibody therapy. (A-B) FT536 significantly reduced the number of lung and liver (not shown) metastases compared to CAR negative iNK control cells in a murine metastatic melanoma model using B16-F10 cells engineered to overexpress human MICA. (C-D) FT536 alone, and in combination with Herceptin, demonstrate significant tumor growth inhibition (TGI) compared to Herceptin alone in an orthotopic xenograft model of human lung adenocarcinoma.ConclusionsCollectively, these studies demonstrate that FT536 is a highly potent, multi-tumor targeting CAR-iNK cell product that is uniform in composition and can be effectively and safely used off-the-shelf for on-demand treatment of multiple solid and hematological malignancies. An IND submission is planned for 2021, with an initial Phase 1 clinical trial to follow.

Identification of the predominant human NK cell effector subset mediating ADCC against HIV-infected targets coated with BNAbs or plasma from PLWH.

The potential of immunotherapy strategies utilizing broadly neutralizing antibodies (BNAbs), such as 3BNC117 and 10-1074, to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating antibody dependent cellular cytotoxicity (ADCC). Utilizing advanced polychromatic flow cytometry, we identified that CD57 positive NK cells from ART-suppressed in People Living With HIV (PLWH) expressed significantly higher levels of the CD16 FcγR receptor, 2B4 ADCC coreceptor, and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC coreceptor (p<0.001, n = 32). Functionally, CD57 positive NK cells from ART-suppressed PLWH with either high or low NKG2C expansion exhibited significantly enhanced degranulation and IFN-γ production against heterologous gp120-coated ADCC targets coated with HIV reference plasma compared to CD57 negative NK cells (p = 0.0029, n = 11). CD57 positive NK cells from control donors lacking NKG2C expansion also exhibited significantly more degranulation and IFN-γ production at every timepoint tested against both heterologous ADCC targets (p = 0.019, n = 9) and HIV-1 infected autologous CD4+ primary T cells coated with BNAbs. Together, our data support CD57 positive and NKG2C positive NK cells as the predominant ADCC effector subsets capable of targeting HIV-infected CD4+ cells in the presence of 3BNC117 and 10-1074 immunotherapy.

Abstract 3260: The preclinical characterization of CS1001, an anti-PD-L1 IgG4 monoclonal antibody and its activity beyond T cell regulation

Abstract CS1001 is a high-affinity, fully human programmed death ligand-1 (PD-L1) blocking IgG4 monoclonal antibody. So far three therapeutic anti-PD-L1 antibodies (atezolizumab, avelumab, durvalumab) have been approved by US FDA in different cancer indications. CS1001 selectively bound to PD-L1 and blocked PD-L1/PD-1 ligation. The crystal structure of CS1001 complexed with human PD-L1 solved at 2.3Å revealed that CS1001 bound perpendicularly to PD-L1 and the binding epitope region well overlapped with the hPD1-binding region. In the Mixed Lymphocyte Reaction (MLR) assay, CS1001 effectively induced the proliferation of CD4+ T lymphocytes and the production of interferon-γ (IFN-γ) and interleukin-2 (IL-2). CS1001 employs IgG4 isotype and lacks antibody-dependent cellular mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Furthermore, the in vivo efficacy of CS1001 in inhibiting tumor growth was demonstrated in the subcutaneous MC38-hPD-L1 murine colon carcinoma (murine MC38 cells expressing human PD-L1) in human PD-1 knock-in mouse (Biocytogen) model. In another dual-humanized animal model, i.e. PD-1/PD-L1 dual knock-in mice implanted with MC38-hPD-L1 tumor (Biocytogen) where human PD-L1 expressed in both tumor and host system, it is interestingly noticed that CS1001 not only enhanced the cytotoxic T cell/regulatory T cell ratio, but also significantly upregulated M1 macrophage &amp; down-regulated MDSC population, suggesting that anti-PD-L1 antibody may play the pro-anti-tumor immune response through modulating myeloid cells that also express PD-L1, thus a non-ADCC/CDC antibody design could be beneficial. CS1001 is currently explored clinically in 5 registration trials as monotherapy or in combination with standard of care therapies. CS1001 monotherapy or combo with chemotherapy or targeted therapy were safe and well tolerated. CS1001 demonstrated promising efficacy in multiple tumor types and support full development of CS1001 as mono/combo therapy for multiple indications in ongoing and planned clinical trials including cHL, ENKTL, GC, EC, and NSCLC. Citation Format: Juan Zhang, Zhenhu Li, Liang Tang, Linlin Liu, Zhaoxiang Ren, Jingshu Ma, Qingmei Shi, Yuanwu Bao, Liang Lu, Yong Zheng, Baotian Yang, Jing Li, Xinzhong Jon Wang. The preclinical characterization of CS1001, an anti-PD-L1 IgG4 monoclonal antibody and its activity beyond T cell regulation [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 3260.

