Rajshekhar Chakraborty & Suzanne Lentzsch

Abstract
Introduction: Systemic AL amyloidosis is a protein-misfolding disorder that is characterized by the deposition of insoluble amyloid fibrils derived from kinetically unstable light chains. Achieving a rapid and deep hematologic response is critical for long-term survival. Areas covered: This review covers the existing and emerging treatment options for systemic AL, divided into anti-plasma cell and fibril-directed therapies. The anti-CD38 monoclonal antibody daratumumab has demonstrated an unprecedented hematologic response rate and will become the new standard-of-care in newly diagnosed patients in combination with CyBorD/VCD. Other plasma cell-directed drugs that have prospective data on safety and efficacy in AL include proteasome inhibitors [bortezomiband ixazomib], immunomodulatory drugs [lenalidomide and pomalidomide], and alkylating agents [melphalan and bendamustine]. A major unmet need is the development of fibril- directed therapies with the goal of eliminating amyloid fibrils that are already deposited in vital organs. Expert opinion: The treatment of newly diagnosed AL in the future will likely include daratumumab-based therapy in conjunction with fibril-directed therapy. The most promising second line drugs are venetoclax [for t(11;14)] and pomalidomide, with several others in the pipeline, including antibody-drug conjugates. Minimal residual disease will emerge as a new endpoint for drug development and will potentially guide treatment duration.

Keywords: Amyloidosis, AL amyloidosis, Daratumumab, Venetoclax, CAEL-101

1. Background
Systemic immunoglobulin light chain amyloidosis [AL Amyloidosis] is a disorder of protein misfolding that is characterized by the deposition of fibrillar protein aggregates derived from immunoglobulin light chains. The amyloidogenic protein [i.e. light chain] typically circulates in the blood and deposits as highly organized protein fibrils in one or more target organs[1]. The protein deposits in amyloidosis are characterized by cross-口 fiber diffraction pattern on X-rays and apple-green birefringence on Congo red staining under polarized light[1-3]. The amyloidogenic protein originates from monoclonal plasma cells in the bone marrow that produces kinetically unstable light chain due to somatic mutations in the light chain variable region[4-6]. This further leads tomisfolding and improper aggregation of light chain, which, together with interaction with the microenvironment, leads to initial nucleation of deposits and oligomer formation. The oligomers serve as a template that eventually helps in the formation of highly organized amyloid fibrils and leads to end organ damage by physical replacement of tissue parenchyma as well as direct cytotoxicity from amyloid deposits[3]. AL is the most common amyloidosis subtype, with an expected approximately 4000 new cases per year in the United States[7,8]. A recent study from the UK National Amyloidosis Center demonstrated an increase in the number of cases by 670% between 1987-99 and 2010-19 time-periods, which the authors attributed to increased awareness, better diagnostics, and advanced cardiac imaging[9]. AL is a diagnosis of the elderly, with the median age at diagnosis being 76 years among Caucasians[8]. However, other studies have shown a mean age of 64 years [±13] among incident cases[10]. The overall survival has improved over the last two decades in both transplant-eligible and transplant-ineligible population due to an expanding therapeutic armamentarium for plasma cell disorders[11]. However, early mortality in patients with advanced cardiac involvement remains a major challenge, with approximately one- fourth of patients succumbing within 6 months of diagnosis[11].

2. Medical need
The major unmet need in AL amyloidosis management is in the following areas:Frontline treatment regimens that can achieve an extremely rapid and deep hematologic response [preferably minimal residual disease [MRD]-negative] in a large proportion of patients,Identify optimal tools for MRD assessment, that can be potentially used for tailoring therapy,Develop safe and effective fibril-directed therapies to target the amyloid protein already deposited in tissues,Decrease early mortality in patients with advanced cardiac involvement,Develop new treatment options for relapsed/refractory AL amyloidosis,Develop biomarker-directed therapies for specific subsets [e.g. BH-3 mimetics for t(11;14)] of AL patients,Determine the role of upfront high dose melphalan-autologous hematopoietic cell transplantation [HDM-AHCT] in eligible patients in the era of daratumumab-
based frontline therapy.

3. Existing paradigm
The two essential pillars of AL amyloidosis management are treatment directed towards the monoclonal plasma cell clone to halt production of amyloidogenic light chain and supportive care by a multidisciplinary team. Historically, the alkylating agent melphalan was one of the first drugs shown to be effective in the treatment of AL amyloidosis in a randomized clinical trial [RCT], with the median overall survival [OS] being 18 months and 5-year OS being 15% with melphalan-prednisone[12]. Another Italian study on oral melphalan and dexamethasone [Mel/Dex] led to a hematologic overall response rate [ORR] of approximately 70% and median OS of 5 years[13]. Patients achieving a hematologic response at 6-month landmark analysis had a significantly superior OS compared to non-responders[13]. Subsequently, an RCT comparing Mel/Dex with High Dose Melphalan-Autologous Hematopoietic Cell Transplantation [HDM-AHCT] demonstrated a significantly worse median OS with the latter [57 vs 22 months; p=0.04] due to a high transplant-related mortality [TRM] at 24%[14], highlighting the importance of careful patientselection. With increased experience and better identification of factors predicting TRM, it has currently decreased to less than 5%, especially at high-volume centers[15,16]. Furthermore, data from the Center for International Blood and Marrow Transplantation Research showed a hematologic complete response [CR] in 37% and renal organ response in 32% of patients transplanted in the most recent cohort[16]. Long-term follow up data from the Mayo Clinic showed a median OS of greater than 10 years with HDM-AHCT, with conditioning dose, Mayo stage, and depth of hematologic response being independent predictors of survival[15]. However, approximately 80% of patients with systemic AL amyloidosis are transplant-ineligible[7], which speaks to the need for effective non-intensive plasma cell-directed therapies.

