Bystander Cytotoxicity Research Articles

Abstract 556: Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression

Abstract An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC50 values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC50 values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p<0.0001) or 40H3-MMAE (p<0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p<0.0001) and 26% with 40H3-MMAE (p<0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs. Citation Format: Sonia Jain, Eric Chun Ho, Kendra A. Porath, Antonella Antignani, David J. FitzGerald, Jann N. Sarkaria. Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 556.

Clonogenic assay to measure bystander cytotoxicity of targeted alpha-particle therapy.

Radiation therapy induces targeted effects in the cells that are irradiated and also non-targeted effects (i.e. bystander effects) in non-irradiated cells that are close to or at short distance (<∼1mm) from irradiated cells. Bystander effects are mediated by intercellular communications and may result in cytotoxic and genotoxic modifications. Their occurrence and relative contribution to the irradiation outcome are influenced by several parameters among which the particle linear energy transfer seems to be prominent. Bystander effects were first observed after external radiation therapy, but have been described also following targeted radionuclide therapy. Therefore, we propose a method to investigate their occurrence in experimental conditions where cells are exposed to radiopharmaceuticals. In this approach, clonogenic cell death is the biological endpoint of the bystander effects caused by irradiation with alpha particles (a potent inducer of the bystander response).

Deciphering the Role of Schwann Cells in Inflammatory Peripheral Neuropathies Post Alphavirus Infection

Old world alphaviruses (e.g., chikungunya) are known to cause severe acute and chronic debilitating arthralgia/arthritis. However, atypical neurological manifestations and, in particular, unexpected cases of acute inflammatory Guillain-Barre syndrome (GBS) have been associated with the arthritogenic alphaviruses. The pathogenesis of alphavirus-associated GBS remains unclear. We herein addressed for the first time the role of Schwann cells (SC) in peripheral neuropathy post-alphaviral infection using the prototypical ONNV alphavirus model. We demonstrated that human SC expressed the recently identified alphavirus receptor MxRA8 and granting viral entry and robust replication. A canonical innate immune response was engaged by ONNV-infected SC with elevated gene expression for RIG-I, MDA5, IFN-β, and ISG15 and inflammatory chemokine CCL5. Transcription levels of prostaglandin E2-metabolizing enzymes including cPLA2α, COX-2, and mPGES-1 were also upregulated in ONNV-infected SC. Counterintuitively, we found that ONNV failed to affect SC regenerative properties as indicated by elevated expression of the pro-myelinating genes MPZ and MBP1 as well as the major pro-myelin transcription factor Egr2. While ONNV infection led to decreased expression of CD55 and CD59, essential to control complement bystander cytotoxicity, it increased TRAIL expression, a major pro-apoptotic T cell signal. Anti-apoptotic Bcl2 transcription levels were also increased in infected SC. Hence, our study provides new insights regarding the remarkable immunomodulatory role of SC of potential importance in the pathogenesis of GBS following alphavirus infection.

CAR-T Cell Infusion Results in Activation of CD160+/NKG2D+/CCL5+ Non-CAR CD8+ Cytotoxic 'Bystander' T Cells in Both Non-Human Primates (NHP) and Patients Receiving B-Cell-Directed CAR-Ts

