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The past and future of CD33 as therapeutic target in acute myeloid leukemia

Blood Reviews

Abstract

CD33 is a myeloid differentiation antigen with endocytic properties. It is broadly expressed on acute myeloid leukemia (AML) blasts and, possibly, some leukemic stem cells and has therefore been exploited as target for therapeutic antibodies for many years. The improved survival seen in many patients when the antibody-drug conjugate, gemtuzumab ozogamicin, is added to conventional chemotherapy validates this approach. However, many attempts with unconjugated or conjugated antibodies have been unsuccessful, highlighting the challenges of targeting CD33 in AML. With the development of improved immunoconjugates and CD33-directed strategies that harness immune effector cells, therapeutics with enhanced efficacy may soon become available. Toxic effects on normal hematopoietic cells may increase in parallel with this increased efficacy and demand new supportive care measures, including possibly rescue with donor cells, to minimize morbidity and mortality from drug-induced cytopenias and to optimize treatment outcomes with these agents in patients with AML.

Keywords: Acute myeloid leukemia, Antibody, Antibody-drug conjugate, Bispecific antibody, BiTE, CD33, Chimeric antigen receptor, Gemtuzumab ozogamicin, Immunotherapy, Radioimmunoconjugate.

1. Introduction

Since the invention of hybridoma technology 4 decades ago, monoclonal antibodies have revolutionized the care of patients with cancer. An increasing number of unconjugated, toxin-loaded, and radiolabeled antibodies have shown anti-tumor efficacy and have been approved for indications in an expanding list of malignancies, including acute myeloid leukemia (AML) [1] and [2]. AML has been a paradigm for the therapeutic use of monoclonal antibodies, in no small part because malignant cells are readily accessible and express well-defined cell surface antigens. Most efforts to date have focused on exploiting CD33 as a target in this disease, and the CD33-directed immunoconjugate, gemtuzumab ozogamicin (GO), was the first anti-cancer antibody-drug conjugate to obtain marketing approval in the U.S. [3] Still, targeting CD33 has proven challenging, as perhaps best reflected by the eventual market withdrawal of GO because of concerns over excess toxicity and lack of efficacy. In this article, we will summarize the biologic characteristics of CD33, emphasizing the properties that make it appealing as a therapeutic target, appraise attempts made thus far with CD33-directed therapies, and discuss current and future therapeutic directions in this field.

2. Physiologic characteristics of CD33

CD33 is a member of the sialic acid-binding immunoglobulin-like lectins (Siglecs), a discrete subset of the immunoglobulin (Ig) superfamily molecules ( Fig. 1 ) [4] and [5]. This 67kD single pass transmembrane glycoprotein is characterized by an amino-terminal V-set Ig-like domain that mediates sialic acid binding and a C2-set Ig-like domain in its extracellular portion [6], [7], and [8]. Alternative splicing of CD33 RNA leads to a shorter isoform that is expressed on the cell surface. This isoform lacks the V-set Ig-like domain as well as the disulfide bond linking the V- and C2-set Ig-like domains. While the biological relevance of this splicing process is unknown, it may be important for the development and use of CD33-directed drugs. Specifically, a dominant epitope, recognized by the majority of initial CD33 antibodies, is located on the V-set Ig-like domain. Thus, some CD33 antibody-based therapeutics will only recognize the full-length but not the shorter splice isoform of CD33 [9] and [10].

gr1

Fig. 1 Structure of CD33. Scheme depicting the domain structure of CD33 as well as individual amino acids that have been implicated in phosphorylation or ubiquitylation events. Abbreviations: C2, C2-set Ig-like domain; P, phospho-; SFKs, Src-family kinases; Ub, ubiquitin; V, V-set Ig-like domain.

The cytoplasmic tail of CD33 contains 2 conserved tyrosine-based inhibitory signaling motifs, which, upon phosphorylation by Src family kinases, provide docking sites for the recruitment and activation of Src homology-2 (SH2) domain-containing tyrosine phosphatases such as SHP-1 and SHP-2. While the signaling events downstream of CD33 remain poorly understood, these phosphatases may not only dephosphorylate CD33 as part of a negative feedback loop but also dephosphorylate and negatively regulate nearby receptors [11], [12], and [13]. Through its SH2 domain, suppressor of cytokine signaling 3 (SOCS3) can compete with SHP-1 or SHP-2 for binding to phosphorylated CD33 and recruit the ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) E3 ubiquitin ligase complex, which ultimately leads to ubiquitylation and results in accelerated proteasomal degradation of CD33 ( Fig. 1 ) [14] .

2.1. CD33 as myeloid differentiation antigen

In healthy individuals, CD33 is primarily a myeloid differentiation antigen with initial expression at the very early stages of myeloid cell development: it is found on normal multipotent myeloid precursors, unipotent colony-forming cells, and maturing granulocytes and monocytes. On CD34+/CD33+ bone marrow cells, CD33 expression has been estimated to average around 8 × 103 molecules/cell, although levels vary widely (1–20 × 103 molecules/cell) [15] . Expression is down-regulated to lower levels on neutrophils (~ 2–2.5 × 103 molecules/cell) and macrophages but retained on circulating monocytes and dendritic cells [15], [16], [17], [18], [19], and [20]. CD33 can also be displayed on subsets of B-cells and activated T- and natural killer (NK) cells [9], [21], [22], [23], [24], [25], [26], and [27]. In contrast, CD33 is not expressed outside the hematopoietic system or on pluripotent hematopoietic stem cells, with the latter indicated both by in vitro studies of normal bone marrow [18], [19], and [28] and by the delayed but durable multi-lineage engraftment after transplantation of CD33-depleted autografts in patients with AML [29] and [30].