The first-generation PI bortezomib currently forms the backbone of frontline therapy in AL amyloidosis. Bortezomib is administered in combination with cyclophosphamide and dexamethasone [CyBorD], based on several reports showing a hematologic ORR of 60- 94%[17,18]. To our knowledge, outcomes of the largest cohort of AL patients treated with frontline CyBorD was recently published by the UK National Amyloidosis Center [n=915], which demonstrated a ≥VGPR rate of approximately 50% and a median OS of 6 years[19]. Cardiac and renal response was noted in 33% and 15% of patients respectively. Notably, among transplant-eligible patients who received frontline CyBorD instead, 78% were alive at 5 years, which is comparable to a 5-year OS of 77% with upfront HDM-AHCT in the CIBMTR study[16]. Furthermore, in patients undergoing upfront HDM-AHCT, administration of a bortezomib-based induction therapy leads to a lower relapse risk and improved PFS at 2-years compared to no induction therapy[20]. Hence, bortezomib-based induction therapy is currently the standard-of-care for most AL patients irrespective of transplant eligibility, with selected patients proceeding to HDM-AHCT for consolidation. Till date, there is no data on maintenance therapy in AL amyloidosis. There are currently no FDA-approved therapies or universally accepted standard-of-care for relapsed/refractory AL amyloidosis. Drugs which are used off-label in this setting are ixazomib, lenalidomide, daratumumab, pomalidomide, and venetoclax [especially fort(11;14)]. Several treatments are emerging in this space which will be discussed in a separate section below.

4.Market review
The global market size for amyloidosis treatment was valued at US$3.6 billion in 2017, with a projected annual growth of approximately 7%[21]. Data from the US has showed an approximately three-fold increase in prevalence of AL amyloidosis between 2007 and 2015, likely due to improving frontline therapy[10]. Since one-third of AL survivors will eventually relapse and need subsequent lines of therapy[22,23], the global market share of AL therapeutics will likely increase in future. The drug pipeline for multiple myeloma, from where most AL treatments are derived, is replete with multiple effective agents including antibody-drug conjugates, BH-3 mimetics, and bispecific T-cell engagers, with the sales expected to double by 2027[24]. Furthermore, with development of prominent fibril-directed therapies, the market share of AL may further expand as several patients achieve a suboptimal organ response and may be candidate for prolonged fibril-directed therapy to improve organ function and potentially quality of life. However, the future of fibril directed therapies will depend upon the success of the randomized trial of CAEL-101. It is worth noting that RCTs of other fibril-directed therapies, such as NEOD001 and green tea extract, were negative. The sales will also be driven in the near future by increased use of frontline daratumumaband several novel agents at relapse,including venetoclax, pomalidomide, ixazomib, and novel immunotherapy including antibody-drug conjugates and bi-specific T-cell engagers. The key players in this area are Janssen, Takeda, Caelum Biosciences, Genentech, Abbvie, Prothena, and Bristol-Myers-Squibb.

5. Current research goals
The current research goals in AL amyloidosis are the following:Establish the best frontline therapy for AL amyloidosis, both in transplant eligible and transplant ineligible patients,Investigate the prognostic impact of t[11;14] in the era of anti-CD38 antibody-based frontline therapy and role of venetoclax in this subgroup which comprises more than half of AL patients,Investigate whether HDM-AHCT needed for all eligible patients in AL amyloidosis, especially with a high rate of deep responses seen with daratumumab-based frontline therapy,Conduct RCTs to determine the best therapy for early relapse in AL patients,Conduct RCTs of fibril-directed therapies,Identify the best method of MRD measurement in AL amyloidosis, prognostic role of MRD, and using MRD as a tool to tailor therap,Investigate the quality of life trajectory and late effects in AL amyloidosis, which is especially important due to a growing number of long-term survivors[25].

6. Scientific rationale
The production of amyloid deposits is divided into three distinct phases and represents an S-shaped growth curve[3]. It includes an initial lag phase which is the flat part of the growth curve, followed by log-phase where high concentrations of pre-formed misfolded fibrils can lead to rapid nucleation and accelerated amyloid formation, and finally the elongation phase, where elongation of amyloid fibrils can continue despite a very low concentration of the precursor protein[3]. This growth curve has two key clinical implications in the treatment and new drug development for AL amyloidosis. First, early diagnosis and treatment initiation is critical before significant deposition of amyloid in vital organs during the log-phase. Second, rapid and deep hematologic response is the key to improve disease outcomes, since amyloid deposition can continue even at a relatively low concentration of abnormal light chains in the elongation phase. Hence, the proximal goal of initial therapy in AL amyloidosis is to rapidly achieve at least a very good partial response [VGPR] and preferably a complete response [CR]. With the availability of several tools to monitor minimal residual disease [MRD] such as multiparameter flow cytometry, next generation sequencing, and mass spectrometry, achievement of MRD-negativity may emerge as a new endpoint for drug development in future.

7. Competitive environment and emerging drugs
A major unmet need in systemic AL amyloidosis is development of highly efficacious non-intensive plasma cell-directed therapies as well as fibril-directed therapies to restore organ function. Several drug classes including newer generation proteasome inhibitors [PIs], immunomodulatory drugs [IMiDs], monoclonal antibodies, standard-dose alkylating agents, and BH-3 mimetics are currently emerging in this space and will be discussed below. Combined targeting of the plasma cell clone and fibrillar amyloid deposits by a combination of monoclonal antibodies has the potential to improve outcomes in future[26]. We will also highlight the shortcomings of currently available therapies and how some of the emerging treatments will address the unmet need in this population. Specific promising compounds currently in development are summarized in Table 1. Data for this table has been obtained from clinicaltrials.gov.

7.1 Plasma cell-directed therapies
7.1.1 Proteasome inhibitors
One of the major toxicities of bortezomib is neuropathy, which can be especially problematic in AL patients with nerve involvement. Hence, newer PIs such as ixazomib and carfilzomib has been tested in this area. The results of published prospective clinical trials of emerging agents is summarized in Table 2 [for all drugs except daratumumab] and Table 3 [for daratumumab].

7.1.1.1 Ixazomib
Ixazomib is an orally bioavailable PI that is currently FDA-approved for the treatment of relapsed/refractory MM and maintenance therapy in MM. The advantages of ixazomib over bortezomib are oral route of administration and lack of significant neurotoxicity. A phase 1/2 trial of ixazomib in relapsed/refractory AL amyloidosis demonstrated a ≥VGPR rate of 43% at the maximal tolerated dose of 4.0 mg weekly, which included CR in 10% and VGPR in 33% of patients[27]. The time to first and best hematologic response was 1.8 and 2.7 months respectively. The median duration of hematologic response was approximately 2 years, with the duration being greater in PI-naïve compared to PI-exposed patients. Common adverse events with ixazomib are gastrointestinal toxicities such as nausea and diarrhea, skin toxicities, and fatigue. Among patients with heart or kidney involvement at baseline, 45% had a cardiac response and 45% had renal response. At a median follow-up of 27 months, the median hematologic PFS in patients treated at the MTD was 15 months. Patient-reported quality of life was maintained during ixazomib treatment. Based on the promising efficacy, safety, and tolerability data of single-agent ixazomib in relapsed/refractory AL amyloidosis, an international phase 3 RCT was launched comparing ixazomib-dexamethasone with physicians’ choice [TOURMALINE-AL1] in patients with 1-2 prior lines of therapy[28]. The primary endpoints were hematologic overall response rate [ORR] and death or vital organ deterioration at the 2-year mark from treatment initiation. Secondary endpoints were overall survival, hematologic CR rate, hematologic/vital organ PFS, time to vital organ deterioration or mortality, duration of hematologic response, and safety.

The options for physicians’ choice in the control arm were dexamethasone alone or in combination with melphalan, cyclophosphamide, thalidomide, or lenalidomide. Unfortunately, the primary endpoints were not met with the hematologic ORR being 53% and 51% in the experimental and control arm respectively [p=NS]. However, ixazomib-dexamethasone was superior in several secondary endpoints including time to vital organ deterioration/death [35 vs 26 months; p=0.012], vital organ PFS [18 vs 11 months; p=0.019], and hematologic CR rate [26% vs 18%]. Furthermore, time to subsequent therapy was significantly longer with ixazomib [27 vs 13 months; p=0.027]. The magnitude of benefit for most time-to-event outcomes with ixazomib was comparable in PI-naïve versus PI-exposed patients[29]. The cumulative incidence of ≥grade 3 adverse events were comparable in the two arms. However, certain non-hematologic toxicities such as nausea, diarrhea, and rash were significantly more frequent in the ixazomib arm. Data on combination of ixazomib with lenalidomide and dexamethasone [IRd] was recently published as a retrospective study by the UK National Amyloidosis Center[30]. Notably, 43% of patients had Mayo stage 3a/3b disease at presentation and 100% were exposed to bortezomib in the past. The hematologic ORR at 3 months was 59%, with CR/VGPR/PR rate being 20.5%/20.5%/17.9%. Cardiac and renal organ response was seen in 6% and 13% respectively. The median PFS and OS was 16 and 29 months respectively. Serious adverse events included infection [40%], fluid overload [33%], cardiac arrhythmia [13%], renal dysfunction [7%], and anemia [7%].

7.1.1.2 Carfilzomib
Carfilzomib is a second-generation irreversible PI, with the major advantage over bortezomib being the lack of neurotoxicity. However, carfilzomib has a significantly higher rate of cardiac and renal toxicity, which makes it challenging to use in AL amyloidosis[31,32]. Furthermore, RCTs did not show superiority of carfilzomib over bortezomib in the frontline setting in multiple myeloma [33,34], which has tempered enthusiasm for testing carfilzomib in frontline AL patients. Carfilzomib was evaluated in relapsed/refractory AL in 28 Mayo stage I/II patients in a phase 1 trial [35]. Notably, patients had a median of 2 prior lines of therapy and 36% were PI-refractory. Heart and kidney were involved in 50% and 64% of patients respectively, with the median baseline NT-pro-BNP being 542 pg/ml. The maximal tolerated dose of carfilzomib was 20/36 mg/m2 in a twice-weekly schedule. The hematologic ORR was 63%, with 46% achieving VGPR or better response. Organ response was seen in 5 patients [3 kidney, 1 GI, and 1 liver]. There were no grade 5 adverse effects. 71% of patients had grade 3/4 AEs, including hypoxia, chest pain, lung infection, hypertension, CHF, and symptomatic ventricular tachycardia. Efficacy of carfilzomib was also reported in a recent series of five patients with amyloidosis-related peripheral or autonomic neuropathy, in whom, bortezomib was contraindicated[36]. Three out of five achieved a CR and two achieved a VGPR, with 80% drop in dFLC at 1 months. Four out of five were Mayo stage II and none had a worsening in cardiac function. Further prospective data is needed before carfilzomib can be safely used in AL amyloidosis. Given several effective and non- cardiotoxic anti-plasma cell therapies in the pipeline, carfilzomib is unlikely to play a major role in the treatment of AL amyloidosis in future.

7.1.2 Immunomodulatory drugs
Prior to the arrival of monoclonal antibodies, immunomodulatory drugs [IMiDs] were widely used as a second line agent in AL amyloidosis, especially in patients who were refractory to PIs. One of the first prospective trials of lenalidomide in relapsed/refractory AL amyloidosis showed a hematologic ORR of 41% and an organ response rate of 23%[37]. However, as shown in subsequent studies, the rate of deep hematologic response [≥VGPR] is relatively low at 25-28%, which translates into a low organ response rate of around 16%[38,39]. Furthermore, an increase in NT-pro-BNP can be observed with IMiDs even in the absence of clinically worsening organ function, which complicates assessment of cardiac response. With availability of 3rd generation IMiD pomalidomide which is active in lenalidomide-refractory myeloma patients, several prospective studies have tested pomalidomide, either as a single agent or in combination with dexamethasone, in relapsed/refractory AL amyloidosis, as discussed below. To our knowledge, three phase 2 clinical trials have reported on the activity of pomalidomide in relapsed/refractory AL amyloidosis, as summarized in Table 2[40-42]. Patients had received a median of 2 prior lines of therapy in these trials. Notably, 21- 48% of patients were either exposed or refractory to lenalidomide and 42-96% were either exposed or refractory to a PI[40-42]. The hematologic ORR and ≥VGPR rate ranged from 48-61% and 18-38% respectively, which compares favorably with the efficacy of lenalidomide, especially in the context of a heavily pretreated patient population. However, the organ response rates are low, with renal response/improvement in 7-17% and cardiac response/improvement in 0-15% of evaluable patients.

