Oncolytic virus-mediated expansion of dual-specific CAR T cells improves efficacy against solid tumors in mice.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)-modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Oncolytic virus-derived type I interferon restricts CAR T cell therapy.

The application of adoptive T cell therapies, including those using chimeric antigen receptor (CAR)-modified T cells, to solid tumors requires combinatorial strategies to overcome immune suppression associated with the tumor microenvironment. Here we test whether the inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment may help to recruit and potentiate the functionality of CAR T cells. Contrary to our hypothesis, VSVmIFNß infection is associated with attrition of murine EGFRvIII CAR T cells in a B16EGFRvIII model, despite inducing a robust proinflammatory shift in the chemokine profile. Mechanistically, type I interferon (IFN) expressed following infection promotes apoptosis, activation, and inhibitory receptor expression, and interferon-insensitive CAR T cells enable combinatorial therapy with VSVmIFNß. Our study uncovers an unexpected mechanism of therapeutic interference, and prompts further investigation into the interaction between CAR T cells and oncolytic viruses to optimize combination therapy.

Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence.

Understanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment-resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immunosurveillance. In the first, the role of TNFα changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)-mediated cytokine response characterized by low IL6 and elevated IFNγ, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNγ, inhibiting both NK-cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFα, which stimulated the growth of MRD tumors. Finally, therapies that blocked PD-1, TNFα, or NK cells delayed or prevented recurrence. These data show how innate immunosurveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identify therapeutic targets in patients with MRD known to be at high risk of relapse. Cancer Immunol Res; 5(11); 1029-45. ©2017 AACR.

Synthesis, Molecular Properties Estimations, and Dual Dopamine D2 and D3 Receptor Activities of Benzothiazole-Based Ligands.

Neurleptic drugs, e.g., aripiprazole, targeting the dopamine D2S and D3 receptors (D2SR and D3R) in the central nervous system are widely used in the treatment of several psychotic and neurodegenerative diseases. Therefore, a new series of benzothiazole-based ligands (3-20) was synthesized by applying the bioisosteric approach derived from the selective D3Rs ligand BP-897 (1) and its structurally related benz[d]imidazole derivative (2). Herein, introduction of the benzothiazole moiety was well tolerated by D2SR and D3R binding sites leading to antagonist affinities in the low nanomolar concentration range at both receptor subtypes. However, all novel compounds showed lower antagonist affinity to D3R when compared to that of 1. Further exploration of different substitution patterns at the benzothiazole heterocycle and the basic 4-phenylpiperazine resulted in the discovery of high dually acting D2SR and D3R ligands. Moreover, the methoxy substitution at 2-position of 4-phenylpiperazine resulted in significantly (22-fold) increased D2SR binding affinity as compared to the parent ligand 1, and improved physicochemical and drug-likeness properties of ligands 3-11. However, the latter structural modifications failed to improve the drug-able properties in ligands having un-substituted 4-phenylpiperazine analogs (12-20). Accordingly, compound 9 showed in addition to high dual affinity at the D2SR and D3R [Ki (hD2SR) = 2.8 ± 0.8 nM; Ki (hD3R) = 3.0 ± 1.6 nM], promising clogS, clogP, LE (hD2SR, hD3R), LipE (hD2SR, hD3R), and drug-likeness score values of -4.7, 4.2, (0.4, 0.4), (4.4, 4.3), and 0.7, respectively. Also, the deaminated analog 10 [Ki (hD2SR) = 3.2 ± 0.4 nM; Ki (hD3R) = 8.5 ± 2.2 nM] revealed clogS, clogP, LE (hD2SR, hD3R), LipE (hD2SR, hD3R) and drug-likeness score values of -4.7, 4.2, (0.4, 0.4), (3.9, 3.5), and 0.4, respectively. The results observed for the newly developed benzothiazole-based ligands 3-20 provide clues for the diversity in structure activity relationships (SARs) at the D2SR and D3R subtypes.

Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)piperidine derivatives as histamine H3R ligands.