Background: Approximately 50% of patients receiving CAR-T cells will relapse, with half of these relapses due to CD19neg antigenic escape. To address this, we have explored an alternative pathway for targeting antigen-negative leukemic cells through activated endogenous CARneg bystander T cells, which could provide additional leukemic control through CAR-independent mechanisms. Here we leverage data from a NHP CAR-T cell model, and from clinical studies, to identify mechanisms of CARneg T cell bystander activation and their potential antileukemic role. Methods: NHP: Anti-CD20 CAR-T cells were transferred into lymphodepleted NHP as previously described (PMID: 29563103). Expansion of these cells resulted in B cell aplasia, and induced clinical Cytokine Release Syndrome (CRS). The transferred CAR-T cells persisted for ~4 weeks, at which point loss of CAR-Ts was followed by B cell recovery. Flow cytometry, scRNA- and scTCR Seq were performed on 5 NHP CAR-T cell recipients. Clinical Samples: Flow cytometry, scRNA- and scTCR-Seq were also performed on T cells from 6 pediatric B-ALL patients receiving Tisangenlecleucel. ScRNA-Seq and scTCR-Seq libraries were aligned with cellranger, preprocessed and analyzed with the Python packages scanpy and scVI. Results: NHP recipients of CD20-CAR T cells demonstrated CAR-T cell maximum expansion on Days 7-11 post-infusion. Coincident with CAR-T cell expansion, we also observed a 5-fold expansion of activated, CD8+ CARneg T cells. To rigorously profile the expanded CARneg T cells, we performed scRNASeq on sorted CARneg T cells from the infused product, at the time of maximum CAR-T proliferation (Day 11), and during the CAR-T contraction phase (Day 20). Longitudinal clone tracking and associated gene expression analysis demonstrated that the CD8 CARneg T cell clones formed a unique cluster that emerged in parallel with expansion of CARpos T cells. This CD8 CARneg population made up <1% of T cells in the blood prior to CAR-T cell infusion or in the infused CAR-T cell product, suggesting evolution and expansion of these cells in the post-CAR-T cell infusion/CRS milieu. These 'bystander' CD8 CARneg T cells demonstrated high expression of the cytokine receptors for IL15 and IL2, and of the cytokines IL-18, IL2 and IFNg, along with expression of canonical cytotoxicity molecules, including Perforin and Granzyme M. In addition, the CARneg CD8 T cells expressed high levels of KLRK1 (NKG2D) and FASL, receptors that have been associated with direct tumor lysis. T cell repertoire analysis demonstrated high TCR diversity, suggesting that these cells were bone fide T cells, and not NK-T or MAIT cells. To better understand this population, we subsetted all clusters enriched for CARneg CD8 T cells at the time of maximum CAR-T cell proliferation and reclustered this data, which enabled the further identification of these CARneg CD8 T cells to express multiple NK markers, including KLRK1 (NGG2D), KLRD1 (CD94), CD160, CCL5, NKG7 (Panel 1), a transcriptional signature that is similar to CD8 T cells recently found to emerge as cytotoxic bystander cells in viral infections (PMID: 31827070), and in the inflammatory tumor microenvironment (PMID: 33468558), where they provide additional anti-viral/tumor control. We next performed scRNA-Seq on samples from 6 patients receiving Tisagenlecleucel CAR-T cells. At the time of maximal CAR-T cell expansion, we identified a CD8 CARneg T cell population in all patients, which mirrored the transcriptional signature originally observed in NHP (Panel 2). To determine whether cytokines released by CRS could induce the transformation of human CD8 CARneg T cells into these highly activated cells, we stimulated primary human T cells with an array of CRS-associated cytokines, and found that the gamma cytokines IL-2 and IL-15 induced a CD8+ T cell population that upregulated a similar group of phenotypic markers (CD160, NKG2D and CCL5) observed in the bystander cells obtained from CAR-T cell patients. Conclusions: These data demonstrate for the first time that a CAR-T cell induced bystander effect is predominantly elicited in CD8 CARneg T cells, in both NHP and patients. These CD8 CARneg T cells express several canonical activation and NK markers in response to CRS-associated cytokines, and also express both NKG2D and FASL, suggesting that they could contribute to anti-leukemic effects through CAR-independent mechanisms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

244 (PB024) - Phase I Biomarker Analysis Results of MORAb-202 (Farletuzumab Ecteribulin) Effects on Vascular Remodeling and Immune Modulation in Patients With Ovarian Cancer

Background: MORAb-202 is an antibody–drug conjugate consisting of the humanized anti-folate receptor-alpha (FRα) monoclonal antibody, farletuzumab, and the potent microtubule dynamics inhibitor, eribulin. It is highly stable in systemic circulation and releases eribulin in the targeted cells. While MORAb-202 is cytotoxic to FRα-expressing cancer cells, it also exerts cytotoxic bystander effects on adjacent tumor and stromal cells, as well as noncytotoxic bystander effects on the tumor microenvironment (TME) in preclinical models.

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

Simple SummaryBystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and may greatly influence therapy outcome. Although the limited data available suggest a role for intercellular gap junction channels, far less is known about the role of connexin hemichannels. Here, we investigated bystander effects induced by photodynamic therapy in syngeneic murine melanoma models in vivo. We determined that (i) photoactivation of a photosensitizer triggered calcium-dependent cell death pathways in both irradiated and bystander tumor cells; (ii) hemichannel activity and adenosine triphosphate release were key factors for the induction of bystander cell death; and (iii) bystander cell killing and antitumor response elicited by photodynamic therapy were greatly enhanced by combination treatment with S-nitrosoglutathione, which promoted hemichannel opening in these experimental conditions. Therefore, these findings in a preclinical model have important translational potential.In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy

Purpose Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. Materials and methods B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. Results In vitro analysis of bystander cytotoxic effects showed that CM killed about 30–40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. Conclusions This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

Tisotumab vedotin versus investigator’s choice chemotherapy in second- or third-line recurrent or metastatic cervical cancer (innovaTV 301/ENGOT-cx12/GOG 3057, trial in progress).

