Omid Vafa

Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge.

The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fcγ receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the “nick” of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fcγ family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.

Avidity confers FcγR binding and immune effector function to aglycosylated immunoglobulin G1.

Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc‐mediated immune effector functions. Human IgGs have an N‐glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N‐linked glycosylation site has been reported to “silence” FcγR‐binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen‐engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes. Copyright

Leveraging SBDD in protein therapeutic development: antibody engineering.

Antibodies make up the largest, growing segment of protein therapeutics in the pharmaceutical and biotechnology industries. The development or engineering of therapeutic antibodies is based to a large extent on our knowledge of antibody structure and requires sophisticated methods that continue to evolve. In this chapter, after a review of what is known about the structure and functional properties of antibodies, the current, state-of-the-art antibody engineering methods are described. These methods include antibody humanization, antigen-affinity optimization, Fc engineering for modulated effector function and extended half-life, and engineering for improved stability and biophysical properties. X-ray crystallographic structures of antibody fragments and their complexes can play a critical role in guiding and, in some cases, accelerating these processes. These approaches represent guidelines for developing antibody therapeutics with the desired affinity, effector function, and biophysical properties.

Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells

BackgroundAmidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein.MethodsTo make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway.ResultsPAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate.ConclusionsPeptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM.