Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Role of metabolism in dimethylnitrosamine-induced immunosuppression: a review.

Dimethylnitrosamine (DMN) has been characterized as a potent hepatotoxin, carcinogen and mutagen. As described below, immunotoxicity should be added to its profile of activity. Although a broad spectrum of immune parameters is affected by DMN, humoral immunity is particularly sensitive. In order for DMN to produce its traditional profile of toxicity it requires metabolic activation to reactive intermediates which alkylate macromolecules. Similarly, DMN also must be metabolized to produce its immunological effects. However, as this review suggests, the metabolism of DMN to an intermediate capable of suppressing the humoral immune response may be qualitatively and/or quantitatively different from that which mediates hepatotoxicity and genotoxicity.

Characterization of the effects of direct alkylators on in vitro immune responses.

Although their mechanism of degradation may differ, both the SN1 alkylators, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitroso-N-methylurea (MNU), and the SN2 alkylators, dimethyl sulfate (DMS) and methyl methanesulfonate (MMS), spontaneously decompose under aqueous conditions to the methyldiazonium ion or a direct methylating intermediate, respectively. Thus, these agents serve as useful probes to investigate the immunosuppressive potential of the putative primary reactive intermediate of dimethylnitrosamine (DMN) metabolism, the methyldiazonium ion. The effects of these direct alkylating agents on the in vitro immune response were characterized. Direct addition of both the SN1 and SN2 alkylators to naive B6C3F1 murine splenocytes produced a dose-dependent suppression of the in vitro antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes (sRBC), T-independent antigen, dinitrophenyl (DNP)-Ficoll, and the polyclonal activator, lipopolysaccharide (LPS). The T-dependent and T-independent responses proved to be more sensitive than the polyclonal response to the effects of these compounds, except for MNNG in which all 3 antibody responses were equally affected. The suppression of the AFC response for all antigens was unaffected by the addition of 2-ME, and was observed at concentrations below those affecting viability, although at the highest concentrations an effect on viability was often observed. The addition of MNNG to the T-dependent AFC response at any time within the first 96 h produced a marked suppression, while the addition of DMS to cultures was only effective in suppressing the AFC response if added within the first 24 h. MNNG and DMS suppressed the proliferative responses to both B-cell (LPS) and T-cell (Concanavalin A; Con A) mitogens, as well as in the mixed lymphocyte response (MLR). In addition, a positive correlation between immunosuppression and DNA damage, as measured by single-strand breaks, was observed. Although these compounds produced suppression of in vitro immune responses, their profile of activity on immunocompetence and DNA damage was different from that associated with DMN and thus, the direct alkylators may not prove to be useful models to elucidate the mechanism of the DMN-induced immunosuppression.

Production of DNA single-strand breaks in unstimulated splenocytes by dimethylnitrosamine.

In an attempt to elucidate the mechanism whereby primary hepatocytes, but not liver S9 homogenates, generate immunosupprssive metabolites of dimethylnitrosamine (DMN), the production of DNA single-strand breaks (SSB) in unstimulated splenocytes was investigated with alkaline-elution analysis. Both hepatocytes and S9 homogenates induced SSB in cultured splenocytes by DMN - minimum detectable doses with the two metabolic activation systems (MAS) were 1 microM and 5 mM, respectively. DNA elution profiles were linear in splenocytes co-cultured with DMN and hepatocytes and convex in splenocytes incubated with DMN and S9 homogenates. Aminoacetonitrile (AAN; 10 mM), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either MAS. Addition of exogenous calf-thymus DNA to the hepatocyte co-culture medium did not affect the production of SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from linear to convex. All of these treatments have been previously shown to block the immunosuppression by DMN in the hepatocyte co-culture system. These results indicate that the immunosuppression by DMN is not related to DNA damage, as measured by the production of SSB, and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.

A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitive to antigen receptor-mediated growth inhibition.

In order to gain a better understanding of the pathways linking receptor immunoglobulin (sIg) crosslinking to downstream B lymphocyte responses, mutants were generated that were defective in sIg-associated signaling pathways. The murine B lymphoma WEHI-231 has proven to be a useful model for studies of sIg-mediated signal transduction. Signaling through sIgM using anti-receptor antibodies (anti-mu) leads to growth arrest and apoptosis of this continuously proliferating cell line. Direct activation of protein kinase C (PKC) with phorbol esters also can mediate this response in WEHI-231. This negative growth response is a useful characteristic that can be exploited to generate signaling mutants that are resistant to the growth-inhibiting effects of anti-mu or phorbol ester. Using this approach, we selected a mutant, PR30-3, in which signaling was blocked downstream of the phorbol diester response element, presumably PKC. Although no longer responsive to phorbol ester stimulation, PR30-3 is not defective in PKC expression or function. Western blot analyses of cellular lysates shows the mutant to express the PKC isoforms alpha, beta, and delta, at levels not markedly different from wild-type WEHI-231. PR30-3 expresses active PKC as shown by its ability to phosphorylate a PKC-specific peptide in vitro. PR30-3 and WEHI-231 express equivalent levels of sIgM expression and nearly indistinguishable second messenger responses in the form of increases in [Ca2+]i and inositol phospholipid hydrolysis. Both PR30-3 and WEHI-231 demonstrate rapid induction of tyrosine kinase activation following sIgM signaling, although there is a reproducible difference in the ability to phosphorylate a 40-kDa substrate in PR30-3. Interestingly, tyrosine phosphorylation of this substrate is induced by phorbol ester stimulation in the wild-type but not the mutant PR30-3. We observed that phorbol ester stimulation of PR30-3 induces the expression of the early response gene c-fos, previously shown to be PKC dependent in this cell line. These results indicate that the signaling component(s) defective in PR30 lie downstream of PKC but upstream of the commitment point for growth inhibition and cell death. Finally, although PR30-3 is resistant to the inhibitory effects of phorbol ester, proliferation is nonetheless still inhibited in response to anti-mu stimulation. These results suggest that the growth inhibitory response of WEHI-231 to anti-mu and phorbol ester involves different pathways.