TPS5596 Background: Doublet chemotherapy (paclitaxel plus either platinum or topotecan) with bevacizumab (if eligible) is recommended for first-line treatment of recurrent (not amenable to curative therapy) or metastatic cervical cancer (r/mCC; Tewari 2014). In the second-line setting, there are limited data for currently available treatment options. Tisotumab vedotin (TV) is an investigational antibody-drug conjugate (ADC) composed of a tissue factor (TF)-directed human monoclonal antibody covalently linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. TV is directed to cells expressing TF and releases MMAE upon internalization, resulting in cell cycle arrest and apoptotic cell death. TV has anti-tumor activity on multiple tumor types and kills tumor cells by direct cytotoxicity, bystander cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and in a manner consistent with immunogenic cell death. In a recent phase 2 pivotal trial (innovaTV 204), TV demonstrated a clinically meaningful objective response rate (ORR) of 24% and median duration of response (DOR) of 8.3 months, as well as a manageable and tolerable safety profile with most adverse events being mild to moderate, in r/mCC patients with disease progression on or after chemotherapy. These findings support further investigation of TV in patients with r/mCC who progress on available first-line treatment options. Methods: The innovaTV 301 trial (NCT04697628) is a global, randomized, open-label, phase 3 clinical trial evaluating the efficacy and safety of TV in patients with previously treated r/mCC. Eligible patients must be ≥18 years, have r/mCC, and have experienced disease progression after receiving 1-2 prior lines of therapy (either standard of care systemic chemotherapy doublet or platinum-based therapy [if eligible; paclitaxel+cisplatin+bevacizumab, paclitaxel+carboplatin+bevacizumab, or paclitaxel+topotecan/nogitecan+bevacizumab]). Approximately 482 patients will be randomized 1:1 to receive 21-day cycles of either TV (2.0 mg/kg IV once every 3 weeks) or investigator’s choice of chemotherapy: topotecan (1 or 1.25 mg/m2 IV; Day 1 [D1] to D5 of each cycle), vinorelbine (30 mg/m2 IV; D1 and D8 of each cycle), gemcitabine (1000 mg/m2 IV; D1 and D8 of each cycle), irinotecan (100 or 125 mg/m2 IV; weekly for 28days, then every 42 days), or pemetrexed (500 mg/m2 IV, D1 of each cycle). The primary endpoint of this trial is overall survival. Key secondary endpoints are progression-free survival, ORR, time to response, DOR, safety, and quality of life outcomes. The study is currently enrolling and will have sites open in the US, EU, Japan, Latin America, Taiwan, Singapore, and South Korea. Clinical trial information: NCT04697628.

A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy.

T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFICANCE: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521.

Abstract SP086: Rationale for combination therapies

Abstract There has been a lot of emerging clinical data in the treatment of early and late stage breast cancer with PD-1/PD-L1 inhibitors in the last 12 months. We have learnt several important points relevant for future development in breast cancer patients. Most important of these is the observation that early-stage disease has a different immune context to advanced disease. This is evidenced by study results that in the neoadjuvant settings PD-(L)1 inhibitors have efficacy in both PD-L1 positive and negative populations, whereas in the advanced setting, efficacy has only been reported in PD-L1 positive patients. It has been hypothesized that chemotherapy, or certain types of chemotherapy, may be more “immunogenic” than others, but thus far, there is scant evidence to support this. Rather it is more likely that the immune context is important. In the early-stage setting, many primary breast cancers (triple negative and HER2-positive) are highly infiltrated with immune cells, the cancers has received no prior treatment, and cancer burden is low, confined to the breast and ultimately completely removed. In contrast, in the advanced setting, the immune infiltrate is low to non-existent, patients often have higher disease burden, hostile tumor microenvironments in multiple organs sites, and have received prior treatment, implying that the cancer has evolved to become treatment resistant, as well as more efficient at suppressing or evading host immune detection. Hence, in the setting of recurrent disease, and likely also in the locally advanced setting, despite chemotherapy usage and objective tumor shrinkage, it is the pre-existing immunity that ultimately dictates the chances of benefit from PD-(L)1 inhibitors. What combinations are likely therefore to be effective in the advanced setting where immunity does not exist? Targeted therapies such as MEK and AKT inhibitors are being evaluated in clinical trials, thus far have proven disappointing. New antibody drug conjugates (ADCs) such as Sacituzumab Govetecan and DS-8201a have shown impressive anti-tumor activity in the advanced setting. However, given their proven cytotoxic bystander effects on nearby cells, it will be essential to evaluate ADCs and immunotherapy sequencing approaches to ensure that the scant amount of pre-existing local TIL is also not destroyed. Interestingly a recent study in advanced HER2-positive breast cancer (KATE2) reported benefit of atezolizumab to trastuzumab emantasine also in only PD-L1 positive patients. Notably T-DM1 does not have prominent bystander effects. Other possibilities to create immunity in the advanced setting include radiotherapy, more potent anti-HER2 agents, as well as genetically engineered T cell receptor or chimeric antigen receptor T cells, and agents targeting T regulatory cells. This task is likely to be a very challenging as it will require more than making “cold” tumors “hot”, as is probable that we will need to find the means that can re-invigorate the whole host immune system in the context of advanced breast cancer. Citation Format: S Loi. Rationale for combination therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP086.