Thromboembolic events, which is a class effect of IMiDs, was seen in 0-7% despite routine thromboprophylaxis with aspirin. Grade 3 or higher adverse events observed in more than 10% of patients were myelosuppression [especially neutropenia], fatigue, respiratory tract infections, including pneumonia or bronchitis, and fluid retention. Notably, a paradoxical increase in BNP after 3 cycles of therapy was observed in 89% of patients without any clinical worsening of CHF, which complicates the assessment of cardiac response[40]. Recently, Milaniet al published the largest series of heavily pretreated AL patients who were treated with pomalidomide and dexamethasone from multiple centers in UK and Europe [n=153][43]. Notably, 65% were refractory to their last line of therapy, 93% were exposed to bortezomib, 81% to lenalidomide, and 75% to oral melphalan. The hematologic ORR after 6 cycles of pomalidomide-dexamethasone was 44%,which included 23% VGPR and 3% CR, comparable to the estimates from prior phase 2 trials. The median overall survival was two-fold higher in patients who achieved a hematologic response compared to those who did not in a 6-month landmark analysis[43]. Daratumumab is a an IgG1-Kappa monoclonal antibody which binds to a unique CD38 epitope on the surface of plasma cells and leads to cell death by several mechanisms including complement dependent cytotoxicity [CDC], antibody dependent cellular cytotoxicity [ADCC], antibody dependent cellular phagocytosis [ADCP], and apoptosis[44,45]. To a lesser extent, it also modulates the enzymatic activity of CD38, which mediates signal transduction in lymphoid cells[46]. Furthermore, daratumumab depletes CD38-positive immunosuppressive regulatory T and B cells as well as myeloid-derived suppressor cells [MDSCs][47], which can potentially modulate the tumor microenvironment.

In heavily pretreated relapsed/refractory MM patients, daratumumab also led to an increase in cytotoxic T-cell number, activity, and clonality[47]. Daratumumab initially demonstrated promising clinical efficacy in relapsed/refractory MM in two phase 1/2 trials[48,49]. A pooled analysis of 148 heavily pre-treated relapsed/refractory MM patients treated at the maximal tolerated dose showed an ORR of 31%[50], which subsequently led to the FDA approval of daratumumab in this setting. Given its promising efficacy in MM, it has also been explored in AL amyloidosis. To our knowledge, the first report of daratumumab efficacy in two patients with advanced systemic AL amyloidosis was demonstrated by Sheretal from the Mayo Clinic[51]. Subsequently, the Stanford group reported on daratumumab efficacy in heavily pretreated AL amyloidosis in 25 patients[52]. They demonstrated a hematologic ORR of 76%, with 60% achieving a VGPR or better response. Furthermore, responses were rapid, with the median time to deepest hematologic response being 1 month [range, 7-188 days]. Despite cardiac involvement in 72% of patients, there was no grade 3 or higher infusion related reaction [IRR] noted in this study. Subsequently, several small retrospective studies demonstrated safety and efficacy of daratumumab, either as a single agent or in combination, in relapsed/refractory AL amyloidosis[53-55]. Recently, five keys studies, including two phase 2 clinical trials and three large retrospective studies, have been published, which highlights the efficacy of daratumumab in relapsed/refractory AL amyloidosis, either as a single agent or in combination[56-60].

These studies have been summarized in Table 3. The median number of prior lines of therapy ranged from 1 to 3 in these studies. The hematologic ORR [PR or better] with daratumumab monotherapy ranged from 55-90%, and the rate of VGPR or better being 48-86%[56-60]. The time-to-hematologic response was one month or less, with the French study demonstrating a VGPR rate of 33% after only one injection of daratumumab[56,59,60]. The rate of MRD-negativity based on multiparameter flow cytometry with a sensitivity of 10-5 was 30%[60]. The cardiac response rate ranged from 22-55% and renal response rate ranged from 31-67%[56- 60]. Based on the modified organ response criteria by Muchtar et al[61], daratumumab led to cardiac VGPR or better in 16-33% and renal VGPR or better in 28-52% of patients[56,57]. Time to best cardiac response and best renal response was 10-17 months and 12-13 months respectively[57,60], which is consistent with prior literature on the timeline of organ response in AL amyloidosis. There was heterogeneity in the duration of therapy, with the French study administering daratumumab for a fixed duration [24 weeks], and studies by the Boston University, Pavia, and Stanford groups administering daratumumab maintenance beyond the 6-month period[56,57,59,60]. The Heidelberg study also reported on outcomes of 62 patients receiving daratumumab with bortezomiband dexamethasone [DVD], which led to a ≥VGPR rate of 66%, cardiac response rate of 28%, and renal response rate of 27%, comparable to the response rates seen with daratumumab monotherapy[58].

However, there were differences in baseline characteristics between the two groups since this was a non-randomized comparison. None of the studies reported grade 3/4 infusion related biomimetic channel reactions [IRRs], except for two patients in the study by the Stanford group[57]. Furthermore, the risk of grade 3/4 infections was lowest [0%] in the French study which had used routine antibacterial prophylaxis with amoxicillin and was highest in the German study with DVD [18%][58,59]. The impact of FISH cytogenetics on outcome was explored in the German study only. Notably, t(11;14) had a favorable impact on hematologic event-free survival [EFS] with daratumumab monotherapy on univariable analysis, whereas +1q21 and hyperdiploidy had a negative impact on overall survival[58]. However, the impact of cytogenetic abnormalities on EFS disappeared on multivariable analysis. Due to promising activity and a manageable safety profile of daratumumab in relapsed/refractory AL amyloidosis, it has been explored in the frontline setting in combination with CyBorD in a randomized fashion [Andromeda Trial; NCT03201965][62]. This trial compared subcutaneous daratumumab in combination with CyBorD versus CyBorD alone in newly diagnosed systemic AL amyloidosis. Notably, the experimental arm receives daratumumab maintenance therapy beyond six months up to a maximum of two years whereas the control arm receives no maintenance therapy after six months of CyBorD. Based on the 28 patient safety run-in results, the hematologic ORR with this combination was 96%,with a ≥VGPR rate of 82%[62]. The median time to first response was 9 days. Toxicity was manageable, with no grade 5 treatment-emergent adverse events and no grade 3 or higher IRRs. At a median follow-up of 18 months, best cardiac, renal, and hepatic response rates were 62%, 67%, and 67% respectively. The primary results of ANDROMEDA trial were recently presented at EHA25, which revealed a hematologic CR rate of 53% and 18% for Dara-CyBorD and CyBorD respectively [Odds Ratio 5.1; p<0.001][63]. Notably, the ≥VGPR rate with Dara-CyBorD was 79%, which is unprecedented in newly diagnosed AL and comparable to the initial safety run-in results.

