Immunogenicity of biopharmaceuticals in laboratory animals.

The potential immunogenicity of new protein therapeutics raises concerns about the possibility of inducing untoward immune reactions in humans. It is generally assumed that all animals will make antibody to human proteins and therefore, there is sentiment among some scientists that this makes the issue of immunogenicity as a safety concern irrelevant. However, recent clinical trials with some proteins have detected the presence of autoantibodies that have resulted in clinical sequelae. These reactions were also observed in preclinical animal studies. In fact, non-human primate and transgenic mouse models can be useful for predicting the relative immunogenicity of human proteins. In addition, the characterization of the immunogenicity of biotechnology molecules provides a practical basis for determining the significance of antibody formation in preclinical safety studies.

Immunogenicity of tissue plasminogen activators in rhesus monkeys: antibody formation and effects on blood level and enzymatic activity.

The immunogenicity of a tissue-type plasminogen activator analog, mt-PA6, consisting of the second kringle and protease domains, was compared to that of the native-sequence protein (nt-PA) in rhesus monkeys. Antibody responses were compared in groups of eight monkeys that were treated by i.v. injection twice, 1 month apart, using doses and regimens chosen to mimic therapy (0.5 mg/kg mt-PA6 bolus, 1.25 mg/kg nt-PA bolus + infusion). An additional group was treated with a 0.5 mg/kg nt-PA bolus. A single positive response was obtained in a monkey treated with 0.5 mg/kg nt-PA after the primary injection. Following the secondary injection, responses were obtained in 1/8, 3/8, and 6/8 monkeys treated with mt-PA6, nt-PA as a bolus, or nt-PA as a bolus + infusion, respectively. Several monkeys were selected to determine whether circulating tPA antibody altered the pharmacokinetics of mt-PA6. Clearance was found to decrease without affecting peak blood levels as antibody concentrations increased from 0.02 to 100 micrograms/ml. In contrast, the peak blood level was reduced by 99% at an antibody concentration of 152 micrograms/ml in a monkey that had been exposed to mt-PA6 in adjuvant 14 months previously. Further, only the serum from this and three other hyperimmunized monkeys inhibited the enzymatic activity of tPA in vitro. It is concluded that mt-PA6 is not more immunogenic than nt-PA in rhesus, and that low levels of antibody are more likely to influence the pharmacokinetic properties of tPA than to inhibit its enzymatic activity. It is unlikely that mt-PA6 would present a serious immunogenic risk in humans.

Immunogenicity of biosynthetic human LysPro insulin compared to native-sequence human and purified porcine insulins in rhesus monkeys immunized over a 6-week period.

Development of insulin antibodies in rhesus monkeys was investigated after immunization with 3 forms of insulin in Freund's adjuvant. Insulins examined included: 1. biosynthetic LysPro insulin (LY275585), a new human insulin analog, 2. biosynthetic native-sequence human insulin, and 3. purified porcine insulin. Male monkeys, 4/insulin type, were immunized weekly over a 6-week period with increasing doses of insulin, ranging from 10 to 100 micrograms/monkey. An ELISA assay was used to measure IgG insulin antibodies in sera collected prior to immunization and 5, 10, and 16 days after final immunization. One monkey had detectable pretreatment levels of antibody. This monkey, which had been assigned to the LysPro insulin treatment group, responded to immunization with a peak antibody level of 20 micrograms/ml. IgG insulin antibody responses were not detected in any of the other monkeys. A passive cutaneous anaphylaxis (PCA) assay was used to measure IgE insulin antibodies in sera collected prior to immunization and 10 days after final immunization. No IgE antibodies were detected in any of the monkeys pre- or post-immunization. Considering that 1. an immunological adjuvant was used, 2. eleven of twelve monkeys failed to develop an antibody response, and 3. the IgG insulin antibody level observed in the single responding monkey was low, it was concluded that these insulins have an extremely weak immunogenic potential in rhesus monkeys. It is suggested that immunization of non-human primates with new therapeutic proteins in adjuvant may be a useful primary screen to determine their immunogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys.

