Inducing Antibody Dependent Cellular Cytotoxicity Research Articles

Abstract 5612: A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study

Abstract Monoclonal antibody (mAb) therapies have become the dominant product class within the biopharmaceutical industry mainly due to their intrinsic capacity to bind endogenous immune receptors and targeted antigens. In fact, this kind of therapeutic agent accounts for one fifth of the FDA’s new drug approvals each year. In addition, their stability and specificity make them the ideal scaffold to develop more complex and efficacious drug modalities such as bispecific antibodies and antibody-drug conjugates. However, in order to advance mAb therapies to the clinic, there are a number of parameters that need to be considered during early-stage development. The purpose of this study was to showcase important preclinical characterization and efficacy experiments aimed at assessing the biological activity, binding profile, mechanism of action and in vivo potency of cetuximab, a mAb therapy. Cetuximab targets EGFR, a well characterized receptor present in the epithelial cell membrane that is overexpressed in several cancer types, such as non-small cell lung cancer, breast cancer and colorectal cancer. In normal tissues EGFR activation initiates several intracellular signaling events involved in development and homeostasis. However, when overexpressed, it stimulates the growth, metastasis and invasion of tumors. For this reason, EGFR has been considered an important target for the development of new drugs. Here we measured the binding affinity of cetuximab to two EGFR expressing cancer cell lines (A-431 and A-549) and its off-target binding to a broad range of full-length human proteins employing Retrogenix Cell Microarray Technology. Using the AlphaLISA system, we observed that cetuximab significantly inhibits EGF binding to EGFR. The consequences of cetuximab treatment on EGF binding and the initiation of the signaling cascade were investigated by looking at the phosphorylation status of EGFR via intracellular staining and flow cytometry. Moreover, we tested the ability of cetuximab to induce Antibody Dependent Cellular Cytotoxicity (ADCC) where the target cell lines were co-cultured with freshly isolated NK cells. ADCC was assessed via both flow cytometry and live cell imaging. Lastly, we studied the efficacy of cetuximab in vivo. A tumor progression mouse model generated from A-431 cells and several Patient Derived Xenografts (PDX) mouse models representing a wide variety of cancers were treated with cetuximab and a significant reduction in tumor growth was observed for most of these cancers. The in vivo efficacy correlated directly with the EGFR expression level determined by IHC. With this case study we have generated a complete and valuable preclinical data package that could be used to advance this mAb therapy into the clinic. Moreover, this study serves as the basis for a streamlined workflow for mAb lead optimization and development as well as comparability studies for biosimilars. Citation Format: David Cobeta Lopez, Kerstin Klingner, Marie Carkill, Robert Nunan, Anya Avrutskaya, Paula Miliani de Marval, Amber Blackwell, Sarah Dawson, Donna Barnes, Jim Freeth, Deborah Bruce, Richard Bazin, René McLaughlin, Julia Schueler, Gemma Moiset, Maria L. Vlaming. A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5612.

Abstract 2102: NFAT2 determines susceptibility of chronic lymphocytic leukemia cells to constitutive and Rituximab-induced NK cell killing

