Thierry Wurch

Strategies and challenges for the next generation of therapeutic antibodies

Antibodies and related products are the fastest growing class of therapeutic agents. By analysing the regulatory approvals of IgG-based biotherapeutic agents in the past 10 years, we can gain insights into the successful strategies used by pharmaceutical companies so far to bring innovative drugs to the market. Many challenges will have to be faced in the next decade to bring more efficient and affordable antibody-based drugs to the clinic. Here, we discuss strategies to select the best therapeutic antigen targets, to optimize the structure of IgG antibodies and to design related or new structures with additional functions.

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins

Importance of the field: Therapeutic properties of many glycoproteins strongly depend on the composition of their glycans. Most of the current approved glycoproteins are produced in mammalian cell lines, which yield mixture of different glycoforms close to the human one but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates. Many alternative glycoprotein productions systems are actively investigated including new-engineered yeast strains, as developed by GlycoFi, a biotech company fully owned by Merck & Co. since 2006. Areas covered in this review: The objectives of this opinion paper is to present a comprehensive overview of the technological breakthrough developed by GlycoFi to produce recombinant human proteins with controlled glycosylation patterns in yeast, in comparison to other glyco-engineering technologies and to discuss the application to large-scale manufacturing of biologicals. What the reader will gain: Research papers and recent review articles on protein glycosylation and glyco-engineering, and in-depth search of the bibliography by the GlycoFis research team, summary of recent meetings discussing the biosimilar topic were analyzed by the authors and will help the reader to gain insight in the field. Take home message: The glyco-engineering technology of the Pichia pastoris N-glycosylation pathway developed by GlycoFi allows producing human proteins with complex N-glycosylation modifications, which are similar to the ones performed in human. Moreover, more homogeneous glycosylation patterns are observed, as opposed to the large heterogeneity of glycan moieties that are found naturally in mammals or in other production systems such as Chinese hamster ovary and NS0 cell lines. These properties associated with the perspective to industrialize the manufacturing process of Pichia makes it a very promising expression system to produce large-scale batches of therapeutics at a lower cost.

IBC’s 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society: December 3–6, 2012, San Diego, CA

The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3–5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4–5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society’s special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5–6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.

6th Annual European Antibody Congress 2010: November 29-December 1, 2010, Geneva, Switzerland.

The 6th European Antibody Congress (EAC), organized by Terrapinn Ltd., was held in Geneva, Switzerland, which was also the location of the 4th and 5th EAC.1,2 As was the case in 2008 and 2009, the EAC was again the largest antibody congress held in Europe, drawing nearly 250 delegates in 2010. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations and representatives from the US Food and Drug Administration (FDA). The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations of golimumab (Simponi®) and canakinumab (Ilaris®) by Johnson & Johnson and Novartis, respectively, updates on antibodies in late clinical development (obinutuzumab/GA101, farletuzumab/MORAb-003 and itolizumab/T1 h, by Glycart/Roche, Morphotek and Biocon, respectively) and success rates for this fast-expanding class of therapeutics (Tufts Center for the Study of Drug Development). Case studies covering clinical progress of girentuximab (Wilex), evaluation of panobacumab (Kenta Biotech), characterization of therapeutic antibody candidates by protein microarrays (Protagen), antibody-drug conjugates (sanofi-aventis, ImmunoGen, Seattle Genetics, Wyeth/Pfizer), radio-immunoconjugates (Bayer Schering Pharma, Université de Nantes) and new scaffolds (Ablynx, AdAlta, Domantis/GlaxoSmithKline, Fresenius, Molecular Partners, Pieris, Scil Proteins, Pfizer, University of Zurich) were presented. Major antibody structural improvements were showcased, including the latest selection engineering of the best isotypes (Abbott, Pfizer, Pierre Fabre), hinge domain (Pierre Fabre), dual antibodies (Abbott), IgG-like bispecific antibodies (Biogen Idec), antibody epitope mapping case studies (Eli Lilly), insights in FcγRII receptor (University of Cambridge), as well as novel tools for antibody fragmentation (Genovis). Improvements of antibody druggability (Abbott, Bayer, Pierre Fabre, Merrimack, Pfizer), enhancing IgG pharmacokinetics (Abbott, Chugai), progress in manufacturing (Genmab, Icosagen Cell Factory, Lonza, Pierre Fabre) and the development of biosimilar antibodies (Biocon, Sandoz, Triskel) were also discussed. Last but not least, identification of monoclonal antibodies (mAbs) against new therapeutic targets (Genentech, Genmab, Imclone/Lilly, Vaccinex) including Notch, cMet, TGFβRII, SEMA4D, novel development in immunotherapy and prophylaxis against influenza (Crucell), anti-tumor activity of immunostimulatory antibodies (MedImmune/Astra Zeneca) and translations to clinical studies including immunogenicity issues (Amgen, Novartis, University of Debrecen) were presented.

9th Annual European Antibody Congress, November 11–13, 2013, Geneva, Switzerland

The annual European Antibody Congress (EAC) has traditionally been the key event for updates on critical scientific advances in the antibody field, and 2013 was no exception. Organized by Terrapinn, the well-attended meeting featured presentations on considerations for developing antibodies and antibody-like therapeutics, with separate tracks for antibody-drug conjugates, naked antibodies, and multispecific antibodies or protein scaffolds. The overall focus of the EAC was current approaches to enhance the functionality of therapeutic antibodies or other targeted proteins, with the ultimate goal being improvement of the safety and efficacy of the molecules as treatments for cancer, immune-mediated disorders and other diseases. Roundtable discussion sessions gave participants opportunities to engage in group discussions with industry leaders from companies such as Genmab, Glenmark Pharmaceuticals, MedImmune, Merrimack Pharmaceuticals, and Pierre Fabre. As the 2013 EAC was co-located with the World Biosimilar Congress, participants also received an update on European Medicines Agency guidelines and thoughts on the future direction and development of biosimilar antibodies in the European Union.

5th European Antibody Congress 2009: November 30–December 2, 2009, Geneva, Switzerland.

The 5th European Antibody Congress (EAC), organized by Terrapin Ltd, was held in Geneva, Switzerland, which was also the location of the 4th EAC.1 With more than 220 delegates, this event was the largest antibody congress held in Europe during 2009. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations. The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations (catumaxomab, certolizumab pegol, rilonacept, ustekinumab and ofatumumab developed by Fresenius, UCB-Celltech, Regeneron, Centocor and Genmab, respectively) and success and attrition rates for this fast expending class of therapeutics. Case studies covering clinical progress in anti-CD20 (Genmab, LFB) and anti-IGF-1R mAbs (Biogen Idec, Imclone, Merck/Pierre Fabre), antibody-drug conjugates (ImmunoGen, Genentech, Seattle Genetics, Wyeth/Pfizer) and new scaffolds (Ablynx, Adnexus/Bristol-Myers Squibb, Domantis/GlaxoSmithKline, Dyax, Molecular Partners, Scil Proteins) were presented. Major antibody structural improvements were showcased, including the latest global developments in 2-in-1 antibodies (Genentech), dual antibodies (Abbott), trifunctional antibodies (Trion Pharma, Fresenius), agonist antibodies (MedImmune, Kyowa Hakko Kirin), Fc-engineered (Centocor, MedImmune), glyco-engineered (Centocor, Kyowa Hakko Kirin, Lonza) aglycosylated IgGs (University of Cambridge) and non-activating formats (Genmab). Improvements of drugability (Pierre Fabre, Pfizer), alternative quantification methods based on mass spectrometry (Novartis, CEA), progress in manufacturing (Biogen Idec, Boehringer-Ingelheim, Merck KG) and patent strategies (Edwards, Angell, Palmer & Dodge) were also discussed. Last but not least, identification of mAbs against new therapeutic targets (Pierre Fabre, Roche, Crucell) and translations to clinical studies (Novartis) were presented, as well as progresses in antibody humanization and engineering (Université de Montpellier, French Army Health Department, Merck-Serono, Pierre Fabre).

8th Annual European Antibody Congress 2012: November 27–28, 2012, Geneva, Switzerland

The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.

Abstract 2448: h224G11, a humanized whole antibody targeting the c-Met receptor, induces c-Met down-regulation and triggers ADCC functions

c-Met is the prototypic member of a sub-family of receptor tyrosine kinases (RTK) which also includes RON and SEA. The c-Met RTK family is structurally different from other RTK families and is the only known high-affinity receptor for hepatocyte growth factor (HGF), also called scatter factor (SF). While the controlled regulation of c-Met and HGF are essential for in mammalian development, tissue maintenance and repair, their deregulation is implicated in the progression of cancers. Aberrant signalling of c-Met can arise by ligand-dependent and independent mechanisms such as over-expression of c-Met, paracrine or autocrine activations, or through gain of function mutations. However, an oligomerization of the c-Met receptor, either in the presence or absence of the ligand, is required to regulate the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. An inappropriate c-Met activation plays a crucial role in tumorigenesis and metastasis. We have generated, a humanized anti-c-Met IgG1 monoclonal antibody (h224G11) that displays fully antagonist activities when used as a whole antibody. The h224G11 Mab exhibits a high affinity for the receptor, blocks HGF binding, inhibits c-Met phosphorylation and c-Met dimerization. These effects are presumably responsible for the inhibition of the major functions of c-Met including cell proliferation, migration, invasion but also cell scattering and angiogenesis, observed in vitro with different cell lines. In addition to these in vitro properties, h224G11 dramatically inhibits in vivo growth of autocrine (U87-MG), partially autophosphorylated (NCI-H441) and c-Met amplified cell lines (MKN45, EBC1 and Hs746T). Recent in vitro experiments demonstrate that h224G11 down-regulates c-Met on many cells including A549 and NCI-H441. Using an ELISA immunoassay, it has been shown that this down-regulation does not result from a shedding process but that h224G11 induces c-Met internalization and degradation. Similar results were obtained in ex-vivo experiments using Hs764T tumors from xenografted mice. In addition to its direct effect on c-Met modulation, the humanized antibody also triggers effector functions. A549, NCI-H441 and Hs746T were used as target cells in an ADCC chromium-released assay with human purified NK cells. In these assays, the h224G11 demonstrates a significant ADCC activity. Taken together the in vitro and in vivo data suggest that the h224G11 antibody targeting c-Met is a promising candidate for the treatment of ligand-dependent and ligand-independent tumors as a single or combined therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2448.

Abstract B127: Single or combined in vivo therapies of cancer with h224G11, a humanized antibody targeting the c‐Met receptor

c‐Met is the prototypic member of a sub‐family of receptor tyrosine kinases (RTK) which also includes RON and SEA. The c‐Met RTK family is structurally distinct from other RTK families and is the only known highaffinity receptor for the hepatocyte growth factor (HGF), also called scatter factor (SF). While the controlled regulation of c‐Met and HGF is essential for in mammalian development, tissue maintenance and repair, their deregulation is implicated in the progression of cancers. Aberrant signalling of c‐Met can arise by ligand‐dependent and independent mechanisms such as over‐expression of c‐Met, paracrine or autocrine activations, or through gain‐of‐function mutations. However, an oligomerization of the c‐Met receptor, either in the presence or absence of the ligand, is required to regulate the binding affinity and binding kinetics of the kinase toward ATP and tyrosine‐containing peptide substrates. An inappropriate c‐Met activation plays a crucial role in tumorigenesis and metastasis. We have generated, a humanized anti‐c‐Met IgG1 monoclonal antibody (h224G11) that display fully antagonist activities. The h224G11 Mab exhibits a high affinity for the receptor, blocks HGF binding, inhibits c‐Met phosphorylation and c‐Met dimerization. These effects are presumably responsible for the inhibition of the major functions of c‐Met including cell proliferation, migration, invasion but also cell scattering and angiogenesis, observed in vitro with different cell lines. In addition to these in vitro properties, h224G11 dramatically inhibits in vivo growth of autocrine (U87‐MG), partially autophosphorylated (NCI‐H441) and c‐Met amplified cell lines (MKN45, EBC1 and Hs746T). A dose effect experiment showed that a single i.v. injection of h224G11 resulted in a significant tumor inhibition with an in vivo EC50 about 9 mg/kg. Ex‐vivo experiments on the Hs746T gastric tumor, demonstrated that h224G11 down‐regulates c‐Met, inhibits cell proliferation, induces a massive apoptosis of tumor cells and abrogates c‐Met phosphorylation. In addition to this powerful effect as a mono‐therapy, an almost complete inhibition of NCI‐H441 (NSCLC) tumor growth was observed when mice were treated with the anti‐c‐Met antibody combined with the tubulin‐binding vinca‐alkaloid vinorelbine (Navelbine®). Likewise, our results showed that a combined h224G11/temozolomide bi‐therapy significantly improved in vivo growth inhibition of U87‐MG tumors compared to the corresponding mono therapies. Taken together the in vitro and in vivo data suggest that the h224G11 antibody targeting c‐Met is a promising candidate for the treatment of ligand‐dependent and ligand‐independent tumors as a single or combined therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B127.