As a continuation of our search for novel histamine H3 receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2-21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their binding properties at human histamine H3 receptor (hH3R). The highest affinities were observed for pentyl derivatives 6-8 (Ki=8.8-23.4nM range) and among them piperidine derivative 6 with Ki=8.8nM. Structures 6, 7 were also classified as antagonists in cAMP accumulation assay (with EC50=157 and 164nM, respectively). Moreover, new compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Seven compounds (2-4, 9, 11, 12 and 20) showed anticonvulsant activity at maximal electroshock (MES) test in the dose of 30mg/kg at 0.5h. In the subcutaneous pentetrazole (scMET) test compound 4 showed protection at 100 and 300mg/kg dose at mice, however compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies were undertaken, to explain affinity of compounds at hH3R (taking into the consideration X-ray analysis of compound 18). In order to estimate "drug-likeness" of selected compounds in silico and experimental evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.

Inhibitory Receptors Induced by VSV Viroimmunotherapy Are Not Necessarily Targets for Improving Treatment Efficacy.

Systemic viroimmunotherapy activates endogenous innate and adaptive immune responses against both viral and tumor antigens. We have shown that therapy with vesicular stomatitis virus (VSV) engineered to express a tumor-associated antigen activates antigen-specific adoptively transferred T cells (adoptive cell therapy, ACT) in vivo to generate effective therapy. The overall goal of this study was to phenotypically characterize the immune response to VSV+ACT therapy and use the information gained to rationally improve combination therapy. We observed rapid expansion of blood CD8+ effector cells acutely following VSV therapy with markedly high expression of the immune checkpoint molecules PD-1 and TIM-3. Using these data, we tested a treatment schedule incorporating mAb immune checkpoint inhibitors with VSV+ACT treatment. Unlike clinical scenarios, we delivered therapy at early time points following tumor establishment and treatment. Our goal was to potentiate the immune response generated by VSV therapy to achieve durable control of metastatic disease. Despite the high frequency of endogenous PD-1+ TIM-3+ CD8+ T cells following virus administration, antibody blockade did not improve survival. These findings provide highly significant information about response kinetics to viroimmunotherapy and juxtapose the clinical use of checkpoint inhibitors against chronically dysfunctional T cells and the acute T cell response to oncolytic viruses.

Immunogenicity of self tumor associated proteins is enhanced through protein truncation.

We showed previously that therapy with Vesicular Stomatitis Virus (VSV) expressing tumor-associated proteins eradicates established tumors. We show here that when cellular cDNA were cloned into VSV which retained their own poly-A signal, viral species emerged in culture which had deleted the cellular poly-A signal and also contained a truncated form of the protein coding sequence. Typically, the truncation occurred such that a Tyrosine-encoding codon was converted into a STOP codon. We believe that the truncation of tumor-associated proteins expressed from VSV in this way occurred to preserve the ability of the virus to replicate efficiently. Truncated cDNA expressed from VSV were significantly more effective than full length cDNA in treating established tumors. Moreover, tumor therapy with truncated cDNA was completely abolished by depletion of CD4+ T cells, whereas therapy with full length cDNA was CD8+ T cell dependent. These data show that the type/potency of antitumor immune responses against self-tumor-associated proteins can be manipulated in vivo through the nature of the self protein (full length or truncated). Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-tumor-associated proteins can be broken through manipulation of protein integrity, allowing for rational design of better self-immunogens for cancer immunotherapy.

(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine H4 receptor ligands.

Within the constantly growing number of histamine H4 (H4R) receptor ligands there is a large group of azine derivatives. A series of novel compounds in the group of 4-methylpiperazine-1,3,5-triazine-2-amines were designed and obtained. Considered structures were modified at the triazine 6-position by introduction of variously substituted arylethenyl moieties. Their affinities to histamine H4 receptors were evaluated in radioligand binding assays with use of Sf9 cells, transiently expressing human H4R. Pharmacological studies results allowed to identify 4-[(E)-2-(3-chlorophenyl)ethenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (Ki = 253 nM) as the most potent compound in the present series.

Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography.

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.

Mutated BRAF Emerges as a Major Effector of Recurrence in a Murine Melanoma Model After Treatment With Immunomodulatory Agents.

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595-605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence.

Aryl-1,3,5-triazine derivatives as histamine H4 receptor ligands.

