First-in-human, phase 1 study of PF-06753512, a vaccine-based immunotherapy regimen (VBIR), in non-metastatic hormone-sensitive biochemical recurrence and metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: This phase 1 study evaluated PF-06753512, a vaccine-based immunotherapy regimen (PrCa VBIR), in two clinical states of prostate cancer (PC), metastatic castration-resistant PC (mCRPC) and biochemical recurrence (BCR). METHODS: For dose escalation, patients with mCRPC received intramuscular PrCa VBIR (adenovirus vector and plasmid DNA expressing prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostate stem cell antigen (PSCA)) with or without immune checkpoint inhibitors (ICIs, tremelimumab 40 or 80 mg with or without sasanlimab 130 or 300 mg, both subcutaneous). For dose expansion, patients with mCRPC received recommended phase 2 dose (RP2D) of PrCa VBIR plus tremelimumab 80 mg and sasanlimab 300 mg; patients with BCR received PrCa VBIR plus tremelimumab 80 mg (Cohort 1B-BCR) or tremelimumab 80 mg plus sasanlimab 130 mg (Cohort 5B-BCR) without androgen deprivation therapy (ADT). The primary endpoint was safety. RESULTS: Ninety-one patients were treated in dose escalation (mCRPC=38) and expansion (BCR=35, mCRPC=18). Overall, treatment-related and immune-related adverse events occurred in 64 (70.3%) and 39 (42.9%) patients, with fatigue (40.7%), influenza-like illness (30.8%), diarrhea (23.1%), and immune-related thyroid dysfunction (19.8%) and rash (15.4%), as the most common. In patients with mCRPC, the objective response rate (ORR, 95% CI) was 5.6% (1.2% to 15.4%) and the median radiographic progression-free survival (rPFS) was 5.6 (3.5 to not estimable) months for all; the ORR was 16.7% (3.6% to 41.4%) and 6-month rPFS rate was 45.5% (24.9% to 64.1%) for those who received RP2D with measurable disease (n=18). 7.4% of patients with mCRPC achieved a ≥50% decline in baseline PSA (PSA-50), with a median duration of 4.6 (1.2-45.2) months. In patients with BCR, 9 (25.7%) achieved PSA-50; the median duration of PSA response was 3.9 (1.9-4.2) and 10.1 (6.9-28.8) months for Cohorts 5B-BCR and 1B-BCR. Overall, antigen specific T-cell response was 88.0% to PSMA, 84.0% to PSA, and 80.0% to PSCA. CONCLUSIONS: PrCa VBIR overall demonstrated safety signals similar to other ICI combination trials; significant side effects were seen in some patients with BCR. It stimulated antigen-specific immunity across all cohorts and resulted in modest antitumor activity in patients with BCR without using ADT. TRIAL REGISTRATION NUMBER: NCT02616185.

Preclinical development of a vaccine-based immunotherapy regimen (VBIR) that induces potent and durable T cell responses to tumor-associated self-antigens.

The development of therapeutic cancer vaccines remains an active area, although previous approaches have yielded disappointing results. We have built on lessons from previous cancer vaccine approaches and immune checkpoint inhibitor research to develop VBIR, a vaccine-based immunotherapy regimen. Assessment of various technologies led to selection of a heterologous vaccine using chimpanzee adenovirus (AdC68) for priming followed by boosts with electroporation of DNA plasmid to deliver T cell antigens to the immune system. We found that priming with AdC68 rapidly activates and expands antigen-specific T cells and does not encounter pre-existing immunity as occurs with the use of a human adenovirus vaccine. The AdC68 vector does, however, induce new anti-virus immune responses, limiting its use for boosting. To circumvent this, boosting with DNA encoding the same antigens can be done repetitively to augment and maintain vaccine responses. Using mouse and monkey models, we found that the activation of both CD4 and CD8 T cells was amplified by combination with anti-CTLA-4 and anti-PD-1 antibodies. These antibodies were administered subcutaneously to target their distribution to vaccination sites and to reduce systemic exposure which may improve their safety. VBIR can break tolerance and activate T cells recognizing tumor-associated self-antigens. This activation lasts more than a year after completing treatment in monkeys, and inhibits tumor growth to a greater degree than is observed using the individual components in mouse cancer models. These results have encouraged the testing of this combination regimen in cancer patients with the aim of increasing responses beyond current therapies.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Distinct Biomarker Profiles and TCR Sequence Diversity Characterize the Response to PD-L1 Blockade in a Mouse Melanoma Model.

