Antibody-directed Cellular Cytotoxicity Research Articles

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

Neutrophils and their friends.

Neutrophils and their friends.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

Combined immunotherapy and radiation induce cellular and humoral immune responses to tumor-associated antigens and prolong survival of patients with advanced cancers.

Immunotherapy is effective in treating patients with a variety of cancers We developed and employed an immunotherapeutic protocol that combined intra-tumor injection of autologous immature dendritic cells , with standard of care radiation to treat patients with a variety of different advanced solid tumors. Pre and post treatment peripheral blood mononuclear cells ( PBMC) were assayed for their capacity to kill autologous tumor cell lines that had been established from patients with different advanced solid tumors. PBMC obtained post treatment were cytotoxic to autologous but not allogeneic tumor cell lines. Cytotoxic activity increased as relative numbers of CD8+-CD56+ dual positive lymphoid cells increased in PBMC obtained post treatment. The autologous tumor cell line directed cytotoxicity was shown to be mediated by both innate and adaptive immune mechanisms. Cytotoxicity of PBMC obtained post treatment was enhanced after co-culture with autologous and not allogeneic tumor cell line lysates. Antibody activity to mesothelin, a tumor associated antigen expressed in a variety of solid tumors, increased in post treatment serum samples from lung cancer patients. These sera mediated antibody directed cellular cytotoxicity ( ADCC) to mesothelin expressing tumor cell lines. SSurvival of 96 patients with a variety advanced cancers patients treated on this protocol over a 3 year period and followed for 1000+ days was significantly increased in patients with low tumor burden ( lesion at <5 sites with diameters < 3cm) (Km= <0.0001) regardless of malignancy. These findings support application of this combination of immunotherapy and radiation in the treatment of cancer patients with advanced disease

Low Dose Cyclophosphamide in Combination with Elotuzumab - a Novel Immunotherapeutic Strategy for Multiple Myeloma

IntroductionCyclophosphamide (CTX) is a widely used anti-neoplastic, performing as an alkylating agent at high doses and immunomodulatory agent at low doses 1.. Combining CTX with monoclonal antibody (mAb) therapy has proven beneficial in potentiating relapsed and/or refractory multiple myeloma (RRMM) therapies, with daratumumab-directed MM cell death enhanced in the presence of CTX 2,3.. Elotuzumab (ELO), the second mAb approved for treating RRMM, promotes MM cell clearance by enhancing macrophage-mediated phagocytosis and CD16- and SLAMF7-directed NK cell cytotoxicity. ELO has been approved for use alongside dexamethasone and lenalidomide 4 or pomalidomide (POM) 5.. However, potential therapeutic benefits of ELO in combination with immunomodulatory drugs such as CTX and POM have yet to be examined. Our research investigates, the efficacy of combining low-dose CTX, alone or in combination with POM, and ELO in enhancing macrophage and NK cell infiltration and function in the MM tumour microenvironment.Materials and MethodsMultiple myeloma cells (MM1S and H929) were treated with low-dose CTX and/or POM for 24hrs, washed to remove residual drug and resuspended in fresh media for tumour cell secretome (TCS) generation. Direct effects of CTX and/or POM on surface expression of checkpoint proteins (PD-1 and CD47) on MM cells was assessed by mean fluorescent intensity (MFI) flow cytometry. CD32/CD64 receptor expression on THP-1 macrophages, NKG2D, CD2, DNAM-1, CD96 and KIR2DL1 receptors on KHYG1 and primary NK cells, were measured using flow cytometry as a measure of activation. Migration of serum-starved, CFSE-labelled macrophages and NK cells towards CTX and/or POM TCS was assessed after 4hrs, with total number of migrated cells quantified using the Accuri flow cytometer. Immune cell function following indirect conditioning of macrophages/NK cells with MM cell TCS was measured by quantifying antibody-directed cellular phagocytosis (ADCP) or antibody-directed cellular cytotoxicity (ADCC), respectively. Conditioned immune cells were co-cultured with MM cells in a 2:1 effector to target ratio for 4hrs in the absence/presence of mAbs (ELO, nivolumab and anti-CD47), after which MM cell clearance was quantified by flow cytometry and presented as relative uptake (ADCP) and cytotoxicity (ADCC). One-way ANOVA statistical analysis was performed, followed by Tukey post hoc tests, with significance recognized at p<0.05.ResultsDirect treatment of MM cells with CTX increased surface expression of immune evading checkpoint proteins PD-1 and CD47 (p<0.05,n=3). POM monotherapy did not alter PD-1/CD47 expression, however dual therapy of CTX and POM supported the CTX-driven effect (p<0.001,n=3). Expression of CD32/CD64 macrophage activation markers was significantly increased on THP-1 cells following CTX-TCS conditioning (p<0.001,n=3). POM altered CD32, but not CD64, however dual treatment with CTX and POM significantly increased expression of both CD32 and CD64 (p<0.001, n=3). Migration of macrophages towards CTX-TCS was enhanced in a dose-dependent manner (p<0.01,n=3). CTX and POM dual therapy supported this CTX driven effect (p<0.001,n=3). Migration trends of both primary and KHYG1 NK cells were also increased towards the secretome from CTX treated MM cells. ADCP and ADCC were increased by CTX alone or in combination with POM (p<0.05, n=3). Effects of CTX on ADCP were not significantly enhanced by ELO, however ELO did significantly augment ADCC by CTX-conditioned primary NK cells (p<0.05,n=3). Given the increased expression of PD-1 and CD47, we investigated if the inclusion of nivolumab and anti-CD47 mAbs potentiated ADCC. Although ADCC was increased in all combinations, there was no significant difference between ELO alone versus ELO in combination with either nivolumab or anti-CD47.ConclusionsLow-dose CTX and POM potentiated the immunomodulatory effects of ELO, with NK-directed cytotoxicity of MM cells enhanced in the presence of this mAb. Our data therefore indicates that the inclusion of low-dose CTX and or POM in combination with ELO could be a novel immunotherapeutic strategy for treating RRMM.

