Michaela Arndt

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody

Despite the availability of antiviral chemotherapy, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections remain a severe global health problem. Of particular concern is the growing incidence of drug resistance in immunocompromised patients, which stresses the urgency to develop new effective treatment alternatives. We have developed a humanized monoclonal antibody (mAb hu2c) that completely abrogates viral cell-to-cell spread, a key mechanism by which HSV-1/2 escapes humoral immune surveillance. Moreover, mAb hu2c neutralized HSV fully independent of complement and/or immune effector cell recruitment in a highly efficient manner. Prophylactic and therapeutic administration of mAb hu2c completely prevented infection-related mortality of severely immunodeficient mice being challenged with a lethal dose of HSV-1. The high neutralization capacity of mAb hu2c was fully maintained toward clinical HSV isolates being multiresistant to standard antiviral drugs, and infection was fully resolved in 7/8 nonobese diabetic/SCID mice being infected with a multidrug resistant HSV-1 patient isolate. Immunohistochemical studies revealed no significant cross-reactivity of the antibody toward human tissues. These features warrant further clinical development of mAb hu2c as an immunotherapeutic compound for the management of severe and particularly drug-resistant HSV infections.

Targeted therapeutic RNases (ImmunoRNases)

Immunotoxins combining antibody specificity with bacterial or plant toxins are limited by their strong immunogenicity and non-specific toxicity. Ribonucleases of the RNase A protein superfamily provide a solution to address these issues because they show potent antineoplastic activity on cell internalization but do not show appreciable immunogenicity or non-specific toxicity. Their therapeutic value is demonstrated by RNase derived from the frog (Rana pipiens), Onconase® (ONC, Alfacell, Inc., New Jersey, USA), the first and only RNase being evaluated in clinical trials at present. Conjugation or fusion of RNases to tumor specific antibodies is a promising approach to further boost tumor cell killing of these compounds. This review focuses on ‘targeted RNases/ImmunoRNases’ as promising novel anticancer therapeutics.

Generation of a highly stable, internalizing anti-CD22 single-chain Fv fragment for targeting Non-Hodgkin's lymphoma

The generation of a single chain Fv (scFv) fragment derived from the anti‐CD22 monoclonal antibody LL2 resulted in a molecule with good antigen binding but very poor stability properties, thus hampering its clinical applicability. Here we report on the construction of an engineered LL2 scFv fragment by rational mutagenesis. The contribution of uncommon wild‐type sequence residues for providing stability to the conserved common core structure of immunoglobulins was examined. Aided by computer homology modeling, 3 destabilizing residues within the core of the wild‐type VH domain were identified. Owing to the conserved nature of the buried core structure, mutagenesis of these sites to respective consensus residues markedly stabilized the molecule but did not influence its antigen binding properties: the engineered scFv MJ‐7 exhibited exceptional biophysical stability with a half‐life not reached after 6 days of incubation in human serum at 37°C, while fully retaining the epitope specificity of the monoclonal antibody, and antigen binding affinity of the wild‐type scFv. Furthermore, both the monoclonal antibody LL2 and the engineered scFv fragment became fully internalized after only 30 min of incubation at 37°C with CD22+ tumor cells. These properties predict scFv MJ‐7 could become a novel powerful tool to selectively deliver cytotoxic agents to malignant CD22+ cells.

A dimeric angiogenin immunofusion protein mediates selective toxicity toward CD22+ tumor cells.

To improve selective cytotoxicity and pharmacokinetics of an anti-CD22 antibody single chain Fv (scFv)-ribonuclease fusion protein, a dimeric derivative was generated. Human angiogenin was fused via a (G4S)3 spacer peptide to the carboxy-terminal end of the stable dimeric anti-CD22 VL-VH zero-linker scFv MLT-7. The dimeric fusion protein and a monovalent counterpart were produced as soluble proteins in the periplasm of Escherichia coli. Comparative studies with homogenously purified fusion proteins revealed that both constructs specifically bound to the target antigen and retained ribonucleolytic activity. However, they exhibited a markedly different capability for killing CD22+ tumor cells. The monomeric construct inhibited protein synthesis of target cells in a dose-dependent manner, but 50% inhibition (IC50) could be achieved only at the highest tested concentration (>350 nM). In contrast, the dimeric fusion protein efficiently killed CD22+ Raji and Daudi tumor cell lines with IC50 values of 74 nM and 118 nM, respectively. These results show that the therapeutic potential of scFv-ANG fusion proteins can be markedly enhanced by engineering dimeric derivatives.

Ribonucleases and immunoRNases as anticancer drugs.

Ribonucleases degrade RNA, now considered an important drug target. The parent member of this protein superfamily is bovine pancreatic RNase A that functions as a digestive enzyme. Other physiological roles and activities have been ascribed to more recently discovered members of this superfamily. Angiogenin was isolated by following angiogenic activity from cell culture media conditioned by colon cancer cells. ONCONASE kills tumor cells in vitro and in vivo and has advanced to a phase IIIb confirmatory clinical trial for the treatment of unresectable malignant mesothelioma. All three of these RNA degrading enzymes have been used to generate immunoRNases; chemical conjugates and ligand-RNase fusion proteins, for cancer therapy. The properties of each of these RNases are described along with the increasingly sophisticated construction of recombinant immunoRNases. The advantages of using RNase as an antibody payload is compared to using plant or bacterial toxins in the construction of immunotoxins, a related strategy for specifically killing malignant cells.

Antibody-Targeted RNase Fusion Proteins (ImmunoRNases) for Cancer Therapy

Ribonucleases (RNases) of the superfamily A exhibit potent antineoplastic activity yet do not mediate appreciable immunogenicity or non-specific toxicity in both animal models and cancer patients. Ranpirnase (Onconase), the first ribonuclease being evaluated as a therapeutic in humans, has progressed to phase III clinical trials in patients with unresectable mesothelioma. Conjugation of RNases to internalizing tumor-targeting monoclonal antibodies was shown to enhance specific cell killing by several orders of magnitude both in vitro and in animal models. In this review we describe the development and current status of genetically engineered 2(nd) generation immunoRNases as promising novel anti-cancer therapeutics.

A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies.

Application of recombinant antibodies in cancer patients.

As a consequence of the invention of the hybridoma technology by Kohler and Milstein (1), many monoclonal antibodies (MAbs) have been evaluated in clinical trials since the early 1980s. Clinical outcomes were generally poor (2–5), with the notable exception of marked tumor responses, including long-term remissions of patients with malignant B-cell lymphoma who were treated with patient-specific antiidiotypic antibodies (6–8). The main factors responsible for these initial shortcomings were related to the immunogenicity of the murine protein, to modulation of targeted antigens, and to the poor ability of these antibodies to sufficiently mediate antibody-dependent effector functions in humans. The advent of recombinant antibody technology led to an enormous revival in the use of antibodies as therapeutic agents in cancer therapy. This review provides a brief historical sketch of the development of recombinant antibodies for immunotherapy of cancer, which is followed by the most significant clinical data, as exemplified by the two clinically most established recombinant antibodies to date. Finally, we will focus on future prospects for antibody-based therapeutic concepts in oncology.