Only a subset of patients responds to immune checkpoint blockade (ICB) in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti-mouse PD-L1 antibody similar to the anti-human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti-PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T-cell infiltration, and T-cell receptor (TCR) signatures in regressing tumors compared with tumors exhibiting no response to anti-PD-L1 treatment. We found that CD8+ T-cell tumor infiltration corresponded to response to treatment, and that anti-PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine-cytokine receptor interaction, and natural killer cell-mediated cytotoxicity. TCR sequence data suggest that an anti-PD-L1-mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy. IMPLICATIONS: Our melanoma model recapitulates the variable response to anti-PD-L1 observed in patients and exhibits biomarkers that characterize early antibody response, including expansion of the TCR repertoire.

In vitro comparison of treatments and commercially available solutions on mortality of Angiostrongylus cantonensis third-stage larvae.

On Hawai'i Island, an increase in human neuroangiostrongyliasis cases has been primarily associated with the accidental ingestion of Angiostrongylus cantonensis L3 in snails or slugs, or potentially, from larvae left behind in the slug's slime or feces. We evaluated more than 40 different treatments in vitro for their ability to kill A. cantonensis larvae with the goal of identifying a safe and effective fruit and vegetable wash in order to reduce the risk of exposure. Our evaluation of treatment lethality was carried out in two phases; initially using motility as an indicator of larval survival after treatment, followed by the development and application of a propidium iodide staining assay to document larval mortality. Treatments tested included common household products, consumer vegetable washes and agricultural crop washes. We found minimal larvicidal efficacy among consumer-grade fruit and vegetable washes, nor among botanical extracts such as those from ginger or garlic, nor acid solutions such as vinegar. Alkaline solutions, on the other hand, as well as oxidizers such as bleach and chlorine dioxide, did show larvicidal potential. Surfactants, a frequent ingredient in detergents that lowers surface tension, had variable results, but dodecylbenzene sulfonic acid as a 70% w/w solution in 2-propanol was very effective, both in terms of the speed and the thoroughness with which it killed A. cantonensis L3 nematodes. Thus, our results suggest promising directions for future investigation.

Postharvest Population Reservoirs of Coffee Berry Borer (Coleoptera: Curculionidae) on Hawai'i Island.

Coffee berry borer, Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), is the most damaging insect pest of coffee worldwide. Old coffee berries (raisins) are widely acknowledged as coffee berry borer reservoirs, yet few studies have attempted to quantify coffee berry borer populations in raisins remaining on farms postharvest. We collected ground and tree raisins at six coffee farms on Hawai'i Island to assess raisin density, infestation, coffee berry borer abundance, and adult mortality in three areas of each farm: trees, driplines (ground below the tree foliage), and center aisles (ground between tree rows). We also assessed infestation of the new season's crop by conducting whole-tree counts of infested green berries. Mean raisin density was significantly higher in the dripline compared to the center aisle and trees (131 vs 17 raisins per m2 and 12 raisins per tree, respectively). Raisin infestation was significantly higher in samples from trees (70%) relative to those from the dripline (22%) and center aisle (18%). Tree raisins had significantly higher coffee berry borer abundance compared to both areas of the ground (20 vs 3-5 coffee berry borer per raisin). Adult mortality was significantly higher on the ground (63-71%) compared to the trees (12%). We also observed a significant positive correlation between ground raisin density and infestation of the new season's crop. Across all farms, we estimated that 49.5% of the total coffee berry borer load was present in dripline raisins, 47.3% in tree raisins, and 3.2% in center aisle raisins. Our findings confirm the importance of whole-farm sanitation in coffee berry borer management by demonstrating the negative impact that poor postharvest control can have on the following season's crop.

Coffee berry borer (Hypothenemus hampei) (Coleoptera: Curculionidae) development across an elevational gradient on Hawai'i Island: Applying laboratory degree-day predictions to natural field populations.

