Search

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express antitumor T cell receptors or chimeric antigen receptors (CARs). Although the potency and specificity of tumor-specific T cells can be manipulated ex vivo, once reinfused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium. Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of antitumor T cells. Antibodies that target PS have been shown to reactivate antitumor immunity by polarizing tumor-associated macrophages into a proinflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. Our lab has previously shown that a PS targeting monoclonal antibody (mch1N11), in combination with transgenic CD4+ T cells that recognize the melanoma antigen Trp1, can regress very advanced melanomas in all treated mice. Here, we further those studies with data showing that a 2nd-generation CAR T cell that binds Trp1 on the surface of B16 melanoma, in combination with mch1N11 can improve antitumor activity and overall survival in B16 tumor-bearing mice. Additionally, in vitro killing assays with antigen-specific T cells sorted from the tumor reveal that mch1N11 enhances the cytolytic function of these T cells against B16 melanoma. Flow cytometry analysis of local immune responses in the tumors of animals treated with tumor-specific T cells and mch1N11 showed a decrease in anti-inflammatory (M2) macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with mch1N11 can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS targeting antibodies with CAR T cell therapy in solid tumors. Citation Format: Sara Schad, Daniel Hirschhorn-Cymerman, Sadna Budhu, Hong Zhong, Xia Yang, Joseph Shan, Steven King, Taha Merghoub, Jedd Wolchok. Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma [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 3568.

Background: Despite the excitement surrounding checkpoint blockade, many tumors remain resistant thus there is a need to sensitize tumors to immunotherapy and augment tumor rejection. Phosphatidylserine (PS) is a phospholipid expressed on the outer surface of apoptotic cells and a variety of immune cell types. PS signaling polarizes macrophages to the M2 phenotype, inhibits TLR signaling and is upregulated on the surface of tumor cells by radiotherapy (RT). RT also promotes infiltration of CD8 T-cells to the tumor microenvironment. Accordingly, RT may improve CD8 T-cell mediated tumor rejection in the context of checkpoint blockade and sensitize tumors to PS antagonism. We hypothesize PS-targeting antibody, mch1n11, in combination with local RT, may be an effective adjuvant or alternative treatment in patients with tumors resistant to checkpoint blockade such as anti-PD-1 (aPD-1). Furthermore, we hypothesize the combination of aPD-1 with mch1n11 and RT will synergize to improve the effector function of CD8 T-cells and enhance tumor elimination. Methods: B16-F10 murine melanoma was injected into the hind limb of C57BL/6 mice. One cohort received triple combination therapy comprised of aPD-1, mch1n11, and RT given simultaneously at an early time point. Another cohort that showed continued tumor growth after therapy with aPD-1 was used to model checkpoint blockade resistance. These mice received delayed treatment with intra-peritoneal mch1n11 in combination with a one-time 15 Gy RT dose targeted to the tumor-bearing hind limb. The aPD-1 resistant cohort was subdivided into two groups: in one cohort aPD-1 was administered prior to and continued with mch1n11+RT therapy. In the other cohort, aPD-1 was discontinued prior to mch1n11+RT administration. Untreated mice, mice treated with aPD-1 alone, mice treated with isotype antibodies, and mice treated without initial aPD-1 therapy that received delayed mch1n11+RT therapy served as controls. Results: Early triple combination therapy results in almost complete tumor elimination and leads to statistically significant prolonged survival. In the aPD-1 resistant cohort, both prior and continuous aPD-1 treatment with mch1n11+RT improved survival compared to all four controls. There was no significant difference in tumor size or survival between the adjuvant and alternative experimental groups. Experiments are ongoing to characterize immune infiltrates of tumors and the individual effects of aPD-1, RT and mch1n11. Conclusion: This preclinical model suggests a phosphatidylserine targeting antibody combined with single dose RT presents an alternative or adjuvant therapy for tumors resistant to checkpoint blockade. The data from this preclinical model will be used to develop a clinical trial for patients with tumors resistant to checkpoint blockade. Citation Format: Rachel Giese, Sadna Budhu, Christopher Barker, Aditi Gupta, Steve King, Joseph Shan, Jedd Wolchok, Taha Merghoub. Phosphatidylserine targeting and radiation improves survival in a mouse tumor model resistant to checkpoint blockade [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 2767.

