Background PD-1/PD-L1 blockade can significantly improve survival across many types of cancer, but only in a minority of patients. To broaden its therapeutic efficacy, several combination partners are now being evaluated together with PD-1/PD-L1 blockade. Agents blocking CD47/SIRPα innate immune checkpoint are one such example, and co-targeting PD-1/PD-L1 and CD47 with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling preferential inhibition of CD47 on PD-L1-positive cells are being tested as an alternative approach. We compare here two distinct bsAbs, based on a common PD-L1 antibody arm, with differing FcgR-enabling effector functions and CD47-binding arm affinities.

Methods An array of fully human bsAbs associating a high affinity PD-L1 arm to CD47 arms with varying affinities were generated using the κλ-body platform.¹ CD47xPD-L1 bsAbs of human IgG1 isotype (CD47 low affinities) or IgG4 isotype (CD47 high affinities) were screened in various binding assays (including to red blood cells (RBC)) and in receptor-blocking assays, and then tested for their Fc-mediated killing and T-cell activation activity (SEA-stimulated PBMC assay). Selected molecules were evaluated in vivo.

Results Both bsAb approaches demonstrated strong blockade of PD-1/PD-L1 interaction and significantly enhanced T-cell activation in vitro. CD47lowxPD-L1 IgG1 bsAbs did not bind to RBC and showed PD-L1-guided inhibition of CD47. ADCP and ADCC experiments with a panel of tumor cell lines expressing various target levels showed superior killing activity with CD47lowxPD-L1 IgG1 bsAbs as compared to the anti-PD-L1 IgG1 mAb, avelumab. On the other hand, CD47highxPD-L1 IgG4 bsAbs showed residual RBC binding and PD-L1-independent blocking of CD47/SIRPα. These CD47high IgG4 bsAbs were able to enhance the anti-tumor activity of anti-tumor-associated antigen (TAA) mAbs in vitro (phagocytosis), and in vivo (Raji lymphoma xenograft model). In addition, anti-tumor activity of mouse CD47xPD-L1 bsAbs in a syngeneic MC38 colon carcinoma model was demonstrated.

Conclusions With the objective of finding the optimal CD47xPD-L1 bsAb design, two approaches targeting CD47 and PD-L1 inhibition were tested. Both the CD47lowxPD-L1 IgG1 bsAbs and CD47highxPD-L1 IgG4 bsAbs were able to mediate enhanced antitumor responses, the former as a standalone treatment, the latter in conjunction with an anti-TAA mAb. To further characterize the CD47lowxPD-L1 and CD47highxPD-L1 bsAbs, lead candidates will be tested in PK and tolerability studies in non-human primates.

References

1. Fischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun 2015 May;6(1):6113.