Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors.

PD-1 checkpoint inhibitors have revolutionized the treatment of patients with different cancer histologies including melanoma, renal cell carcinoma, and non-small cell lung carcinoma. However, only a subset of patients show a dramatic clinical response to treatment. Despite intense biomarker discovery efforts, no single robust, prognostic correlation has emerged as a valid outcome predictor. Immune competent, pet dogs develop spontaneous tumors that share similar features to human cancers including chromosome aberrations, molecular subtypes, immune signatures, tumor heterogeneity, metastatic behavior, and chemotherapeutic response. As such, they represent a valuable parallel patient population in which to investigate predictive biomarkers of checkpoint inhibition. However, the lack of a validated, non-immunogenic, canine anti-PD-1 antibody for pre-clinical use hinders this comparative approach and prevents potential clinical benefits of PD-1 blockade being realized in the veterinary clinic. To address this, fully canine single-chain variable fragments (scFvs) that bind canine (c)PD-1 were isolated from a comprehensive canine scFv phage display library. Lead candidates were identified that bound with high affinity to cPD-1 and inhibited its interaction with canine PD-L1 (cPD-L1). The lead scFv candidate re-formatted into a fully canine IgGD reversed the inhibitory effects of cPD-1:cPD-L1 interaction on canine chimeric antigen receptor (CAR) T cell function. In vivo administration showed no toxicity and revealed favorable pharmacokinetics for a reasonable dosing schedule. These results pave the way for clinical trials with anti-cPD-1 in canine cancer patients to investigate predictive biomarkers and combination regimens to inform human clinical trials and bring a promising checkpoint inhibitor into the veterinary armamentarium.

Development of a fully canine anti-canine CTLA4 monoclonal antibody for comparative translational research in dogs with spontaneous tumors.

The immune checkpoint inhibitor (ICI) ipilimumab has revolutionized the treatment of patients with different cancer histologies, including melanoma, renal cell carcinoma, and non-small cell lung carcinoma. However, only a subset of patients shows dramatic clinical responses to treatment. Despite intense biomarker discovery efforts linked to clinical trials using CTLA4 checkpoint blockade, no single prognostic correlate has emerged as a valid predictor of outcome. Client-owned, immune competent, pet dogs develop spontaneous tumors that exhibit similar features to human cancers, including shared chromosome aberrations, molecular subtypes, immune signatures, tumor heterogeneity, metastatic behavior, and response to chemotherapy. As such, they represent a valuable parallel patient population in which to investigate novel predictive biomarkers and rational therapeutic ICI combinations. However, the lack of validated, non-immunogenic, canine ICIs for preclinical use hinders this comparative approach. To address this, fully canine single-chain variable fragments (scFvs) that bind canine CTLA4 were isolated from a comprehensive canine scFv phage display library. A lead candidate for clinical development was selected based on its subnanomolar binding affinity to canine CTLA4 and its ability to prevent CTLA4 binding to CD80/CD86 and promote T cell proliferation and effector function. In vivo mouse studies revealed pharmacokinetics similar to isotype control IgG with no evidence of short-term adverse effects. This work paves the way for in vivo analysis of the first fully canine, anti-canine CTLA4 antibody to promote anti-tumor immunity in dogs with immune-responsive cancers and provide an important comparative tool to investigate correlative biomarkers of response and mechanisms of resistance to CTLA4 checkpoint inhibition.

Generation and Validation of an Antibody to Canine CD19 for Diagnostic and Future Therapeutic Purposes.

