Challenges and gaps in immunosafety evaluation of therapeutics: An IQ DruSafe survey.

Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.

Successful Development of Nonclinical Anti-Drug Antibody Assays to Support Zinpentraxin Alfa Reproductive Toxicology Studies.

Immunogenicity assessment is an essential part of biotherapeutic drug development. While the immune response in animals is not always representative of the human immune response, immunogenicity data obtained in animal models is still informative for the evaluation of drug exposure and safety. The most common assay format used for the detection of anti-drug antibodies (ADAs) in preclinical and clinical studies is the bridging format. The advantage of this method is that it can detect all antibody isotypes generated against the therapeutic. However, the method development can be time-consuming and labor-intensive, due to the need for labeling of the drug which is used both as capture and detection. Various generic ADA assays have been successfully implemented to overcome these disadvantages and to enable faster assay development timelines to support nonclinical toxicology studies. Here, we describe the challenges in the development of an assay to detect antibodies to zinpentraxin alfa, a recombinant human pentraxin-2, in rabbit and rat toxicology studies. Our initial efforts to develop a bridging assay failed, prompting us to develop a method adapted from generic assay formats to detect anti-zinpentraxin alfa antibodies in the serum of different species with minimal optimization. However, while the general assay format remained similar, assay reagents were adapted between the different species, resulting in the development of two distinct assays for the detection of ADAs in rat and rabbit. Here, we share the final development/validation data and the immunogenicity study results. Our work highlights the need for the evaluation of alternate assay formats when evaluating novel drug modalities.

Nonclinical pharmacokinetics, pharmacodynamics and safety assessment of a FLT3L-Fc molecule for cancer immunotherapy.

FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.

Repeat-dose and embryo-fetal developmental toxicity of zinpentraxin alfa.

Zinpentraxin alfa is a recombinant human pentraxin-2 (PTX-2) developed for the treatment of various fibrotic diseases with the hypothesis that supplementing endogenous PTX-2 levels through intravenous administration should increase its regulatory capacity in circulation and at the site of disease, thereby promoting healing and reducing fibrosis. Zinpentraxin alfa has been studied in various clinical trials, particularly in patients with idiopathic pulmonary fibrosis, where it has demonstrated efficacy in slowing decline in lung function in a phase 2 study. In the present investigation, we summarize findings from 14-day repeat-dose toxicity studies in rats and cynomolgus monkeys supporting early clinical development of zinpentraxin alfa. In addition, we also describe the findings from the embryo-fetal developmental (EFD) studies conducted in rats and rabbits, since the intended fibrosis patient population may include patients of childbearing potential. Zinpentraxin alfa was well tolerated by rats and monkeys in general toxicity studies with no treatment-related adverse effects, as well as by pregnant rats over the same dose range in a definitive EFD study. In contrast, substantial toxicity was observed in a rabbit dose-range-finder EFD study. Zinpentraxin alfa was poorly tolerated by pregnant rabbits and effects on the dams correlated with post-implantation fetal losses. The disparate effects of zinpentraxin alfa on embryo-fetal development between the two species suggests a potential unknown biological function of PTX-2 in pregnancy in the rabbit, which may be relevant to humans. Our findings warrant the consideration for highly effective contraceptive measures to avoid pregnancy in patients enrolled in clinical studies with zinpentraxin alfa.

Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

In Vivo Assessment of Antibody-Dependent Enhancement of Influenza B Infection.