2.2. Putative functions of CD33

Increasing evidence suggests a role for CD33 and related Siglecs in the modulation of inflammatory and immune responses through a dampening effect on tyrosine kinase-driven signaling pathways [31] and [32]. For example, in vitro studies have demonstrated that CD33 constitutively suppresses the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-8 by human monocytes in a sialic acid ligand-dependent and SOCS3-dependent manner [33] . Conversely, reduction of cell surface CD33 (e.g. via SOCS3 activity or RNA interference) or interruption of sialic acid binding can increase p38 mitogen-activated protein kinase (MAPK) activity and enhance cytokine secretion as well as cytokine-induced cellular proliferation [14] and [33]. Engagement with CD33 antibodies has similarly been shown to affect cytokine and chemokine secretion by monocytes, although both increases [34] or decreases [33] have been observed in vitro.

2.3. Endocytic properties of CD33

A therapeutically important characteristic of CD33 is its internalization when bound by bivalent antibodies; this process is slower than that observed with other cell surface antigens such as the transferrin receptor [35], [36], [37], [38], [39], [40], [41], [42], and [43]. Mechanistic studies indicate that endocytosis is largely limited and determined by the intracellular domain of CD33 and may be regulated by tyrosine phosphorylation and perhaps ubiquitylation of the cytoplasmic tail, although some internalization of CD33/antibody complexes occurs in a phosphorylation-independent manner [41], [42], and [43]. Related to this endocytic property, engagement of CD33 with bivalent antibodies leads to a decrease (“modulation”) of CD33 cell surface levels [35], [40], and [44]. Although new CD33 sites are continuously expressed [40] , this feature could reduce the efficacy of CD33-directed therapeutics because of the reduction of available target binding sites.

3. CD33 in AML and other malignancies

Depending on how antigen positivity is defined, CD33 is found on at least a subset of blasts in nearly all AML patients [45] and [46], consistent with its characteristic as a myeloid differentiation antigen. Although surface levels show considerable inter-patient variability (> 2-log fold) [15], [45], and [46], CD33 expression is relatively limited with an average of ~ 104 molecules/AML blast [15] and [47] and is typically even lower in immature (e.g. CD34+/CD38-/CD123+ or CD34+/CD38-) cell subsets [46] and [48]. CD33 expression correlates with disease characteristics: for example, expression is homogeneous and typically bright in acute promyelocytic leukemias (APL) [49] and [50]. High levels of CD33 are also associated with NPM1 as well as high allelic FLT3/ITD mutations, while expression is generally low with core-binding factor translocations [45], [46], and [51]. Likely at least partially related to such associations, it has been a recurrent observation in pediatric trials that, on average, patients whose AML blasts display high CD33 levels experience inferior disease-free and overall survival when treated with conventional chemotherapy that does not include CD33-targeted agents [52] and [53]; similar data from adult patients is currently not available. In AML patients, CD33 can also be detected as soluble protein in the circulation and may provide some prognostic information [54] and [55]; however, its role as predictive biomarker has not been studied in detail. It is also unclear as to what degree, if any, soluble CD33 might interfere with the therapeutic efficacy of CD33 antibodies, although some in vitro evidence suggests that soluble CD33 may not impact the activity of CD33-targeted immunotherapy [56] .

Besides its broad expression on AML blasts, a main impetus to pursue CD33 as a drug target came from the notion that the stem cells underlying some AMLs could be CD33+, implying that CD33-directed therapy could potentially eradicate malignant stem and/or progenitor cells in such cases while sparing normal hematopoietic stem cells [57] and [58]. This possibility was first indicated by classic X chromosome inactivation studies, which found clonal dominance limited to granulocytes and monocytes in a subset of leukemias, suggesting that expansion of the malignant clone could occur at the committed CD33+ myeloid precursor cell stage [59] and [60]. To test the assumption that CD33- precursors would be predominantly or completely normal in some of these cases, CD33-depleted specimens from a small number of patients with such leukemias were placed in long-term culture together with irradiated allogeneic stroma cells; over time, CD33- precursors from some patients indeed generated colony-forming cells with X chromosome inactivation patterns consistent with predominantly non-clonal hematopoiesis [61] and [62]. The demonstration of CD33 expression on AML-initiating cells in immunodeficient mice [63] has further been used to argue that human AML stem/progenitor cells may display this antigen in at least some cases.

In addition to its expression in AML, CD33 is found not only on abnormal cells in other myeloid neoplasms (e.g. myelodysplastic syndromes and myeloproliferative neoplasms) but also on subsets of B-cell and T-cell acute lymphoblastic leukemias (ALL)/lymphoblastic lymphomas [15] and [64], perhaps consistent with its occasional expression on normal non-myeloid cells [21], [22], [23], [24], [25], [26], and [27]. This expression pattern has led to the use of CD33-directed therapeutics in patients with malignancies other than AML, including myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, and ALLs [35], [65], [66], [67], [68], [69], [70], and [71].