The cardiac and renal responses with Dara-CyBorD and CyBorD were 42% and 22% [cardiac] and 54% and 27% [renal] respectively. At a median follow-up of 11 months, the composite endpoint of major organ deterioration and PFS favored the Dara-CyBorD arm [HR 0.58; p=0.0224]. The most common grade 3/4 AEs in Dara-CyBorD and CyBorD arms were lymphopenia [13% and 10% respectively], pneumonia [8% and 4% respectively], diarrhea [6% and 4% respectively], CHF [6% and 5% respectively], neutropenia [5% and 3% respectively], syncope [5% and 6% respectively], and peripheral edema [3% and 6% respectively]. Notably, daratumumab was administered subcutaneously in this trial, which likely led to a low incidence of infusion-related reactions [7%; all grade 1-2]. The total number of deaths were comparable in both arms. This trial will pave the way for Dara-CyBorD to be the new standard-of-care in newly diagnosed AL amyloidosis irrespective of transplant eligibility. Isatuximab is a novel chimeric IgG1-Kappa anti-CD38 monoclonal antibody, which targets a unique CD38 epitope and elicits anti-plasma cell activity through several mechanisms including CDC, ADCC, ADCP, and apoptosis[64]. Based on robust activity of isatuximab in relapsed/refractory MM[65], it is currently being investigated in AL amyloidosis [NCT03499808] and monoclonal gammopathy of renal significance[66]. Elotuzumab is an IgG1-Kappa monoclonal antibody targeting signaling lymphocytic activation molecule family member F7 [SLAMF7], also known as cell-surface glycoprotein CD2 subset 1 [CS1], on the surface of plasma cells.

Elotuzumab, in combination with lenalidomide, is approved in relapsed/refractory MM based on the phase 3 ELOQUENT-2 trial[67]. To our knowledge, prospective studies on efficacy of elotuzumab in AL amyloidosis are lacking at this time and clinical trials are ongoing [NCT03252600]. However, it is important to note that elotuzumab does not have single- agent activity in MM[68], especially since achieving a rapid and deep response is the key in AL amyloidosis. 7.1.3.4 Antibody-drug conjugates The anti-CD38 antibody-drug conjugate [ADCs] Duostatin 5.2 or STI-6129 [Sorrento Therapeutics] is currently being tested in relapsed/refractory AL in phase 1 trial [NCT04316442]. STI-6129 consists of a fully humanized monoclonal antibody against CD38 that is linked to a cytotoxic payload named Duostatin 5.2 which is a microtubule inhibitor via a non-polyethylene glycol linker[69]. It showed string pre-clinical activity in MM cell lines. With success of B-Cell Maturation Antigen [BCMA]-directed ADCs in relapsed/refractory MM with high single-agent activity[70], these may be further tested in the relapsed/refractory AL space in future, especially among patients relapsing after anti-CD38 mAB-based therapy. An important opportunity to develop biomarker-guided therapy in AL is the use of BH-3 mimetics in t[11;14] patients. Resisting cell death by tipping the balance towards pro-survival or anti-apoptotic proteins is a hallmark of cancer cells[71]. Pro-apoptotic [BAX, BAD, BIM, BID, and NOXA] and anti-apoptotic [BCL-2, BCL-XL, BCL-W, and MCL-1] proteins in the BCL-2 family are the critical regulators of the cellular apoptotic machinery[72].

The pro-apoptotic members of the broader BCL-2 family, which promote cell death, are known as BH3 [BCL-2 homology domain 3]-only proteins. Venetoclax, a BH3 mimetic, is an orally bioavailable and selective inhibitor of the anti-apoptotic protein BCL-2, which inhibits the growth of BCL-2 dependent tumors and has demonstrated robust clinical activity in several hematologic malignancies[73,74]. Initial preclinical studies showed activity of BH-3 mimetic agents in myeloma cell lines harboring t(11;14), which had a high BCL-2/MCL-1 ratio[75]. The investigators also screened the public expression database in myeloma patients, which demonstrated a significantly higher BCL-2/MCL-1 ratio in t(11;14) and hyperdiploid myeloma cells compared to read more those with other cytogenetic abnormalities[75]. Furthermore, functional BH3 profiling assays have demonstrated dependence of MM cell lines on BCL-2 for survival[76]. One of the first trials of single agent venetoclax in relapsed/refractory MM showed an overall response rate [ORR] of 21%, with 15% achieving a very good partial response [VGPR] or better[77]. Notably, the ORR and ≥VGPR rate was 40% and 27% respectively in the t(11;14) subgroup. However, in the subsequent BELLINI trial, which randomized patients with relapsed/refractory myeloma [irrespective of t(11;14) status] to bortezomib and dexamethasone with or without venetoclax, a higher mortality rate was observed in the venetoclax arm despite a superior response rate and progression-free survival [PFS][78]. The major causes of death were infection, progressive disease, or combination of both. Interestingly, in patients with t(11;14) or high BCL-2 by quantitative PCR, the risk/benefit profile was favorable for venetoclax, with markedly increased response rate and PFS and no adverse impact on mortality[79].