Glioblastoma multiforme (GBM) is one of the most intractable cancers in humans, and yet, in the past decade, incremental advances in the treatment of brain tumors have begun to suggest that effective therapies may be on the hori- zon. Here we review the latest treatments available to patients and focus on a promising radiotherapeutic strategy that em- ploys the isotope 131 Iodine conjugated to an antibody that binds the necrotic core found in all solid tumors. Historically, GBM patients who relapse have a median survival time of no more than 24 weeks; however, the Tumor Necrosis Therapy discussed here has already provided a good quality of life for several patients years beyond the historical median survival time. The cases of two long-term survivors are reviewed, and data are presented to show that initial post-treatment as- sessments of tumor progression actually turned out to be tumor necrosis and inflammation. Given the current lack of im- aging modalities that can distinguish between tumor progression and pseudoprogression, these cases further highlight the challenges faced by physicians in differentiating disease progression or recurrence from necrosis.

Meeting abstracts Bavituximab is a novel chimeric IgG1 monoclonal antibody targeting the membrane phospholipid, phosphatidylserine (PS), externalized on the luminal surface of endothelium in tumors, tumor cells, and tumor exosomes under stressor conditions in the tumor microenvironment. PS exposure

The primary aim of this study was to evaluate the biodistribution and toxicity of 131I-chimeric(ch) TNT-1/B monoclonal antibody (MAB), which binds to intracellular antigens of necrotic regions within tumors, in patients with advanced colon or colorectal cancer. The rationale for targeting areas of tumor necrosis is the observation that necrotic lesions are more abundant in cancer lesions than in surrounding tissues.Cohorts of patients with advanced colon or colorectal cancer were administered a one-time 30-60-minute intravenous (i.v.) infusion of 131I-chTNT-1/B at doses ranging from 12.95 to 66.23 MBq/kg (0.35-1.79 mCi/kg).The dose-limiting toxicity, experienced at 66.23 MBq/kg (1.79 mCi/kg) 131I-chTNT-1/B MAB, was myelosuppression. Two (2) patients at the 66.23-MBq/kg (1.79 mCi/kg) dose level had both grade 3 thrombocytopenia and grade 3 neutropenia that persisted for at least 2 weeks but were reversible. The maximum tolerated dose was 58.09 MBq/kg (1.57 mCi/kg) 131I-chTNT-1/B MAB. Of the 21 patients, one developed a moderate human antichimeric antibody (HACA) response and 6 developed low HACA responses.The infusion of 131I-chTNT-1/B MAB was well tolerated, without significant nonhematological toxicity. No patient obtained a complete or partial response, based on tumor cross-product response criteria. Tumor localization was seen in patients with dose levels at, and exceeding, 50.23 MBq/kg (1.36 mCi/kg) 131I-chTNT-1/B MAB.