Engineered antigen-specific T cells secreting broadly neutralizing antibodies: combining innate and adaptive immune response against HIV

Background & Aim Over 36 million people worldwide live with HIV, and while antiretroviral therapy (ART) suppresses viremia, it cannot cure infection, because of persistently infected cells that evade immunity and re-seed infection after the cessation of treatment. These patients are thus on life-long therapy, but they remain at increased risk for comorbidities. A more definitive cure based on elimination of persistent cells is thus needed. Two promising strategies to eliminate these cells include adoptive transfer of HIV-specific T cells and passive immunization with HIV-specific broadly neutralizing antibodies capable of mediating antibody dependent cellular cytotoxicity (ADCC). The simultaneous use of both approaches as the basis of a single immunotherapeutic has never been explored. Methods, Results & Conclusion We modified HIV-specific T cells so they are able to express a neutralizing antibody (10-1074) directed against HIV envelope to elicit ADCC. HIV-specific T cells (HST) were expanded from healthy donors by priming T cells with antigen presenting cells expressing overlapping HIV antigens. T cells retained specificity against HIV antigens gag, pol and nef (112±67 IFNg SFC/1 × 105 cells) following transduction (28±21%) with a retroviral vector encoding the 10-1074 antibody. Transduced HIV T cells secreted 10-1074 antibodies (117±90 ng/mL) which displayed ADCC activity (10 +/-4%) and, maintained antigen-specific T cell function. Antibody secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication compared to non-transduced HST (see Figure). In summary, HST can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadening the target range of this immunotherapy.

Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

PS1158 IBRUTINIB PLUS RITUXIMUB IN STEROID REFRACTORY AUTOIMMUNE HEMOLYTIC ANEMIA: ELABORATION OF TREATMENT APPROACH (ISRAEL): AN INTERIM ANALYSIS OF THE ROHS TRIAL

Background:Autoimmune hemolytic anemia (AIHA) and pure red cell aplasia (PRCA) are well recognized autoimmune complications (AIC) of chronic lymphocytic leukemia (CLL). Both AIHA and PRCA complicate patient management, have significant impact on quality of life and can be fatal. Ibrutinib is an irreversible inhibitor of BTK and ITK (interleukin‐2 inducible tyrosine kinase), mediating antibody dependent cellular cytotoxicity, which makes it attractive in treatment of AIC in the context of CLL. We present a pre‐planned interim analysis of a multicentre trial evaluating efficacy of ibrutinib in AIC.Aims:The primary objective of this study is to evaluate efficacy (response rate and its duration) of ibrutinib in combination with rituximab for steroid refractory AIHA and PRCA in patients with CLL.Methods:ISRAEL is an open‐label, multicentre, single arm, two staged study of combination of ibrutinib and rituximab in patients with AIHA/PRCA and underlying CLL. In stage I (induction) patients received ibrutinib 420 mg/day and rituximab (9 weekly with subsequent 3 monthly infusions); in stage II (maintenance) – ibrutinib was only given until relapse, progression or unacceptable toxicity. Inclusion criteria included proven diagnosis of CLL per iwCLL guidelines, the presence of clinically significant hemolysis with positive direct antiglobulin test (DAT) in case of AIHA, the presence of erythroblastopenia, confirmed by immunohistochemical staining with Glycophorin A and platelet level &gt;100.000/mkl in case of PRCA, relapse of AIHA/PRCA after steroids, splenectomy or rituximab or refractoriness to glucocorticosteroid hormones.Results:Thirty three patients with CLL and AIC have been enrolled since February, 2017. The median age was 66 years (range 50 ‐ 82), 14 (42%) were female. 30 patients had AIHA and 3 PRCA. The median hemoglobin level at day of 1st infusion was 8,2 g/dL (range 6,5 – 11,3). Twenty two patients (66%) were transfusion‐dependent. Five patients (15%) had deletion 17p and 8 (24%) deletion 11q. Patients had received a median of 2 (range 1 – 6) lines of prior treatment for AIC, including steroids (n = 31), rituximab (n = 6), splenectomy (n = 3), RCD/R‐CVP (n = 13), alemtuzumab (n = 1), BR (n = 7) and other (n = 8). At time of reporting, 22 patients have completed induction. The median duration of ibrutinib treatment was 281 days (range 22 – 673). Adverse events were reported from 31 participants (94%). Of the 351 AEs, 321 (91,5%) were graded as CTCAE grade 1 – 2 and 30 (8,5%) as grade &gt;3. The latter were represented by infections (n = 7), neutropenia (n = 7), arrhythmias (n = 4), arterial hypertension (n = 7), myalgias (n = 3). Two cases of sudden death were reported. Response of AIHA was evident in all patients. By day 141 4 (21%) patients achieved a DAT‐ve CR, 10 (53%) DAT+ve CR, 1 – DAT‐ve PR and 4 (21%) DAT+ve PR. All PR patients achieved transfusion independence. Two patients with PRCA achieved a CR by days 84 and 150, and 1 patient did not respond. Response of CLL, assessed at day 225 by IWCLL 2008 criteria was available in 19 patients. Seven patients (37%) achieved a CR, including one MRD‐negative case. Twelve patients (63%) were classified as PRs because of residual bone marrow infiltration (n = 12) along with persistent splenomegaly (n = 3) or persistent neutropenia (n = 2). No patients have relapsed so far.Summary/Conclusion:The combination of ibrutinib and rituximab demonstrated high activity in the treatment of patients with relapsed and steroid refractory AIHA with underlying CLL. The safety profile of IR combination corresponded to published data.