These findings restricted further development of venetoclax mostly in multiple myeloma patients harboring t(11;14). Interestingly, t(11;14) is the most common primary cytogenetic abnormality in AL amyloidosis and present in approximately 50% of patients[80,81], which makes it an attractive target and forms the rationale for pursuing clinical trials of venetoclax-based regimens. To our knowledge, the first report of activity of venetoclax-based regimen [Venetoclax-Bortezomib-Dexamethasone] in AL amyloidosis was published by Leung et al[82]. The patient in this report had plateaued at a partial hematologic response on CyBorD but subsequently achieved a complete response after initiating venetoclax. Subsequently, multiple case reports demonstrated the activity of both single agent venetoclax as well as combinations in AL amyloidosis[83-86]. Our experience with venetoclax-based combination regimens in relapsed/refractory AL amyloidosis from a multicenter retrospective study showed a ≥VGPR rate of 67% [unpublished data]. Although t(11;14) patients had a superior response rate [≥VGPR in 80%], clinically meaningful responses were seen in non-t(11;14) patients as well [ORR of 56% and ≥VGPR of 44%]. Another study from the Mayo Clinic reported on the efficacy of venetoclax-based regimens in 12 patients with relapsed/refractory AL amyloidosis, 11 of whom had t(11;14)[87].

Seven out of 12 patients received venetoclax alone or in combination with dexamethasone. All patients had prior exposure to PIs and one-third were exposed to daratumumab. Heart and kidney involvement were present in 50% and 75% of patients respectively. VGPR or better response was achieved in seven out of eight evaluable patients. One out of fourevaluable patients with cardiac involvement and two out of six with renal involvement achieved cardiac and renal response respectively. Mild gastrointestinal toxicity in the form of loose stools was seen in six and upper respiratory tract involvement in two patients. No treatment-related mortality was observed. Traditionally, melphalan and cyclophosphamide were the only alkylating agents which formed the backbone of treatment regimens for AL amyloidosis. Recently, bendamustine showed encouraging activity in relapsed/refractory AL[88,89]. In patients with non-Hodgkin lymphoma, bendamustine was demonstrated to not show cross- resistance to other cytotoxic drugs[90]. A phase II study of bendamustine- dexamethasone in relapsed/refractory AL showed a hematologic ORR of 57%, which included CR in 11% and VGPR in 18% of patients[89]. The overall organ response in evaluable patients was 29%, including renal response in 46% and cardiac response in 13%. At a median follow-up of 15 months, the median PFS and OS was 11 and 18 months respectively[89]. The most common therapy-related severe AEs were myelosuppression, fatigue and nausea/vomiting.

Propylene glycol-free melphalan [Evomela] is being tested for conditioning prior to HDM-AHCT since the co-solvent propylene glycol has been implicated in several peri- transplant complications including nephrotoxicity and cardiotoxicity. A phase II study of Evomela [140 or 200 mg/m2] by the BU amyloidosis group showed rates of peri- transplant cardiac arrhythmia and post-transplant response rate comparable to historical controls, with potentially less kidney toxicity[91]. Additional studies are currently ongoing in both MM and AL amyloidosis. Melflufen, which is a peptidase-potentiated alkylating agent and active in relapsed/refractory MM, has shown preclinical evidence of efficacy in AL amyloidosis[92], and is being pursued in early phase clinical trials. Although rare, IgM amyloidosis is a distinct clinical entity that is associated most commonly with lymphoplasmacytic lymphoma [LPL], followed by plasma cell neoplasm and other low-grade B-cell non-Hodgkin’s lymphoma[93]. IgM amyloidosis has a low incidence of t(11;14) and a high incidence of MYD88L265P mutations[93,94]. Treatment in IgM amyloidosis should be directed at the clonal disease identified on bone marrow morphology, such as rituximab or ibrutinib-based therapy in LPL and bortezomib-based therapy in pure plasma cell neoplasm. AL amyloidosis [both light chain and IgM] can rarely be associated with non-lymphoplasmacytic lymphoma, including marginal zone lymphoma, indolent B-cell lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma[95]. Treatment in such cases should be directed to the respective malignancy.

Although plasma cell-directed therapies are effective in halting further production of amyloidogenic light chain, they are not effective in eliminating the amyloid protein already deposited in tissues. Hence, therapies targeting the amyloid fibrils deposited in vital organs such as heart, kidneys, and liver are urgently needed to reverse end-organ damage and potentially improve quality of life. Using mouse hybridoma models, Abeet al were the first to demonstrate the feasibility of developing anti-light chain monoclonal antibodies with specificity for both constant region and variable region epitopes in kappa and lambda light chains[96]. Subsequently, their group developed the 11-1F4 monoclonal antibody, which had a promising efficacy in mouse amyloidoma model[97]. After injecting mice with amyloid proteins obtained from spleen or liver of AL patients, the investigators administered anti- light chain monoclonal antibody with specificity for a unique light chain epitope. Compared to controls, mice which were injected with the antibody had a rapid resolution of amyloidoma irrespective of the fibril isotype. Furthermore, neutrophilic infiltration into the amyloidoma was noted in mice with amyloid response or resolution. The 11-1F4 antibody was subsequently chimerized for clinical use[98], and was then tested in a first-in-human Phase 1a/b trial[99]. In the phase 1a portion, patients received a single intravenous infusion of 11-1F4 and four weekly infusions was administered in the phase 1b potion. The dose escalation design was used in both phase 1a and 1b potions, with successive doses ranging from 0.5-500 mg/m2. A total of 27 patients were enrolled, with eight in phase 1a and 19 in phase 1b. No drug-related grade 4 or higher adverse events or dose-limiting toxicities were noted.

Of note, skin biopsy in a patient with drug-related rash demonstrated 11-1F4 antibody binding to amyloid fibrils with an associated neutrophilic infiltrate. Five out of eight [63%] evaluable patients in the phase 1a portion and 11 out of 18 [61%] evaluable patients in phase 1b achieved an organ response,with the median-time-to-organ response being two weeks. Faster organ response was achieved at higher doses. Global Longitudinal Strain [GLS] was measured as a part of this trial at screening and at 12 weeks[100]. Among 10 patients with cardiac involvement, a significant improvement in GLS was noted from -15.58 ± -4.14% at screening to -17.37 ± -3.53% at week 12 [p = 0.004]. GLS was improved in nine out of 10 patients with cardiac involvement, with the only patient without improvement having received a low dose [5 mg/m2]. The Pearson correlation coefficient between NT-pro- BNP response and GLS response was 0.345, indicating a moderate correlation. Currently, a phase 2 open-label study of CAEL-101 in combination with standard-of- care plasma cell-directed therapy is actively recruiting [NCT04304144],and will determine the safety, tolerability, and recommended dose for a global phase 3 RCT which will randomize patients with newly diagnosed AL amyloidosis with advanced cardiac involvement to CAEL-101 or placebo in combination with plasma cell-directed therapy. NEOD001 is a humanized form of murine monoclonal antibody 2A4 which binds to a unique epitope on misfolded light chains[101].