4. Past efforts with therapeutic CD33 antibodies in AML

Over the last 25 years, numerous attempts have been made to exploit CD33 as target for therapeutic antibodies in patients with AML (and, by extension, other tumors displaying cell surface CD33). While most efforts have focused on antibody-drug conjugates, in particular GO, other studies have involved unconjugated CD33 antibodies or CD33 antibodies linked to alternative toxins or radionuclides ( Table 1 ).

Table 1 Overview of CD33-directed therapeutics.

Therapeutic Characteristics Development stage Clinical results Comments
Unconjugated Antibodies        
 Lintuzumab (HuM195; SGN-33) Humanized IgG1 Ab Clinical (phase 3) Very modest activity as single agent; failed to improve survival when added to conventional chemotherapeutics Clinical development terminated in 2010
 MAb 33.1/BI 836858 Fully human IgG1 Ab engineered to have increased ADCC Clinical (phase 1) Not reported yet  
Antibody-drug conjugates        
 GO Humanized IgG4 Ab/calicheamicin-γ1 derivative Clinical (phase 3) Highly efficacious in APL and some activity in non-APL as single agent; improves survival when added to induction therapy in newly diagnosed AML Market withdrawal in most countries in 2010 for lack of efficacy and concern for toxicity
 AVE9633 (huMy9-6-DM4) Humanized IgG1 Ab/maytansinoid derivative Clinical (phase 1) Very modest activity as single agent Clinical development terminated
 HuM-195/rGel Humanized IgG1 Ab/recombinant gelonin Clinical (phase 1) Very modest activity as single agent  
 SGN-CD33A Humanized IgG1 Ab/PBD dimer Clinical (phase 1) Not reported yet Improved linker/conjugation technology; highly potent toxin with activity in GO-resistant AML models
 scFvCD33:sTRAIL Single-chain variable region Ab fragment/sTRAIL Preclinical --- Limited data suggesting better stability, activity, and selectivity than GO
Radiolabeled Antibodies        
 β-emitters (e.g. [131] I, [90] Y) Murine or humanized Ab/β-emitter Clinical (phase 1) Cytoreductive effects; can be given alone or in conjunction with transplant conditioning Logistically complex
 α-emitters (e.g. 225Ac, 213Bi) Lintuzumab/α-emitter Clinical (phase 2) Can be given safely; has anti-AML activity Logistically complex
Other        
 Bispecific Ab (e.g. AMG 330) 2 different scFvs fused in tandem via flexible linker to bring CD33 + cell to immune effector cell (e.g. via CD3) Preclinical --- Very high in vitro cytolytic activity
 CAR T-cell immunotherapy T-cells engineered to express CAR ± co-stimulatory endodomains Clinical (phase 1) Not reported yet Very high in vitro cytolytic activity

Abbreviations: Ab, antibody; ADCC, antibody-dependent cell-mediated cytotoxicity; APL, acute promyelocytic leukemia; CAR, chimeric antigen receptor; PBD, pyrrolobenzodiazepine; scFv, single-chain variable fragment; sTRAIL, soluble tumor necrosis factor-related apoptosis-inducing ligand.

4.1. Unconjugated antibodies

In vitro studies have demonstrated that crosslinking of CD33 on AML cells via bivalent antibodies can inhibit cell proliferation and induce apoptosis [72] and [73]. Nevertheless, the clinical results with unconjugated CD33 antibodies have so far been disappointing. In initial studies with an unconjugated murine anti-CD33 antibody (M195), only few patients had transient decreases in peripheral blast counts at a saturating or supra-saturating dose [35] . Subsequent studies employed a humanized version of M195, lintuzumab (HuM195; SGN-33), which had > 8-fold higher binding avidity than M195 and, unlike M195, demonstrated antibody-dependent cell-mediated cytotoxicity (ADCC) [36] and [74]. Still, while limited studies pointed toward some activity in APL when used in in patients with minimal residual disease [75] , lintuzumab has very modest activity as single agent in non-APL AML even at supra-saturating doses that fully blocked CD33 binding sites throughout a 4-week period, with the infrequent achievement of complete or partial remissions limited to patients with low tumor burden [76] and [77]. Efficacy can perhaps be increased if supra-saturating doses are given repeatedly, as suggested by a small trial in which very high doses of lintuzumab were given weekly for 5 weeks and then every other week for patients with clinical benefit [78] . In two larger randomized studies, however, lintuzumab failed to improve survival when added to mitoxantrone, etoposide, and cytarabine in adults with relapsed/refractory AML [79] or when added to low-dose cytarabine in older patients with untreated AML [80] , and the clinical development of this antibody was eventually terminated in 2010.

