Immunoconjugates and new molecular targets in hairy cell leukemia

Abstract

Hairy cell leukemia (HCL) is a B-cell malignancy that in its classic form is exquisitely sensitive to single-agent purine analog therapy, but that is associated in many patients with late relapse and eventual purine analog resistance. Minimal residual disease, which is present in most patients achieving complete remission with purine analogs, retains Ags that are ideal for targeted therapy. Rituximab, which targets CD20, is active as a single agent, particularly if combined with purine analogs. Recombinant immunotoxins targeting either CD25 or CD22 and containing truncated Pseudomonas exotoxin have achieved major responses in relapsed/refractory HCL. Moxetumomab pasudotox in phase 1 testing achieved responses in 86% of such patients (complete in 46%) without dose limiting toxicity and often without MRD. Soluble CD22 has been used for improved detection and monitoring of HCL, particularly the poor-prognosis variant that lacks CD25. Ig rearrangements unique for each HCL patient have been cloned, sequenced, and followed by real-time quantitative PCR using sequence-specific reagents. Analysis of these rearrangements has identified an unmutated IGVH4-34-expressing poor-prognosis variant with immunophenotypic characteristics of either classic or variant HCL. The BRAF V600E mutation, reported in 50% of melanomas, is present in > 85% of HCL cases that are both classic and express rearrangements other than IGVH4-34, making HCL a potential target for specific inhibitors of BRAF V600E. Additional targets are being defined in both classic and variant HCL, which should improve both detection and therapy.

Figure 1.. Intoxication of CD22⁺ B cells by moxetumomab pasudotox.

The recombinant immunotoxin is composed of VH and VL (green), which are disulfide bonded together by cysteines replacing Arg44 of VH and Gly100 of VL. The carboxy terminus of VH is fused to the toxin. VH contains a mutation of SSY to THW at positions 100, 100a, and 100b, respectively. The toxin PE38 is composed of domain 2 (amino acids 253–364, yellow), 1a (amino acids 381–399, not shown), and the catalytic domain 3 (amino acids 400–613, red). After the variable domains bind to CD22, the immunotoxin internalizes into a coated pit by endocytosis, domain 2 is proteolytically cleaved between Arg279 and Gly280 by Furin, the carboxy terminus of the toxin traffics to the endoplasmic reticulum using the KDEL receptor (light green), and then, once in the cytosol, the enzymatic domain within domain III ADP ribosylates EF2, leading to protein synthesis inhibition and apoptotic cell death.

Figure 2.. Domains of PE.

The domains of PE include the binding domain (1a); amino acids 1–252; and domains II, Ib, and III, as noted in Figure 1. C3 is a connector (ASGGPE) between VH and the toxin. To allow recombinant immunotoxins to bind selectively to target cells, the VH and VL domains of the mAb are connected to a truncated form of the toxin devoid of its binding domain.

Figure 3.

Response of HCL to moxetumomab pasudotox in phase 1 testing.