Joyce Mordenti

Monoclonal antibody therapy of human cancer: taking the HER2 protooncogene to the clinic.

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function.In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-α sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer.In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.

Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and fab antibodies in rhesus monkeys following intravitreal administration

Access of recombinant proteins to the retina following intravitreal administration is poorly understood. A study was conducted in male Rhesus monkeys (15 to 28 mo of age; 2.8-3.3 kg) in order to compare the intraocular tissue distribution, pharmacokinetics, and safety of 125Iodine (I)-labeled full-length humanized rhuMAb HER2 antibody (148 kD) and of 125I-labeled humanized rhuMAb vascular endothelial growth factor Fab antibody (48.3 kD) following bilateral bolus intravitreal injection on day 0 (5 animals/group). The dose administered to each eye was 25 μg (9-10 μCi) in 50 μl. Animals were euthanatized on day 0 (1 hr postdose) and on days 1, 4, 7, and 14. Safety assessment included direct ophthalmoscopy, intraocular pressure measurements, clinical observations, body weight, and hematology and clinical chemistry panels. Blood and vitreous samples were collected daily (blood only) and at necropsy for pharmacokinetics and analysis for antibodies to the test materials; the ocular tissue distribution of the test material was evaluated by microautoradiography. All animals completed the study. Microautoradiography demonstrated that the full-length antibody did not penetrate the inner limiting membrane of the retina at any of the time points examined. In contrast, the Fab antibody fragment diffused through the neural retina to the retinal pigment epithelial layer at the 1-hr time point and persisted in this location for up to 7 days. Systemic exposure to test material was low but variable: the highest plasma concentration of the full-length antibody was 20.3 ng/ml, whereas plasma concentrations for the Fab antibody remained below the limit of quantitation (i.e., <7.8 ng/ml). An immune response to the test material was not evident in either treatment group. The half-life in vitreous was 5.6 days for the full-length antibody and 3.2 days for the Fab antibody. The shorter intravitreal half-life of the Fab antibody is related to its smaller size and its significant diffusion through the retinal layers. The differences in pharmacokinetics and tissue distribution that are noted between the full-length and Fab antibodies in this study identify potential therapeutic approaches that may be exploited in specific disease conditions.

Interspecies scaling of clearance and volume of distribution data for five therapeutic proteins

The clearance and volume of distribution of five human proteins (recombinant CD4, CD4 immuno-globulin G, growth hormone, tissue-plasminogen activator, and relaxin) in humans and laboratory animals were analyzed as a function of body weight using allometric scaling techniques. These proteins cover a 16-fold range of molecular weight (6 to 98 kD), are produced by recombinant or synthetic methods, and may be cleared by different mechanisms. The analyses revealed that the clearance and volume data for each protein were satisfactorily described by an allometric equation (Y = a Wb). The allometric exponent (b) for clearance (ml/min) ranged from 0.65 to 0.84, the allometric exponent for the initial volume of distribution (ml) ranged from 0.83 to 1.05, and the allometric exponent for the volume of distribution at steady state (ml) ranged from 0.84 to 1.02. Exponent values from 0.6 to 0.8 for clearance and 0.8 to 1.0 for volumes are frequently cited for small molecules and are expected based on empirical interspecies relationships. When the preclinical data were analyzed separately, the pre-clinical allometric relationships were usually predictive of the human results. These findings indicate that the clearance and volume of distribution of select biomacromolecules follow well-defined, size-related physiologic relationships, and preclinical pharmacokinetic studies provide reasonable estimates of human disposition. Employing this methodology during the early phases of drug development may provide a more rational basis for dose selection in the clinical environment.

Efficacy and concentration-response of murine anti-VEGF monoclonal antibody in tumor-bearing mice and extrapolation to humans.

