Antibody engineering to improve manufacturability.

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

Functional premature polyadenylation signals and aberrant splicing within a recombinant protein coding sequence limit expression.

Recombinant glycoproteins can be produced at high levels in permanently transfected mammalian cells using expression vectors with strong viral promoters. CHO-K1 cell lines developed to produce the recombinant complement activator blocking protein, CAB-2 (a fusion of membrane co-factor protein, MCP, and decay accelerating factor, DAF), showed unexpectedly low expression. Northern blot analysis revealed that in addition to the expected 2300 base CAB-2 mRNA species, these cell lines expressed 790 and 1500 base mRNA species accounting for ~50% and ~10% of the total CAB-2 mRNA, respectively. RT-PCR studies established that the 1500 base species resulted from aberrant splicing from within the DAF region of the CAB-2 coding sequence to a site within the 3' untranslated region. 3' RACE analysis confirmed that the 790 base species resulted from premature polyadenylation at an AATAAA site within the MCP coding region of CAB-2. Another prematurely polyadenylated species, not observed on Northern blots, was observed in the DAF region by 3' RACE. Analysis of human tissues and cell lines revealed that these internal polyadenylation signals in native MCP and DAF coding regions also generated prematurely polyadenylated mRNAs. Genetic modification of these functional RNA processing elements within the CAB-2 gene eliminated the aberrant mRNA species and significantly increased recombinant CAB-2 expression. These results illustrate that protein expression can be limited by aberrant mRNA processing and demonstrate the importance of identifying and eliminating these mRNA processing signals from within coding DNA to maximize recombinant protein expression.

Kinetic approach to pathway attenuation using XOMA 052, a regulatory therapeutic antibody that modulates interleukin-1beta activity.

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.

An allosteric antibody to the leptin receptor reduces body weight and reverses the diabetic phenotype in the Lep(ob) /Lep(ob) mouse.

OBJECTIVE: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. METHODS: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. RESULTS: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. CONCLUSIONS: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency.

In vitro and in vivo pharmacology and pharmacokinetics of a human engineered monoclonal antibody to epithelial cell adhesion molecule.

ING-1(heMAb), a Human Engineered monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 microg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21.

rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin. rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23. Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus. To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids. A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI). These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure). Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities. In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21. In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease. We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.

Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

XOMA 052, a potent, high-affinity monoclonal antibody for the treatment of IL-1ß-mediated diseases.

Interleukin-1ß (IL-1ß) is a potent mediator of inflammatory responses and plays a role in the differentiation of a number of lymphoid cells. In several inflammatory and autoimmune diseases, serum levels of IL-1ß are elevated and correlate with disease development and severity. The central role of the IL-1 pathway in several diseases has been validated by inhibitors currently in clinical development or approved by the FDA. However, the need to effectively modulate IL-1ß-mediated local inflammation with the systemic delivery of an efficacious, safe and convenient drug still exists. To meet these challenges, we developed XOMA 052 (gevokizumab), a potent anti-IL-1ß neutralizing antibody that was designed in silico and humanized using Human Engineering™ technology. XOMA 052 has a 300 femtomolar binding affinity for human IL-1ß and an in vitro potency in the low picomolar range. XOMA 052 binds to a unique IL-1ß epitope where residues critical for binding have been identified. We have previously reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse model thought to be driven by low levels of chronic inflammation. We report here that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect of exogenously administered human IL-1ß and blocking peritonitis in a mouse model of acute gout. Based on its high potency, novel mechanism of action, long half-life, and high affinity, XOMA 052 provides a new strategy for the treatment of a number of inflammatory, autoimmune and metabolic diseases in which the role of IL-1ß is central to pathogenesis.