4.2. Antibody-drug conjugates

4.2.1. GO
4.2.1.1. Development and early clinical testing of GO

Because of the endocytic property of CD33, many efforts have been made to arm CD33 antibodies to improve their therapeutic efficacy. By far the biggest clinical experience has been with GO, a humanized IgG4 kappa CD33 antibody that is conjugated to a disulfide derivative of calicheamicin-γ1 (N-acetyl gamma calicheamicin dimethyl hydrazide) via a hydrolyzable linker to allow for rapid release of the toxic moiety under acidic conditions in lysosomes contained within the target tumor cell ( Fig. 2 ). Calicheamicins have been shown to bind in the minor grove of DNA and, after activation by cellular thiols, initiate single- and double-stranded breaks. This DNA damage elicits a strong cellular response with cell cycle arrest followed by either DNA repair or, if damage is overwhelming, apoptosis and cell death, predominantly through mitochondrial pathways with cytochrome c release, involvement of Bcl-2 family proteins, and caspase activation [58], [81], and [82].

gr2

Fig. 2 Schematic structure of GO. The humanized IgG4 CD33 antibody is conjugated to the calicheamicin-γ1 derivative via a hybrid 4-(4′-acetylphenoxy)butanoic acid linker. GO has approximately 50% of the antibody loaded with 4–6 mol of the toxic moiety per mole of antibody; the remaining 50% of the antibody molecules are unconjugated. source: Reprinted from Current Opinion in Pharmacology [165] with permission from Elsevier.

GO was given accelerated U.S. marketing approval in 2000 for treatment of adults > 60 years of age with CD33+ AML in first relapse who were not candidates for standard cytotoxic chemotherapy. Approval was based on interim data from 3 open-label, multicenter single-arm trials showing an overall response rate (complete remission [CR] and CR with incomplete platelet count recovery [CRp]) of almost 30% in 142 adults with CD33+de novo AML in first relapse who typically received two 9 mg/m2 doses of GO 14 days apart [3] and [83]; this dose was chosen because it provided complete saturation of CD33 binding sites without dose-limiting non-hematologic toxicities in an earlier phase 1 study [84] . The final report on 277 patients confirmed the early results in this patient population with an overall response rate of approximately 25%, although remission durations were relatively short [85] .

4.2.1.2. Phase 2 experience with GO

Subsequently, several phase 2 studies investigated GO in unselected patients with newly diagnosed and/or relapsed/refractory non-APL AML. While they confirmed single agent activity of GO, the overall response rates have usually not exceeded 25–35% and were occasionally quite disappointing, particularly in heavily pretreated patients [86], [87], [88], [89], [90], and [91]. Of note, in most of the non-APL AML studies, GO was given at 9 mg/m2 every 2 weeks. However, new CD33 binding sites continuously arise and surface CD33 levels return to pretreatment levels within 72 h after antibody administration [40] and [74]. Hence, repeated administration of lower, (near-) saturating doses of GO every 3 days may enhance intracellular drug accumulation. The Acute Leukemia French Association (ALFA) group used such a fractionated dosing schedule and showed it to have promising efficacy and acceptable toxicity [92] and [93], although no direct comparisons have been conducted to demonstrate superiority over the traditional administration schedule. In contrast to its limited effectiveness in non-APL AML, GO + all-trans retinoic acid (ATRA) produced CR and relapse-free survival rates similar to those seen with an anthracycline + ATRA in newly diagnosed APL [94] and [95], while single agent GO was found to routinely eliminate evidence of APL in patients with molecular relapse of APL [96], [97], and [98]. The distinctive sensitivity of APL cells to GO likely results from the disease's CD33-rich nature and lack of significant drug transporter activity.

Because GO is subject to drug pump-mediated extrusion from cells, several studies have combined GO with agents that block drug efflux, but these as well as other studies combining GO with conventional therapeutics or alternative types of chemosensitizers have been hampered by small sample sizes and absence of control groups, and have produced mixed results that are difficult to interpret [86], [87], [88], [89], [90], [91], and [99].

4.2.1.3. Randomized trials of GO as add-on to intensive AML induction chemotherapy

A clearer picture has emerged from 5 large, randomized studies conducted in Europe and the U.S. that have investigated GO as an addition to conventional chemotherapy in adults with newly diagnosed AML. In 4 of the studies (MRC/NCRI AML15 and AML16, ALFA0701, and GOELAMS AML 2006 IR), the use of GO during induction resulted in statistically significantly improved survival in similar subsets of patients [100], [101], [102], and [103], particularly those with “favorable” prognosis AML (as defined by core-binding factor translocations) or those with normal cytogenetics, the majority of whom have been reproducibly shown to live longer if given GO. GO also improved outcome when combined with FLAG-Ida [100] , suggesting that GO was not acting merely as a non-specific means of intensification of standard-dose cytarabine-containing induction therapy. There was no effect of GO on survival in the 5th trial, SWOG S0106 [104] . Unlike the European trials, however, in which identical doses of conventional chemotherapeutics were used in the +/− GO arms, S0106 used a lower anthracycline dose with GO than the control arm, possibly accounting for some of the differences in outcomes between these trials. Furthermore, even in S0106 longer survival was seen in patients with core-binding factor AML. A recent individual patient meta-analysis of these 5 randomized trials indicated that the addition of GO significantly reduced the risk of relapse (hazard ratio [HR] = 0.80 [95% confidence interval, 0.72–0.89], P = 0.00006), leading to improved relapse-free survival (HR = 0.84 [0.76–0.94], P = 0.001) and overall survival (HR = 0.89 [0.82–0.97], P = 0.01) despite a slightly greater early mortality (P = 0.08). In contrast to the improvement in survival, the addition of GO did not change the remission rates during induction. As suggested by the individual studies, there was a highly significant interaction between the treatment effect and cytogenetic risk group, with the benefit of GO being primarily seen in patients with favorable-risk disease (HR = 0.50 [0.33–0.77], P = 0.001) and, to a lesser degree, intermediate-risk disease (HR = 0.85 [0.76–0.96], P = 0.007) but not those with adverse-risk disease (HR = 1.04 [0.86–1.25], P = 0.7) [105] . These findings in adult patients are complemented by recent data from a large randomized pediatric trial (COG-AAML0531) in over 1000 individuals < 30 years of age, in whom the addition of GO to conventional intensive chemotherapy was associated with a significantly improved event-free survival (HR = 0.83 [0.70–0.99], P = 0.04) and relapse-free survival (HR = 0.74 [0.60–0.93], P = 0.01) due to a reduction in relapse risk, whereas there was no difference in response rates or overall survival. In contrast to the adult trials, the benefit with GO was observed across all risk groups [106] .

