(M1530-02-12) Characterizing Interactions of RO7297089, an Anti-B-Cell Maturation Antigen (BCMA)/CD16A Bispecific Tetravalent Antibody with Shed Soluble CD16A Antigen in Plasma In Vitro

Purpose: RO7297089 is an anti-B-cell maturation antigen (BCMA)/ anti-CD16A bispecific tetravalent antibody under development as a potential therapeutic to treat multiple myeloma. Engagement of both BCMA on malignant plasma cells and CD16A on NK cells triggers NK cell-mediated killing of BCMA-expressing malignant plasma cells, a process known as antibody-dependent cellular cytotoxicity (ADCC) (Cai et al. 2022).  However, shed antigens (both BCMA and CD16A) in plasma could form complexes with RO7297089 which may prevent the alignment of the two targeted cells, as such, impacting PK and efficacy.  To better understand the interaction of RO7297089 with the shed targets, we have investigated the complex formation of RO7297089 with CD16A soluble antigen in animal and human plasma using the radiolabeling approach.

Methods: Anti-BCMA antibody, as control, and anti-BCMA/anti-CD16A bispecific tetravalent antibody were first radiolabeled with iodine-125, respectively. To determine the specificity of binding to soluble CD16A, the radiolabeled antibodies were incubated with and without soluble CD16A in PBS. Next, the stoichiometry of the binding was characterized by titrating the soluble CD16A with anti-BCMA/anti-CD16A. Finally, the radiolabeled anti-BCMA/anti-CD16A was incubated in mouse, Cynomolgus monkey (Cyno), and human plasma to determine the complex formation with endogenous soluble CD16A in plasma. All samples from the experiments were analyzed for any biotransformation using size-exclusion high-performance liquid chromatography (SE-HPLC) with an in-line radio-detector.

Results: The SE-HPLC analysis showed that anti-BCMA has a retention time of 16.5 minutes and anti-BCMA/anti-CD16A has a retention time of 15.7 minutes in the absence of soluble CD16A in PBS.  Incubation of [¹²⁵I]-anti-BCMA with spiked soluble CD16A antigen in PBS did not show any shift whereas incubation of [¹²⁵I]-anti-BCMA/anti-CD16A with soluble CD16A antigen revealed a complete shift of retention time from 15.7 minute to 14.0 minutes, suggesting the complex formation between anti-BCMA/anti-CD16A and soluble CD16A antigen but not for anti-BCMA antibody.  The titration of soluble CD16A antigen and anti-BCMA/ant-CD16A displayed a 2:1 ratio for CD16A binding to anti-BCMA/ant-CD16A.  Finally, incubation of [¹²⁵I]-anti-BCMA/anti-CD16A in mouse plasma (non-binding species) did not show any complex formation; in contrast, incubation of [¹²⁵I]-anti-BCMA/anti-CD16A in human and cyno plasma (binding species) revealed the complex formation with the elution time corresponding to soluble CD16A complex formation.  It was estimated that healthy human plasma appears to have 7.5 to 22.5 nM of shed CD16A antigen based on the 2:1 antigen to antibody ratio.

Conclusion: In summary, we have characterized the interactions of anti-BCMA/anti-CD16A tetravalent bispecific antibody with soluble CD16A antigen using radiolabeling approach, and demonstrated that anti-BCMA/anti-CD16A could form complex with CD16A at 1:2 ratio. Furthermore, anti-BCMA/anti-CD16A could form complex with endogenous CD16A in cyno and human plasma (cross binding) but not in mouse plasma (non-binding). These data imply that the shed soluble antigen may interfere with therapeutic antibody’s activity and PK due to the complex formation. Therefore, it should be taken into consideration for such interference for drug development.

References: Cai H, Kakiuchi-Kiyota S, Hendricks R, Zhong S, Liu L, Adedeji AO, Chan P, Schutten MM, Kamath AV, Ovacik MA. Nonclinical Pharmacokinetics, Pharmacodynamics, and Translational Model of RO7297089, A Novel Anti-BCMA/CD16A Bispecific Tetravalent Antibody for the Treatment of Multiple Myeloma. AAPS J. 2022 Sep 20;24(6):100. doi: 10.1208/s12248-022-00744-8.