Novel Auristatins with High Bystander and Cytotoxic Activities in Drug Efflux-positive Tumor Models.

Auristatins, a class of clinically validated anti-tubulin agents utilized as payloads in antibody-drug conjugates, are generally classified by their membrane permeability and the extent of cytotoxic bystander activity on neighboring cells after targeted delivery. The drugs typically fall within two categories: membrane permeable monomethyl auristatin E-type molecules with high bystander activities and susceptibility to efflux pumps, or charged and less permeable monomethyl auristatin F (MMAF) analogs with low bystander activities and resistance to efflux pumps. Herein, we report the development of novel auristatins that combine the attributes of each class by having both bystander activity and cytotoxicity on multidrug-resistant (MDR+) cell lines. Structure-based design focused on the hydrophobic functionalization of the N-terminal N-methylvaline of the MMAF scaffold to increase cell permeability. The resulting structure-activity relationships of the new auristatins demonstrate that optimization of hydrophobicity and structure can lead to highly active free drugs and antibody-drug conjugates with in vivo bystander activities.

Glypican-3-Targeted Alpha Particle Therapy for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is expressed in 75% of hepatocellular carcinoma (HCC), but not normal liver, making it a promising HCC therapeutic target. GC33 is a full-length humanized monoclonal IgG1 specific to GPC3 that can localize to HCC in vivo. GC33 alone failed to demonstrate therapeutic efficacy when evaluated in patients with HCC; however, we posit that cytotoxic functionalization of the antibody with therapeutic radionuclides, may be warranted. Alpha particles, which are emitted by radioisotopes such as Actinium-225 (Ac-225) exhibit high linear energy transfer and short pathlength that, when targeted to tumors, can effectively kill cancer and limit bystander cytotoxicity. Macropa, an 18-member heterocyclic crown ether, can stably chelate Ac-225 at room temperature. Here, we synthesized and evaluated the efficacy of [225Ac]Ac–Macropa–GC33 in mice engrafted with the GPC3-expressing human liver cancer cell line HepG2. Following a pilot dose-finding study, mice (n = 10 per group) were treated with (1) PBS, (2) mass-equivalent unmodified GC33, (3) 18.5 kBq [225Ac]Ac–Macropa–IgG1 (isotype control), (4) 9.25 kBq [225Ac]Ac–Macropa–GC33, and (5) 18.5 kBq [225Ac]Ac–Macropa–GC33. While significant toxicity was observed in all groups receiving radioconjugates, the 9.25 kBq [225Ac]Ac–Macropa–GC33 group demonstrated a modest survival advantage compared to PBS (p = 0.0012) and 18.5 kBq [225Ac]Ac–IgG1 (p = 0.0412). Hematological analysis demonstrated a marked, rapid reduction in white blood cells in all radioconjugate-treated groups compared to the PBS and unmodified GC33 control groups. Our studies highlight a significant disadvantage of using directly-labeled biomolecules with long blood circulation times for TAT. Strategies to mitigate such treatment toxicity include dose fractionation, pretargeting, and using smaller targeting ligands.

Abstract OT1-07-02: A phase 3, multicenter, randomized, open-label trial of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) vs investigator’s choice in HER2-low breast cancer (DESTINY-Breast04)