The B-cell coreceptor, CD19 is a transmembrane protein expressed throughout B-cell ontogeny from pro-B cell to plasmablast. It plays an important role in B-cell development and function and is an attractive target for antibody-directed immunotherapies against B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (B-NHL) in humans. With the rapid development of next-generation immunotherapies aimed at improving therapeutic efficacy, there is a pressing need for a clinically relevant, immune-competent, spontaneous animal model to derisk these new approaches and inform human immunotherapy clinical trials. Pet dogs develop spontaneous B-cell malignancies, including B-NHL and leukemias that share comparable oncogenic pathways and similar immunosuppressive features to human B-cell malignancies. Despite treatment with multiagent chemotherapy, durable remissions in canine B-NHL are rare and most dogs succumb to their disease within 1 year of diagnosis. Here we report the development and validation of an anti-canine CD19-targeting monoclonal antibody and its single-chain derivatives, which enable next-generation CD19-targeted immunotherapies to be developed and evaluated in client-owned dogs with spontaneous B-NHL. These future in vivo studies aim to provide important information regarding the safety and therapeutic efficacy of CD19-targeted mono- and combination therapies and identify correlative biomarkers of response that will help to inform human clinical trial design. In addition, development of canine CD19-targeted immunotherapies aims to provide better therapeutic options for pet dogs diagnosed with B-cell malignancies.

Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma.

Multiple rodent and primate preclinical studies have advanced CAR T cells into the clinic. However, no single model accurately reflects the challenges of effective CAR T therapy in human cancer patients. To evaluate the effectiveness of next-generation CAR T cells that aim to overcome barriers to durable tumor elimination, we developed a system to evaluate CAR T cells in pet dogs with spontaneous cancer. Here we report on this system and the results of a pilot trial using CAR T cells to treat canine diffuse large B cell lymphoma (DLBCL). We designed and manufactured CD20-targeting, second-generation canine CAR T cells for functional evaluation in vitro and in vivo using lentivectors to parallel human CAR T cell manufacturing. A first-in-species trial of five dogs with DLBCL treated with CAR T was undertaken. Canine CAR T cells functioned in an antigen-specific manner and killed CD20+ targets. Circulating CAR T cells were detectable post-infusion, however, induction of canine anti-mouse antibodies (CAMA) was associated with CAR T cell loss. Specific selection pressure on CD20+ tumors was observed following CAR T cell therapy, culminating in antigen escape and emergence of CD20-disease. Patient survival times correlated with ex vivo product expansion. Altering product manufacturing improved transduction efficiency and skewed toward a memory-like phenotype of canine CAR T cells. Manufacturing of functional canine CAR T cells using a lentivector is feasible. Comparable challenges to effective CAR T cell therapy exist, indicating their relevance in informing future human clinical trial design.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step.

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Elimination of ALDH+ breast tumor initiating cells by docosahexanoic acid and/or gamma tocotrienol through SHP-1 inhibition of Stat3 signaling.

Study investigated the ability of docosahexaenoic acid (DHA) alone and in combination with gamma-tocotrienol (γT3) to eliminate aldehyde dehydrogenase positive (ALDH+) cells and to inhibit mammosphere formation, biomarker and functional assay for tumor initiating cells (TICs), respectively, in human triple negative breast cancer cells (TNBCs), and investigated possible mechanisms of action. DHA upregulated Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) protein levels and suppressed levels of phosphorylated signal transducer and activator of transcription-3 (pStat3) and its downstream mediators c-Myc, and cyclin D1. siRNA to SHP-1 enhanced the percentage of ALDH+ cells and Stat-3 signaling, as well as inhibited, in part, the ability of DHA to reduce the percentage of ALDH+ cells and Stat-3 signaling. γT3 alone and in combination with DHA reduced ALDH+ TNBCs, up-regulated SHP-1 protein levels, and suppressed Stat-3 signaling. Taken together, data demonstrate the anti-TIC potential of achievable concentrations of DHA alone as well as in combination with γT3.

Targeting tumor-associated myeloid cells for cancer immunotherapy.

Tumor-associated myeloid cells undermine the therapeutic efficacy of cancer immunotherapy by their inhibitory properties on immune effector cells. Development of therapeutic agents to deplete suppressive myeloid cells in tumor microenvironment requires identification of cell-specific targets. A competitive phage display technique on live cells paves the way to discovery of such a target.

Discovery of potent anticancer agent HJC0416, an orally bioavailable small molecule inhibitor of signal transducer and activator of transcription 3 (STAT3).

