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Targeted immunotherapy for hairy cell leukemia.

In Journal of Clinical Oncology, Kreitman et al report the efficacy of anti-CD22 monoclonal antibody (mAb) conjugated to Pseudomonas exotoxin (CAT-8015) in patients with relapsed/refractory hairy cell leukemia (HCL). More importantly, the study highlights important aspects related to the preclinical and clinical development of mAbtoxin conjugates as targeted therapies for patients with cancer. The first step when designing an antibody-based therapy is the rational selection of the target. In this regard, CD22, a member of the sialic acid–binding immunoglobulin-like lectin family of adhesion molecules, has several features that make it an effective target for treatment of HCL. First, CD22 is a lineage-restricted B cell–specific antigen that is expressed on the surface of mature B lymphocytes but is absent on other normal tissues, early progenitor B cells, and hematopoietic stem cells, which therefore limits on-target toxicity to the mature B-cell pool and reduces the risk of significant myelotoxicity. Second, CD22 is expressed on 60% to 80% of B-cell lymphomas and leukemias, but CD22 is most highly expressed in HCL cells, followed by marginal zone lymphoma and chronic lymphocytic leukemia cells. Third, antigen shedding is minimal with CD22, and circulating soluble CD22 does not seem to interfere with the cytotoxicity of immunotoxins. Initial attempts to target CD22 with unconjugated mAbs alone had modest therapeutic value in patients with B-cell malignancies. Because CD22 undergoes constitutive endocytosis, and ligation with anti-CD22 mAb markedly accelerates internalization (t1/2 1 hour), 8,9 unconjugated mAbs, which rely on complementand antibody-dependent cell-mediated cytotoxicity, seem inadequate to generate significant targeted cellular killing. By contrast, rapid internalization of mAb targeting CD22 makes it ideal as a target for immunotoxin-based therapy. Immunotoxins are typically composed of a variable domain derived from a monoclonal antibody fused to a bacterial or plant toxin, resulting in cell death when internalized (Fig 1). The immunotoxin used by Kreitman et al—RFB4(dsFV)-PE38—contains the variable heavy and light chains of the anti-CD22 monoclonal antibody RFB4 linked by a disulfide bond, fused via a peptide linker to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). The binding of the immunotoxin to CD22 leads to endocytosis of the immunotoxin/ CD22 complex. The translocation domain of the toxin is then processed, followed by binding of the carboxy terminus of the toxin to the intracellular KDEL receptor, which carries it to the endoplasmic reticulum. Finally, the toxin is translocated to the cytosol, where it causes cell death by at least two different mechanisms: ADP ribosylation of elongation factor 2, leading to protein synthesis inhibition, and activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase cleavage and resultant downregulation of antiapoptotic proteins. Once the ideal target and therapeutic strategies are identified, several factors need to be considered to successfully translate these therapies to the clinical setting. These include stabilization of the immunotoxin constructs, enhancement of target affinity, minimization of nontarget toxicities and immunogenicity of the toxins, and optimization of the cost and complexity of production, all of which are highlighted in the generation of the recombinant immunotoxin RFB4(dsFV)-PE38. To stabilize the variable domain of the antibody and to make it more suitable for clinical development, cysteine residues were engineered into framework regions of the variable heavy and light chains, which were then linked by a disulfide bond. Investigators at the National Cancer Institute further modified the mAb using phage display and hotspot mutagenesis of complementary-determining region 3 (CDR3) to generate an immunotoxin with 14-fold increase in affinity for CD22. Optimization of target affinity is a critical issue for CD22 for two reasons. First, the majority of surface CD22 on B cells is masked, unable to bind exogenous probes, but the limited accessibility of surface CD22 can be overcome by higher-affinity ligands. Second, CD22 expression level varies among the different B-cell malignancies, and CD22 expression density has been shown to be a determinant of response to anti-CD22 immunotoxins. However, using modified higher-affinity anti-CD22 immunotoxins, a 10to 100-fold increase in potency has been reported in primary CLL cells. Thus, phage display and hotspot mutagenesis techniques reliably generate a more active immunotoxin and represent a more cost-effective strategy to increase ligand affinity compared with codon-based mutagenesis, CDR walking, error-prone replication, and synthetic CDR construction. The identification and modification of the toxins are as critical as the selection of target when designing immunotoxins. The use of whole plant and bacterial toxins is largely limited by inefficient cellular processing, release of the toxin from the mAb, nonspecific binding to normal cells, and development of vascular leak syndrome. Subsequent generations of immunotoxins have been designed to contain only the elements required to kill tumor cells. The resultant immunotoxins, because of their relatively small molecular size, better penetrate tumor masses, reduce adverse effects by shortening the time to interact with nontarget tissues, and reduce the cost and complexity of production in large quantities. Lastly, to decrease immunogenicity caused by neutralizing antibodies against a portion of a bacterial protein and to allow repeated treatments, several approaches are currently being JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 15 MAY 2