4.2.1.4. Other randomized trials with GO

While these randomized studies strongly support a role of GO as addition to conventional intensive chemotherapy in many patients, the value of GO may be schedule-dependent, particularly in older individuals, as suggested by recent data from the EORTC/GIMEMA AML-17 study, in which sequential rather than concomitant use of GO and standard chemotherapy in patients aged 61–75 years with newly diagnosed AML provided no clear benefit as compared to standard chemotherapy alone, and was too toxic for those ≥ 70 years of age [107] . Furthermore, given a large randomized study that found a doubling of the CR rate but no survival advantage when GO was added to low-dose cytarabine, the utility of GO as an adjunct to therapy less intense than 3 + 7 remains in doubt [108] . Several randomized studies have also tested the value of GO during post-remission therapy in pediatric and adult patients, but so far no benefit has been demonstrated either when integrated into consolidation therapy or used alone as “maintenance” therapy [100], [104], [109], [110], and [111].

4.2.1.5. Withdrawal of GO from commercial market

Thus, experience with GO has been mixed. In fact, because of the lack of pre-specified overall improvement in outcome and slight increase in early deaths with GO in SWOG S0106, which was conducted to fulfill the post-approval commitment of the drug manufacturer to the U.S. Food and Drug Administration (FDA), the drug was withdrawn from the U.S. and European markets and is currently available in the U.S. only under a compassionate-use protocol. Several articles have appeared suggesting that this decision was misguided [112], [113], [114], and [115], unrealistically emphasizing an average result when AML's principal characteristic is its clinical and genetic/epigenetic heterogeneity. Unlike other countries, GO remains commercially available in Japan, where it has received full regulatory approval [116] .

4.2.2. Other toxin-loaded CD33 antibodies

While clinical experience with GO validates CD33 as a target for antibody-drug conjugates, the results with other toxin-loaded CD33 antibodies have been disappointing. For example, AVE9633 (huMy9-6-DM4), a humanized IgG1 CD33 antibody that is conjugated to a thiol-containing maytansinoid derivative, a potent tubulin inhibitor, was tested in 3 phase 1 studies in a total of 54 adults with refractory/relapsed AML. Only very modest activity was observed, and 2 of the 3 explored dosing schedules were discontinued early because of drug inactivity at doses significantly higher than CD33 saturation, and the clinical development of AVE9633 was subsequently terminated [117] . Similarly modest clinical activity was observed in initial studies with an immunoconjugate carrying recombinant gelonin (HuM-195/rGel) [118] .

4.3. Radiolabeled antibodies

Because of the radiosensitive nature of AML [119], [120], and [121], radionuclides are an attractive alternative to toxins to increase the efficacy of CD33 antibodies. Several studies have explored such antibodies as carriers for β- as well as α-emitters in AML, but no well-controlled studies have been conducted thus far, and it remains to be determined whether they can improve patient outcomes. Still, early studies using 131I – a radionuclide chosen for its ready availability, low cost, and simple radiochemistry – demonstrated the feasibility of using radiolabeled CD33 antibodies alone or as part of conditioning regimens for hematopoietic cell transplantation. When used alone, cytoreductive effects on AML cells were seen in many patients, and profound cytopenias occurred with higher doses of the radionuclide. In conjunction with conditioning regimens, remissions were achieved in the majority of patients transplanted without engraftment complications and with little toxicity related to the radioisotope except perhaps some increase in liver toxicity [35], [65], [68], [122], [123], and [124]. These investigations with 131I identified important shortcomings of radiolabeled CD33 antibodies, in particular the limited amount of radiation that could be delivered because of the low expression of CD33 – a limitation that could partly be overcome by repeated dosing in 2–3 day intervals [35] and [74] – and the short-lasting intracellular radionuclide retention [35], [37], and [122].