In a continuing effort to develop orally bioavailable small-molecule STAT3 inhibitors as potential therapeutic agents for human cancer, a series of novel diversified analogues based on our identified lead compound HJC0149 (1) (5-chloro-N-(1,1-dioxo-1H-1λ(6)-benzo[b]thiophen-6-yl)-2-hydroxybenzamide, Eur. J. Med. Chem. 2013, 62, 498-507) have been rationally designed, synthesized, and pharmacologically evaluated. Molecular docking studies and biological characterization supported our earlier findings that the O-alkylamino-tethered side chain on the hydroxyl group is an effective and essential structural determinant for improving biological activities and druglike properties of these molecules. Compounds with such modifications exhibited potent antiproliferative effects against breast and pancreatic cancer cell lines with IC50 values from low micromolar to nanomolar range. Among them, the newly discovered STAT3 inhibitor 12 (HJC0416) displayed an intriguing anticancer profile both in vitro and in vivo (i.p. & p.o.). More importantly, HJC0416 is an orally bioavailable anticancer agent as a promising candidate for further development.

Transcription Factor STAT3 as a Novel Molecular Target for Cancer Prevention.

Signal Transducers and Activators of Transcription (STATs) are a family of transcription factors that regulate cell proliferation, differentiation, apoptosis, immune and inflammatory responses, and angiogenesis. Cumulative evidence has established that STAT3 has a critical role in the development of multiple cancer types. Because it is constitutively activated during disease progression and metastasis in a variety of cancers, STAT3 has promise as a drug target for cancer therapeutics. Recently, STAT3 was found to have an important role in maintaining cancer stem cells in vitro and in mouse tumor models, suggesting STAT3 is integrally involved in tumor initiation, progression and maintenance. STAT3 has been traditionally considered as nontargetable or undruggable, and the lag in developing effective STAT3 inhibitors contributes to the current lack of FDA-approved STAT3 inhibitors. Recent advances in cancer biology and drug discovery efforts have shed light on targeting STAT3 globally and/or specifically for cancer therapy. In this review, we summarize current literature and discuss the potential importance of STAT3 as a novel target for cancer prevention and of STAT3 inhibitors as effective chemopreventive agents.

Oridonin ring A-based diverse constructions of enone functionality: identification of novel dienone analogues effective for highly aggressive breast cancer by inducing apoptosis.

Oridonin (1) has attracted considerable attention in recent years because of its unique and safe anticancer pharmacological profile. Nevertheless, it exhibits moderate to poor effects against highly aggressive cancers including triple-negative and drug-resistant breast cancer cells. Herein, we report the rational design and synthesis of novel dienone derivatives with an additional α,ß-unsaturated ketone system diversely installed in the A-ring based on this class of natural scaffold that features dense functionalities and stereochemistry-rich frameworks. Efficient and regioselective enone construction strategies have been established. Meanwhile, a unique 3,7-rearrangement reaction was identified to furnish an unprecedented dienone scaffold. Intriguingly, these new analogues have been demonstrated to significantly induce apoptosis and inhibit colony formation with superior antitumor effects against aggressive and drug-resistant breast cancer cells in vitro and in vivo while also exhibiting comparable or lower toxicity to normal human mammary epithelial cells in comparison with 1.

Synthesis and screening of novel vitamin E derivatives for anticancer functions.

α-TEA, RRR-α-tocopherol ether linked acetic acid, exhibits potent anticancer actions in vitro and in vivo; whereas, the parent molecule has no anticancer activity. In this study, we incorporated fluorine at the chroman head and/or ether linkage between the chroman head and phytyl tail of α-TEA as well as RRR-α-tocopherol to synthesize 6 vitamin E derivatives, and evaluated the anticancer actions in vitro for ability to induce cell death by apoptosis of human MCF-7 and MDA-MB-231 breast cancer cell lines and mouse mammary cancer cell line 66cl-4GFP. All derivatives, with the exception of compound 12, exhibited anticancer properties. The modified α-TEA ether-type phytyl group exhibited the highest pro-apoptotic activity in comparison with α-TEA as well as other vitamin E derivatives.

Distinct roles of different forms of vitamin E in DHA-induced apoptosis in triple-negative breast cancer cells.

SCOPE: Docosahexaenoic acid (DHA) has been shown to exhibit anticancer actions in vitro and in vivo in a variety of cancers. Here, we investigated the role for DHA in inducing apoptosis in triple-negative breast cancer (TNBC) and studied the mechanisms of action. METHODS AND RESULTS: DHA induces apoptosis as detected by Annexin V-FITC/PI assay as well as induces cleavage of caspase-8 and -9, endoplasmic reticulum stress (ERS), and elevated levels of death receptor-5 (DR5) protein expression as detected by western blot assays. Chemical inhibitors of caspase-8 and -9 and small interfering RNAs (siRNAs) show DHA to induce ERS/CHOP/DR5-mediated caspase-8 and -9 dependent apoptosis. Furthermore, DHA induces elevated cellular levels of reactive oxygen species (ROS) and antioxidant; RRR-α-tocopherol (αT) blocked DHA-induced apoptotic events. In contrast to the antagonistic impact of αT, gamma-tocotrienol (γT3) was demonstrated to cooperate with DHA in inducing apoptotic events in TNBC cells. CONCLUSION: Data, for the first time, demonstrate that DHA induces apoptosis in TNBC cells via activation of ERS/CHOP/DR5-mediated caspase-8 and -9 dependent pro-apoptotic events, and that different forms of vitamin E exhibit distinct effects on DHA-induced apoptosis; namely, inhibition by αT and enhancement by γT3.

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway.

Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) induces apoptosis in MCF-7 and HCC-1954 human breast cancer cells in a dose- and time-dependent manner. α-TEA induces increased levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor-5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE-like inhibitory protein (c-FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c-FLIP (L), caspase-8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase-9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α-TEA to enhance DR5 protein levels, downregulate c-FLIP(L) protein levels and induce apoptosis. Combination of α-TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c-FLIP (L) protein levels. siRNA knockdown of c-FLIP produces a low level of spontaneous apoptosis and enhances α-TEA- and TRAIL-induced apoptosis. Taken together, these studies show that α-TEA induces TRAIL/DR5 mitochondria-dependent apoptosis in human breast cancer cells, and that TRAIL/DR5-dependent increases in DR5 and decreases in c-FLIP expression are triggered by TRAIL or α-TEA treatments.

α-TEA-induced death receptor dependent apoptosis involves activation of acid sphingomyelinase and elevated ceramide-enriched cell surface membranes.

BACKGROUND: Alpha-tocopherol ether-linked acetic acid (α-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent and selective apoptosis-inducing agent for human cancer cells in vivo and in vitro. α-TEA induces apoptosis via activation of extrinsic death receptors Fas (CD95) and DR5, JNK/p73/Noxa pathways, and suppression of anti-apoptotic mediators Akt, ERK, c-FLIP and survivin in breast, ovarian and prostate cancer cells. RESULTS: In this study, we demonstrate that α-TEA induces the accumulation of cell surface membrane ceramide, leading to co-localization with Fas, DR5, and FADD, followed by activation of caspases-8 and -9 and apoptosis in human MDA-MB-231 breast cancer cells. α-TEA treatment leads to increased acid sphingomyelinase (ASMase) activity by 30 min, peaking at 4 hrs, which is correlated with ASMase translocation from cytosol to the cell surface membrane. Functional knockdown of ASMase with either the chemical inhibitor, desipramine, or siRNA markedly reduces α-TEA-induced cell surface membrane accumulation of ceramide and its co-localization with Fas, DR5, and FADD, cleavage of caspases-8 and -9 and apoptosis, suggesting an early and critical role for ASMase in α-TEA-induced apoptosis. Consistent with cell culture data, immunohistochemical analyses of tumor tissues taken from α-TEA treated nude mice bearing MDA-MB-231 xenografts show increased levels of cell surface membrane ceramide in comparison to tumor tissues from control animals. CONCLUSION: Taken together, these studies demonstrate that ASMase activation and membrane ceramide accumulation are early events contributing to α-TEA-induced apoptosis in vitro and perhaps in vivo.