Immunogenicity of Immunotoxins Containing Pseudomonas Exotoxin A: Causes, Consequences, and Mitigation

Abstract

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Keywords: B cell epitopes; LMB-100; T cell epitopes; anti-drug antibodies (ADA); moxetumomab pasudotox; neutralizing antibodies; recombinant immunotoxins.

Figure 1

Models and structural models of conjugated and recombinant immunotoxins. (A) OVB3-PE is composed of a mouse IgG chemically conjugated to full length Pseudomonas exotoxin A (PE) using a linker. (B) LMB-1 consists of a mouse IgG chemically conjugated via a lysine residue to a 40 kDa fragment of PE that contains domain II (gray), domain III (yellow), and domain Ib (not shown). (C) SS1P consists of the disulfide-stabilized (ds) heavy chain Fv (V_H; magenta) and light chain Fv (V_L; cyan) of the antibody fragment SS1. The V_H is linked to PE38 that contains domains II and III but Ia is deleted. (D) Lysosome resistance (LR) immunotoxin. The ds-Fv of SS1P is linked to a 24-kDa fragment consisting of domain III of PE38 (termed PE24) (E) SS1-LO10R. A 24-kDa fragment of PE24 with 6-point mutations in domain III designed to suppress binding to B cell receptors. Point mutations are marked with red balls. (F) LMB-100 consists of a humanized Fab linked to LO10R PE24 toxin fragment and 6 point mutations as in E. (G) LMB-T20. PE24 with 6-point mutations in domain III designed to diminish T cell epitopes. (H) LMB-T14. PE24 with 10-point mutations in domain III designed to diminish B and T cell epitopes. All images are based on the structures of native PE and IgG. Images (C–H) were adapted from Mazor et al. (37).

Figure 2

Impact of ADA on maximal concentration of LMB-100 (C_max) in blood of patients. C_max and ADA measurements were performed as described in (25). C_max and ADA results were obtained from (25). C_max values were log transformed and fitted to an asymmetric sigmodial, 5-parameter curve fit. Dotted line represents EC₅₀.

Figure 3

Approaches to mitigate immunogenicity of PE-based recombinant immunotoxins.

Figure 4

Overlap of experimental T cell epitopes and in silico HLA binding prediction. Twenty three T cell epitope were identified in PE38 by experimental T cell activation assays using 50 PBMC donors. In silico predicted binders in PE38 were predicted using the IEDB consensus HLA class II binding algorithm and 15 HLA-DR alleles. A peptide was considered a potential epitope using a threshold of (A) top 30 predicted binders and (B) top 56 predicted binders (50%). Peptides 8 and 9 were false negatively predicted using both thresholds.