Induce Tumor Cell Lysis Research Articles

Oncolytic intralesional therapy for metastatic melanoma.

In-transit metastasis (ITM) develop in approximately 1 in 10 patients with melanoma and the disease course can vary widely. Surgical resection is the gold-standard treatment; however, ITM are often surgically unresectable due to size, distribution, and/or anatomic involvement. Oncolytic viral therapies are one category of non-surgical treatment options available for ITM. They induce tumor cell lysis and systemic anti-tumor activity through selective infection of tumor cells by naturally occurring or genetically modified factors. While there are numerous oncolytic viral therapies in various stages of development for the treatment of ITM, this discussion focuses on the mechanism and available literature for the two most established herpes virus-based therapies.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen.

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

Abstract 6524: A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) shows potent anti-tumor effect through tumor-directed T cell activation

Abstract 4-1BB (TNFSF9, CD137) is a member of the tumor necrosis factor receptor superfamily that functions as a potent costimulator to immune cells, especially T-cell. Although current 4-1BB-targeted strong agonistic antibody exhibit a considerable anti-tumor effect through T cell activation, it has a limitation of liver toxicity. To overcome the challenges of targeting the 4-1BB, we developed a bispecific antibody that can induce tumor-directed T cell activation. A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) targets human epidermal growth factor receptor 2 (HER2) and 4-1BB simultaneously and binds to both targets with high affinity. YH32367 (ABL105) exhibited a strong activation of 4-1BB signal in tumor cells expressing HER2 such as NCI-N87, BT474 and HCC1954 cell lines. In co-culture assay using human peripheral blood mononuclear cell (hPBMC) and HER2-expressing tumor cells, YH32367 (ABL105) stimulated IFN-γ secretion from hPBMC and thereby induced tumor cell lysis. The anti-tumor efficacy of YH32367 (ABL105) was evaluated in HCC1954-bearing hPBMC engrafted humanized mice and MC38/hHER2-bearing h4-1BB knock-in mice. YH32367 (ABL105) had more potent efficacy on tumor growth inhibition in both mouse models compared to the equimolar dosing of trastuzumab. Peripheral T cell increase was not observed in these studies. In particular, YH32367 (ABL105) showed a tumor regression in MC38/hHER2-bearing h4-1BB knock-in mice. In conclusion, YH32367 (ABL105) significantly inhibited tumor growth in a HER2 expression dependent manner and showed potent in vivo efficacy through tumor-directed T cell activation without non-specific systemic T-cell activation. Citation Format: Moo Young Song, Eun-Jung Lee, Hyejin Chung, Yangsoon Lee, Young Bong Park, Min Hyeok Jee, KyeongBae Kim, Mikyeong Ju, Jun Kyung Lee, Byung Hyun Choi, Ju Young Park, DaeWon Kim, Junhwan Kim, Sujin Ahn, Kyoung Kyu Ahn, Kyeongsu Park, Uijung Jung, Wonjun Son, Jaehyun Eom, Eunjung Kim, Donghoon Yeom, Jinhyung Ahn, Daehae Song, Byungje Sung, Weon-kyoo You, Jong Gyun Kim, Se-Woong Oh, Jaeho Jung. A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) shows potent anti-tumor effect through tumor-directed T cell activation [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 6524.

Abstract 4541: QL315, A CD3 bispecific antibody redirecting T cell activation to the tumor stroma antigen LRRC15