In mouse amyloidoma models, NEOD001 binds to the misfolded light chain aggregates while sparing normally folded light chains and subsequently leads to phagocyte-mediated clearance of amyloid deposits[101]. Given its potential to eliminate amyloid protein deposited in tissues, it was initially tested in a phase I/II trial on patients with previously treated light chain amyloidosis and persistent organ dysfunction[102]. In the dose escalation component of the study, no serious adverse events, dose limiting toxicity, or drug discontinuation due to adverse events were noted. Cardiac and renal response was noted in 57% and 60% of evaluable patients. Notably, a 48% reduction in NT-pro-BNP and 62% reduction in proteinuria was observed compared to baseline. Based on the promising safety and efficacy results, a phase III RCT was launched [VITAL Amyloidosis study; NCT02312206], which randomized patientsto NEOD001 versus placebo in combination with standard-of-care plasma cell directed therapy in newly diagnosed AL amyloidosis. The primary endpoint of the study was time to a composite of all-cause mortality or centrally adjudicated cardiac hospitalization. Unfortunately, the study was terminated dueto futility after 103 events [HR for NEOD001 vs placebo 0.835; 95% CI, 0.5799- 1.2011; p=0.330][103]. Similarly, the phase 2b randomized PRONTO study which compared NEOD001 versus placebo in patients with systemic AL amyloidosis who were in a stable hematologic response with persistent cardiac dysfunction was also a negative study. The proportion of patients achieving a cardiac organ response [primary endpoint] was 39.4% and 47.6% in NEOD001 and placebo respectively[104]. RAIN trial was another randomized placebo-controlled phase 2b study comparing NEOD001 versus placebo in patients with systemic AL amyloidosis who have a stable hematologic response but persistent kidney dysfunction, defined as proteinuria>500 mg/day[105]. The primary endpoint of this study was achievement of a renal organ response after 12 months of treatment. Due to negative results of VITAL and PRONTO studies, further development of NEOD001 was discontinued and RAIN trial was halted as well.

After the trial was halted,the study chair broke the randomization code and examined results on 11 patients [6 received NEOD001 and 5 received placebo][106]. Two out of 6 in the NEOD001 arm and 1 out of 5 in placebo arm achieved a renal response. Notably, a post-hoc analysis of VITAL study for all-cause mortality stratified by Mayo 2012 stage at diagnosis showed a benefit with NEOD001 in 77 patients with Mayo Stage IV disease [HR 0.498; 95% CI, 0.2404-1.0304; p=0.0556][103]. There was no major safety signal for NEOD001 noted in this study. Based on the promising results in patients with advanced cardiac involvement, further studies are warranted in this population which has a high risk of early mortality and poor outcome despite novel plasma cell-directed therapies. Serum amyloid protein [SAP] is bound to all forms of amyloid deposits [not specific to AL] and protects amyloid fibrils from proteolysis[107]. Hence, targeting SAP by monoclonal antibodies is a rational approach for eliminating amyloid fibrils deposited in organs. Indeed, pre-clinical studies in mice showed that administration of anti-human SAP antibodies leads to clearance of amyloid deposits in the viscera by triggering a complement-mediated macrophage-derived giant cell reaction[108]. However, an important caveat to this approach is that there is circulating SAP which needs to be depleted such that the anti-SAP antibodies are able to bind only to the SAP present in organ amyloid deposits. This approach was tested in an open label dose escalation phase I trial in 15 amyloidosis patients, 8 of whom had AL[109]. Circulating human SAP was depleted by the drug CPHPC [(R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo- hexanoyl]pyrrolidine-2-carboxylic acid] prior to the infusion of humanized monoclonal IgG1-κ anti-SAP antibody. Notably, patients with cardiac involvement were not included in this study. No serious AEs were noted.

In patients who had received more than 200 mg of the antibody dose, a decrease in liver stiffness was noted by transient elastography, accompanied with an improvement in liver function and decrease in liver amyloid burden by imaging. A reduction in the renal and lymph node amyloid burden was also noted. Subsequently, it was tested in an expanded cohort of 23 patients with systemic amyloidosis[110]. Dose-dependent amyloid clearance from liver, kidneys, and spleen was demonstrated using scintigraphy, with the main adverse event being rash. However, further development of this compound has been terminated currently due to a change in the risk/benefit profile. Preclinical studies have demonstrated anti-fibril activity of doxycycline, including disruption of recombinant light chain fibril formation and reduction in the number of intact fibrils after exposure to doxycycline ex vivo[111]. A retrospective study from the UK National Amyloidosis Center on 76 patients treated with upfront bortezomib-based regimens showed a significantly higher 1-year survival in patients treated with doxycycline compared to those who were not [84% vs 58% respectively; p=0.012][112]. Another phase II study testing doxycycline for 1 year along with anti-plasma cell therapy demonstrated low 1-year mortality compared to historical data [20%],high transplant utilization rate [60%], and no grade 2 or higher toxicities[113]. Currently, a randomized phase II/III trial of doxycycline versus standard supportive therapy in newly diagnosed cardiac AL amyloidosis is ongoing in the context of bortezomib-based frontline therapy [NCT03474458], which will provide a definitive answer on the efficacy of doxycycline. regenerative medicine EGCG is a compound that is found in green tea, with preclinical evidence showing that it can disrupt formation of amyloid fibril[114]. However, a randomized trial TAME-AL [A Trial for the Treatment of Cardiac AL-Amyloidosis With the Green Tea Compound EGCG] did not show any benefit of EGCG on reducing left ventricular mass over placebo[115].

