Factors that Determine Sensitivity and Resistances of Tumor Cells Towards Antibody-Targeted Protein Toxins

Abstract

Recombinant immunotoxins are composed of antibody-derived targeting entities fused to truncated toxins. Pseudomonas toxins inactivate eEF2 by ADPribosylation and are potent antitumoral agents in clinical development. The sensitivity of tumor cells towards such fusion proteins, and hence their therapeutic efficacy, is influenced by multiple factors: (i) access to tumor cells, (ii) target antigen binding and internalization, (iii) entry into the cytosol, (iv) enzymatic modification of the intracellular target eEF2, and (v) induction of apoptosis. Parameters that affect these steps and hence modulate sensitivity include: (i) protein stability and immunogenicity, (ii) presence, density and internalization of target antigen, (iii) cellular factors involved in processing, routing or translocation of the toxin, (iv) factors involved in diphthamide synthesis on eEF2, and (v) factors that influence cellular susceptibility towards apoptosis. This chapter describes sensitivity or resistance factors for Pseudomonas exotoxin -derived immunotoxins that were identified experimentally and/ or observed in clinical studies.

Influence of DPH1 and DPH5 Protein Variants on the Synthesis of Diphthamide, the Target of ADPRibosylating Toxins

“…Promoter methylation of DPH1 and DPH4 genes correlated with reduced sensitivity of cells to targeted toxins that contain truncated derivatives of Pseudomonas Exotoxin A (PE) or DT as cytotoxic payload [13,14,15]. Also, recombinant tumor (and yeast) cells harboring DPH gene knockouts were resistant to PE, DT and immunotoxins, confirming the essentiality of DPH functionality for immunotoxin therapy [1,16,20,22]…”

“…Diphthamide is a conserved modified histidine on eukaryotic translation elongation factor 2 (eEF2) and the target of ADP-ribosylating Diphtheria toxin (DT) and Pseudomonas Exotoxin A (PE) [1,2,3,4,5]. Derivatives of these toxins are applied as cytotoxic payloads in targeted tumor therapy, their activity is strictly dependent on the presence of diphthamide [6,7,8,9,10].…”

“…Diphthamide is generated by the concert and successive action of seven proteins [1,15,16,17,18,19,20,21]: combined functionality of DPH1, DPH2, DPH3 and DPH4 is required to attach 3-amino-3-carboxypropyl (ACP) to H715 of human eEF2. This ACP-eEF2 becomes subsequently converted by the methyltransferase DPH5 to diphthine.…”

“…Only diphthamide-eEF2 serves as target for ADP-ribosylating toxins (even the ‘almost complete’ diphthine modification is not recognized by toxins). Therefore, individual functionality of each DPH enzyme influences the diphthamide status of cells and thereby the potency of targeted toxins: reduced activity or loss of DPH functionality renders tumor cells less susceptible or resistant to ADP-ribosylating toxins and tumor-targeted immunotoxins ([1,11,14,15,21,22], Figure 1a).…”

Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress

“…The post-translational diphthamide modification of eukaryotic translation elongation factor eEF2 is highly conserved in eukaryotes as well as in the archaeal eEF2 counterpart [1] , [2] , [3] , [4] , [5] . It consists of a histidine in elongation factor 2 (His 715 in human eEF2), modified by the concert action of diphthamide synthesis enzymes encoded by DPH genes DPH1–7 in humans, [6] , [7] , [8] , [9] , [10] , [11] , [12] .…”