The development of a neovascular supply (angiogenesis) is a major aspect of tumorigenesis. Recent work has indicated that vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. In vitro and in vivo studies have demonstrated that an anti-VEGF antibody is capable of suppressing the growth of human tumor cell lines. The following study was conducted in tumor-bearing nude mice to evaluate the concentration-response relationship of murine anti-VEGF monoclonal antibody (muMAb VEGF) so that an efficacious plasma concentration of the recombinant humanized form (rhuMAb VEGF) in cancer patients could be estimated. (This study was included in our Investigational New Drug application to support the clinical dosing regimen and projected human safety factors for the toxicology program.) Additionally, the growth dynamics of the tumors were evaluated as a function of dose to explore whether a mechanismic interpretation of tumor growth inhibition by muMAb VEGF is possible. On day 1, A673 human rhabdomyosarcoma cells (2 X 106 cells/mouse) were injected subcutaneously in 188 beige nude mice (16-24 g). Treatment with muMAb VEGF (0.05-5.0 mg/kg; n = 24/group), phosphate-buffered saline (n = 10), or anti-gp120 isotype-matched control antibody (5.0 mg/kg; n =10) began 24 hr later. Each animal received intraperitoneal injections of test material twice weekly for 4 wk. Immediately prior to each dose, 2 mice from each muMAb VEGF group were selected randomly, and plasma was collected for pharmacokinetic evaluation; at the end of the study, samples were collected from all animals for pharmacokinetic evaluation. Tumor dimensions were recorded weekly, and at the end of the study, tumor weight and dimensions were recorded. Satisfactory tumor suppression in nude mice was achieved at muMAb VEGF doses of ≥2.5 mg/kg, where the average trough muMAb VEGF plasma concentration was 30 μg/ml (concentrations in individual animals >10 μg/ml). Assuming the pharmacokinetics of rhuMAb VEGF in patients will resemble the pharmacokinetics of a similar humanized anticancer monoclonal antibodies, a clinical dosing regimen was designed to maintain the rhuMAb VEGF plasma concentration in this efficacious range. This study shows an approach that can be used to estimate a human dosing regimen from preclinical pharmacokinetic/pharmacodynamic data. Because we have just initiated clinical trials with rhuMAb VEGF, we cannot judge clinical outcome in relation to these preclinical predictions; nonetheless, it is hoped that by sharing our approach and thought processes with other investigators we can assist the discovery and development of anticancer therapeutics.

Reproductive toxicity testing of therapeutic biotechnology agents

Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105 (J.WH.); Miles, Inc., Elkhart, Indiana 46515 (K.G.H.); USFDA, Rockville, Maryland 20852 (M.A.S., J.A.C.); California Regional Primate Research Center, Davis, California 95616 (A.G.H.); Hoffman-LaRoche, Inc., Nutley, New Jersey 07110 (N.D.A.); Skyline Technology Consulting Group, Znc., San Francisco, California 94133 (A.H.C.K.); and Genentech, Znc., South San Francisco, California 94080 (J.M.)

Pharmacokinetics and Tissue Distribution of Recombinant Human Transforming Growth Factor Beta1 After Topical and Intravenous Administration in Male Rats

Recombinant human transforming growth factor beta (rhTGF-β1) enhances the healing process after topical application to various animal wound models. A detailed pharmacokinetic and tissue distribution study was performed to support the clinical development of rhTGF-β1 for wound healing indications. Rats received radioiodinated or unlabeled rhTGF-β1 as an intravenous (iv) bolus or as a topical formulation applied to a full thickness wound. Plasma concentrations of TGF-β1 were estimated from TCA-precipitable radioactivity or were measured by ELISA. Following iv administration, the initial half-life was rapid (<11 min), regardless of whether radi-olabeled or unlabeled rhTGF-β1 was used. The terminal half-life was long (163 min) when the test material was radioiodinated and administered as a trace dose and relatively short (≤61 min) when given at high doses and assayed by ELISA. Analysis of plasma radioactivity by SDS-PAGE revealed a time-dependent clearance of the 25-kDa parent molecule without a significant appearance of lower molecular weight radiolabeled metabolites. The majority of the radioactivity was associated with highly perfused organs, known iodide elimination pathways, and the thyroid at 1 and 8 hr after iv injection. After topical administration of a high dose (0.8 mg/kg), no immunoreactive TGF-β1 was detectable in plasma samples taken over a 48-hr period. However, trace amounts (≤0.05 ng/mL) of acid-precipitable radioactivity were detected in plasma after topical application of [125I]rhTGF-β1 (1 µg/kg, 126 µCi/kg). A significant portion (35%) of the [125I]rhTGF-β1 persisted intact (25 kDa) at the wound site 24 hr after application. In conclusion, rhTGF-β1 was rapidly cleared after iv bolus administration and distributed primarily to the liver, lungs, kidney, and spleen. Little systemic exposure was observed after applying a single topical dose of rhTGF-β1 to a wound, and the intact molecule persisted at the wound site.

