Morphotek Technology

Generation of Therapeutic Immunoconjugates via Residue Specific Conjugation Technology RESPECT Utilizing a Native Cysteine in the Light Chain

The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be utilized for site-specific conjugation. An efficient process for the purification of highly conjugatable antibody was developed. The unpaired C80 was shown to be efficiently conjugated using a number of different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.

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Rapid High-throughput Cloning and Stable Expression of Monoclonal Antibodies

The rapid production of monoclonal antibodies (mAbs) isolated from an immunized organism is essential for the development of therapeutic and diagnostic reagents, and critical in the support of vaccine studies. Traditional methods to produce and screen mAbs, however, are inefficient due to genomic instability and low mAb expression of resulting cell lines. An ideal mAb cloning and expression platform would consist of a single, high-throughput, cloning step followed by rapid generation of a continuous source of recombinant mAbs. To this end, a protocol was developed to decrease the time, cost and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Throughput for the entire process-plasmid assembly through transient transfections and selection of stable cell lines-was increased by removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol, and utilizing bicistronic plasmids to generate stable, semi-clonal, cell pools during the stable cell-line process. These changes reduced the time required by an individual to clone, express, and select stable cell lines in a high-throughput format from 6 to 12 months to only 4 to 6 weeks.

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Novel Antibody Probes for the Characterization of Endosialin/TEM-1

Endosialin/TEM-1 (Tumor Endothelial Marker-1) is a cell surface protein whose restricted expression in malignant cells of mesenchymal origin and tumor-associated pericytes has resulted in its pursuit as a therapeutic target and marker for the diagnosis of cancer. The development of immune-based reagents that can target the various extracellular domains (ECD) of endosialin/TEM-1 from patient tissue and experimental systems may aid in further elucidating the role of this target in promoting and supporting tumorigenesis and optimize methods for best applying these agents to treat endosialin/TEM-1 positive cancers. Novel monoclonal antibodies (mAbs) were generated using full-length ECD region of the protein as an immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as molecular tools for functional studies as well as diagnostic reagents able to robustly detect endosialin/TEM-1 using an array of diagnotstic platforms and methods. A number of endosialin/TEM-1 specific monoclonal antibodies (mAbs) were generated that are able to target the distinct structural motifs within the ECD of the potein as well as detect rodent and human endosialin/TEM-1 orthologs. In addition, these reagents were used to generate a diagnostic assay that can detect soluble endosialin/TEM-1 (sEND) in the plasma of patients as a method for analyzing the steady-state levels of sEND in patients with or without disease as well as during therapeutic treatment.

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Enabling Freeze-Thaw Stablity of PBS-Based Formulations of a Monoclonal Antibody

Phosphate-buffered saline (PBS) is often used in research and development to formulate proteins, but is not typically a preferred vehicle for biopharmaceutical manufacturing due to freeze-thaw instability. Crystallization, as well as the rate and frequency of freeze-thaw cycles, are key drivers of PBS-formulated biopharmaceutical agent degradation, including for monoclonal antibodies. Given the critical need for stability in formulation development, a three-part study was conducted to explore potential agents to stabilize PBS-formulated biopharmaceutical agents under various freeze-thaw protocols. The study showed that the addition of polyols such as 5% sucrose and 3% sorbitol can inhibit aggregation during a single freeze-thaw cycle. Additionally, the study found that a high sodium chloride level and a slow freeze-thaw cycle were key drivers of PBS-formulated biopharmaceutical agent instability. Adding a nonionic surfactant, polysorbate-80, and increasing the protein level within the formulation aided stability during the freeze-thaw cycle. Incorporating these formulation and freeze-thaw protocol changes into the formulation deveopment process may potentially enable PBS-based biopharmaceutical agent formulations to be used from discovery to commercialization.

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Characterization of the Human Folate Receptor Alpha Via Novel Antibody-Based Probes

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full-length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration.

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Targeted Therapeutic Medicines

Importance of the field: The number of disease-associated protein targets has significantly increased over the past decade due to advances in molecular and cellular biology technologies, human genetic mapping efforts and information gathered from the human genome project. The identification of gene products that appear to be involved in supporting the underlying cause of disease has offered the biopharmaceutical industry an opportunity to develop compounds that can specifically target these molecules. This targeted therapy may, in turn, improve therapeutic responses and lower the risk of unwanted side effects that are commonly seen in traditional small chemical-based medicines.

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Monoclonal Antibodies: A Morphing Landscape for Therapeutics

The concept of using antibodies as therapeutics to cure human diseases was postulated nearly 100 years ago by Paul Ehrlich and subsequently enabled by the discovery of hybridoma technology by Kohler and Milstein in 1975. While the use of monoclonal antibodies (mAbs) as drugs that can specifically target a disease-associated antigen is compelling, it has taken a quarter century for these molecules to be adopted as bona fide therapeutic agents. Despite their slow pursuit in drug development during the pioneering years, it is now estimated that there are nearly 500 mAb-based therapies in development. Major factors that have influenced the acceptance of monoclonal antibodies as therapeutics include their drug safety profiles, technological advancements for facilitating mAb discovery and development, and market success.

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Whole-Genome Evolution Technology

Over the past decade, therapeutic proteins and monoclonal antibodies (mAbs) have become one of the most successful classes of pharmaceutical agents because they can replace or block specific targets associated with disease.

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Enhancing Therapeutic Antibodies and Titer Yields of Mammalian Cell Lines

The monoclonal antibodies (MAbs) represent the fastest growing segment of the biopharmaceutical market, with sales that totaled greater than US $3 billion in 2002 and have been projected to grow up to over $5 billion by 2005. A survey by the Pharmaceutical Research and Manufacturers of America of 144 companies lists 75 MAbs among the 371 biotechnology-based medicines currently in clinical development (1). The dramatic success of antibodies as therapeutics has created substantial value for companies developing such products (2, 3).

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Rapid Generation of Plant Traits via Regulation of DNA Mismatch Repair

The reversible inhibition of DNA repair is a novel approach to maximize genetic diversity within a plant’s genome in order to generate offspring exhibiting important de novo output traits. This process is based on the inhibition of the evolutionarily conserved mismatch repair (MMR) system. In this process, a human dominant negative MMR gene allele is introduced into the germline of a target plant, yielding progeny that can be screened to identify variants with commercially important agronomic output traits. Using this novel strategy, we generated MMR-deficient Arabidopsis thaliana plants that showed genome-wide instability of nucleotide repeats associated with chromosomal microsatellites, in addition to base substitution mutations.

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Morphogenics as a Tool for Target Discovery and Drug Development

Mutations in DNA mismatch repair (MMR) genes lead to genetically hypermutable cells. Germline mutations in MMR genes in man have been linked to the genetic predisposition to hereditary nonpolyposis colon cancer and a number of other inherited and sporadic malignancies. The ability to modulate the MMR process (referred to as morphogenics) in model systems offers a powerful tool for generating functional diversity in cells and multicellular organisms via the perpetual genome-wide accumulation of randomized point and slippage mutation(s).

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A Naturally Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator Phenotype

Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germline mutations in MMR genes predispose kindreds to HNPCC suggest a “two-hit” inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

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