Immunogenicity of therapeutic recombinant immunotoxins

Abstract

Recombinant immunotoxins (RITs) are chimeric proteins designed to treat cancer. They are made up of an Fv or Fab that targets an antigen on a cancer cell fused to a 38-kDa portion of Pseudomonas exotoxin A (PE38). Because PE38 is a bacterial protein, it is highly immunogenic in patients with solid tumors that have normal immune systems, but much less immunogenic in patients with hematologic malignancies where the immune system is suppressed. RITs have shown efficacy in refractory hairy cell leukemia and in some children with acute lymphoblastic leukemia, but have been much less effective in solid tumors, because neutralizing antibodies develop and prevent additional treatment cycles. In this paper we will (i) review data from clinical trials describing the immunogenicity of PE38 in different patient populations; (ii) review results from clinical trials using different immunosuppressive drugs; and (iii) describe our efforts to make new less-immunogenic RITs by identifying and removing T- and B-cell epitopes to hide the RIT from the immune system.

Keywords: anti-drug antibodies; immunotherapy; mesothelin; mesothelioma; neutralizing antibodies.

Figure 1. Structural models of RITs

The RIT SS1P consists of the disulfide-stabilized V_H and V_L polypeptide chains of the Fv from the antimesothelin monoclonal antibody SS1 coupled to a 38-kDa fragment of PE38. (A) SS1P. The Fv (cyan and magenta) is recombinantly connected to PE38, which is divided into domain II (gray), domain III (yellow), and part of domain Ib from native PE38. (B) SS1-LR-GGS. Deletion of domain II with GGS linker between the linker and the domain III. (C) SS1-LO10R. PE24 with six point mutations in domain III designed to eliminate binding to B cell receptor. (D) LMB-T20. PE24 with six point mutations in domain III designed to diminish T cell epitopes. All models are hypothetical arrangements based on the structures of native PE and immunoglobulin G; they do not represent actual structure determinations

Figure 2. Neutralizing antibody formation for PE38 RITs

Summary of immunogenicity rate during treatment cycles from nine clinical trials. Immunogenicity rate was evaluated using functional Nab assay with a cut point of 75% neutralization in all trials (17, 19, 22, 27, 28, 38) and unpublished data. (A) HCL (n=146), ALL (n=49) and CLL patients (n=50) treated with Moxetumomab paseudotox or BL22. (B) Patients treated with SS1P by bolus (QOD x 3) (n=21) or continues infusion (n=24) over the course of 10 days. (C) Immunogenicity of LMB-2 in hematological (n=35) and melanoma patients (n=7). (D) Immunogenicity in patients treated with SS1P as a monotherapy (n=21) or in SS1P combined with Pentostatin and Cyclophosphamide (n=11). All patients (with the exaptation of continues infusion group) were treated in a similar schedule of 3x QOD bolus per cycle, and 21-day intervals between cycles.

Figure 3. Antibody mediated response to protein immunogen

(Step 1) The RIT is engulfed by an APC and inside the APC, the RIT is processed in endosomal compartments and some high affinity peptides bind to HLA II molecules. The peptides- HLA class II complex is transported to the membrane of the APC and is presented. A specific T cell receptor recognizes the complex and interacts with the APC via co stimulatory signals resulting in T cell activation and differentiation. (Step 2) At the same time, B cells that have a B cell receptor that can recognize conformational epitopes on PE38 encounters PE38. Crosslinking of the B cell receptor initiates an activation cascade of the B cell and homing to the lymph node. The activated B cell also processes whole antigen and presents PE38 derived peptides. Once a cognate B and T_H cell meet co-activation will occur. T_H2 cells start secreting cytokines that help the B cell to activate. (Step 3) Activated B cell start affinity maturation and (Step 4) class switching and evolve to a memory or a secreting plasma cell.

Figure 4. Antigenicity of SS1P and SS1-LO10 to human antisera

Ratio of binding of SS1-LO10 to binding of SS1P from 20 patients treated with SS1P. p<0.0001, Wilcoxon signed rank test.

Figure 5. T cell activation of SS1P and LMB-T20

Stimulation of PBMC from 10 donors with LMB-T20 and SS1P show a decrease in T cell activation. PBMC from 10 naïve donors were stimulated with either SS1P or LMB-T20. After 14 days of in vitro expansion cells that were re-stimulated with either 111 peptides spanning the sequence of PE38 or 76 peptides spanning the sequence of LMB-T20. T cell activation was detected using IL-2 ELISpot. All positive peptides were assayed twice for each PBMC sample and each assay was run in triplicate. Response strength is shown in the Spot Forming cell/million cells (SFC/1E6) ladder on the right. Black stars represent peptides that were deleted in LMB-T20 and red stars represent peptides that are different from wild type. This figure to be published in Mazor et al., Mol. Cancer Res., In press (75).