8. Potential development issues
The improvement in outcome of AL amyloidosis has been possible thus far due to effective plasma cell-directed therapies, which can turnoff the production of amyloidogenic light chain by targeting monoclonal plasma cells in the bone marrow. Given a rich and diverse pipeline of anti-plasma cell therapy, the therapeutic space in relapsed/refractory AL is highly competitive and will likely favor treatments that have limited organ toxicities and no adverse impact on quality of life. For example, chimeric antigen receptor T-cell [CART] therapy or bi-specific T-cell engagers [BiTEs] targeting B-cell maturation antigen, which is highly efficacious in relapsed myeloma, may not be suitable for AL patients due to toxicities like cytokine release syndrome, which may be lethal in patients with severely compromised organ function. However, CAR T and BiTEs may have a role in younger transplant-eligible AL patients as it may offer durable responses. Similarly, drugs that are notable to achieve a rapid and deep response and preferably MRD-negativity may not be suitable for AL where the depth of hematologic response is the most important predictor of organ function and survival. Furthermore, an important drug development issue in AL is a high risk of early mortality in patients with advanced cardiac involvement who are typically excluded from clinical trials and are the ones most in need for novel agents. Another important issue is selection of appropriate endpoints. It remains to be seen whether the addition of fibril-directed therapies and daratumumab to frontline AL regimen will decrease the rate of early mortality in patients with advanced Mayo stage disease.

9. Conclusion
The current treatment paradigm in systemic AL amyloidosis includes prompt initiation of bortezomib-based plasma cell-directed therapy to halt further production of the amyloidogenic light chain. With the positive results of ANDROMEDA trial, daratumumab, in combination with CyBorD will become the new standard of care for newly diagnosed patients in near future. Results from the randomized trial on CAEL-101 will inform whether fibril directed therapy will have a role in frontline treatment. Carefully selected patients who are eligible for HDM-AHCT should proceed to transplant as
frontline therapy, with a strong consideration of bortezomib-based induction prior to transplant. There is no universally accepted standard-of-care for relapsed/refractory AL amyloidosis. Drugs that demonstrated promising activity in this space are daratumumab, ixazomib, pomalidomide, venetoclax, and bendamustine.

10. Expert opinion
Rapid drug development in multiple myeloma in the past two decades resulted in availability of multiple therapeutic options for targeting the plasma cell clone in AL amyloidosis. Introduction of PI bortezomib to the therapeutic armamentarium of AL was a major advance that led to deep hematologic responses that translated into an improved survival. Advantages of ixazomib over bortezomib are lack of peripheral neuropathy and oral route of administration. Although TOURMALINE-AL1 did not achieve its primary endpoint, ixazomib-dexamethasone was clearly superior with respect to the secondary time-to-event endpoints. However, an important caveat was the absence of daratumumab as an option in the control arm, which has the best efficacy data in relapsed/refractory AL amyloidosis at present. Going forward, ixazomib will have a role in relapsed/refractory AL amyloidosis, especially as daratumumab moves to the frontline setting. However, since deep responses are limited with single agent ixazomib [≥VGPR≈40%], it will likely be explored in combination with other active agents without overlapping toxicities. Among novel agents with activity in relapsed/refractory AL amyloidosis, daratumumab is the most efficacious, with unprecedented rates of deep hematologic response [≥VGPR] of up to 86%. Furthermore, it is able to achieve responses rapidly within weeks, which is critical for halting end-organ damage, especially during the log phase of amyloid growth curve. Indeed, the organ response rates seen with daratumumab-based regimens in relapsed/refractory AL amyloidosis are significantly higher compared to other drug classes such as IMiDs and alkylating agents. With positive results of the ANDROMEDA trial, daratumumab will likely be approved in the frontline setting in near future.

Whether efficacy of daratumumab in the relapsed/refractory setting will be maintained in the era of frontline daratumumab use remains to be seen. Furthermore, with deep responses including MRD-negativity in the newly diagnosed setting with Dara-CyBorD regimen, the role of HDM-AHCT could be challenged, especially in the absence of RCT data for transplant and the small but significant risk of treatment-related mortality. One of the major limitations of using IMiDs, either as a single agent or with dexamethasone, is low rates of rapid and deep hematologic response, translating into a low rate of organ response, compared to other novel agents such as monoclonal antibodies and BH3 mimetics. Furthermore, NT pro-BNP progression, both in the setting of true organ progression and without clinically worsening CHF, complicates the assessment of organ response in patients with cardiac involvement. Hence, going forward, IMiDs may be suitable for selected patients with an indolent or biochemical relapse without clinically significant CHF. Although data on venetoclax in relapsed/refractory AL amyloidosis mostly comes from retrospective studies at this time, the efficacy in t(11;14) cohort is unprecedented with a ≥VGPR rate in the upwards of 80% in a heavily pre-treated population. Given the unexpected high mortality in venetoclax arm among unselected relapsed/refractory MM patients in BELLINI trial, the clinical trial of venetoclax- dexamethasone in relapsed/refractory AL [NCT03000660] is currently on hold. However, recent update from the BELLINI trial showed a favorable risk-benefit profile in t(11;14) subgroup[116].

Hence, future clinical trials of venetoclax in AL amyloidosis should focus on t(11;14) patients which comprise 50-60% of AL population. Given the retrospective nature of currently available studies on venetoclax and short follow-up, the durability of response and optimal treatment duration remains unclear. Our current treatment approach is depicted in Figure 1. In summary, outcomes are expected to improve in the near future once daratumumab moves to frontline setting in combination with bortezomib-based therapy. MRD assessment will also play a role in tailoring therapy, especially with a rich pipeline of drugs that are effective in this population. None of the available drugs meaningfully improve outcomes of patients with stage IIIb [2004] or Stage IV [2012] AL amyloidosis and these patients are typically excluded from most clinical trials. It remains to be seen whether early mortality in such patients would be mitigated by fibril-directed therapies such as CAEL-101. Finally, since AL is a systemic disease, coordinated care by a multidisciplinary team including organ-specific specialists with expertise in amyloidosis, preferably at high volume centers, will remain crucial for superior outcomes.