Correction: Panjideh et al. Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861. Toxins 2022, 14, 478.

The authors wish to make corrections to their paper [...].

Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861.

Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt's lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies.

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed.

Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma-based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T-cell antigen CD3 that efficiently prevents tumor growth in a mouse B-cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B-cell non-Hodgkin and lymphocyte-predominant Hodgkin lymphoma, as well as on a subset of CD4(+) T cells known as follicular T-helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co-stimulation-independent activation of CD8(+) and CD4(+) T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN-γ, TNF-α, IL-6 and IL-10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B-cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody-based therapies in the treatment of B-cell malignancies.

Targeted tumor therapy with a fusion protein of an antiangiogenic human recombinant scFv and yeast cytosine deaminase.

In adults, endothelial cell division occurs only in wound healing, during menstruation, or in diseases such as wet age-related macular degeneration or development of benign or malignant tissues. Angiogenesis is one of the major requirements to supply the fast developing tumor tissue with oxygen and nutrients, and enables it to spread into other tissues far from its origin. We selected the extradomain B (ED-B), a splice variant of fibronectin, which is exclusively expressed in ovaries, uterus, during wound healing, and in tumor tissues, as a target for the development of an innovative antiangiogenic, prodrug-based targeted tumor therapy approach. We designed a fusion protein termed L19CDy-His, consisting of the antibody single chain fragment L19 for targeting ED-B and yeast cytosine deaminase for the conversion of 5-fluorocytosine into cytotoxic 5-fluorouracil. We purified high amounts of the fusion protein from Pichia pastoris that is stable, enzymatically active, and retains 75% of its activity after incubation with human plasma for up to 72 hours. The binding of L19CDy-His to ED-B was confirmed by an enzyme-linked immunosorbent assay and quantified by surface plasmon resonance spectroscopy determining a KD value of 81±7 nM. L19CDy-His successfully decreased cell survival of the murine ED-B-expressing teratocarcinoma cell line F9 upon addition of the prodrug 5-fluorocytosine. Our data demonstrate the suitability of targeting ED-B by L19CDy-His for effective prodrug-based tumor therapy.

Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer.

In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131I labeled A33scFv::CDy. 131I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [131I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [131I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [131I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [131I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5-fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.

Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants.

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting.

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.

Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer.

Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDS-polyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.