A theoretical safety concern proposed in the influenza literature is that therapeutic antiviral antibodies could have the potential for antibody-dependent enhancement (ADE) of infection and disease. ADE may occur when virus-specific antibodies at subtherapeutic, nonneutralizing concentrations facilitate virus uptake and, in some cases, enhance replication, which can lead to an exacerbation of virus-mediated disease. Alternatively, ADE may occur due to antibody-dependent complement activation exacerbating virus-mediated disease in the absence of increased replication. As a result of this theoretical safety concern, safety assessment of anti-influenza antibodies may include an in vivo evaluation of ADE of infection and/or disease. These studies were conducted to investigate the potential of MHAB5553A, a broadly specific, neutralizing therapeutic anti-influenza B antibody, to elicit ADE of infection and disease in mouse models of influenza B infection. In parallel studies, female DBA/2J mice were infected with either influenza B/Victoria/504/2000 or influenza B/Brisbane/60/2008 representing distinct lineages. Assessment of ADE was based on an integration of results from multiple endpoints, including infectious lung viral titers and genomes, body weight, mortality, lung weight, and histopathology. In these studies, the high dose of 15 mg/kg MHAB5553A resulted in substantial attenuation of influenza pneumonia, with more modest effects at 1.5 mg/kg; whereas MHAB5553A treatment at 0.15 or 0.015 mg/kg was generally comparable to vehicle-treated controls. Our results demonstrate that MHAB5553A across a broad range of doses did not enhance primary influenza B infection or disease in this model, and represent a nonclinical de-risking of the ADE potential with this antibody.

T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.

Activation of the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.

Perspective on a Modified Developmental and Reproductive Toxicity Testing Strategy for Cancer Immunotherapy.

The intent of cancer immunotherapy (CIT) is to generate and enhance T-cell responses against tumors. The tumor microenvironment establishes several inhibitory pathways that lead to suppression of the local immune response, which is permissive for tumor growth. The efficacy of different CITs, alone and in combination, stems from reinvigorating the tumor immune response via several mechanisms, including costimulatory agonists, checkpoint inhibitors, and vaccines. However, immune responses to other antigens (self and foreign) may also be enhanced, resulting in potentially undesired effects. In outbred mammalian pregnancies, the fetus expresses paternally derived alloantigens that are recognized as foreign by the maternal immune system. If unchecked or enhanced, maternal immunity to these alloantigens represents a developmental and reproductive risk and thus is a general liability for cancer immunotherapeutic molecules. We propose a tiered approach to confirm this mechanistic reproductive liability for CIT molecules. A rodent allopregnancy model is based on breeding 2 different strains of mice so that paternally derived alloantigens are expressed by the fetus. When tested with a cross-reactive biotherapeutic, small molecule drug, or surrogate molecule, this model should reveal on-target reproductive liabilities if the pathway is involved in maintaining pregnancy. Alternatively, allopregnancy models with genetically modified mice can be interrogated for exquisitely specific biotherapeutics with restricted species reactivity. The allopregnancy model represents a relatively straightforward approach to confirm an expected on-target reproductive risk for CIT molecules. For biotherapeutics, it could potentially replace more complex developmental and reproductive toxicity testing in nonhuman primates when a pregnancy hazard is confirmed or expected.

T cell LFA-1 engagement induces HuR-dependent cytokine mRNA stabilization through a Vav-1, Rac1/2, p38MAPK and MKK3 signaling cascade.

BACKGROUND: Engagement of the ß2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile ß-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice. CONCLUSIONS/SIGNIFICANCE: Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.

Cannabinoid-mediated elevation of intracellular calcium: a structure-activity relationship.

