mized, which improved the survival of AML engrafted mice, providing a platform for preclinical examination of CSL362. Since the compromised NK cell function of immune-deficient mice may underestimate the efficacy of CSL362, human buffy coat-derived NK cells were expanded ex vivo for adoptive transfer into AML xenografted mice. CSL362 demonstrated additional efficacy against AML xenografts in combination with chemotherapy and adoptively transferred human NK (huNK) cells, supporting its further development in the clinic.
Background: Pembrolizumab (Pembro), an anti-PD-1 immune checkpoint inhibitor, has been approved for the treatment of a variety of cancers including melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and urothelial carcinoma. Pembro was recently evaluated in HER2- breast cancer patients in the neoadjuvant I-SPY 2 TRIAL and graduated in the triple negative (TN), HR+HER2-, and HER2- signatures. HER2- patients were randomized to receive Pembro+paclitaxel followed by doxorubicin/cyclophosphamide (P+T -> AC) vs. T -> AC. We and others have shown that TN breast cancers tend to have high numbers of immune infiltrates, including T cells and tumor associated macrophages (TAMs). We evaluated expression signatures representing 14 immune cell types (TILs, T cells, CD8 T cells, exhausted T cells, Th1, Tregs, cytotoxic cells, NK, NK CD56dim, dendritic cells, mast cells, B cells, macrophages, and neutrophils) as specific predictors of response to Pembro. Methods: Data from 248 patients (Pembro: 69; controls: 179) were available. Pre-treatment biopsies were assayed using Agilent gene expression arrays. Signature scores are calculated by averaging cell type specific genes. All I-SPY 2 qualifying biomarker analyses follow a pre-specified analysis plan. We used logistic modeling to assess biomarker performance. A biomarker is considered a specific predictor of Pembro response if it associates with response in the Pembro arm but not the control arm, and if the biomarker x treatment interaction is significant (likelihood ratio test, p<0.05). This analysis is also performed adjusting for HR status as covariates, and within receptor subsets. For successful biomarkers, we use Bayesian modeling to estimate the pCR rates of 'predicted sensitive' patients in each arm. Our statistics are descriptive rather than inferential and do not adjust for multiplicities of other biomarkers outside this study. Results: 10 out of the 14 cell-type signatures tested are associated with response in the Pembro arm. Higher expression levels of 9 of these cell-type signatures are associated with higher pCR rates (T cells, exhausted T cells, Th1, cytotoxic cells, NK, NK CD56dim, dendritic cells, B cells, and macrophages), whereas higher mast cell signature expression is associated with non-pCR. Interestingly, many of these same signatures also associate or trend towards association with response in the control arm; and in a model adjusting for HR status, only 3 of these signatures (Th1, B cells and dendritic cells) show significant interaction with treatment. Within the whole population and the TN subtype, the dendritic cell signature is the strongest predictor of specific response to Pembro (OR/1SD: 4.04 and 4.4, LR p < 0.001 overall and in TN). Although other immune signatures (T cells, exhausted T cells, NK, and macrophages) also associate with response in the Pembro arm in the TN subtype, only the dendritic cell and Th1 signatures have a significant interaction with treatment. In contrast, in the HR+HER2- subtype, only 3 signatures (Th1, B cells, and mast cells) associate with response to Pembro; but none of these signatures have significant interaction with treatment. Of note, in both the Pembro and control arms, HR+HER2- patients with higher average mast cell marker expression have lower pCR rates (OR/1SD: 0.33 and 0.51, LRp: 0.006 and 0.04 in Pembro and control arm). Conclusion: As expected, multiple immune cell expression signatures are predictive of response in the Pembro arm; but only dendritic cells and Th1 cells are specific to Pembro in both the population as a whole and the TN subtype. Interestingly, the presence of mast cells may impede response, especially in HR+HER2- patients. Correlation of these signatures with multiplex-IF immune markers is pending. Citation Format: Campbell M, Yau C, Borowsky A, Vandenberg S, Wolf D, Rimm D, Nanda R, Liu M, Brown-Swigart L, Hirst G, Asare S, van't Veer L, Yee D, DeMichele A, Berry D, Esserman L. Analysis of immune infiltrates (assessed via multiplex fluorescence immunohistochemistry) and immune gene expression signatures as predictors of response to the checkpoint inhibitor pembrolizumab in the neoadjuvant I-SPY 2 trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-08.
