Harnessing phagocytes for amyloid clearance: Development of a novel bispecific phagocyte engager (LC Fibril x CD64) for AL amyloidosis Free

AL amyloidosis is an incurable plasma cell disorder where misfolded light chains (LC) form toxic fibrils that deposit in organs, leading to dysfunction and death. Current chemotherapies suppress new amyloidogenic LCs production by targeting clonal plasma cells but do not eliminate existing amyloid, which continue to damage organs. Anselamimab (ch11-1F4, CAEL101) is an AL amyloid fibril-specific antibody promoting amyloid clearance via antibody-dependent phagocytosis through binding to Fc receptor (FcR) on phagocyte (Ashfaque et al., 2025). Phase 3 trials showed meaningful clinical benefit in a patient subgroup but missed the primary endpoint overall. Unfortunately, the therapeutic efficacy of monoclonal antibodies is limited due to competition with abundant endogenous IgG (7-16 mg/mL in serum) for FcR binding on effector cells (Bauer-Smith et al., 2023). To circumvent this problem, we developed a bispecific phagocyte engager BiPE (LC fibril × CD64) that bypasses Fc competition by directly recruiting phagocytes through high-affinity FcγRI/CD64 binding to enhance amyloid clearance and improve outcomes in AL amyloidosis.

We engineered eight BiPE constructs by combining the scFv or Fab fragments of the anti-LC amyloid antibody ch11-1F4 with the anti-FcγRI/CD64 antibody H22, which binds phagocyte-specific hFcγRI/CD64 with high affinity at a site distinct from the Fc-binding domain. The constructs were produced in CHO cells and purified first with Protein A chromatography, then by size exclusion chromatography if needed. Top BiPE candidates were selected based on binding affinities to amyloid and FcγRI/CD64, and compared to the parental antibodies, ch11-1F4 and H22. One Fab-IgG format BiPE showed the highest binding to κ4 Len amyloid fibrils (EC₅₀= 2.68×10^-11 M), comparable to ch11-1F4 (EC₅₀= 1.69×10^-11 M) by ELISA. FcγRI/CD64 binding, assessed by SPR, showed the BiPE KD of 2.24×10^-10 M, also comparable to H22 (KD = 2.69×10^-10 M). In contrast, Fc-FcR-based binding of traditional hIgGs, hIgG1κ isotype control and ch11-1F4, was markedly lower (KDs = 2.70×10^-9 M and 1.23×10^-9 M, respectively).

Next, the phagocyte binding affinities were measured using flow cytometry assays after incubating the antibodies with human THP-1 cells. As expected, BiPE showed enhanced phagocyte binding affinity (EC₅₀= 2.12×10^-10 M) comparable to the parental H22 (EC₅₀= 4.09×10^-10 M), which is much higher than the traditional IgGs: hIgG1κ isotype control (EC₅₀= 7.74×10^-10 M) and ch11-1F4 (EC₅₀= 9.54×10^-10 M).

To verify distinct phagocyte-binding mechanisms and mimic physiological conditions with high endogenous IgG, we performed binding assays using THP-1 cells or human PBMCs in the presence of either Fc blocker or excess human IgG. Binding of ch11-1F4 and BiPE was assessed by flow cytometry using TAMRA-conjugated Len(1-22) peptide, the epitope recognized by 11-1F4 Fab arms. As expected, ch11-1F4 binding to both THP-1 cells and primary phagocytes was completely abrogated by Fc blocker and substantially reduced by IgG competition, consistent with Fc-mediated phagocyte engagement. In contrast, BiPE exhibited significantly stronger phagocyte binding that remained unaffected by Fc blockade or IgG competition, confirming its Fc-independent, high-affinity targeting of phagocytes and resistance to endogenous IgG.

BiPE's amyloid clearance activity was evaluated using a phagocytosis assay. pHrodo Red-labeled κ4-Len amyloid fibrils were pre-incubated with hIgG1 control, ch11-1F4, or BiPE, then added to THP-1 macrophages with or without Fc blocker or 20% human serum to mimic IgG competition. After 3 hours, red fluorescence was measured to indicate phagocytic uptake. As expected, ch11-1F4-induced phagocytosis was abolished by Fc blockade and substantially suppressed by serum IgG. In contrast, BiPE induced significantly enhanced phagocytosis that remained unaffected by Fc blocker or IgG competition. Similar results with primary human phagocytes supported BiPE's superior Fc-independent recruitment and amyloid clearance, resistant to serum IgG interference.

We developed a bispecific antibody (LC fibril × CD64) that harnesses phagocytes via Fc-independent binding, which is resistant to IgG competition. By overcoming a key limitation of traditional antibodies, this approach enhances amyloid clearance and offers strong therapeutic potential for AL amyloidosis, including those on concurrent monoclonal antibody therapies.