Abstract Genetic loss of the transcription factor NFAT2 (NFATc1), known as important player in lymphocyte development, causes an aggressive course of CLL, as it enhances BCR signaling and leads to highly malignant and proliferative disease (Märklin et al. Nat Commun 2017). To investigate whether and how NFAT2 affects treatment resistance in CLL, we generated a CRISPR/Cas9 based NFAT2 knockout (KO) in MEC-1 CLL cells to model aggressive CLL. In immunodeficient NSG-mice, the NFAT2 KO cells showed a ten-fold higher engraftment in the bone marrow after three weeks compared to scrambled (SCR) controls. Cytotoxicity assays revealed a profoundly higher resistance of NFAT2 KO cells to NK cell killing without detectable differences at the level of recognition as revealed by analyses of NK cell activation, degranulation and IFNy release, which excluded resistance to NK cells was due to altered immunogenicity. As CLL patients are routinely treated with anti-CD20 antibody Rituximab, which mediates its therapeutic efficacy in large part by inducing antibody dependent cellular cytotoxicity (ADCC) of NK cells via secretion of perforin and granzymes. When we exposed the target cells to the cytotoxic content isolated out of NK cells, MEC-1 NFAT2 KO cells showed higher resistance for membrane permeabilization compared to SCR controls. In line, perforin blockade equalized susceptibility to membrane permeabilization of both, NFAT2 KO cells and controls, confirming the involvement of NFAT2 in maintaining membrane integrity. In addition, caspase 3/7 activation was reduced in NFAT2 KO cells upon exposure to NK cell granule content as well as upon treatment with staurosporine. This shows an important role of NFAT2 for resistance of the leukemic cells at early stages of apoptosis. The results on the role of NFAT2 in resistance to NK effector function were confirmed with primary CLL patient cells, which were grouped according to high and low NFAT2 expression followed by treatment with NK cell content. Our results demonstrate that loss of NFAT2 allows CLLs cells to evade NK cell effector function. This holds true for both, constitutive cytotoxicity and therapeutically induced ADCC and is due to lowered susceptibility of the leukemic cells to perforin mediated membrane permeabilization and intrinsic resistance to granzyme induced apoptosis. Thus, NFAT2 loss, which correlates with aggressive clinical course in patients, facilitates resistance of CLL cells to CD20 antibody-based treatment. Citation Format: Samuel J. Holzmayer, Sarah M. Greiner, Kuebra Kaban, Jonas S. Heitmann, Helmut R. Salih, Melanie Maerklin. NFAT2 determines susceptibility of chronic lymphocytic leukemia cells to constitutive and Rituximab-induced NK cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2102.

Mouse CD38-Specific Heavy Chain Antibodies Inhibit CD38 GDPR-Cyclase Activity and Mediate Cytotoxicity Against Tumor Cells.

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

Daratumumab Efficiently Targets NK/T Cell Lymphoma with High CD38 Expression

CD38 expression can be detected on a majority of NK/T cell lymphoma (NKTCL). Based on the clinical data of 94 patients with ENKTL, high CD38 surface expression was an adverse prognostic factor for progression free survival, and these patients had significantly inferior outcomes (Wang, 2015). This corroborates with our own studies showing that relapsed and refractory NKTCL patients almost always exhibit high CD38 expression, thereby proposing that it may be worthwhile to target CD38 as a novel therapeutic option.Daratumumab is a human anti-CD38 monoclonal antibody which has shown significant anti-cancer activity in multiple myeloma. In this study, we investigated the potential cytotoxic effects and mechanisms of actions of Daratumumab in a panel of 10 NKTCL cell lines. Firstly, we identified that cell lines can be stratified into CD38Hi, CD38Mid and CD38Lo as quantified by mRNA expression and CD38 protein surface expression which was qualitatively analysed by FACS and IHC. Daratumumab is able to induce antibody dependent cellular cytotoxicity (ADCC) in a CD38-dependent manner and in a dose-dependent fashion. We also observe a greater induction of antibody dependent cellular phagocytosis (ADCP) activation in correlation with CD38 expression. Furthermore, complement-dependent cytotoxicity (CDC) was significantly triggered by Daratumumab in CD38Hi and CD38Mid cells. However, we found evidence of an additional level of regulation provided by the expression of complement inhibitory proteins(CIPs) CD46, CD55 and CD59 on NKTCL cell lines which can limit and inhibit the induction of CDC regardless of CD38 surface expression levels.All-trans retinoic acid (ATRA) is a vitamin A derivative that binds to retinoic acid receptor alpha leading to the upregulation of CD38 expression. Treatment with ATRA increases CD38 mRNA and surface protein expression in all CD38Hi-Lo NKTCL cell lines. Interestingly CD38Lo cell lines showed the greatest fold increase of CD38 mRNA and surface protein expression. Notably, addition of ATRA to SNK-10, a CD38LoCD55Hi cell line, prior to Daratumumab treatment was able to improve the induction of ADCC but not CDC. This suggests a critical role for CIPS in mediating the induction of CDC in NKTCL.Subsequent investigation for the efficacy of Daratumumab in vivo is currently being performed.We also discovered a synergistic cytotoxic effect in NKTCL CD38Hi cell lines mediated by L-Asparaginase (a component of the current chemotherapeutic regimen in NKTCL) treatment in combination with Daratumumab. Altogether,these results propose a rationale therapeutic option for single or combined Daratumumab treatment in the clinic especially for relapsed and refractory patients. DisclosuresJin:Janssen China R&D: Employment. Yu:Janssen China R&D: Employment. Chen:Janssen China R&D: Employment. Yang:Janssen China R&D: Employment. Chng:Janssen China R&D: Research Funding.