Abstract 1497: A unique strategy to arm iPSC-derived CAR-T cells to perform antibody-directed cellular cytotoxicity to facilitate multi-antigen targeting

Abstract Although chimeric antigen receptor (CAR)-T cells have been transformational for several liquid tumors, their effectiveness in treating a variety of solid tumors has been limited in part because the heterogeneity of tumor-associated antigens found in many bulky tumors represents a major obstacle to the development and the delivery of effective CAR-T cell therapies in cancer. Here we demonstrate that iPSC-derived CAR-T (CAR-iT) cells are an ideal off-the-shelf approach that can be engineered to perform antibody-dependent cell-mediated cytotoxicity (ADCC) to facilitate multi-antigen targeting with the introduction of various monoclonal antibodies (mAbs). Under this strategy, while the CAR is equipped to predominantly target antigen 1, targeting additional tumor associated antigens through the use of various mAbs can be leveraged through ADCC. To this end, CAR-iT cells were genetically edited at the iPSC stage to uniformly express a novel high-affinity variant (158V), non-cleavable CD16A (hnCD16) Fc receptor to recognize and bind to the Fc fragment of mAbs and facilitate ADCC. When combined with therapeutic mAbs targeting HER2, EGFR, or PDL1, the cytolytic efficacy of hnCD16+ CAR-iT cells was significantly enhanced compared to parental CAR-iT cells in several tumor models, including ovarian, HER2+ breast, and triple-negative breast cancers, underscoring the flexibility of a combined CAR and ADCC – mediated targeting approach. Importantly, although both CD16A and CAR signaling share common CD3ζ pathways, simultaneous activation did not appreciably attenuate the activation and effector function of CAR-iT cells. Moreover, hnCD16+ CAR-iT cells demonstrated enhanced control in an established triple negative breast cancer solid tumor xenograft model. The control of antigenically heterogenous tumors by hnCD16+ CAR-iT cells was also significantly improved when compared to parental CAR-iT cells. To confirm selectivity and avoidance of immunoglobulin interference with functional output, various in vitro cellular cytotoxicity assays were conducted with IgG1 to confirm that hnCD16 requires crosslinking to elicit ADCC while maintaining full potentiation of CAR function. Additional in vitro and in vivo studies are ongoing and will be presented. Collectively, the data demonstrates that CAR-iT cells can be further modified with hnCD16 Fc receptor to elicit ADCC and the combination of CAR and hnCD16 is a unique approach to tackling tumor heterogeneity often found in bulky tumors. Citation Format: Martin Hosking, Soheila Shirinbak, Joy Grant, Wen-I Yeh, Yijia Pan, Mochtar Pribadi, Amit Mehta, Amanda Yzaguirre, Philip Chu, Shohreh Sikaroodi, Angela Gentile, Bruce Walcheck, Jianming Wu, Anhco Nguyen, Bahram Valamehr. A unique strategy to arm iPSC-derived CAR-T cells to perform antibody-directed cellular cytotoxicity to facilitate multi-antigen targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1497.