A series of novel 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazine derivatives with different aryl substituents in the 6-position was designed, synthesized and evaluated for histamine H4 receptor (H4R) affinity in Sf9 cells expressing human H4R co-expressed with G-protein subunits. Triazine derivative 8 with a 6-(p-chlorophenyl) substituent showed the highest affinity with hH4R Ki value of 203 nM and was classified as an antagonist in cAMP accumulation assay. This compound, identified as a new lead structure, demonstrated also anti-inflammatory properties in preliminary studies in mice (carrageenan-induced edema test) and neither possessed significant antiproliferative activity, nor modulated CYP3A4 activity up to concentration of 25 µM. In order to discuss structure-activity relationships molecular modeling and docking studies were undertaken.

The histamine H4 receptor: targeting inflammatory disorders.

The discovery of the histamine H(4) receptor has added a new chapter to the century of extensive biogenic amine research. The human histamine H(4) receptor is mainly expressed in cells of the human immune system (e.g. mast cells, eosinophils, monocytes, dendritic cells, T cells) and mediates several effects on chemotaxis with numerous cell types. The distinct expression pattern and the immunomodulatory role highlight its physiological relevance in inflammatory and immunological processes. Inflammatory conditions, e.g. allergy, asthma and autoimmune diseases, were for a long time thought to be mainly mediated by activation of the histamine H(1) receptor subtype. However, in the treatment of diseases as chronic pruritus, asthma and allergic rhinitis the use of histamine H(1) receptor antagonists is unsatisfying. Selective H(4) receptor ligands and/or synergism of histamine H(1) and H(4) receptor modulation may be more effective in such pathophysiological conditions. Promising preclinical studies underline its role as an attractive target in the treatment of inflammatory and autoimmune disorders. Meanwhile, first histamine H(4) receptor antagonist has reached clinical phases for the treatment of respiratory diseases.

N-Alkenyl and cycloalkyl carbamates as dual acting histamine H3 and H4 receptor ligands.

Previous studies have shown that several imidazole derivatives possess affinity to histamine H(3) and H(4) receptors. Continuing our study on structural requirements responsible for affinity and selectivity for H(3)/H(4) receptor subtypes, two series of 3-(1H-imidazol-4-yl)propyl carbamates were prepared: a series of unsaturated alkyl derivatives (1-9) and a series possessing a cycloalkyl group different distances to the carbamate moiety (10-13). The compounds were tested for their affinities at the human histamine H(3) receptor, stably expressed in CHO-K1 cells. Compounds 1, 2, 5-7, 10-13 were investigated for their affinities at the human histamine H(4) receptor co-expressed with Gα(i2) and Gß(1)γ(2) subunits in Sf9 cells. To expand the pharmacological profile, compounds were further tested for their H(3) receptor antagonist activity on guinea pig ileum and in vivo after oral administration to mice. All tested compounds exhibited good affinity for the human histamine H(3) receptor with K(i) values in the range from 14 to 194nM. All compounds were active in vivo after peroral administration (p.o.) to Swiss mice, thus demonstrating their ability to cross the blood-brain barrier. The most potent H(3) receptor ligand of these series was compound 5, 3-(1H-imidazol-4-yl)propyl pent-4-enylcarbamate with the highest affinity (K(i)=14nM). Additionally, compound 3 showed remarkable central nervous system (CNS) H(3)R activity, increasing the N(τ)-methylhistamine levels in mice with an ED(50) value of 0.55mg/kg, p.o. evidencing therefore, a twofold increase of inverse agonist/antagonist potency compared to the reference inverse agonist/antagonist thioperamide. In this study, the imidazole propyloxy carbamate moiety was kept constant. The different lipophilic moieties connected to the carbamate functionality in the eastern part of the molecule had a range of influences on the human H(4) receptor affinity (154-1326nM).

Receptor-specific functional efficacies of alkyl imidazoles as dual histamine H3/H4 receptor ligands.

