Mahrukh Huseni

Anti-CD20/CD3 T cell–dependent bispecific antibody for the treatment of B cell malignancies

Anti-CD20/CD3 T cell–dependent bispecific antibodies may be useful for the treatment of B cell malignancies. Two-headed cancer therapy Immunotherapeutic approaches harness either humoral (antibody-mediated) or cellular (T cell–mediated) immunity to fight cancer. Sun et al. combine these approaches by designing a CD3/CD20 TDB (T cell–dependent bispecific), a dual-targeted antibody that recruits T cells to CD20-expressing cells. Their humanized TDB induces T cells to kill primary patient leukemia and lymphoma cells both in vitro and in a mouse model and can deplete CD20-expressing B cells in a macaque model with similar properties as conventional antibodies. If these data hold true in clinical studies, this CD20/CD3 TDB could add to our expanding arsenal of cancer immunotherapeutics. Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell–recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell–targeting anti-CD20/CD3 T cell–dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using “knobs-into-holes” technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Anti-EGFL7 antibodies enhance stress-induced endothelial cell death and anti-VEGF efficacy

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma

We describe results from IMmotion150, a randomized phase 2 study of atezolizumab (anti-PD-L1) alone or combined with bevacizumab (anti-VEGF) versus sunitinib in 305 patients with treatment-naive metastatic renal cell carcinoma. Co-primary endpoints were progression-free survival (PFS) in intent-to-treat and PD-L1+ populations. Intent-to-treat PFS hazard ratios for atezolizumab + bevacizumab or atezolizumab monotherapy versus sunitinib were 1.0 (95% confidence interval (CI), 0.69–1.45) and 1.19 (95% CI, 0.82–1.71), respectively; PD-L1+ PFS hazard ratios were 0.64 (95% CI, 0.38–1.08) and 1.03 (95% CI, 0.63–1.67), respectively. Exploratory biomarker analyses indicated that tumor mutation and neoantigen burden were not associated with PFS. Angiogenesis, T-effector/IFN-γ response, and myeloid inflammatory gene expression signatures were strongly and differentially associated with PFS within and across the treatments. These molecular profiles suggest that prediction of outcomes with anti-VEGF and immunotherapy may be possible and offer mechanistic insights into how blocking VEGF may overcome resistance to immune checkpoint blockade.An exploratory randomized controlled clinical trial of renal cell carcinoma identifies molecular patterns distinguishing responders to immune checkpoint blockade alone or combined with angiogenesis inhibitor versus angiogenesis inhibitor alone.

Abstract CT081: Molecular correlates of differential response to Atezolizumab +/- Bevacizumab vs Sunitnib in a Phase II study in untreated metastatic renal cell carcinoma (RCC) patients

Background: The addition of bevacizumab (bev) to atezolizumab (atezo) has demonstrated enhanced anti-tumor immune responses in pts with solid tumors (Wallin 2016). In IMmotion150 (NCT01984242), a phase II trial that compared atezo+/-bev vs sunitinib (sun) in untreated mRCC, encouraging antitumor activity of atezo+bev vs sun was observed in PD-L1 expressing tumors. We performed integrated tumor genomic analyses to correlate molecular signatures with clinical outcomes. Methods: PD-L1 status on tumor infiltrating immune cells (IC) was assessed with the SP142 IHC assay (IC0, IC1, IC2/3) (n=297). Exploratory analyses included mutation evaluation by WES (n=170) and gene expression analysis by RNA-Seq (n=263). Established gene signatures at median (high) expression levels representing T effector and IFNγ response (Teff) and angiogenesis (Ang) were evaluated in relation to PFS (RECIST v1.1 by independent review). Results: PFS was longer in PD-L1 IC2/3 and in PD-L1 IC1/2/3 in atezo+bev pts vs sun pts and in PD-L1 IC2/3 in atezo pts vs sun pts. High Teff signature expression was associated with PD-L1 IHC and longer PFS in atezo+bev pts vs sun pts. High Ang expression was associated with improved clinical activity in the sun arm; but not the atezo+bev arm. Atezo+bev had improved PFS vs sun in the Ang low subset. Additional data exploring association of high prevalence mutations with clinical outcome will be presented. Conclusions: These data indicate that the addition of bev to atezo may improve clinical benefit in patients with pre-existing anti-tumor immunity (as determined by high Teff score or PD-L1 IHC) compared to sun. Molecular profiles identified in these analyses suggest that prediction of differential outcomes to VEGF TKI and immunotherapy may be possible in front line mRCC. These results will be further explored in the ongoing phase III study IMmotion151 (NCT02420821). Citation Format: David McDermott, Mahrukh Huseni, Brian Rini, Robert Motzer, Michael Atkins, Berard Escudier, Dorothee Nickles, Zach Boyd, Shruthi Sampath, Jennifer Doss, Ning Leng, Christina Schiff, Daniel S. Chen, Gregg Fine, Thomas Powles, Priti S. Hegde. Molecular correlates of differential response to Atezolizumab +/- Bevacizumab vs Sunitnib in a Phase II study in untreated metastatic renal cell carcinoma (RCC) patients [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 CT081. doi:10.1158/1538-7445.AM2017-CT081

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho- protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.MethodsWe conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.ResultsThe phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.ConclusionsWe developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

CD8+ T cell infiltration in breast and colon cancer: A histologic and statistical analysis