Abstract CT162: ACCEPT: A phase Ib/II combination of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with diffuse large B-cell lymphoma (DLBCL)

Abstract Background DLBCL is the most common non-Hodgkin lymphoma (NHL) comprising one third of all adult NHL cases, accounting for an annual incidence in the UK of approximately 5,000 patients. R-CHOP has been accepted as the international standard first-line regimen, with cure rates of around 60-70%. There is a need to further improve efficacy of first line treatment. Chronic and tonic B-cell receptor (BCR) signaling has been identified as a feature of DLBCL. Preclinical models of this lymphoma have demonstrated cytotoxicity through interruption of BCR-signaling. Inhibition of Bruton's tyrosine kinase (BTK), downstream of the BCR has proven active clinical trials in DLBCL, particularly in the activated B-cell subtype. The second generation BTK inhibitor, acalabrutinib, has demonstrated improved target specificity and bioavailability, with potential for better efficacy and tolerability compared to earlier agents. It is appealing to combine this targeted agent with R-CHOP in untreated de novo DLBCL to understand its safety profile and efficacy. Method Patients must be 16 years or older, treatment naive with histologically confirmed DLBCL. All will receive 6 cycles of R-CHOP chemotherapy on a standard 21-day schedule, with the addition of acalabrutinib in cycles 2-6, to allow determination of the subtype by whole transcriptome gene expression profiling during cycle 1. This will be followed by a continuation phase of acalabrutinib only, for 2 cycles of 28 days. The primary objective of the phase I is to establish a recommended phase II dose of acalabrutinib in combination with R-CHOP. The primary objective of the phase II is to assess activity of the combination (by overall response rate) and determine additional safety information. Secondary endpoints include duration of response, PFS and OS and their relation to the cell of origin subtype, and BTK receptor occupancy in peripheral blood mononuclear cells and tumor measured by a drug analogue enzyme-linked immunosorbent assay. The effect of acalabrutinib on antibody-directed cellular cytotoxicity mediated by rituximab will be measured in vitro during treatment. The trial is coordinated by the Cancer Research UK Southampton Clinical Trials Unit. It will recruit up to 24 patients during phase I in a modified classical 6+6 design, and 15 patients during phase II. This is an investigator initiated study that has been granted free access to investigational medicinal product, trial Management and translational study support through a grant from Acerta Pharma B.V. (IST-LY-801) and has endorsement from Cancer Research UK (CRUKDE/16/006). Trial registration: ISRCTN11965217 Progress Phase I of the study opened in April 2017 and has recruited 6 eligible patients to the first cohort at a dose of 100mg acalabrutinib once daily, with a further 4 patients recruited to the second cohort at a dose of 100mg twice daily. Citation Format: Peter W M Johnson, Joshua Caddy, Tom Cumin, Bridgen Merton, Tom Maishman, Angelic Galanopoulou, Gareth Griffiths, Andrew Davies. ACCEPT: A phase Ib/II combination of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT162.

Abstract 3755: Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy

Abstract The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (&amp;gt;95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (&amp;gt;95% CD16+ hnCD16-NK vs. &amp;lt;10% CD16+ PB-derived NK cell, upon target cell-mediated activation), and demonstrated enhanced antibody binding compared to PB-derived NK cells expressing the low-affinity variant. In addition to increased expression of the cytolytic molecules perforin and granzyme B and enhanced direct cytotoxicity against tumor targets, hnCD16-NK cells displayed superior ADCC capacity and cytokine production in response to CD16 stimulation. Importantly, manufacture of hnCD16-NK cells was proven to be highly scalable, delivering up to 107 fold expansion over a 35 day period. The maintained proliferative capacity can be in part associated with longer telomere length seen in hnCD16-NK cells. Furthermore, deletion of classical human leukocyte antigen molecules and ectopic expression of immunosuppressive proteins engineered at the iPSC level provided the ability of hnCD16-NK cells to potentially overcome the host histocompatibility barrier and to improve persistence in the allogeneic setting. In conclusion, the preclinical data presented herein highlight the therapeutic value of hnCD16-iNK cells as an ideal ADCC-mediated “off-the-shelf” NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity, manufacturing reliability and preclinical efficacy. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [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 3755. doi:10.1158/1538-7445.AM2017-3755

