Development of a high-affinity antibody specific to the predominant λ light chain amyloid subtype for AL amyloidosis immunotherapy

Abstract

Abstract BACKGROUND Although current quadruplet anti-plasma cell treatments effectively eliminate the production of new amyloidogenic light chains (LCs) in AL amyloidosis, pre-existing fibrils remain unaffected and continue to affect organ function (Sanchorawala et al N Engl J Med 2024). Birtamimab (humanized 2A4), a fibril-targeting monoclonal antibody (mAb), showed clinical benefit in only a subset of patients enrolled in the phase 3 VITAL trial (NCT02312206) and did not meet primary endpoints in the subsequent AFFIRM-AL trial (NCT04973137). Another anti-fibril mAb, anselamimab (chimeric 11-1F4), similarly showed clinical benefit in only a subset of patients in the CARES phase 3 trials (NCT04512235, NCT04504825). Such mixed results are not unexpected: birtamimab was developed against serum amyloid A and anselamimab against κ LC amyloid, but 80% of AL amyloidosis cases involve λ LCs. To address this majority patient population, we developed a novel high-affinity monoclonal antibody specifically targeting λ LCs. METHODS AND RESULTS BALB/C mice were cross-immunized with both recombinant λ6 Wil fibril and patient-derived cardiac λ amyloid fibrils. Hybridoma clones were screened based on the binding affinities to both immunogens by ELISA assay. A novel antibody clone, 1F10 mAb, was identified with superior binding affinity for λ LC amyloid and no cross-reactivity to soluble κ or λ LCs. Cross-reactivity to κ4 Len fibril and soluble κ and λ LCs was also assessed by ELISA. 2A4 and 11-1F4 mouse mAbs were included as controls. 1F10 mAb demonstrated high-affinity binding to λ6 Wil fibril (EC50=7.42x10-10 M) and λ patient amyloid (3.11x10-8 M), in contrast to 11-1F4 mAb (EC50=1.24x10-7 and 5.51x10-7 M), and 2A4 (EC50= 2.3x10-6M and undetectable). The 1F10 mAb also exhibited moderate binding to κ4 Len fibrils (EC50=7.52x10-9 M). To confirm the amyloid-specific binding of the 1F10 mAb, we performed immunofluorescent staining on renal biopsies from patients with λ AL amyloidosis (n = 11). Antigen retrieval on paraffin slides was done using Pronase treatment (Nasr et al Kidney Int 2006). Co-staining was performed using FITC-anti-λ LC and 1F10 mAb with Cy3-anti-mouse IgG. Thioflavin T (ThT) staining was done on an adjacent section to verify amyloid deposits. The 1F10 mAb demonstrated strong colocalization with ThT and anti-λ LC signals in glomeruli, vasculature, and/or tubulointerstitium, confirming its high sensitivity and specificity for λ amyloid, as validated by expert pathologist review (M.S.). The λ amyloid fibril clearance efficacy of the 1F10 mAb was investigated by phagocytosis assay using RAW264.7 macrophages. pHrodo-red labeled λ6 Wil fibrils were incubated with mIgG, 1F10, 11-1F4 or 2A4 mAbs and then added to RAW264.7 cells. The phagocytosis rate was quantified based on the red fluorescence intensity reading after 3 hours. The 1F10 mAb exhibited superior phagocytic efficacy on λ6 Wil fibrils than both 11-1F4 and 2A4 (P<0.0001 by 2-way ANOVA). 1F10 in vivo λ amyloid clearance activity was assessed using a subcutaneous AL amyloidoma mouse model. Dylight755-labeled Wil fibrils were mixed with 20% Matrigel and subcutaneously injected into BALB/C mice (5mg/mouse). On day 2, the mice were randomized (n=5/group) and received intravenous injection of 11-1F4, 1F10, or mIgG2a isotype mAb (100ug/mouse) 3 times weekly. Amyloid clearance rate was monitored by in vivo fluorescence imaging. 1F10 induced significantly accelerated amyloid clearance compared to 11-1F4 and mIgG2a isotype mAb (1F10 vs IgG2a: P=0.022; 1F10 vs 11-1F4: P=0.038 by two-way ANOVA). In contrast, no difference was observed between 11-1F4 and mIgG2a. Next, the 1F10 mAb was successfully chimerized to a human IgG1 backbone (ch1F10 mAb). As expected, ch1F10 mAb retained the same binding affinities to λ fibrils, superior to ch11-1F4 mAb, and moderate binding to κ4 Len fibrils. Ch1F10 mAb also showed significantly higher phagocytic activity than ch11-1F4 mAb against λ6 Wil amyloid using human THP-1 macrophages (P<0.0001, one-way ANOVA). CONCLUSIONSWe developed the novel λ-light chain specific monoclonal antibody 1F10, which demonstrated high-affinity binding and clearance activity. Given the current lack of efficient fibril targeting therapies for this majority patient population, the 1F10 mAb represents a promising candidate for advancing subtype-specific precision immunotherapy in AL amyloidosis.