Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy

Abstract

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

Keywords: LMB-2; Tregs; anticancer therapy; denileukin diftitox; fusion protein toxins; immunotherapy; melanoma; targeted toxins.

Figure 1.. Cartoon depicting antigens and receptors abundant on Treg cells.

CD3: T cell co-receptor which associates with the T cell receptor (TCR) and ζ-chain (zeta-chain) to generate the TCR complex that leads to T cell activation; CD4: Glycoprotein originally known as leu-3 or T4; CD25: IL-2 receptor alpha chain; CTLA4: Cytotoxic T-lymphocyte-associated protein 4 (also known as CD152); FoxP3, forkhead box protein P3; GITR, glucocorticoid-induced TNFR-related protein; LAG3: Lymphocyte-activation gene 3; PD1: Programmed cell death protein 1 (also known as CD279); TIM3: T-cell immunoglobulin and mucin-domain containing-3; TIGIT: T cell immunoreceptor with Ig and ITIM domains.

Figure 2.. Current strategies to block or inhibit tumor-associated Treg cells.

(A) In humans, an anti-CD25 mAb, daclizumab, has been used to deplete Treg cells. More recently, the recombinant diphtheria toxin-IL-2 fusion protein denileukin diftitox (Ontak^®) has been used to target T cells with high CD25 expression. Upon internalization, diphtheria toxin irreversibly inhibits protein synthesis, ultimately triggering cell death. (B) Inhibition of Treg via targeting of CTLA-4, PD-1, OX40 or GITR alleviates the suppressor activity of these cells on effector CD8 cells. (C) A peptide inhibitor of Foxp3 known as P60-D2A-S5A impairs immunosuppressive activity of murine and human-derived Treg cells leading to enhanced effector T cell function. (D) Treg cells produce adenosine via catabolism of ATP by extracellular ectonucleotidases CD39 and CD73. Adenosine is an immunosuppressive metabolite that may participate in the immunosuppressive activity of Foxp3+ T cells. Inhibitors of CD39 (POM1 and IPH2) and of CD73 (MEDI9447 and BMS-986179) are depicted in red. (E) TLR ligands can directly reverse Treg cell suppressive function.

Figure 3.. Cartoon depiction of domains present in Treg-targeting bacterial fusion toxins.

(A) LMB2 is a Treg-targeting fusion protein comprised of a single-chain Fv fragment of an anti-CD25 monoclonal antibody fused to a truncated form of Pseudomonas exotoxin A. The 252 amino acid anti-CD25-targeting single chain Fv fragment, anti-Tac-dsFv, is shown in green. The 361 amino acid Pseudomonas exotoxin A fragment, PE38, comprising domain II (membrane translocation) and domain III (ADP-ribosyltransferase catalytic domain) of exotoxin A is shown in blue. (B) The Treg-cell targeting diphtheria toxin-CCR4 single-chain fold-back diabody fusion protein is shown. The fusion protein is comprised of the N-terminal 390 amino acids of diphtheria toxin which contain the ADP-ribosyltransferase catalytic domain and the transmembrane domain (shown in blue) fused to a dimeric, codon optimized anti-human CCR4 scFv shown in green. The bivalent scFv is comprised of paired VL (variable light chain) VH (variable heavy chains) separated by single or repeated pentapeptide linkers (G₄S or Gly₄Ser) as shown. (C) The structures of DAB389IL-2 (denileukin diftitox or Ontak^®) and DAB389IL-2(V6A) are shown. These Treg-targeting fusion toxins contain the N-terminal 390 amino acids of diphtheria toxin with its ADP-ribosyltransferase catalytic domain and transmembrane domain (shown in blue) fused to IL-2 (green) which is the cognate ligand of CD25. DAB389IL-2(V6A) is a second-generation version of DAB389IL-2 which harbors a valine-to-alanine substitution at residue 6 (V6A) within vascular leak syndrome motif 1 and elicits significantly reduced levels of vascular leak in vitro and in animal models.

Figure 4.. Cartoon depictions of the mechanism of action of s-DAB389IL-2 and s-DAB389IL-4 which are produced as secreted proteins using Corynebacterium diphtheriae and are soluble, fully monomeric versions of DAB389IL-2 and DAB389IL-4.

Cartoon depiction of Treg-targeting action of s-DABIL-2 (targeting the IL-2 receptor, CD25) and the MDSC-, TAM-, and tumor-targeting action of s-DABIL-4 (targeting the IL-4 receptor, CD124 [Parveen et al., unpublished data]). Both targeted toxins bind to their cognate receptor, undergo receptor-mediated endocytosis with vesicular acidification and subsequently deliver their catalytic domains (red) to the cytosol of the targeted cell. The catalytic domain, a potent ADP-ribosyltransferase of elongation factor-2 (EF-2), enzymatically shuts down protein synthesis leading to apoptotic cell death.

Figure 5.. Antitumor effects of s-DABIL-2(V6A)-His₆ when used as monotherapy and as dual sequential therapy with anti-PD1 in the mouse B16-F10 syngeneic melanoma tumor model.

Groups C57BL/six mice (eight mice per group) were inoculated with B16-F10 murine melanoma cells on day 0. They were treated with intraperitoneal doses on the indicated days of: (A) anti-PD1 isotype control antibody, (B) 10 μg doses of purified s-DABIL-2(V6A)-His₆, abbreviated V6A (C) 100 μg doses of anti-PD1 antibody or (D) a sequential combination of purified s-DABIL-2(V6A)-His₆ followed by anti-PD1 antibody. Tumor volume was measured with electronic callipers.