Abstract 611: Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGFβ1 (TGFβ TRAP)

Abstract Background: Evasion of antitumor immunity is widely regarded as a hallmark of cancer. Tumor cells can avoid immune destruction by upregulating the immune checkpoint molecule PD-L1, and antibodies targeting the PD-1/PD-L1 axis have demonstrated clinical efficacy in several tumor types. Antitumor immunity can also be suppressed by TGFβ, a pleiotropic cytokine that reduces innate and adaptive antitumor immune responses. Additionally, TGFβ impacts tumor cells by activating a differentiation program called epithelial-mesenchymal transition (EMT), in which tumor cells lose epithelial features and gain mesenchymal characteristics. The gain of mesenchymal traits is associated with metastasis, stemness, and resistance to multiple therapeutic modalities, including chemotherapy and radiation. The objective of this work is to investigate the therapeutic potential of M7824 in non-small cell lung cancer (NSCLC). M7824 is a first-in-class bi-functional immunotherapeutic agent designed to target two negative regulatory pathways in immunosuppression. It is a novel fusion protein comprised of the extracellular domain of human TGFβRII covalently linked to the C-terminus of the Fc domain of the fully human anti-PD-L1 IgG1 antibody avelumab. Methodology: In vitro assays were conducted using NSCLC cell lines treated with TGFβ1 alone or in combination with avelumab or M7824 to assess the ability of M7824 to revert features of tumor cell EMT, including molecular marker expression, cell proliferation, and response to various chemotherapies. The ability of M7824 to mediate antibody-directed cellular cytotoxicity (ADCC) of TGFβ1-treated cells in vitro was also assessed. Results: Treatment with TGFβ1 induced mesenchymal marker expression, reduced cell proliferation, and enhanced resistance to docetaxel, paclitaxel, and gemcitabine. Treatment with M7824, but not avelumab, was able to both prevent and revert these effects of TGFβ1. M7824 also exhibited the ability to mediate ADCC of TGFβ1-treated cells in vitro. Conclusions: M7824 effectively antagonizes TGFβ1-mediated tumor cell mesenchymalization, including resistance to chemotherapy. These results suggest that M7824 has the potential to relieve immunosuppression and block treatment-resistance induced by TGFβ1 in patients with NSCLC. Citation Format: Justin M. David, Jeffrey Schlom, Yan Lan, Claudia Palena. Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGF&amp;#9461 (TGFβ&amp;#946 TRAP) [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 611. doi:10.1158/1538-7445.AM2017-611

Abstract POSTER-THER-1433: Overcoming resistance to antibody targeted therapy in ovarian cancer

