(PA-028) Development of anti-AL amyloidosis CAR Phagocyte Therapy

Current anti-plasma cells therapies for AL amyloidosis reduce new fibril formation but do not remove existing deposits, which continue to damage organs. CAEL-101(c11-1F4), a fibril-specific monoclonal antibody in Phase 3A/B trials, promotes amyloid clearance via phagocytosis. However, the use of anti-CD38 antibodies in AL amyloidosis treatment and the recent failure of Birtamimab raised concerns that antibody-dependent phagocytosis for amyloid clearance may be impaired by high serum levels of anti-CD38 antibodies competing for Fc receptor binding. To address this, we developed a first-in-class human CAR phagocyte therapy based on 11-1F4 that directly targets amyloid deposits, bypassing Fc competition and enhancing amyloid clearance.

Methods:

The anti-AL amyloid CAR lentiviral construct was designed to include 11-1F4 scFv and FcRγ signaling domain with GFP tag. Irrelevant CD19-CAR-GFP or empty G4S-GFP construct (with scFv deleted) were used as controls. Transduced RAW264.7 or human CD34⁺ cells were sorted by GFP⁺ flow. Human CAR-phagocytes were then differentiated from the transduced CD34⁺ cells using M-CSF. CAR-P phagocytic activity was assessed using pHrodo Red-labeled AL amyloid fibrils, visualized by fluorescence microscopy. For in vivo assay, Dylight 755-labeled Len fibrils (5 mg/mouse) and luciferase-expressing 11-1F4-CAR or control phagocytes (2 × 10⁷ cells/mouse) were s.c. co-injected into NSG-SGM3 mice (N=5/group). Amyloid clearance and CAR cell persistence were tracked using fluorescence and BLI.

Results:

11-1F4-CAR–transduced RAW264.7 macrophages exhibited significantly enhanced phagocytic activity against λ6 Wil and κ4 Len amyloid fibrils compared to controls (****P < 0.0001). To support clinical translation and scalable manufacturing, we utilized human CD34⁺ hematopoietic stem cells for lentiviral CAR-transduction and achieved a transduction rate of > 90%. The cells were expanded ex vivo and then differentiated into CAR phagocytes with M-CSF. Flow cytometry analysis confirmed expression of characteristic phagocyte markers, including CD14, CD16, CD64, CD68, CD86, CD206, CD11c, and HLA-DR. In phagocytosis assays, CD34⁺-derived 11-1F4-CAR phagocytes showed significantly increased engulfment of λ6 Wil and κ4 Len fibrils compared to the controls (****P < 0.0001).

For in vivo evaluation, Dylight 755-labeled Len fibrils were co-injected with luciferase-expressing CAR phagocytes into NSG-SGM3 mice. BLI imaging confirmed CAR phagocyte persistence for up to 5 weeks. Co-injection with 11-1F4-CAR phagocytes significantly accelerated amyloid clearance compared to controls (*P < 0.05), reducing the median clearance time from 7.1 days (control) to 4.8 days, supporting their therapeutic potential.

Conclusions:

We developed the first-in-class anti-AL amyloidosis CAR phagocytes using CD34⁺ cells and confirmed their amyloid-targeting activities both in vitro and in vivo. By bypassing serum IgG Fc competition, CAR phagocytes represent a promising novel cell therapy for AL amyloidosis.