Discovery and development of COVID-19 vaccines and therapeutics: nonclinical perspectives.

The development and regulatory review of BNT162b2, a COVID-19 vaccine, and PaxlovidTM (nirmatrelvir tablets/ritonavir tablets), a COVID-19 therapeutic, are benchmarks for accelerated innovation during a global pandemic. Rapid choice of the SARS-CoV-2 spike protein and main protease (Mpro) as targets for the vaccine and therapeutic, respectively, leveraged the available knowledge of the biology of SARS-CoV-2 and related viruses. The nonclinical immunogenicity and safety of BNT162b2 was rigorously assessed. Likewise, a comprehensive nonclinical safety assessment was conducted for the therapeutic candidates, lufotrelvir (PF-07304814) and nirmatrelvir (PF-07321332). The development and regulatory review of BNT162b2 and Paxlovid was enabled through close collaboration of the pharmaceutical industry with regulatory agencies and public health organizations. This experience highlights approaches that could be adopted for pandemic preparedness including risk-based investment strategies, conduct of activities in parallel that normally are conducted sequentially, quick kill decisions, simultaneous evaluation of multiple candidates, and use of flexible, established vaccine platforms.

Safety, Tolerability, and Pharmacokinetics of Single and Multiple Ascending Intravenous Infusions of PF-07304814 (Lufotrelvir) in Participants Hospitalized With COVID-19.

Background: An urgent need remains for antiviral therapies to treat patients hospitalized with COVID-19. PF-07304814-the prodrug (lufotrelvir) and its active moiety (PF-00835231)-is a potent inhibitor of the SARS-CoV-2 3CL protease. Method: Eligible participants were 18 to 79 years old and hospitalized with confirmed COVID-19. This first-in-human phase 1b study was designed with 2 groups: single ascending dose (SAD) and multiple ascending dose (MAD). Participants could receive local standard-of-care therapy. In SAD, participants were randomized to receive a 24-hour infusion of lufotrelvir/placebo. In MAD, participants were randomized to receive a 120-hour infusion of lufotrelvir/placebo. The primary endpoint was to assess the safety and tolerability of lufotrelvir. The secondary endpoint was to evaluate the pharmacokinetics of lufotrelvir and PF-00835231. Results: In SAD, participants were randomized to receive 250 mg lufotrelvir (n = 2), 500 mg lufotrelvir (n = 2), or placebo (n = 4) by continuous 24-hour infusion. In MAD, participants were randomized to receive 250 mg lufotrelvir (n = 7), 500 mg lufotrelvir (n = 6), or placebo (n = 4) by continuous 120-hour infusion. No adverse events or serious adverse events were considered related to lufotrelvir. At doses of 250 and 500 mg, concentrations for the prodrug lufotrelvir and active moiety PF-00835231 increased in a dose-related manner. Unbound concentrations of the lufotrelvir active metabolite reached steady state approximately 2- and 4-fold that of in vitro EC90 following 250- and 500-mg doses, respectively. Conclusions: These safety and pharmacokinetic findings support the continued evaluation of lufotrelvir in clinical studies. Clinical Trials Registration. ClinicalTrials.gov NCT04535167.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Thymic Lymphomas in a 6-Month rasH2-Tg Mouse Carcinogenicity Study With the RORγt Inverse Agonist, BMS-986251.

