Immunohematological test reagent for pre-analytical depletion of anti-CD47 antibody from patient plasma

Background

CD47 is an immune checkpoint that promotes tumor cell evasion as a "don"t eat me" signal. Consequently, anti-functional antibodies against CD47 are being tested as immune checkpoint inhibitors in a variety of malignant indications. Since, besides tumor cells, erythrocytes also express CD47, these antibodies cause pan-agglutination in the indirect antiglobulin test (IAT) with most available test systems, thus interfering with antibody screening and differentiation.

Methods

Using lentiviral vectors, the murine suspension cell MEL was engineered to express CD47 at a density of approximately 700,000 molecules per cell. These cells will henceforth be referred to as "Magrosorb". Patient plasma was spiked with the anti-CD47 antibody Magrolimab, then subjected to pre-incubation as indicated, subsequently co-incubated with test erythrocytes in LISS buffer on conventional gel agglutination cards. Shown left to right are untreated plasma, plasma treated with parental cells (not CD47-transduced) and Magrosorb cells. Negativity of the right-most plasma for anti-RBC immune antibodies is apparent (Figure 1).

Results

Human plasma spiked with Magrolimab, currently the leading candidate as clinical CD47 antagonist resulted in 4+ positive reactions during antibody screening on conventional gel agglutination cards. Incubation of 1x107 Magrosorb cells in 25 µl of Magrolimab-spiked plasma and subsequent removal of Magrosorb cells by centrifugation depleted Magrolimab, resulting in a cleanly negative antibody screening test. In selected spiked plasma from antibody patients, these blood group antibodies became readily specifically detectable. We also demonstrate the possibility to stabilize the cells in fixating buffers, providing the possibility of intermittent batch production and distribution of test cells.

Conclusion

Medicinal antibodies causing pan-agglutination of test erythrocytes and thus obscure diagnostics provide a novel challenge for immunohematology testing. High antigen expression on non-human cells allows specific depletion of these medicinal antibodies from patient plasma without interfering with underlying specific immune antibodies. The system provides a readily adaptable tool for other medicinal or immune antibodies.

Offenlegung Interessenkonflikt:

There is no conflict of interest.