Abstract 532: A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 activity

Abstract Background Adaptive immunity to cancer cells has been shown to form a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether anti-tumor antibodies could be isolated from a patient with metastatic melanoma that was tumor-free for 6 years following adoptive transfer of ex vivo expanded autologous T cells (Verdegaal 2011). Methods Peripheral blood memory B cells were immortalized using AIMM's immmortalisation technology (ectopic Bcl-6 and Bcl-xL; Kwakkenbos 2010) expression and analyzed for the presence of tumor-reactive B cells. Results AT1412 antibody was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 was found to bind the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities and disease progression. The crystal structure of the CD9 large extracellular loop in complex with an AT1412 Fab fragment revealed that AT1412 binds the CD9 epitope in an extended and unfolded conformation. In addition to melanoma, AT1412 binds other tumor types including gastric, colon- and pancreatic cancer. AT1412 was shown to induce antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of cancer cells. In addition, AT1412 demonstrated activation of monocytes, most likely via activation of CD9 on platelets-bound monocytes. In mice carrying a human immune system (HIS-mice; van Lent 2010) AT1412 strongly enhanced tumor rejection of A375 and SKMEL-5 tumor cells. AT1412 treatment was shown to enhance tumor infiltration of CD8 T cells and macrophages. AT1412 efficacy was further synergistically enhanced when combined with an anti-PD1 antibody (nivolumab). Other groups have shown that anti-CD9 antibodies can induce tumor rejection in mice. However, these antibodies could not be advanced due to induction of platelet aggregation. We show that AT1412 does not induce aggregation of both human and cynomolgus platelets. Also affinity matured versions of AT1412 do not induce platelet aggregation, supporting that AT1412 binds a unique epitope on CD9. In addition, we studied the safety of AT1412 during a one month exposure in a weekly administration in cynomolgus monkeys. Besides a transient thrombocytopenia AT1412 was well tolerated up to 10 mg/kg antibody (highest dose tested) and did not lead to other adverse events nor were any coagulation factors affected Conclusions Taken together, applying AIMM's proprietary B-cell immortalization technology we isolated antibody AT1412 that targets a unique epitope on CD9. AT1412 was shown to induce tumor rejection as a single agent and enhances the activity of anti-PD-1 antibodies, while not inducing platelet aggregation. One month exposure of AT1412 in monkeys indicated that AT1412 can be safely administered. Preclinical development of AT1412 is ongoing to initiate clinical evaluation in 2020. Funding Dutch Cancer Society, grant UVA 2010-4822 Citation Format: Remko Schotte, Julien Villaudy, Martijn Kedde, Wouter Pos, Koen Wagner, Viviana Neviani, Daniel Go, Etsuko Yasuda, Christien Fatmawati, Els Verdegaal, Susan van Hal, Yvonne Claasen, Hans van Eenennaam, Pauline van Helden, Piet Gros, Sjoerd van der Burg, Hergen Spits. A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 activity [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 532.

Identifying host-intrinsic resistance mechanisms to therapeutic anti-tumor antibody

Abstract Monoclonal antibody (mAb) therapies have greatly improved outcomes for many cancer subtypes, many of which target tumor-associated antigens (TAA). Trastuzumab targets the TAA human epidermal growth factor 2 (HER2), inducing antibody dependent cellular cytotoxicity and phagocytosis (ADCC/ADCP) as well as interfering with receptor signaling. Despite its reputation as the gold standard treatment for HER2-overexpressing cancers, response rates remain low in metastatic breast cancer (≤30% as a monotherapy, ≤50% with chemotherapy). Multiple studies suggest induction of endogenous anti-tumor immunity is a determinant of therapeutic success. Utilizing the genetically heterogeneous Diversity Outbred (DO) mouse model, we seek to identify host-intrinsic mechanisms of resistance to TAA-mediated immunotherapy. We find a divergent response to αNeu (HER2 homolog) mAb clone 7.16.4 in (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic TUBO mammary carcinoma. We find host genetic background regulates anti-tumor immune activity. ~25% showed complete response (CR) to therapy, ~60% a partial response (PR), and ~15% failed to respond (NR). CR mice were protected from a contralateral rechallenge, suggesting adaptive anti-tumor immunity was promoted by mAb therapy. Peripheral anti-tumor IgG, measured by flow cytometry, and IFNγ producing T cells, measured via ELISpot, also trend toward CR>PR>NR. Genetic linkage analysis using R/qtl2 identified loci associated with robust response to mAb therapy. Genes within these loci may represent potential routes of ingress toward addressing innate and acquired resistance during anti-TAA mAb therapy, particularly as regulators of immunogenicity of ADCC and ADCP. NIH R37 CA220482.

The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide.

Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an "off-the-shelf" therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab.

Prediction for the Risk of Gvhd after Allogeneic HSCT in Patients with ATL Treated By Mogamulizumab

Introduction: Mogamulizumab (Mog) is an anti-CCR4 (C-C motif chemokine receptor 4) antibody, which induce antibody dependent cellular cytotoxicity against cells expressing CCR4. Administration of Mog before allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with adult T-cell leukemia/lymphoma (ATL) is considered to raise the risk of graft versus host disease (GVHD). Since not only ATL cells but regulatory T cells (Tregs) express CCR4, both are targeted by Mog. Tregs play a crucial role in maintaining and suppressing excessive immune responses especially in GVHD, and the depletion of Tregs by Mog is considered to result in the progression of GVHD. The aim of this study is to elucidate the characteristics of GVHD observed in patients treated by Mog, and further tried to predict the risk of GVHD by incorporating the information of serum concentration of Mog, the number of Tregs, and the alteration in Treg fractions (naïve/resting Treg; nTreg, effector Treg; eTreg). Patients and Methods: In this study, we analyzed 25 ATL patients who received allo-HSCT. The serum concentration of Mog was measured by SRL Medisearch (Tokyo, Japan). The fraction of Tregs in peripheral blood mononuclear cells (PBMNCs) were analyzed by flow cytometry (BD FACSCalibur, BD biosciences). Statistical analysis (the cumulative incidence of Grade II-IV GVHD and the number of Tregs) were performed with EZR (Easy R) software (Jichi Medical University, Saitama, Japan). Results: In comparison to allo-HSCT without Mog (Mog-, n=14), the risk of Grade II-IV GVHD was increased in the allo-HSCT with Mog (Mog+, n=11). Stage 3 or more skin GVHD was observed in 4 patients of Mog+ (36 %) and 1 patient of Mog- (7 %), respectively. Notably, stage 3 or more gut GVHD was observed in 6 patients of Mog+ (54 %), and 4 patients died with GVHD or infectious complications, whereas 3 Mog- (21 %) experienced non-fatal gut GVHD. Moreover, cytomegalovirus enteritis was observed only in the Mog+ cases. Mog was detectable in serum for approximately 6 months after the last administration (cut off level; 5.0 ng/ml), and the number of Tregs, especially eTreg (Foxp3 high, CD45RA low) was continuously suppressed during this period. In the Mog+ case, the number of eTregs recovered 180 days after allo-HSCT in most cases. Among 7 patients who received Mog before allo-HSCT, 5 patients who developed gut GVHD were performed allo-HSCT on average 52 (19-87) days after the last administration of Mog. The serum level of Mog was 100-1000 ng/ml in the period, and this concentration of Mog was considered to be critical for the onset of severe GVHD. Conclusion: Use of mogamulizumab before allo-HSCT for ATL patient is a significant risk for severe GVHD, especially gut GVHD. Although the number of patients in this study is limited, our results suggest that the serum concentration of mogamulizumab (≥10 ng/ml) and the number of eTregs in PB at the time of allo-HSCT may enable to predict the risk of developing GVHD. Disclosures Yoshimitsu: Sanofi: Speakers Bureau; Chugai: Speakers Bureau; Takeda: Speakers Bureau; Bristol-Myer-Squibb,: Speakers Bureau; Shire: Speakers Bureau; Novartis: Speakers Bureau. Ishitsuka:Daiichi Sankyo: Consultancy, Honoraria; Alexion: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Eisai: Honoraria, Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; mundiharma: Honoraria; MSD: Research Funding; Celgene: Honoraria; Shire: Honoraria; Mochida: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Eli Lilly: Research Funding; Genzyme: Honoraria; sanofi: Honoraria; Bristol-Myers Squibb: Honoraria; sanofi: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Genzyme: Honoraria; Alexion: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; mundiharma: Honoraria; Eisai: Honoraria, Research Funding; Taiho Pharmaceutical: Honoraria, Research Funding; Mochida: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria; Shire: Honoraria; Otsuka Pharmaceutical: Honoraria; Novartis: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Teijin Pharma: Research Funding; Pfizer: Honoraria; Yakult: Research Funding; Astellas Pharma: Honoraria, Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Janssen Pharmaceutical: Honoraria; Celgene: Honoraria; Teijin Pharma: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Yakult: Research Funding.

