Antiangiogenic chimeric anti-endoglin (CD105) antibody: pharmacokinetics and immunogenicity in nonhuman primates and effects of doxorubicin

Abstract

We generated a human/mouse chimeric antibody c-SN6j of human IgG1 isotype from a murine anti-human endoglin (EDG) monoclonal antibody (mAb) SN6j that suppressed angiogenesis, tumor growth and metastasis in mice. We determined pharmacokinetics (PKs) and immunogenicity of c-SN6j in monkeys after multiple i.v. injections. A dose-escalation study was performed by administration of c-SN6j into six monkeys at the dose of 1 mg, 3 mg and 10 mg per kg body weight. In addition, both c-SN6j (3 mg/kg) and doxorubicin (0.275 mg/kg) were injected into two monkeys. c-SN6j and doxorubicin were injected twice a week for 3 weeks. We developed a unique and sensitive ELISA by sequentially targeting the common and idiotypic epitopes of c-SN6j-Fv to quantify plasma c-SN6j. Application of the ELISA showed that increasing the c-SN6j dose resulted in a proportional increase in the circulating c-SN6j after the first injection. In addition, the estimated area under the curve (AUC) for the first injection of c-SN6j is proportional to dose. We carried out detailed analyses of PKs of c-SN6j during and after the repeated injections. Our model of PKs fitted the empirical data well. Addition of doxorubicin modulated the PK parameters. We developed two ELISAs to separately determine the immune responses to the murine part and the human part of c-SN6j in monkeys. Interestingly, the murine part induced a weaker immune response than the human part. Doxorubicin potentiated the immune responses. Increasing the dose of c-SN6j increased plasma levels of c-SN6j but did not increase the immune responses to c-SN6j.

Fig. 1

A standard curve for the circulating c-SN6j as measured by a double-antibody sandwich ELISA. Serial dilutions of the purified c-SN6j were used in the assay

Fig. 2

Pharmacokinetic profiles for plasma c-SN6j in eight monkeys. The fitted curve for the whole time course and raw data points (indicated by circles) are displayed for each monkey. All monkeys were injected six times, at the time points indicted by the arrows, with the indicated amount of c-SN6j. Monkeys G and H received doxorubicin in addition to c-SN6j. c-SN6j and doxorubicin were given i.v

Fig. 3

Standard curves for anti-c-SN6j antibodies as measured by two double-antigen sandwich ELISAs. Serial dilutions of the affinity-purified anti-SN6j-F(ab’)₂ antibodies (a) and anti-human IgG-Fc antibodies (b) were used in the assays to separately evaluate the immune responses to the mouse part and human part of c-SN6j, respectively

Fig. 4

Immune responses to the mouse part (open circle) and human part (crosses) of c-SN6j during and after six injections of c-SN6j into monkeys A – H. c-SN6j was given i.v. at a dose of 1 mg (monkeys A and B), 3 mg (monkeys C, D, G and H) or 10 mg (monkeys E and F) per kg BW. Monkeys G and H received i.v. administration of doxorubicin (0.275 mg/kg BW) in addition to c-SN6j. Preimmune serum of each monkey was included as a negative control in the assays