The prevalence of cytotoxic tumor infiltrating lymphocytes (TILs) has demonstrated prognostic value in multiple tumor types. In particular, CD8 counts (in combination with CD3 and CD45RO) have been shown to be superior to traditional UICC staging in colon cancer patients and higher total CD8 counts have been associated with better survival in breast cancer patients. However, immune infiltrate heterogeneity can lead to potentially significant misrepresentations of marker prevalence in routine histologic sections. We examined step sections of breast and colorectal cancer samples for CD8+ T cell prevalence by standard chromogenic immunohistochemistry to determine marker variability and inform practice of T cell biomarker assessment in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Stained sections were digitally imaged and CD8+ lymphocytes within defined regions of interest (ROI) including the tumor and surrounding stroma were enumerated. Statistical analyses of CD8+ cell count variability using a linear model/ANOVA framework between patients as well as between levels within a patient sample were performed. Our results show that CD8+ T-cell distribution is highly homogeneous within a standard tissue sample in both colorectal and breast carcinomas. As such, cytotoxic T cell prevalence by immunohistochemistry on a single level or even from a subsample of biopsy fragments taken from that level can be considered representative of cytotoxic T cell infiltration for the entire tumor section within the block. These findings support the technical validity of biomarker strategies relying on CD8 immunohistochemistry.

Prevalence analysis of OX40-positive cell populations in solid tumors.

Meeting abstracts Increased numbers of OX40+ T cells within the primary tumor of colorectal cancer (CRC) patients has been associated with improved survival. Similarly, OX40 expression in sentinel lymph nodes is inversely associated with tumor grade and clinical stage in melanoma patients. However

A CD4/Foxp3/OX40 triple immunofluorescence assay determines association between T cell immune subsets and outcome in colorectal cancer

Purpose Increased numbers of OX40+ T cells in the tumor microenvironment of colorectal cancer (CRC) patients have been associated with improved outcome. However, the OX40+ T cell population is heterogeneous and includes, among others, CD4+Foxp3+ regulatory T cells (Treg) as well as CD4+Foxp3- effector T cells (Teff). In this study, we used a novel triple immunofluorescence assay for CD4, FoxP3 and OX40 to determine the relationship between tumor-associated CD4+ T cell subsets and patient disease stage as well as outcome in CRC. Methods

Publisher Correction: Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma

In the version of this article originally published, there was an error in Fig. 2n. The top line of the HR comparison chart originally was Atezo + bev vs sun. It should have been Atezo + bev vs atezo. The error has been corrected in the HTML and PDF versions of this article.

Abstract CT097: A first-in-human phase I dose escalation study of the OX40 agonist MOXR0916 in patients with refractory solid tumors

Background: OX40 is a co-stimulatory receptor that is transiently expressed by T cells upon antigen recognition. In murine models, OX40 engagement by an agonist anti-OX40 antibody can promote durable tumor regression associated with co-stimulation of effector T cells and reduction of regulatory T cells. MOXR0916 is a humanized effector-competent agonist IgG1 monoclonal antibody that targets OX40. Methods: A Phase I, open-label, multicenter study was conducted to evaluate the safety and pharmacokinetics (PK) of MOXR0916 in patients (pts) with locally advanced or metastatic refractory solid tumors that have progressed after available standard therapy. MOXR0916 was administered at fixed doses every 3 weeks (q3w), and treatment beyond RECIST progression was permitted in the absence of clinical deterioration. A 3+3 dose-escalation was conducted in immunotherapy-naive pts with a 21-day window to evaluate dose-limiting toxicity (DLT). A dedicated expansion cohort enrolled pts who consented to serial tumor biopsies, enabling immune profiling by immunohistochemistry and gene expression methods. In the biopsy cohort, prior immunotherapy with adequate washout was permitted, provided there was no history of Grade (G) ?3 immune-mediated adverse events (AEs). Results: Enrollment in the dose-finding phase of the trial is complete, with 34 pts treated across 10 dose escalation cohorts (dose levels 0.2-1200 mg) and 36 pts treated in the serial biopsy cohort (dose levels 3.2-600 mg). While NSCLC (n = 8), clear cell RCC (n = 6), melanoma (n = 2), and bladder (n = 2) were represented, less immunogenic tumor types predominated. The median number of prior regimens for metastatic disease was 2 (range 0-9); 4 pts had received prior checkpoint inhibitors. As of 11 Jan 2016, no DLTs, G4/5 AEs attributed to study treatment, or AEs leading to treatment discontinuation were reported. The majority of treatment-related AEs were G1 in severity; 4 related G3 events (autoimmune hepatitis responsive to steroids, worsening dyspnea in a pt with malignant pleural effusions, hypertension, and fatigue) were reported. At doses ?40 mg q3w, PK was linear and sustained peripheral blood OX40 receptor saturation was achieved. Tumor pharmacodynamic (PD) biomarker modulation supportive of the mechanism of action was observed in a subset of pts. Eleven of 70 pts (16%) have been treated with MOXR0916 for > 6 months (?9 cycles) with a best response of stable disease per RECIST v1.1. Updated clinical data will be presented. Conclusions: In a heterogeneous, refractory population, MOXR0916 was well-tolerated at all doses evaluated. The recommended dose and schedule based on PK and OX40 receptor saturation is 300 mg q3w. Tumor PD modulation and evidence of prolonged stable disease support the ongoing expansion phase to evaluate anti-tumor activity in select indications and Phase Ib trial in combination with atezolizumab (anti-PD-L1). Citation Format: Aaron R. Hansen, Jeffrey R. Infante, Grant McArthur, Michael S. Gordon, Alexander M. Lesokhin, Ann-Lee Stayner, Todd M. Bauer, Shahneen Sandhu, Frank Tsai, Alexandra Snyder, Deepa S. Subramaniam, Jeong Kim, Eric Stefanich, Chi-Chung Li, Jane Ruppel, Maria Anderson, Houston Gilbert, Bruce McCall, Mahrukh A. Huseni, Ina Rhee, Michael Pishvaian. A first-in-human phase I dose escalation study of the OX40 agonist MOXR0916 in patients with refractory solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT097.