Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

Abstract

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

Fig. 1A

Schematic representation of the structures of Pseudomonas aeruginosa exotoxin A (ETA) and an ETA-based single-chain antibody-toxin. ETA consists of an N-terminal cell binding domain (Ia), an internal translocation domain (II), and a C-terminal enzymatic domain (III) facilitating ADP-ribosylation of eukaryotic elongation factor 2. By replacement of domain Ia with a scFv antibody fragment, chimeric antibody-toxins with novel target cell specificity are derived. B Specificity of scFv(FRP5)-ETA antibody-toxin. ErbB2-overexpressing SKBR3 and ErbB2-negative MDA-MB468 human breast carcinoma cells were incubated for 24 h with 1 μg/ml ErbB2-specific scFv(FRP5)-ETA (lower panels) or PBS (upper panels) before analysis by light microscopy. C Antimetastatic activity of scFv(FRP5)-ETA in vivo. Balb/c mice were injected intravenously with 10⁵ murine renal carcinoma (Renca) cells stably transfected with lacZ and human c-erbB2 constructs (RLZ/ErbB2) at day 0. Animals were treated i.v. with 20 µg/dose of scFv(FRP5)-ETA from days 1–10. Control animals received PBS. Mice were sacrificed at day 28, lungs were excised and analyzed for pulmonary metastasis upon X-gal staining [27]

Fig. 2A

Schematic representation of the T-cell receptor (TCR) complex. The TCR consists of the α/β dimer recognizing antigenic peptides presented by MHC molecules. Complexed with the TCR are the invariant chains of the CD3 complex CD3γ, CD3δ, CD3ε, and the ζ chains. The invariant chains couple the antigen-recognizing TCR proteins to intracellular signaling molecules such as the ZAP-70 protein tyrosine kinase (PTK) that binds to phosphorylated immunoreceptor tyrosine–based activation motifs (boxed). B Single-chain chimeric antigen receptor consisting of a target-cell specific scFv antibody fragment comprising antibody heavy (VH) and light chain (VL) variable domains fused via a flexible hinge region to transmembrane and intracellular regions of the TCR-associated ζ chain. Alternative designs for chimeric antigen receptors are mentioned in the text

Fig. 3A–D

Specific killing of ErbB2-overexpressing tumor cells by NK-92 natural killer cells expressing an ErbB2-specific chimeric antigen receptor. Human SKBR3 breast carcinoma cells were incubated with ErbB2-specific NK-92-scFv(FRP5)-ζ (A, C) or parental NK-92 cells (B, D) at a low effector to target ratio of 1:1. Cells were analyzed by light microscopy after incubation at 37°C for 90 min (A, B) and 29 h (C, D). Adherent cells represent SKBR3. NK-92 and NK-92-scFv(FRP5)-ζ cells are characterized by their smaller size and rounded shape. E Influence of antigen receptor density on cell killing activity. Selected populations of NK-92 cells expressing different levels of ErbB2-specific antigen receptors that either employ murine or human ζ chains as signal-transducing elements were established by FACS and magnetic beads sorting. Cytolytic activity of isolated cell populations was then analyzed in europium (Eu³⁺) release assays. SKBR3 target cells were labeled with Eu³⁺ solution by electroporation followed by incubation for 2 h with NK-92-scFv(FRP5)-ζ cells (open symbols) or parental NK-92 transduced with empty vector (mock) at an effector to target (E/T) ratio of 5:1. Release of Eu³⁺ complexes into the medium was determined by fluorimetric analysis and served as a measure of target cell lysis