Abstract Background: Human epidermal growth factor receptor 2 (HER2)-targeted therapies have greatly improved survival in subjects with HER2-positive breast cancer (BC). However, there are many more subjects with BC with HER2-low expression (immunohistochemistry [IHC] 1+ or 2+/in situ hybridization negative), for whom no HER2-targeted therapies are approved. [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is an antibody-drug conjugate with a humanized HER2 antibody, peptide-based cleavable linker, and topoisomerase I inhibitor payload. It has a drug-to-antibody ratio of 7 to 8, and the membrane permeability of the cleaved payload induces a cytotoxic bystander effect. In an ongoing, phase 1 study, T-DXd demonstrated an objective response rate (ORR) of 44.2% (19/43) with a manageable safety profile in heavily pretreated, advanced HER2-low BC (Modi et al, SABCS 2018). Results from the pivotal, phase 2 study of subjects with HER2-positive BC previously treated with [ado-] trastuzumab emtansine (DESTINY-Breast01) confirmed the activity observed in the phase 1 trial and will be presented at the meeting (Krop, et al). Here, we describe the phase 3 trial evaluating T-DXd in subjects with HER2-low BC. Study Description: DESTINY-Breast04 is a randomized, phase 3, 2-arm, open-label, multicenter trial comparing the efficacy and safety of T-DXd with investigator’s choice of treatment in HER2-low (IHC1+ or IHC2+/ISH−), unresectable and/or metastatic BC. The trial started in December 2018 and is recruiting subjects from 223 sites in North America, Europe, and Asia. Approximately 540 subjects (480 hormone receptor [HR]+ and 60 HR-) will be randomized (2:1) to T-DXd (5.4 mg/kg intravenously every 3 weeks) or investigator’s choice (capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel). Randomization will be stratified by HER2 IHC status, number of prior treatments, and prior CDK4/6 inhibitor therapy/HR status. HER2 IHC will be assessed and confirmed by central testing based on archival samples. The primary efficacy endpoint is progression-free survival (PFS) per RECIST v1.1, determined by blinded independent central review. Secondary efficacy endpoints include investigator-assessed PFS, overall survival, ORR, and duration of response. Safety endpoints include treatment-emergent adverse events (AEs), serious AEs, and AEs of special interest (including interstitial lung disease/pneumonitis, cardiotoxicity, and infusion-related reactions). For further information about this trial, visit ClinicalTrials.gov (NCT03734029). Citation Format: Shanu Modi, Shoichiro Ohtani, Caleb Lee, Yibin Wang, Kapil Saxena, David A. Cameron. A phase 3, multicenter, randomized, open-label trial of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) vs investigator’s choice in HER2-low breast cancer (DESTINY-Breast04) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT1-07-02.

Abstract P1-18-12: A phase 1, multicenter, open-label study to assess the effect of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) on QTc and pharmacokinetics in subjects with HER2-expressing metastatic and/or unresectable breast cancer

Abstract Background [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is a novel antibody-drug conjugate with a humanized anti-HER2 antibody, peptide-based cleavable linker, and topoisomerase I inhibitor payload (MAAA-1181a). T-DXd has a drug-to-antibody ratio of ≈ 8, and the membrane permeability of the cleaved payload induces a cytotoxic bystander effect. In the phase 1 study (NCT02564900), T-DXd 5.4 and 6.4 mg/kg had a manageable safety profile and confirmed objective response rates (ORRs) of 59.5% in subjects with advanced HER2-positive breast cancer (BC) and 44% in subjects with BC with low HER2 expression. Methods This phase 1 study of T-DXd assessing the QTc interval (by 12-lead ECG) and pharmacokinetics (PK) in subjects with HER2-expressing metastatic BC (NCT03366428) was conducted at 7 study sites in Japan. Subjects received T-DXd 6.4 mg/kg IV infusion once every 3 weeks. Data cutoff (Dec 5, 2018) occurred after all subjects completed ≥ 3 cycles (C) or discontinued. Results 51 subjects enrolled and received T-DXd; all were female and Asian. Median age was 56 y (range, 31-79; ≥ 65 y, 25%); 8% were HER2-positive (IHC3+ or IHC2+/ISH+) and 92% were HER2-low (IHC1+, 73%; IHC 2+/ISH−, 12%); 75% were estrogen-receptor positive. Prior therapies included trastuzumab (24%), CDK4/6i (22%), T-DM1 (16%), and pertuzumab (16%); 67% had received &amp;gt; 5 lines of therapy. At data cutoff, 63% of subjects remained on treatment; primary reasons for discontinuation were disease progression (29%) and adverse events (AEs, 4%; grade [Gr] 2 pneumonitis and Gr 2 LVEF decrease). Median duration of survival follow-up was 4.8 months (range, 1.4-8.9). T-DXd 6.4 mg/kg was not associated with a clinically meaningful QTcF prolongation (change of &amp;gt; 10 ms); the upper bound of the 90% CI was &amp;lt; 10 ms at all assessments. The PK profile of total anti-HER2 antibody was similar to that of T-DXd; MAAA-1181a exposure was low. Some accumulation (35%) of T-DXd was observed from C 1-3 (Table); mean accumulation ratio for AUCtau at C 3 was consistent with the observed t1/2 of T-DXd. All subjects had treatment-emergent AEs (TEAEs; 67% Gr ≥ 3), most of which were gastrointestinal or hematologic disorders and primarily Gr 1 or 2. Most common Gr ≥ 3 TEAEs were neutrophil count decreased (45%), white blood cell count decreased (24%), anemia (10%), platelet count decreased (8%), lymphocyte count decreased (8%), and fatigue (6%). 2 subjects had serious TEAEs (Gr 2 nausea [drug related] and Gr 3 femur fracture [not drug related]). 1 subject had ILD (Gr 2 pneumonitis) and 4 had Gr 1 electrocardiogram QT prolonged. No grade 5 events occurred. The unconfirmed ORR (CR + PR) was 39% (95% CI, 26%-54%), with responses seen in HR+ (37%), HR− (46%), and HER2-low (IHC 1+, 43%; IHC 2+/ISH−, 20%) BC. Median time to response was 2.9 months (range, 1.2-4.4), and the duration of response was not yet reached. The disease control rate (CR + PR + SD ≥ 5 weeks from first dosing date) was 84% (95% CI, 82%-99%). Conclusion T-DXd 6.4 mg/kg was not associated with clinically relevant QTcF prolongation; the accumulation of T-DXd from C 1-3 was consistent with the elimination t1/2. T-DXd had a manageable safety profile, consistent with other studies. T-DXd showed preliminary antitumor activity with clinically meaningful responses in heavily pretreated subjects with metastatic HER2-expressing BC (mostly HER2-low), including HR+ and HR− disease. Pharmacokinetic Parameters for 3 Analytes by CycleMean (SD)Median (range)Cmax, μg/mLAUCtau, μg·d/mLt1/2, dTmax, hT-DXdCycle 1 (n = 51)179 (112)677 (141)5.82 (1.11)2.08 (1.55-7.02)Cycle 3 (n = 37)154 (23.3)905 (189)7.40 (1.48)2.12 (0.70-7.15)AR Cycle 3/ Cycle 1-1.35 (0.150)--Total Anti-HER2 AntibodyCycle 1 (n = 51)165 (110)752 (185)6.35 (2.01)2.07 (1.48-7.02)Cycle 3 (n = 37)142 (27.0)1030 (256)8.27 (1.97)2.07 (0.60-7.15)AR Cycle 3/ Cycle 1-1.36 (0.219)--MAAA-1181aCmax, ng/mLAUCtau, ng·d/mLt1/2, dTmax, hCycle 1 (n = 51)12.6 (4.49)39.0 (11.2)5.74 (1.29)6.93 (3.88-191.47)Cycle 3 (n = 37)9.60 (3.89)41.5 (13.8)6.57 (1.81)6.92 (1.95-70.65)AR Cycle 3/ Cycle 1-1.09 (0.194)--AR, accumulation ratio; AUCtau, area under plasma concentration-time curve over dosing interval; Cmax, maximum concentration; HER2, human epidermal growth factor receptor 2; t1/2, half-life; Tmax, time to maximum concentration. Citation Format: Toshinari Yamashita, Akihiko Shimomura, Toshimi Takano, Yasuaki Sagara, Junichiro Watanabe, Eriko Tokunaga, Kunika Kikumori, Emi Kamiyama, Takahiro Kamio, Ryo Nakamura, Tetsu Shinkai, Shunji Takahashi. A phase 1, multicenter, open-label study to assess the effect of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) on QTc and pharmacokinetics in subjects with HER2-expressing metastatic and/or unresectable breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-12.

