ALBERT

Description: The use of immunotherapy (IT) in cancer has recently resulted in impressive responses. Yet, their usages, especially those involving cytokine therapies, have also resulted in the induction of severe systemic toxicities often not previously characterized in their preclinical animal studies. Our laboratory has demonstrated that treatment of EL4 lymphoma with a combination immunotherapy consisting of monoclonal agonistic antibody CD40 and IL-2 resulted in significant anti-tumor responses leading to overall increases in survival using young inbred mice (2-6 months of age). Yet, the majority of hematological cancers occur in aged individuals, with most diagnoses occurring past the age of 60. We now demonstrate that systemic immunotherapy administration in aged (〉16 months of age), but not young, mice resulted in the induction of a rapid cytokine release syndrome, also known as “cytokine storm”, culminating in multi-organ damage (liver, lung, and gut) leading to rapid lethality by day 2 of treatment. Aging is accompanied by an overall redistribution of body mass, with a decrease in lean muscle mass and increase in adiposity. Similarly, we found that normal aging in inbred mice housed under specific pathogen free conditions was accompanied by increases in visceral fat that was similar to young obese (ob/ob or diet-induced obese [DIO]) mice. We therefore assessed the impact of aging and obesity on inflammatory responses to cancer immunotherapeutics. We determined the effects of increased body fat on systemic immunotherapy tolerance in aged mice compared to young obese mice. Both young ob/ob and DIO generated pro-inflammatory cytokine (ie TNFa and IL6) levels and organ pathologies comparable to aged WT mice following immunotherapy, culminating in rapid lethality after several days of therapy. We observed that young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNFa+ macrophages in comparison to young lean mice with immunotherapy. Administration of aCD40/IL-2 with macrophage depletion or TNF-blockade prevented the development of cytokine storms within young obese mice, providing protection from lethality, suggesting that the toxicity was macrophage mediated through increases in TNFa. Calorie-restricted aged mice contain less visceral fat and upon aCD40/IL-2 administration displayed reduced cytokine levels, protection from organ pathology, and protection from lethality. Our data demonstrates adiposity as a critical factor in the age-associated inflammatory pathologic responses to systemic anti-cancer immunotherapy and may have a significant impact when immunotherapy is used clinically within cancer therapy regimens. These data also underscore the critical importance of taking aging and body fat into consideration with preclinical assessments. Disclosures No relevant conflicts of interest to declare.

Description: Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and over-expressed on several tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPalpha) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPalpha interaction and induces phagocytosis of hematologic and solid tumor cells, but also exhibits several unique functional properties. The first property is the ability of AO-176 to induce direct tumor cytotoxic cell death in hematologic (ex. Jurkat, Raji and Molt-4) as well as solid human tumor cell lines by a cell autonomous mechanism (not ADCC). Secondly, AO-176 exhibits preferential binding to tumor versus normal cells, including red blood cells (RBCs), T cells, endothelial cells, skeletal muscle cells and epithelial cells. A0-176 also does not affect the function of any of these primary cells when assayed ex vivo. The negligible binding of AO-176 to RBCs versus hematologic (ex. Jurkat, Raji or Molt-4) or solid tumor cells is particularly profound and different from other reported anti-CD47 antibodies. AO-176 also does not induce hemagglutination of RBCs. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. Consistent with this attribute, AO-176 was well tolerated in cynomolgus monkeys with no adverse effects in general nor with respect to RBCs which was consistent with ex vivo results. A third novel property of AO-176 is its enhanced binding to tumor cells at acidic pH. Because the microenvironment of leukemic bone marrow and solid tumors has an acidic pH, this enhanced binding of AO-176 at low pH has the potential added advantage of tumor-specific targeting. Lastly, we show that AO-176 demonstrates dose-dependent anti-tumor activity in hematologic and solid tumor xenograft models. Taken together, the unique properties and anti-tumor activity of our next generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPalpha axis targeting agents as it progresses to clinical development. Disclosures No relevant conflicts of interest to declare.