To reduce off-target toxicity and allow selective leukemia cell killing, e.g. in the setting of MRD, later studies explored the use of high energy, short-range α-emitters such as 213Bi or 225Ac, which decays with a half-life of 10 days and generates therapeutic, short-lived daughter α-particles (221Fr, 217At, and 213Bi). Despite similar internalization, bismuth radioimmunoconjugates are retained about 2–3 fold better than those containing [131] I, likely due to binding to transferrin and other intracellular proteins, and show efficient killing of CD33+ target cells with as little as 2 bismuth atoms bound per cell [125] and [126]. Initial trials with 213Bi or 225Ac radiolabeled CD33 antibodies indicate that they can be given safely and have anti-leukemia activity [66], [127], [128], and [129], and studies with a 225Ac-labeled antibody alone or with conventional chemotherapy are currently ongoing (NCT00672165, NCT01756677) [130] .

5. Limitations and toxicities of CD33-directed therapy

Immunotherapy of AML targeting CD33 has come of age. Undoubtedly, improved survival of subsets of patients observed with GO supports the concept that CD33 is a suitable target in AML, although it is currently unclear whether this benefit is due to the elimination of CD33+ AML stem cells (and is thus limited to leukemias arising from CD33+ AML stem cells) or due to efficient “debulking” of mature CD33+ progeny with eradication or control of underlying CD33- or CD33+ stem cells by other, e.g. immunological, means [58] and [82]. However, many attempts with unconjugated or armed antibodies have been disappointing, highlighting the challenges of utilizing CD33-directed anti-cancer therapeutics. Several factors may underlie the poor clinical activity of the agents that have been tested clinically thus far, with most important limitations depending on the specifics of the immunotherapeutic. For example, for unconjugated antibodies, CD33's relatively low cell surface density will in many cases limit ADCC. Preclinical studies with GO demonstrate that CD33 expression levels are also limiting for the activity of antibody-drug conjugates [41] . For toxin-loaded antibodies, however, this problem is compounded by non-uniform conjugation of the toxin with the antibody, as exemplified by GO, in which 50% of the antibody molecules remained unlabeled after the conjugation process, thereby limiting the amount of toxin deposited onto the surface of CD33+ cells [82] . The low target expression then combines with slow internalization kinetics of CD33/antibody complexes and further limits intracellular accumulation of antibody-delivered payloads. After their cleavage, toxins may become substrates for drug transporters (e.g. P-glycoprotein and MRP1 in the case of GO [82] ) and be extruded from the AML cell before they can exert any cytotoxic effects. Such limitations may explain why GO is ineffective in many patients even though a highly potent toxin is delivered [58] and [82]. And finally, for radiolabeled antibodies, the endocytic property of CD33 and subsequent metabolization shortens the time during which the CD33+ cell is exposed to the radionuclide and increases radiation to non-targeted tissues, complicating the clinical use of such constructs.

Because of the expression of CD33 on normal hematopoietic cells, myelosuppression is an expected toxicity of effective CD33-targeted therapy, as exemplified by the invariable development of neutropenia and thrombocytopenia seen with GO [85] . A characteristic, life-threatening adverse event with GO is sinusoidal obstruction syndrome (SOS; also known as veno-occlusive disease [VOD]), which is characterized clinically by tender hepatomegaly, portal hypertension, fluid retention, weight gain as well as ascites and encephalopathy at later stages [83], [85], [131], and [132]. SOS is more likely when the drug is given at higher doses or in combination with a hepatotoxic agent, and was observed in the initial studies most commonly when GO was used within a few months before allogeneic hematopoietic cell transplantation [133] and [134]. Its etiology remains somewhat elusive, although toxic effects on cells in hepatic sinusoids are likely underlying this pathological process: proposed mechanisms include exposure of these cells to the unconjugated calicheamicin-γ1 derivative, or uptake of GO by CD33+ Kupffer cells or other cell populations in the liver [135] , perhaps including hepatocytes which some studies have suggested to express CD33 [136] . Unlike myelosuppression, however, it is conceivable that SOS is an adverse event that is relatively specific to GO as it appears primarily related to the toxin utilized with this antibody-drug conjugate, and may thus not be seen to that degree with other CD33-directed therapeutics.

6. Emerging strategies to improve CD33-directed therapy

With the market withdrawal of GO in most countries, there is currently no CD33-directed drug commercially available for the treatment of AML. However, with the demonstration of survival improvement seen in many patients with GO, there is renewed interest in therapeutics that target CD33 ( Table 1 ).

6.1. Unconjugated antibodies

Efforts are ongoing to generate engineered unconjugated CD33 antibodies with increased affinity to CD16a (FcγRIIIa) that have better ADCC against human AML cells than lintuzumab [137] and [138]. A fully human, Fc-engineered IgG1 antibody with these properties (MAb 33.1, BI 836858) has recently entered clinical testing (NCT01690624).