The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex

ABSTRACTHere we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.

Impact of pre-existing immunity on the induction of functional cross-reactive anti-hemagglutinin stalk antibodies following vaccination with an AS03 adjuvanted pandemic H1N1 vaccine

The 2009 pandemic H1N1 (A(H1N1)pdm09) virus had a highly divergent hemagglutinin (HA) compared to pre-2009 seasonal H1N1 strains. Most peoples were immunologically naïve to the A(H1N1)pdm09, although hospital workers were exposed early in the pandemic before pandemic vaccines became available. Here, we evaluated how pre-existing antibodies influence the induction of cross-functional HA stalk antibodies following A(H1N1)pdm09 vaccination.Fifty-six healthcare workers vaccinated with AS03 adjuvanted A(H1N1)pdm09 vaccine were chosen by their pre-vaccination priming status (primed HI titers ≥ 40 or unprimed < 40). We analyzed the HA head- and stalk-specific serum IgG subclasses at pre- and 21 days post-vaccination. We also assessed the functionality of the HA stalk-specific antibodies to neutralize virus and mediate antibody dependent cellular cytotoxicity (ADCC).Primed individuals had higher pre-existing HA head- and stalk-specific IgG1, as well as higher ADCC functionality of stalk antibodies. However, following vaccination with the adjuvanted pandemic vaccine, only the quantity of HA head specific IgG1 antibodies were significantly higher than in unprimed individuals. The priming status did not impact upon the cross-reactive HA stalk specific IgG antibodies or their ability to neutralize virus or induce ADCC post-vaccination. In conclusion, a single dose of AS03 adjuvanted pandemic vaccine elicited similar levels of functional antibodies in naïve and primed individuals. These findings are important for understanding the immunological factors that impact or modulate pandemic vaccine responses in humans.

Abstract 4151: Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically

Abstract Among several resistant mechanisms of trastuzumab for HER2+ breast cancer patients, little is clear on resistant mechanism of trastuzumab mediated ADCC. In the current study, we investigated the role of Vacuolar-ATPase subunit ATP6V1B1 on the resistant mechanism of trastuzumab mediated ADCC in HER2+ human breast cancer cell line SK-BR-3. To establish the trastuzumab mediated ADCC resistant SK-BR-3, PBMCs are co-cultured with 1µg/ml of trastuzumab and SK-BR-3 for 4 hours. We removed CD45 positive PBMC by using MACS separation system and obtained ADCC resistant SK-BR-3. After we expanded the resistant cells and repeated these processes several times, we established the ADCC resistant SK-BR-3. The cytotoxicity of ADCC resistant SK-BR-3, which was evaluated by LDH assay, was significantly reduced as compared with that of normal SK-BR-3 (38.7% and 67.4%, respectively). To sort candidate genes for the resistance, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. We investigated ATP6V1B1 mRNA level of SK-BR-3 and resistant cell by using real time PCR and found the 50% reduction of ATP6V1B1 mRNA level in ADCC resistant cells. To evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, we established ATP6V1B1 knockout SKBR3 by using CRISPR/Cas9 system. We cultured from small numbers of the knock out cells and expand to obtain completely knock out cells. We confirmed that the ATP6V1B1 knockout cell line was knocked out by western blotting and immunohistochemistry. We found that ATP6V1B1-knockout SK-BR-3 were significantly less ADCC activity as compared to control SK-BR-3(67.4% and 21.2%, respectively). Furthermore, as the same as the result of ADCC activity, we found that ATP6V1B1-knockout SK-BR-3 was less AICC (antibody-independent cellular cytotoxicity) activity as compared to control SK-BR-3(41.2% and 12.5%, respectively). Based on the in vitro findings, we evaluated the role of ATP6V1B1 expression in HER2 positive breast cancer patients who are treated with neoadjuvant chemotherapy (NAC) containing trastuzumab. Biopsy samples before NAC were stained with ATP6V1B1 immunohistochemically. Four pathological complete response (pCR) cases after NAC and 4 non pCR cases were included. We found that non pCR cases showed significantly week expression of the ATP6V1B1 protein as compared to that of pCR cases. ATP6V1B1 gene is one of the isoforms of the Vacuolar (V)-ATPase which is located in intracellular membrane that regulate the cytosolic pH and membrane trafficking. It is hypothesized that ATP6V1B1 is involved in somewhere through the process of cytotoxicity mediated by perforin and granzymes and now we are trying to explore the mechanisms further. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Takeshi Kotake, Masahiro Hirata, Fengling Pu, Keiko Sakamoto, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2017-4151

