RBC-Specific CD47 Pruning Confers Protection and Underlies the Transient Anemia in Patients Treated with Anti-CD47 Antibody 5F9

CD47 is an anti-phagocytic (i.e. "don't eat me") signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential.

We previously established that administration of 5F9 clears a subset of aged RBCs, resulting in a compensatory reticulocytosis. This occurs transiently, predominantly after the first dose, and does not worsen with subsequent doses. These findings, aligned with the observations of Oldenborg et al., which established that removal of aged RBCs, is dependent on the balance between the accumulating pro-phagocytic signals and loss of inhibitory anti-phagocytic signals. We therefore hypothesized that an initial lower 5F9 "priming dose", sufficient to trigger clearance of aged RBCs, would yield an overall younger pool of RBCs that are less vulnerable to subsequent higher 5F9 therapeutic "maintenance doses". Indeed, this priming and maintenance dosing strategy has substantially mitigated the on-target anemia and has allowed for 5F9 treatment in multiple clinical trials and indications.

To further probe this observed selective RBC clearance, we employed a receptor occupancy assay to quantitate the CD47 expression on the cell surface of RBCs, leukocytes (WBCs), and acute myeloid leukemia (AML) blasts. We discovered that the priming dose of 5F9 not only triggered clearance of a subset of RBCs, but also resulted in a near complete loss of CD47 - a phenomenon we term CD47 pruning. This effect was RBC-specific, as WBCs and AML bone marrow (BM) blasts did not exhibit this same CD47 pruning (Fig 1).

To explore the functional consequence of these unexpected clinical findings, we investigated whether we could recapitulate them in a preclinical mouse model. Similar to the clinical setting, anti-mouse CD47 blocking Ab treatments exhibited a similar pruning effect and transient anemia. Using this model, we asked whether, in the context of anti-CD47 treatment, these pruned RBCs were protected compared to unpruned RBCs. We transfused fluorescently labeled RBCs from CD47 Ab-untreated and CD47-treated mice into animals receiving continuous anti-CD47 treatment. We observed that CD47-pruned RBCs exhibited a significantly longer half-life compared to unpruned RBCs, suggesting that CD47 pruning is protective for RBCs.

To distinguish whether this protection is cell-intrinsic or cell-extrinsic, we transfused fluorescently labeled RBCs into CD47 Ab-untreated and -treated mice and observed that CD47-pruned RBCs are rapidly cleared in untreated recipients, suggesting that this treatment-induced protection of RBCs is cell extrinsic and a host-based alteration. Through depletion of distinct immune subsets by chemical depletion, antibody depletion, splenectomy, and partial hepatectomies, we demonstrated that host immune effector cells (e.g. red-pulp macrophages, neutrophils, T cells etc.) were not necessary for this pruning phenomenon. Critically, we also found that this CD47 pruning and resulting tolerance was Fc-independent.

In order to understand whether these findings were generalizable to other anti-human CD47 blocking agents, we conducted similar studies in double humanized huCD47 and huSIRPa micewith additional agents and observed this same CD47 pruning and tolerance phenomenon. Thereby suggesting this represents a class-effect of this emerging potential therapy class. Notably, these clinical and pre-clinical findings have been RBC-specific and have not precluded anti-tumor efficacy. The loss of CD47 on RBCs after the priming dose also suggests that the potential risk of CD47 Ab-mediated RBC agglutination following subsequent maintenance dosing is substantially reduced.

Unlike prior RBC antigen modulation reports, to our knowledge, these findings represent a novel RBC antigen depletion phenomenon that is independent of the known RBC regulatory compartments, including the spleen, liver, major immune effectors cells, complement, and is critically Fc-independent. These findings provide fundamental insight into the mechanism underlying how anti-CD47 Abs are tolerated without impairing therapeutic efficacy.

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