The relative concentrations of antibodies produced in monkeys against three forms of human growth hormone (hGH) were determined using an antigen-specific avidin/biotin ELISA assay. Monkeys were treated in two separate 90-day studies with recombinant methionyl-hGH (met-hGH) and pituitary-derived hGH (pit-hGH) (Study 1) and recombinant natural sequence hGH (Study 2). The lowest dose was equal to the expected therapeutic dose of 0.1 IU/kg. Sixty-nine percent of monkeys treated with pit-hGH and 81% of those treated with met-hGH developed detectable anti-hGH responses. The magnitudes of the responses exhibited wide animal to animal variability, were not markedly related to dose or sex, and were lower than levels obtained in monkeys immunized with hGH in Freund's adjuvant. In contrast, the incidence of antibody responses in monkeys treated with natural sequence hGH was lower (23% in one experiment and 5% in a replicate experiment) and took longer to develop. Antibody concentrations were lower, on average, than in those animals treated with met- or pit-hGH. These results are in accord with those observed clinically, thus supporting the use of the monkey model to predict the relative immunogenicity of some proteins in humans.

Somatotropin antibody formation in cows treated with a recombinant bovine somatotropin over two lactations.

Blood plasma from cows treated with somidobove, a form of recombinant bovine somatotropin, was assayed for development of antibodies against the protein. Forty-three Holstein cows, selected from an animal safety study, were monitored. Cows were divided into four groups and treated with placebo, 960, 2880, or 4800 mg somidobove per dose at 28-d intervals during two successive lactation periods. Blood plasma was collected at intervals prior to and during the lactations, and levels of IgG antibody reactive with somidobove were determined in an enzyme-linked immunosorbent assay. Virtually all of the cows treated with somidobove developed low levels (less than 40 micrograms/ml) of antibody against somidobove. One or two cows from each group responded with some-what higher levels, ranging from 40 to 200 micrograms/ml. Responses generally increased during the first 3 mo of treatment, then decreased, and remained constant with continued treatment. There was no sign of a memory response within or among the lactation periods, and no adverse health effects or decreases in lactational performance were associated with antibody production.

Delayed type hypersensitivity in zinc deficient adult mice: impairment and restoration of responsivity to dinitrofluorobenzene.

Seven-week-old A/J mice were divided into three uniform groups and fed: 1) a zinc deficient diet (0.6 micrograms Zn/g) ad libitum (deficient); 2) a zinc adequate diet (55 micrograms Zn/g) ad libitum (control); and 3) a zinc adequate diet (55 micrograms An/g) with intake restricted to the average daily amount consumed by the zinc deficient group (restricted). After 26 days on the diets, part of the mice from each dietary group were percutaneously sensitized with dinitrofluorobenzene (DNFB) to assess of restricted mice to DNFB [Stimulation Index (S.I.) of 1.98] was only moderately reduced compared to controls (S.I. of 2.53). Zinc deficient mice, on the other hand, gave a very poor response to DNFB (S.I. of 1.23). Thus, DTH is yet another branch of the immune system affected by a suboptimal intake of dietary zinc. To assess the ability of deficient mice to regenerate DTH responsivity the remaining deficient mice were refed diets containing adequate zinc (55 micrograms Zn/g). After 21 days of nutritional repletion, previously zinc deficient mice gave DTH responses nearly identical to that of control mice.

Regeneration of T-cell helper function in zinc-deficient adult mice.

Diets deficient in zinc cause rapid atrophy of the thymus and loss of T-cell helper function in the young adult A/J mouse. Because zinc deficiency, as well as other nutritional deficiences, causes extensive damage to the immune system, the question arose as to whether zinc-deficient mice could repair the thymus and fully regenerate T-cell helper function if returned to diets containing adequate amounts of zinc. Five-week-old A/J female mice were fed either a zinc-deficient (<1 mug of Zn per g) or a zinc-adequate (50 mug of Zn per g) diet for 31 days. Histological examination of thymuses from the zinc-deficient mice revealed that the cortex was preferentially involuted and the thymus was about one-third of normal size. The direct plaque-forming cells produced per mouse spleen in response to immunization with sheep erythrocytes was 34% of normal; indirect plaque-forming cells were 18% of normal (Jerne plaque assay). After the deficient mice had been fed a zinc-adequate diet for 1 week, their response was nearly normal, except that the indirect response was 68% of controls; in this same period, the thymuses of these mice had quadrupled in size and exhibited a greatly enlarged cortex repopulated with immature thymocytes. By 2 weeks, the thymuses of the previously zinc-deficient mice were normal in size and appearance; however, there was a slight increases in numbers of indirect plaque-forming cells. By 4 weeks, the thymus weights, direct and indirect plaque-forming cell counts, and secondary response of the previously deficient mice were normal. Mice that were nearly athymic after 45 days of dietary zinc deficiency were also able to fully reconstruct the thymus and regenerate T-cell helper function. The data show that the zinc-deficient young adult mouse has the capacity to fully restore the T-cell-dependent antibody-mediated responses upon nutritional repletion.