3599 Introduction: Despite the development of many new agents for the treatment of acute myelogenous leukemia (AML), the prognosis for most patients remains dismal. The interleukin-3 receptor α chain (IL-3Rα, CD123) is highly expressed on the surface of acute and chronic leukemias compared with normal hematopoietic stem cells, providing a rational therapeutic target. CSL362 is a fully humanized, second generation, neutralizing monoclonal antibody targeting CD123 that contains a modified Fc region to enhance human natural killer (huNK) cell-mediated antibody dependent cellular cytotoxicity (ADCC). The purpose of this study was to test the in vivo efficacy of CSL362 in combination with an induction-type chemotherapy regimen and adoptive transfer of huNK cells against xenograft models of human AML. Methods: Direct patient explants of 5 AML samples with defined genetic lesions were established as continuous xenografts in sub-lethally irradiated immune-deficient NOD/SCID or NSG mice. In order to complement the clinical development of CSL362 we optimized an induction-type regimen of cytarabine (AraC, 25 mg/kg IP daily × 4 × 2 weeks) combined with daunorubicin (DNR, 0.62 mg/kg IV weekly × 2 weeks) to simulate the complex biology of AML in a post-chemotherapy setting. For adoptive transplantation into AML engrafted mice, primary huNK cells were expanded 600- to 700-fold in vitro by culturing on feeder cells in the presence of IL-2 for 21 days. In efficacy experiments, an event was defined a priori to occur when the % human CD45 cells in the peripheral blood (%huCD45) reached 25%. Results: Human AML xenografts consistently and predictably produced progressive and disseminating disease in the peripheral blood, bone marrow, spleen, liver, kidneys, lungs and brain of mice between 42 and 200 days post-transplantation. Disease progression represented by increasing percentages of human CD45+ cells in the peripheral blood (%huCD45) inversely correlated with murine red blood cell and platelet counts, and mouse hematocrit and hemoglobin levels. The immunophenotype and gene expression profiles of each xenograft were comparable with the respective original patient samples, indicating that the xenografts provide a valid representation of the human disease. In vitro co-cultures of AML xenograft cells with CD56+/CD16+ huNK cells (5 h at a 1:10 ratio) resulted in an average AML cell death of 18% (range 7–38%). The addition of CSL362 (10 μg/ml) resulted in an average 26% (range 2–64%) increase in AML cell death compared with huNK alone. In vivo AraC/DNR treatment significantly extended the event-free survival (EFS) of mice engrafted with 3/3 AML xenografts by between 32 and 41 days compared with vehicle-treated control mice (P < 0.0001). The addition of CSL362 (300μg IP thrice weekly × 2) significantly extended the median EFS of AraC/DNR-treated mice by 5.9 days (P = 0.002). Moreover, while the adoptive transfer of huNK cells (20 million IV × 2) resulted in no significant survival advantage, huNK cells administered with CSL362 further prolonged mouse EFS by 15.2 days compared with chemotherapy alone (P = 0.0004), providing evidence of therapeutic enhancement. Conclusion: AML xenografts established in NOD/SCID or NSG mice from direct patient explants provide a clinically relevant experimental model for preclinical drug testing, and reflect the immunophenotype and gene expression profiles of the original biopsy specimen. CSL362 treatment of AML xenografted mice augments the efficacy of an induction-type therapy, with additional therapeutic enhancement achieved by the adoptive transfer of huNK cells. These data support the rationale for CSL362 treatment of AML patients post remission induction in an upcoming clinical trial. Disclosures: Lee: CSL Limited: Research Funding. Yee:CSL Limited: Research Funding. Busfield:CSL Limited: Employment. Vairo:CSL Limited: Employment. Lock:CSL Limited: Consultancy, Research Funding.
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