Antibody fusion proteins: anti-CD22 recombinant immunotoxin moxetumomab pasudotox

Abstract

Recombinant immunotoxins are fusion proteins that contain the cytotoxic portion of a protein toxin fused to the Fv portion of an antibody. The Fv binds to an antigen on a target cell and brings the toxin into the cell interior, where it arrests protein synthesis and initiates the apoptotic cascade. Moxetumomab pasudotox, previously called HA22 or CAT-8015, is a recombinant immunotoxin composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38-kDa fragment of Pseudomonas exotoxin A, called PE38. Moxetumomab pasudotox is an improved, more active form of a predecessor recombinant immunotoxin, BL22 (also called CAT-3888), which produced complete remission in relapsed/refractory hairy cell leukemia (HCL), but it had a <20% response rate in chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), diseases in which the leukemic cells contain much lower numbers of CD22 target sites. Compared with BL22, moxetumomab pasudotox is up to 50-fold more active on lymphoma cell lines and leukemic cells from patients with CLL and HCL. A phase I trial was recently completed in HCL patients, who achieved response rates similar to those obtained with BL22 but without dose-limiting toxicity. In addition to further testing in HCL, moxetumomab pasudotox is being evaluated in phase I trials in patients with CLL, B-cell lymphomas, and childhood ALL. Moreover, protein engineering is being used to increase its activity, decrease nonspecific side effects, and remove B-cell epitopes.

Figure 1. Intoxication of cells by moxetumomab pasudotox

On the left is an illustration of the structure of PE, PE38 and recombinant immunotoxin moxetumomab pasudotox designed to kill CD22-expressing cells. On the right is a cartoon showing the steps required for the entry and cell killing by moxetumomab pasudotox and similar recombinant immunotoxins containing PE38. After internalization, PE undergoes proteolysis and disulfide-bond reduction to separate the catalytic domain III from the binding domain Ia (, –61). PE38 undergoes both removal of the carboxy terminal lysine residue (18) and processing between residues 279 and 280, resulting in a 37 kDa carboxy terminal toxin fragment ending in the residues REDL. This fragment is believed to be transported intracellularly via the KDEL receptor from the Golgi to the ER (21), where it translocates to the cytosol, resulting in ADP-ribosylation of elongation factor 2 (9), leading to apoptotic cell death (25).

Figure 2. Recombinant immunotoxins in use or under development

BL22 was reported in 1997 (37) as RFB4(dsFv)-PE38 and was later called CAT-3888. VL and VH are disulfide-bonded together using engineered cysteine residues replacing Arg44 of VH and Gly100 of VL. In 2002, BL22 was mutated to HA22 (later called CAT-8015 and moxetumomab pasudotox) by changing SSY to THW at positions 100, 100a and 100b of VH (45), represented here by horizontal red bars. In 2009, the deletion mutant HA22-LR was reported where PE amino acids 251–394 were replaced by 274–284 containing the Furin cleavage site (50). Most recently, in 2011, HA22-LR-8M, a mutant of HA22-LR, was reported to contain 8 mutations, D406A, R432G, R467A, R490A, R513A, E548A, K590S and Q592A (59).

Figure 3. Structure of HA22-LR-8M

Ribbon structures show the locations of the mutations in HA22-LR-8M and the deletion from HA22 used in HA22-LR and HA22-LR-8M. Adapted from Onda et al. (59).