Development of Anti-Amyloid 11-1F4- CAR Phagocytes for Treatment of AL Amyloidosis

Introduction

Light chain (AL) amyloidosis is an incurable, rare monoclonal plasma cell dyscrasia closely related to multiple myeloma. Clonal plasma cells produce excessive misfolded monoclonal immunoglobulin light chains, which form extracellular insoluble fibrils, causing functional damage in organs and eventually leading to death. Current treatments target clonal plasma cells to suppress the synthesis of new amyloidogenic light chains; however, existing fibrillar deposits remain unaffected, continuing to cause persistent organ damage (Merlini et al, 2018). Anselamimab (11-1F4, CAEL-101) is an AL amyloid fibril specific monoclonal antibody currently being tested in phase 3 clinical trials, which triggers amyloid clearance via phagocytosis. To further improve AL amyloid clearance efficacy, we designed a first-in-class human anti-AL amyloidosis CAR macrophage therapy expressing 11-1F4 scFv.

Methods and Results

The first-generation anti-amyloid CAR lentiviral construct was designed to consist of an extracellular domain from 11-1F4 scFv sequences and an intracellular FcR gamma signaling domain with a C-terminal GFP tag. Mouse macrophage cell line RAW264.7 or mouse bone marrow derived macrophages (BMDMs) were transduced with either 11-1F4-CAR-GFP or GFP-CAAX control (Morrissey et al, 2018). The cell surface expression of 11-1F4-CAR-GFP fusion protein was confirmed under fluorescence microscope. To assess the phagocytosis efficacy of AL amyloid, recombinant amyloidogenic lambda 6 light chain Wil-fibril was covalently labeled with pH-sensitive fluorescence dye pHrodo red-STP ester, which only fluoresces in the acidic microenvironment of the phagolysosome. After 1-4 hours of incubation with GFP-CAAX control or 11-1F4-CAR-GFP transduced RAW264.7 cells or BMDMs, the phagocytosis of amyloid was assessed by red fluorescence signal for acidification of pHrodo red-labeled Wil fibrils under fluorescence microscope. 11-1F4-CAR-GFP macrophages exhibited significantly increased phagocytic activity compared to the GFP-CAAX control cells (**P<0.01).

Gene editing in human primary macrophages is challenging, and macrophages do not expand, making the production of sufficient CAR-transduced macrophages for in vivo study and future therapies inefficient. Therefore, we utilized lentiviral transduction of human CD34⁺ hematopoietic stem cells, achieving over 90% efficacy. Transduced cells were expanded ex vivo by 3 orders of magnitude over a period of 3 weeks before differentiating into phagocytes upon stimulation with M-CSF. Flow cytometry analysis of the differentiated cells revealed a typical monocytic phenotype, including CD45, CD33, CD14, CD64, and CD172a expressions. In vitro amyloid phagocytosis assay using GFP-CAAX vs 11-1F4-CAR-GFP CD34⁺ derived phagocytes confirmed that 11-1F4-CAR significantly increased AL amyloid phagocytosis(**P<0.01).

We next investigated the in vivo amyloid targeting activity of 11-1F4-CAR CD34⁺- derived macrophages using a humanized AL amyoidoma mouse model. Firefly luciferase co-transduced 11-1F4-CAR-GFP or GFP-CAAX control macrophages were i.p.injected into NSG-SGM3 mice (N=5 per group). Wil-amyloid-fibrils were labeled with DyLight 755 fluorescence dye and i.p.injected into both groups (5 mg per mouse). The amyloid burden was monitored by in vivo fluorescence imaging. The distribution and persistence of the human macrophages in vivo were monitored by bioluminescence imaging (BLI). Dual fluorescence and BLI imaging data demonstrated the co-localization of human macrophages with amyloid, indicating successful AL amyloid targeting in vivo. The persistent in vivo survival of human macrophages was confirmed by BLI imaging for up to 3 weeks until the mice were euthanized. Dual imaging of dissected amyloid nodules and tissues showed that both GFP-CAAX control and 11-1F4-CAR-GFP macrophages were recruited to the amyloidoma nodules, with higher amyloid recruitment efficacy of 11-1F-CAR-GFP macrophages compared to the control CAR-GFP macrophages.

Conclusions

We developed first-in-class anti-amyloidosis human CAR macrophages using CD34⁺ hematopoietic stem cells and confirmed their amyloid-targeting activities both in vitro and in vivo. Our data suggest that anti-AL amyloid CAR macrophages could be a promising novel cell therapy for AL amyloidosis.

Li:Bristol Myers Squibb: Current Employment. Mapara:CRISPR/Vertex: Consultancy; Incyte: Consultancy; bluebird bio: Consultancy; Caelum: Current holder of stock options in a privately-held company; Ossium: Consultancy. Lentzsch:Caelum Bioscience: Patents & Royalties: CAEL-101; Pfizer: Other: Advisory Board; Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Other: Advisory Board; Sanofi: Other: Advisory Board, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Other: Advisory Board; Takeda: Other: Advisory Board; PeerView: Honoraria; Clinical Care Options (CCO): Honoraria; RedMed: Honoraria; Bio Ascend: Honoraria; Medscape: Honoraria; Sanofi: Other: Advisory Board, Research Funding; Zentalis: Research Funding.