Complement-independent bystander injury in AQP4-IgG seropositive neuromyelitis optica produced by antibody-dependent cellular cytotoxicity

Cellular injury in AQP4-IgG seropositive neuromyelitis spectrum disorder (herein called NMO) involves AQP4-IgG binding to astrocytes, resulting in astrocyte injury by complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) mechanisms. The rapid disease progression, severe tissue damage, and abundant leukocyte infiltration seen in some NMO patients suggest a more direct mechanism for demyelination and neurologic deficit than secondary injury from astrocyte loss. Here, we report evidence for an ‘ADCC bystander mechanism’ in NMO involving injury to nearby cells by leukocytes following their activation by AQP4-bound AQP4-IgG on astrocytes. In model cocultures containing AQP4-expressing and null CHO cells, AQP4-IgG and complement killed bystander null cells to ~ 100 μm away from AQP4-expressing cells; AQP4-IgG and NK cells produced bystander killing to ~ 300 μm, with perforin deposition seen on injured null cells. Bystander cytotoxicity was also seen with neutrophil-mediated ADCC and in astrocyte-neuron cocultures. Mechanistic studies, including real-time imaging, suggested that leukocytes activated by an AQP4-dependent ADCC mechanism injure bystander cells by direct targeted exocytosis on neighboring cells and not by diffusion of soluble granule contents. In support of this conclusion, ADCC bystander injury was preferentially reduced by an RGDS peptide that inhibits integrin adhesion. Evidence for ADCC bystander injury to oligodendrocytes and neurons was also found in mice following intracerebral injection of AQP4-IgG and NK cells, which was inhibited by RGDS peptide. These results establish a novel cellular pathogenesis mechanism in AQP4-IgG seropositive NMO and provide evidence that inflammatory mechanisms can cause widespread tissue damage in NMO independently of the secondary effects from astrocyte loss.