This laboratory has reported previously that Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and cannabinol (CBN) robustly elevate intracellular calcium ([Ca(2+)](i)) in resting human and murine T cells, whereas CP55,940 [5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol], a high-affinity ligand for CB1 and CB2, does not. In light of our previous studies, the objective of the present investigation was to examine the ability of various cannabinoid compounds to elevate [Ca(2+)](i) in the CB2 receptor-expressing human peripheral blood acute lymphoid leukemia T cell line and the dependence of structural similarity to Delta(9)-THC therein. The present studies demonstrate that CBN and HU-210 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol], both tricyclic and in that respect structurally similar to Delta(9)-THC, elevate [Ca(2+)](i). The [Ca(2+)](i) elevation elicited by both CBN and HU-210 was attenuated upon removal of extracellular calcium and upon pretreatment with SK&F96365 [1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole], an inhibitor of receptor-operated cation channels. In addition, pretreatment with either CB1 or CB2 receptor antagonists attenuated the CBN- and HU-210-mediated [Ca(2+)](i) elevation. Further investigation of the dependence of Delta(9)-THC, CBN, and HU-210 on cannabinoid receptors using splenocytes from wild-type and CB1(-/-)/CB2(-/-) mice showed that the [Ca(2+)](i) elevation elicited by all three tricyclic cannabinoids was independent of CB1 and CB2. Moreover, both the CB1 and CB2 receptor antagonists attenuated that rise in [Ca(2+)](i) elicited by the tricyclic cannabinoids in the wild-type and CB1(-/-)/CB2(-/-) mouse splenocytes. Taken together, the present results demonstrate that classic tricyclic cannabinoids with structural similarity to Delta(9)-THC elicit a robust influx of calcium in T cells putatively through receptor-operated cation channels in a manner sensitive to the cannabinoid receptor antagonists, but independent of the CB1 and CB2 receptors.

Induction of intracellular calcium elevation by Delta9-tetrahydrocannabinol in T cells involves TRPC1 channels.

We have reported previously that Delta9-tetrahydrocannabinol (Delta9-THC) treatment of resting human and murine splenic T cells robustly elevated intracellular calcium ([Ca2+]i). The objective of the present investigation was to examine the putative role of [Ca2+]i store depletion and store-operated calcium (SOC) and receptor-operated cation (ROC) channels in the mechanism by which Delta9-THC increases [Ca2+]i in the cannabinoid-2 receptor-expressing human peripheral blood-acute lymphoid leukemia (HPB-ALL) human T cell line. By using the smooth endoplasmic reticulum Ca2+-ATPase pump inhibitor, thapsigargin, and the ryanodine receptor antagonist, 8-bromo-cyclic adenosine diphosphate ribose, we demonstrate that the Delta9-THC-mediated elevation in [Ca2+]i occurs independently of [Ca2+]i store depletion. Furthermore, the ROC channel inhibitor, SK&F 96365 was more efficacious at attenuating the Delta9-THC-mediated elevation in [Ca2+]i than SOC channel inhibitors, 2-aminoethoxydiphenyl borate and La3+. Recently, several members of the transient receptor potential canonical (TRPC) channel subfamily have been suggested to operate as SOC or ROC channels. In the present studies, treatment of HPB-ALL cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeant analog of diacylglycerol (DAG), which gates several members of the TRPC channel subfamily, rapidly elevated [Ca2+]i, as well as prevented a subsequent, additive elevation in [Ca2+]i by Delta9-THC, independent of protein kinase C. Reverse transcriptase-polymerase chain reaction analysis for TRPC1-7 showed that HPB-ALL cells express detectable mRNA levels of only TRPC1. Finally, small interference RNA knockdown of TRPC1 attenuated the Delta9-THC-mediated elevation of [Ca2+]i. Collectively, these results suggest that Delta9-THC-induced elevation in [Ca2+]i is attributable entirely to extracellular calcium influx, which is independent of [Ca2+]i store depletion, and is mediated, at least partially, through the DAG-sensitive TRPC1 channels.

2-Arachidonoyl-glycerol suppresses interferon-gamma production in phorbol ester/ionomycin-activated mouse splenocytes independent of CB1 or CB2.

2-Arachidonoyl-glycerol (2-AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin-2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2-AG on interferon-gamma (IFN-gamma) expression and associated upstream signaling events. Pretreatment of splenocytes with 2-AG markedly suppressed phorbol 12-myristate 13-acetate plus calcium ionophore (PMA/Io)-induced IFN-gamma secretion. In addition, 2-AG suppressed IFN-gamma steady-state mRNA expression in a concentration-dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1(-/-)/CB2(-/-) knockout mice were used. No difference in the magnitude of IFN-gamma suppression by 2-AG in wild-type versus CB1/CB2 null mice was observed. Time-of-addition studies revealed that 2-AG treatment up to 12 h post-cellular activation resulted in suppression of IFN-gamma, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2-AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin-induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN-gamma production. Indeed, Io partially attenuated the 2-AG-induced suppression of PMA/Io-stimulated IFN-gamma production. Taken together, these data demonstrate that 2-AG suppresses IFN-gamma expression in murine splenocytes in a CB receptor-independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.