Coffee berry borer (CBB, Hypothenemus hampei) (Coleoptera: Curculionidae: Scolytinae) is the most destructive pest of coffee worldwide. Information on CBB development times can be used to predict the initiation of new infestation cycles early in the coffee-growing season and thus inform the timing of insecticide applications. While laboratory estimates of CBB development under constant conditions exist, they have not been applied under the heterogeneous environmental conditions that characterize many coffee-growing regions. We measured CBB development times and abundance in commercial coffee farms across an elevational gradient on Hawai'i Island and applied thermal accumulation models from previous laboratory studies to test their fit to field data. Artificial lures were used to infest coffee berries at five farms ranging in elevation from 279-792 m, and weather variables were monitored at macro (farm-level) and micro (branch-level) scales. CBB development was followed in the field from the time of initial berry infestation by the founding female through the development of F1 mature adults. Mean development time from egg to adult across all sites was 38.5 ± 3.46 days, while the mean time required for the completion of a full life cycle (from time of infestation to presence of mature F1 females) was 50.9 ± 3.35 days. Development time increased with increasing elevation and decreasing temperature. Using macro-scale temperature data and two different estimates for the lower temperature threshold (14.9°C and 13.9°C), we estimated a mean requirement of 332 ± 14 degree-days and 386 ± 16 degree-days, respectively, from the time of berry infestation to the initiation of a new reproductive cycle in mature coffee berries. Similar estimates were obtained using micro-scale temperature data, indicating that macro-scale temperature monitoring is sufficient for life-cycle prediction. We also present a model relating elevation to number of CBB generations per month. Our findings suggest that CBB development times from laboratory studies are generally applicable to field conditions on Hawai'i Island and can be used as a decision support tool to improve IPM strategies for this worldwide pest of coffee.

Turning the corner on therapeutic cancer vaccines.

Recent advances in several areas are rekindling interest and enabling progress in the development of therapeutic cancer vaccines. These advances have been made in target selection, vaccine technology, and methods for reversing the immunosuppressive mechanisms exploited by cancers. Studies testing different tumor antigens have revealed target properties that yield high tumor versus normal cell specificity and adequate immunogenicity to affect clinical efficacy. A few tumor-associated antigens, normal host proteins that are abnormally expressed in cancer cells, have been demonstrated to serve as good targets for immunotherapies, although many do not possess the needed specificity or immunogenicity. Neoantigens, which arise from mutated proteins in cancer cells, are truly cancer-specific and can be highly immunogenic, though the vast majority are unique to each patient's cancer and thus require development of personalized therapies. Lessons from previous cancer vaccine expeditions are teaching us the type and magnitude of immune responses needed, as well as vaccine technologies that can achieve these responses. For example, we are learning which vaccine approaches elicit the potent, balanced, and durable CD4 plus CD8 T cell expansion necessary for clinical efficacy. Exploration of interactions between the immune system and cancer has elucidated the adaptations that enable cancer cells to suppress and evade immune attack. This has led to breakthroughs in the development of new drugs, and, subsequently, to opportunities to combine these with cancer vaccines and dramatically increase patient responses. Here we review this recent progress, highlighting key steps that are bringing the promise of therapeutic cancer vaccines within reach.

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Integrated Pest Management of Coffee Berry Borer in Hawaii and Puerto Rico: Current Status and Prospects.

The coffee berry borer (CBB), Hypothenemus hampei, is the most significant insect pest of coffee worldwide. Since CBB was detected in Puerto Rico in 2007 and Hawaii in 2010, coffee growers from these islands are facing increased costs, reduced coffee quality, and increased pest management challenges. Here, we outline the CBB situation, and summarize the findings of growers, researchers, and extension professionals working with CBB in Hawaii. Recommendations for the Integrated Pest Management (IPM) program for CBB in Hawaiian Islands and Puerto Rico include: (1) establish a CBB monitoring program, (2) synchronize applications of insecticides with peak flight activity of CBB especially during the early coffee season, (3) conduct efficient strip-picking as soon as possible after harvest and perform pre-harvest sanitation picks in CBB hotspots if needed, (4) establish protocols to prevent the escape of CBB from processing areas and when transporting berries during harvest, and (5) stump prune by blocks. Progress achieved includes the introduction of the mycoinsecticide Beauveria bassiana to coffee plantations, the coordination of area-wide CBB surveys, the establishment and augmentation of native beetle predators, and an observed reduction of CBB populations and increased coffee quality where IPM programs were established. However, CBB remains a challenge for coffee growers due to regional variability in CBB pressures, high costs, and labor issues, including a lack of training and awareness of CBB management practices among growers.