Abstract 221: Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro

Abstract Tisotumab vedotin is an antibody-drug conjugate composed of a human IgG targeting tissue factor (TF), a valine citruline linker, and the microtubule disrupting agent monomethyl auristatin E (MMAE). TF is aberrantly expressed in solid tumors and is thought to contribute to tumor progression by enhancing tumor growth, neo-angiogenesis and metastatic potential through local activation of coagulation and protease-activated receptor-2 (PAR-2) signaling. Tisotumab vedotin was previously shown to induce cytotoxicity through MMAE- and Fc-dependent mechanisms. In addition, tisotumab vedotin inhibited PAR-2 signaling in TF-positive tumor cells. Here we aimed to elucidate additional effector mechanisms by assessing the capacity of tisotumab vedotin to induce immunogenic cell death (ICD), bystander cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP) in vitro. ICD was assessed by incubating A431, MDA-MB-231, and HPAFII cells with tisotumab vedotin, and measuring ER stress by western blot, ATP secretion by reporter assay, and HMGB1 release by ELISA. Bystander cytotoxicity was assessed by incubating mixed cultures of TF+ (MDA-MB-231) and TF- (A549) tumor cells with tisotumab vedotin and quantifying viable cells by flow cytometry. To assess ADCP, PKH26-labeled BxPC-3 or A431 cells were incubated with human monocyte derived macrophages in presence of tisotumab vedotin. ADCP activity was analyzed by measuring the fraction of macrophages that double stained with PKH26 and anti-CD11c antibody using flow cytometry. Tisotumab vedotin induced key hallmarks of ICD including induction of ER stress, ATP secretion, and release of HMGB1, which have been shown to facilitate antitumor immunity. In co-cultures of TF+ and TF- tumor cells, tisotumab vedotin induced cytotoxicity in both cell types, even at a target-positive to target-negative cellular ratio of 1:15. This was mediated through bystander cytotoxicity, as demonstrated by the lack of cytotoxicity in monocultures of TF- cells. Finally, ADCP was observed with macrophages derived from multiple donors, with double-positive macrophages comprising up to 50% of total macrophages. In summary, ICD, bystander cytotoxicity, and ADCP were identified as novel effector mechanisms of tisotumab vedotin in vitro. Combining the present study with previous results, tisotumab vedotin induces TF-dependent anti-tumor activity through 1) MMAE-mediated effector mechanisms, including MMAE-mediated direct and bystander cytotoxicity, and induction of ICD; 2) Fc-mediated effector mechanisms, including ADCC and ADCP; and 3) Fab-mediated inhibition of PAR-2 dependent signaling. Tisotumab vedotin is currently being investigated in a variety of solid tumors (NCT03245736, NCT03485209, NCT03438396, NCT03657043). Citation Format: Stephen C. Alley, Jeffrey R. Harris, Anthony Cao, Elke Gresnigt-van den Heuvel, Jyoti Velayudhan, David Satijn, Sandra Verploegen, Teresa Dominguez, Esther C. Breij. Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro [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 221.

A phase III, multicenter, randomized, open label trial of [fam-] trastuzumab deruxtecan (DS-8201a) versus investigator’s choice in HER2-low breast cancer.

TPS1102 Background: Human epidermal growth factor receptor 2 (HER2)-targeted therapies have greatly improved survival in HER2+ breast cancer (BC). However, there are many more BC patients with HER2-low expression (immunohistochemistry [IHC] 1+ or 2+/ in situ hybridization-negative), for whom no HER2-targeted therapies are approved. [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is an antibody-drug conjugate with a humanized HER2 antibody, peptide-based cleavable linker, and a topoisomerase I inhibitor payload. It has a drug-to-antibody ratio of ~8 and the membrane permeability of the cleaved payload enables a cytotoxic bystander effect. In an ongoing phase 1 trial, T-DXd demonstrated an objective response rate (ORR) of 44.2% (19/43) with a manageable safety profile in heavily pretreated, advanced HER2-low BC (Oct 2018 data cutoff; Modi et al, SABCS 2018). Methods: DESTINY-Breast04 is a randomized, phase 3, 2-arm, open-label, multicenter study comparing efficacy and safety of T-DXd vs physician’s choice in HER2-low, unresectable and/or metastatic BC. Approximately 540 subjects will be randomized 2:1 to T-DXd (5.4 mg/kg intravenous every 3 weeks) or physician’s choice (capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel). Randomization is stratified by HER2 IHC status, number of prior treatments, and prior CDK4/6 inhibitor therapy/hormone receptor (HR) status. HER2 IHC will be assessed and confirmed by central testing based on archival samples. The primary efficacy endpoint is progression-free survival (PFS) per mRECIST v1.1 determined by blinded independent central review. Secondary efficacy endpoints are investigator-assessed PFS, overall survival, confirmed ORR, and duration of response. Safety endpoints include serious adverse events (AEs), treatment-emergent AEs, and AEs of special interest (interstitial lung disease/pneumonitis, cardiotoxicity, and infusion-related reactions). Primary PFS analysis will occur when 318 events are observed in HR+ subjects; providing 90% power to detect a hazard ratio of 0.68 with 1-sided α of 0.025. Enrollment began in Dec 2018. Clinical trial information: NCT03734029.