AT1412, a Patient-Derived Antibody in Development for the Treatment of CD9 Positive Precursor B-Acute Lymphoblastic Leukemia

Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy, remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19 targeting immunotherapy has significantly improved patient outcomes in (relapsed) B-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. Therefore an ongoing urgency remains for the identification of additional or alternative immunotherapeutic targets for the treatment of ALL. AT1412 is an antibody that was identified from the peripheral blood memory B cell pool of a patient cured of metastatic melanoma after adoptive T-cell therapy, using a B cell immortalization technology (AIMSelect) with ectopic Bcl-6 and Bcl-xL expression as described previously [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo [manuscript submitted]. In addition to melanoma, AT1412 binds other tumor types including B-ALL, gastric, colon- and pancreatic cancer. The target of AT1412 is the tetraspanin CD9, which is expressed by more than half of all B-ALL. Expression of CD9 has been correlated with adverse prognosis [Liang et al. Cancer Biomark. 2018]. We assessed binding of this human CD9 antibody to a panel of ALL cell lines using flow cytometry. Binding of AT1412 to the B-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on the CD9 levels that we detected using a commercial CD9 antibody. AT1412 induced antibody dependent cellular cytotoxicity (ADCC) on these cells, in line with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. Importantly, these findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=30). AT1412 showed binding to 61% of B-ALL samples but not to T-ALL samples. The potential of AT1412 to induce ADCC was tested on patient samples from the same panel. Remarkably, AT1412 induced ADCC of all B-ALL samples it bound to (8 out of 14) and of none of the T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were supported by the observation that AT1412 induced B-ALL cell death when a freshly drawn whole bone marrow sample from a patient with newly diagnosed B-ALL was cocultured with AT1412. AT1412-induced cell death of B-ALL blasts occurred without affecting the monocytic, granulocytic and lymphocytic populations. This cell death was not observed when this patient's ALL blasts were incubated with AML-targeting antibodies. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the bone marrow sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human B-ALL. Taken together, the majority of precursor B-ALL blasts express CD9 and expression of CD9 is associated with a dismal outcome. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Disclosures De Jong: AIMM Therapeutics: Employment. Levie:AIMM Therapeutics: Employment. Schotte:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Pos:AIMM Therapeutics: Patents & Royalties: Patent WO2017119811A1. Go:AIMM Therapeutics: Employment, Patents & Royalties: Patent WO2017119811A1. Yasuda:AIMM Therapeutics: Employment, Equity Ownership. Cercel:AIMM Therapeutics: Employment. van Hal-van Veen:AIMM Therapeutics: Employment. Frankin:AIMM Therapeutics: Employment. Villaudy:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Helden:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Eenennaam:AIMM Therapeutics: Employment. Spits:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Hazenberg:AIMM Therapeutics: Other: Employment/equity of partner/spouse.

CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a “safe” mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an “off-the-shelf”cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab.Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients.Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing.Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with “off-the-shelf” CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. [Display omitted] DisclosuresSarkar:Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Induction of tumor cell-based ADCC resistance to create new tumor cell survival and therapy resistance mechanisms.

130 Background: EGFR overexpression & amplification is seen in many types of cancer. Cetuximab is an EGFR targeting monoclonal antibody that acts by signal perturbation, complement activation & inducing antibody dependent cellular cytotoxicity(ADCC) by recruiting effector cells. Cetuximab resistance has been extensively investigated, but it has proven difficult to study the molecular mechanisms underlying ADCC resistance because such models are not readily available. Methods: We established an EGFR-targeted ADCC resistance model system using NK92-CD16V effector cells, Cetuximab, and the A431 EGFR-expressing squamous cell carcinoma cell line. Continuous ADCC challenge of A431 (26+ passages) yielded an ADCC-resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. To further understand the evolution of resistance to ADCC, we explored the ADCCr cell surface signature, gene expression profile & ability to activate NK cells. Results: We find that ADCCr cells(when compared to its ADCC-sensitive(ADCCs) counterpart) have a modest reduction of EGFR expression, a distinctive transcriptional profile highlighted by overexpression of histone & interferon related genes, altered cell surface expression of important cell adhesion molecules, reduced in vitro proliferation and in vivo growth & resistance that is specific to the formation and function of immune synapses. ADCCr can be partially reversed by the exposure to an inhibitor of the histone acetyl transferase p300 subunit. Interestingly, the ADCCr cell line has reduced sensitivity (compared with ADCCs) to antimetabolite, DNA-intercalating and ABC transporter-regulated cytotoxic agents.The PD-L1 immune checkpoint does not modulate ADCC in this model system. Conclusions: These findings suggest that the induction of resistance to EGFR-targeted immune attack in this model system creates broadly useful survival and potential therapy resistance mechanisms.

A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells.

The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.

A prospective cancer chemo-immunotherapy approach mediated by synergistic CD326 targeted porous silicon nanovectors

Combination therapy via nanoparticulate systems has already been proposed as a synergistic approach for cancer treatment. Herein, undecylenic acid modified thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs) loaded with sorafenib and surface-biofunctionalized with anti-CD326 antibody (Ab) were developed for cancer chemo-immunotherapy in MCF-7 and MDA-MB-231 breast cancer cells. The cytocompatibility study showed no significant toxicity for the bare and antibody-conjugated UnTHCPSi (Un-Ab) NPs at concentrations lower than 200 μg·mL−1. Compared to the bare UnTHCPSi, Un-Ab NPs loaded with sorafenib reduced the premature drug release in plasma, increasing the probability of proper drug targeting. In addition, high cellular interaction and subsequent internalization of the Un-Ab NPs into the cells expressing CD326 antigen demonstrated the possibility of improving antigen-mediated endocytosis via CD326 targeting. While an in vitro antitumor study revealed a higher inhibitory effect of the sorafenib-loaded Un-Ab NPs compared to the drug-loaded UnTHCPSi NPs in the CD326 positive MCF-7 cells, there was no difference in the anti-proliferation impact of both the abovementioned NPs in the CD326 negative MDA-MB-231 cells, suggesting CD326 as an appropriate receptor for Ab-mediated drug delivery. It was also shown that the anti-CD326 Ab can act as an immunotherapeutic agent by inducing antibody dependent cellular cytotoxicity and enhancing the interaction of effector immune and cancer cells for subsequent phagocytosis and cytokine secretion. Hence, the developed nanovectors can be applied for simultaneous tumor-selective drug targeting and immunotherapy.

Abstract 2512: PF-04605412 induces cytokine activation and marginalization of NK cells and macrophages in patients with solid tumor

Abstract Introduction: Integrin Δ5α1 is a fibronectin receptor involved in cell adhesion, proliferation, and survival expressed by cancer cells in addition to tumor-associated macrophages, fibroblasts, and endothelial cells. PF-04605412 is a fully human IgG1 mAb against integrin α1Δ5, with enhanced FcαRIII binding capacity, currently in Phase 1. Pre-clinical studies indicate this antibody has anti-angiogenic properties and is capable of inducing antibody dependent cellular cytotoxicity. We report on correlative biological studies designed to understand the underlying mechanisms for infusion reactions which occurred to some patients during the dose escalation. Methods: This sub-study includes 9 patients (5 female, 4 males; median age 58, ECOG PS 0-1) who received doses of PF-04605412 from 7.5 mg to 34 mg as 2 hrs infusion. Cytokine-like infusion reactions was observed at the first dose level (7.5 mg). To mitigate symptoms, subsequent patients were pre-medicated and the infusion time was extended to two hours. An extensive cytokine panel was assessed at time points corresponding to pharmacokinetic (PK) analysis. Circulating monocytes (CD14+/CD16+) and NK (CD56+/CD16+) cells were evaluated by flow cytometry. Cell-bound PF-04605412 was detected using Fab’2 anti-human antibody. PK analysis has been conducted in four patients to date (7.5 mg (n=3) and 11.25 mg (n=1). Results: Infusion reactions with premedications were mild and included chills, fever, rigors and hypotension (grades 1-2). A decline in white blood cell subsets occurred at the onset of fevers and chills, independent of the dose administered, and normalized within 24 hours post infusion. Activation of cell populations was evident by an increase in percentage of cells expressing CD16 at 24 hour post infusion compared to baseline. Persistent (up to 24 hours) in vivo cell-bound PF-04605412 was observed with the Fab’2 anti-human antibody in all patients. Cmax values for the 7.5 mg and 11.25 mg doses were 671 ng/mL and 933 ng/mL, respectively. PF-04605412 serum concentrations were below quantitation limit 24 hours post infusion at these two doses. Pro inflammatory mediators such as IL 6, IL 8, and TNF alpha typically increased within 1-1.5 hours post infusion and normalized over 10-24 hours. Marked increases were seen in several patients with the first infusion while this was reduced with subsequent doses. There was not a direct relationship between adverse events and the magnitude of cytokine increase. Conclusions: PF-04605412 leads to robust cellular activation and cytokine induction resulting from Fc- FcαRIII engagement of effector cells. Biopsies of metastatic sites pre- and post- treatment will be examined for evidence of effector cell accumulation at the tumor site and will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2512. doi:1538-7445.AM2012-2512

Abstract C221: Glycoengineered anti-EGFR (GA201) elicits enhanced ADCC responses by NK cells from colorectal cancer patients despite tumor-associated impairments to natural cytotoxicity.

Abstract Background: One component of the therapeutic efficacy of IgG1 monoclonal antibodies (mAb) is their ability to induce antibody dependent cellular cytotoxicity (ADCC) by NK cells and other immune effectors bearing the low-affinity Fc RIIIa (CD16) receptor. Clinical studies establish the relevance of ADCC finding widespread polymorphisms that encode a very low affinity CD16 (158F/F) predict limited clinical responses to Cetuximab or Rituximab. Now, mAbs with increased affinity for CD16 can be produced using GlycoMab technology. The altered glycosylation pattern in the Fc domain of these antibodies enhances CD16-binding and ADCC irrespective of genotype. Yet, the potential for enhanced ADCC in patients with advanced colorectal carcinoma (CRC) has not been demonstrated. Considering the potential for tumor-associated immune impairments and treatments-associated leukopenia, we enumerated and characterized NK cells from patients prior to chemotherapy, on active therapy, or following two-lines of standard treatment and compared responsiveness to standard and glycoengineered anti-EGFR mAbs. Methodology: PBMCs were isolated from blood of age-matched healthy donors and 100 patients: either at presentation with metastatic disease; during chemotherapy with FOLFOX, FOLFIRI, or Irinotecan; or at disease progression >4 weeks following second line failure. Laboratory tests included: immunophenotyping for CD3,CD8,CD45,CD4 and CD3,CD56/16,CD45,CD19 markers; measurements of CD16- and NKG2D-expressing NK cells; CD16 158 genotype; and assessment of K562-killing (natural cytotoxicity) and degranulation (CD107+) of NK cells among cryopreserved PBMC in response to K562 or EGFR+ A431 cells with 10 g/ml GlycoMAb anti-EGFR (GA201), wild-type GA201, Panitumumab or Cetuximab. Results: For each group of patients, the proportion of NK cells among lymphocytes remained similar to controls. However, NK cells counts were reduced about half in patients (affected primarily by disease and only slightly by treatment). Per NK cell killing capacity of K562 was substantially impaired in patients prior to therapy and most profoundly among the post-chemotherapy group. ADCC responses were similarly impaired but to lesser degrees. In all cohorts tested, GlycoMAb anti-EGFR was superior to cetuximab in activating more NK cells. The superior ADCC activation of NK cells by GlycoMAb was most pronounced in the patients with chemotherapy-resistant disease. Conclusion: Chemotherapy does not induce gross alterations in the immune phenotype of CRC patients. At all stages of treatment, patients retain CD16+ NK cells capable of ADCC. The impairments in the NK functional responses tested are likely due to host/tumor interaction and not a consequence of treatment. Despite impairments, the best activation of NK cells was always in response to GlycoMAb GA201 for all individuals tested. These findings support testing for enhanced ADCC by GA201 in clinical trials of CRC patients following two lines of chemotherapy or in combination with chemotherapy. Acknowledgements: Simon Hollingsworth, current affiliation Astra Zeneca UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C221.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Rapid High-Resolution Characterization of Functionally Important Monoclonal Antibody N-Glycans by Capillary Electrophoresis