BMS-986251 is a retinoid-related orphan receptor γt (RORγt) inverse agonist that was in development for the treatment of autoimmune diseases. RORγt is a nuclear hormone receptor and transcription factor that is involved in the differentiation and function of T helper 17 cells. RORγt-deficient (constitutive or conditional) mice develop thymic lymphomas with >50% mortality at 4 months, whereas heterozygous mice are normal. A 6-month study was conducted in rasH2-Tg hemizygous mice to assess the potential carcinogenicity of BMS-986251. BMS-986251 was administered once daily by oral gavage to groups of 27 mice/sex at doses of 0 (water control), 0 (vehicle control), 5, 25, or 75 mg/kg. The positive control, N-methyl-N-nitrosourea, was administered by a single intraperitoneal injection to 15 mice/sex at a dose of 75 mg/kg. There were no tumors attributed to BMS-986251 except for thymic lymphomas. Thymic lymphoma was observed in 1 male (3.7%) and 3 females (11.1%) at the mid dose, and 6 females (22.2%) at the high dose. No lymphomas were observed in the negative control groups whereas the incidence of lymphomas in the positive control group was 47-60%. The incidence of thymic lymphomas in the BMS-986251-treated groups was higher than published literature and test facility historical control data. Furthermore, increased thymic lymphoid cellularity (lymphoid hyperplasia) was observed at the mid dose in males and at all doses in females. Since lymphoid hyperplasia may represent a preneoplastic change, a no-effect dose for potential tumor induction was not identified in this study. These results led to the discontinuation of BMS-986251 and underscore the challenges in targeting RORγt for drug development.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

CD28 Superagonistic Activation of T Cells Induces a Tumor Cell-Like Metabolic Program.

CD28 superagonist (CD28SA), a therapeutic immunomodulatory monoclonal antibody triggered rapid and exaggerated activation of CD4+ effector memory T cells (TEMs) in humans with unwanted serious adverse effects. It is well known that distinct metabolic programs determine the fate and responses of immune cells. In this study, we show that human CD4+ TEMs stimulated with CD28SA adopt a metabolic program similar to those of tumor cells with enhanced glucose utilization, lipid biosynthesis, and proliferation in hypoxic conditions. Identification of metabolic profiles underlying hyperactive T cell activation would provide a platform to test safety of immunostimulatory antibodies.

A Combined In Vitro/In Silico Approach to Identifying Off-Target Receptor Toxicity.

Many xenobiotics can bind to off-target receptors and cause toxicity via the dysregulation of downstream transcription factors. Identification of subsequent off-target toxicity in these chemicals has often required extensive chemical testing in animal models. An alternative, integrated in vitro/in silico approach for predicting toxic off-target functional responses is presented to refine in vitro receptor identification and reduce the burden on in vivo testing. As part of the methodology, mathematical modeling is used to mechanistically describe processes that regulate transcriptional activity following receptor-ligand binding informed by transcription factor signaling assays. Critical reactions in the signaling cascade are identified to highlight potential perturbation points in the biochemical network that can guide and optimize additional in vitro testing. A physiologically based pharmacokinetic model provides information on the timing and localization of different levels of receptor activation informing whole-body toxic potential resulting from off-target binding.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Development of interferon beta-neutralising antibodies in multiple sclerosis--a systematic review and meta-analysis.

PURPOSE: Interferon beta (IFN-ß) is the drug of choice for treatment of relapsing forms of multiple sclerosis and is known to reduce the frequency and severity of relapses. This systematic review determines the occurrence of neutralising antibodies (NAbs) against different formulations of IFN-ß: IFN-ß-1a Avonex™, IFN-ß-1a Rebif™ and IFN-ß-1b Betaferon/Betaseron™. METHODS: The databases used in the review included MEDLINE Ovid (from 1950 to March 2015), Embase Ovid (from 1980 to March 2015), CENTRAL on The Cochrane Library (2011, Issue 4) and ClinicalTrials.gov (from 1997 to March 2015). All studies that compared the efficacy of the different formulations of IFN-ß in patients with relapsing forms of multiple sclerosis including IFN-ß-1a Avonex™, IFN-ß-1a Rebif™, IFN-ß-1b Betaferon/Betaseron™ and IFN-ß-1b Extavia™ were included. RESULTS: Assessment of randomised controlled trials demonstrated that Avonex™ was 76% less likely than Rebif™ to lead to the formation of NAbs. Avonex™ was 88% less likely than Betaferon/Betaseron™ to lead to the formation of NAbs. Similar findings were also observed in the non-randomised controlled studies, with Avonex™ having the lowest risk. The formation of NAbs was dose dependent: Avonex™ at 30 µg was 64% less risky than Avonex™ at 60 µg. CONCLUSIONS: Our data show that 2.0-18.9% of patients developed NAbs to Avonex™, 16.5-35.4% of patients developed NAbs to Rebif™ and 27.3-53.3% of patients developed NAbs to Betaferon/Betaseron™.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.

Heme oxygenase-1 regulates dendritic cell function through modulation of p38 MAPK-CREB/ATF1 signaling.

Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis.

Immunoglobulin G1 and immunoglobulin G4 antibodies in multiple sclerosis patients treated with IFNß interact with the endogenous cytokine and activate complement.

A subset of patients with relapsing-remitting multiple sclerosis (RRMS) on therapy with interferon beta (IFNß) develop neutralising anti-drug antibodies (ADA) resulting in reduced, or loss of, therapeutic efficacy. The aims were to characterise the relative contributions of anti-IFNß antibody isotypes to drug neutralising activity, ability of these antibodies to cross-react with endogenous IFNß, to form immune complexes and activate complement. IFNß-specific ADA were measured in plasma from RRMS patients treated with IFNß1a (Rebif(®)). Neutralisation of endogenous and therapeutic IFNß by ADA was determined by IFNß bioassay. IFNß-ADA profile was predominantly comprised of IgG1 and IgG4 antibody isotypes. The contribution of IgG4-ADA towards neutralising activity was found to be minimal. Neutralising IFNß-ADA blocks endogenous IFNß activity. ADA interaction with therapeutic IFNß results in immune complex formation and complement activation. In summary, IgG1 and IgG4 IFNß-ADA have the ability to neutralise therapeutic and endogenous protein and to activate complement.

Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMP-responsive element binding protein/activating transcription factor 1 signaling.

Nrf2 is a redox-responsive transcription factor that has been implicated in the regulation of DC immune function. Loss of Nrf2 results in increased co-stimulatory molecule expression, enhanced T cell stimulatory capacity, and increased reactive oxygen species (ROS) levels in murine immature DCs (iDCs). It is unknown whether altered immune function of Nrf2-deficient DCs (Nrf2(-/-) iDCs) is due to elevated ROS levels. Furthermore, it is unclear which intracellular signaling pathways are involved in Nrf2-mediated regulation of DC function. Using antioxidant vitamins to reset ROS levels in Nrf2(-/-) iDCs, we show that elevated ROS is not responsible for the altered phenotype and function of these DCs. Pharmacological inhibitors were used to explore the role of key MAPKs in mediating the altered phenotype and function in Nrf2(-/-) iDCs. We demonstrate that the increased co-stimulatory molecule expression (MHC II and CD86) and antigen-specific T cell activation capacity observed in Nrf2(-/-) iDCs was reversed by inhibition of p38 MAPK but not JNK. Importantly, we provide evidence for increased phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor 1 (ATF1), transcription factors that are downstream of p38 MAPK. The increased phosphorylation of CREB/ATF1 in Nrf2(-/-) iDCs was sensitive to p38 MAPK inhibition. We also show data to implicate heme oxygenase-1 as a potential molecular link between Nrf2 and CREB/ATF1. These results indicate that dysregulation of p38 MAPK-CREB/ATF1 signaling axis underlies the altered function and phenotype in Nrf2-deficient DCs. Our findings provide new insights into the mechanisms by which Nrf2 mediates regulation of DC function.

Challenges and approaches for the development of safer immunomodulatory biologics.

Immunomodulatory biologics, which render their therapeutic effects by modulating or harnessing immune responses, have proven their therapeutic utility in several complex conditions including cancer and autoimmune diseases. However, unwanted adverse reactions--including serious infections, malignancy, cytokine release syndrome, anaphylaxis and hypersensitivity as well as immunogenicity--pose a challenge to the development of new (and safer) immunomodulatory biologics. In this article, we assess the safety issues associated with immunomodulatory biologics and discuss the current approaches for predicting and mitigating adverse reactions associated with their use. We also outline how these approaches can inform the development of safer immunomodulatory biologics.

