Modified Anti-PstS1 Bi-specific antibodies unlock potent protection against tuberculosis.

The role of antibodies in the host response against Mycobacterium tuberculosis (M. tb) bacteria is still poorly understood. We previously isolated two monoclonal antibodies (mAbs), p4-36 and p4-163, from an M. tb infected donor that target two non-overlapping epitopes on PstS1, a subunit of the M. tb phosphate transporter. Although these antibodies reduced lung bacterial burden in mice (30-40% reduction in CFU), their efficacy remained modest for therapeutic application. Here, we employed a rational antibody engineering approach to further enhance their anti-M. tb potency. Affinity maturation of p4-163 yielded p4-163LR, a variant with superior binding to PstS1 and improved recognition of live, attenuated M. tb. Surprisingly, p4-163LR alone did not confer enhanced protection against virulent M. tb in vivo. However, the generation of a bispecific antibody combining p4-36 and p4-163LR (Bi-S 36/163LR) significantly improved bacterial binding and antibody-dependent cellular phagocytosis (ADCP). Notably, prophylactic administration of Bi-S 36/163LR led to a ~ 1 log reduction in lung bacterial burden compared to control animals treated with isotype control. These findings define a novel, structure-guided strategy to amplify the functional capacity of natural anti-M. tb antibodies and highlight bispecific antibody platforms as promising candidates for host-directed tuberculosis immunotherapy.