Engineering humanized antibody framework sequences for optimal site-specific conjugation of cytotoxins.

The prevailing techniques used to generate antibody-drug conjugates (ADCs) involve random conjugation of the linker-drug to multiple lysines or cysteines in the antibody. Engineering natural and non-natural amino acids into an antibody has been demonstrated to be an effective strategy to produce homogeneous ADC products with defined drug-to-antibody ratios. We recently reported an efficient residue-specific conjugation technology (RESPECT) where thiol-reactive payloads can be efficiently conjugated to a native unpaired cysteine in position 80 (C80) of rabbit light chains. Deimmunizing the rabbit variable domains through humanization is necessary to reduce the risk of anti-drug antibody responses in patients. However, we found that first-generation humanized RESPECT ADCs showed high levels of aggregation and low conjugation efficiency. We correlated these negative properties to the phenylalanine at position 83 present in most human variable kappa frameworks. When position 83 was substituted with selected amino acids, conjugation was restored and aggregation was reduced to levels similar to the chimeric ADC. This engineering strategy allows for development of second-generation humanized RESPECT ADCs with desirable biopharmaceutical properties.

Generation of therapeutic immunoconjugates via Residue-Specific Conjugation Technology (RESPECT) utilizing a native cysteine in the light chain framework of Oryctolagus cuniculus.

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 used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several 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.

The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity.

Because of its high mortality rate, ovarian cancer is a leading cause of death among women and a highly unmet medical need. New therapeutic agents that are effective and well tolerated are needed and cancer antigen-specific monoclonal antibodies that have direct pharmacologic effects or can stimulate immunological responses represent a promising class of agents for the treatment of this disease. The human folate receptor α (FOLR1), which is overexpressed in ovarian cancer but largely absent in normal tissues, appears to play a role in the transformed phenotype in ovarian cancer, cisplatin sensitivity, and growth in depleted folate conditions and therefore has potential as a target for passive immunotherapy. The anti-FOLR1 monoclonal antibody MORAb-003 (farletuzumab) was previously shown to elicit antibody dependent cellular cytotoxicity (ADCC) and inhibit tumor growth of human tumor xenografts in nude mice. Because of its promising preclinical profile, farletuzumab has been evaluated in clinical trials as a potential therapeutic agent for ovarian cancer. In this report, we demonstrated that farletuzumab's antitumor effect against an experimental model of ovarian cancer is mediated by its ADCC activity.

Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin.

Novel therapeutic agents that are safe and effective are needed for the treatment of pancreatic, ovarian, lung adenocarcinomas and mesotheliomas. Mesothelin is a glycosyl-phosphatidyl inositol (GPI)-linked membrane protein of 40 kDa over-expressed in all pancreatic adenocarcinoma and mesothelioma, in >70% of ovarian adenocarcinoma, and in non-small cell lung and colorectal cancers. The biological functions of mesothelin are not known, although it appears to be involved in cell adhesion via its interaction with MUC16. We have recently developed MORAb-009, a mouse-human chimeric IgG1kappa monoclonal antibody with an affinity of 1.5 nM for human mesothelin. Here we provide evidence that MORAb-009 prevents adhesion of mesothelin-bearing tumor cells to MUC16 positive cells and can elicit cell-mediated cytotoxicity on mesothelin-bearing tumor cells. Treatment that included MORAb-009 in combination with chemotherapy led to a marked reduction in tumor growth of mesothelin-expressing tumors in nude mice compared to chemotherapy or MORAb-009 treatment alone. No adverse effects of MORAb-009 were noted during toxicology studies conducted in non-human primates. The preclinical data obtained from our studies warrants pursuing clinical testing of MORAb-009. We have in fact initiated a Phase I clinical study enrolling patients with mesothelin-positive pancreatic, mesothelioma, non-small cell lung and ovarian cancers.

Preclinical evaluation of MORAb-003, a humanized monoclonal antibody antagonizing folate receptor-alpha.

The highly restricted distribution of human folate receptor-alpha (FRalpha) in normal tissues and its high expression in some tumors, along with its putative role in tumor cell transformation, make this antigen a suitable target for antigen-specific, monoclonal antibody-based immunotherapy for oncology indications. We have developed a therapeutic humanized monoclonal antibody with high affinity for FRalpha, named MORAb-003, which was derived from the optimization of the LK26 antibody using a whole cell genetic evolution platform. Here we show that MORAb-003 possesses novel, growth-inhibitory functions on cells overexpressing FRalpha. In addition, MORAb-003 elicited robust antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro, and inhibited growth of human ovarian tumor xenografts in nude mice. Because of its multimodal activity in vitro and its safe toxicology profile in non-human primates, MORAb-003 development has recently been advanced to clinical trials involving ovarian cancer patients.

Human antibodies for immunotherapy development generated via a human B cell hybridoma technology.

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.

A controlled trial of intermittent enteral nutrient supplementation in maintenance hemodialysis patients.

OBJECTIVE: This controlled trial was undertaken to evaluate the benefits of short-term enteral nutrient supplementation in maintenance hemodialysis (MHD) patients using a high-calorie and high-protein blend formula (low-cost home-prepared [HP] blend or a commercially available supplement) and to study its effect on selected parameters of nutritional status. The acceptability and palatability of the HP blend formula, ease of use, and cost were also assessed in comparison with the commercial nutritional supplement (CNS). DESIGN: Randomized controlled trial. SETTING: Hemodialysis (HD) unit of a tertiary referral care hospital in Southern India. PATIENTS: Nondiabetic adult MHD patients with no intercurrent illness, on regular thrice weekly MHD for at least 1 month before recruitment, with a body mass index (BMI) <20 and a serum albumin level of <4.0 g/dL. Patients were randomized into control group and experimental group, the latter in turn to recieve either CNS or HP blend. INTERVENTION: The control group received appropriate monitoring, including dietary recall and counselling for the prescribed diet (protein intake of 1.2 g/kgIBW/d and energy of 35 to 45 kcal/kgIBW/d) but no specific post-HD supplement. Patients in the supplement group received the respective supplement post-HD (providing 500 kcal and 15 g protein) for 1 month in addition to the monitored diet prescription. MAIN OUTCOME MEASURES: (1) Nutritional status parameters, BMI and serum albumin; (2) functional status on a 10-point Karnofsky scale; (3) adverse metabolic effects, hyperphosphatemia at start and end of study; and (4) subjective scoring for appetite, and acceptability of and tolerance to supplement. RESULTS: Both groups showed an improvement in dry weight and BMI. In addition, the supplement group showed a significant increase in serum albumin level and functional scoring. Mild hyperphosphatemia occurred in the supplement group. An increase in baseline food intake was seen in the control group, but not in the supplemented group. No intolerance was reported to either supplement. CONCLUSION: Enteral nutrient supplementation was shown to bring about a significant improvement in serum albumin level even in a short-term study. Use of an HP supplement was beneficial, acceptable, and inexpensive.