Treatment of malignant glioma is therapeutically challenging. Despite improvements in neurosurgery, radiotherapy and chemotherapy, few patients diagnosed with anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM) (WHO grades 3 and 4, respectively) will live beyond 2 years. Poor survival is due to the highly invasive nature and protected location of these tumours. Most malignant gliomas cannot be completely resected or irradiated due to their ability to infiltrate diffusely into normal brain tissue. Brain tissue is protected from the systemic circulation via the blood-brain barrier (BBB), which impedes entry of water-soluble chemotherapeutic agents into the tumour at therapeutic concentrations. (131)I-chTNT-1/B mAb (Cotara) employs an innovative strategy to treat the invasive portion of the tumour and the core lesion. (131)I-chTNT-1/B mAb is a genetically engineered, radiolabelled, chimeric monoclonal antibody specific for a universal intracellular antigen (i.e., DNA/histone H1 complex) exposed in the necrotic core of malignant gliomas. This antigen provides an abundant, insoluble, non-diffusible anchor for the mAb. Once localised to necrotic regions of the tumour, (131)I-chTNT-1/B mAb delivers a cytotoxic dose of (131)I radiation to the core lesion. (131)I-chTNT-1/B mAb is delivered via convection-enhanced delivery in order to maximise coverage to the tumour and the invasive front of the glial tumour. The clinical experience to date with (131)I-chTNT-1/B mAb is presented.

OBJECTIVE: We report the safety and feasibility of using convection-enhanced delivery to administer Cotara (Peregrine Pharmaceuticals, Inc., Tustin, CA), a novel radioimmunotherapeutic agent, to patients with malignant glioma. METHODS: Between April 1998 and November 2002, 51 patients with histologically confirmed malignant glioma received Cotara by convection-enhanced delivery. Most patients (88%) were treated with Cotara targeting tumor volume-dependent, single or multiple administrations of activity ranging from 0.5 to 3.0 mCi/cm3 of baseline clinical target volume. Two weeks after infusion, single-photon emission computed tomographic imaging determined the spatial distribution of Cotara. Patients were followed for as long as 41 months (average follow-up, 5 mo). Safety was evaluated on the basis of incidence of procedure-related, neurological, and systemic adverse events. Feasibility was evaluated in a subset of patients on the basis of the correlation between the prescribed activity and the actual activity administered to the targeted region. RESULTS: Fifty-one patients, 37 with recurrent glioblastoma multiforme, 8 with newly diagnosed glioblastoma multiforme, and 6 with recurrent anaplastic astrocytomas, were treated. Average tumor volume was 36 ± 27.6 cm3 (range, 5–168 cm3). Of the 67 infusions, 13 (19%), 52 (78%), and 2 (3%) delivered less than 90%, 100 ± 10%, and more than 110%, respectively, of the prescribed administered activity to the targeted region. Treatment-emergent, drug-related central nervous system adverse events included brain edema (16%), hemiparesis (14%), and headache (14%). Systemic adverse events were mild. Several patients had objective responses to Cotara. CONCLUSION: The majority of Cotara infusions delivered between 90 and 110% of the prescribed administered activity to the targeted region. This method of administration has an acceptable safety profile compared with literature reports of other therapeutics delivered by convection-enhanced delivery.

Background SUNRISE, a global, double-bind, Phase III trial of docetaxel (D) plus bavituximab (B) or D plus placebo (P) in previously treated non-squamous non-small cell lung cancer (NSCLC), demonstrated similar overall survival (OS) in both treatment arms. Immune correlate analyses including pre-treatment IFN-γ levels in blood and tumor tissue were used to potentially identify prognostic and/or predictive correlation with clinical outcome. Methods Serum was isolated from all randomized NSCLC patients at screening, periodically during treatment and at disease progression for evaluation of IFN-γ levels using the SimoaTM assay (Myriad RBM, Austin, TX, USA). Available archival tissue was also tested for 91- immune gene activation markers, including IFN-γ by the Fluidigm-based gene-expression platform (Sirona Dx, Lake Oswego, OR, USA). Kaplan-Meier statistical methods and Cox proportion hazards models were utilized to evaluate and contrast the correlation of peripheral and intratumoral IFN-γ levels with OS. Patients were classified paradoxically as IFN-γ "low" with a favorable disease prognosis versus "high" associated with more aggressive disease based on the median. Results Pretreatment serum results were available for 582 out of the 597 randomized patients. Each patient was classified to be pre-treatment IFN-γ high or low (< cut-off) using cut-off 0.093 pg/ml, which is the median IFN-γ value in the D+B group. Median overall survival (mOS) in all patients with IFN-γ low is 11.3 months (95% confidence interval [CI], 10.1-13.5) versus 10.4 months (95% CI, 8.4-11.3) in all IFN-γ high; p=0.047. mOS of D+B arm is 11.6 months (95% CI, 10.2-13.9) and 11.1 months (95% CI, 9.1-14.7) in the D group; p=0.982 for IFN-γ low. mOS of D+B arm is 9.0 months (95% CI, 6.7-11.2) and 10.6 months (95% CI, 8.9-13.0) in the D group; p=0.252 for IFN-γ high. With the limited intratumoral IFN-γ gene expression data (n=33), no statistically significant correlation with OS was observed. Conclusions Correlative approaches identified low peripheral low IFN-γ at pretreatment as a biomarker of interest correlating with more favorable clinical outcomes and is consistent with the hypothesis that bavituximab may demonstrate more immunomodulatory effects in patients with “immune cold” tumors. Citation Format: Nikoletta Kallinteris, Leora Horn, Min Tang, Tobias Guennel, Shen Yin, Jennifer Lai, Joseph Shan, Rachel E. Sanborn. IFN-γ analysis in blood and tissue as a potential prognostic and/or predictive biomarker [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 CT159. doi:10.1158/1538-7445.AM2017-CT159

11603 Background: SUNRISE, a global, double-bind, Phase III trial of docetaxel (D) plus bavituximab (B) or D plus placebo (P) in previously treated non-squamous non-small cell lung cancer, demonstrated similar overall survival (OS) in both treatment arms. Biomarkers including pre-treatment PD-L1 expression are being retrospectively assessed in on-going exploratory analyses. Methods: Archival tissue obtained at the time of diagnosis was requested but not required in the SUNRISE trial. FFPE slides were stained with a panel of lymphoid cell markers: CD3+, CD8+, FoxP3+, PD-L1+, CD163+, CK+ and DAPI using a 6-plex quantitative immunohistochemistry (IHC) assay (OPAL, PerkinElmer, Hopkinton, MA, USA). Baseline PD-L1 expression was retrospectively scored on tumor cells (TC) as a percentage of PD-L1 expressing tumor cells: TC3≥50%, TC2≥5% and < 50%, TC1≥1% and < 5%, and TC0 < 1%. Cox regression models for PD-L1 IHC subgroup populations were used for correlation with OS. Results: In the subset of patients with available diagnostic biopsies (110 out of 597 randomized patients), the prevalence of PD-L1 expression was 5% for TC3, 18% for TC2/3, 35% for TC1/2/3, 65% for TC0. Median OS (mOS) of the D+B arm is 11.5 months (TC0, < 1%) and 6.0 months (TC1/2/3, ≥1%) with HR 0.38 (95% CI, 0.19-0.76); p-value = 0.004. mOS of the D+P arm is 11.1 months (TC0, < 1%) and 10.4 months (TC1/2/3, ≥1%) with HR 0.93 (95% CI, 0.47-1.87); p value = 0.844. Conclusions: Baseline PD-L1 expression in a subset of SUNRISE patients demonstrated that PD-L1 expression (TC0) was associated with a significantly prolonged OS compared to positive PD-L1 expression (TC1/2/3) in patients receiving D+B. No difference in OS was observed in the D+P group by PD-L1 expression. These observations are consistent with the hypothesis that bavituximab may demonstrate more effect in PD-L1 negative or low expressing “immune cold” tumors. Clinical trial information: NCT01999673.

