RATIONALEAccurate quantification of methionine oxidation in therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS) is challenging due to the potential artifacts introduced during sample preparation and analysis in the peptide mapping workflow. In this study, a systematic approach for optimization of the peptide mapping procedure to achieve reliable quantification of endogenous methionine oxidation in monoclonal antibodies was developed.METHODSThe approach is based on usage of a stable-isotope-labeled reporter peptide, identical in sequence to the tryptic peptide of an IgG1 monoclonal antibody containing the methionine residue most prone to oxidation. This approach was applied to evaluating various desalting procedures, and tested on nanoLC/MS, microLC/MS and UPLC/MS for the peptide mapping analysis of a model monoclonal antibody IgG1 sensitive to oxidation.RESULTSSeveral steps in the peptide mapping procedure with LC/MS detection at which protein oxidation occurred were identified and optimized using the reference stable-isotope-labeled peptide. Thus, reliable quantification of methionine oxidation in the target monoclonal antibody was validated.CONCLUSIONSThe methodology which utilizes the reference stable-isotope-labeled reporter peptide is applicable to monoclonal antibody oxidation analysis and could be extended to other biotherapeutics once oxidation-prone methionine(s) in the protein sequence are identified. Copyright © 2016 John Wiley & Sons, Ltd.

While phosphate-buffered saline (PBS) is commonly used in research and early development, significant freezethaw instability of PBS-formulated biotherapeutics has been established; crystallization of dibasic sodium phosphate is known as a major driver for degradation. The authors have identified an approach to stabilize PBSbased formulation for a monoclonal antibody (mAb). The effect of sodium chloride, nonionic surfactant, protein concentration, polyols, and freezing rates on freeze-thaw stability of a mAb was studied in 12 PBS-based formulations using six different freezing protocols. In formulations not containing polyols, aggregation, the primary degradation pathway, was observed for all freezing protocols, except freezing at -20 °C. Statistical analysis indicated high salt concentration as the most significant destabilizing factor followed by slow freezing at 1 °C/min, while higher protein concentration and polysorbate-80 had a stabilizing effect. Formulations containing polyols displayed no increase in aggregation for all freezing protocols. The antibody formulated in PBS containing polyols demonstrated stability on freeze-thaw stress at higher concentrations of both sodium chloride and protein. However, for polyol-containing formulations with low concentrations of both protein and salt, an increase in % polydispersity (due to submicron particle formation) was found by dynamic light scattering. The study demonstrates a systematic approach to stabilize PBS-formulated mAbs against freeze-thaw degradation.

While phosphate-buffered saline (PBS) is commonly used in research and early development, significant freeze-thaw instability of PBS-formulated biotherapeutics has been established; crystallization of dibasic sodium phosphate is known as a major driver for degradation. The authors have identified an approach to stabilize PBS-based formulation for a monoclonal antibody (mAb). The effect of sodium chloride, nonionic surfactant, protein concentration, polyols, and freezing rates on freeze-thaw stability of a mAb was studied in 12 PBS-based formulations using six different freezing protocols. In formulations not containing polyols, aggregation, the primary degradation pathway, was observed for all freezing protocols, except freezing at -20 degrees C. Statistical analysis indicated high salt concentration as the most significant destabilizing factor followed by slow freezing at 1 degrees C/min, while higher protein concentration and polysorbate-80 had a stabilizing effect. Formulations containing polyols displayed no increase in aggregation for all freezing protocols. The antibody formulated in PBS containing polyols demonstrated stability on freeze-thaw stress at higher concentrations of both sodium chloride and protein. However, for polyol-containing formulations with low concentrations of both protein and salt, an increase in % polydispersity (due to submicron particle formation) was found by dynamic light scattering. The study demonstrates a systematic approach to stabilize PBS-formulated mAbs against freeze-thaw degradation.

Phosphate buffered saline (PBS) is a physiologically suitable formulation vehicle often used for biologics in research and preclinical studies, but it is notorious for freeze-thaw instability. Here, the authors report differential scanning calorimetry (DSC) analysis of PBS-based formulations of a monoclonal antibody (mAb) designed to prevent protein aggregation upon frozen storage. Flash freezing at -90 degrees C followed by slow heating at 2 degrees C/min drastically increased the sensitivity of the method and enabled detection of a transition temperature (Tg*) occurring immediately after the glass transition temperature (Tg'). A Tg* as low as -73.6 degrees C was detected for the mAb solution in PBS, which decreased by 25 degrees C with increase in protein concentration from 5 to 20 mg/mL. Lowering the concentration of sodium chloride (NaCI) to 30 mM dramatically increased Tg* by 40 degrees C. Addition of polyols also resulted in a marked increase in Tg* value, likely due to blending of miscible excipients. To the authors' knowledge, the current study is the first report on DSC of a mAb formulated in PBS to demonstrate an effective means of increasing glass transition temperature, making frozen storage of mAbs in PBS-based formulations kinetically stable at temperatures of practical use. The results provide a mechanistic understanding of the behavior of PBS-based compositions on freeze-thaw stress and long-term frozen storage (as reported in Part I and Part II of this series, respectively).

BACKGROUND:Staphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.METHODS:To develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.RESULTS:The high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.CONCLUSION:We have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.