Abstract Leucine-rich repeat containing 15 (LRRC15) antigen is highly expressed on both stromal fibroblasts of multiple solid tumors and on cancer cells of mesenchymal origin. As its expression is very low in normal tissues, targeting LRRC15 has shown therapeutic potential as an antibody-drug conjugate (Demetri, ASCO 2019), which could lead to significant tumor inhibition by solely targeting stromal LRRC15 expression. In this study, we have developed and rank ordered an array of IgG-based CD3 bispecific molecules based on biophysical and biologic attributes and anti-cancer efficiency through T-cell engagement and redirection/activation against LRRC15-expressing tumors. The selected lead, QL315, robustly and conditionally induced T cell activation and proliferation only in the presence of LRRC15-expressing cancer cells with an EC50 in a picomolar range in vitro but not in the coculture with LRRC15-negative cancer cells. Moreover, QL315 strongly induced tumor cell lysis in vitro and led to significant tumor regression and T cell expansion in a mouse xenograft model. Importantly, toxicologic evaluation of QL315 in cynomolgus monkeys showed antibody-like stability and half-life, and good tolerability with no changes of circulating blood cell homeostasis and organ damage. At highest levels, liver enzymes, as well as cytokine release, were transiently elevated but returned to normal within one week of treatment. In conclusion, these data demonstrate drug-like properties, promising anti-tumor capability and good tolerability of the anti-LRRC15/CD3 bispecific antibody, QL315, indicating its potential as a therapeutic option in the treatment of solid tumors of diverse origin by targeting either tumor stromal micro environment or patient cancers expressing LRRC15. Citation Format: Monica Jin, Aaron Kurtzman, Weiwei W. Prior, Allan Chan, Anna McClain, Shenda Gu, Irene Tang, Zijie Cai, Lauren Schwimmer, Hieu Tran, Shihao Chen. QL315, A CD3 bispecific antibody redirecting T cell activation to the tumor stroma antigen LRRC15 [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 4541.

Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs.

Blockade of CD47-Sirpα Interactions Improved Myeloid Effector Cell Mediated Lymphoma Cell Killing By the HLA-DR Antibody Apolizumab

Background:Apolizumab (APO) is a humanized IgG1 antibody targeting a HLA-DRβ chain specific epitope, which is expressed by approx. 50 % of B cell lymphomas. The antibody's clinical development was hampered by toxicity, potentially mediated by its strong CDC activity. However, previous studies demonstrated that APO efficiently triggered tumor cell killing by myeloid cells, especially by granulocytes. Myeloid effector cell engagement is strongly regulated by CD47-SIRPα interactions, and blockade of this axis has been demonstrated to improve the efficacy of therapeutic antibodies. Hence, our goal was to characterize the influence of CD47-SIRPα blockade on myeloid cell engagement by APO. Methods: Expression of the APO epitope, CD20 and CD47 on different B lymphoma cell lines was measured by flow cytometry. The capacity of APO and Rituximab (RTX) to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) by mononuclear (MNC) or polymorphonuclear (PMN) effector cells was analysed by 51Cr release assays. Antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages was analysed by fluorescence microscopy. PMN-mediated ADCC and macrophage-mediated ADCP experiments were performed in the presence or absence of the CD47 blocking antibody 5F9-IgG2σ to analyse the impact of CD47-SIRPα blockade on APO-mediated lymphoma cell killing by myeloid cells. Results: As anticipated we observed binding of APO on approx. 50% of the tested B lymphoma cell lines, including Raji (Burkitt's lymphoma), MINO (mantle cell lymphoma) and Carnaval (double hit DLBCL). Focusing on these cell lines, APO's CDC activity was more potent (Raji) or similar (Mino) compared to RTX. Furthermore, APO significantly induced tumor cell lysis by PMN or MNC in 51Cr release assays and achieved higher lysis rates in comparison to RTX. Blocking CD47-SIRPα interactions by 5F9-IgG2σ resulted in significantly increased APO-induced ADCC by PMN against all tested B cell lymphoma lines (Mino: 28.8 ± 10.2 % vs. 41.8 ± 13.3 %; Carnaval: 37.9 ± 17.9 % vs. 55.6 ± 17.6 %; Raji: 23.6 ± 11.7 % vs. 52.4 ± 19.4 % for APO alone vs. APO + 5F9-IgG2σ, respectively, p < 0.05 by ANOVA). However, no ADCC by PMN and RTX was observed - even in the presence of CD47-SIRPα blockade. Moreover, APO mediated similar or even stronger ADCP by macrophages compared to RTX, which could be further enhanced by 5F9-IgG2σ (Figure 1). Conclusions: APO's potent myeloid cell activation could be further enhanced by disruption of CD47-SIRPα interactions. Our observations suggest to generate isotype variants of APO with lower CDC but preserved or enhanced ADCC activity. Subsequently, a combination of APO and CD47-SIRPα blocking strategies may improve B cell lymphoma immunotherapy. Disclosures No relevant conflicts of interest to declare.

Abstract 3206: Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors

Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3206.

Novel anti-EGFRvIII bispecific T cell engager (BiTE) antibody construct in glioblastoma (GBM): Trial in progress of AMG 596 in patients with recurrent or newly diagnosed disease.

TPS2071 Background: GBM is the most aggressive primary brain tumor in adults and is extremely difficult to treat. Patients with GBM tend to progress rapidly within weeks or months. Median overall survival is only 12–15 months despite aggressive treatment, and less than 5% of patients survive 5 years. GBM also severely impacts quality of life and cognitive function. Approximately 50% of GBM tumors test positive for amplification or mutation of the epidermal growth factor receptor (EGFR), the most common of which is the EGFRvIII gain-of-function mutation. AMG 596 is a bispecific T cell engager (BiTE®) antibody construct designed to crosslink and engage CD3-positive T cells to EGFRvIII-positive tumor cells, inducing tumor cell lysis and T cell proliferation. A clinical trial is being conducted for this novel immunotherapy agent in patients with EGFRvIII-positive GBM. Methods: NCT03296696 is a phase 1, first-in-human, open-label, sequential dose-escalation and dose-expansion study evaluating the safety, tolerability, and pharmacokinetics and pharmacodynamics (PK/PD) of AMG 596 in patients with EGFRvIII-positive GBM. AMG 596 is administered via continuous intravenous infusion. The study is expected to enroll approximately 82 patients total and comprises two groups (Group 1: patients with recurrent GBM; Group 2: patients with newly diagnosed GBM in the maintenance treatment phase after standard of care). Key inclusion criteria include: male or female; ≥ 18 years of age; with pathologically documented and diagnosed grade IV GBM; Eastern Cooperative Oncology Group performance status ≤ 1; life expectancy ≥ 3 months per study investigator; and acceptable renal, hematological, and hepatic function. The primary endpoint evaluates the safety and tolerability of AMG 596 via collection of treatment-emergent adverse events. Additional endpoints include objective response rate per modified Response Assessment in Neuro-Oncology Criteria and PK/PD analyses of AMG 596 in serum. The study began enrolling patients in April 2018 and enrollment is ongoing. For more information, please contact Amgen Medical Information: medinfo@amgen.com. Clinical trial information: NCT03296696.

Focal cryotherapy: step by step technique description

ABSTRACTIntroduction and objective:Focal cryotherapy emerged as an efficient option to treat favorable and localized prostate cancer (PCa). The purpose of this video is to describe the procedure step by step.Materials and methods:We present the case of a 68 year-old man with localized PCa in the anterior aspect of the prostate.Results:The procedure is performed under general anesthesia, with the patient in lithotomy position. Briefly, the equipment utilized includes the cryotherapy console coupled with an ultrasound system, argon and helium gas bottles, cryoprobes, temperature probes and an urethral warming catheter. The procedure starts with a real-time trans-rectal prostate ultrasound, which is used to outline the prostate, the urethra and the rectal wall. The cryoprobes are pretested and placed in to the prostate through the perineum, following a grid template, along with the temperature sensors under ultrasound guidance. A cystoscopy confirms the right positioning of the needles and the urethral warming catheter is installed. Thereafter, the freeze sequence with argon gas is started, achieving extremely low temperatures (-40°C) to induce tumor cell lysis. Sequentially, the thawing cycle is performed using helium gas. This process is repeated one time. Results among several series showed a biochemical disease-free survival between 71-93% at 9-70 month- follow-up, incontinence rates between 0-3.6% and erectile dysfunction between 0-42% (1–5).Conclusions:Focal cryotherapy is a feasible procedure to treat anterior PCa that may offer minimal morbidity, allowing good cancer control and better functional outcomes when compared to whole-gland treatment.

Fighting Cancer with Mathematics and Viruses.

After decades of research, oncolytic virotherapy has recently advanced to clinical application, and currently a multitude of novel agents and combination treatments are being evaluated for cancer therapy. Oncolytic agents preferentially replicate in tumor cells, inducing tumor cell lysis and complex antitumor effects, such as innate and adaptive immune responses and the destruction of tumor vasculature. With the availability of different vector platforms and the potential of both genetic engineering and combination regimens to enhance particular aspects of safety and efficacy, the identification of optimal treatments for patient subpopulations or even individual patients becomes a top priority. Mathematical modeling can provide support in this arena by making use of experimental and clinical data to generate hypotheses about the mechanisms underlying complex biology and, ultimately, predict optimal treatment protocols. Increasingly complex models can be applied to account for therapeutically relevant parameters such as components of the immune system. In this review, we describe current developments in oncolytic virotherapy and mathematical modeling to discuss the benefit of integrating different modeling approaches into biological and clinical experimentation. Conclusively, we propose a mutual combination of these research fields to increase the value of the preclinical development and the therapeutic efficacy of the resulting treatments.