Characterization of the N-glycosylation present in the Fc region of therapeutic monoclonal antibodies requires rapid, high-resolution separation methods to guarantee product safety and efficacy during all stages of process development. Determination of fucosylated oligosaccharides is particularly important during clone selection, product characterization, and lot release as fucose has been shown to adversely affect the ability of mAbs to induce antibody dependent cellular cytotoxicity (ADCC). Here, we apply a general capillary electrophoresis optimization strategy to separate functionally relevant fucosylated and afucosylated glycans on mononclonal antibody products in the presence of several high mannose oligosaccharides. The N-glycans chosen represent those most commonly reported on CHO cell derived therapeutic antibodies. A rapid (<7 min) high-resolution separation of 12 commonly reported and functionally important IgG glycans was developed by systematically evaluating the effects of selectivity (boric acid) and efficiency (linear polyacrylamide) enhancing additives. The approach can be used to rapidly optimize capillary electrophoresis separation of other glycan mixtures. Following optimization, the method was applied to overnight sample processing for automated 96 well plate-based glycosylation analyses of two nonproprietary therapeutic monoclonal antibodies, demonstrating ruggedness and suitability for high-throughput process and product monitoring applications.

Granulocyte-macrophage colony stimulating factor in autologous bone marrow transplantation: augmentation of graft versus tumor effect via antibody dependent cellular cytotoxicity.

Several immunomodulatory approaches have been explored with the aim of inducing a graft versus tumor effect (GVT) in autologous bone marrow transplantation (ABMT). Granulocyte-macrophage colony stimulation factor (GM-CSF) has been reported to induce antibody dependent cellular cytotoxicity (ADCC) via stimulation of peripheral blood neutrophils, lymphocytes, and monocytes. We investigated the role of GM-CSF in inducing ADCC via bone marrow (BM) macrophages against murine and human tumor cells both in vitro and vivo. Our data shows that stimulation of murine BM macrophages with GM-CSF induced a potent ADCC against a murine melanoma in vitro. Treatment of tumor bearing mice with a combination of GM-CSF and antibody against melanoma resulted in a significant reduction in the dissemination of melanoma both in a nontransplant as well as in a transplantation setting. Adoptive transfer of BM macrophages obtained from animals undergoing treatment with GM-CSF plus antibody significantly reduced the spread of tumor in secondary recipients; this effect was seen only in mice undergoing bone marrow transplantation. GM-CSF treatment of human BM macrophages induced a significant ADCC against a human melanoma and a lymphoma in vitro, as well as against a human lymphoma implanted in nude mice. Treatment with GM-CSF alone or with antibody alone was ineffective in controlling the dissemination of tumors both in transplantation as well as in nontransplant situations. These observations indicate that treatment with GM-CSF plus tumor specific monoclonal antibodies after ABMT may induce a GVT effect and bring about the eradication of residual disease.