Description: AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

Description: Mantle cell lymphoma (MCL), a B-cell non-Hodgkin lymphoma, remains incurable with current treatment modalities. Bruton tyrosine kinase (BTK), a key component of the early BCR signaling pathway, has emerged as a promising therapeutic target. Ibrutinib, a specific inhibitor that binds covalently to the active site of BTK at cysteine 481, has been approved for the treatment of MCL. In a recent Phase II study, treatment for relapsed or refractory MCL with Ibrutinib alone achieved a response rate of 68%. Although this response rate is striking, approximately one third of patients show primary resistance, and acquired resistance with a C481S mutation in BTK can also develop. To overcome primary and acquired resistance to BTK inhibition, a combinatory strategy that targets multiple pathways is needed. Here, we focused on the BCL2 anti-apoptotic pathway. Our previous study established FBXO10 as the E3 ubiquitin ligase that targets BCL2 for proteasomal degradation and revealed reduced levels of FBXO10 expression in MCL cell lines. To examine BCL2 and FBXO10 expression in patient samples, we performed immunohistochemical (IHC) analysis in a tissue microarray (TMA) that contained 62 MCL cases. Our data showed high levels of BCL2 expression but no or low FBXO10 expression in MCL cases. Based on this initial expression analysis, we hypothesized that a defect in FBXO10-mediated proteasomal degradation contributes to high BCL2 expression in MCL. To test this hypothesis, we genetically manipulated FBXO10 expression in MCL cell lines. Flow cytometric analysis revealed that overexpression of FBXO10 resulted in reduced BCL2 expression. Inversely, we silenced endogenous FBXO10 by shRNA. Indeed, after blocking protein synthesis by cycloheximide, the amount of BCL2 protein was sustained in these FBXO10 shRNA-expressing cells. These results indicate that BCL2 is the predominantly expressed anti-apoptotic protein in MCL, due to a specific defect in its proteasomal degradation. Inhibition of BCL2 by the specific inhibitor ABT-199 killed cancer cells in culture and also inhibited xenografted tumor growth. BCL2 is a target gene of the BTK/canonical NF-κB signaling pathway. We next analyzed BTK expression in the above TMA and found that BTK was highly expressed and positively correlated with BCL2 expression in these cases. BTK inhibition reduced BCL2 expression as shown in the BCR-dependent cell lines Jeko and Mino. RNA-seq analysis confirmed that a set of anti-apoptotic genes (e.g. BCL2, BCL-XL and DAD1) was downregulated by BTK shRNA. The downregulated genes also included those that are critical for B cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Elevated BCL2, however, can also result from other mechanisms, such as amplification. Indeed, this genomic alteration is present in some MCL cell lines, including Granta-519 and Z138 used for this study. Since these cell lines are insensitive or less sensitive to Ibrutinib, we hypothesized that BCL2 upregulation is a mechanism underlying primary resistance to Ibrutinib, and thus targeting BCL2 by ABT-199 increases or restores sensitivity of these cells to Ibrutinib. The results supported this hypothesis and demonstrated a synergistic effect of ABT-199 and Ibrutinib on growth inhibition of Granta-519 and Z138 cells both in vitro and in vivo. In summary, this study elucidated mechanisms of BCL2 overexpression and association with the BCR/BTK signaling pathway in MCL. Our data provided a mechanistic rationale for co-targeting of these two oncogenic pathways by ABT-199 and Ibrutinib as a new therapeutic strategy in a pre-clinical setting. Most importantly, the findings indicate that this combination has a synergistic effect on the killing of both BCR-dependent and independent MCL cells. Since these two signaling pathways are deregulated in DLBCL and follicular lymphoma, co-targeting by Ibrutinib and ABT-199 may provide an attractive therapeutic strategy for these additional lymphoma patients as well. Disclosures Kahl: Roche/Genentech: Consultancy; Seattle Genetics: Consultancy; Millennium: Consultancy; Cell Therapeutics: Consultancy; Celgene: Consultancy; Infinity: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy.

Description: T cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematologic malignancy comprising 15% of pediatric and 25% of adult cases of ALL. With current treatment options, T-ALL survival rates have reached 50-60% in adults and 85% in children. Despite great strides in the treatment of this subset of ALL, T-ALL still shows resistance to first-line therapies in over 50% of adults and 25% of children, and relapse is often chemorefractory. Mutations in the Janus Activating Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways and overexpression of the B cell lymphoma-2 (Bcl-2) protein are highly linked to the progression of T-ALL, and to the resistance of a number of available treatments for lymphoid malignancies. JAK/STAT is one of the main cytokine signaling pathways involved in hematopoietic cell growth and development. Indeed, the JAK/STAT pathway is often constitutively activated by T-ALL. The Bcl-2 pathway also plays an important role in cell survival. Overexpression of Bcl-2, an anti-apoptotic member of the Bcl-2 family, promotes cell survival by binding and neutralizing pro-apoptotic members. Inhibition of key proteins in both of these pathways has been greatly explored individually, but little is known about their combined effects on T-ALL. It is hypothesized that T-ALL manipulates both of these pathways as a means of escaping individual inhibition of either JAK/STAT or Bcl-2. Our hypothesis is that inhibiting both the JAK/STAT and Bcl-2 pathways with two small molecule inhibitors; Ruxolitinib (JAK 1/2 inhibitor) and Venetoclax (BH3 mimetic targeting Bcl-2), will inhibit T-ALL growth and survival. Proliferation of T-ALL was assessed by MTT assay and viability was measured by trypan blue and flow cytometry at 24, 48 and 72-hour time points post-treatment. Single-drug dose responses were conducted for both inhibitors. Six doses of both Ruxolitinib and Venetoclax were tested from a range of 0.156uM-5uM for Ruxolitinib and 1.56nM-50nM of Venetoclax. A response was seen for the three highest doses of both inhibitors (1.25uM, 2.5uM, and 5uM for Ruxolitinib and 12.5nM, 25nM, 50nM for Venetoclax). However, a synergistic effect was only achieved when combining 1.25uM Ruxolitinib with 25nM Venetoclax or 2.5uM Ruxolitinib with 12.5nM or 25nM Venetoclax. The combination dose of 1.25uM Ruxolitinib and 25nM Venetoclax demonstrated the greatest combined synergistic effect (CI

Description: Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8+ T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8+ T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8+ T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8+ CD25− cells within the tumor site. These findings demonstrate that memory CD8+ T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.