Personalizing Amyloidosis Therapy with Real Time PET Imaging of Fibril-Reactive Monoclonal Antibody Cael-101

Background AL amyloidosis is the most common type of systemic amyloidosis in western countries and has a poor prognosis, with a median survival of 12 to 18 months. Despite the improved prognosis gained by eliminating the offending plasma cell clone, mortality remains high due to multi-organ dysfunction caused by persistent, insoluble amyloid fibril deposits. The amyloid fibril-reactive murine monoclonal antibody 11-1F4 was designed to target amyloid deposits by directly binding to a conformational epitope present on human light-chain amyloid fibrils. The murine form of this antibody has demonstrated potential to bind amyloid in mice and humans (Blood. 2010 116: 2241) and to clear insoluble fibrils in mice with induced human AL amyloidomas, demonstrating the feasibility of using immunotherapy to elicit rapid destruction of amyloid fibrils. Of great translational importance, a chimeric form of 11-1F4 was produced (CAEL-101) and recently demonstrated therapeutic potential in an open-label, dose-escalation phase 1a/b study where 67% of patients with cardiac or renal amyloidosis demonstrated organ response. There was a statistically significant change in Global Longitudinal Strain with 9/10 patients showing improvement (p=0.004). Since we have shown that CAEL-101 successfully improved organ function, the overall goal of this work is for the first time to explore the diagnostic potential of CAEL-101 radiolabeled with a positron emitting radioisotope for systemic amyloidosis as well as to explore its use as a companion biomarker to stratify patients for CAEL-101 immunotherapy.

Methods We obtained human amyloid extracts from the heart (κ1), liver (κ1), spleen (λ1) and kidney (λ6). Lyophilized human amyloid extracts were suspended in 25ml of sterile PBS and homogenized for 3 minutes and centrifuged at 12,000g for 30 minutes. 100mg of the resulting pellet was resuspended in sterile saline. Balb/c mice were then injected subcutaneously with amyloid extract. For imaging experiments, cGMP grade CAEL-101 was radiolabeled with ¹²⁴I, a positron emitting radioisotope used for PET imaging, with the standard iodegen reaction. Approximately 5 days after human amyloid extract was implanted to form subcutaneous amyloidomas, animals were injected with 200μCi of [¹²⁴I]CAEL-101 and imaged 1 and 4 days post injection using an Inveon microPET scanner. SUVmax for amyloidomas and contralateral background were obtained by drawing regions of interest in the PMOD software package and calculating tumor-to-background (T:B) ratios at 1 and 4 days post tracer infusion.

Results We found that [¹²⁴I]CAEL-101 successfully imaged 100% of mice bearing human amyloid extracts (κ1, λ1 and λ6 subtypes derived from heart, liver, spleen, and kidney). Human amyloidomas were visualized at both at 1 and 4 days post tracer infusion, with significantly increasing T:B radio by day 4, as expected when imaging large molecular weight antibodies. T:B ratios ranged from 2.1 to 4.2 at 4 days. Mice implanted with κ subtypes demonstrated significantly better in vivo T:B ratios (4.1 +/- 0.20), compared to λ subtypes (2.8 +/- 0.46), although all amyloidomas exhibited T:B uptake > 2.1, which would be clinically significant.

Conclusions We have demonstrated for the first time the potential of using radiolabeled CAEL-101 as a companion diagnostic to image real-time targeting of human amyloidosis in vivo. This is highly translatable due to the fact that CAEL-101 has shown great promise in early stage clinical trials to clear insoluble amyloid plaques. Importantly, we successfully used PET imaging to visualize cardiac derived amyloid fibrils from AL amyloidosis patients. Therefore, we anticipate that dedicated gated cardiac PET/CT imaging of radiolabeled CAEL-101 will be successful at visualizing cardiac amyloid deposits in patients, especially with the rich blood flow in cardiac tissue and newer generation highly sensitive, high resolution digital PET scanners, in contrast to the non-cardiac optimized whole body scans used in prior studies with antibody-based PET. Given that we were able to image 100% of implanted human amyloidomas derived from heart, spleen, liver and kidney consisting of both κ and λ subtypes, we envision using CAEL-101 PET imaging to (1) diagnose systemic amyloidosis, (2) stratify patients for CAEL-101 immunotherapy, and (3) quantify peripheral organ amyloid fibril deposition pre and post anti-amyloid therapy.

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