Characterization of the MN gp120 HIV-1 Vaccine: Antigen Binding to Alum

AbstractPurpose. The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. Methods. Several observations suggest that the gpl20-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. Results. Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. Conclusions. These observations indicate that both the antigen and the adjuvant require optimization together.

Phase I study of continuous-infusion soluble CD4 as a single agent and in combination with oral dideoxyinosine therapy in children with symptomatic human immunodeficiency virus infection

To determine the safety and pharmacokinetics of recombinant soluble CD4 (sCD4) administered by continuous intravenous infusion to children with symptomatic human immunodeficiency virus type 1 infection, we conducted a phase I study at the National Cancer Institute. Three dose levels of sCD4 were evaluated: 100, 300, and 1000 micrograms/kg per day. After an initial 12 weeks of treatment with sCD4 alone, dideoxyinosine at a dose of 90 mg/m2 every 8 hours was added and subjects were observed for an additional 12 weeks. Combination therapy was continued in patients in whom it was well tolerated. In addition to toxicity and pharmacokinetic monitoring, surrogate markers of antiviral activity were evaluated. Eleven children were enrolled in the study. During the 12 weeks of treatment with sCD4 alone, and during subsequent sCD4 plus dideoxyinosine combination therapy, no significant toxic reaction attributable to sCD4 or dideoxyinosine was encountered. Low-level anti-CD4 antibodies developed in two patients. Steady-state sCD4 levels increased proportionately at higher doses. The CD4 cell counts and serum p24 antigen levels did not provide evidence of antiviral activity. We conclude that sCD4 was well tolerated at doses up to 1000 micrograms/kg per day when administered by continuous intravenous infusion; however, evidence of in vivo antiviral activity was not observed in this study.

The Pharmacokinetics of Recombinant Human Relaxin in Nonpregnant Women After Intravenous, Intravaginal, and Intracervical Administration

The pharmacokinetics of recombinant human relaxin (rhRlx) after intravenous (iv) bolus administration and the absorption of rhRlx after intracervical or intravaginal administration were determined in nonpregnant women. The study was conducted in two parts. In part I, 25 women received 0.01 mg/kg rhRlx iv. After a minimum 7-day washout period, these women were dosed intracervically (n = 10) or intravaginally (n = 15) with 0.75 or 1.5 mg rhRlx, respectively, in 3% methylcellulose gel. Part II was a double-blind, randomized, three-way crossover study in 26 women. At 1-month intervals, each woman received one of three intravaginal treatments consisting of 0 (placebo), 1, or 6 mg rhRlx in 3% methylcellulose gel. The serum concentrations of relaxin following iv administration were described as the sum of three exponentials. The mean (±SD) initial, intermediate, and terminal half-lives were 0.09 ± 0.04, 0.72 ± 0.11, and 4.6 ± 1.2 hr, respectively. Most of the area under the curve was associated with the intermediate half-life. The weight-normalized clearance was 170 ± 50 mL/hr/kg. The observed peak concentration was 98 ± 29 ng/mL, and the weight-normalized initial volume of distribution was 78 ± 40 mL/kg, which is approximately equivalent to the serum volume. If central compartment elimination was assumed, the volume of distribution at steady state (Vss/W) was 280 ± 100 mL/kg, which is approximately equivalent to extracellular fluid volume. Vss/W could be as large as 1300 ± 400 mL/kg without this assumption. After intravaginal administration of the placebo gel, endogenous relaxin concentrations were evident (i.e., ≥20 pg/mL) in 9 of the 26 women (maximum concentrations, 23–234 pg/mL). A similar proportion of women (approximately 35–40%) exhibited measurable serum concentrations of relaxin following intravaginal rhRlx treatment; this proportion increased to 90% following intracervical rhRlx treatment. For both routes of administration, the maximum serum concentrations of relaxin were usually within the range of values observed for endogenous relaxin, suggesting that the absorption of rhRlx was minimal.

Estimation of Permanence Time, Exit Time, Dilution Factor, and Steady-State Volume of Distribution

General solutions for exit time, permanence time, dilution factor, and volume of distribution at steady state are derived for compartmental and noncompartmental systems. These derivations require that the systems are linear and state-determined. Unique values for these parameters cannot be determined when the site of elimination is not known; in this case the parameters can be defined by a range. Interpretation of this range and its significance and use in clinical situations are illustrated with two examples.