The death receptor Fas mediates local bystander killing of antigen-negative variants by antigen-specific CD8 T cells in a heterogeneous tumor

Abstract Antigen-specificity is one of the hallmarks of the adaptive immune system, and CD8 T cells are particularly crucial for eliminating malignant cells with aberrant expression of tumor-associated self-antigens or neoantigens. We established a high-throughput pooled CRISPR/Cas9 screen for functionally assessing cancer cell genes involved in modulating the cytotoxic activity of antigen-specific CD8 T cells. As validation of our approach, we identified many genes known to be altered in cancers refractory to immunotherapy, including MHC component B2m and the checkpoint molecule Cd274. Interestingly, the cell surface death receptor Fas was identified as a strong hit in mediating the killing of antigen-positive lymphoma and was further verified with breast cancer, despite the availability of other cytotoxic effectors such as perforin/granzymes. Using a 2-antigen system, we surprisingly observed that Fas was also mediating the killing of neighboring GFP-negative cancer cells by T cells highly specific for a GFP epitope. This phenomenon was not dependent on MHC Class I expression but was potentiated by interferon gamma, which is known to upregulate Fas expression. Even for T cells that were previously primed, this bystander cytotoxicity required the presence of neighboring antigen-positive cells, suggesting a very hyperlocal and temporally regulated off-target effect that was not dependent on natural killer cells in vivo. We believe this phenomenon to be crucial to the durable remissions seen with highly antigen-specific approaches such as CAR-T cell therapy. Modulating this pathway may be a viable approach for preventing immune escape of rare antigen-loss variants or treating heterogeneous tumors with no clear single target antigen.

RA-839, a selective agonist of Nrf2/ARE pathway, exerts potent anti-rotaviral efficacy in vitro

Acute watery diarrhea due to Rotavirus (RV) infection is associated with high infantile morbidity and mortality in countries with compromised socio-economic backgrounds. Although showing promising trends in developed countries, the efficacy of currently licensed RV vaccines is sub-optimal in socio-economically poor settings with high disease burden. Currently, there are no approved anti-rotaviral drugs adjunct to classical vaccination program. Interestingly, dissecting host-rotavirus interaction has yielded novel, non-mutable host determinants which can be subjected to interventions by selective small molecules. The present study was undertaken to evaluate the anti-RV potential of RA-839, a recently discovered small molecule with potent and highly selective agonistic activity towards cellular redox stress-sensitive Nuclear factor erytheroid-derived-2-like 2 (Nrf2)/Antioxidant Response Element (ARE) pathway. In vitro studies revealed that RA-839 inhibits RV RNA and protein expression, viroplasm formation, yield of virion progeny and virus-induced cytopathy independent of RV strains, RV-permissive cell lines and without bystander cytotoxicity. Anti-RV potency of RA-839 was subsequently identified to be independent of stochastic Interferon (IFN) stimulation but to be dependent on RA-839's ability to stimulate Nrf2/ARE signaling. Interestingly, anti-rotaviral effects of RA-839 were also mimicked by 2-Cyano-3, 12-dioxo-oleana-1, 9(11)-dien-28-oic acid methyl ester (CDDO-Me) and Hemin, two classical pharmacological activators of Nrf2/ARE pathway. Overall, this study highlights that RA-839 is a potent antagonist of RV propagation in vitro and can be developed as anti-rotaviral therapeutics.

New path to treating pancreatic cancer: TRAIL gene delivery targeting the fibroblast-enriched tumor microenvironment

Gene therapy has shown promise in antitumor strategies for advanced cancer. However, efficient and safe delivery of potent therapeutic gene expressing in specific tumor tissues remains elusive, especially when there exist stromal obstacles. Here we report a non-viral gene delivery approach targeting pancreatic stellate cells (PSCs) as the transfection host in the fibroblast-enriched tumor microenvironment of pancreatic cancer. Plasmid DNA (pDNA) encapsulated in branched polyethylenemine (BPEI) was found to selectively transfect PSCs rather than pancreatic cancer cells and other fibroblast cell lines. Mechanism investigations reveal that the highly expressed fibroblast growth factor receptors (FGFRs) in PSCs facilitated the cellular uptake of polyplexes in PSCs. This delivery platform carrying gene encoding of TNF-related apoptosis-inducing ligand (TRAIL) displayed effective by-stander effect and tumor cell-selective cytotoxicity. More importantly, the therapeutic efficacy was proved in a PSC-enriched orthotopic pancreatic tumor model. Thus, this gene delivery strategy smartly converts PSCs as the microenvironment obstacle for drug delivery into the producer and reservoir of selective tumor-killing proteins.