6.2. Antibody-drug conjugates

Given the limitation with GO discussed above, obvious technological advancements over GO would include improvements in conjugation and linker technology as well as use of highly potent toxins that are poor substrates for drug transporters. These improvements have been implemented in SGN-CD33A, a humanized CD33 antibody with engineered cysteines that carries a synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker ( Fig. 3 ) [139] . Preclinical studies have demonstrated that SGN-CD33A is more potent than GO against human AML cell lines and primary AML cells and, unlike GO, maintains activity in models of multidrug resistant disease. Based on these results, SGN-CD33A has recently entered the early phase of clinical testing (NCT01902329). Several years ago, in vitro studies have also suggested that a CD33 single-chain variable fragment antibody linked to soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) could provide a stable immunoconjugate with better anti-AML activity than GO [140] , but so far no clinical experience with this or a similar construct has been reported.

gr3

Fig. 3 Schematic structure of SGN-CD33A. The humanized IgG1 CD33 antibody is engineered to contain a cysteine at position 239 on the heavy chain for site-specific drug loading (“h2H12ec”). This modification allows attachment of a PBD dimer via a maleimidocaproyl-valine-alanine dipeptide linker with high precision and homogeneity, averaging 1.9 PBD moieties per antibody. source: Reprinted from Blood [139] with permission from The American Society of Hematology.

6.3. Bispecific antibodies

A much-pursued strategy to improve the efficacy of anti-tumor antibodies is the use of bispecific constructs that also recognize an immune effector cell antigen such as CD3 on T-cells or CD16 on NK cells to harness the immune system in the elimination of cancer cells. Many types of such antibodies have been explored over the last 3 decades, but their clinical success was limited by suboptimal effector cell recruitment, requirement for high effector-to-target (E:T) ratios and antibody concentrations, and production challenges [141] and [142], As evidenced by recent data on T-cell-directed constructs, these shortcomings may not pertain to small bispecific antibodies that combine the minimal binding domains of the two different antibodies on one polypeptide chain. Binding the invariant epsilon subunit of CD3, they bring polyclonal CD3+ T-cells in close proximity of target tumor cells and force formation of an immunological lytic synapse that triggers lymphocyte activation and proliferation as well as serial destruction of attached tumor cells through perforin/granzyme-mediated induction of apoptosis at low E:T ratio in an HLA-independent manner [142], [143], and [144]. Therapeutic utility of these molecules for acute leukemias is suggested by emerging results from small studies with the CD19/CD3 BiTE (Bispecific T-cell Engager) antibody, blinatumomab, showing a 70–80% response rate and high relapse-free survival rates among adults with CD19+ ALL that persisted or relapsed after chemotherapy [145] and [146].

Developing small bispecific antibodies targeting CD33 for AML is a logical consequence extending from the clinical experience with GO and the encouraging data obtained with bispecific antibodies in ALL. As a first step toward that goal, we and others have recently demonstrated that a CD33/CD3-directed drug ( Fig. 4 ) built on the BiTE platform (AMG 330) that recognizes a linear epitope in the V-set Ig-like domain of CD33 is highly potent in causing cytolysis of CD33+ AML cell lines or primary human AML cells in the presence of healthy donor T-cells or autologous T-cells from AML patients at low E:T ratio in vitro and in immunodeficient mice [44], [46], [56], and [147]. Target antigen density, antibody dose, and E:T ratio were identified as critical determinants for the activity of AMG 330. In contrast, cytolysis was not affected by drug transporter activity and, unlike bivalent antibodies, AMG 330 did not modulate CD33 expression after continued exposure [44] . The activity of AMG 330 was also not significantly impacted by soluble CD33 [56] . Besides AMG 330, other CD33/CD3-targeting small bispecific antibodies have been generated and confirm the high in vitro efficiency of these constructs in redirecting human T-cells toward CD33+ AML cells [148] and [149]. None of these single chain constructs have entered clinical trials, and while they already appear very potent in preclinical studies, further improvements may be possible. For example, a modular targeting system that not only brings CD33+ AML cells together with CD3+ T-cells but also provides co-stimulation to redirected T-cells via CD137 (4-1BB) yielded more efficient cytolysis of AML cells expressing low levels of CD33 [150] . Moreover, several groups are exploring dual targeting of tumor cells (e.g. CD33/CD123 or CD33/CD19) [151] and [152] and dual targeting of immune effector cells (e.g. CD3 and NKG2D) [153] , and are investigating the use of other single immune effector cell antigens (e.g. CD16) [154], [155], and [156] instead of CD3.

gr4

Fig. 4 Single-chain bispecific CD33/CD3 antibody. Schematic structure of single-chain bispecific CD33/CD3 antibody and simplified proposed mode of action of drug-induced, T-cell mediated cytolysis. Abbreviation: Ab, antibody.

6.4. Chimeric antigen receptor (CAR) T-cell immunotherapy

An alternative strategy to bispecific antibodies to bring immune effector cells into juxtaposition with leukemia cells to hasten their elimination is adoptive immunotherapy using T-cells genetically engineered to express chimeric antigen receptors (CARs). CARs are hybrid single-chain receptor constructs containing an extracellular tumor antigen-recognizing domain linked to an intracellular component comprised of the CD3 zeta chain as primary domain with or without additional co-stimulatory endodomains (e.g. CD28, CD137, OX40, etc.) that, together, activate immune effector cells upon binding to the tumor antigen [157] . Complete clinical responses were observed in small series of ALL patients treated with T-cells engineered to express CARs recognizing CD19, providing proof-of-principle evidence for the potential value of this therapeutic strategy in acute leukemias [157] . Recent studies have demonstrated the feasibility of generating various types of T-cells modified to express CD33-directed CARs [158], [159], [160], and [161]. In vitro as well as in immunodeficient mice, such cells very efficiently reduce the burden of human AML cell lines and primary AML cells; however, not surprisingly, these cells have also high efficacy against normal hematopoietic progenitor cells [158], [160], and [161]. A first clinical trial testing CD33-directed CAR T-cell immunotherapy is currently ongoing (NCT01864902).