A human monoclonal antibody that is recombinantly expressed and specifically binds to the granulocyte and monocyte colony stimulating factor (GM-CSF), wherein said antibody comprises a heavy chain comprising a CDR1 having a SEQ No. ID: 40, a CDR2 with a SEQ ID NO: 41, a CDR3 with a SEQ ID NO: 42, as well as a light chain comprising a CDR1 with a SEQ ID NO: 43, a CDR2 with a SEQ ID NO: 44 and a CDR3 with a SEQ ID NO: 45, and the binding affinity of said human anti-GM-CSF antibody has a dissociation constant (KD) of less than 1x10-10 M.

The genomics era has provided valuable information on the content of the human genome, including the structure and chromosomal location of many disease-associated loci. This wealth of information has resulted in the identification of novel targets that are amenable to biopharmaceutical product development, leading to an overall enhanced pace of therapeutic development for a broad array of disease indications. Historically, small chemical entities have been designed as therapies to gene products that are encoded by intracellular proteins, enzymes and channels. With the advent and development of biologically based (protein- and cell-based) entities (BBEs) therapies, it is now possible to create molecules that have exquisite specificity for disease targets and spare unwanted pharmacologic activity against normal tissues. In addition to the specificity, BBEs generally have lower toxicity profiles when compared with small chemical entities, making biologically-based therapeutic approaches even more attractive. One of the difficulties that has been encountered with BBEs is the ability to produce compounds with maximal pharmacologic activity as well as establishing systems that can manufacture these complex biological molecules in sufficient quantities to meet clinical demand. This review discusses a broad enabling platform technology called morphogenics that can rapidly yield robust systems that can overcome present manufacturing shortfalls as well as accelerate the development of highly efficacious BBEs from those with insufficient pharmacological activity.

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.

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.

La presente invention concernes des lignees d'hybridomes secretant des anticorps monoclonaux humains avec une haute specificite de liaison et d'activite biologique, notamment une activite neutralisante contre le facteur de stimulation de colonies de granulocytes, et des procede de generation des lignees d'hybridomes. L'invention concerne egalement des antigenes et des epitopes cibles. Les anticorps peuvent etre utilises dans des procedes therapeutiques, par exemple, dans le traitement du cancer, de maladie infectieuse, et de maladie autoimmunitaire.

Endosialin/TEM1 was originally discovered as a human embryonic fibroblast-specific antigen and was later found to be differentially expressed in tumor stroma and endothelium. Endosialin/TEM1 overexpression has been observed in many cancers of various tissue origin, including colon, breast, pancreatic, and lung. The knockout (KO) mouse model showed the absence of endosialin/TEM1 expression reduced growth, invasion, and metastasis of human tumor xenografts. In addition, lack of endosialin/TEM1 led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels. This abnormal angiogenic response could be responsible for the reduced tumor growth and invasion observed in endosialin/TEM1 KO mice, suggesting a role for endosialin/TEM1 in controlling the interaction among tumor cells, endothelia, and stromal matrix. Here we report the identification of fibronectin (FN) and collagen types I and IV as specific ligands for endosialin/TEM1. More importantly, cells expressing endosialin/TEM1 exhibit enhanced adhesion to FN as well as enhanced migration through matrigel, although these properties could be blocked by a humanized antibody directed against human endosialin/TEM1. Our results pinpoint to a molecular mechanism by which expression of endosialin/TEM1 in the tumor stroma and endothelium may support tumor progression and invasion.

654 Background: Mesothelin is a glycosyl-phosphatidyl inositol-linked membrane protein that is consistently over-expressed in pancreatic cancer and mesothelioma, and some ovarian, non-small cell lung and colorectal cancers. Mesothelin is involved in cellular attachment and binds to MUC16. MORAb-009 is an IgG1κ monoclonal antibody directed against mesothelin with demonstrated bimodal anti-tumor activity on mesothelin-bearing tumor cells, through prevention of adhesion of mesothelin bearing tumor cells to MUC16 positive cells, and through cytotoxic immune-effector functions. In preclinical studies, MORAb-009 has shown anti-tumor activity in xenograft models. Methods: MORAb-009 was tested in Antibody Dependent Cellular Cytotoxicity (ADCC) assays and heterotypic cell adhesion assays. For the ADCC assay, mesothelin bearing target cells were incubated with MORAb-009 or control antibody. Normal peripheral blood mononuclear cells (PBMC) were added in defined ratios. The amount of LDH release was measured to determine the percent of cytotoxicity. Non-mesothelin bearing target cells were used as a negative control. In the heterotypic cellular adhesion assay, mesothelin-bearing cells were loaded with fluorescein and allowed to bind to a confluent monolayer of either MUC16 positive or negative cells, in the presence of MORAb-009 or irrelevant control antibody. The amount of bound fluorescing cells was quantitated. MORAb-009 was also tested in xenograft experiments against mesothelin-bearing human tumors. Results: In ADCC experiments, MORAb-009 was very effective in mediating cellular cytotoxicity. Appropriate controls demonstrated that the effect was specific for MORAb-009 and mesothelin. In the heterotypic cell adhesion assay, MORAb-009 effectively blocked mesothelin-expressing cells from binding to a MUC16 positive monolayer. (See figure). In mouse xenograft experiments, MORAb-009 had anti-tumor effect as a single agent and caused dramatic tumor regressions in combination with gemcitabine. Non- human primate toxicology studies did not demonstrate any toxicity. Conclusions: MORAb-009, a monoclonal antibody against mesothelin, has preclinical activity against mesothelin positive tumor cells in mediating ADCC, preventing adhesion to MUC16 positive monolayers, and in xenograft experiments. MORAb-009 is entering clinical studies in the treatment of mesothelin positive tumors. Preliminary data will be presented.

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.