Histamine H(3) and H(4) receptors are highly related G protein-coupled receptors. Preclinical and clinical data strongly suggest the potential therapeutic application of selectively acting histamine H(4) receptor ligands to inflammatory conditions but also hint at a certain interference of the two receptors in diseases attended with itch and pain. The aim of this investigation was to identify dual acting ligands as pharmacological tools. Receptor binding profiles of ω-(1H-imidazol-4-yl)alkyl derivatives structurally defined as amides, carbamates, esters, ethers, ketones and ureas were evaluated with respect to their potencies at histamine H(3) and H(4) receptors. A two-step screening method based on in vitro radioligand binding studies and functional [(35)S]GTPγS binding experiments was performed. The examined series of imidazole-containing compounds displayed both, selective histamine H(4) receptor and dual acting histamine H(3)/H(4) receptor ligands. Slight structural modifications caused major differences in selectivity profiles and on functional properties at the human histamine H(4) receptor. N-(3-(1H-Imidazol-4-yl)propyl)-2-cyclohexylacetamide 11 was identified as most potent and selective human histamine H(4) receptor ligand in this series (K(i)=45nM). Amide- and ether-containing structures consistently exhibited partial agonist efficacies, whereas ureas, ketones, esters and carbamates mainly acted as antagonists and inverse agonists. We identified novel dual acting histamine H(3)/H(4) receptor ligands with varying efficacies at the histamine H(4) receptor subtype, whereas histamine H(3) receptor antagonism was kept constant, e.g. 3-(1H-imidazol-4-yl)propyl (cyclohexylmethyl)carbamate 19 or 4-(3-(3-phenylpropylthio)propyl)-1H-imidazole 30. These compounds state valuable pharmacological tools in studies of diseases, in which histamine H(3) and H(4) receptor signalling contributes to pathophysiological conditions.

Kojic acid derivatives as histamine H(3) receptor ligands.

The histamine H(3) receptor (H(3)R) is a promising target in the development of new compounds for the treatment of mainly centrally occurring diseases. However, emerging novel therapeutic concepts have been introduced and some indications in the H(3)R field, e.g. migraine, pain or allergic rhinitis, might take advantage of peripherally acting ligands. In this work, kojic acid-derived structural elements were inserted into a well established H(3)R antagonist/inverse agonist scaffold to investigate the bioisosteric potential of γ-pyranones with respect to the different moieties of the H(3)R pharmacophore. The most affine compounds showed receptor binding in the low nanomolar concentration range. Evaluation and comparison of kojic acid-containing ligands and their corresponding phenyl analogues (3-7) revealed that the newly integrated scaffold greatly influences chemical properties (S Log P, topological polar surface area (tPSA)) and hence, potentially modifies the pharmacokinetic profile of the different derivatives. Benzyl-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)methanamine ligands 3 and 4 belong to the centrally acting diamine-based class of H(3)R antagonist/inverse agonist, whereas kojic acid analogues 6 and 7 might act peripherally. The latter compounds state promising lead structures in the development of H(3)R ligands with a modified profile of action.

First metal-containing histamine H3 receptor ligands.

Iron-containing ligands targeting the human histamine H(3) receptor (hH(3)R) were prepared. The compounds contain ferrocene sandwich complexes coupled via different linkers to a basic hH(3)R antagonist/inverse agonist pharmacophore. In a click chemistry approach, a triazole was successfully inserted as a new linking element. Two ferrocenylmethylamines and a ferrocenyltriazole were the most affine hH(3)R ligands within this series, showing receptor binding in the nano- and subnanomolar concentration range.

Histamine H3 and H4 receptor affinity of branched 3-(1H-imidazol-4-yl)propyl N-alkylcarbamates.

A series of imidazole-containing (non-)chiral carbamates were tested at human histamine H(3) receptor (H(3)R). All compounds displayed K(i) values below 100 nM. A trend for a stereoselectivity at human H(3)R was observed for the chiral alpha-branched ligands. Selected compounds were also tested at human histamine H(4) receptor and showed moderate to weak affinities (118-1460 nM).

2,4-Diaminopyrimidines as histamine H4 receptor ligands--Scaffold optimization and pharmacological characterization.

The human histamine H(4) receptor (hH(4)R) is a promising new target in the therapy of inflammatory diseases and disorders of the immune system. For the development of new H(4)R antagonists a broad ligand-based virtual screening was performed resulting in two hits. The dissection of their common annelated aromatic core into its heteromonocyclic components showed that 2,4-diaminopyrimidine is a potent hH(4)R affinity scaffold, which was comprehensively investigated. Structure-activity relationship studies revealed that slight structural changes evoke extensive differences in functional activities and potencies: while o- and p-substituted benzyl amines mainly showed partial agonism, m-substituted and rigidified ones exhibited inverse agonist efficacy.