Cannabinoid receptor-mediated regulation of intracellular calcium by delta(9)-tetrahydrocannabinol in resting T cells.

Cannabinoids exhibit broad immune modulating activity by targeting many cell types within the immune system, including T cells, which exhibit sensitivity, as evidenced by altered activation, proliferation, and cytokine expression. As a result of the critical role calcium plays in T cell function coupled with previous findings demonstrating disruption of the calcium-regulated transcription factor, nuclear factor of activated T cells, by cannabinoid treatment, the objective of the present investigation was to perform an initial characterization of the role of the cannabinoid receptors in the regulation of the intracellular calcium concentration ([Ca(2+)](i)) by delta(9)-tetrahydrocannabinol (delta(9)-THC) in T lymphocytes. Here, we demonstrate that delta(9)-THC robustly elevates [Ca(2+)](i) in purified murine splenic T cells and in the human peripheral blood acute lymphoid leukemia (HPB-ALL) human T cell line but only minimally elevates [Ca(2+)](i) in Jurkat E6-1 (dysfunctional cannabinoid receptor 2-expressing) human T cells. Removal of extracellular calcium severely attenuated the delta(9)-THC-mediated rise in [Ca(2+)](i) in murine splenic T cells and HPB-ALL cells. Pretreatment with cannabinoid receptor antagonists, SR144528 and/or SR141716A, led to an attenuation of delta(9)-THC-mediated elevation in [Ca(2+)](i) in splenic T cells and HPB-ALL cells but not in Jurkat E6-1 cells. Furthermore, pretreatment of HPB-ALL cells with SR144528 antagonized the small rise in [Ca(2+)](i) elicited by delta(9)-THC in the absence of extracellular calcium. These findings suggest that delta(9)-THC induces an influx of extracellular calcium in resting T cells in a cannabinoid receptor-dependent manner.

Cannabinol enhancement of interleukin-2 (IL-2) expression by T cells is associated with an increase in IL-2 distal nuclear factor of activated T cell activity.

It has been demonstrated previously that cannabinol (CBN) differentially modulates interleukin-2 (IL-2) protein secretion by T cells with a corresponding change in extracellular signal-regulated kinase activity. The objective of the present studies was to further investigate the molecular mechanism by which CBN enhances IL-2 gene expression using the EL4 T cell line. We demonstrate here that steady-state IL-2 mRNA expression was significantly enhanced by CBN in a concentration-dependent manner in EL4 cells activated with suboptimal concentrations of phorbol-12-myristate-13-acetate (2-10 nM). Concordantly, a marked increase was observed in nuclear factor of activated T cells (NF-AT) DNA binding activity to the IL-2 distal NF-AT site, but not to nuclear factor for immunoglobulin kappa chain in B cells or activator protein 1 motifs. Transient transfection of EL4 cells with a reporter gene under the control of multiple IL-2 distal NF-AT motifs exhibited increased transcriptional activity by CBN in suboptimally activated cells. In addition, the CBN-mediated enhancement of IL-2 protein secretion and the transcriptional activity of the IL-2 distal NF-AT reporter gene was abrogated by the calcium/calmodulin-dependent protein kinase inhibitor KN93, but not by the CB2 receptor antagonist SR144528. Enhancement of IL-2 was also demonstrated with CP55940, Delta(9)-tetrahydrocannabinol, and cannabidiol, thus suggesting that the phenomenon is not unique to CBN. Collectively, these results suggest that increased IL-2 secretion by CBN is mediated through the enhancement of IL-2 gene transcription by activation of NF-AT in a CB1/CB2-independent manner.