Flight Activity and Field Infestation Relationships for Coffee Berry Borer in Commercial Coffee Plantations in Kona and Kau Districts, Hawaii.

The coffee berry borer (CBB), Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae) is a recent invader to Hawaii. To date, limited information regarding the seasonal phenology of this pest on the islands limits the implementation of integrated control strategies. As part of a coffee farmer training program, we monitored CBB flight activity in 15 coffee plantations (Kona and Kau Districts) over 10 mo with methanol-ethanol (3:1 ratio) baited traps. Concurrently, we quantified CBB infestation and penetration rates inside developing coffee berries through the end of harvest. Approximately 1 million CBB were captured, with the highest activity (e.g., >500 CBB/trap/wk) in December through February, coinciding with end of main regional harvesting periods. Relatively high activity (>250 CBB/trap/wk) was also observed during berry development, in May and June (Kona) and June and July (Kau). Field infestation rates were higher overall in Kau (9.6 ± 1.1%) compared with coffee plantations in Kona (4.7 ± 0.4%). Linear regression investigated relationships between CBB trap data and berry infestation rates. Trap catch data generally correlated better with the proportion of shallow entries (AB position) compared with deeper penetrations (CD position) or total infestation. Pearson correlation coefficients based on different parameters (i.e., region, altitude, and berry phenology) revealed positive and mostly significant correlations between these variables (R values 0.410 to 0.837). Timing peak flight activity of CBB with insecticide applications will help coffee growers improve pest control. The ability of trap data to calculate reliable economic (action) thresholds for the CBB is discussed.

Targeting ADAM17 inhibits human colorectal adenocarcinoma progression and tumor-initiating cell frequency.

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody-Drug Conjugate Targeting 5T4.

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.

Antibody-Drug Conjugates Bearing Pyrrolobenzodiazepine or Tubulysin Payloads Are Immunomodulatory and Synergize with Multiple Immunotherapies.

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody-drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. Cancer Res; 77(10); 2686-98. ©2017 AACR.

5T4-Targeted Therapy Ablates Cancer Stem Cells and Prevents Recurrence of Head and Neck Squamous Cell Carcinoma.

Purpose: Locoregional recurrence is a frequent treatment outcome for patients with advanced head and neck squamous cell carcinoma (HNSCC). Emerging evidence suggests that tumor recurrence is mediated by a small subpopulation of uniquely tumorigenic cells, that is, cancer stem cells (CSC), that are resistant to conventional chemotherapy, endowed with self-renewal and multipotency.Experimental Design: Here, we evaluated the efficacy of MEDI0641, a novel antibody-drug conjugate targeted to 5T4 and carrying a DNA-damaging "payload" (pyrrolobenzodiazepine) in preclinical models of HNSCC.Results: Analysis of a tissue microarray containing 77 HNSCC with follow-up of up to 12 years revealed that patients with 5T4high tumors displayed lower overall survival than those with 5T4low tumors (P = 0.038). 5T4 is more highly expressed in head and neck CSC (ALDHhighCD44high) than in control cells (non-CSC). Treatment with MEDI0641 caused a significant reduction in the CSC fraction in HNSCC cells (UM-SCC-11B, UM-SCC-22B) in vitro Notably, a single intravenous dose of 1 mg/kg MEDI0641 caused long-lasting tumor regression in three patient-derived xenograft (PDX) models of HNSCC. MEDI0641 ablated CSC in the PDX-SCC-M0 model, reduced it by five-fold in the PDX-SCC-M1, and two-fold in the PDX-SCC-M11 model. Importantly, mice (n = 12) treated with neoadjuvant, single administration of MEDI0641 prior to surgical tumor removal showed no recurrence for more than 200 days, whereas the control group had 7 recurrences (in 12 mice; P = 0.0047).Conclusions: Collectively, these findings demonstrate that an anti-5T4 antibody-drug conjugate reduces the fraction of CSCs and prevents local recurrence and suggest a novel therapeutic approach for patients with HNSCC. Clin Cancer Res; 23(10); 2516-27. ©2016 AACR.