Immunogenicity to biologics: mechanisms, prediction and reduction.

Currently, there is a significant rise in the development and clinical use of a unique class of pharmaceuticals termed as Biopharmaceuticals or Biologics, in the management of a range of disease conditions with, remarkable therapeutic benefits. However, there is an equally growing concern regarding development of adverse effects like immunogenicity in the form of anti-drug antibodies (ADA) production and hypersensitivity. Immunogenicity to biologics represents a significant hurdle in the continuing therapy of patients in a number of disease settings. Efforts focussed on the identification of factors that contribute towards the onset of immunogenic response to biologics have led to reductions in the incidence of immunogenicity. An in-depth understanding of the cellular and molecular mechanism underpinning immunogenic responses will likely improve the safety profile of biologics. This review addresses the mechanistic basis of ADA generation to biologics, with emphasis on the role of antigen processing and presentation in this process. The article also addresses the potential contribution of complement system in augmenting or modulating this response. Identifying specific factors that influences processing and presentation of biologic-derived antigens in different genotype and disease background may offer additional options for intervention in the immunogenic process and consequently, the management of immunogenicity to biologics.

Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors.

Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage-sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of I(h) and the underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund's adjuvant (CFA), we demonstrate increased excitability of C- but not Aδ-nociceptors, 5 to 7 days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation-induced nociceptor hyperexcitability is associated, in C- but not Aδ-nociceptors, with increases in the mean I(h) amplitude/density and in the proportion of I(h) expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4-negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2- immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective I(h) antagonist, ZD7288. Taken together, the findings suggest that C- but not Aδ-nociceptors sustain chronic inflammatory pain and that I(h)/HCN2 channels contribute to inflammation-induced C-nociceptor hyperexcitability.

Loss of transcription factor nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) leads to dysregulation of immune functions, redox homeostasis, and intracellular signaling in dendritic cells.

Dendritic cells (DCs) are critical mediators of immunity and immune tolerance by orchestrating multiple aspects of T cell activation and function. Immature DCs (iDCs) expressing low levels of co-stimulatory receptors are highly efficient at antigen capture but are poor activators of T cells. Maturation of DCs is associated with increased expression of co-stimulatory molecules. Co-stimulatory receptor gene expression is regulated by intracellular redox, NF-κB, and MAPK pathways and by histone deacetylase (HDAC) activity. The transcription factor, Nrf2, is important for maintaining intracellular glutathione (GSH) levels and redox homeostasis and has been implicated in modulating DC co-stimulatory receptor expression. It is unclear whether Nrf2 mediates this effect by GSH-dependent mechanisms and whether it influences DC signaling pathways. Using bone marrow-derived iDCs from Nrf2(+/+) and Nrf2(-/-) mice, we demonstrate that Nrf2(-/-) iDCs have lower basal GSH levels, enhanced co-stimulatory receptor expression, impaired phagocytic functions, and increased antigen-specific CD8 T cell stimulation capacity. Interestingly, lowering GSH levels in Nrf2(+/+) iDCs did not recapitulate the Nrf2(-/-) iDC phenotype. Loss of Nrf2 resulted in elevated basal levels of reactive oxygen species but did not affect basal NF-κB activity or p38 MAPK phosphorylation. Using pharmacological inhibitors, we demonstrate that enhanced co-stimulatory receptor phenotype of Nrf2(-/-) iDC does not require ERK activity but is dependent on HDAC activity, indicating a potential interaction between Nrf2 function and HDAC. These results suggest that Nrf2 activity is required to counter rises in intracellular reactive oxygen species and to regulate pathways that control DC co-stimulatory receptor expression.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism.

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.