Abstract 5671: AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma

Abstract Multiple myeloma (MM) is the second most common haematological cancer and is characterized by the accumulation of neoplastic plasma cells in the bone marrow and production of high levels of monoclonal immunoglobulin (M-protein). While historically considered incurable, recent approvals and ongoing clinical trials with monoclonal antibodies (mAbs) targeting surface antigens promise greatly improved outcomes and have heralded a new era of MM treatment in which immunotherapies are expected to take center stage. However, an unmet need remains as patients eventually relapse and/or become refractory to currently available treatments. Consequently, novel immunotherapeutic approaches are needed to provide improved treatment options to MM patients. Among the currently explored targets, B-cell maturation antigen (BCMA, CD269) is considered to be particularly attractive due to its limited expression on healthy tissues and almost universal expression on myeloma cells in the majority of patients. Natural killer (NK) cells are cytotoxic effectors of the innate immune system capable of rapidly eradicating infected and transformed cells. The cytolytic activity of NK-cells can be used therapeutically to induce tumor cell lysis by direct engagement of the activating receptor CD16A (FcγRIIIa) using mAbs (ADCC). Despite similar mechanisms of target cell lysis, activation of NK-cells is not associated with the systemic symptoms of high level cytokine release as seen with direct T-cell engagement. Hence, it is considered a potent immunotherapeutic approach with reduced toxicity and a well-manageable safety profile. NK-cells readily infiltrate bone marrow and are thought to contribute to the efficacy of current myeloma treatments. Therefore, redirecting NK-cell cytotoxicity to malignant plasma cells appears to be a suitable therapeutic approach for MM. Here we describe the characterization of AFM26, a novel tetravalent bispecific tandem diabody (TandAb) that specifically targets BCMA and CD16A with high affinity and induces potent and efficacious myeloma cell lysis. AFM26 incorporates an affinity-improved anti-CD16A domain and interacts bivalently with NK-cells, resulting in high avidity and prolonged cell surface retention that is largely unaffected by the presence of polyclonal IgG. Notably, AFM26 potently induces NK-cell-mediated in vitro lysis of target cells expressing low levels of BCMA at low effector:target ratios, even in presence of polyclonal IgG. This may suggest that AFM26, in contrast to classical mAbs, retains ADCC activity at low antibody concentrations in presence of serum IgG and despite high levels of IgG M-protein occurring in about half of MM patients. AFM26 exhibits high protein stability, full cross-reactivity with cynomolgus antigens (BCMA and CD16A) and does not bind APRIL and TACI, two functionally related receptors. These data suggest that TandAb AFM26 is a promising and highly potent drug candidate for MM treatment. Citation Format: Thorsten Gantke, Uwe Reusch, Kristina Ellwanger, Ivica Fucek, Michael Weichel, Martin Treder. AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma [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 5671. doi:10.1158/1538-7445.AM2017-5671

Sipuleucel-T (sip-T) induced lytic CD8+ T cell responses to target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

162 Background: Sip-T is an FDA-approved immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic CRPC. Sip-T is manufactured from autologous peripheral blood mononuclear cells cultured with the immunogen PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage colony-stimulating factor. After sip-T, antibody and T cell responses to PA2024 and/or PAP correlate with improved survival. To further elucidate the mechanism of sip-T–induced immune responses, we evaluated the proliferative and lytic ability of PA2024- and PAP-specific CD8+ T cells. Methods: Mononuclear blood cells were labeled with the membrane dye carboxyfluorescein succinimidyl ester (CFSE) and cultured with PA2024 or PAP. In vitro proliferative and lytic CD8+ (cytotoxic T lymphocyte [CTL]) T cell responses to these antigens were evaluated by flow cytometry. For proliferation, progressive dilution of CFSE was measured. For CTL activity, the loss of intracellular granzyme B (GzB), indicating exocytosis of this apoptosis-mediating enzyme, was assessed. Samples were from 2 sip-T clinical trials STAND (NCT01431391) and STRIDE (NCT01981122), hormone-sensitive and CRPC pts, respectively. Results: Six wk after sip-T administration, CD8+ PAP- and PA2024-specific responses were observed (n=14 pts assessed). The magnitude of PA2024-specific CD8+ proliferative responses was greater than that for PAP-specific responses. CD8+ T cells from a subset of pts who exhibited PA2024- and/or PAP-specific proliferative responses were assessed for lytic ability. After in vitro antigen stimulation, CTL activity in all evaluated samples (n=14, PA2024; n=13, PAP) was demonstrated by a significant decrease (p<0.05) in intracellular GzB relative to a no-antigen control. Conclusions: Sip-T induced CD8+ CTL proliferation against the target antigens PAP and PA2024. Moreover, antigen-specific CTL activity provides the first direct evidence that sip-T can induce tumor cell lysis. These antigen-specific CD8+ lytic abilities were observed within 6 wk following sip-T, suggesting rapidly generated immune responses. Clinical trial information: NCT01431391; NCT01981122.

Abstract B67: Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo

Abstract Recent success of T cell based cancer immunotherapy approaches like chimeric antigen receptor T cells (CAR-Ts) and especially bispecific T cell engagers (BiTEs) has suggested great potential for cancer therapy. However, these technologies have several drawbacks like high risks for side-effects, such as autoimmunity, as well as inappropriate T cell activation due to a non-natural synapse. Hence, we sought to use antibody-mediated delivery of viral peptides to tumor cells in order to mimic viral infection and induce tumor cell lysis by virus-specific cytotoxic CD8+ T cells. Here, we generated immunoconjugates called antibody-targeted pathogen-derived peptides (ATPPs), where mature virus-derived MHC class I peptides from Influenza A or Epstein-Barr virus were conjugated to a tumor-associated antigen-specific antibody via a disulfide bond. Using optical imaging in combination with fluorescence resonance energy transfer, we revealed that after ATPP binding to the target antigen and subsequent internalization, the peptides are released in endosomes and loaded onto recycling MHC class I complexes. These peptide-MHC complexes are subsequently presented on the cell surface and mediate activation of human peptide-specific CD8+ T cells as shown by Interferon-γ release using enzyme-linked immunosorbent assay. Moreover, ATPP-loaded cancer cells from different tumor types were efficiently killed in vitro by human CD8+ T cells in a dose-dependent manner, reaching up to 100% killing after 24h at 0,13nM with an effector-to-target ratio of 3:1. Tumor cell death was assessed by the xCELLigence system as well as lactate dehydrogenase quantification in the supernatant. Interestingly, ATPPs were about 1000-times more potent than free peptide, while antibody alone, a non-cleavable ATPP construct, as well as peptides conjugated to a non-binding antibody exhibited no effects. Importantly, all used antibodies carried the P329G/LALA mutation for abolished Fc-receptor binding. In addition, we performed an experimental subcutaneous xenograft in vivo study with PDL1-expressing MDA-MB231 breast cancer cells in NOG mice in combination with anti-PD1 treatment. We observed that ATPPs successfully recruited adoptively transferred human peptide-specific CD8+ T cells into the tumor and induced 60% tumor growth inhibition of an established tumor after 3 weeks in the anti-PD1 combination setting. Altogether, we generated first in class immunoconjugates for antibody-targeted loading of cancer cells with foreign MHC-I restricted peptide antigens. Our results demonstrate potent ATPP-mediated antitumor efficacy in vitro and in vivo, by loading tumor cells with viral peptide antigens and redirecting virus-specific cytotoxic T cells against cancer. Citation Format: Julian Peter Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke. Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B67.

Abstract IA38: Engineered T cell-receptor based therapy of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) presents a formidable challenge for effective therapies given the robust desmoplasia, immunosuppressive elements and relatively few coding mutations characteristic of this disease. We demonstrate the safety and therapeutic activity of T cells engineered to express an affinity-enhanced T cell receptor (TCR) targeting the naturally overexpressed self/tumor antigen Mesothelin in an autochthonous, genetically engineered mouse model of PDA (KPC). Notably, despite the hostile tumor microenvironment surrounding PDA, the engineered T cells readily infiltrate the desmoplastic tumor, induce tumor cell lysis and remodel the tumor extracellular matrix without causing detectable toxicity in normal tissues. The transferred T cells persist in PDA, but antitumor activity is transient, as the tumor-infiltrating donor T cells upregulate a panel of inhibitory receptors and become progressively dysfunctional specifically in PDA that is due to both TCR-dependent and independent mechanisms of inhibition. This obstacle to sustained therapeutic activity can be overcome by serial T cell infusions, which mediate objective responses and a near doubling of KPC survival without overt toxicities. Similarly engineered human T cells recognize and lyse PDA cells in vitro, further supporting the clinical advancement of this TCR-based strategy for the treatment of human PDA. Citation Format: Ingunn M. Stromnes, Thomas M. Schmitt, Scott J. Brockenbrough, Hieu Nguyen, Carlos Cuevas, Ayaka Hulbert, Jennifer Hotes, Ashley Dotson, Sunil R. Hingorani, Philip D. Greenberg. Engineered T cell-receptor based therapy of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA38.

Abstract PR08: Enhanced affinity T cell receptor based therapy can surmount immune and physical barriers to treat pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that employs numerous mechanisms of immune suppression. Here we demonstrate the therapeutic activity and safety of an affinity-enhanced T cell receptor (TCR)-based therapy targeting a naturally overexpressed self/tumor antigen in an autochthonous, genetically engineered mouse model of PDA (KPC). T cells engineered to recognize Mesothelin infiltrated the desmoplastic tumor, induced tumor cell lysis and remodeled the extracellular matrix without apparent toxicities to normal tissues. However, this anti-tumor activity was transient despite T cell persistence in PDA. Donor T cells progressively upregulated inhibitory receptors and became dysfunctional specifically in the tumor microenvironment, indicating selective immunosuppression at the tumor site. However, the T cell dysfunction in PDA also was, in part, independent of antigen specificity and the observed upregulation of inhibitory receptors. Nevertheless, a second infusion of engineered T cells still preferentially accumulated in PDA and increased tumor epithelial cell death indicating that PDA remains susceptible to the effects of serial T cell infusions. Finally, we demonstrate that serial T cell infusions significantly increased survival in KPC mice enrolled with invasive PDA indicating that the observed anti-tumor activity can be associated with therapeutic benefits to the host. These results support the clinical advancement of TCR-based therapies for treatment of PDA. This abstract is also presented as Poster B20. Citation Format: Ingunn M. Stromnes, Scott J. Brockenbrough, Thomas M. Schmitt, Sunil R. Hingorani, Philip D. Greenberg. Enhanced affinity T cell receptor based therapy can surmount immune and physical barriers to treat pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR08.

Re-exposure to blinatumomab after CD19-positive relapse: Experience from three trials in patients (pts) with relapsed/refractory B-precursor acute lymphoblastic leukemia (R/R ALL).

7051 Background: Blinatumomab is a bispecific T-cell engager antibody construct designed to link cytotoxic T cells and CD19-positive B cells, inducing tumor cell lysis. In this combined analysis, w...

Abstract 474: Complete regression and long-term specific protective immune responses obtained in rodent tumor models after intratumoral treatment with LTX-315 .

Abstract LTX-315 is a de novo designed peptide derived from a naturally occurring host defence peptide. LTX-315 has the potential to induce long-term specific protective immune responses by stimulating immune cells, inducing tumor cell lysis with subsequent release of danger signals (e.g. HMGB1) and tumor associated antigens (TAA`s). A complete tumor regression has been obtained in several syngenic rodent tumor models by intratumoral (i.t.) injection with LTX-315. The effect was T- cell- dependent since the intervention was inefficient in immune-deficient animals. Studies on treated tumor tissue confirmed infiltration of immune cells and a switch in the cytokine profile towards a Th1 response. Successfully treated animals were protected against re-challenge with the tumor cell type treated, but not against other types of tumor cells. Moreover, tumor resistance could be adoptively transferred by spleen cells from LTX-315-treated animals. The resistance was abrogated by depletion of T- lymphocytes. Additional studies also indicate that LTX-315`s potential to locally activate the innate immune system by the immunogenic stressing of cells, in addition to the subsequent release of endogenous adjuvants and natural danger signals, provides a strong rationale for using LTX-315 as an adjuvant for vaccines based on tumor-associated antigens (TAA) and for combination with other types of immune–modulatory therapies. LTX-315 is currently being tested in a Phase I dose escalation clinical study and may represents a novel strategy for personalized in situ vaccination against cancer. Citation Format: Øystein Rekdal, Gunnar Kvalheim, Pål-Dag Line, Bent Rolstad, Ketil Camilio, Gerd Berge, Janne Nestvold, Mengyu Wang, Jihua Shi, Ali Areffard, Baldur Sveinbjørnsson. Complete regression and long-term specific protective immune responses obtained in rodent tumor models after intratumoral treatment with LTX-315 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2013-474

Abstract 3522: Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma

Abstract NK cells are professional cytotoxic immune effector cells and, unlike cytotoxic T cells, exist in a constitutively activated state not requiring pre- or co-stimulation. Thus, NK cells are attractive effectors to employ for cancer immunotherapy with bispecific antibodies. To improve the recruitment and activation of NK cells for lysis of tumor cells, we isolated a human antibody that was highly specific for the A isoform of FcαRIII (CD16A) and displayed substantially lower dissociation constant than native or Fc-optimized mAbs. The variable domains of this antibody were used to construct a tetravalent bispecific antibody, termed TandAb, consisting solely of antibody variable domains, with two valencies for CD16A (on effector cells) and two for CD30 (on target cells). CD30 represents a validated tumor target which is expressed on Hodgkin's Reed-Sternberg lymphoma cells, and on some activated immune cells. The binding properties of the TandAb and its efficacy for inducing tumor cell lysis were compared to those of a native anti-CD30 mAb, a mAb with enhanced Fc receptor binding properties, and an anti-CD30 bispecific diabody, all of which comprised the same anti-CD30 variable domains as the TandAb. Due to its bivalency for CD16A, the TandAb was retained longer on NK cells than the IgGs or the bispecific diabody, and relatively high concentrations of IgG were necessary to displace it from NK cells. The TandAb bound equally well to both CD16A alleles (158F, 158V) indicating a potentially broader patient response than that achieved with current therapeutic mAbs. The TandAb did not induce any systemic activation of NK cells even though it's binding was bivalent to CD16A. In a cytotoxicity assay, the TandAb displayed higher potency (EC50) and higher efficacy (maximal lysis) than the IgGs or the bispecific diabody. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies revealed half-lives for the TandAb up to 22.9 h after repeated dosing. No MTD was reached in a single dose escalation study to a maximum dose of 100 mg/kg. A pivotal 28 days toxicity study in which animals were dosed repeatedly up to 10 mg/kg every other day revealed an excellent safety profile. Several immunological parameters were assessed indicating no signs of immunotoxicity or systemic cytokine release. In conclusion, TandAbs may provide a more effective treatment for tumors that are refractory to treatment with IgGs and is currently being evaluated in the Phase I clinical trial. 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 3522. doi:1538-7445.AM2012-3522

Development of an oncolytic herpes simplex virus using a tumor-specific HIF-responsive promoter

We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic Herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, ICP4, was placed under the regulation of a HIF-responsive promoter and then introduced into the thymidine kinase locus (UL23) of HSV d120 which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV were isolated and their derivation from d120 was verified by expression of a truncated, nonfunctional form of ICP4 protein. Disruption of the UL23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported- reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.

Efficacy of oncolytic reovirus against human breast cancer cells

Human reovirus can replicate and induce tumor cell lysis in several cancer types with an activated Ras signaling pathway. The aim of this study was to investigate the oncolytic effect of reovirus against breast cancer cells, and clarify the relationship between the susceptibility to reovirus and HER2 expression, which is associated with the Ras signaling pathway. Reovirus (serotype 3), 6 human breast cancer cell lines and normal mammary gland epithelial cell line were used in this study. The mRNA expression of HER2 receptor was examined by RT-PCR, and the protein of HER2 and activated Ras protein were examined by a Western blot analysis. In vitro, the cytopathic effects, viral protein synthesis, and cell viability on infection by reovirus were examined. Reovirus can infect all the 6 examined cancer cell lines, but the control cell line was not susceptible to the reovirus infection. The cytopathic effect appeared from day 1 after infection, and a 50% or greater cytotolysis was demonstrated at day 7 after infection. The Ras activities in all examined cell lines were higher than those in the control cell line. No relationship was observed in the susceptibility to the reovirus and HER2 expression. The increased Ras activity itself, regardless of the HER2 expression, may therefore play an important role in the susceptibility to reovirus of breast cancer cells. As a result, breast cancer may thus become a candidate target for oncolytic reovirus therapy.