7. Conclusion and future challenges

The survival of some patients with AML has substantially improved over the last 3–4 decades. However, this success is largely due to advancements in supportive care, while the drugs themselves have changed little [162], [163], and [164]. Nevertheless, among the few new drugs that have shown benefit is GO, demonstrating the validity of selecting CD33 as therapeutic target in this disease. Considering the significant limitations of this immunoconjugate with regard to drug labeling and extrusion of the toxic moiety by drug transporters, it is likely that the true potential of CD33-based therapies has not yet been reached. Emerging preclinical results with several novel CD33 antibody-based therapeutics suggest that they are less limited by low CD33 abundance or drug transporter activity, and might be active against a much broader subset of AMLs than GO. It is thus conceivable that these agents could prove valuable in a high proportion of AML patients. As made abundantly clear from the GO experience, efforts should be made from the beginning to identify the subsets that will most likely benefit from individual agents with less emphasis placed on mean survival in all patients.

With increasing activity against CD33+ cells, toxic effects on normal hematopoietic cells may increase. While the most primitive normal hematopoietic stem cells may be devoid of CD33, this antigen is widely expressed on hematopoietic progenitor cell populations. Some of these cells may have been protected from GO through low CD33 expression or drug transporters, leading to more limited and generally manageable cytopenias. Conceivably, patients may experience more prolonged cytopenias with CD33-targeted drugs that are highly effective even at very low CD33 expression levels and in cells with drug transporter activity. Effects of improved CD33-directed therapeutics on normal hematopoietic cells may therefore be substantially greater than those seen with GO and require excellent supportive care strategies or rescue with donor hematopoietic cells to minimize morbidity and mortality. Anticipated cytopenias may prove a particular hurdle for strategies employing T-cells engineered to express CD33-targeted CARs, as forming myeloid cells could be continuously destroyed, and may require the use of suicide genes or myeloablative conditioning to avoid permanent cytopenias in these patients. On the other hand, CD33 expression on activated T-cells or NK cells may pose a challenge for bispecific antibodies and, particularly, CAR-modified T-cells, although available data with AMG 330 suggest that the degree of CD33 neoexpression may be limited [56] .

Undoubtedly, the many new agents on the horizon denote a new era in the use of CD33-directed immunotherapies. Many questions will need to be addressed in future studies, but these therapeutics may offer new tools for a disease for which the outcomes remain generally unsatisfactory.

8. Practice points

 

  • CD33 is found on at least a subset of leukemic blasts in nearly all AML patients, and may be expressed on AML stem cells in some
  • CD33 has been explored as therapeutic target with unconjugated and armed antibodies for over 25 years
  • Numerous treatment attempts have been unsuccessful, but the improved survival seen in many patients when GO is added to conventional chemotherapy validates CD33 as therapeutic target in AML
  • Low antigen expression levels and slow antibody internalization offer challenges for the use of CD33 antibodies
  • Several novel CD33-targeted therapeutics that may overcome some of the limitation of earlier therapeutics are currently in preclinical and early clinical development

9. Research agenda

 

  • Which subset of AML patients is suitable for CD33-targeted therapy?
  • For which disease stage should CD33-targeted therapies be used?
  • Is CD33-directed therapy effectively eliminating AML stem cells in some patients?
  • What are anticipated resistance mechanisms to novel CD33-targeted therapeutics, and how can they be overcome?
  • How are novel CD33-directed agents best combined with conventional therapeutics?
  • How extensive will suppressive effects on normal hematopoiesis be, and what supportive care is needed to allow safe administration of highly effective CD33-targeted drugs?
  • Does soluble CD33 interfere with the efficacy of CD33-targeted therapeutics? If so, could this be overcome, e.g. by pretreatment with unconjugated CD33 antibody or use of external affinity columns?
  • Can CD33 expression be increased on AML cells pharmacologically in vivo, and can this augment the activity of CD33-directed therapeutics?
  • Can novel radionuclides improve the efficacy of radiolabeled CD33 antibodies?

Conflict of interest statement

R.B.W. has received research funding from Amgen, Inc., and Seattle Genetics, Inc., and has served as a consultant for Seattle Genetics, Inc. G.S.L. and E.H.E. declare no competing conflict of interest.

Acknowledgment

This work was supported by a grant from the Alex's Lemonade Stand Foundation (to R.B.W.). R.B.W. is a Leukemia & Lymphoma Society Scholar in Clinical Research.

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Footnotes

a Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

b Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA

c Department of Epidemiology, University of Washington, Seattle, WA, USA

lowast Corresponding author at: Clinical Research Division, Fred Hutchinson Cancer Research Center; 1100 Fairview Ave N, D2-190; Seattle, WA 98109-1024, USA. Tel.: + 1 206 667 3599; fax: + 1 206 667 5255.