e23091 Background: Bavituximab is a chimeric monoclonal antibody that targets the membrane phospholipid phosphatidylserine (PS) exposed on endothelial cells and cancer cells in solid tumors. Our previous studies showed that bavituximab enhances the activation of CD8+ TILs that correlates with increased cytokine production by lymphoid and myeloid cells in lung cancer with low PD-L1 expression suggesting that the interruption of the PD-1/PD-L1 axis by nivolumab may enhance the bavituximab effect in tumors. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay, where 3D tumor microspheres were treated with bavituximab or nivolumab alone or in combination at 10 mg/ml for 36 hours. At the end of the treatment, a multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines, including human IFNg, in culture media as a surrogate of TIL activation. In addition, a gene expression analysis using a NanoString platform containing probes to quantitate 770 immune function genes. Results: Preliminary results indicate the combination treatment with bavituximab and nivolumab led to increased expression of genes involved in M1 polarization of tumor associated macrophages in a subpopulation of lung tumors that closely correlated with release of cytokines such as MIP1b (CCL4) which is a chemoattractant for natural killer cells, monocytes and a variety of other cells involved in tumor immune response. Conclusions: This lung patient derived ex-vivo approach indicates that bavituximab in combination with nivolumab may enhance immune response. This response likely involves M1 polarization of tumor associated macrophages and suggests potential clinical implications in the treatment of lung cancer.

Bavituximab is a monoclonal antibody directed against the membrane phospholipid phosphatidylserine exposed on the outer leaflet of tumor and vascular endothelial cells of the tumor microenvironment. Bavituximab modulates the tumor microenvironment by blocking PS-mediated immune suppression and activating cytotoxic T lymphocyte anti-tumor responses. In this study, we tested the immunomodulatory effect of bavituximab using a proprietary 3D ex vivo tumor microsphere technology. Upon obtaining informed consent, fresh tumor tissue from lung cancer patients were collected at the time of surgical resection. Tissue was processed for characterization of the tumor microenvironment and potential immunosuppressive mechanisms such as expression of PD-1, CTLA4, LAG3, TIM3, BTLA, and Adenosine A2AR. 3D tumor microspheres were prepared and cells were treated ex vivo with f(ab)’2 version of bavituximab, bavituximab, docetaxel, and a combination of bavituximab and docetaxel for 36 hours within the 3D tumor microsphere simulating an intact tumor microenvironment made up of tumor infiltrating lymphocytes (TIL) and myeloid cells. At the end of the treatment, TILs were analyzed by flow cytometry for cell activation and changes in CD4, CD8 and Treg (CD25+/CD127-) subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines, including human IFN-gamma in culture media as a surrogate of TIL activation. Preliminary results indicate the combination of bavituximab and docetaxel can induce TIL activation as demonstrated by a significant increase in IFN-gamma secretion when compared to tumors treated with control or either agent alone. Flow cytometry analysis revealed that this effect was associated with low PD-1 expression on CD8 cells, but did not correlate with other known immune-modulating receptors. This lung patient derived ex-vivo approach indicates that bavituximab in combination with docetaxel can elicit a tumor specific immune response in human adenocarcinoma of the lung. This effect involves, at least in part, activation of CD8+ TIL and increased inflammatory cytokine production by lymphoid and myeloid cells. In addition, we have observed low PD1 expression as a potential prognostic biomarker of positive response to bavituximab treatment. Citation Format: Soner Altiok, Melanie Mediavilla-Valera, Jenny Kreahling, David Noyes, Tiffany N. Razabdouski, Nikoletta L. Kallinteris, Joseph Shan, Scott Antonia. Bavituximab modulates tumor microenvironment and activates CD8+ tumor infiltrating lymphocytes in a patient-derived 3D ex vivo system of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 274. doi:10.1158/1538-7445.AM2015-274

2035 Background: GBM is the most common and clinically aggressive primary brain tumor. For recurrent GBM, treatment by surgical resection, radiation therapy, and chemotherapy remains palliative, an...

2039 Background: Interstitial 131I-chTNT-1/B MAb (Cotara) is a radioiodinated chimeric monoclonal antibody specific for DNA and histone H1 complex, which is exposed in the necrotic areas of gliomas...