Targeting CD73 in the tumor microenvironment with MEDI9447.

MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).

Effective combination therapies in preclinical endocrine resistant breast cancer models harboring ER mutations.

Although endocrine therapy is successfully used to treat patients with estrogen receptor (ER) positive breast cancer, a substantial proportion of this population will relapse. Several mechanisms of acquired resistance have been described including activation of the mTOR pathway, increased activity of CDK4 and activating mutations in ER. Using a patient derived xenograft model harboring a common activating ER ligand binding domain mutation (D538G), we evaluated several combinatorial strategies using the selective estrogen receptor degrader (SERD) fulvestrant in combination with chromatin modifying agents, and CDK4/6 and mTOR inhibitors. In this model, fulvestrant binds WT and MT ER, reduces ER protein levels, and downregulated ER target gene expression. Addition of JQ1 or vorinostat to fulvestrant resulted in tumor regression (41% and 22% regression, respectively) though no efficacy was seen when either agent was given alone. Interestingly, although the CDK4/6 inhibitor palbociclib and mTOR inhibitor everolimus were efficacious as monotherapies, long-term delayed tumor growth was only observed when co-administered with fulvestrant. This observation was consistent with a greater inhibition of compensatory signaling when palbociclib and everolimus were co-dosed with fulvestrant. The addition of fulvestrant to JQ1, vorinostat, everolimus and palbociclib also significantly reduced lung metastatic burden as compared to monotherapy. The combination potential of fulvestrant with palbociclib or everolimus were confirmed in an MCF7 CRISPR model harboring the Y537S ER activating mutation. Taken together, these data suggest that fulvestrant may have an important role in the treatment of ER positive breast cancer with acquired ER mutations.

Therapeutic Efficacy of Cancer Stem Cell Vaccines in the Adjuvant Setting.

Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem-like cells (CSC) may be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical evidence that this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous cell cancer and D5 melanoma. Vaccination of mice with an ALDH(high) SCC7 CSC-DC vaccine after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged host survival. This effect was augmented significantly by simultaneous administration of anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma, treatment of mice with ALDH(high) CSC-DC vaccination inhibited primary tumor growth, reduced spontaneous lung metastases, and increased host survival. In this setting, CCR10 and its ligands were downregulated on ALDH(high) D5 CSCs and in lung tissues, respectively, after vaccination with ALDH(high) D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell migration in vitro and metastasis in vivo T cells harvested from mice vaccinated with ALDH(high) D5 CSC-DC selectively killed ALDH(high) D5 CSCs, with additional evidence of humoral immunologic engagement and a reduction in ALDH(high) cells in residual tumors. Overall, our results offered a preclinical proof of concept for the use of ALDH(high) CSC-DC vaccines in the adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary metastasis, as compared with traditional DC vaccines, with increased host survival further accentuated by simultaneous PD-L1 blockade. Cancer Res; 76(16); 4661-72. ©2016 AACR.

Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics.

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

IL-6 Inhibition With MEDI5117 Decreases The Fraction of Head and Neck Cancer Stem Cells and Prevents Tumor Recurrence.

Head and neck squamous cell carcinomas (HNSCC) exhibit a small population of uniquely tumorigenic cancer stem cells (CSC) endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDH(high)CD44(high) cells). Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117) using three low-passage patient-derived xenograft (PDX) models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05). This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11). Low dose MEDI5117 (3 mg/kg) consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001), PDX-SCC-M1 (P < .001), PDX-SCC-M11 (P = .04). Interestingly, high dose MEDI5117 (30 mg/kg) decreased the CSC fraction in the PDX-SCC-M11 model (P = .002), but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDH(high)CD44(high) cells cultured in ultra-low attachment plates (P < .05), supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC.