Multiplexing of receptor occupancy measurements for pharmacodynamic biomarker assessment of biopharmaceuticals.
BACKGROUND
Receptor occupancy (RO) assays measure drug target engagement, and are used as pharmacodynamic (PD) biomarkers. RO assays are commonly performed by flow cytometry and often require multiplexing for assessment of multiple PD biomarkers when specimen volumes are limited. We present multiplexed RO assays for an IGF1R-EGFR bispecific antibody (Bs-Ab) and a CTLA4-Ig recombinant fusion protein to demonstrate key considerations for accurate RO assessment.


METHODS
RO in cynomolgus monkeys was determined in whole blood using flow cytometry. Free and total receptors were measured using anti-receptor fluorescence-labeled detection reagents, competitive and noncompetitive to drug, respectively.


RESULTS
RO of IGF1R was examined as PD for Bs-Ab, since IGF1R was expressed on blood cells. Multiplexed measurements of free and total IGF1R showed that IGF1R expression measured by total receptor was highly variable, impacting interpretation of free-IGF1R. Normalization of free-over-total IGF1R measurements compensated for variability of receptor expression allowing for accurate RO assessment. RO of CTLA4-Ig, a recombinant fusion protein targeting CD80 and CD86 receptors, was multiplexed to simultaneously measure target engagements for both receptors. Both RO methods demonstrated specificity of receptor measurements without cross-reactivity to each other in multiplexed formats. RO methods were used for evaluation of PD activity of Bs-Ab and CTLA4-Ig in cynomolgus monkeys. In both cases, RO results showed dose-dependent target engagement, corresponding well to the pharmacokinetics.


CONCLUSIONS
Multiplexed RO methods allowed accurate assessment of PD activity for Bs-Ab and CTLA4-Ig, facilitating development of these biopharmaceuticals from preclinical to clinical stages.
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Inna
I
Vainshtein
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Amy K
AK
Schneider
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Bo
B
Sun
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Martin
M
Schwickart
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Lorin K
LK
Roskos
Clinical Pharmacology & DMPK, Medimmune LLC, Mountain View, California, 94043.
Meina
M
Liang
D018033:Antibodies, Bispecific / Q000276:immunology* / Q000627:therapeutic use; D018122:B7-1 Antigen / Q000276:immunology / Q000627:therapeutic use; D015415:Biomarkers; D060908:CTLA-4 Antigen / Q000276:immunology; D066246:ErbB Receptors / Q000276:immunology* / Q000627:therapeutic use; D005434:Flow Cytometry; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology; D007166:Immunosuppressive Agents / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use; D017526:Receptor, IGF Type 1; D017451:Receptors, Somatomedin / Q000276:immunology* / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000627:therapeutic use
D018033:Antibodies, Bispecific; D018122:B7-1 Antigen; D015415:Biomarkers; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C000597236:IGF1R protein, human; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D017526:Receptor, IGF Type 1
CD80; CD86; IGF1R; biopharmaceutical; flow cytometry; pharmacodynamic biomarker; receptor occupancy