A Functional Role for Spontaneously Occurring Natural Anti-Transthyretin Antibodies from Patients with Transthyretin Cardiac Amyloidosis

Transthyretin cardiac amyloidosis (ATTR-CA) is a progressive disease that results from the accumulation of transthyretin (TTR) fibrils in the extracellular space of the myocardium, leading to heart failure.1 The pathogenesis of ATTR-CA results from a reduction in cardiac compliance due to amyloid fibril deposition, but recent observations suggest that soluble TTR intermediate oligomers are toxic to cardiomyocytes and contribute to myocardial functional compromise.2 Recently, proof-of-mechanism and proof-of-concept have been established for the ability of monoclonal antibodies to induce Fc gamma-mediated clearance of extracellular beta-amyloid in patients with Alzheimer's disease.3 This led to the Food and Drug Administration approval of a drug that clears amyloid plaques and attenuates cognitive decline.3 In pre-clinical studies, several monoclonal antibodies have been shown to mediate Fc gamma-dependent clearance of aggregated TTR by macrophages in experimental models.4-6 Very recently, an initial phase I study demonstrated the safety of this approach and provided initial hints of efficacy in reducing imaging-related pathology via magnetic resonance imaging and scintigraphy.7 In a recent preliminary observation, antibodies binding to TTR were described for the first time in two patients with ATTR-CA, and were associated with spontaneous clinical recovery and regression of imaging-related findings.8 However, this intriguing finding has not yet established a mechanistic role for these naturally occurring antibodies in facilitating amyloid removal. We aimed to characterize in detail the spontaneously occurring purified antibodies to TTR oligomers and fibrils and test their related functional activities in vitro and in cellular and experimental models, supporting their potential involvement in the spontaneous regression of ATTR amyloidosis. A cohort of 13 patients with ATTR-CA was compared to a control group comprising 11 patients with heart failure in whom ATTR amyloidosis was ruled out. An ELISA based on coating plates with misfolded TTR (misTTR) protein was performed to determine antibody levels. Blood samples were defined as positive for anti-TTR antibodies when optical density values were a mean + 2 standard deviations of the median from control patients. After confirming the presence of anti-TTR antibodies in two positive patients, we performed affinity purification of the sera with beads conjugated with misTTR. The purified IgG was tested for reactivity to native, oligomeric, and aggregated TTR using ELISA and dot blot. Purified anti-TTR antibodies were evaluated for their ability to promote the uptake of fluorescently labelled misTTR by human macrophages.2 A cell viability/toxicity assay was conducted in cardiomyocytes to study the protective role of anti-TTR antibodies.2 Clearance of aggregated fluorescently labelled TTR extracted from human cardiac tissue in a murine ATTR plug model was assessed using fluorescent imaging.9 Clinical data for the two patients were retrospectively collected from the electronic medical records of the heart failure clinic, Kaplan Medical Center. In this study, we report the presence of high titers of anti-TTR antibodies in two male patients with ATTR-CA, aged 71 and 80 years. These patients were clinically classified as having New York Heart Association (NYHA) functional class III–IV. Both were diagnosed with ATTR-CA based on their clinical presentation, echocardiographic features of a hypertrophic phenotype, and 99mTc-pyrophosphate scintigraphy showing Perugini grade 3 uptake. Purified IgG autoantibodies from the patients were reactive to aggregated ATTR (Figure 1A). Purified anti-TTR IgG antibodies (Figure 1B–D) enhanced the uptake of misTTR by macrophages (Figure 1E) and attenuated misTTR-mediated cardiomyocyte toxicity (Figure 1F). Both patients derived anti-TTR antibodies exhibited in vivo clearance of fluorescently labelled cardiac-derived TTR using in vivo imaging (Figure 1G,H). Microscale thermophoresis did not reveal higher levels of misTTR in the sera of these two patients. During clinical follow-up, both patients with high anti-TTR antibodies exhibited clinical and echocardiographic improvements, evidenced by reductions in cardiac hypertrophy and improvements in ventricular diastolic parameters. The first patient, classified as NYHA functional class III at diagnosis, received optimal medical therapy for heart failure throughout follow-up, with gradual clinical improvement. Approximately 5 years into follow-up and 3 years post-diagnosis, disease-modifying treatment was initiated. By the end of follow-up, the patient showed marked clinical improvement, with moderate improvement to NYHA functional class I–II and stress echocardiography showing a peak exercise capacity of 6.2 METs. Echocardiographic evaluation revealed a reduction in left ventricular end-diastolic diameter (LVEDD) from 64 mm at baseline to 58 mm after 7 years of follow-up. Septal and posterior wall thickness