Abstract Targeted therapies utilizing monoclonal antibodies (mAbs) are now the major class of successful therapeutics for treating a variety of malignancies. Mechanistically, mAbs can have direct antitumoral activity but often their effectiveness relies upon antibody-dependent cellular cytotoxicity (ADCC). For instance, Trastuzumab is a therapeutic monoclonal antibody directed against HER2 that is well-tolerated and useful in the treatment of HER2-positive breast cancer, where efficacy is mediated in part by ADCC. In contrast, Trastuzumab is relatively ineffective in the treatment of epithelial ovarian cancer (EOC) where virtually all cancer cells express detectable levels of HER2. Poor response to mAb therapy in EOC and other types of cancers may be explained in part by diminished numbers and cytotoxic potential of NK cells in cancer patients, compared to healthy individuals. Therefore, reports of poor antitumor effect of Ab-therapy in cancer led us to hypothesize that the development of potent effector cells with the capacity to bind tumor-bound mAb and mediate strong antibody-directed cellular cytotoxicity would markedly improve the efficacy of mAb-targeted therapy for EOC. In order to expand applications for T cell-based immunotherapy and to enhance ADCC, we developed novel effector T cells engineered to express Fc binding immune receptors (FcIR) containing human Fc receptor of low FcγRIIIA (CD16), intermediate FcγRIIA (CD32) or high affinity FcγRI (CD64) molecules fused to intracellular TCR and co-stimulatory signaling domains in order to enable cytotoxic T cells to mediate strong mAb-directed cytotoxicity against antigen-expressing tumor cells. Following FcIR gene transduction, all forms of FcγRs were efficiently expressed on the surface of primary human T cells which allowed these cells to be armed with mAb. Trastuzumab-armed FcIR T cells specifically recognized HER2+ cancer cells, as did unarmed FcIRs but only when the cancer cells were first pre-bound with Trastuzumab. Addition of a CD28 cytoplasmic domain juxtaposed to the TCR CD3z signaling moiety increased IFN-g secretion by FcIR-28z transduced T cells following encounter with antigen-bound mAb on the cancer cell surface. Notably, T cells expressing a high affinity FcIRI (CD64) demonstrated the greatest specific anti-tumor reactivity in comparison to cells expressing FcγRIIA (CD32) or FcγRIIIA (CD16) FcIRs. In addition, FcIRI (CD64) T cells exhibited stronger specific lytic activity than NK cells, even at low antibody concentrations. Further, co-administration of FcIRI (CD64) FcIR-28z T cells with immunotherapeutic mAb, Trastuzumab, exerted strong antitumor activity in vivo, completely eliminating HER2+ tumor. In summary, our results show that ADCC can be enhanced by human T cells engineered to express an FcIR and that this novel approach may overcome issues of resistance to mAb-targeted therapies including those utilizing Trastuzumab. Citation Format: Katarzyna Urbanska, Mathilde Poussin, Caitlin Stashwick, Jenessa B. Smith, Daniel J. Powell Jr. Overcoming resistance to antibody targeted therapy in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1433.

Evaluation of Otlertuzumab (TRU-016), an Anti-CD37 ADAPTIRTM Therapeutic in Preclinical Combination Studies with Kinase Inhibitors and a Next Generation Anti-CD20 Mab in Vitro and in Animal Models of Non-Hodgkin’s Lymphoma

Background: CD37 is a 50-55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. Otlertuzumab is a novel humanized anti-CD37 therapeutic, built on the ADAPTIRTM (modular protein technology) platform that has been shown to mediate caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be distinct from CD20 therapies. In addition, otlertuzumab results in killing through antibody directed cellular cytotoxicity (ADCC), mediated in part by NK cells. The therapeutic potential of otlertuzumab in the treatment of chronic lymphocytic leukemia (CLL) is currently being investigated in Phase 2 clinical studies in combination with bendamustine or rituximab. Preclinical vitro and in vivocombination studies for NHL to evaluate otlertuzumab in combination with other emerging drugs including kinase inhibitors (PI3K and BTK) and the next generation anti-CD20 mAb obinituzumab are reported here.Methods: The ability of otlertuzumab to interact and increase malignant B cell killing with kinase inhibitors was investigated, including a pan PI3K inhibitor (LY294002), a PI3K delta inhibitor (CAL101(GS-1101, idelalisib)), a PI3K delta/gamma inhibitor (IPI-145, (INK1197)) and an inhibitor of BTK (PCI-32765). Combination studies were assayed in vitro using the Minos (mantle cell lymphoma), DoHH-2 (follicular lymphoma) and Ramos (Burkitt’s B cell lymphoma) cell lines. In addition, studies were performed in vitro to test the combination of otlertuzumab and obinituzumab. Individual drugs were tested alone or in combination with otlertuzumab. Combination index analyses were performed for drug combinations over the 20-90% effect levels. To determine whether in vitro synergy could be repeated in vivo, the tumor line with the best in vitro combination characteristics was utilized in xenograft tumor models and treated with otlertuzumab ± LY294002 or otlertuzumab ± PCI-32765 or otlertuzumab ± obinituzumab.Results: Combination index analyses determined that the killing effects of otlertuzumab were synergistic with the pan PI3K inhibitor LY294002, demonstrating comparable results in all three cell lines tested in vitro. The combination of otlertuzumab and the BTK inhibitor PCI-32765 demonstrated synergy in vitro with the Ramos and Minos cell lines. The PI3K delta inhibitor (idelalisib) also demonstrated synergistic activity with all three cell lines when tested in combination with otlertuzumab in vitro. Finally, combination index analyses determined that the killing effects of otlertuzumab were synergistic with the obinituzumab, demonstrating comparable results in all three cell lines tested in vitro.In vivo, the combination of otlertuzumab plus the pan PI3K inhibitor (LY294002) or otlertuzumab plus obinituzumab resulted in greater efficacy relative to each agent alone in the DoHH-2 xenograft tumor models. The combination of otlertuzumab with PCI-32765 resulted in significant delay of tumor outgrowth compared to PCI-32765 alone in the MINO xenograft model of NHL. In vivo results indicated that the in vitro synergy results were applicable to a more complex in vivodisease model.Conclusions: Otlertuzumab tested in combination with multiple kinase inhibitors or next generation anti-CD20 had increased cell killing of NHL cell lines in vitro over that observed for each agent alone. Furthermore, the combinations of otlertuzumab with either obinituzumab, LY294002 or PCI-32765 displayed greater anti-tumor activity in vivo than each of the agents alone. These results provide preclinical rationale for the potential combinations of otlertuzumab with several emerging therapeutics for the treatment of NHL and related B-cell malignancies, including CLL. DisclosuresGross:Emergent BioSolutions Inc: Employment.

Metabolic Engineering of Monoclonal Antibody Carbohydrates for Antibody–Drug Conjugation

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

Phase II study of irinotecan plus panitumumab as second-line therapy for patients with advanced esophageal adenocarcinoma (EAC).

TPS206 Background: Esophageal carcinoma is highly lethal and the incidence is increasing, especially the subtype of EAC. Our group demonstrated that overexpression of the epidermal growth factor receptor (EGFR) correlates with lower survival in EAC. Panitumumab (P) is a high affinity fully human IgG2 monoclonal antibody directed against human EGFR. Cetuximab combined with irinotecan (I) is effective for second-line treatment of advanced colorectal cancer (CRC). P alone is active in EGFR expressing CRC. Our group has empiric evidence for the activity of cetuximab + I. As such, we composed this phase II study of P + I as second-line therapy for advanced EAC. Methods: The primary endpoint is overall response (OR) as measured by RECIST (imaging every 2 cycles). Secondary endpoints include overall survival, safety/toxicity and correlative studies. Patients with a history of one prior treatment will be treated with P 9 mg/m2 on day 1 and I 125 mg/m2 on days 1 and 8 of each 21 day cycle to a maximum of 6 cycles. Inclusion criteria include confirmed EAC, measurable disease, no prior I or P, PS <2 and normal organ function. Exclusion criteria include use of CYP 3A4 inhibitors, prior anti-EGFR therapy, uncontrolled intercurrent illness. A Simon two-stage design uses power of 80% and a type I error of 0.05. 18 patients will be tested in the first stage, and the trial will be terminated if 2 or fewer respond. In the second stage, a total of 43 patients will be studied. Correlative Studies: Research blood and tissue will be obtained pre-treatment and during the third week of cycle 2. Blood will be studied for the role of FcgR receptors on the effector cell surface leading to antibody-directed cellular cytotoxicity. Tumor tissues will be analyzed for pharmacodynamic (PD) and pharmacogenomic (PG) effects by measurement of EGFR pathway components using FISH and IHC and by kras mutation analysis. Enrollment: The study recently opened at UPMC, and two patients are enrolled. Significance: Along with the primary endpoint of OR, the goal of this trial is to utilize sequential tumor and blood sampling to study PD and PG correlates. The goal is to improve patient selection for targeted therapies in future trials. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen

Personalized immunotherapy: a siren myth?

10.2217/PME.09.39 © 2009 Future Medicine Ltd “...the antigenic characteristics of tumor cells can be perceived by the innate and cognate immune systems, which represent the extrinsic barriers against tumor progression, or at least a substantial component of the host–tumor equilibrium.” Antoine Tesniere1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Guido Kroemer1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Thomas Tursz Departement de medecine, Institut Gustave Roussy, Villejuif, France

A phase II trial of trastuzumab in combination with low-dose interleukin-2 (IL-2) in patients (PTS) with metastatic breast cancer (MBC) who have previously failed trastuzumab

Trastuzumab mediates the lysis of HER2-expressing breast cancer cell lines by interleukin-2 (IL-2) primed natural killer (NK) cells. We hypothesized that IL-2 would augment the anti-tumor effects of trastuzumab in MBC in patients who had progressed on or within 12 months of receiving a trastuzumab-containing regimen. Secondary objectives were to measure antibody-directed cellular cytotoxicity (ADCC) against HER2 over-expressing target cells, and to measure serum cytokines. Patients received trastuzumab (4 mg/kg intravenously (IV)) every 2 weeks in combination with daily low-dose IL-2 (1 million IU/m(2) subcutaneously (SC)) and pulsed intermediate-dose IL-2 (12 million IU/m(2) SC). Samples were analyzed for NK cell expansion and ADCC against a HER2-positive breast cancer cell line. In addition, interferon-gamma (IFN-gamma), mRNA expression in peripheral blood mononuclear cells (PBMC) and the following serum cytokines were measured: IFN-gamma, monokine-induced by IFN-gamma (MIG), and interferon-inducible protein ten (IP-10). The median number of treatment cycles was four (range 1-23) and the treatment was well tolerated. There were no objective responses. NK cells were not expanded and ADCC was not enhanced. Eight (62%) patients had a twofold or higher increase in mRNA transcript for IFN-gamma, two (15%) patients had elevated serum levels of IFN-gamma and 12 (92%) had increases angiogenic MIG and IP-10. In trastuzumab-refractory patients adding IL-2 did not produce responses and did not result in NK cell expansion. However, these patients had the ability to respond to IL-2 as evidenced by increases in IFN-gamma transcripts and chemokines. The lack of NK cell expansion may explain the absence of clinical benefit.

A phase II trial of trastuzumab and low dose interleukin-2 in patients with metastatic breast cancer who have previously failed trastuzumab

3028 Background: Trastuzumab (TZB) mediates lysis of Her2-pos. breast cancer cells by interleukin-2 (IL2) primed natural killer (NK) cells. We hypothesized that IL2 would augment the effects of TZB. The objective of this study was to determine the response rate to and toxicity of low dose IL2 plus TZB in patients with Her2-pos. metastatic breast cancer who had progressed within 12 months of receiving TZB. Also, we measured the ability of patient (pt) peripheral blood mononuclear cells (PBMC) to produce cytokines and conduct antibody-directed cellular cytotoxicity (ADCC) against Her2-pos. target cells. Methods: Pts received low (1 million IU/m2) and intermediate dose (12 million IU/m2) IL2 plus TZB (4 mg/kg) in each cycle. Low dose IL2 was given on days 2–7 and days 12–21 of cycle 1, and days 4–14 of later cycles. Intermediate dose IL2 was given on days 9–11 of cycle 1, and on days 1–3 of later cycles. TZB was given on day 1 and 8 of cycle 1, and on day 1 of later cycles. Pt plasma and PBMCs were analyzed for levels of serum cytokines and ADCC against a TZB-coated cell line, respectively. Results: Thirteen pts with median age of 52 (range 30–71), and a median of 1 (range 1–2) prior TZB-containing regimens were enrolled. The median number of cycles completed was 4. Five pts had grade 3 or greater toxicities, including fever, nausea, vomiting, diarrhea, dyspnea, and hypercalcemia. Twelve pts had progression of disease, and 1 pt withdrew consent. ADCC of pt PBMCs against a TZB-coated cell line was enhanced in only 1 pt. Two pts had elevated plasma levels of interferon-gamma (IFNg), and one of these pts had a 20-fold increase in IFNg transcript levels. The antiangiogenic chemokines MIG and IP-10 rose significantly over baseline in 11 pts. Conclusions: TZB given with low and intermediate pulse-dose IL2 did not produce a robust immune or clinical response in this pt population. A clinical trial in a TZB-naive population may help to determine the immune effects of this combination of IL2 plus TZB. No significant financial relationships to disclose.

Induction of Adaptive Anti-HER2/neu Immune Responses in a Phase 1B/2 Trial of 2B1 Bispecific Murine Monoclonal Antibody in Metastatic Breast Cancer (E3194): A Trial Coordinated by the Eastern Cooperative Oncology Group

2B1 is a bispecific murine monoclonal antibody that binds to the extracellular domains of HER2/neu and FcgammaRIII. 2B1 efficiently promotes the lysis of tumor cells overexpressing HER2/neu by natural killer cells and mononuclear phagocytes that express the FcgammaRIII A isoform. Here, we report the results of E3194, a phase 1B/2 trial conducted by the Eastern Cooperative Oncology Group that employed 2B1 therapy in 20 women with metastatic breast cancer. The median age was 51 years. All but 1 patient had received prior chemotherapy. After the first dose, 3 of the initial 8 patients experienced dose-limiting toxicities that required dose-reduction. The nature of these dose-limiting toxicities resulted in a reduced dose from 2.5 mg/m/d to 1 mg/m/d in the remaining 12 patients. Objective antitumor responses were not seen. However, 2B1 therapy induced adaptive immune responses to both intracellular and extracellular domains of HER2/neu. Even though 2B1 antibody therapy did not show activity in metastatic breast cancer at the current administered doses, the ability of this antibody to induce detectable immune responses against an important tumor antigen has implications for understanding the mechanisms by which antibodies that mediate antibody-directed cellular cytotoxicity may exert their clinical antitumor effects.

FcγRI-triggered Generation of Arachidonic Acid and Eicosanoids Requires iPLA2 but Not cPLA2 in Human Monocytic Cells

Aggregation of receptors for immunoglobulin G (FcgammaRs) on myeloid cells activates a series of events that are key in the inflammatory response and that can ultimately lead to targeted cell killing by antibody-directed cellular cytotoxicity. Generation of lipid-derived proinflammatory mediators is an important component of the integrated cellular response mediated by receptors for the constant region of immunoglobulins (Fc). We have demonstrated previously that, in interferon-gamma-primed U937 cells, the high affinity receptor for IgG, FcgammaRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D, sphingosine kinase, calcium transients, and protein kinase C isoforms, leading to the activation of the NADPH-oxidative burst. Here, we investigate the nature of the phospholipase that regulates arachidonic acid and eicosanoid production. Our data show that FcgammaRI couples to iPLA(2)beta for the release of arachidonic acid and the generation of leukotriene B(4) and prostaglandin E(2). Activation of iPLA(2)beta was protein kinase C-dependent; on the other hand, platelet-activating factor triggered cPLA(2)alpha by means of the mitogen-activated protein kinase pathway. These studies demonstrate that intracellular PLA(2)s can be selectively regulated by different stimuli and suggest a critical role for iPLA(2)beta in the intracellular signaling cascades initiated by FcgammaRI and its functional role in the generation of key inflammatory mediators.

Monoclonal antibody therapeutics and apoptosis.

The potential for disease-specific targeting and low toxicity profiles have made monoclonal antibodies attractive therapeutic drug candidates. Antibody-mediated target cell killing is frequently associated with immune effector mechanisms such as antibody-directed cellular cytotoxicity, but they can also be induced by apoptotic processes. Antibody-directed mechanisms, including antigen crosslinking, activation of death receptors, and blockade of ligand-receptor growth or survival pathways, can elicit the induction of apoptosis in targeted cells. Depending on their mechanism of action, monoclonal antibodies can induce targeted cell-specific killing alone or can enhance target cell susceptibility to chemo- or radiotherapeutics by effecting the modulation of antiapoptotic pathways. This review will focus on the mechanisms by which antibodies are capable of eliciting programmed cell death either directly or indirectly within tumor cells.

FcγRI Coupling to Phospholipase D Initiates Sphingosine Kinase-mediated Calcium Mobilization and Vesicular Trafficking

Aggregation of receptors specific for the constant region of immunoglobulin G activates a repertoire of monocyte responses that can lead ultimately to targeted cell killing via antibody-directed cellular cytotoxicity. The high affinity receptor, FcgammaRI, contains no recognized signaling motif in its cytoplasmic tail but rather utilizes the gamma-chain of FcepsilonRI as an accessory molecule to recruit tyrosine kinases for signal transduction. We show here that, in a human monocytic cell line primed with interferon-gamma, FcgammaRI mobilizes intracellular calcium stores using a novel pathway that involves tyrosine kinase coupling to phospholipase D and resultant downstream activation of sphingosine kinase. Moreover, FcgammaRI is not coupled to phospholipase C; hence, calcium release from intracellular stores occurred in the absence of any measurable rise in inositol triphosphate. Finally, as this novel activation pathway is also shown to be responsible for mediating the vesicular trafficking of internalized immune complexes for degradation, it is likely to play a key role in controlling intracellular events triggered by FcgammaRI.