A new bispecific antibody targeting non-overlapping epitopes on IGF2: design, in vitro characterization and pharmacokinetics in macaques

Abstract

The insulin-like growth factor 2 (IGF2) is an important target for cancer therapy. We have previously proposed an approach for fast and irreversible removal of IGF2 from the circulation by using monoclonal antibodies (mAbs) that bind to two or more non-overlapping epitopes on the same molecule. We provided initial evidence for the formation of oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (FcγRs) with high avidity using an antibody domain with relatively low affinity as one of the anti-IGF2 mAbs. Recently, we identified a mAb, m708.5, in a scFv format which binds to both IGF2 and IGF1 with very high (pM) affinity. Interestingly, and rather surprisingly, this mAb did not compete with our other high affinity mAb, m610.27, for binding to IGF2. Therefore, we generated a new bispecific mAb, m67, by combining m708.5 and m610.27. As expected m67 potently inhibited binding of IGF2 to cells expressing the IGF1R and its phosphorylation, and resulted in formation of multimolecular complexes when incubated with IGF2 and bound with high avidity to cells expressing FcγRII; the complexes were internalized in a macrophage-like cell line. However, although m67 exhibited a reasonably long half-life (6.4 ± 0.6 days) in cynomolgus macaques and high stability in serum, its administration to three animals did not result in any measurable decrease in the IGF2 concentration likely due to the complexity of the IGF2 interactions in the blood and the relatively low (2mg/kg) dose of the mAb leading to a relatively low maximal blood concentration of 120nM. In spite of the lack of effect on the IGF2 concentration in this particular experimental setup, m67 exhibited good drugability properties and could be highly effective in other animal models and in humans. Studies with animal models of cancer are ongoing to evaluate the potential of m67 as a new candidate mAb-based therapeutic.

Keywords: Bispecific antibodies; Cynomolgus macaques; Half-life; IGF ligand.

Figure 1. Diagram of IGF2-bispecific antibody m67

The two arms of m67 are m610.27 (recognizing long and mature IGF2) and m708.5 (cross-reactive to IGF1/2), both are fully human monoclonal antibodies. The two antibodies were constructed in single chain format and placed in front of heavy chain CH1 and kappa light chain constant domain. The specificity and epitopes of the two antibodies are indicated.

Figure 2. Competition of m610 with m708.5 in binding to IGF2

(A) IGF2 was directly coated on the ELISA plate. Bound scFv m610 was detected by HRP-conjugated anti-Flag antibody in the presence of fixed concentration of antibody competitors (IgG1 m708.5 or IgG1 m102.4. (B) IgG1 m610.27 was coated on the ELISA plate and IGF2 was captured by coated IgG1 m610.27. Bound scFv m708.5 or VH m630.3 was detected by HRP-conjugated anti-Flag tag antibody.

Figure 3. Stability of m67 in human sera

m67 was incubated with human sera at 37°C for 9 days and then tested for binding to IGF1 and IGF2 by ELISA.

Figure 4. m67 inhibited bindings of IGF1 (A) and IGF2 (B) to MCF7 cells

MCF-7 cells were incubated with 5 nM biotinylated IGF1 or 1 nM biotinylated IGF2 in the absence or presence of antibodies. Bound biotinylated IGF1 or 2 was detected by streptavidin-PE. Cells incubated with streptavidin-PE conjugate only as the background control are in black. Cells incubated with biotin-IGF1 or 2 are in red. Those for biotin-IGF1 or 2 with antibodies are in blue.

Figure 5. Inhibition of IGF1R and IR phosphorylation

MCF-7 cells were starved in serum free medium for 5 h first, followed by addition of treatment medium with 1 nM IGF1 or 5 nM IGF2 with indicated concentrations of antibodies. Thirty minutes later cells were chilled and lysed. (A) IGF1R was immunoprecipitated and the phosphorylated IGF1R was detected with a phosphor-tyrosine specific antibody. The total amount of IGF1R were detected by the same polyclonal antibody used for the immunoprecipitation. (B) IR was immunoprecipitated and detected as following above methods.

Figure 6. Size-exclusion chromatography analysis of m67/IGFs complex.<

br>m67, the mixture of m67 and IGF2, or the mixture of m67/IGF2 plus IGF1 was analyzed by Superdex 200 10/300G column.

Figure 7. Binding of antibodies to BJAB Cells in the presence of IGF2

Bound biotinylated IGF1 or 2 was detected by Streptavidin-PE. Cells incubated with Streptavidin-PE conjugate are shown in black as the background control. Cells incubated with IGF2 only are in red. Those for IGF2 with antibodies are in blue.

Figure 8. Endocytosis of m67-IGF2 by macrophage-like U937 Cells

Bound antibodies were detected by FITC-conjugated goat F(ab′)2 anti-human Fc IgG antibody. The black histograms are the blank cells incubated with the secondary antibody only. The histograms for cells incubated with antibody alone, the mixture of antibody and IGF2, and the mixture of the antibody, IGF2 and Cytochalasin D are in red, blue and purple, respectively.

Figure 9. Plasma concentration-time course plot of m67

A single injection of m67 at 2 mg/kg dose was administered. The result shown was the average of data from 3 animals. The inset shows the plot at within 24 hours after m67 injection.

Figure 10. A Plasma concentration-time course plot of total IGF2

IGF2 was dissociated from binding proteins and antibody with acid/ethanol treatment, then detected with capture ELISA. Concentrations from three animals were shown individually. B, Plasma concentration-time course plot of total IGF2 within 24 hours after m67 injection.

Figure 10. A Plasma concentration-time course plot of total IGF2

IGF2 was dissociated from binding proteins and antibody with acid/ethanol treatment, then detected with capture ELISA. Concentrations from three animals were shown individually. B, Plasma concentration-time course plot of total IGF2 within 24 hours after m67 injection.

Figure 11. Body weights of animals during the study remained steady

Body weight of the monkeys were taken two days before the injection of m67 and weekly after the injection.