decreased from 11 to 10 mm. Diastolic function improved, transitioning from grade III diastolic dysfunction with elevated filling pressures to grade I diastolic dysfunction. Additionally, the severity of mitral regurgitation improved by the end of follow-up. Despite these improvements, left ventricular systolic function remained severely impaired. The second patient remained in NYHA functional class III throughout the follow-up period but reported subjective clinical improvement and mildly increased exercise tolerance. Disease-modifying therapy was initiated but discontinued after no significant change in functional status, which remained at class III. Echocardiographic assessment showed a decrease in LVEDD from 58 mm to 50 mm, with a mild reduction in septal thickness from 16 to 15 mm. Although grade III diastolic dysfunction persisted, there was a notable reduction in the E/e' ratio. Herein, we identified, purified, and characterized IgG antibodies binding misfolded forms of TTR in two patients with ATTR-CA who exhibited spontaneous clinical and echocardiographic stabilization. This finding follows a recent preliminary observation showing the presence of these natural antibodies in improved ATTR-CA patients.8 We initially tested the nature of the target of these naturally occurring IgG antibodies and found that they bound soluble oligomers as well as mature TTR fibrils and exhibited cross-reactivity to native, non-modified TTR. This application of a wider epitope coverage for detection of anti-TTR antibodies explains the apparent difference in the occurrence of the antibodies between our study and the paper by Fontana et al.8 TTR undergoes a series of changes yielding soluble intermediates and aggregated TTR forms, both of which appear to possess functional and toxic properties.4 By assaying both misfolded forms of TTR, we were able to track a larger percentage of patients (2/13) that exhibited immune reactivity to the protein. Cumulative data suggest that while mature extracellular fibril infiltration of the myocardium results in reduced compliance and consequent heart failure with preserved ejection fraction, soluble oligomers induce cardiomyocyte toxicity.2 We have previously shown that monoclonal antibodies raised against misTTR induced Fc gamma-mediated uptake of aggregated TTR by macrophages and attenuated cardiomyocyte toxicity.4 These findings were corroborated by in vivo clearance of TTR plugs in experimental models.4, 9 We hypothesized that TTR-binding IgG antibodies from the two patients with clinical stabilization would possess functional properties explaining this potential causal relationship. This assumption is not necessarily obvious, as spontaneously occurring antibodies are much less specific than monoclonal antibodies in their epitope recognition and likely target various misfolded forms of TTR rather than a single sequence. Affinity-purified antibodies from the two patients promoted uptake of misTTR by macrophages, and Fc gamma blockade abolished this effect. These misTTR-binding antibodies also attenuated toxicity to cardiomyocytes induced by soluble TTR oligomers. When delivered in vivo, patient-derived anti-TTR IgG antibodies rapidly cleared subcutaneously implanted Matrigel plugs infiltrated with fluorescently labelled misTTR, suggesting their capability to induce amyloid debulking in vivo. Future clinical trials should focus on elucidating the precise molecular mechanisms by which antibodies facilitate the clearance of misTTR and confer protection against TTR-induced toxicity. This includes characterizing the specific epitopes recognized by these antibodies and delineating their interactions with immune effector cells, which remain inadequately understood. Furthermore, to enhance the robustness of comparative analyses, subsequent studies should include additional control groups, such as healthy individuals and patients with other subtypes of amyloidosis, alongside the heart failure cohort without ATTR amyloidosis. This broader control framework may yield more comprehensive insights into disease pathophysiology and therapeutic efficacy across different patient populations. This study is also limited by its small sample size (n = 13), which reflects the rarity of patients with high-titer anti-TTR antibodies and their clinical stabilization. Larger studies are necessary to validate these findings. In conclusion, we describe, for the first time, a protective functional role for a subset of natural IgG antibodies binding pathological forms of TTR in patients with clinically stabilized ATTR-CA. These antibodies appear to promote the clearance of misTTR and protect against its toxicity to cardiomyocytes in cellular systems. These findings are consistent with pre-clinical and preliminary clinical data showing that immunotherapy with monoclonal antibodies targeting misTTR results in clearance of extracellular TTR. Insights into the underlying epitopes targeted by these antibodies may refine the development of future immunotherapies for patients with ATTR-CA, given the functional role of these antibodies. Conflict of interest: none declared.