Highly efficient destruction of squamous carcinoma cells of the head and neck by photochemical internalization of Ranpirnase.

INTRODUCTION: Photochemical Internalization is a novel drug delivery technology for cancer treatment based on the principle of Photodynamic Treatment. Using a photosensitizer that locates in endocytic vesicles membranes of tumor cells, Photochemical internalization enables cytosolic release of endocytosed antitumor agents in a site-specific manner. The purpose of the present in-vitro study was to explore whether Photochemical Internalization is able to enhance the efficacy of Ranpirnase, a cytotoxic amphibian ribonuclease, for eradication of squamous cell carcinoma of the head and neck. METHODS: Cell viability was measured in 8 primary human cell lines of squamous cell carcinoma of the head and neck after treatment with Ranpirnase and Photochemical Internalization. For Photochemical Internalization the photosensitizer disulfonated tetraphenyl porphine was incubated with tumor cells followed by exposure to blue light (435 nm). RESULTS: Our study demonstrates significant enhancement of antitumor activity of Ranpirnase by Photochemical Internalization. Treatment responses were heterogeneous between the primary cancer cell lines. Combining Photochemical Internalization with Ranpirnase resulted in 4.6 to 1,940-fold increased cytotoxicity when compared with the ribonuclease alone (P < 0.05). CONCLUSION: Cytotoxicity of Ranpirnase can be markedly enhanced by Photochemical Internalization in squamous cell carcinoma of the head and neck.

High treatment efficacy by dual targeting of Burkitt's lymphoma xenografted mice with a (177)Lu-based CD22-specific radioimmunoconjugate and rituximab.

PURPOSE: Dual-targeted therapy has been shown to be a promising treatment option in recurrent and/or refractory B-cell non-Hodgkin's lymphoma (B-NHL). We generated radioimmunoconjugates (RICs) comprising either a novel humanized anti-CD22 monoclonal antibody, huRFB4, or rituximab, and the low-energy ß-emitter (177)Lu. Both RICs were evaluated as single agents in a human Burkitt's lymphoma xenograft mouse model. To increase the therapeutic efficacy of the anti-CD22 RIC, combination therapy with unlabelled anti-CD20 rituximab was explored. METHODS: The binding activity of CHX-A″-DTPA-conjugated antibodies to target cells was analysed by flow cytometry. To assess tumour targeting of (177)Lu-labelled antibodies, in vivo biodistribution experiments were performed. For radioimmunotherapy (RIT) studies, non-obese diabetic recombination activating gene-1 (NOD-Rag1 (null) ) interleukin-2 receptor common gamma chain (IL2rγ (null) ) null mice (NRG mice) were xenografted subcutaneously with Raji Burkitt's lymphoma cells. (177)Lu-conjugated antibodies were administered at a single dose of 9.5 MBq per mouse. For dual-targeted therapy, rituximab was injected at weekly intervals (0.5 - 1.0 mg). Tumour accumulation of RICs was monitored by planar scintigraphy. RESULTS: Conjugation of CHX-A"-DTPA resulted in highly stable RICs with excellent antigen-binding properties. Biodistribution experiments revealed higher tumour uptake of the (177)Lu-labelled anti-CD22 IgG than of (177)Lu-labelled rituximab. Treatment with (177)Lu-conjugated huRFB4 resulted in increased tumour growth inhibition and significantly longer survival than treatment with (177)Lu-conjugated rituximab. The therapeutic efficacy of the anti-CD22 RIC could be markedly enhanced by combination with unlabelled rituximab. CONCLUSION: These findings suggest that dual targeting with (177)Lu-based CD22-specific RIT in combination with rituximab is a promising new treatment option for refractory B-NHL.

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.

Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity.

Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.

Impact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus.

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.

High-level production of a humanized immunoRNase fusion protein from stably transfected myeloma cells.

ImmunoRNases represent a highly attractive alternative to conventional immunotoxins for cancer therapy. Quantitative production of immunoRNases in appropriate expression systems, however, remains a major challenge for further clinical development of these novel compounds. Here we describe a method for high-level production and purification of a fully functional immunoRNase fusion protein from supernatants of stably transfected mammalian cells.

Preclinical evaluation of a diabody-based (177)Lu-radioimmunoconjugate for CD22-directed radioimmunotherapy in a non-Hodgkin lymphoma mouse model.

Radioimmunotherapy is considered as treatment option in recurrent and/or refractory B-cell non-Hodgkin lymphoma (B-NHL). To overcome the dose limiting bone marrow toxicity of IgG-based radioimmunoconjugates (RICs), we modified a humanized diabody with 5-, 10-, or 20-kDa polyethylene glycol (PEG) for CD22-targeted radioimmunotherapy using the low-energy ß-emitter lutetium-177 ((177)Lu). A favorable pharmacokinetic profile was observed for the 10-kDa-PEG-diabody in nude mice being xenografted with subcutaneous human Burkitt lymphoma. Even at high doses of 16 MBq this diabody RIC was well tolerated by NOD Rag1(null) IL2rγ(null) (NRG) mice and did not reveal signs of organ long-term toxicity 80 days post injection. Combination therapy of the diabody RIC with unconjugated anti-CD20 Rituximab demonstrated therapeutic efficacy in established disseminated mantle cell lymphoma xenograft models. When compared with the combination of the IgG formatted (177)Lu anti-CD22 antibody and Rituximab, dual targeted therapy with the diabody RIC achieved an improved reduction of disease burden in the first nine days following treatment. The data indicate that the PEGylated anti-CD22 diabody may have potential for extending the repertoire of radiopharmaceuticals for the treatment of patients with B-NHL.

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.

An EGF receptor targeting Ranpirnase-diabody fusion protein mediates potent antitumour activity in vitro and in vivo.

Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase(®)) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m(2). We have in the present study generated a dimeric anti-EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers.

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers.

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.

A fusogenic dengue virus-derived peptide enhances antitumor efficacy of an antibody-ribonuclease fusion protein targeting the EGF receptor.

Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.

Generation of "LYmph Node Derived Antibody Libraries" (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected "LYmph Node Derived Antibody Libraries" (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

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.

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.

Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme.

We report on the generation and functional characterization of a humanized immunoenzyme comprising a stable humanized single chain Fv (scFv) with grafted specificity of the anti-CD22 murine monoclonal antibody RFB4 and the human ribonuclease angiogenin (ANG). The fusion protein produced from transiently transfected mammalian Chinese hamster ovary cells could easily be purified to homogeneity, retained full ribonucleolytic activity, and efficiently killed CD22(+) tumour cells with an IC(50) of 56 nmol/l. In contrast, incubation of tumour cells with either ANG or scFv alone did not result in any cytotoxicity. Potent receptor-mediated killing of target cells, expected lack of extracellular toxicity, predictable low immunogenic potential, and ease of production, suggest that this novel immunoenzyme has potential for the immunotherapy of CD22(+) malignancies.

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 homogeneously 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.

Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein.

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22(+) tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V(L)36(Leu-->Tyr)) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K(D)=0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22(+) tumor cell lines with high efficacy (IC(50)=3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.

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 degrees 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 degrees 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.

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment.

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37 degrees C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.