Preexisting Antibodies to an F(ab')2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment

Abstract

Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab')2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab')2 CDR using anti-DLL4 F(ab')2 and a control F(ab')2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

Figure 1

Specificity of signal reduction in the anti-DLL4 F(ab′)₂ ATA assay by different competitor molecules. Anti-DLL4 F(ab′)₂ competitor will reduce all reactivity in the ATA assay. Control F(ab′)₂ will not reduce anti-DLL4 F(ab′)₂ CDR reactivity but will reduce F(ab′)₂ framework and hinge reactivity. Anti-DLL4 full length antibody will reduce anti-DLL4 CDR and framework reactivity, but not hinge reactivity.

Figure 2

Preexisting ATAs are directed primarily at the F(ab′)₂ hinge, as shown by the reactivity reduction by competition with anti-DLL4 F(ab′)₂, Herceptin F(ab′)₂, or anti-DLL4 full length antibody in the bridging ELISA format. In the bridging ATA assay using 50 μg/mL competitor (in-well concentration) and a 1/20 sample dilution, full length antibody causes very little signal reduction; however the F(ab′)₂ form of the same antibody reduces the signal almost to background. An F(ab′)₂ form of Herceptin, which has the same framework as anti-DLL4 but with a different CDR, also reduces the signal, indicating that the signal is due to anti-hinge antibodies.

Figure 3

Comparative reactivity of cynomolgus monkey serum samples with anti-DLL4 Fab or anti-DLL4 F(ab′)₂ directly coated on a well (direct binding format). More sample reactivity is observed with the F(ab′)₂ coat.

Figure 4

Representative anti-DLL4 F(ab′)₂ bridging ATA assay titer curves from three cynomolgus monkeys sampled weekly, three samples per animal total. The signals were consistent within animal but varied widely between animals.

Figure 5

Response-time plots of ATA titer to the whole F(ab′)₂ molecule and anti-CDR ATA positivity status by dose group. Solid light gray bar: pretreatment titer (average of two samples). Solid blue bar: posttreatment titer for anti-CDR negative sample. Horizontal hatched red: posttreatment titer for anti-CDR positive sample. Diagonal hatched yellow bar: posttreatment titer for anti-CDR indeterminate sample.