Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression (original) (raw)
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Rapid optimization and prototyping for therapeutic antibody-like molecules
mAbs, 2013
Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. the design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. the challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (erbB3). A MM-141 proof-of-concept (poC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-erbB3 single-chain variable fragments (scFvs). these optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. these re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.
Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment
Protein Engineering Design & Selection, 2006
The chimeric anti-CD30 IgG 1 , cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys!Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.
Engineering antibodies for clinical applications
Trends in Biotechnology, 2007
Molecular engineering has contributed immensely to the clinical success of antibodies in recent years. The modular structure of antibodies has permitted their modification in numerous ways, to meet various clinical requirements. With the help of antibody engineering, it has been possible to modify the molecular size, pharmacokinetics, immunogenicity, binding affinity, specificity and effector function of antibodies. In addition, fusion proteins of antibodies with various proteins and peptides have yielded targeted biological modifiers, toxins and imaging agents. This review focuses on the recent trends in antibody engineering for improving their clinical utility.
Antibodies, 2019
Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational ...
Current progress in innovative engineered antibodies
Protein & Cell, 2017
As of May 1, 2017, 74 antibody-based molecules have been approved by a regulatory authority in a major market. Additionally, there are 70 and 575 antibodybased molecules in phase III and phase I/II clinical trials, respectively. These total 719 antibody-based clinical stage molecules include 493 naked IgGs, 87 antibodydrug conjugates, 61 bispecific antibodies, 37 total Fc fusion proteins, 17 radioimmunoglobulins, 13 antibody fragments, and 11 immunocytokines. New uses for these antibodies are being discovered each year. For oncology, many of the exciting new approaches involve antibody modulation of T-cells. There are over 80 antibodies in clinical trials targeting T cell checkpoints, 26 T-cellredirected bispecific antibodies, and 145 chimeric antigen receptor (CAR) cell-based candidates (all currently in phase I or II clinical trials), totaling more than 250 T cell interacting clinical stage antibody-based candidates. Finally, significant progress has been made recently on routes of delivery, including delivery of proteins across the blood-brain barrier, oral delivery to the gut, delivery to the cellular cytosol, and gene-and viral-based delivery of antibodies. Thus, there are currently at least 864 antibody-based clinical stage molecules or cells, with incredible diversity in how they are constructed and what activities they impart. These are followed by a next wave of novel molecules, approaches, and new methods and routes of delivery, demonstrating that the field of antibody-based biologics is very innovative and diverse in its approaches to fulfill their promise to treat unmet medical needs.
mAbs, 2020
The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables aviditydriven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementaritydetermining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant dietinduced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.
Engineering Antibodies for Clinical Applications in Cancer
Tumor Biology, 2004
The 'magic bullet' concept predicted over a century ago that antibodies would be used to target cancer therapy. Since then initial problems that were related to specificity, purity and immungenicity of antibody-based reagents have slowly been overcome due to developments in technology and increased knowledge. As a result, antibodies are in use for many clinical applications and now comprise the second largest category of medicines in clinical development after vaccines. For antibody-based cancer therapeutics the last 20 years have met with an explosion of knowledge about the biology of the disease and potential targets as well as new technology which allows cloning and manipulation of multifunctional antibody-based molecules. However, the focus still remains on developing therapeutics that will have potential for treating cancer in people and this is efficiently assessed in mechanistic clinical trials that feed back to the laboratory for further development. This review illustrates the mechanistic approach to making new molecules for antibody imaging and therapy of cancer. It is illustrated by examples of radioimmunotherapy and antibody-directed enzyme prodrug therapy developed by the authors.
Antibody engineering: facing new challenges in cancer therapy
Acta Pharmacologica Sinica, 2005
Antibody-based therapeutics are beginning to realize the promise enclosed in their early denomination as "magic bullets". Initial disappointment has turned into clinical and commercial success, and engineered antibodies currently represent over 30% of biopharmaceuticals in clinical trials. Recent structural and functional data have allowed the design of a new generation of therapeutic antibodies, with strategies ranging from complement-mediated and antibody-dependant cellular cytotoxicity enhancement to improved cytotoxic payloads using toxins, drugs, radionucleids and viral delivery. This review considers the structure of different types of recombinant antibodies, their mechanism of action and how their efficacy has been increased using a broad array of approaches. We will also focus on the additional benefits offered by the use of gene therapy methods for the in vivo production of therapeutic antibodies.
Design of therapeutic proteins with enhanced stability
Proceedings of the National Academy of Sciences, 2009
Therapeutic proteins such as antibodies constitute the most rapidly growing class of pharmaceuticals for use in diverse clinical settings including cancer, chronic inflammatory diseases, kidney transplantation, cardiovascular medicine, and infectious diseases. Unfortunately, they tend to aggregate when stored under the concentrated conditions required in their usage. Aggregation leads to a decrease in antibody activity and could elicit an immunological response. Using full antibody atomistic molecular dynamics simulations, we identify the antibody regions prone to aggregation by using a technology that we developed called spatial aggregation propensity (SAP). SAP identifies the location and size of these aggregation prone regions, and allows us to perform target mutations of those regions to engineer antibodies for stability. We apply this method to therapeutic antibodies and demonstrate the significantly enhanced stability of our mutants compared with the wild type. The technology described here could be used to incorporate developability in a rational way during the screening of antibodies in the discovery phase for several diseases.
Enhanced antibody half-life improves in vivo activity
Nature Biotechnology, 2010
The well-established role of FcRn in IgG turnover has been the foundation for Fc engineering efforts aimed at improving the pharmacokinetic properties of therapeutic antibodies 1,2. Despite contrary results about the relationship between FcRn affinity and half-life 3,4 , several such efforts at pharmacokinetic engineering in nonhuman primates, whose FcRn is similar to that of humans, have demonstrated that engineered antibody variants have a prolonged half-life 5-8. Yet, although the successful extension of half-life in pharmacokinetic experiments bodes well for the prospect of improving clinical dosing, a critical gap remains. For half-life extension technologies to be of practical use, efficacy of a biotherapeutic with longer half-life must be preserved at longer dosing intervals. Although the relationship between drug exposure and efficacy is well-established, this correlation has not thus far been established for antibodies engineered for longer half-life. We coupled rational design methods with high-throughput protein screening to engineer a series of Fc variants with greater affinity for human FcRn. Variants were constructed in the context of the humanized anti-vascular endothelial growth factor (VEGF) IgG1 antibody bevacizumab 9 (Avastin), which is currently approved for the treatment of colorectal, lung, breast and renal cancers. A description of the construction, production and binding studies of the antibodies is provided in Supplementary Methods. As FcRn binds IgG at the lower pH of the early endosome (pH 6.0-6.5) but not at the higher pH of blood (pH 7.4), we used Biacore to screen antibodies for binding to human FcRn at pH 6.0. Our engineered variants demonstrated between 3-and 20-fold greater binding to FcRn at pH 6.0, with improvements enhanced antibody half-life improves in vivo activity