Endogenous expression of delta on the surface of WEHI-231. Characterization of its expression and signaling properties.

WEHI-231 is a murine B cell lymphoma that has been used extensively as a model for the immature stage B cell and its functional response to Ag receptor cross-linking as a model for immature B cell tolerance. This cell line expresses sIgM, CD5, and FcR gamma, but lacks the B cell-specific isoform of CD45 (B220). This study demonstrates for the first time that WEHI-231, in contrast to classically defined immature B cells, expresses delta on its surface. Analysis of delta on WEHI-231 revealed structural differences with respect to that on BAL-17 or primary splenic B cells. Although the m.w. of delta on the latter two B cell populations was similar, delta on WEHI-231 manifested a marked increase in its apparent m.w. deduced by SDS-PAGE. This difference was found to be due primarily to differential N-linked glycosylation. Signal transduction through the endogenous sIgD on WEHI-231 was investigated. In contrast to cross-linking of sIgM, cross-linking of the endogenous surface IgD on WEHI-231 was unable to generate a negative growth response in these cells. This inability may be due to uncoupling from normal surface Ig signaling pathways. The signaling properties of the endogenous sIgD on WEHI-231 differ from that on primary B cells and other sIgD-expressing cell lines. Whereas sIgD on splenic B lymphocytes or the mature B cell line BAL-17 is coupled to inositol phospholipid hydrolysis and calcium mobilization, cross-linking of sIgD on WEHI-231 failed to elicit these events, although induced changes in tyrosine phosphorylation were observed. Thus, endogenous expression of surface IgD on WEHI-231 is inconsistent with its representing the classically defined immature stage B cell. The structural and signaling differences associated with delta on these cells suggest the potential for developmentally regulated delta function and model for study of sIgD signal transduction.

Differential effects of coadministration of aminoacetonitrile on immunosuppression and hepatotoxicity produced by dimethylnitrosamine.

Aminoacetonitrile (AAN) has been reported to be a dimethylnitrosamine (DMN) demethylase inhibitor which prevents the metabolic activation of DMN to a hepatotoxin. The purpose of this investigation was to determine if AAN pretreatment, which ameliorates hepatotoxicity, would also prevent the immunotoxicity associated with DMN exposure. Female B6C3F1 mice were exposed to either 3 or 6 mg/kg of DMN (in saline) or saline i.p. for 7 consecutive days. The animals were also treated (i.p.) twice daily, 1 hr before and 6 hr after DMN exposure, with either, 10, 30 or 100 mg/kg of AAN (in saline) or saline. Mice were sensitized with sheep red blood cells i.v. on day 8. On day 12, body and organ weights were determined, serum chemistry and histopathology were evaluated and day 4 immunoglobulin M antibody response was measured. Hepatotoxicity caused by DMN, as reflected by an increase in body weight attributed to the production of ascites, a 485.7% increase in the serum glutamic pyruvic transaminase levels and histopathology was reversed by doses of AAN as low as 10 mg/kg. Conversely, doses of AAN as high as 100 mg/kg were unable to reverse the suppression of the antibody forming cells response to sheep red blood cells produced by DMN. The results of this investigation indicate that DMN-induced immunosuppression and hepatotoxicity can be separated and that an immunosuppressive metabolite of DMN is produced by an AAN-insensitive pathway.

Prevention of immunotoxin-induced immunogenicity by coadministration with CTLA4Ig enhances antitumor efficacy.

Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not become part of standard cancer therapy because of factors that include their inherent immunogenicity, which limits the duration of therapy. To address this issue, we evaluated in preclinical models the concomitant use of the immunosuppressive agent CTLA4Ig and BR96 sFv-PE40, a single-chain immunotoxin that binds to carcinoma cells expressing Le(y). Cotreatment with CTLA4Ig, an inhibitor of the CD28/CTLA4-CD80/CD86 costimulation pathway, blocked the production of Abs against BR96 sFv-PE40 in immunocompetent rodents and dogs. It also blocked hypersensitivity reactions in rats carrying colon carcinoma allografts during a second course of BR96 sFv-PE40 therapy, and the cotreatment with CTLA4Ig resulted in enhanced antitumor activity. Cotreatment with CTLA4Ig also prevented hypersensitivity reactions induced by repeat dosing of BR96 sFv-PE40 (q3dx5) in dogs. The production of anti-BR96-sFv-PE40 Abs was decreased in CTLA4Ig-cotreated rodents and dogs resulting in increased plasma levels of BR96 sFv-PE40 relative to non-CTLA4Ig-cotreated animals. These data show that cotreatment of immunotoxins with CTLA4Ig, by inhibiting the production of anti-immunotoxin Abs, can extend the duration of BR96 sFv-PE40 therapy to give greater exposure, reduced toxicities, and increased efficacy.

BR96 sFv-PE40 immunotoxin: nonclinical safety assessment.

BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewis(y)-related antigens. Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans. Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40. In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative). Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats. These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts. In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas. Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis. After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative). No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewis(y)-expressing dogs. Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function. The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40.