Development of Novel Cis-Receptor Occupancy Method for Bispecific Antibodies

Introduction: Johnson & Johnson Innovative Medicine developed a bispecific antibody (BsAb) targeting receptors X and Y that blocks multiple B cell signaling pathways through tethering X and Y together on the membrane of the same cell. Connecting the two receptors is necessary to unlock anti-X x anti-Y’s novel functionalities to treat a variety of B cell mediated autoimmune diseases. However, traditional RO methods cannot differentiate each receptor’s occupancy in this case. A cisRO assay was developed to assess the receptor occupancy of this BsAb on receptors X and Y individually on a given cell.

Methods: We carefully screened the optimal clone and/or dose of recombinant proteins and anti-idiotype (ID) for both arms of the BsAb, as well as anti-human IgG (hIgG) using wild type (WT), X or Y knockout (KO) CARNAVAL cell lines and human whole blood. With the availability of these tools, we designed a 6-tube (see below) flow cytometry assay:

Tube 1 (T1): spiked with BsAb and detected with anti-hIgG to measure receptors (X and/or Y) occupied by BsAb (double or single bound).

Tube 2 (T2): spiked with BsAb and detected with anti-X’s anti-ID to measure Y receptor occupied by single bound BsAb.

Tube 3 (T3): spiked with BsAb and detected with anti-Y’s anti-ID to measure X receptor occupied by single bound BsAb.

Tube 4 (T4): saturated with excess BsAb and detected with anti-hIgG to measure total X and Y receptors occupied by double or single bound BsAb.

Tube 5 (T5): saturated with excess BsAb and detected with anti-X’s anti-ID to measure Y receptor occupied by single bound BsAb.

Tube 6 (T6): saturated with excess BsAb and detected wit anti-Y’s anti-ID to measure X receptor occupied by single bound BsAb.

Using data from the 6 tubes above and a series of calculation, we can evaluate percentage of bound receptors, percentage of bound receptors occupied by double bound BsAb, and percentage of bound X or Y receptor occupied by double bound BsAb.

Results: In vitro assessment using human whole blood was done to detect the binding of receptors X and Y on B cells. At saturating concentration, all X and Y receptors are occupied by BsAb. About 50% of X receptors are occupied by BsAb that is also bound to Y; the rest of X receptors are occupied by single bound BsAb. Almost all the Y receptors are occupied by double bound BsAb. BsAbs are either double bound or X single bound.

Conclusion: With this 6-tube cisRO strategy, we can detect cisRO for same cell targeted BsAb and provide precise information for further PK PD analysis.