Terminally differentiated CD8 effector T cells have NK-like features and are potent mediators of HIV-specific ADCC

Abstract Chronic viral infection and persistent antigenic stimulation is associated with a rise of late-stage differentiated CD8 T cells including a unique subset of CD8 T cells expressing FCg receptor IIIa (CD16). Expansion of CD16+ CD8 T cells is observed in other chronic diseases like HCV, but not HIV-1 infection. CD16 mediates antibody dependent cellular cytotoxicity (ADCC), most notably on NK cells, and is associated with protection from HIV-1 acquisition. Using cryopreserved PBMC from healthy and HIV-1 chronically infected donors from Uganda, we compared phenotypic, transcriptional, and functional changes in CD16+ CD8 T cells. We found that chronic, untreated HIV-1 infection is associated with elevated numbers of CD45RA+CD57+ terminal effector CD16+ CD8 T cells compared to healthy individuals, and display a distinct transcriptional profile in between conventional CD8 T cells and NK cells, characterized by high IKZF2 and KLRF1, and low expression of IL7R. This transcriptional profile translates into a NKp80+ IL-7Rα-surface phenotype and high expression of the Helios transcription factor. Additionally, CD16+ CD8 T cells are capable of mediating ADCC at levels similar to that of NK cells. These CD16+ CD8 T cells are highly activated and correlate positively with expansion of the CD8 T cell compartment and plasma levels of soluble factors of antiviral immunity and inflammation, such as IP-10, TNF, IL-6, and TNFRII. These data indicate that terminally differentiated, effector CD8 T cells acquire innate, NK cell-like characteristics during chronic HIV-1 infection, and suggest that HIV-specific ADCC is part of the repertoire of functions CD8 T cells use to combat the ability of HIV-1 to escape the TCR-specific, anti-viral effects of CD8 T cells.

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

P103 Crosslinking immunogenetics to pharmacogenetics: Fc[formula omitted]RIIIa polymorphism predicts response to Rituximab therapy in mantle cell lymphoma

Aim Mantle cell lymphoma (MCL) and Diffuse large B cell lymphoma (DLBCL) are the most aggressive forms of B-cell lymphoma. Rituximab, a monoclonal anti-CD20 antibody is extensively used as a part of the frontline R-CHOP therapy for treatment of B-cell lymphoma. Rituximab triggers lymphoma cell killing through NK cell mediated antibody dependent cellular cytotoxicity (ADCC). The binding of Rituximab to Fc γ RIIIa during ADCC is influenced by Fc γ RIIIa (158 V/F; Valine to Phenylalanine) polymorphism, where −158 V allele has a high affinity for antibody binding. In the present investigation, we assessed the influence of Fc γ RIIIa 158 V/F polymorphism on the overall survival of MCL and DLBCL patients treated with R-CHOP therapy. Methods A total of 146 R-CHOP treated lymphoma patients (MCL = 42; DLBCL = 104) were accrued. Genotyping of Fc γ RIIIa −158 V/F polymorphism was performed on DNA extracted from diagnostic specimens by resequencing of PCR amplified regions using Sanger’s method. Survival analysis was performed using multivariate cox proportional hazards model. Results MCL patients carrying Fc γ RIIIa ‘V’ genotypes (VV/VF) had significantly better overall survival after R-CHOP therapy in comparison to MCL patients carrying FF genotypes (HR = 0.45, 95% CI = .55–1.1, Fig. 1). However, no correlation was observed in DLBCL patients treated with R-CHOP therapy. Conclusion Fc γ RIIIa −158 V allele (VV/VF genotypes) are predictive of survival of Mantle cell lymphoma patients receiving Rituximab (R-CHOP) therapy. The findings may lead to improved selection of lymphoma patients who will benefit from Rituximab therapy. Download : Download high-res image (150KB) Download : Download full-size image